MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 41, Number 2, 2022
ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2021.0065
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Establishment of Novel Anti-Mouse CCR3 Monoclonal
Antibodies (C3Mab-6 and C3Mab-7) by N-terminal
Peptide Immunization

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

The CC chemokine receptor 3 (CCR3) is a member of the G protein-coupled receptor family that is highly
expressed in eosinophils and basophils. CCR3 has been proposed as a therapeutic target for human immunodeficiency virus and allergy diagnosis. Therefore, in this study, we developed specific and sensitive
monoclonal antibodies (mAbs) for mouse CCR3 (mCCR3), which are useful for flow cytometry by peptide
immunization. The established anti-mCCR3 mAbs, C3Mab-6 (rat IgG1, kappa) and C3Mab-7 (rat IgG1, kappa),
reacted with mCCR3-overexpressed Chinese hamster ovary-K1 (CHO/mCCR3), in addition to mCCR3endogenously expressed cell lines, such as P388 (mouse lymphoid neoplasma) and J774-1 (mouse macrophagelike) through flow cytometry. Kinetic analyses using flow cytometry indicated that the dissociation constants(KDs) of C3Mab-6 for CHO/mCCR3, P388, and J774-1 cells were 8.7 · 10-9 M, 1.4 · 10-7 M, and 1.7 · 10-7 M,
respectively, whereas the KDs of C3Mab-7 for these cell lines were 3.7 · 10-9 M, 5.1 · 10-7 M, and 3.1 · 10-7 M,
respectively. Results also indicated that C3Mab-6 and C3Mab-7 are useful for detecting cells expressing CCR3
through flow cytometry, thereby making them potentially beneficial for treating CCR3-expressing cells.
Keywords: mouse CCR3, monoclonal antibody, peptide immunization

tometry successfully, using the N-terminal peptides of GPCR
as an immunogen.(7,8) In contrast, immunization with cells
expressing target GPCRs is a suitable approach against other
GPCRs, such as chemokine receptors.(9–12) Besides, GPCRs
expressed on plasma membranes have native conformations.
Therefore, developed mAbs can react with native GPCRs on
plasma membrane, but not with the denatured one.
The CC chemokine receptor 3 (CCR3), also known as
CD193, is a member of the GPCR, which is highly expressedin eosinophils, basophils, and airway epithelial cells.(13,14)
Since CCR3 has been proposed as a therapeutic target forhuman immunodeficiency virus(15–17) and allergic diseases,(18–21) the development of sensitive mAbs for CCR3
has been desired. Many anti-chemokine receptor mAbs
available for flow cytometry are commercially available.
Most of them are developed through the immunization ofwhole cells or purified proteins.
However, only a few mAbs available for flow cytometry
have been developed, using immunizing peptides, for

Introduction

G

protein-coupled receptors (GPCRs) are cellsurface signal transmission receptor proteins that transfer
extracellular signals, such as light, hormones, and chemokines
to intracellular molecules.(1) They play crucial roles in thepathogenesis of diseases, including cancer, inflammation, andinfection.(2–4) Therefore, GPCRs are important targets for drug
development.(5) Alternatively, although anti-GPCR monoclonal antibodies (mAbs) are valuable tools for therapy and diagnosis, the development of anti-GPCR mAbs is challenging.
GPCRs are seven-transmembrane protein that possesses thesmall extracellular loop and N-terminal regions, which are
antibody accessible regions.
Moreover, GPCRs have complicated three-dimensional
structures, which makes their purification difficult.(6) Therefore, selecting the starting material to be used for an immunogen is important to generate anti-GPCR mAbs. Several
studies have also generated mAbs applicable to flow cy-

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

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ANTI-MOUSE CCR3 MONOCLONAL ANTIBODY

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instance, anti-CCR2 mAb (clone SN707; Thermo Fisher
Scientific, Inc., Waltham, MA) and anti-CCR5 mAb (clone
T21/8; Boster Bio, Pleasanton, CA). The synthesized peptide
immunization is a simple strategy and possesses several advantages to generate anti-CCR mAbs since the N-terminal
regions are not conserved among the families, linearly exposed on the cell surface.(22)
Furthermore, the neutralizing activity could be expected
when the N-terminal region is a ligand-binding domain. The
N-terminal region of CCR3 is a binding site for eotaxin,
which is a ligand for CCR3.(23,24) Therefore, anti-CCR3
mAbs targeting the N-terminal region of CCR3 will be usefulas an antagonistic antibody. In this study, we developed novelanti-mouse CCR3 (mCCR3) mAbs, using N-terminal peptide
immunization methods, after which we investigated its usefulness for flow cytometry, and then determined the dissociation constant to mCCR3-expressed cell lines.
Materials and Methods
PeptidesEurofins Genomics KK (Tokyo, Japan) synthesized apartial sequence of the N-terminal extracellular region ofmCCR3 (Accession No. NP_034044.3), using cysteine as its
C-terminus (mCCR3p1-19C; sequence: MAFNTDEIKTVVESFETTPC). Subsequently, the keyhole limpet hemocyanin (KLH) was conjugated at the C-terminus of the peptide(mCCR3p1-19C-KLH).
Preparation of cell lines

Chinese hamster ovary (CHO)-K1 and P3X63Ag8U.1(P3U1) cells were obtained from the American Type Culture
Collection (Manassas, VA). P388 (mouse lymphoid neoplasma) and J774-1 (mouse macrophage-like) cells wereobtained from the Cell Resource Center for Biomedical Research Institute of Development, Aging and Cancer Tohoku
University (Miyagi, Japan). The synthesized DNA (Eurofins
Genomics KK) encoding mCCR3 (Accession No.
NM_009914.4) was subsequently subcloned into a pCAGBle vector (FUJIFILM Wako Pure Chemical Corporation,
Osaka, Japan). Then, the mCCR3 plasmid was transfected
into CHO-K1 and LN229 cells, using a Neon transfection
system (Thermo Fisher Scientific, Inc.).
Afterward, stable transfectants were established through cellsorting using a cell sorter (SH800; Sony Corp., Tokyo, Japan),after which cultivation in a medium, containing 0.5 mg/mL of
Zeocin (InvivoGen, San Diego, CA) was conducted. CHO-K1,
P3U1, mCCR3-overexpressed CHO-K1 (CHO/mCCR3), P388,
and J774-1 were cultured in a Roswell Park Memorial Institute(RPMI) 1640 medium (Nacalai Tesque, Inc., Kyoto, Japan)
with 10% heat-inactivated fetal bovine serum (Thermo Fisher
Scientific, Inc.), 100 U/mL of penicillin, 100 lg/mL of streptomycin, and 0.25 lg/mL of amphotericin B (Nacalai Tesque,Inc.). Cells were grown in a humidified incubator at 37C, at an
atmosphere of 5% CO2 and 95% air.
Antibodies

An anti-mCCR3 mAb (clone J073E5) was purchased from
BioLegend (San Diego, CA). A secondary Alexa Fluor 488conjugated anti-rat IgG was purchased from Cell Signaling
Technology, Inc., (Danvers, MA) as well.

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Production of hybridomas

A 5-week-old Sprague–Dawley (SD) rat was purchasedfrom CLEA Japan (Tokyo, Japan). Animals were housed
under specific pathogen-free conditions. All animal experiments were also conducted according to relevant guidelines
and regulations to minimize animal suffering and distress in
the laboratory. The Animal Care and Use Committee of
Tohoku University (Permit No. 2019NiA-001) approved
animal experiments. The rat was monitored daily for healthduring the full 4-week duration of the experiment. A reduction of >25% of the total body weight was defined as a humane endpoint. During sacrifice, the rat was euthanizedthrough cervical dislocation, after which death was verified
through respiratory and cardiac arrest.
To develop mAbs against mCCR3, one SD rat was intraperitoneally immunized, using 100 lg of mCCR3p1-19CKLH peptide with Imject Alum (Thermo Fisher Scientific,Inc.). The procedure included three additional immunization,
which was followed by a final booster intraperitoneal injection, administered 2 days before the harvest of spleen cells.
Harvested spleen cells were subsequently fused with P3U1
cells, using PEG1500 (Roche Diagnostics, Indianapolis, IN),
after which hybridomas were grown in an RPMI 1640 medium supplemented with hypoxanthine, aminopterin, andthymidine for the selection (Thermo Fisher Scientific, Inc.).
Supernatants were subsequently screened with the p1-19C
peptide of mCCR3, using enzyme-linked immunosorbent
assay (ELISA), after flow cytometry, using CHO/mCCR3,
CHO-K1, P388, and J774–1 cells.
ELISA

The synthesized peptide, mCCR3p1-19C, was immobilized on Nunc Maxisorp 96-well immunoplates (Thermo Fisher Scientific, Inc.) at a concentration of 1 lg/mL for
30 minutes at 37C. After washing with phosphate-bufferedsaline (PBS) containing 0.05% Tween20 (PBST; Nacalai
Tesque, Inc.), wells were blocked with 1% bovine serum
albumin (BSA)-containing PBST for 30 minutes at 37C.
Plates were then incubated with supernatants of hybridomas, followed by a peroxidase-conjugated anti-rat immunoglobulins (1:2000 diluted; Sigma-Aldrich Corp., St.
Louis, MO). Next, enzymatic reactions were conducted, using an ELISA POD Substrate TMB Kit (Nacalai Tesque,Inc.), after measurement of the optical density at 655 nm,
using an iMark microplate reader (Bio-Rad Laboratories,
Inc., Berkeley, CA).
Purification of mAbs

The cultured supernatants of C3Mab-6 or C3Mab-7
producing-hybridomas were collected through centrifugation
at 2330 x g for 5 minutes, followed by filtration using Steritop(0.22 mm, Merck KGaA, Darmstadt, Germany). Filtered supernatants were subsequently applied to 1 mL Ab-Capcher
ExTra (ProteNova Co., Ltd, Kagawa, Japan). After washingwith PBS, bound antibodies were eluted with an IgG Elution
Buffer (Thermo Fisher Scientific, Inc.), followed by an immediate neutralization of eluates, using 1M Tris-HCl. Finally, eluates were concentrated, after which PBS was used toreplace the elution buffer, using Amicon Ultra (Merck
KGaA).

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ASANO ET AL.

Flow cytometric analyses

CHO-K1, CHO/mCCR3, P388, and J774-1 were harvested
after an exposure to 1 mM ethylenediaminetetraacetic acid
(EDTA, Nacalai Tesque, Inc.). Cells were subsequently washed with 0.1% BSA in PBS and treated with 0.01, 0.1, 1, and10 lg/mL of primary mAbs for 30 minutes at 4C. Then, cells
were treated with Alexa Fluor 488-conjugated anti-rat IgG(1:2000; Cell Signaling Technology, Inc.), after which fluorescence data were collected, using the BD FACSLyric(BD Biosciences, Franklin Lakes, NJ).

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Determination of dissociation constants (KDs)through flow cytometry

CHO/mCCR3, P388, and J774-1 were suspended in
100 lL of serially diluted anti-mCCR3 mAbs, after which
50 lL of Alexa Fluor 488-conjugated anti-rat IgG (1:200;
Cell Signaling Technology, Inc.) was added. Afterward,
fluorescence data were collected, using BD FACSLyric. The
KDs were subsequently calculated by fitting saturation binding curves to the built-in; one-site binding models in
GraphPad PRISM 8 (GraphPad Software, Inc., La Jolla, CA).
Results
Development of anti-mCCR3 mAbs
by peptide immunization

To develop anti-mCCR3 mAbs, one SD rat was immunized with an mCCR3p1-19C-KLH peptide (Fig. 1A). Spleen
was then excised from the rat, after which splenocytes werefused with myeloma P3U1 cells (Fig. 1B). Developed hybridomas were subsequently seeded into 20 of 96-well plates
and cultivated for 6 days. Afterward, wells in which cultured
supernatants were positive for the mCCR3p1-19C peptide
through ELISA were selected, followed by the selection ofmCCR3-expressing cell-reactive and CHO-K1-nonreactive
supernatants, using flow cytometry (Fig. 1C).
Although the ELISA screening approach identified strong
signals from mCCR3p1-19C peptide-immobilized wells, nosignal was detected from control wells in 92 of 1914 wells(4.81%). The flow cytometric screening approach, therefore,
identified strong signals from CHO/mCCR3 cells, and a weakor no signal from CHO-K1 cells in 31/92 (33.7%). In addition, strong signals were identified from P388 and J774-1
cells in 8 of the 31 hybridoma supernatants identified in theprevious step (25.8%). After limiting dilution and several
additional screenings, anti-mCCR3 mAbs, C3Mab-6 (rat
IgG1, kappa), and C3Mab-7 (rat IgG1, kappa) were finallyestablished (Fig. 1D).
Flow cytometric analysis

We conducted flow cytometry, using three anti-mCCR3
mAbs; C3Mab-6, C3Mab-7, and the commercially available
anti-mCCR3 mAb (clone J073E5 from BioLegend: positive
control) against mCCR3-expressing cell lines; CHO/mCCR3,
P388, and J774-1. Although all three mAbs dose-dependently
recognized CHO/mCCR3 cells at 10, 1, 0.1, and 0.01 lg/mL(Fig. 2A), parental CHO-K1 cells were not recognized(Fig. 2B). Also, although C3Mab-7 and J073E5 did not reactwith P388 cells at 0.01 lg/mL (Fig. 2C, middle and rightpanel), C3Mab-6 reacted with the cells even at 0.01 lg/mL

FIG. 1. A schematic illustration showing the productionof anti-mCCR3 mAbs. (A) The mCCR3p1-19C-KLH peptide was immunized into an SD rat, using an intraperitoneal
injection. (B) Spleen cells were then fused with P3U1 cells.
(C) Subsequently, culture supernatants were screened
through ELISA and flow cytometry to select anti-mCCR3
mAb-producing hybridomas. (D) After limiting dilution andsome additional screenings, anti-mCCR3 mAbs were finallyestablished. ELISA, enzyme-linked immunosorbent assay;
mAbs, monoclonal antibodies.

(Fig. 2C, left panel). All three mAbs, therefore, recognized
J774-1 cells at 10 and 1 lg/mL (Fig. 2C). Hence, these results
suggested that C3Mab-6 and C3Mab-7 were specific for
mCCR3, thereby making then useful for detecting endogenous
mCCR3 through flow cytometry.
Kinetic analysis of the interaction of C3Mab-6
and C3Mab-7 with mCCR3-expressed cells
through flow cytometry

To assess the apparent KDs of C3Mab-6 and C3Mab-7 with
mCCR3-expressing cells, we conducted kinetic analysis of
the interaction of C3Mab-6 and C3Mab-7 with CHO/mCCR3,
P388, and J774-1, using flow cytometry. The geometric meanof the fluorescence intensity was then plotted versus the

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ANTI-MOUSE CCR3 MONOCLONAL ANTIBODY

FIG. 2. Flow cytometry using anti-mCCR3. CHO/mCCR3 (A), CHO-K1 (B), P388 (C), and J774-1 (D) cells were treatedwith 0.01 mg/mL (dashed-dotted line), 0.1 mg/mL (dotted line), 1 mg/mL (dashed line), and 10 mg/mL (solid line) of C3Mab-6(left panels), C3Mab-7 (middle panels), or J073E5 (right panels), followed by treatment with Alexa Fluor 488-conjugatedanti-rat IgG. Filled, without first Ab as negative controls. CHO, Chinese hamster ovary.

concentration of C3Mab-6 or C3Mab-7, after fitting throughone-site binding models in GraphPad PRISM 8. The KDs of
C3Mab-6 for CHO/mCCR3, P388, and J774-1 cells were
determined as 8.7 · 10-9 M, 1.4 · 10-7 M, and 1.7 · 10-7 M,
respectively. The KDs of C3Mab-7 for these cell lines were
3.7 · 10-9 M, 5.1 · 10-7 M, and 3.1 · 10-7 M, respectively(Fig. 3). These results, therefore, indicate that C3Mab-6 orC3Mab-7 possesses a high affinity for CHO/mCCR3, with amoderate affinity for P388 and J774-1 cells.

Discussion

GPCRs expressed on the plasma membrane have native
conformations. Therefore, immunization with cells, which
express target GPCRs, is a suitable approach against GPCRs,such as chemokine receptors.(9–12) By using the Cell-Based
Immunization and Screening (CBIS) method, in whichantigen-expressing cell lines are used for immunization andscreening, we have established useful mAbs against many

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FIG. 3. Determination of the KDs of C3Mab-6 and C3Mab-7 against mCCR3-expressing cells. CHO/mCCR3 (A) wassuspended in 100 mL serially diluted C3Mab-6 or C3Mab-7 (0.006–100 mg/mL). Then, P388 (B) and J774-1 (C) weresuspended in 100 mL serially diluted C3Mab-6 or C3Mab-7 (0.06–1000 mg/mL). The Alexa Fluor 488-conjugated anti-rat
IgG was subsequently added, after which fluorescence data were obtained, using BD FACSLyric.
membrane proteins, including GPCRs, such as CCR3,(11)CCR8,(12) CCR9,(10) and other membrane proteins, such as
podoplanin,(25–28) CD20,(29,30) CD44,(31) EpCAM,(32)
TROP2,(33) EGFR,(34) and HER3(35) in previous studies.
Although developed mAbs through the CBIS method reacts with native GPCRs on plasma membranes, solubilized
and denatured proteins have remained unreactive. Therefore,
they are useful for flow cytometry. In contrast, many of them
are not useful for ELISA and Western blot. In this study, wedeveloped novel anti-CCR3 mAbs, C3Mab-6 and C3Mab-7,
through immunization with the N-terminal peptide ofmCCR3. As observed, C3Mab-6 and C3Mab-7 were useful
not only for ELISA but also for flow cytometry. This result,
therefore, proposes that the N-terminal region of mCCR3
forms a linear structure.
Structural analyses showed that eotaxin binds to the
N-terminus of human CCR3 (residues 8–23).(23,24) The KDs
of CCR3’s binding to eotaxin-1, -2, and -3 are 2.1, 9.7,
1.2 · 10-9 M, obtained from competitive radioligand binding
assays using CCR3-expressing CHO cells, respectively.(36)
Because eotaxin, C3Mab-6, and C3Mab-7 recognize
N-terminal region of mCCR3, we need further investigation

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ANTI-MOUSE CCR3 MONOCLONAL ANTIBODY

to reveal the essential amino acids recognized by C3Mab-6
and C3Mab-7.(24) In the future study, it is interesting
to evaluate the neutralizing activity of C3Mab-6 andC3Mab-7 to eotaxin.
In the future study, we will investigate activities ofantibody-dependent cellular cytotoxicity (ADCC) andcomplement-dependent cytotoxicity (CDC). Since rat IgG1
does not possess ADCC and CDC activities, we will also
convert the subclass of C3Mab-6 and C3Mab-7 into mouse
IgG2a, then evaluate their ADCC and CDC activities. This
antibody is useful for treating mCCR3-expressing cancers
and depleting mCCR3-expressing eosinophils and basophils.
In a previous study, we developed other anti-mCCR3
mAbs; C3Mab-2 and C3Mab-3, using the CBIS method, andshowed their usefulness in flow cytometry.(11) As observed,
C3Mab-3, C3Mab-6, and C3Mab-7 recognized both EDTAtreated and trypsin-treated CHO/mCCR3 cells. In contrast,
C3Mab-2 recognized only EDTA-treated cells, but not
trypsin-treated cells. These results suggest that the epitope of
C3Mab-2 will be different from that of other three mAbs, andaffected by trypsin. In contrast, at least the N-terminal 19
amino acids, recognized by C3Mab-6 and C3Mab-7, were notaffected by trypsin. Future studies are required to identify the
epitope of C3Mab-2 and C3Mab-3.
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.), JP21am0401013 (to Y.K.),
and JP21am0101078 (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
MiyagiJapan
E-mail: yukinarikato@med.tohoku.ac.jp
Received: December 17, 2021
Accepted: February 14, 2022