MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 41, Number 1, 2022
ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2021.0050
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C3Mab-2: An Anti-Mouse CCR3 Monoclonal
Antibody for Immunocytochemistry

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Masaki Saito,1 Yasuhiro Harigae,1 Guanjie Li,1 Teizo Asano,2 Tomohiro Tanaka,2Hiroyuki Suzuki,1 Mika K. Kaneko,2 and Yukinari Kato1,2

The C–C motif chemokine receptor 3 (CCR3) is a G protein-coupled receptor activated by eotaxin-1-3, MCP-2-4,
and RANTES. CCR3 is associated with allergic diseases and cancer development and is highly expressedin eosinophils, basophils, and cancer cells. Besides, research on the physiological roles of CCR3 is
ongoing. Thus, specific monoclonal antibodies (mAbs) for CCR3 would be useful for diagnostic andtherapeutic purposes and for unraveling the function of CCR3. We previously developed an anti-mouse
CCR3 (mCCR3) mAb (C3Mab-2; rat IgG2b, kappa) using the Cell-Based Immunization and Screening
method and showed that C3Mab-2 could detect endogenous and exogenous mCCR3 in flow cytometry. In
this study, we showed that C3Mab-2 and its recombinant antibody (recC3Mab-2f) specifically recognizedendogenous mCCR3 in P388 (a mouse lymphocyte-like cell line) and J774-1 (a mouse macrophage-like cellline) cells and are usable in immunocytochemistry.
Keywords: mouse CCR3, C3Mab-2, monoclonal antibody, immunocytochemical analysis

tions of target molecules. Therefore, developing mAbs thatspecifically recognize endogenous CCR3 in multiple applications would be advantageous, although the production ofmAbs for GPCR remains notoriously difficult.
Using the Cell-Based Immunization and Screening(CBIS) method, we have developed mAbs that target transmembrane proteins, such as podoplanin,(10,11) CD133,(12)EpCAM,(13) PD-L1,(14) and several GPCRs, such as CCR8(15)and CCR9.(16) We also previously developed an anti-mouse
CCR3 (mCCR3) mAb (C3Mab-2; rat IgG2b, kappa) throughthe CBIS method and showed that C3Mab-2 was usable inflow cytometry.(17) Furthermore, this study demonstrates that
C3Mab-2 is usable in immunocytochemistry against endogenous mCCR3 in cultured lymphocyte-like cells andmacrophage-like cells.

IntroductionT

he C–C motif chemokine receptor 3 (CCR3) is a seventransmembrane G protein-coupled receptor (GPCR)
activated by eotaxin-1/CCL11, eotaxin-2/CCL24, eotaxin-3/
CCL26, MCP-2/CCL8, MCP-3/CCL7, MCP-4/CCL13, andRANTES/CCL5.(1) CCR3 is expressed by eosinophils, basophils, T helper 2 (TH2) cells, and airway smooth muscle
cells(2–5); it contributes to the recruitment/migration of cells
at allergic inflammation and asthma sites. Moreover, CCR3
participates in cancer development. Cancer cells from breast
cancer, glioblastoma, lung adenocarcinoma, and renal cell
carcinoma express high CCR3 levels, increasing their proliferation, migration, and invasion.(6–9) Therefore, CCR3 canbe a diagnostic and therapeutic target molecule for allergic
diseases and cancer.
Developing diagnostic and therapeutic methods requires a
precise understanding of the physiological roles of CCR3.
However, the physiological roles of CCR3 remain unclear.
In general, monoclonal antibodies (mAbs) help explore
diagnostic and therapeutic methods and elucidate the func-

Materials and Methods
Cell lines

We purchased Chinese hamster ovary (CHO)-K1 cells
from the American Type Culture Collection (Manassas, VA).

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

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We previously reported the development of the CHO-K1 cellline stably expressing mCCR3 (CHO/mCCR3).(17) The Cell
Resource Center for Biomedical Research Institute ofDevelopment, Aging and Cancer, Tohoku University(Miyagi, Japan) provided the mouse lymphocyte-like cellline (P388 cells) and mouse macrophage-like cell line ( J7741 cells). We cultured the CHO-K1, CHO/mCCR3, P388, andJ774-1 cells in Roswell Park Memorial Institute (RPMI) 1640
medium (Nacalai Tesque, Inc., Kyoto, Japan) supplemented
with 10% heat-inactivated fetal bovine serum (Thermo Fisher
Scientific, Inc., Waltham, MA), 100 U/mL of penicillin,
100 lg/mL of streptomycin, and 0.25 lg/mL of amphotericin B (Nacalai Tesque, Inc.). We maintained the cells in ahumidified atmosphere at 37C with 5% CO2.

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Antibodies

We previously described C3Mab-2 development.(17) To
produce recombinant C3Mab-2 (recC3Mab-2f), we subcloned variable (VH) and constant (CH) regions of heavy chain

SAITO ET AL.

cDNAs of C3Mab-2 into the pCAG-Neo vector along withvariable (VL) and constant (CL) regions of light chain cDNAs
of C3Mab-2 into the pCAG-Ble vector (FUJIFILM Wako
Pure Chemical Corporation, Osaka, Japan). We transfected
C3Mab-2 vectors into BINDS-09 cells (www.med-tohokuantibody.com/topics/001_paper_cell.htm) using the ExpiCHO
Expression System (Thermo Fisher Scientific, Inc.). We purified the resulting mAb (recC3Mab-2f) using an Ab-Capcher(ProteNova, Kagawa, Japan). We purchased an anti-mCCR3
mAb (clone J073E5) from BioLegend (San Diego, CA) andAlexa Fluor 488-conjugated anti-rat IgG from Cell Signaling
Technology, Inc., (Danvers, MA).
Immunocytochemistry

We conducted immunocytochemistry in two ways: a conventional way (prefix method) and a modified way (postfix
method). In the prefix method, we fixed CHO-K1, CHO/
mCCR3, P388, and J774-1 cells with 4% paraformaldehyde(PFA) in phosphate-buffered saline (PBS) for 10 minutes

FIG. 1. Immunocytochemical experiment against endogenous mCCR3 through the prefix method. P388 cells (A) andJ774-1 cells (B) fixed with 4% PFA were incubated with buffer control, 10 mg/mL of J073E5, 10 mg/mL of C3Mab-2, or
10 mg/mL of recC3Mab-2f for 1 hour, followed by incubation with Alexa Fluor 488-conjugated anti-rat IgG and DAPI for 45
minutes. Scale bars: 20 mm. DAPI, 4¢,6-diamidino-2-phenylindole; mCCR3, mouse C–C motif chemokine receptor 3; PFA,
paraformaldehyde.

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ANTI-MOUSE CCR3 MAB FOR IMMUNOCYTOCHEMISTRY

and quenched with 50 mM NH4Cl in PBS supplemented with
0.2 mM Ca2+ and 2 mM Mg2+ (PBSc/m) for 10 minutes atroom temperature. Next, we blocked the cells using blocking buffer (PBSc/m supplemented with 0.5% bovine serum
albumin) for 30 minutes and incubated them with 10 lg/mLof primary antibodies for 1 hour and then with Alexa Fluor
488-conjugated anti-rat IgG (1:400) for 45 minutes at room
temperature. We stained the cell nuclei with 4¢,6-diamidino2-phenylindole (DAPI) (Thermo Fisher Scientific, Inc.).
In the postfix method, we blocked the CHO-K1, CHO/
mCCR3, P388, and J774-1 cells using a chilled blocking
buffer for 30 minutes on ice. We further incubated the cellswith chilled primary antibodies (10 lg/mL) for 1 hour,
followed by chilled secondary antibody (1:400) for 45
minutes on ice. We then fixed the cells with 4% PFA in PBS
for 10 minutes on ice and an additional 15 minutes at room
temperature. Finally, we treated the cells with PBSc/m
supplemented with DAPI for 10 minutes at room temperature. We acquired the fluorescence images using a
40 · objective on a BZ-X800 digital microscope (Keyence,
Osaka, Japan).

47
Results
Immunocytochemical analysis of CHO/mCCR3 cells
using C3Mab-2 and recC3Mab-2f

We previously showed that C3Mab-2 allowed to weakly
detect CHO/mCCR3 cells using flow cytometry.(17) First,
to investigate the binding of C3Mab-2 (10 lg/mL) andrecC3Mab-2f (10 lg/mL) to exogenous mCCR3, we fixed
CHO/mCCR3 cells in 4% PFA and conducted immunocytochemistry (prefix method). Unexpectedly, C3Mab-2 andrecC3Mab-2f did not detect exogenous mCCR3 in CHO/
mCCR3 cells (Supplementary Fig. S1A). Both antibodies
showed no signal in the parental CHO-K1 cells (Supplementary Fig. S1B). By contrast, a commercially available
anti-mCCR3 antibody ( J073E5, 10 lg/mL) clearly detected
the mCCR3 (Supplementary Fig. S1A), but it showed strong
nonspecific signals in CHO-K1 cells (Supplementary
Fig. S1B).
We wondered whether the PFA fixation changed thestructure of cell surface proteins in CHO-K1 cells, causing
the nonspecific signals observed with J073E5. To address this

FIG. 2. Immunocytochemical experiment against endogenous mCCR3 through the postfix method. P388 cells (A) andJ774-1 cells (B) were incubated with buffer control, 10 mg/mL of J073E5, 10 mg/mL of C3Mab-2, or 10 mg/mL of recC3Mab2f for 1 hour, followed by incubation with Alexa Fluor 488-conjugated anti-rat IgG for 45 minutes on ice. The cells were
fixed with 4% PFA and stained with DAPI. Scale bars: 20 mm.

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issue, we labeled living cells with J073E5 on ice and thecorresponding secondary antibody before fixation with 4%
PFA (postfix method). Consequently, J073E5 recognized
mCCR3 in CHO/mCCR3 cells (Supplementary Fig. S2A)
without visible nonspecific signals in CHO-K1 cells (Supplementary Fig. S2B), suggesting that, through the postfix
method, J073E5 detected mCCR3 specifically. By contrast,
both C3Mab-2 and recC3Mab-2f did not bind to mCCR3 in
CHO/mCCR3 using this method (Supplementary Fig. S2B).
These results suggest that C3Mab-2 and recC3Mab-2f could
not detect exogenous mCCR3 in an immunocytochemistry
experiment.

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Immunocytochemical analysis of P388 and J774-1
cells using C3Mab-2 and recC3Mab-2f

Next, we attempted to label endogenous mCCR3 with
C3Mab-2 (10 lg/mL) and recC3Mab-2f (10 lg/mL) throughthe prefix method. Consequently, both antibodies recognized
mCCR3 in the 4% PFA-fixed P388 and J774-1 cells (Fig. 1A,
B). C3Mab-2 and recC3Mab-2f also detected endogenous
mCCR3 in living P388 and J774-1 cells through the postfix
method, suggesting that C3Mab-2 and recC3Mab-2f recognize the endogenous mCCR3 located on the cell surface(Fig. 2A, B). Although J073E5 showed nonspecific strong dot
signals in P388 and J774-1 cells through the prefix method(Fig. 1A, B), it displayed specific but weak signals in both celllines through the postfix method (Fig. 2A, B). These results
suggest that C3Mab-2 and recC3Mab-2f can detect endogenous mCCR3 in immunocytochemistry experiments.

SAITO ET AL.

Some studies have demonstrated that antibodies against
CCR3 or its ligands can be therapeutic agents against inflammatory diseases. Blocking CCR3 activity with CCR3 mAbs
reduces eosinophilic inflammation, attenuating asthmatic
response, gastroenteritis, and pulmonary inflammation.(18–20)
An anti-CCL24 mAb suppresses the CCL24/CCR3 axis,
repairing liver fibrosis and inflammation.(21) This study
showed that C3Mab-2 and recC3Mab-2f react with endogenous
mCCR3 in P388 and J774-1 cells, suggesting that both antibodies are usable for diagnosis and/or allergic inflammation
and asthma medical treatment development in mouse models.
Moreover, CCR3 regulates tumor progression. CCL7
promotes the migration of oral squamous cell carcinoma cells
through CCR1 and CCR3. Anti-CCR1 and anti-CCR3 mAbs
block CCR1 and CCR3, suppressing migration.(22) The CCR3
inhibitor SB328437 inhibited clear-cell renal cell carcinoma progression through the autocrine-regulatory loop of
gankyrin/STAT3/CCL24/CCR3 in vitro and in vivo.(23)
Future studies should investigate (1) whether C3Mab-2 andrecC3Mab-2f recognize endogenous CCR3 expressed bymouse cancer cells, (2) whether they suppress CCR3mediated signal transduction, and (3) whether they show
antitumor activity in vitro and in vivo.
In conclusion, our study reveals that C3Mab-2 andrecC3Mab-2f are useful for detecting endogenous mCCR3
through immunocytochemistry experiments.
Author Disclosure Statement

No competing financial interests exist.

Discussion

Funding Information

This study demonstrated that C3Mab-2 and recC3Mab-2f
could detect endogenous mCCR3 in the PFA-fixed P388 andJ774-1 cells in immunocytochemistry experiments. Moreover, C3Mab-2 and recC3Mab-2f recognized endogenous
mCCR3 in living P388 and J774-1 cells, indicating that both
antibodies bind to the extracellular antigen region of the cellsurface mCCR3. By contrast, C3Mab-2 and recC3Mab-2f did
not allow the detection of exogenously expressed mCCR3 in
CHO/mCCR3 cells. Although C3Mab-2 detected mCCR3
in CHO/mCCR3 cells through flow cytometry, the fluorescence intensity was weaker than with J073E5.(17) We supposethat immunocytochemical methods are not sensitive enoughto detect exogenous mCCR3 recognition by C3Mab-2. However, we cannot exclude the possibility that C3Mab-2 andrecC3Mab-2f can recognize exogenous mCCR3 expressedin other cell lines.
This study compared the immunolabeling of C3Mab-2
and recC3Mab-2f with that of J073E5, although J073E5 is
only applicable to flow cytometry according to the manufacturer’s technical datasheet. Regarding the strong nonspecific signals caused by J073E5 in PFA-fixed cells (prefix
method) (Fig. 1 and Supplementary Fig. S1), we speculatethat PFA altered the morphology of cell surface proteins,which interacted nonspecifically with J073E5. In this study,
we can exclude an effect of residual PFA because ammonium
chloride quenched it and because C3Mab-2 and recC3Mab-2f
showed no nonspecific signal. By contrast, J073E5 specifically detected mCCR3 when we immunolabeled before fixing with PFA (postfix method) (Fig. 2 and Supplementary
Fig. S2).

This research was supported in part by Japan Agency
for Medical Research and Development (AMED) underGrant Nos. JP21am0401013 (to Y.K.) and JP21am0101078(to Y.K.), and by the Japan Society for the Promotionof Science ( JSPS) Grants-in-Aid for Scientific Research(KAKENHI) Grant Nos. 21K06059 (to M.S.), 21K15523(to T.A.), 21K20789 (to T.T.), 21K07168 (to M.K.K.), and19K07705 (to Y.K.).
Supplementary Material

Supplementary Figure S1
Supplementary Figure S2
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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: October 12, 2021
Accepted: January 11, 2022