MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 40, Number 3, 2021
ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2021.0009

Development of Anti-Mouse CC Chemokine Receptor 3
Monoclonal Antibodies for Flow Cytometry

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Teizo Asano,1 Ren Nanamiya,1 Junko Takei,1 Takuro Nakamura,1 Miyuki Yanaka,1 Hideki Hosono,1Tomohiro Tanaka,1 Masato Sano,1 Mika K. Kaneko,1 and Yukinari Kato1,2

CC chemokine receptor 3 (CCR3), also known as CD193, belongs to class A of G protein-coupled receptors andis present in high levels in eosinophils, basophils, and airway epithelial cells. CCR3 is considered the therapeutic target for human immunodeficiency virus (HIV) infections and allergic diseases; therefore, the development of sensitive monoclonal antibodies (mAbs) for CCR3 has been desired. This study aimed to establish a
specific and sensitive mAb against mouse CCR3 (mCCR3) useful for flow cytometry analysis by employing the
Cell-Based Immunization and Screening (CBIS) method. The generated anti-mCCR3 mAb, C3Mab-2 (rat
IgG2b, kappa), was found to react with mCCR3-overexpressed Chinese hamster ovary-K1 (CHO/mCCR3) cells,
according to flow cytometric analysis. Also, it reacted with P388 (mouse lymphoid neoplasm) or J774-1 (mouse
macrophage-like) cells, which express endogenous mCCR3. Taken together, C3Mab-2, generated by the CBIS
method, can be a valuable tool for detecting mCCR3 on the surface of mouse cells.
Keywords: CCR3, monoclonal antibody, CBIS, flow cytometry, allergic diseases

Introduction

C

hemokines are small cytokines that induce cell
movement in response to a chemokine gradient; theyplay an essential role in cell migration.(1) They also have arole in allergic inflammatory diseases, human immunodeficiency virus (HIV)-associated diseases, and cancers.(2,3)
Chemokines are classified into four subfamilies: CXC, CC,
CX3C, and XC. All chemokines bind to and activate G
protein-coupled receptors (GPCRs).(1,4) Chemokine receptors are divided into four families that correspond to theirligand subfamilies: CXC chemokine receptor (CXCR), CC
chemokine receptor (CCR), CX3C chemokine receptor, and
XC chemokine receptor.
CC chemokine receptor 3 (CCR3), also known as CD193,
is a receptor for the CC family of chemokines, such as eotaxin, MCP-3, and RANTES. CCR3, first cloned and identified as a murine(5) or human(6) eosinophil CCR, belongs to
the class A of GPCR. CCR3 is present at high levels in eosinophils, basophils, and microglial cells, as well as in subsets
of mast, Th2, and airway epithelial cells. It is also present in
CD31-positive endothelial cells in the choroidal neovascularization membranes of eyes with wet age-related maculardegeneration.(7–10) CCR3 supports HIV Env-mediated syncytia formation, and eotaxin as its ligand can reduce theefficiency of HIV-1 infection.(9,11,12) Moreover, CCR3 is
1
2

important for recruiting eosinophils into the lung; thus, it canbe used to induce airway hyper-responsiveness in a murine
model of allergic asthma. CCR3 also contributes to ocularallergy and, therefore, is regarded as the therapeutic target for
HIV and allergic diseases.(13–16)
The development of anti-CCR3-specific monoclonal antibodies (mAbs) is necessary for allergic disease, HIV therapy, and pathological diagnosis. In this study, we havesuccessfully developed a novel anti-CCR3 mAb using the
Cell-Based Immunization and Screening (CBIS) method.(17,18) We have also investigated the usefulness of antiCCR3 mAb for flow cytometry analysis.
Materials and Methods
Cell lines

Chinese hamster ovary K1 (CHO-K1) and P3X63Ag8U.1(P3U1) were supplied by the American Type Culture Collection (Manassas, VA). P388 (mouse lymphoid neoplasm)
and J774-1 (mouse macrophage-like) cells were obtained
from Cell Resource Center for Biomedical Research Instituteof Development, Aging and Cancer, Tohoku University(Miyagi, Japan). The synthesized DNA (Eurofins Genomics
KK, Tokyo, Japan) encoding mouse CCR3 (mCCR3) (accession no. NM_009914.4) was subcloned into a pCAG-Ble
vector (FUJIFILM Wako Pure Chemical Corporation, Osaka,

Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, Sendai, Japan.
New Industry Creation Hatchery Center, Tohoku University, Sendai, Japan.

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Japan). The mCCR3 plasmid was transfected using the Neon
Transfection System (Thermo Fisher Scientific, Inc., Waltham, MA). Stable transfectants were identified using a cell
sorter (SH800; Sony Corp., Tokyo, Japan) and cultured in a
medium containing 0.5 mg/mL of zeocin (InvivoGen, San
Diego, CA).
CHO-K1, P3U1, CHO/mCCR3, P388, and J774-1 were
cultured in a Roswell Park Memorial Institute (RPMI) 1640
medium (Nacalai Tesque, Inc., Kyoto, Japan) supplemented
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.) in a humidified incubator at 37C with 5% CO2.

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Antibodies

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

One 6-week-old Sprague Dawley (SD) rat (CLEA Japan,
Tokyo, Japan) was housed under specific pathogen-free
conditions. All animal experiments were performed according to relevant guidelines and regulations to minimize animal
suffering and distress. The animal experiments were approved by the Animal Care and Use Committee of Tohoku
University (permit number: 2019NiA-001). The rat wasmonitored daily for health during the 4-week experiment.
A reduction of >25% of the total body weight was defined as ahumane end point. The rat was euthanized by cervical dislocation, and its death was verified by respiratory and cardiac
arrest. We employed the absorption step-added CBIS (abCBIS) method to develop mAbs against mCCR3. In brief, an
SD rat was immunized with 1 · 109 CHO/mCCR3 cells with
the Imject Alum (Thermo Fisher Scientific, Inc.) by intraperitoneal injection. The procedure included three additional
immunizations, followed by a final booster intraperitoneal
injection administered 2 days before the harvest of spleencells. The harvested spleen cells were subsequently fusedwith P3U1 cells using PEG1500 (Roche Diagnostics, Indianapolis, IN). The resultant hybridomas were grown in an
RPMI medium supplemented with hypoxanthine, aminopterin, and thymidine for selection (Thermo Fisher Scientific,Inc.). Culture supernatants were mixed with CHO-K1 cells
for 30 minutes to absorb the antibodies that react to CHO-K1
cells. The supernatants were then collected for centrifugationand screened with CHO/mCCR3 cells by flow cytometry to
identify the hybridomas producing mCCR3-specific mAbs.
Flow cytometry

The CHO/mCCR3 cells were harvested after a brief incubation with 1 mM ethylenediaminetetraacetic acid (Nacalai Tesque, Inc.). They were then washed with 0.1% bovine
serum albumin in phosphate-buffered saline, treated withprimary mAbs for 30 minutes at 4C, and incubated with
Alexa Fluor 488-conjugated anti-rat IgG (1:2000; Cell Signaling Technology, Inc.). The fluorescence data were collected using the SA3800 Cell Analyzer (Sony Corp.).

ASANO ET AL.
Determination of antibody-binding affinity
by flow cytometry

P388 and J774-1 were suspended in 100 lL of seriallydiluted anti-mCCR3 mAbs, and Alexa Fluor 488-conjugatedanti-rat IgG (1:200; Cell Signaling Technology, Inc.) was
added. The fluorescence data were collected using the BD
FACSLyric (BD Biosciences, Franklin Lakes, NJ). The dissociation constant (KD) was calculated by fitting binding
isotherms to built-in one-site binding models in GraphPad
PRISM 8 (GraphPad Software, Inc., La Jolla, CA).
Results
Creation of anti-mCCR3 mAbs
using the abCBIS method

We employed the abCBIS method to develop anti-mCCR3
mAbs and using the stable mCCR3 transfectants for both the
immunization and flow cytometric screening (Fig. 1). In thisstudy, one rat was immunized with CHO/mCCR3 cells overexpressing mCCR3 to develop anti-mCCR3 mAbs. The developed hybridomas were seeded into 96-well plates andcultivated for 6 days. The culture supernatants from the wells
were mixed with CHO-K1 cells to absorb the CHO-K1reacting antibodies. The supernatants were again collected
after centrifugation.
Wells positive for CHO/mCCR3 were selected using flow
cytometry. The first screening method observed the supernatant from 48 of the 956 wells with hybridomas (5.02%) displayed strong antibody-binding signals with the CHO/mCCR3cells and weak or no signals with the CHO–K1 cells. Then, the48 hybridoma clones were tested with P388 (lymphoid neoplasms) and J774-1 (macrophage-like cell lines) cells in thesecond screening. The supernatants from 5 of the 48 hybridoma culture (10.4%) exhibited robust antibody-binding signals
with P388 and J774-1 cells. After limiting dilution and several
additional screenings, an anti-mCCR3 mAb, C3Mab-2 (rat
IgG2b, kappa), was finally established.
Flow cytometry analysis

We performed flow cytometry using C3Mab-2 against
CHO/mCCR3, P388, J774-1, and CHO-K1 cells. C3Mab-2
recognized the CHO/mCCR3 cells in a dose-dependent manner(Fig. 2A), but not the parental CHO-K1 cells (Fig. 2B). Anotheranti-mCCR3 mAb (clone J073E5 from BioLegend), which
acted as the positive control, also recognized CHO/mCCR3 in adose-dependent manner (Fig. 2A), but not CHO-K1 (Fig. 2B).
Although C3Mab-2 reacted with CHO/mCCR3 cells at>0.1 lg/mL, J073E5 reacted with CHO/mCCR3 cells even at
0.01 lg/mL (Fig. 2A). C3Mab-2 also detected the endogenous
mCCR3 on P388 (Fig. 2C) and J774-1 cells (Fig. 2D) in a dosedependent manner. These results suggest that C3Mab-2 is
specific for mCCR3 and is useful for detecting endogenous
mCCR3 in flow cytometric analysis.
The reaction of C3Mab-2 to CHO/mCCR3 was lost whentrypsin was used for harvesting CHO/mCCR3 cells (data notshown), indicating that the epitope of C3Mab-2 is trypsinsensitive. In contrast, the reaction of J073E5 to CHO/mCCR3
was observed even when trypsin was used for harvesting
CHO/mCCR3 cells (data not shown). These results indicatethat the epitope of C3Mab-2 is different from that of J073E5.

ANTI-MOUSE CCR3

109

tometry. The geometric mean of fluorescence intensity wasplotted versus the concentrations of C3Mab-2 and fitted by
one-site binding models. The KD of C3Mab-2 for P388 andJ774-1 was determined to be 2.1 · 10-7 M and 3.7 · 10-7 M,
respectively, indicating the moderate affinity of C3Mab-2 formCCR3-expressing cells.

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Discussion

FIG. 1. Production of anti-mCCR3 mAbs. Procedure of
abCBIS method. CHO/mCCR3 cells were immunized into SD
rat using intraperitoneal injection. Screening was performed
using flow cytometry. CBIS, Cell-Based Immunization andScreening; mAbs, monoclonal antibodies; CHO/mCCR3,
Chinese hamster ovary/mouse CC chemokine receptor 3; SD,
Sprague Dawley.
Determination of the binding affinity using C3Mab-2
against P388 and J774-1 cells by flow cytometry

We conducted a kinetic analysis to assess the binding affinity of C3Mab-2 to P388 and J774-1 cells using flow cy-

Purified proteins and peptides are usually used as immunogens to generate antibodies. It takes much time and effort tocollect purified proteins, especially membrane proteins. Although peptides are commercially available and easy to obtain,
the mAbs generated by immunizing with peptides often do notrecognize the native proteins.(19) Although many commercially available anti-mCCR3 polyclonal antibodies are usefulin Western blot, immunohistochemistry, and enzyme-linked
immunosorbent assay (ELISA), they may not be effective inflow cytometry. Of the seven anti-mCCR3 mAbs that we
commercially obtained, only five are useful in flow cytometry(Supplementary Table S1). The CBIS method, which uses
antigen-expressing cell lines for both immunization andscreening, can help us effectively develop mAbs that are usefulin flow cytometry. We recently have succeeded in developing
numerous useful mAbs that target membrane proteins, including podoplanin,(20–23) CD20,(24) CD44,(25) and TROP2.(26)In this study, we have used the modified CBIS method,
abCBIS, and successfully generated a novel anti-mCCR3
mAb, C3Mab-2, which is useful for flow cytometry. Grimaldi
et al. previously reported that anti-mCCR3 mAbs weregenerated by immunizing rats with Y3/mCCR3 cells andscreening with NIH3T3/mCCR3 and NIH3T3 cells.(27)
Likewise, different kinds of cells, such as Y3 and NIH3T3,
should be used for immunization and screening, respectively,
because using parental cells for immunization may produce
nonspecific antibodies that may hinder the screening forspecific anti-mCCR3 antibodies. In this study, the same parental cells, such as CHO-K1 cells, were used for immunization and screening in the abCBIS method. We could obtainspecific mAbs against targets because almost all the nonspecific antibodies were removed during the absorption step.
In this study, we successfully established a novel anti-mCCR3
mAb, C3Mab-2, which is useful for flow cytometry using the
abCBIS method. Notably, C3Mab-2 reacted with endogenous
mCCR3 in P388 and J774-1 cells in the same way as the J073E5
mAb (BioLegend). In the future study, we will determine
whether C3Mab-2 is useful for Western blot, immunohistochemistry, and ELISA because there is no anti-mCCR3 mAb
that is useful in all the applications (Supplementary Table S1).
Chemokines and their receptors are attractive therapeutictargets; some drugs that target chemokine receptors havebeen launched. For example, Maraviroc is a CCR5 antagonist. CCR5 binds to gp120, which is on the surface of the HIV
envelope and essential for virus entry into cells.(28–30)
Therefore, maraviroc is used as an antiretroviral drug to block
HIV entry. Plerixafor is a CXCR4 antagonist and an immunostimulant used in patients with lymphoma and multiple
myeloma. Mogamulizumab, a mAb against CCR4, is used as
an anticancer drug.(31,32) The anti-mCCR3 mAbs effective in
multipurpose analyses will also allow us to explore mCCR3mediated allergic diseases in a mouse model, and might leadto the development of CCR3-targeting drugs.

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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 (solid line), 0.1 mg/mL (dashed line), 1 mg/mL (dotted line), and 10 mg/mL (dashed-dotted line) of C3Mab-2 orJ073E5 (BioLegend), followed by treatment with Alexa Fluor 488-conjugated anti-rat IgG; filled, negative control.
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ANTI-MOUSE CCR3
Author Disclosure Statement

T.A., R.N., J.T., T.N., M.Y., H.H., T.T., and M.S. performed experiments; M.K.K. designed the experiments; T.A.
and Y.K. wrote the article.
Funding Information

This research was supported in part by AMED under Grant
Numbers JP20am0401013 (Y.K.) and JP20am0101078(Y.K.), and by JSPS KAKENHI Grant Numbers 20K16322(M.S.) and 19K07705 (Y.K.).
Supplementary Material

Supplementary Table S1Downloaded by Yukinari Kato from www.liebertpub.com at 06/23/21. For personal use only.

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Address correspondence to:
Yukinari Kato
New Industry Creation Hatchery Center
Tohoku University
2–1, Seiryo-machi, Aoba-ku
Sendai 980-8575
MiyagiJapan
E-mail: yukinarikato@med.tohoku.ac.jp
Received: February 16, 2021
Accepted: February 24, 2021