MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 41, Number 6, 2022
ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2022.0021
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Development of a Novel Anti-Mouse CCR6
Monoclonal Antibody (C6Mab-13)
by N-Terminal Peptide Immunization
Teizo Asano,1 Tomohiro Tanaka,1 Hiroyuki Suzuki,2 Guanjie Li,2 Ren Nanamiya,1Nami Tateyama,1 Yu Isoda,1 Yuki Okada,2 Hiyori Kobayashi,2
Takeo Yoshikawa,3 Mika K. Kaneko,1 and Yukinari Kato1–3

The CC chemokine receptor 6 (CCR6) is a G protein-coupled receptor family member that is highly expressedin B lymphocytes, certain subsets of effector and memory T cells, and immature dendritic cells. CCR6 has only
one chemokine ligand, CCL20. The CCL20–CCR6 axis has been recognized as a therapeutic target for autoimmune diseases and tumor. This study developed specific monoclonal antibodies (mAbs) against mouse CCR6(mCCR6) using the peptide immunization method. The established anti-mCCR6 mAb, C6Mab-13 (rat IgG1,
kappa), reacted with mCCR6-overexpressed Chinese hamster ovary-K1 (CHO/mCCR6), and mCCR6endogenously expressed P388 (mouse lymphoid neoplasma) and J774-1 (mouse macrophage-like) cells in flow
cytometry. The dissociation constant (KD) of C6Mab-13 for CHO/mCCR6 cells was determined to be 2.8 · 10-9
M, indicating that C6Mab-13 binds to mCCR6 with high affinity. In summary, C6Mab-13 is useful for detecting
mCCR6-expressing cells through flow cytometry.
Keywords: mouse CCR6, monoclonal antibody, peptide immunization
IntroductionT

he CC chemokine receptor 6 (CCR6), also known as
CD196, is a member of the G protein-coupled receptor(GPCR) family consisting of an extracellular N-terminus,
seven membrane-spanning regions, and a cytoplasmic Cterminus.1 CCR6 was first cloned in human and is a 374
amino acid-long protein.2 CCR6 is expressed in B lymphocytes, certain subsets of effector and memory T cells, andimmature dendritic cells, but not in monocytes, natural killer
cells, and granulocytes.2–5 The ligand of CCR6, CCL20/
macrophage inflammatory protein-3a, is increased in inflammatory and/or autoimmune conditions.6 CCL20 isinvolved in the recruitment of Th17 cells to sites of inflammation.7 Several studies using CCR6-knockout mice or
CCR6 inhibitors indicate the potential of CCR6 as a therapeutic target for autoimmune diseases.6,8
Furthermore, CCL20 is elevated in several tumor types,
including breast, hepatocellular, pancreatic, and colorectal
carcinomas.9,10 Similarly, its expression is observed intumor-associated macrophages, which are a significant sourceof the ligands in the tumors.11,12 CCR6 expression has beenshown on tumor cells. For instance, an autocrine stimulationof their proliferation and migration by the CCL20–CCR6
pathway plays a crucial role in tumor progression.13,14 Furthermore, CCL20 in tumors recruits regulatory T (Treg) cells
and Th17 in the stroma, resulting in tumor immune evasion.15–17 In addition, it has been reported that high expression of CCR6 and CCL20 in tumors correlates with poor
prognosis.18–21
Previously, we have developed monoclonal antibodies(mAbs) against membrane proteins, including podoplanin,22–25
CD20,26,27 CD44,28,29 EpCAM,30 TROP2,31 EGFR,32
HER2,33 and HER3.34 Furthermore, we have succeededin the development of some anti-GPCR mAbs, including
anti-mouse CCR2 mAb,35 anti-mouse CCR3 mAbs,36–38 antimouse CCR4 mAb,39 anti-mouse CCR8 mAbs,40–42 antimouse CXCR6 mAb,43 and anti-human CCR9 mAb.44 In thisstudy, we established a novel anti-mouse CCR6 (mCCR6)
mAb using the N-terminal peptide immunization methodand determined the dissociation constant (KD) using mCCR6expressed cell lines by flow cytometry.

Departments of 1Antibody Drug Development, 2Molecular Pharmacology, and 3Pharmacology, Tohoku University Graduate Schoolof Medicine, Sendai, Miyagi, Japan.

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Materials and Methods

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Cell lines

Chinese hamster ovary (CHO)-K1 and P3X63Ag8U.1(P3U1) cells were obtained from the American Type Culture
Collection (Manassas, VA). Mouse lymphoid neoplasma(P388) and mouse macrophage-like ( J774-1) cells were obtained 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 mCCR6 (Accession
No. NM_001190333.1) was subcloned into a pCAG-Ble
vector (FUJIFILM Wako Pure Chemical Corporation, Osaka,
Japan). CHO-K1 cells were transfected with plasmids bearing mCCR6 with tags (PA tag at N-terminus, and RAP and
MAP tags at C-terminus) using the Neon Transfection System (Thermo Fisher Scientific Inc., Waltham, MA). Stabletransfectants were established through cell sorting using acell 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, mCCR6-overexpressed CHO-K1 (CHO/
mCCR6), P388, and J774-1 cells were cultured in a Roswell
Park Memorial Institute (RPMI) 1640 medium (Nacalai
Tesque, Inc., Kyoto, Japan), which was supplemented with
10% heat-inactivated fetal bovine serum (Thermo Fisher
Scientific Inc.), 100 U/mL penicillin, 100 lg/mL streptomycin, and 0.25 lg/mL amphotericin B (Nacalai Tesque, Inc.).
Cells were grown in a humidified incubator at 37C with anatmosphere of 5% carbon dioxide and 95% air.

ASANO ET AL.

To develop mAbs against mCCR6, one rat was intraperitoneally immunized, using 300 lg mCCR6p1-19C-KLH
peptide with Imject Alum (Thermo Fisher Scientific Inc.).
The procedure included three additional immunization sessions, which was followed by a final booster intraperitoneal
injection, administered 2 days before the harvest of spleencells. 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 mCCR6p119C peptide, using enzyme-linked immunosorbent assay(ELISA), after flow cytometry, using CHO/mCCR6, CHO-K1,
P388, and J774-1 cells.

Antibodies

An anti-mCCR6 mAb (clone 29-2L17) was bought from
BioLegend (San Diego, CA). Secondary Alexa Fluor 488conjugated anti-rat IgG and Alexa Fluor 488-conjugated antiArmenian hamster IgG were bought from Cell Signaling
Technology, Inc. (Danvers, MA).
PeptidesEurofins Genomics KK (Tokyo, Japan) synthesized a partial
sequence of the N-terminal extracellular region of mCCR6(1-MNSTESYFGTDDYDNTEYY-19) plus C-terminal cysteine (mCCR6p1-19C). Subsequently, the keyhole limpet
hemocyanin (KLH) was conjugated at the C-terminus of thepeptide (mCCR6p1-19C-KLH).
Production of hybridomas

A 7-week-old Sprague–Dawley (SD) rat was purchasedfrom CLEA Japan (Tokyo, Japan), and was housed under
specific pathogen-free conditions. All animal experiments
were conducted according to relevant guidelines and regulations to minimize animal suffering and distress in the laboratory. Animal experiments were approved by the Animal
Care and Use Committee of Tohoku University (Permit No.
2019NiA-001). The rat was monitored daily for their healthduring the full 4-week duration of the experiment. A reduction of more than 25% of total body weight was defined as ahumane endpoint. The rat was euthanized through cervicaldislocation and death was verified through respiratory andcardiac arrest.

FIG. 1. Schematic illustration of the production of antimCCR6 mAbs. (A) An SD rat was immunized with Nterminal mCCR6 peptide using an intraperitoneal injection.
(B) Spleen cells were fused with P3U1 cells. (C) Culture
supernatants were screened with ELISA and flow cytometry
to select hybridomas that are producing anti-mCCR6 mAbs.
(D) After limiting dilution and some additional screenings,
anti-mCCR6 mAbs were finally established. CCR6, CC
chemokine receptor 6; CHO, Chinese hamster ovary; ELISA,
enzyme-linked immunosorbent assay; mAbs, monoclonal
antibodies; mCCR6, mouse CCR6; SD, Sprague–Dawley.

DEVELOPMENT OF ANTI-MOUSE CCR6 MAB
ELISA

of 1 lg/mL for 30 min at 37C. After washing with phosphate-buffered saline (PBS) containing 0.05% Tween20(PBST; Nacalai Tesque, Inc.), wells were blocked with 1%
bovine serum albumin (BSA)-containing PBST for 30 min at
37C. Plates were then incubated with supernatants of

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The synthesized peptide (MNSTESYFGTDDYDNTE
YYC), was immobilized on Nunc Maxisorp 96 well immunoplates (Thermo Fisher Scientific Inc.) at a concentration

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FIG. 2. Flow cytometric analyses using C6Mab-13. CHO/mCCR6, CHO-K1, P388, and J774-1 cells were treated with 10,
1, 0.1, or 0.01 mg/mL of C6Mab-13, followed by treatment with Alexa Fluor 488-conjugated anti-rat IgG. Fluorescence data
were obtained using SA3800 Cell Analyzer. Black line, negative control.

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Flow cytometry

Cells were harvested after a brief exposure to 1 mM
ethylenediaminetetraacetic acid (Nacalai Tesque, Inc.). They
were washed with 0.1% BSA in PBS and treated with 10,
1, 0.1, or 0.01 lg/mL primary mAbs for 30 minutes at 4C.
The cells were then treated with Alexa Fluor 488-conjugatedDownloaded by Yukinari Kato from www.liebertpub.com at 12/28/22. For personal use only.

hybridomas, followed by peroxidase-conjugated anti-rat
immunoglobulins (1:20000 diluted; Sigma-Aldrich Corp., St.
Louis, MO). Next, enzymatic reactions were conducted,using ELISA POD Substrate TMB Kit (Nacalai Tesque, Inc.),
followed by measurement of the optical density at 655 nm,
using iMark microplate reader (Bio-Rad Laboratories, Inc.,
Berkeley, CA).

ASANO ET AL.

FIG. 3. Flow cytometric analyses using 29-2L17. CHO/mCCR6, CHO-K1, P388, and J774-1 cells were treated with 10, 1,
0.1, or 0.01 mg/mL of 29-2L17, followed by treatment with Alexa Fluor 488-conjugated anti-Armenian hamster IgG.
Fluorescence data were obtained using SA3800 Cell Analyzer. Black line, negative control.

DEVELOPMENT OF ANTI-MOUSE CCR6 MAB

347

anti-rat IgG (1:1000) or Alexa Fluor 488-conjugated antiArmenian hamster IgG (1:1000). Fluorescence data wereobtained using SA3800 Cell Analyzer (Sony Corp.).
Determination of KD by flow cytometry

The CHO/mCCR6 cells were suspended in 100 lL of seriallydiluted anti-mCCR6 mAbs and then 50 lL of Alexa Fluor 488conjugated anti-rat IgG (1:200) or Alexa Fluor 488-conjugatedanti-Armenian hamster IgG (1:200) was added. Fluorescencedata were obtained using the BD FACSLyric (BD Biosciences,Franklin Lakes). The KD was calculated by fitting saturation
binding curves to built-in one-site binding models in GraphPad
Prism 6 (GraphPad Software, Inc., La Jolla).
Results

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Establishment of anti-mCCR6 mAbs
using the peptide immunization method

To develop anti-mCCR6 mAbs, we employed the peptide
immunization method (Fig. 1). One SD rat was immunized with
mCCR6p1-19C-KLH (Fig. 1A). The spleen of the rat was excised, and splenocytes were subsequently fused with myeloma
cells using PEG1500 (Fig. 1B). The developed hybridomas
were seeded into 96-well plates and cultivated for 7 days.
Culture supernatants that were positive for mCCR6p119C peptide were screened using ELISA (Fig. 1C). The firstscreening approach identified signals from mCCR6p1-19C
in 477 out of 1916 wells (24.9%). We selected the top 82
wells for second screening. The second screening procedure
identified signals from CHO/mCCR6 cells in 20 out of the
82 hybridoma supernatants identified in the previous step
(24.4%). After limiting dilution and several additional flow
cytometric screenings, an anti-mCCR6 mAb, C6Mab-13 (rat
IgG1, kappa) was eventually established (Fig. 1D).
Flow cytometry analysis

We performed flow cytometry using C6Mab-13, and commercially available anti-mCCR6 mAb (clone 29-2L17)
against CHO/mCCR6, CHO-K1, P388, and J774-1 cells.
C6Mab-13 dose-dependently recognized CHO/mCCR6 cells
at 10, 1, 0.1, and 0.01 lg/mL, whereas C6Mab-13 did notreact to CHO-K1 cells even at 10 lg/mL (Fig. 2). C6Mab-13
also reacted to P388 and J774-1 cells at 10, 1, and 0.1 lg/mL,
and did not react at 0.01 lg/mL (Fig. 2). Another antimCCR6 mAb (clone 29-2L17) also recognized CHO/mCCR6and J774-1 cells in a dose-dependent manner at 10, 1, 0.1, and0.01 lg/mL, and P388 cells at 10, 1, and 0.1 lg/mL, but not
CHO-K1 cells (Fig. 3). These results suggest that C6Mab-13
is useful for detecting exogenously and endogenously expressed mCCR6 using flow cytometry.
Determination of the KD between anti-mCCR6 mAbs
and CHO/mCCR6 cells by flow cytometry

To assess the KD of C6Mab-13 and 29-2L17 with mCCR6expressing cells, we performed the kinetic analysis of the interactions of C6Mab-13 and 29-2L17 with CHO/mCCR6 cells
using flow cytometry. The geometric mean of fluorescence intensity was plotted versus the concentrations of C6Mab-13 or
29-2L17, and fitted by one-site binding models in GraphPad
Prism 6. The KD of C6Mab-13 for CHO/mCCR6 cells wasdetermined to be 2.8 · 10-9 M (Fig. 4A). In addition, the KD of

FIG. 4. Determination of the binding affinity of C6Mab-13
and 29-2L17. CHO/mCCR6 cells were suspended in 100 mL
serially diluted C6Mab-13 (0.00031–5 mg/mL) (A) or 292L17 (0.00061–10 mg/mL) (B). Subsequently, Alexa Fluor
488-conjugated anti-rat IgG and anti-Armenian hamster IgG
were added, respectively. Fluorescence data were collectedusing the FACSLyric.
29-2L17 for CHO/mCCR6 cells was determined to be
8.2 · 10-9 M (Fig. 4B). These results show that C6Mab-13 and29-2L17 possesses a high affinity for CHO/mCCR6 cells.
Discussion

The development of the mAbs for GPCRs is difficult because of the structural complexity of GPCRs. The Cell-Based
Immunization and Screening (CBIS) method is a usefultechnique to develop mAbs against complex structured antigens, including GPCRs (CCR3, CCR8, and CCR9)38,42,44
and four-transmembrane protein (CD20).26,27 We previously
developed an anti-human CCR9 mAb (clone C9Mab-1) byusing CBIS method and determined that C9Mab-1 epitope islocated on the N-terminus of the human CCR9.44,45 Therefore, we have developed mAbs against GPCRs, including
mouse CCR2,35 mouse CCR3,37 mouse CCR4,39 and mouse
CXCR643 by N-terminal peptide immunization.
In this study, we developed a novel mAb against mCCR6(C6Mab-13) recognizes exogenous and endogenous mCCR6
by flow cytometry (Fig. 2). Although most mAbs, developed
using the peptide immunization method, react with peptide,
they will not react with native GPCRs on plasma membranes.
Therefore, they are useful for ELISA and Western blot, butmany of them are not useful for flow cytometry. In thisstudy, we demonstrated that C6Mab-13 can be available forflow cytometric analysis. Moreover, C6Mab-13 binds to CHO/
mCCR6 cells with high affinity (Fig. 4A).

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348

CCL20 makes interactions with all three extracellulardomains of CCR6 and N-terminal residues from Tyr27 toLeu38. In contrast, N-terminal residues before Tyr27 in
CCR6 are not involved in CCL20 interaction.46 The major
sites of interaction on CCR6 are the second extracellulardomain and N-terminal residues from Tyr27 to Leu38 in
CCR6. The epitope of C6Mab-13 is located from 1st to 19th
amino acids of mCCR6; therefore, C6Mab-13 might notpossess neutralizing activity for CCL20.
The recruitment of Th17 cells through the CCL20–CCR6
pathway plays a critical role in the development of autoimmune diseases, including inflammatory bowel disease,
psoriasis, rheumatoid arthritis, and multiple sclerosis.6 Therefore, antibody-mediated inhibition of CCR6-positive cells
migration to the sites of inflammation could control thediseases. In future study, we will try to develop the mAbs,
which have neutralizing activity for CCL20 by using peptide
immunization method.
The CCR6-positive Treg cells infiltrating tumors have beenreported to play a critical role in the suppression of tumor immunity.47 Therefore, the depletion of Treg cells and/or blocking
of infiltration of Treg cells into tumor tissues by modulating theCCR6-CCL20 axis are promising strategies of treatment.7,9Recently, Robert et al reported that bispecific antibodies against
CCR6 and CXCR3 effectively block cell chemotaxis and induce specific antibody-dependent cell-mediated cytotoxicity(ADCC).48 We will convert the subclass of C6Mab-13 into
mouse IgG2a to investigate whether C6Mab-13 possesses the
ADCC activity in vitro and evaluate the depletion of Treg
cells and antitumor activities in vivo animal models.
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 Y.K.), JP22am0401013 (toY.K.), JP22bm1004001 (to Y.K.), JP22ck0106730 (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, Miyagi 980-8575
JapanE-mail: yukinari.kato.e6@tohoku.ac.jp
Received: May 22, 2022
Accepted: September 30, 2022