MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 42, Number 2, 2023
ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2022.0034
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Epitope Mapping of Anti-Mouse CCR3 Monoclonal
Antibodies (C3Mab-6 and C3Mab-7)

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Nami Tateyama,1,* Teizo Asano,1,* Tomohiro Tanaka,2 Yu Isoda,1 Yuki Okada,2 Hiyori Kobayashi,2 Guanjie Li,2Ren Nanamiya,1 Takeo Yoshikawa,3 Mika K. Kaneko,1,2 Hiroyuki Suzuki,1,2 and Yukinari Kato1–3

One of G protein-coupled receptors, CC chemokine receptor 3 (CCR3), is expressed in eosinophils, basophils, asubset of Th2 lymphocytes, mast cells, and airway epithelial cells. CCR3 levels in the serum of colorectal
cancer patients are significantly higher than in control groups. Moreover, CCR3 is essential for recruiting
eosinophils into the lung. Therefore, CCR3 is considered both a therapeutic target for colorectal cancer andallergic diseases. Previously, we established anti-mouse CCR3 (mCCR3) monoclonal antibodies (mAbs),
C3Mab-6 (rat IgG1, kappa) and C3Mab-7 (rat IgG1, kappa), by immunizing a rat with an N-terminal peptide ofmCCR3. These mAbs can be used in flow cytometry and enzyme-linked immunosorbent assays. In this study,
we performed the epitope mapping of C3Mab-6 and C3Mab-7 using alanine scanning. The reactivity between
these mAbs and point mutants of mCCR3 were analyzed using flow cytometry. The results indicated that Phe3,Asn4, Thr5, Asp6, Glu7, Lys9, Thr10, and Glu13 of mCCR3 are essential for C3Mab-6 binding, whereas Phe15
and Glu16 are essential for C3Mab-7 binding.
Keywords: mouse CCR3, monoclonal antibody, epitope, flow cytometry, alanine scanning

cancer,17 breast cancer,18 and oral squamous cell carcinomas.19 Therefore, CCR3 is regarded as the therapeutic target
for both allergic diseases and cancers.9,15,20–22
Monoclonal antibodies (mAbs) are used for the treatment
of inflammatory diseases and cancers.23–26 Many therapeuticmAbs possess neutralizing activity through the blockade
between the targets and their ligands.27 The epitope identification of mAbs is important to elucidate the pharmacological function of mAbs, and is essential to avoid unexpectedcross-reactivity. Previously, we produced novel anti-mouse
CCR3 (mCCR3) mAbs, C3Mab-6 (rat IgG1, kappa), andC3Mab-7 (rat IgG1, kappa) by immunizing a rat with themCCR3 N-terminal peptide.28 C3Mab-6 and C3Mab-7 reacted not only with mCCR3-overexpressed Chinese hamster
ovary-K1 (CHO/mCCR3) cells, but also P388 (mouse lymphoid neoplasma) and J774-1 (mouse macrophage-like) cells,
which express mCCR3 endogenously in flow cytometry.
However, the critical amino acids of mCCR3 for the bindingof C3Mab-6 and C3Mab-7 have not been determined.

IntroductionT

he CC chemokine receptor 3 (CCR3), also known as
CD193, is a member of the G protein-coupled receptor.1
CCR3 is a seven-transmembrane domain receptor with four
extracellular domains, and the N-terminal region of CCR3 islocated in extracellular, whereas the C-terminal region is located in intracellular. CCR3 is expressed on the surface ofeosinophils, basophils, a subset of Th2 lymphocytes, mastcells, and airway epithelial cells.2–6 Some CC chemokines,
such as eotaxin-1, eotaxin-2, eotaxin-3, MCP-3, and RANTES,
are known to be ligands for CCR3.7–9
CCR3 and its ligands play an important role in recruiting
eosinophils into the lung, inducing airway hyperresponsiveness in a murine model of allergic asthma.10–13
CCR3 also contributes to ocular allergy.14 Moreover, CCR3
levels in the serum of colorectal cancer patients are significantly higher than in control groups.15,16 High expression
levels of eotaxins occur in some tumors such as colorectal

Departments of 1Antibody Drug Development, 2Molecular Pharmacology, and 3Pharmacology, Tohoku University Graduate School ofMedicine, Sendai, Japan.
*These authors contributed equally to this study.

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FIG. 1. Determination of the C3Mab-6 and C3Mab-7 epitope for mCCR3 by flow cytometry. C3Mab-6 (1 mg/mL; grayline), C3Mab-7 (1 mg/mL; gray line), or NZ-1 (1 mg/mL; gray line) were treated with mCCR3 point mutants-overexpressed
CHO-K1 cells for 30 minutes at 4C, followed by the addition of the secondary antibody. The black line represents thenegative control. CHO-K1, Chinese hamster ovary-K1; mCCR3, mouse CC chemokine receptor 3.
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In this study, we determined the binding epitope ofC3Mab-6 and C3Mab-7 to mCCR3 using alanine scanning byflow cytometry.29–34

TATEYAMA ET AL.

E7A, I8A, K9A, T10A, V11A, V12A, E13A, S14A, F15A,E16A, T17A, T18A, P19A, and Y20A) were generated using
QuikChange Lightning Site-Directed Mutagenesis Kits (Agilent Technologies Inc., Santa Clara, CA, USA).

Materials and Methods
Cell lines

The pEX-A2J2 plasmid subcloned mCCR3 (accession No.
NM_009914.4) was purchased from Eurofins Genomics KK(Tokyo, Japan).28,35–37 Substitutions of amino acids to alanine
in the mCCR3 sequence were conducted by QuikChangeLightning Site-Directed Mutagenesis Kits (Agilent Technologies, Inc., Santa Clara, CA). PCR fragments bearing the desired
mutations were inserted into the pCAG-Ble vector (FUJIFILM
Wako Pure Chemical Corporation, Osaka, Japan) and a PA16
tag38–41 was added at the N-terminus using the In-Fusion HDCloning Kit (Takara Bio, Inc., Shiga, Japan). The alanine(glycine) substitution mutants (A2G, F3A, N4A, T5A, D6A,

CHO-K1 cells were obtained from the America Type Culture Collection (ATCC, Manassas, VA). The CHO/mCCR3cells were produced in our previous study.37 mCCR3 point
mutant plasmids were transfected into CHO-K1 cells using theNeon Transfection System (Thermo Fisher Scientific, Inc.,Waltham, MA). Stable transfectants were selected using a cellsorter (SH800; Sony Corp., Tokyo, Japan). CHO/mCCR3 celland stable transfectants were cultured in 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 streptomycin, andDownloaded by Yukinari Kato from www.liebertpub.com at 04/27/23. For personal use only.

Plasmids of mCCR3 point mutants

FIG. 2. Schematic illustration of C3Mab-6 and C3Mab-7 epitope for mCCR3. (A) C3Mab-6 epitope for mCCR3 involves
Phe3, Asn4, Thr5, Asp6, Glu7, Lys9, Thr10, and Glu13 of mCCR3. (B) C3Mab-7 epitope for mCCR3 involves Phe15 andGlu16 of mCCR3.

EPITOPE OF ANTI-MCCR3 MAB

Cells were harvested after brief exposure to 0.25% trypsin/1 mM ethylenediaminetetraacetic acid (Nacalai Tesque,Inc.). After washing with 0.1% bovine serum albumin inphosphate-buffered saline, cells were treated with C3Mab-6,C3Mab-7, or NZ-1 (1 lg/mL) for 30 minutes at 4C and subsequently with Alexa Fluor 488-conjugated anti-rat IgG (1:1000;Cell Signaling Technology, Inc., Danvers, MA). Fluorescencedata were collected using EC800 Cell Analyzer (Sony Corp.).

N-terminus of human CCR3 (residues 8–23).42,43 Therefore,
it is expected that both C3Mab-6 and C3Mab-7 may competewith eotaxin for binding to mCCR3.
Zhu et al. showed that the dissociation constants (KD) ofCCR3’s binding to eotaxin-1, -2, and -3 are 2.1, 9.7, and1.2 · 10-9 M, respectively.44 We previously showed thatC3Mab-6 and C3Mab-7 bound to CHO/mCCR3 with KD of
8.7 · 10-9 M and 3.7 · 10-9 M, respectively.28 The KD values ofC3Mab-6 and C3Mab-7 to CHO/mCCR3 were comparable withthat of eotaxin. Therefore, we expect that C3Mab-6 and C3Mab7 treatments would block eotaxin’s binding to CHO/mCCR3.
In future studies, the neutralizing activities of C3Mab-6 andC3Mab-7 should be further assessed. Moreover, these mAbs
will be useful for depleting mCCR3-expressing eosinophils
and basophils, and targeting mCCR3-positive cancers.

Results

Author Disclosure Statement

0.25 lg/mL amphotericin B (Nacalai Tesque, Inc.) at 37C in ahumidified atmosphere containing 5% CO2. The stabletransfectants were cultivated in a medium containing
0.5 mg/mL Zeocin (InvivoGen, San Diego, CA).
Flow cytometry

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To investigate the epitope of C3Mab-6 and C3Mab-7 formCCR3, we conducted alanine scanning. We constructed 19
alanine substitution mutants of mCCR3. Mutant proteins
were stably expressed on CHO-K1 cells, and the reactivities
against C3Mab-6, C3Mab-7, or anti-PA16 tag mAb (NZ-1)
were analyzed using flow cytometry.
First, we investigated the cell surface expression of mCCR3mutants on CHO-K1 cells using NZ-1. The reactivities ofNZ-1 against each mCCR3 mutant were confirmed in all themutants (Fig. 1, right). Since PA16 tag was not conjugated inthe wild-type mCCR3, NZ-1 did not react with it (Fig. 1, right).
Next, we examined the reactivities of C3Mab-6 and C3Mab-7
against mCCR3 mutants. C3Mab-6 reacted with 11 mutants(A2G, I8A, V11A, V12A, S14A, F15A, E16A, T17A, T18A,P19A, and Y20A) and wild-type mCCR3 (Fig. 1, left).
In contrast, C3Mab-6 did not react with 8 mutants (F3A, N4A,T5A, D6A, E7A, K9A, T10A, and E13A) (Fig. 1, left). C3Mab-7
reacted with 17 mutants (A2G, F3A, N4A, T5A, D6A, E7A, I8A,K9A, T10A, V11A, V12A, E13A, S14A, T17A, T18A, P19A,and Y20A) and wild-type mCCR3, whereas 2 mutants (F15Aand E16A) lost their reactivity to C3Mab-7 (Fig. 1, middle).
These results indicated that eight amino acids of mCCR3 (Phe3,Asn4, Thr5, Asp6, Glu7, Lys9, Thr10, and Glu13) are critical for
C3Mab-6 binding (Fig. 2A), whereas two amino acids (Phe15and Glu16) are critical for that on C3Mab-7 (Fig. 2B).
Discussion

Therapeutic mAbs possesses biological activities, including neutralizing activity against the physiological ligands.27
The epitope determination in the target protein is necessary tounderstand the properties of mAbs. Previously, we established two anti-mCCR3 mAbs, C3Mab-6 and C3Mab-7,
which were established by immunizing the N-terminal peptide of mCCR3 (1-MAFNTDEIKTVVESFETTP-19).28
Therefore, the binding epitope of C3Mab-6 and C3Mab-7 areincluded within the residues.
In this study, we performed the epitope mapping ofC3Mab-6 and C3Mab-7 using alanine scanning of themCCR3 N-terminal domain. The results showed that C3Mab6 and C3Mab-7 have different binding regions of mCCR3(Fig. 1). The C3Mab-6 epitope is mainly located in the firsthalf of the N-terminal region, whereas that of C3Mab-7 islocated in another half of the N-terminal region (Fig. 2).
Structural analyses showed that eotaxin binds to theNo competing financial interests exist.
Funding Information

This research was supported in part by Japan Agency for
Medical Research and Development (AMED) under grantnos. JP22ama121008 (to Y.K.), JP22am0401013 (to Y.K.),
JP22bm1004001 (to Y.K.), JP22ck0106730 (to Y.K.), andJP21am0101078 (to Y.K.), and by the Japan Society for thePromotion of Science ( JSPS) Grants-in-Aid for Scientific Research (KAKENHI) grant nos. 21K15523 (to T.A.), 21K20789(to T.T.), 22K06995 (to H.S.), 21K07168 (to M.K.K.), and22K07224 (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: yukinari.kato.e6@tohoku.ac.jp
Received: September 26, 2022
Accepted: March 21, 2023