MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 41, Number 2, 2022
ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2021.0062
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C3Mab-3: A Monoclonal Antibody for Mouse CC
Chemokine Receptor 3 for Flow Cytometry

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Teizo Asano,1 Hiroyuki Suzuki,2 Tomohiro Tanaka,1 Masaki Saito,2 Guanjie Li,2
Nohara Goto,2 Ren Nanamiya,1 Mika K. Kaneko,1 and Yukinari Kato1,2

CC chemokine receptor 3 (CCR3) belongs to the G protein-coupled receptor family and is highly expressed ineosinophils and basophils. CCR3 is essential for recruiting eosinophils into the lung. Moreover, CCR3 wasfound in the serum of colorectal cancer patients higher than in the control group. Therefore, CCR3 will be auseful target for asthma and colorectal cancer diagnosis. This study developed a specific and sensitive
monoclonal antibody (mAb) for mouse CCR3 (mCCR3), which is useful for flow cytometry using the CellBased Immunization and Screening method. The established anti-mCCR3 mAb, C3Mab-3 (rat IgG2a, kappa),
reacted with mCCR3-overexpressed Chinese hamster ovary-K1 (CHO/mCCR3) cells through flow cytometry.
C3Mab-3 also reacted with P388 (mouse lymphoid neoplasma) and J774-1 (mouse macrophage-like) cells,
which express mCCR3 endogenously. Kinetic analyses using flow cytometry indicated that KDs of C3Mab-3 for
CHO/mCCR3, P388, and J774-1 cells were 4.3 · 10-8 M, 2.6 · 10-7 M, and 2.4 · 10-7 M, respectively. C3Mab-3
could be a valuable tool for elucidating mCCR3-related biological response using flow cytometry.
Keywords: mouse CCR3, monoclonal antibody, CBIS

Chemokines are classified into four subfamilies: CC, CXC,
CX3C, and XC.(16,17) CC type chemokines, such as eotaxin
family (CCL11, CCL24, and CCL26), Monocyte Chemotactic Protein-3 (MCP-3; CCL7), and regulated on activation,
normal T cell expressed and secreted (RANTES; CCL5), are
known as ligands for CCR3. CCR3 support HIV Envmediated syncytia formation, and eotaxin inhibits the efficiency of HIV-1 infection.(2,18,19) CCR3 also contributes toocular allergy.(20) Moreover, CCR3 is essential for the recruitment of eosinophils into the lung and airway hyperresponsiveness in a murine model of allergic asthma.(21–24)
A recent study indicated that the CCR3 concentration in
the serum of colorectal cancer patients was significantly higher
than the control group.(25) Therefore, CCR3 is one of the mostimportant chemokine receptors in asthma and will be useful as abiomarker for diagnosing colorectal cancer. Moreover, single
and repeated intrathecal administration of SB328437, CCR3
antagonists, decreased mechanical and thermal hypersensitivity
caused by nerve injury, and blockade of CCR3 by SB328437
enhanced morphine and buprenorphine effectiveness; therefore,
CCR3 will be a promising target for neuropathic pain therapy.(26)Introduction

C

C chemokine receptor 3 (CCR3), also known
as CD193, belongs to the G protein-coupled receptor(GPCR) family of membrane proteins.(1) CCR3 is highly
expressed in eosinophils and basophils, and its expression has
also been reported in subsets of mast and Th2 cells, airway
epithelial cells, and endothelial cells.(2–6) CCR3 was firstcloned and identified as mouse(7) and human(8) eosinophil
chemokine receptors in 1995. Chemokines contribute toallergic inflammatory disease, cancer, and human immunodeficiency virus (HIV)-associated diseases.(9–11)
Therefore, chemokines and their receptors are attractive
diagnostics and therapeutic targets, and some chemokineand chemokine receptor-targeted drugs have been launched.
For example, plerixafor is the CXCR4 receptor antagonist.
Plerixafor is an immunostimulant and is used for patientswith lymphoma and multiple myeloma. Mogamulizumab, amonoclonal antibody (mAb) against CCR4, is used as an
anticancer drug.(12,13) Maraviroc, a CCR5 antagonist, is used
as an antiretroviral drug that blocks HIV entry.(10,14,15)
1
2

Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, Sendai, Miyagi, Japan.
Department of Molecular Pharmacology, Tohoku University Graduate School of Medicine, Sendai, Miyagi, Japan.

74

ANTI-MOUSE CCR3 MAB

The development of specific anti-CCR3 mAbs has beenrequired for allergic and HIV therapy and pathological diagnosis of age-related macular degeneration. However, thedevelopment of anti-GPCR mAbs has been challenging because GPCRs have complicated three-dimensional structures, and have a small extracellular region. Purification of
GPCRs for an antigen is complex.(27) In this study, a novelanti-mouse CCR3 (mCCR3) mAb was developed using thecell-based immunization and screening (CBIS) method(28–33)
and investigated its usefulness for flow cytometry analysis, and determined the dissociation constant between thenovel anti-mCCR3 mAb and mCCR3-expressed cell lines.
Materials and Methods

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Cell lines

Chinese hamster ovary (CHO)-K1, LN229, 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). Synthesized DNA (Eurofins
Genomics KK, Tokyo, Japan) encoding mCCR3 (Accession
No. NM_009914.4) was subcloned into a pCAG-Ble vector(FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan).
mCCR3 plasmid was transfected into CHO-K1 and LN229
cells using the Neon transfection system (Thermo Fisher Scientific Inc., Waltham, MA). Stable transfectants were established through cell sorting using a cell sorter (SH800; Sony
Corp., Tokyo, Japan) and cultivation in a medium containing
0.5 mg/mL of Zeocin (InvivoGen, San Diego, CA).
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). LN229 and mCCR3-overexpressed
LN229 (LN229/mCCR3) were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Nacalai Tesque, Inc.). RPMI
1640 and DMEM were supplemented with 10% heatinactivated fetal bovine serum (Thermo Fisher ScientificInc.), 100 U/mL penicillin, 100 lg/mL streptomycin, and0.25 lg/mL amphotericin B (Nacalai Tesque, Inc.). Cells
were grown in a humidified incubator at 37C with an atmosphere of 5% CO2 and 95% air.

75

tion of more than 25% of total body weight was defined as ahumane endpoint. A rat was euthanized through cervicaldislocation, and death was verified through respiratory andcardiac arrest.
To develop mAbs against mCCR3, the CBIS method was
employed. In brief, one SD rat was immunized with LN229/
mCCR3 cells (1 · 109) by the intraperitoneal route together
with the Inject Alum (Thermo Fisher Scientific Inc.). The
procedure included three additional immunization, 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). The hybridomas weregrown in an RPMI 1640 medium supplemented with hypoxanthine, aminopterin, and thymidine for selection (Thermo Fisher
Scientific Inc.). Flow cytometry was used to screen the supernatants with CHO/mCCR3, CHO-K1, P388, and J774-1 cells.

Antibodies

An anti-mCCR3 mAb (clone J073E5) was bought from
BioLegend (San Diego, CA). Secondary Alexa Fluor 488conjugated anti-rat IgG was bought from Cell Signaling
Technology, Inc. (Danvers, MA).
Production of hybridomas

Sprague Dawley (SD) rats (6 weeks old) were purchasedfrom CLEA Japan (Tokyo, Japan). Animals were housed
under specific pathogen-free conditions. All animal experiments were conducted according to relevant guidelines andregulations to minimize animal suffering and distress in thelaboratory. Animal experiments were approved by the Animal Care and Use Committee of Tohoku University (Permit
No. 2019NiA-001). A rat was monitored daily for healthduring the full 4-week duration of the experiment. A reduc-

FIG. 1. Schematic illustration of the production of antimCCR3 mAbs. (A) LN229/mCCR3 cells were immunized
into an SD rat using an intraperitoneal injection. (B) Spleencells were fused with P3U1 cells. (C) Culture supernatants
were screened with CHO/mCCR3 using flow cytometry to
select hybridomas that are producing anti-mCCR3 mAb.
(D) After limiting dilution and many additional screenings,
an anti-mCCR3 mAb was finally established. CCR3, CC chemokine receptor 3; CHO, Chinese hamster ovary; mAb, monoclonal antibody; mCCR3, mouse CCR3; SD, Sprague Dawley.

76
Flow cytometry

Determination of dissociation constants(KDs) by flow cytometry

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

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Cells were harvested after a brief exposure to 0.25% trypsin
and 1 mM ethylenediaminetetraacetic acid (Nacalai Tesque,Inc.). They were washed with 0.1% bovine serum albumin in
phosphate-buffered saline and treated with primary mAbs for
30 minutes at 4C. The cells were then treated with Alexa Fluor
488-conjugated anti-rat IgG (1:2000; Cell Signaling Technology, Inc.). Fluorescence data were obtained using BD FACSLyric (BD Biosciences, Franklin Lakes, NJ).

ASANO ET AL.

FIG. 2. Flow cytometry using anti-mCCR3 mAbs. CHO/mCCR3 (A), CHO-K1 (B), P388 (C), and J774-1 (D) cells weretreated with 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-3 (left panels) or J073E5 (right panels), followed by treatment with Alexa Fluor 488-conjugated anti-rat IgG. Filled,
without 1st Ab as negative controls.

ANTI-MOUSE CCR3 MAB

77

Results

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The development of anti-mCCR3 mAbs using
the CBIS method

To develop anti-mCCR3 mAbs, we employed CBIS method. CBIS method includes the immunization of mCCR3overexpressed cells and the high-throughput hybridoma
screening using flow cytometry (Fig. 1). In this study, one
rat was immunized with LN229/mCCR3 cells (Fig. 1A).
The spleen was excised from the rat, and splenocytes werefused with P3U1 cells (Fig. 1B). Developed hybridomas
were seeded into 96-well plates and cultivated for 6 days.
Culture supernatants positive for CHO/mCCR3 were screenedusing flow cytometry (Fig. 1C).
The first screening approach identified strong signals from
CHO/mCCR3 cells and weak or no signals from CHO-K1
cells in 30 of 956 wells (3.14%). The second screening procedure identified strong signals from P388 and J774-1 cells in
26 of the 30 hybridoma supernatants identified in the previous
step (86.7%). After limiting dilution and several additional
screenings, an anti-mCCR3 mAb, C3Mab-3 (rat IgG2a, kappa)
was finally established (Fig. 1D).
Flow cytometry analysis

We performed flow cytometry using C3Mab-3 against
CHO/mCCR3, CHO-K1, P388, and J774-1. C3Mab-3 dosedependently recognized CHO/mCCR3 cells at 10, 1, and
0.1 lg/mL (Fig. 2A, left panel), but not parental CHO-K1
cells (Fig. 2B, left panel). Another anti-mCCR3 mAb (clone
J073E5 from BioLegend: positive control) also recognized
CHO/mCCR3 in a dose-dependent manner (Fig. 2A, rightpanel), but not CHO-K1 (Fig. 2B, right panel). Although
C3Mab-3 did not react with CHO/mCCR3 at 0.01 lg/mL,
J073E5 reacted with it even at 0.01 lg/mL (Fig. 2A). C3Mab3 and J073E5 also recognized P388 cells (Fig. 2C) and J774-1
cells (Fig. 2D) expressing endogenous mCCR3 in a dosedependent manner. These results suggested that C3Mab-3 is
specific for mCCR3 and is useful for detecting endogenous
mCCR3 using flow cytometry.
Determination of the KDs using C3Mab-3 against
mCCR3-expressing cells by flow cytometry

To assess the KD of C3Mab-3 with mCCR3-expressed
cells, we performed kinetic analysis of the interaction of
C3Mab-3 with CHO/mCCR3, P388, and J774-1 using flow
cytometry. The geometric mean of fluorescence intensity wasplotted versus the concentration of C3Mab-3 and fitted by
one-site binding models in GraphPad PRISM 8. The KDs of
C3Mab-3 for CHO/mCCR3, P388, and J774-1 cells were
determined to be 4.3 · 10-8 M (Fig. 3A), 2.6 · 10-7 M(Fig. 3B), and 2.4 · 10-7 M (Fig. 3C), respectively. These
results show that C3Mab-3 possesses moderate affinity for
CHO/mCCR3 cells and low affinity for P388 and J774-1.
Discussion

GPCRs respond to different external signals, such as
light, hormones, and chemokines, and transmit extracellularsignals to intracellular molecules. GPCR-driven signaling
pathways are involved in physiological function and thepathogenesis of several diseases. Therefore, GPCRs are

FIG. 3. Determination of the KDs of C3Mab-3 against
mCCR3-expressing cells. CHO/mCCR3 (A) was suspended
in 100 mL serially diluted C3Mab-3 (0.006–100 mg/mL).
P388 (B) and J774-1 (C) were suspended in 100 mL seriallydiluted C3Mab-3 (0.06–1000 mg/mL). Alexa Fluor 488conjugated anti-rat IgG was then added. Fluorescence data
were obtained using BD FACSLyric.
therapeutic targets. Therefore, anti-GPCR mAbs are a valuable tool for therapy and diagnosis. To generate the antibodies, purified proteins or synthesized peptides are often
used as an immunogen. However, the purification of GPCR iscomplex, and consumes much time and effort. Althoughsynthesized peptides are easy to obtain, established mAbs
using immunizing peptides often do not recognize proteinsthat have a native conformation.(34)
Previously, we have succeeded in the development of
useful mAbs against membrane proteins, including CCR3,(31)Downloaded by Yukinari Kato from www.liebertpub.com at 04/26/22. For personal use only.

78

ASANO ET AL.

CCR8,(32) CCR9,(33) podoplanin,(35–38) CD20,(39,40) CD44,(30)
EpCAM,(41) TROP2,(42) EGFR,(43) and HER3,(44) using
CBIS method in which antigen-expressing cell lines are used
for immunization and screening. In this study, the novel antimCCR3 mAb, C3Mab-3, which is useful for flow cytometry
using the CBIS method. C3Mab-3 reacted with endogenous
mCCR3 in P388 and J774-1 cells similar to the J073E5(BioLegend). In contrast, C3 Mab-3 reacted with CHO/
mCCR3 cells, but this reactivity is weaker than J073E5 with
CHO/mCCR3 (Fig. 2).
In a previous study, other anti-mCCR3 mAbs, C3Mab-2,
was developed using immunizing CHO/mCCR3 cell lines.(31)
The reactivities of C3Mab-3 with CHO/mCCR3, P388, andJ774-1 cell lines are comparable with that of C3Mab-2 withthese cell lines. In addition, KDs between C3Mab-3 and thesecell lines are also comparable with KDs between C3Mab-2
and them. However, C3Mab-2 cannot detect trypsin-treated
CHO/mCCR3 (data not shown). Otherwise, C3Mab-3 can reactwith trypsin-treated CHO/mCCR3. This result indicates that
C3Mab-3 and C3Mab-2 recognize different regions on mCCR3.
In future studies, the epitope regions of C3Mab-3 and C3Mab-2
will need to be determined. Moreover, it will be confirmed
whether C3Mab-3 is useful for Western blotting, immunohistochemistry, and enzyme-linked immunosorbent assay.
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 Yukinari Kato), JP21am0401013(to Yukinari Kato), and JP21am0101078 (to Yukinari Kato).
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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, Miyagi 980-8575
JapanE-mail: yukinarikato@med.tohoku.ac.jp
Received: December 3, 2021
Accepted: February 14, 2022