antibodies
ArticleEpitope Mapping of Anti-Mouse CCR3 Monoclonal Antibodies
Using Flow Cytometry
Nami Tateyama 1,† , Teizo Asano 1,† , Hiroyuki Suzuki 2, * , Guanjie Li 2 , Takeo Yoshikawa 3 ,Tomohiro Tanaka 1 , Mika K. Kaneko 1 and Yukinari Kato 1,2,3, *
1

2

3

*
†

Citation: Tateyama, N.; Asano, T.;
Suzuki, H.; Li, G.; Yoshikawa, T.;
Tanaka, T.; Kaneko, M.K.; Kato, Y.
Epitope Mapping of Anti-Mouse
CCR3 Monoclonal Antibodies Using

Department of Antibody Drug Development, Tohoku University Graduate School of Medicine,
Sendai 980-8575, Japan
Department of Molecular Pharmacology, Tohoku University Graduate School of Medicine,
Sendai 980-8575, Japan
Department of Pharmacology, Tohoku University Graduate School of Medicine, Sendai 980-8575, Japan
Correspondence: hiroyuki.suzuki.b4@tohoku.ac.jp (H.S.); yukinari.kato.e6@tohoku.ac.jp (Y.K.);
Tel.: +81-22-717-8207 (H.S. & Y.K.)
These authors contributed equally to this work.

Abstract: The CC chemokine receptor 3 (CCR3) is a receptor for CC chemokines, including CCL5/
RANTES, CCL7/MCP-3, and CCL11/eotaxin. CCR3 is expressed on the surface of eosinophils, basophils, a subset of Th2 lymphocytes, mast cells, and airway epithelial cells. CCR3 and its ligands are
involved in airway hyperresponsiveness in allergic asthma, ocular allergies, and cancers. Therefore,
CCR3 is an attractive target for those therapies. Previously, anti-mouse CCR3 (mCCR3) monoclonal
antibodies (mAbs), C3 Mab-3 (rat IgG2a , kappa), and C3 Mab-4 (rat IgG2a , kappa) were developed using the Cell-Based Immunization and Screening (CBIS) method. In this study, the binding epitope ofthese mAbs was investigated using flow cytometry. A CCR3 extracellular domain-substituted mutant
analysis showed that C3 Mab-3, C3 Mab-4, and a commercially available mAb (J073E5) recognized
the N-terminal region (amino acids 1–38) of mCCR3. Next, alanine scanning was conducted in the
N-terminal region. The results revealed that the Ala2, Phe3, Asn4, and Thr5 of mCCR3 are involvedin C3 Mab-3 binding, whereas Ala2, Phe3, and Thr5 are essential to C3 Mab-4 binding, and Ala2 andPhe3 are crucial to J073E5 binding. These results reveal the involvement of the N-terminus of mCCR3
in the recognition of C3 Mab-3, C3 Mab-4, and J073E5.

Flow Cytometry. Antibodies 2022, 11,75. https://doi.org/10.3390/

Keywords: mouse CCR3; monoclonal antibody; epitope mapping; alanine scanning; flow cytometry

antib11040075
Academic Editor: Tianlei Ying
Received: 2 November 2022
Accepted: 29 November 2022
Published: 2 December 2022
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims inpublished maps and institutional affiliations.

Copyright: © 2022 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms andconditions of the Creative Commons
Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

1. Introduction
Chemokines are a family of small cytokines secreted by cells, and they play essential
roles in cell migration, inflammation, and immune responses by binding to chemokine
receptors [1–4]. The CC chemokine receptor 3 (CCR3) is a receptor for CC chemokines,
including CCL5/RANTES, CCL7/MCP-3, and CCL11/eotaxin [5–7]. CCR3 is expressedon the surface of eosinophils, basophils, a subset of Th2 lymphocytes, mast cells, andairway epithelial cells [8–13]. CCR3 is a family of G-protein-coupled receptors (GPCRs)
that transduce extracellular signals to intracellular signaling molecules [14]. The CCR3
signaling pathway is critical in eosinophil migration [15,16]. It has been reported that
CCR3 and its ligands can cause airway hyperresponsiveness in a murine allergic asthma
model [17–20], contributing to ocular allergies [21]. Moreover, elevated eotaxin expression
has been observed in colorectal cancer [22], breast cancer [23], and oral squamous cell
carcinomas [24]. Therefore, CCR3 and its ligands are therapeutic targets for allergic diseases
and cancers [7,25].
GPCR has seven transmembrane domains; four extracellular regions, including an
N-terminal region (amino acids [aa] 1–38); and three extracellular loops (ECL1; aa 96–111,
ECL2; aa 176–207, and ECL3; aa 269–285). Previously, monoclonal antibodies (mAbs) were

Antibodies 2022, 11, 75. https://doi.org/10.3390/antib11040075

https://www.mdpi.com/journal/antibodies

Antibodies 2022, 11, 75

2 of 11developed against GPCRs, including an anti-mouse CCR2 mAb [26], an anti-human CCR2
mAb [27], an anti-mouse CCR3 (mCCR3) mAb [28–30], an anti-mouse CCR4 mAb [31],an anti-mouse CCR8 mAb [32–34], an anti-human CCR9 mAb [35], and an anti-mouse
CXCR6 mAb [36]. The binding epitopes of anti-CCR2, CCR4, CCR9, and CXCR6 mAbs,
which were established using the peptide immunization method, were determined using
an enzyme-linked immunosorbent assay (ELISA) [37–39]. However, anti-mCCR3 mAbs,
C3 Mab-3 (rat IgG2a , kappa) and C3 Mab-4 (rat IgG2a , kappa), were established using the
Cell-Based Immunization and Screening (CBIS) method [30]. We encountered difficulty
in determining the epitopes by ELISA in mAbs developed using the CBIS method [40,41].
Therefore, flow cytometry could have better coverage than ELISA for epitope mapping.
In this study, the epitope mapping of anti-mCCR3 mAbs was conducted using flow
cytometry, utilizing the extracellular region substitution and the alanine scanning methods
to clarify the features of C3 Mab-3 and C3 Mab-4.
2. Materials and Methods
2.1. Cell Lines
Chinese hamster ovary (CHO)-K1 cells were obtained from the America Type Culture
Collection (ATCC, Manassas, VA, USA). The CHO/mCCR3 cells were produced in ourprevious study [28]. The chimera and the point mutant plasmids were transfected into
CHO-K1 cells using the Neon Transfection System (Thermo Fisher Scientific Inc., Waltham,
MA, USA). Stable transfectants were selected using a cell sorter (SH800; Sony Biotechnology
Inc., Tokyo, Japan). The cells were cultured in Roswell Park Memorial Institute (RPMI) 1640
medium (Nacalai Tesque, Inc., Kyoto, Japan) supplemented with 10% heat-inactivated fetal
bovine serum (FBS) (Thermo Fisher Scientific Inc.), 100 units/mL of penicillin, 100 µg/mL
streptomycin, and 0.25 µg/mL amphotericin B (Nacalai Tesque, Inc.) at 37 ◦ C in a humidified atmosphere containing 5% CO2 . The stable transfectants were cultivated in a medium
containing 0.5 mg/mL Zeocin (InvivoGen, San Diego, CA, USA).
2.2. Plasmid Construction
Synthesized DNA (Eurofins Genomics KK, Tokyo, Japan) encoding mCCR3 (Accession
No.: NM_009914.4) [28–30] and mouse CCR8 (mCCR8; Accession No.: NM_007720.2) [32–34]
were subcloned into a pCAG-Ble vector (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan). Chimeric mutants mCCR8 (mCCR3p1-38), mCCR8 (mCCR3p96-111), mCCR8(mCCR3p176-207), and mCCR8 (mCCR3p269-285) were produced with a RAP [42,43] anda MAP tag [44,45] at their C-terminus using a HotStar HiFidelity polymerase kit (Qiagen
Inc., Hilden, Germany). Alanine (glycine) substitutions in the mCCR3 N-terminal region
were conducted using QuikChange Lightning Site-Directed Mutagenesis Kits (Agilent
Technologies Inc., Santa Clara, CA, USA). PCR fragments bearing the desired mutations
were inserted into the pCAG-Ble vector (FUJIFILM Wako Pure Chemical Corporation)using an In-Fusion HD Cloning Kit (TaKaRa Bio, Inc., Shiga, Japan).
2.3. Antibodies
C3 Mab-4 was established using the CBIS method together with C3 Mab-3 [30]. C3 Mab7 was established using N-terminal peptide immunization as described previously [29]. An
anti-mCCR3 mAb (clone J073E5) was purchased from BioLegend (San Diego, CA, USA). A
secondary Alexa Fluor 488-conjugated anti-rat IgG was also purchased from Cell Signaling
Technology, Inc. (Danvers, MA, USA).
2.4. Flow Cytometry
The cells were harvested after a brief exposure to 0.25% trypsin/1 mM ethylenediaminetetraacetic acid (Nacalai Tesque, Inc.). After washing with 0.1% bovine serum
albumin in phosphate-buffered saline, the cells were treated with primary mAbs (1 µg/mL)
for 30 min at 4 ◦ C and subsequently with Alexa Fluor 488-conjugated anti-rat IgG (1:1000;

2.4. Flow Cytometry

Antibodies 2022, 11, 75

The cells were harvested after a brief exposure to 0.25% trypsin/1 mM ethylenediaminetetraacetic acid (Nacalai Tesque, Inc.). After washing with 0.1% bovine serum albumin in phosphate-buffered saline, the cells were treated with primary mAbs (1 μg/mL) for
3 of 1130 min at 4 °C and subsequently with Alexa Fluor 488-conjugated anti-rat IgG (1:1000; Cell
Signaling Technology, Inc., Danvers, MA, USA). Fluorescence data were obtained using
an EC800 Cell Analyzer (Sony Biotechnology Inc.).
Cell Signaling Technology, Inc., Danvers, MA, USA). Fluorescence data were obtained
using
an EC800 Cell Analyzer (Sony Biotechnology Inc.).
3. Results
3.1.
Determination of the Epitope of Anti-mCCR3 mAbs Using Flow Cytometry and Chimeric
3.
Results
Proteins
3.1. Determination of the Epitope of Anti-mCCR3 mAbs Using Flow Cytometry and ChimericProteins
C3Mab-3 and C3Mab-4 were established using the CBIS method, and they are applicableCto
flow cytometry[29].were
To investigate
binding
epitopesof Cand
C3Mab3Mab-3
and C3 Mab-4
establishedthe
using
the CBIS
method,theyandare applica3 Mab-3
4, we
on four [29].
extracellularregions
ofbinding
mCCR3,epitopes
namely,of
the
(aa
ble
to focusedflow cytometry
To investigate
theC3N-terminal
Mab-3 and region
C3 Mab-4,1-38),
ECL1(aa
96-111),ECL2(aa
176-207),
and
ECL3
(aa
269-285).
The
four
extracellular
we focused on four extracellular regions of mCCR3, namely, the N-terminal region (aa 1-38),
regionsmCCR3ECL2
were(aasubstituted
corresponding
ofextracellular
mCCR8, which
posECL1(aaof96-111),
176-207),into
and theECL3(aa 269-285).regions
The fourregions
sessesan aminoacid structure
to that of mCCR3.
shown inFigure1, mCCR8
ofmCCR3
were substituted
intosimilar
the corresponding
regions As
of mCCR8,
which
possessesanaminoacid structure
similarto that of mCCR3.mCCR8
As shown
in Figure 1, mCCR8and(mCCR3p1(mCCR3p1-38),mCCR8(mCCR3p96-111),(mCCR3p176-207),
mCCR8
38),
mCCR8 (mCCR3p96-111),
mCCR8
mCCR8
(mCCR3p269-285)(mCCR3p269-285)
were generated.
The(mCCR3p176-207),
chimeric proteins andweretransiently
expressed on
weregenerated.
The
chimeric
proteins were
oncommercially
the CHO-K1 cells,
andthe CHO-K1
cells,
andtheir reactivities
to Ctransiently
Mab-4, andavailable
3Mab-3, C3expressedtheirreactivities
toC
Mab-3,
C
Mab-4,
andcommerciallyavailable
J073E5
wereanalyzed
J073E5 were analyzed
cytometry. As shown in Figure 2, C3Mab-3, C3Mab-4,
3 using flow
3
using
flow cytometry.
As shown
in mCCR8
Figure 2,(mCCR3p1-38).
C3 Mab-3, C3 Mab-4,
and J073E5
slightly
reactedand J073E5
slightly reacted
with
In contrast,
they
did notreact
with
In contrast,
they did not react with
mCCR8(mCCR3p269-285).
(mCCR3p96-111),
with mCCR8
mCCR8 (mCCR3p1-38).
(mCCR3p96-111),mCCR8 (mCCR3p176-207),or mCCR8mCCR8(mCCR3p176-207),or mCCR8(mCCR3p269-285).
reactedcells.
withThese
mCCR3,
They reactedwith mCCR3, which
was stably
overexpressedThey
in CHO-K1
rewhichwas stably
overexpressedin CHO-K1
cells.isThese
resultsby
showthat thesults showthat theN-terminal regionof mCCR3
recognized
C3Mab-3,C3N-terminal
Mab-4, andregionJ073E5.of mCCR3 is recognized by C3 Mab-3, C3 Mab-4, and J073E5.

Figure 1.
1. Schematic
Schematic illustration
illustration ofof mCCR8mCCR8 andand mCCR3mCCR3 chimericchimeric proteins.
proteins. The
The fourfour extracellular
extracellular
Figureregions of mCCR3, namely, (A) the N-terminal region (aa 1–38), (B) ECL1 (aa 96–111), (C) ECL2 (aaregions of mCCR3, namely, (A) the N-terminal region (aa 1–38), (B) ECL1 (aa 96–111), (C) ECL2 (aa176–207), and (D) ECL3 (aa 269–285), were substituted into the corresponding regions of mCCR8.
176–207), and (D) ECL3 (aa 269–285), were substituted into the corresponding regions of mCCR8.
ECL, extracellular loop, aa; amino acids.
ECL, extracellular loop, aa; amino acids.

Antibodies 2022,
2022, 11,
11, x75FOR PEER REVIEW
Antibodies

11
4 4ofof11

FigureFigure 2.
2. Determination
Determination ofof thethe epitope
epitope ofof anti-mCCR3
anti-mCCR3 mAbs
mAbs using
using flow
flow cytometry
cytometry andand chimericchimeric
proteins.
C
3Mab-3 (1 μg/mL) (A), C3Mab-4 (1 μg/mL) (B), and J073E5 (1 μg/mL) (C) were treatedproteins. C3 Mab-3 (1 µg/mL) (A), C3 Mab-4 (1 µg/mL) (B), and J073E5 (1 µg/mL) (C) were treatedwith◦ C, followedwith CHO-K1
CHO-K1 cells
cells thatthat transiently
transiently expressed
expressed chimericchimeric proteinsproteins forfor 3030 minmin atat 44 °C,
followed byby thetheaddition of Alexa 488-conjugated anti-rat IgG. Red lines show the cells with anti-mCCR3 mAbs
addition of Alexa 488-conjugated anti-rat IgG. Red lines show the cells with anti-mCCR3 mAbs
treatment, and black lines show cells without anti-mCCR3 mAbs treatment as a negative control.
treatment, and black lines show cells without anti-mCCR3 mAbs treatment as a negative control.

3.2.
3.2. Determination
Determination ofof thethe CC33Mab-3
Mab-3Epitope
EpitopeUsing
UsingFlow
FlowCytometry
Cytometryand
andAlanine
AlanineScanning
Scanning
Next,
Next, alanine
alanine scanningscanning waswas conductedconducted inin thethe N-terminal
N-terminalregion,
region,except
exceptfor
forCys28.
Cys28.
Thirty-six
of of
mCCR3
were
constructed,
and andthe mutant
proThirty-sixalanine
alaninesubstitution
substitutionmutants
mutants
mCCR3
were
constructed,
the mutant
teins
were
transiently
expressed
onon
thetheCHO-K1proteins
weretransiently
expressed
CHO-K1cells.
cells.The
Thereactivity
reactivityagainst
against C
C33Mab-3,
Mab-3,
C
cytometry. As
Asshownshownin
inFigure
Figure3A,
3A,CC33Mab-3
MabC33Mab-4,
Mab-4,andand J073E5
J073E5 was
was assessed
assessed using flow cytometry.
3did
didnotnotreact
reactwithwithfourfourmutants
mutants(A2G,(A2G,F3A,
F3A,N4A,
N4A, andand T5A).
T5A). In contrast, C33Mab-3
reactedMab-3 reacted
withwith thethe otherother 32
32 mutants.
mutants. These
These results
results show
show thatthat four
four residues
residues (Ala2,(Ala2, Phe3,
Phe3, Asn4,
Asn4, andandThr5)Thr5) ofof mCCR3
mCCR3 areare important
important for C33Mab-3
Mab-3 binding
binding (Figure(Figure 3B).
3B). CC33Mab-4
Mab-4 did
did not
not react
react
with
(A2G, F3A,
F3A,andandT5A)
T5A)but
butreacted
reactedwith
withthetheothers
others(Figure(Figure4A),
indicatwith three mutants (A2G,
4A),
indicating
3Mab-4ing
three
residues(Ala2,
Phe3,
Thr5)
of mCCR3
importantbindthatthat
three
residues(Ala2,
Phe3,
andandThr5)
of mCCR3
areareimportant
forfor
C3 C
Mab-4
binding
ing(FigureJ073E5react
mutants(A2G
F3A)
reactedwith(Figure4B).4B).
J073E5
did did
not not
react
withwith
twotwomutants(A2G
andandF3A)
but but
reactedwiththeothers(Figure5A),5A),
indicatingthat that
two residues(Ala2(Ala2and Phe3)of mCCR3
are importanttheothers(Figureindicating
two residues
and Phe3)of mCCR3
are imfor J073E5(Figure(Figure
5B). The5B).
cellThesurfaceexpressionof the mCCR3
mutants
on theportant
for binding
J073E5 binding
cell surface
expressionof the mCCR3
mutants
CHO-K1
cells wasusing using
the anti-mCCR3
mAb, mAb,
C3 Mab-7.
It hasItalreadybeen
on
the CHO-K1
cellsconfirmed
was confirmed
the anti-mCCR3
C3Mab-7.
has alreadyconfirmedthat Phe15
and Glu16
are essential
for C3forMab-7
binding(manuscript
submitted).
been
confirmedthat Phe15
and Glu16
are essential
C3Mab-7binding(manuscript
subWe couldWe
confirm
cell surface
expressionof four mutants(A2G,
F3A, (A2G,
N4A, andofmitted).
couldtheconfirmthe cellsurface expressionof fourmutants
F3A,T5A)
N4A,
mCCR3
using
C
Mab-7
(Figure6).
and T5A) of mCCR3
using C3Mab-7 (Figure 6).
32022,
11, 752022, 11, xAntibodies
FOR PEER
REVIEW

5 of 115 of 11

Figure 3. Determination
the C3Mab-3 epitope
flow cytometry and alanine scanning. (A)Figure 3.ofDeterminationof the Cusing
3 Mab-3 epitope using flow cytometry and alanine scanning.
C3Mab-3 (1 μg/mL)
wastreatedwith
CHO-K1cells
thattransiently
expressed
mutant proteinsfor mutant pro(A) C3 Mab-3 (1 µg/mL) was treated with
CHO-K1 cells
that transiently
expressed
30 min at 4 °C, followedbytheadditionofAlexa
488-conjugatedanti-rat
IgG.
Red
lines
show
the
teins for 30 min at 4 ◦ C, followed by the addition of Alexa 488-conjugated anti-rat IgG. Red lines
cells with C3Mab-3 treatment, and black lines show cells without Ab treatment as a negative control.
show the cells with C3 Mab-3 treatment, and black lines show cells without Ab treatment as a negative(B) The C3Mab-3 epitope for mCCR3 involves Ala2, Phe3, Asn4, and Thr5 of mCCR3.
control. (B) The C3 Mab-3 epitope for mCCR3 involves Ala2, Phe3, Asn4, and Thr5 of mCCR3.

2022,
11, 752022, 11, xAntibodies
FOR PEER
REVIEW

6 of 11

6 of 11

Figure 4. Determination of the C3Mab-4 epitope using flow cytometry and alanine scanning. (A)Figure 4. Determination of the C3 Mab-4 epitope using flow cytometry and alanine scanning.
C3Mab-4 (1 μg/mL) was treated with CHO-K1 cells that transiently expressed mutant proteins for(A) C3 Mab-4 (1 µg/mL) was treated with CHO-K1 cells that transiently expressed mutant pro30 min at 4 °C, followed by the additionof Alexa 488-conjugated anti-rat IgG. Red lines show theteins for 30 min at 4 ◦ C, followed by the addition of Alexa 488-conjugated anti-rat IgG. Red lines
cells with C3Mab-4 treatment, and black lines show cells without Ab treatment as a negative control.
show the cells with C Mab-4 treatment, and black lines show cells without Ab treatment as a negative(B) The C3Mab-4 epitope for mCCR33 involves Ala2, Phe3, and Thr5 of mCCR3.
control. (B) The C3 Mab-4 epitope for mCCR3 involves Ala2, Phe3, and Thr5 of mCCR3.

11, 75
2022, 11, x Antibodies
FOR PEER2022,
REVIEW

7 of 11

7 of 11

Figure 5. Determination
the J073E5 of
epitopeusingflow using
cytometry
and alanine
scanning.
(A) (A) J073E5
Figure 5. ofDetermination
the J073E5
epitopeflow cytometry
and alanine
scanning.
J073E5 (1 μg/mL) was treated with CHO-K1 cells that transiently expressed mutant proteins for 30(1 µg/mL) was treated with CHO-K1 cells that transiently expressed mutant proteins for 30 min at
min at 4 °C, followedby the addition of Alexa 488-conjugated anti-rat IgG. Red lines show the cells
4 ◦ C, followed by the addition of Alexa 488-conjugated anti-rat IgG. Red lines show the cells with
with J073E5 treatment, and black lines show cells without Ab treatment as a negative control. (B)J073E5 treatment, and black lines show cells without Ab treatment as a negative control. (B) The
The J073E5 epitope for mCCR3 involves Ala2 and Phe3 of mCCR3.
J073E5 epitope for mCCR3 involves Ala2 and Phe3 of mCCR3.

AntibodiesAntibodies
2022, 11, x2022,
FOR11,
PEER
75 REVIEW

8 of 118 of 11

Figure 6.Figure
Cell surface
of mCCR3
on CHO-K1
cells using
flow
cytometry.
C3Mab-C Mab-7
6. Cellexpression
surface expressionof mutants
mCCR3 mutantson CHO-K1
cells
using
flow cytometry.
3
7 (1 μg/mL)
wastreatedwith
CHO-K1cells
thattransiently
expressed
mutantproteins
for30
min(1 µg/mL) was treated with CHO-K1 cells that transiently expressed mutant proteins
forat30 min at
4 °C, followedby the addition of Alexa 488-conjugated anti-rat IgG. Red lines show the cells with
4 ◦ C, followed by the addition of Alexa 488-conjugated anti-rat IgG. Red lines show the cells with
C3Mab-7 treatment, and black lines show cells without Ab treatment as a negative control.
C3 Mab-7 treatment, and black lines show cells without Ab treatment as a negative control.

4. Discussion
4. Discussion
We previously
establishedvarious various
mAbs against
proteinsproteinsusing the
CBISthe CBIS
We previously
establishedmAbs membrane
against membraneusing
method.method.
BecauseBecause
mAbs sometimes
recognize
conformational
epitopes,
they
can
be
mAbs sometimes recognize conformational epitopes, they canapbe applied
plied toto
flow
blotting or
orELISA.
ELISA.Two
Twoanti-mCCR3
anti-mCCR3
mAbs
flowcytometry
cytometrybut
butnotnotto
to Western
Western blotting
mAbs
examined
3Mab-4,
examined
thisstudy,
C3Mab-3
andwereestablishedestablishedusing
usingthetheCBIS
CBISmethod
method[30]. We
in in
this
study,
C3 Mab-3
andC3C
Mab-4,
were[30]. Weattempted
attemptedtotoidentify
identifytheir
theirepitopes
epitopesusing
usingsynthetic
syntheticpeptides
peptidesandandELISA.
ELISA.However,
Howthey
ever, theydidnotrecognizethesynthetic
peptides,includingthemCCR3
N-terminal
redid not recognize the synthetic peptides, including the mCCR3 N-terminal region (aa 1–19),
gion (aawhich
1–19),contains
which contains
the epitope
determinedusing
flow cytometry(Figures
3 Thesethe epitope
determinedusing flow
cytometry(Figures3 and 4).
and 4). Thesesuggestthat
the residues
participate
in the formationof conforma-epitopes
resultsresults
suggestthat theresidues
participate
in the formationof conformationaltional epitopes
and/or undergo
post-translational
modification
the cellFurthermore,
surface. Fur-we could
and/or undergo
post-translational
modificationon the cellon
surface.
thermore,
could notthe ofpossibility
theasfirstMet
as their epitopes.
Inof
theCcasenotwe
excludetheexclude
possibility
the first of
Met
their
epitopes.
In the case
3 Mab-3 andC3 Mab-4
Asn4 and
Thr5andare Thr5
involved
in recognition.
AlthoughAlthough
Asn and Thr are
3Mab-4 epitopes,of C3Mab-3
and Cepitopes,
Asn4
are involved
in recognition.
be N- and
respectively,
there are noreports
onreports
the glycosylation
Asn andknown
Thr aretoknown
to beO-glycosylated,
N- and O-glycosylated,
respectively,
there
are no
on
of
the
Asn4
and
Thr5
of
mCCR3.
Furtherstudiesarerequiredtoanalyzetheinvolvement
the glycosylation of the Asn4 and Thr5 of mCCR3. Further studies are required to analyze
the involvement of the post-translational modification of these residues in the recognition
by C3Mab-3 and C3Mab-4.

Antibodies 2022, 11, 75

9 of 11of the post-translational modification of these residues in the recognition by C3 Mab-3 andC3 Mab-4.
Other anti-mCCR3 mAbs, C3 Mab-6, and C3 Mab-7, were also developed via mCCR3
N-terminal peptide immunization [29]. It was found that they could recognize the synthetic
peptide of the mCCR3 N-terminal region (aa 1-19) using ELISA and cell-surface-expressed
mCCR3 using flow cytometry. Furthermore, the Phe3, Asn4, Thr5, Asp6, Glu7, Lys9, Thr10,
and Glu13 of mCCR3 were determined to be C3 Mab-6 epitope, whereas Phe15 and Glu16
were determined to be C3 Mab-7 epitope (manuscript submitted). These results indicatethat C3 Mab-6 and C3 Mab-7 recognize both the naked N-terminal peptide and cell-surfaceexpressed mCCR3. Further studies are essential to understand the difference in mCCR3
recognition between C3 Mab-3/C3 Mab-4 and C3 Mab-6/C3 Mab-7.
It has been reported that a CCR3 ligand, CCL11/eotaxin, binds to the N-terminal
region of CCR3 [46,47]. Therefore, our established anti-mCCR3 mAbs could compete withligand binding to mCCR3 and have neutralizing activity. Shen et al. reported that antiCCR3 mAb could significantly suppress airway eosinophilia and mucus overproduction inasthmatic mice; therefore, the blockage of the CCR3 axis may be an attractive strategy forasthma therapy [48]. In future studies, we would like to examine the neutralizing activitiesof these anti-mCCR3 mAbs.
Author Contributions: Conceptualization, M.K.K. and Y.K.; Funding acquisition, Y.K.; Investigation,
N.T., T.A., G.L., T.Y., T.T. and M.K.K.; Methodology, M.K.K.; Supervision, Y.K.; Writing—original draft,
N.T., T.A. and H.S.; Writing—review & editing, H.S. and Y.K. All authors agreed to be accountable
for all aspects of the research in ensuring that the accuracy or integrity of any part of the work is
appropriately investigated and resolved. All authors have read and agreed to the published versionof the manuscript.
Funding: 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.), and JP21am0101078 (to Y.K.), and by the Japan
Society for the Promotion of Science (JSPS) Grants-in-Aid for Scientific Research (KAKENHI) grantnos. 21K20789 (to T.T.), 22K06995 (to H.S.), 22K15523 (to T.A.), 22K07168 (to M.K.K.), and 22K07224(to Y.K.).
Data Availability Statement: Data is contained within the article.
Acknowledgments: The authors would like to thank Saori Okuno and Saori Handa (Departmentof Antibody Drug Development, Tohoku University Graduate School of Medicine) for technicalassistance in the experiments.
Conflicts of Interest: The authors declare no conflict of interest involving this article.

References
1.
2.
3.
4.
5.

6.
7.
8.

Kufareva, I.; Salanga, C.L.; Handel, T.M. Chemokine and chemokine receptor structure and interactions: Implications fortherapeutic strategies. Immunol. Cell Biol. 2015, 93, 372–383. [CrossRef] [PubMed]
Zlotnik, A.; Yoshie, O. Chemokines: A New Classification System and Their Role in Immunity. Immunity 2000, 12, 121–127.
[CrossRef] [PubMed]
Charo, I.F.; Ransohoff, R.M. The Many Roles of Chemokines and Chemokine Receptors in Inflammation. N. Engl. J. Med. 2006,
354, 610–621. [CrossRef] [PubMed]
Murphy, P.M.; Baggiolini, M.; Charo, I.F.; Hébert, C.A.; Horuk, R.; Matsushima, K.; Miller, L.H.; Oppenheim, J.J.; Power, C.A.
International union of pharmacology. XXII. Nomenclature for chemokine receptors. Pharmacol. Rev. 2000, 52, 145–176. [PubMed]
Wise, E.L.; Duchesnes, C.; da Fonseca, P.; Allen, R.A.; Williams, T.J.; Pease, J.E. Small Molecule Receptor Agonists and Antagonistsof CCR3 Provide Insight into Mechanisms of Chemokine Receptor Activation. J. Biol. Chem. 2007, 282, 27935–27943. [CrossRef][PubMed]Bisset, L.R.; Schmid-Grendelmeier, P. Chemokines and their receptors in the pathogenesis of allergic asthma: Progress andperspective. Curr. Opin. Pulm. Med. 2005, 11, 35–42. [CrossRef]
Mackay, C.R. Moving targets: Cell migration inhibitors as new anti-inflammatory therapies. Nat. Immunol. 2008, 9, 988–998.
[CrossRef]
Willems, L.I.; Ijzerman, A.P. Small molecule antagonists for chemokine CCR3 receptors. Med. Res. Rev. 2010, 30, 778–817.
[CrossRef]

Antibodies 2022, 11, 75

9.
10.
11.

12.
13.
14.
15.

16.

17.

18.
19.
20.
21.
22.
23.
24.
25.
26.

27.

28.

29.

30.

31.

32.

33.

10 of 11

Stellato, C.; Brummet, M.E.; Plitt, J.R.; Shahabuddin, S.; Baroody, F.M.; Liu, M.C.; Ponath, P.D.; Beck, L.A. Cutting Edge: Expressionof the C-C Chemokine Receptor CCR3 in Human Airway Epithelial Cells. J. Immunol. 2001, 166, 1457–1461. [CrossRef]
He, J.; Chen, Y.; Farzan, M.; Choe, H.; Ohagen, A.; Gartner, S.; Busciglio, J.; Yang, X.; Hofmann, W.; Newman, W.; et al. CCR3 andCCR5 are co-receptors for HIV-1 infection of microglia. Nature 1997, 385, 645–649. [CrossRef]
Heath, H.; Qin, S.; Rao, P.; Wu, L.; LaRosa, G.; Kassam, N.; Ponath, P.D.; Mackay, C. Chemokine receptor usage by humaneosinophils. The importance of CCR3 demonstrated using an antagonistic monoclonal antibody. J. Clin. Investig. 1997, 99, 178–184.
[CrossRef] [PubMed]
Sallusto, F.; Mackay, C.R.; Lanzavecchia, A. Selective Expression of the Eotaxin Receptor CCR3 by Human T Helper 2 Cells.
Science 1997, 277, 2005–2007. [CrossRef] [PubMed]
New, D.C.; Wong, Y.H. CC chemokine receptor-coupled signalling pathways. Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao
2003, 35, 779–788. [PubMed]
Kroeze, W.K.; Sheffler, D.J.; Roth, B.L. G-protein-coupled receptors at a glance. J. Cell Sci. 2003, 116, 4867–4869. [CrossRef][PubMed]Kampen, G.T.; Stafford, S.; Adachi, T.; Jinquan, T.; Quan, S.; Grant, J.A.; Skov, P.S.; Poulsen, L.K.; Alam, R. Eotaxin induces
degranulation and chemotaxis of eosinophils through the activation of ERK2 and p38 mitogen-activated protein kinases. Blood
2000, 95, 1911–1917. [CrossRef] [PubMed]
Zhu, Y.; Bertics, P.J. Chemoattractant-Induced Signaling via the Ras–ERK and PI3K–Akt Networks, along with Leukotriene C4
Release, Is Dependent on the Tyrosine Kinase Lyn in IL-5– and IL-3–Primed Human Blood Eosinophils. J. Immunol. 2011, 186,
516–526. [CrossRef]
Fulkerson, P.C.; Fischetti, C.A.; McBride, M.L.; Hassman, L.M.; Hogan, S.P.; Rothenberg, M.E. A central regulatory role for
eosinophils and the eotaxin/CCR3 axis in chronic experimental allergic airway inflammation. Proc. Natl. Acad. Sci. USA 2006,
103, 16418–16423. [CrossRef]
Fukuda, K.; Kuo, C.-H.; Morohoshi, K.; Liu, F.-T.; Ono, S.J. The murine CCR3 receptor regulates both eosinophilia and hyperresponsiveness in IgE-mediated allergic conjunctivitis. Br. J. Ophthalmol. 2012, 96, 1132–1136. [CrossRef]
Chantry, D.; Burgess, L.E. Chemokines in Allergy. Curr. Drug Targets-Inflamm. Allergy 2002, 1, 109–116. [CrossRef]
Pease, J.E. Asthma, Allergy and Chemokines. Curr. Drug Targets 2006, 7, 3–12. [CrossRef]
Miyazaki, D.; Nakamura, T.; Komatsu, N.; Nawata, N.; Ikeda, Y.; Inoue, Y.; Higashi, H.; Ono, S.J. Roles of Chemokines in Ocular
Allergy and Possible Therapeutic Strategies. Cornea 2004, 23, S48–S54. [CrossRef] [PubMed]
Cho, H.; Lim, S.-J.; Won, K.Y.; Bae, G.; Kim, G.Y.; Min, J.W.; Noh, B.-J. Eosinophils in Colorectal Neoplasms Associated withExpression of CCL11 and CCL24. J. Pathol. Transl. Med. 2016, 50, 45–51. [CrossRef] [PubMed]
Thomas, J.K.; Mir, H.; Kapur, N.; Bae, S.; Singh, S. CC chemokines are differentially expressed in Breast Cancer and are associatedwith disparity in overall survival. Sci. Rep. 2019, 9, 4014. [CrossRef] [PubMed]
Lorena, S.; Oliveira, D.; Dorta, R.; Landman, G.; Kowalski, L. Eotaxin expression in oral squamous cell carcinomas with andwithout tumour associated tissue eosinophilia. Oral Dis. 2003, 9, 279–283. [CrossRef]
Gerard, C.; Rollins, B.J. Chemokines and disease. Nat. Immunol. 2001, 2, 108–115. [CrossRef] [PubMed]
Tanaka, T.; Li, G.; Asano, T.; Saito, M.; Kaneko, M.K.; Suzuki, H.; Kato, Y. Development of a Novel Anti-Mouse CCR2 Monoclonal
Antibody (C2 Mab-6) by N-Terminal Peptide Immunization. Monoclon. Antibodies Immunodiagn. Immunother. 2022, 41, 80–86.
[CrossRef]
Tanaka, T.; Li, G.; Saito, M.; Suzuki, H.; Asano, T.; Kaneko, M.K.; Kato, Y. Development of an Anti-human CCR2 Monoclonal
Antibody (C2 Mab-9) by N-Terminal Peptide Immunization. Monoclon. Antibodies Immunodiagn. Immunother. 2022, 41, 188–193.
[CrossRef]
Asano, T.; Nanamiya, R.; Takei, J.; Nakamura, T.; Yanaka, M.; Hosono, H.; Tanaka, T.; Sano, M.; Kaneko, M.K.; Kato, Y.
Development of Anti-Mouse CC Chemokine Receptor 3 Monoclonal Antibodies for Flow Cytometry. Monoclon. Antibodies
Immunodiagn. Immunother. 2021, 40, 107–112. [CrossRef]
Asano, T.; Suzuki, H.; Goto, N.; Tanaka, T.; Kaneko, M.K.; Kato, Y. Establishment of Novel Anti-Mouse CCR3 Monoclonal
Antibodies (C3 Mab-6 and C3 Mab-7) by N-terminal Peptide Immunization. Monoclon. Antibodies Immunodiagn. Immunother. 2022,
41, 94–100. [CrossRef]
Asano, T.; Suzuki, H.; Tanaka, T.; Saito, M.; Li, G.; Goto, N.; Nanamiya, R.; Kaneko, M.K.; Kato, Y. C3 Mab-3: A Monoclonal
Antibody for Mouse CC Chemokine Receptor 3 for Flow Cytometry. Monoclon. Antibodies Immunodiagn. Immunother. 2022, 41,
74–79. [CrossRef]
Takei, J.; Suzuki, H.; Asano, T.; Tanaka, T.; Kaneko, M.K.; Kato, Y. Development of a Novel Anti-Mouse CCR4 Monoclonal
Antibody (C4 Mab-1) by N-Terminal Peptide Immunization. Monoclon. Antibodies Immunodiagn. Immunother. 2022, 41, 87–93.
[CrossRef] [PubMed]
Saito, M.; Suzuki, H.; Tanaka, T.; Asano, T.; Kaneko, M.K.; Kato, Y. Development of an Anti-Mouse CCR8 Monoclonal Antibody(C8 Mab-1) for Flow Cytometry and Immunocytochemistry. Monoclon. Antibodies Immunodiagn. Immunother. 2022. online ahead ofprint. [CrossRef] [PubMed]
Suzuki, H.; Saito, M.; Asano, T.; Tanaka, T.; Kitamura, K.; Kudo, Y.; Kaneko, M.K.; Kato, Y. C8 Mab-3: An Anti-Mouse CCR8
Monoclonal Antibody for Immunocytochemistry. Monoclon. Antibodies Immunodiagn. Immunother. 2022, 41, 110–114. [CrossRef][PubMed]Antibodies 2022, 11, 75

34.

35.

36.
37.
38.
39.

40.
41.

42.

43.
44.
45.
46.
47.

48.

11 of 11

Tanaka, T.; Nanamiya, R.; Takei, J.; Nakamura, T.; Yanaka, M.; Hosono, H.; Sano, M.; Asano, T.; Kaneko, M.K.; Kato, Y.
Development of Anti-Mouse CC Chemokine Receptor 8 Monoclonal Antibodies for Flow Cytometry. Monoclon. Antibodies
Immunodiagn. Immunother. 2021, 40, 65–70. [CrossRef] [PubMed]
Nanamiya, R.; Takei, J.; Asano, T.; Tanaka, T.; Sano, M.; Nakamura, T.; Yanaka, M.; Hosono, H.; Kaneko, M.K.; Kato, Y.
Development of Anti-Human CC Chemokine Receptor 9 Monoclonal Antibodies for Flow Cytometry. Monoclon. Antibodies
Immunodiagn. Immunother. 2021, 40, 101–106. [CrossRef] [PubMed]
Kitamura, K.; Suzuki, H.; Kaneko, M.K.; Kato, Y. Cx6 Mab-1: A Novel Anti-Mouse CXCR6 Monoclonal Antibody Established by
N-Terminal Peptide Immunization. Monoclon. Antibodies Immunodiagn. Immunother. 2022, 41, 133–141. [CrossRef] [PubMed]
Tanaka, T.; Li, G.; Asano, T.; Kaneko, M.K.; Suzuki, H.; Kato, Y. Epitope Mapping of the Anti-Human CCR2 Monoclonal Antibody
C2 Mab-9. Monoclon. Antibodies Immunodiagn. Immunother. 2022, 41, 150–156. [CrossRef] [PubMed]
Asano, T.; Suzuki, H.; Tanaka, T.; Kaneko, M.K.; Kato, Y. Identification of the Binding Epitope of an Anti-mouse CCR4 Monoclonal
Antibody, C4 Mab-1. Monoclon. Antibodies Immunodiagn. Immunother. 2022, 41, 214–220. [CrossRef]
Takei, J.; Asano, T.; Li, G.; Saito, M.; Suzuki, H.; Kaneko, M.K.; Kato, Y. Epitope Mapping of an Anti-Human CCR9 Monoclonal
Antibody (C9 Mab-1) Using Enzyme-Linked Immunosorbent Assay. Monoclon. Antibodies Immunodiagn. Immunother. 2021, 40,
239–242. [CrossRef]
Li, G.; Suzuki, H.; Asano, T.; Tanaka, T.; Suzuki, H.; Kaneko, M.K.; Kato, Y. Development of a Novel Anti-EpCAM Monoclonal
Antibody for Various Applications. Antibodies 2022, 11, 41. [CrossRef]
Itai, S.; Fujii, Y.; Nakamura, T.; Chang, Y.-W.; Yanaka, M.; Saidoh, N.; Handa, S.; Suzuki, H.; Harada, H.; Yamada, S.; et al.
Establishment of CMab-43, a Sensitive and Specific Anti-CD133 Monoclonal Antibody, for Immunohistochemistry. Monoclon.
Antibodies Immunodiagn. Immunother. 2017, 36, 231–235. [CrossRef] [PubMed]
Fujii, Y.; Kaneko, M.K.; Ogasawara, S.; Yamada, S.; Yanaka, M.; Nakamura, T.; Saidoh, N.; Yoshida, K.; Honma, R.; Kato, Y.
Development of RAP Tag, a Novel Tagging System for Protein Detection and Purification. Monoclon. Antibodies Immunodiagn.
Immunother. 2017, 36, 68–71. [CrossRef] [PubMed]
Miura, K.; Yoshida, H.; Nosaki, S.; Kaneko, M.K.; Kato, Y. RAP Tag and PMab-2 Antibody: A Tagging System for Detecting andPurifying Proteins in Plant Cells. Front. Plant Sci. 2020, 11, 510444. [CrossRef] [PubMed]
Fujii, Y.; Kaneko, M.K.; Kato, Y. MAP Tag: A Novel Tagging System for Protein Purification and Detection. Monoclon. Antibodies
Immunodiagn. Immunother. 2016, 35, 293–299. [CrossRef] [PubMed]
Wakasa, A.; Kaneko, M.K.; Kato, Y.; Takagi, J.; Arimori, T. Site-specific epitope insertion into recombinant proteins using the MAP
tag system. J. Biochem. 2020, 168, 375–384. [CrossRef] [PubMed]
Ye, J.; Kohli, L.L.; Stone, M.J. Characterization of binding between the chemokine eotaxin and peptides derived from thechemokine receptor CCR3. J. Biol. Chem. 2000, 275, 27250–27257. [CrossRef] [PubMed]
Millard, C.J.; Ludeman, J.P.; Canals, M.; Bridgford, J.L.; Hinds, M.G.; Clayton, D.J.; Christopoulos, A.; Payne, R.J.; Stone,
M.J. Structural Basis of Receptor Sulfotyrosine Recognition by a CC Chemokine: The N-Terminal Region of CCR3 Bound to
CCL11/Eotaxin-1. Structure 2014, 22, 1571–1581. [CrossRef] [PubMed]
Shen, H.-H.; Xu, F.; Zhang, G.-S.; Wang, S.-B.; Xu, W.-H. CCR3 monoclonal antibody inhibits airway eosinophilic inflammationand mucus overproduction in a mouse model of asthma. Acta Pharmacol. Sin. 2006, 27, 1594–1599. [CrossRef]