MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 42, Number 4, 2023
ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2023.0004

Original Articles
Open camera or QR reader andscan code to access this article
and other resources online.

Establishment of a Novel Anti-Human CCR6 Monoclonal
Antibody C6Mab-19 with the High Binding Affinity
in Flow Cytometry

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Tomohiro Tanaka,* Kaishi Kitamura,* Hiroyuki Suzuki,* Mika K. Kaneko, and Yukinari Kato

CC chemokine receptor 6 (CCR6) is a member of the G-protein-coupled receptor family that is highly expressed in B lymphocytes, effector and memory T cells, regulatory T cells, and immature dendritic cells. CCR6
has been revealed to have important functions in many pathological conditions, such as cancer, intestinal bowel
disease, psoriasis, and autoimmune diseases. The only CCR6 chemokine ligand, CC motif chemokine ligand 20(CCL20), is also involved in pathogenesis by interacting with CCR6. The CCL20/CCR6 axis is drawing
attention as an attractive therapeutic target for various diseases. In this study, we developed novel monoclonal
antibodies (mAbs) against human CCR6 (hCCR6) using the peptide immunization method, which are applicable to flow cytometry and immunohistochemistry. The established anti-hCCR6 mAb, clone C6Mab-19(mouse IgG1, kappa), reacted with hCCR6-overexpressed Chinese hamster ovary-K1 (CHO/hCCR6), humanliver carcinoma (HepG2), and human differentiated hepatoma (HuH-7) cells in flow cytometry. The dissociation
constant (KD) of C6Mab-19 was determined as 3.0 · 10-10 M for CHO/hCCR6, 6.9 · 10-10 M for HepG2, and
1.8 · 10-10 M for HuH-7. Thus, C6Mab-19 could bind to exogenously and endogenously expressed hCCR6 withextremely high affinity. Furthermore, C6Mab-19 could stain formalin-fixed paraffin-embedded lymph node
tissues from a patient with non-Hodgkin lymphoma by immunohistochemistry. Therefore, C6Mab-19 is suitablefor detecting hCCR6-expressing cells and tissues and could be useful for pathological analysis and diagnosis.
Keywords: CCR6, monoclonal antibody, peptide immunization, flow cytometry, immunohistochemistry

Introduction

C

hemokines are chemotactic cytokines with a small
molecular weight (8,000–12,000) and are classified into
four categories: CC, CXC, CX3C, and XC subfamilies, according to the number and position of cysteine residues intheir N-terminus.1,2 Approximately 50 chemokines, with diverse functions, have been discovered so far. Chemokines
were originally thought to function in the leukocyte development, differentiation, and migration, but they are now
known to be deeply involved in the pathogenesis of numerous
diseases, including inflammatory diseases and cancers.3,4
Chemokines exert their functions by binding to corresponding seven-transmembrane G-protein-coupled receptors(GPCRs). This chemokine-GPCR interaction is important forthe physiological functions and pathogenesis of cells.5
In 1997, the CC chemokine receptor 6 (CCR6) was discovered as a specific receptor for CC motif chemokine ligand
20 (CCL20).6 CCR6 has been reported to be expressed intissues, including intestinal mucosa, lung mucosa, and lymph
nodes, as well as B and T cells. CCL20, also known as macrophage inflammatory protein-3a or liver and activationregulated chemokine, is expressed in tissues, such as the liver,lung, and lymph nodes. CCL20 is expressed in immune-related
cells, such as dendritic cells, Th17 cells, natural killer cells, andB cells.4 CCL20 functions in cooperation with CCR6.
The binding mode of CCL20 and CCR6 has been structurally elucidated.7 CCL20 has the shortest N-terminus

Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, Sendai, Japan.
This article was previously published in preprint.org (doi: 10.20944/preprints202303.0181.v1).
*These authors contributed equally to this work.

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118

among chemokines and binds to a shallow surface region of
CCR6. CCL20 and/or CCR6 are involved in the progressionof numerous pathologies.8 CCL20/CCR6-activated M1
macrophages cause adenoid hypertrophy by adenoid epithelium inflammation. An imiquimod-induced psoriasis model
reported that skin-homing CCR6+ gdLow T cells strongly
produce interleukin-17A and cause inflammation.9 Furthermore, a CCR6 antagonist ameliorates inflammatory bowel
disease by regulating immune cell chemotaxis.10 These reports indicate that CCL20/CCR6 contributes to pathogenesisby controlling immune cells.
Many reports have shown the effects of CCL20/CCR6 intumors.11 The regulatory T (Treg) cells, which act as a suppressive factor of cancer immunity, interact with CCL20 in
the tumor microenvironment via CCR6 on Treg and contribute to cancer progression and poor prognoses, such as
oral, head and neck squamous carcinomas, and hepatocellular
carcinomas.12–14 CCL20, which is secreted by macrophages
and stromal fibroblasts, promotes cancer cells or Th17 cell
behavior.15–17 CCL20/CCR6 appears to play roles in managing immune cells around tumors. CCR6-expressing chimeric antigen receptor-T cells (CAR-T) have been shown tobe effective in lung cancer models.18 These findings make the
CCL20/CCR6 axis an attractive therapeutic target for various
diseases.
Previously, we have developed various monoclonal antibodies (mAbs) against mouse and human chemokine
receptors, including an anti-mouse CCR2 mAb,19 antimouse CCR3 mAbs,20–23 an anti-mouse CCR4 mAb,24 antimouse CCR6 mAbs,25 anti-mouse CCR8 mAbs,26 ananti-mouse CCR9 mAb,27 an anti-mouse CXCR6 mAb,28 ananti-human CCR2 mAb,29 and anti-human CCR9 mAbs.30,31
In this study, we developed anti-human CCR6 (hCCR6)
mAbs using the N-terminal peptide immunization method.
Materials and Methods
Cell lines

Human liver carcinoma (HepG2) and human differentiatedhepatoma (HuH-7) were obtained from the Cell Resource
Center for Biomedical Research Institute of Development,
Aging and Cancer at Tohoku University. Chinese hamster
ovary (CHO)-K1 and P3X63Ag8U.1 (P3U1) were obtained
from the American Type Culture Collection (Manassas, VA).
The expression plasmid of hCCR6 (pCMV6neo-hCCR6Myc-DDK; Accession No.: NM_004367) was purchasedfrom OriGene Technologies, Inc. (Rockville, MD). A Neon
Transfection System (Thermo Fisher Scientific, Inc., Waltham, MA) was used to transfect the hCCR6 plasmid into
CHO-K1 cells. Stable transfectants were established throughcell sorting using a cell sorter (SH800; Sony Corp., Tokyo,
Japan) and cultivation in a medium containing 0.5 mg/mL of
G418 (Nacalai Tesque, Inc., Kyoto, Japan).
CHO-K1, P3U1, hCCR6-overexpressed CHO-K1(CHO/hCCR6), and HuH-7 were cultured in the Roswell
Park Memorial Institute (RPMI)-1640 medium (Nacalai
Tesque, Inc.). HepG2 cells were cultured in Dulbecco’s
modified Eagle’s medium (DMEM; Nacalai Tesque, Inc.).
RPMI-1640 medium and DMEM were supplemented with
10% heat-inactivated fetal bovine serum (FBS; Thermo
Fisher Scientific, Inc.), 100 units/mL penicillin, 100 lg/mL
streptomycin, and 0.25 lg/mL amphotericin B (Nacalai

TANAKA ET AL.

Tesque, Inc.). Cells were grown in a humidified incubator at
37C with an atmosphere of 5% CO2 and 95% air.
Antibodies

An anti-hCCR6 mAb (clone G034E3) was purchased from
BioLegend (San Diego, CA). Secondary Alexa Fluor 488conjugated anti-mouse IgG was purchased from Cell Signaling Technology, Inc. (Danvers, MA).
PeptidesA partial sequence of the N-terminal extracellular region of
hCCR6 (1-MNFSDVFDSSEDYFVSVNT-19) plus C-terminal
cysteine (hCCR6p1-19C) was synthesized by Eurofins
Genomics KK (Tokyo, Japan). Subsequently, the keyhole
limpet hemocyanin (KLH) was conjugated at the C-terminusof the peptide (hCCR6p1-19C-KLH).
Production of hybridomas

The two 5-week-old BALB/c mice were purchased from
CLEA Japan (Tokyo, Japan) and were housed under specificpathogen-free conditions. All animal experiments wereconducted following relevant guidelines and regulations tominimize animal suffering and distress in the laboratory. The
Animal Care and Use Committee of Tohoku University(Permit number: 2019NiA-001) approved the animal experiments. The mice were monitored daily for their health during
the full 4-week experiment duration. A humane end point was
defined as a reduction of >25% of total body weight. The
mice were euthanized through cervical dislocation, and death
was verified through respiratory and cardiac arrest.
The mice were intraperitoneally immunized using 100 lg
of the hCCR6p1-19C-KLH peptide with Imject Alum
(Thermo Fisher Scientific, Inc.) to develop mAbs against
hCCR6. The procedure included three additional immunizations, followed by a final booster intraperitoneal injection,
2 days before harvesting the spleen cells. PEG1500 (Roche
Diagnostics, Indianapolis, IN) was used to subsequently fuseharvested spleen cells with P3U1 cells. Afterward, hybridomas were grown in the RPMI-1640 medium, supplemented
with 10% heat-inactivated FBS, 100 units/mL penicillin,
100 lg/mL streptomycin, 0.25 lg/mL amphotericin B, andhypoxanthine/aminopterin/thymidine (HAT) for the selection (Thermo Fisher Scientific, Inc.).
Enzyme-linked immunosorbent assay (ELISA) with the
hCCR6p1-19C peptide was used to subsequently screen supernatants, followed by flow cytometry, using CHO/hCCR6,
CHO-K1, HepG2, and HuH-7.
Enzyme-linked immunosorbent assay

The hCCR6p1-19C peptide was immobilized on Nunc
MaxiSorp 96-well immunoplates (Thermo Fisher Scientific,Inc.) at a concentration of 1 lg/mL for 30 minutes 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 minutes at 37C. The plates wereincubated with hybridoma supernatants, followed byperoxidase-conjugatedanti-mouse
immunoglobulins(1:2,000 diluted; Agilent Technologies, Inc., Santa Clara,
CA). Enzymatic reactions were performed using the ELISAESTABLISHMENT OF ANTI-HCCR6 MAB

POD Substrate TMB Kit (Nacalai Tesque, Inc.). Opticaldensity was measured at 655 nm using an iMark Microplate
Reader (Bio-Rad Laboratories, Inc., Berkeley, CA).

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K1 cells. An anti-hCCR6 mAb, C6Mab-19 (mouse IgG1,
kappa), was finally established after the single-cell cloning of
hybridoma by limiting dilution and several additional flow
cytometric screenings (Fig. 1D).

Flow cytometric analysis

Cells were harvested after a brief exposure to 0.25%
trypsin and 1 mM of ethylenediaminetetraacetic acid (Nacalai Tesque, Inc.). They were washed with 0.1% BSA in PBS
and treated with 1, 0.1, 0.01, or 0.001 lg/mL of anti-hCCR6
mAbs for 30 minutes at 4C. Then, the cells were treated with
Alexa Fluor 488-conjugated anti-mouse IgG (1:2,000).
Fluorescence data were obtained using SA3800 Cell Analyzer (Sony Corp.).

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Determination of KD by flow cytometry

The CHO/hCCR6, HepG2, and HuH-7 cells were suspended in 100 lL of serially diluted anti-hCCR6 mAbs, andthen, 50 lL of Alexa Fluor 488-conjugated anti-mouse IgG(1:200) was added. The BD FACSLyric (BD Biosciences,Franklin Lakes) was used to obtain fluorescence data. 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).
Immunohistochemical analysis using C6Mab-19
against non-Hodgkin lymphoma tissue

A formalin-fixed paraffin-embedded (FFPE) section oflymph nodes from non-Hodgkin lymphoma patients (Catalog
No. T2235161-1; BioChain Institute, Inc., Newark, CA) was
autoclaved in citrate buffer (pH6.0; Nichirei Biosciences, Inc.,Tokyo, Japan) for 20 minutes. Then, the section was thenincubated with 0.3 lg/mL of C6Mab-19 for 1 hour at room
temperature, followed by treatment of an Envision+ kit(Agilent Technologies, Inc.) for 30 minutes at room temperature. The color was developed using 3,3¢-diaminobenzidine
tetrahydrochloride (DAB; Agilent Technologies, Inc.) for 1
minute at room temperature, and the section was then counterstained with hematoxylin (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan).
Results
Establishment of anti-hCCR6 mAbs using the peptide
immunization method

In this study, we employed the peptide immunization
method to develop anti-hCCR6 mAbs (Fig. 1). Two mice
were immunized with hCCR6p1-19C-KLH (Fig. 1A). The
spleens of the mice were excised, and splenocytes were
subsequently fused with myeloma cells (P3U1) using
PEG1500 (Fig. 1B). The produced hybridomas were seeded
into 96-well plates and cultivated in HAT-contained medium
for 7 days.
Hybridoma culture supernatants, which were positive forthe hCCR6p1-19C peptide, were screened using ELISA(Fig. 1C). The first screening approach identified signals fromhCCR6p1-19C in 134 out of 958 wells (14.0%). We selected
the top 86 positive wells for the second screening. The second
screening procedure using flow cytometry identified signals
against CHO/hCCR6 cells in 49 out of the 86 hybridoma
supernatants (57.0%). They did not react with parental CHO-

Flow cytometric analysis of C6Mab-19
to hCCR6-expressing cells

We conducted flow cytometry using C6Mab-19 and acommercially available anti-hCCR6 mAb (clone G034E3)
against CHO/hCCR6, CHO-K1, HepG2, and HuH-7 cells.
We selected HepG2 and HuH-7 cells for endogenously
hCCR6-expressing cells because CCR6 expression was elevated in the human hepatocellular carcinoma cells in previous study.32 C6Mab-19 dose dependently recognized
CHO/hCCR6 cells at 1, 0.1, 0.01, and 0.001 lg/mL (Fig. 2A),
while C6Mab-19 did not react to CHO-K1 parental cells evenat 1 lg/mL (Fig. 2B). Additionally, C6Mab-19 possesses
higher sensitivity than G034E3, especially at 0.1 and0.01 lg/mL, although G034E3 recognized CHO/hCCR6 in adose-dependent manner at 1, 0.1, 0.01, and 0.001 lg/mL(Fig. 2A, C). Moreover, C6Mab-19 also reacted to endogenously hCCR6-expressing cell lines, such as HepG2 andHuH-7 (Fig. 2D, E). C6Mab-19 reacted to HepG2 at 1 and
0.1 lg/mL (Fig. 2D) and reacted to HuH-7 at a low concentration of 0.01 lg/mL (Fig. 2E). These results suggest that
C6Mab-19 is applicable for detecting exogenously and endogenously expressed hCCR6 using flow cytometry.
Determination of the KD for anti-hCCR6 mAbs against
hCCR6-expressing cells by flow cytometry

We performed the kinetic analysis using flow cytometry toassess the KD of anti-hCCR6 mAbs, such as C6Mab-19 andG034E3, for hCCR6-expressing cells. The KD of C6Mab-19
for CHO/hCCR6 cells was 3.0 · 10-10 M, as shown in
Figure 3A. In contrast, the KD of G034E3 for CHO/hCCR6cells was 1.5 · 10-9 M (Fig. 3B), indicating a much higher
binding affinity of C6Mab-19 than that of G034E3.
Next, we analyzed the KD of C6Mab-19 against endogenously hCCR6-expressing HuH-7 and HepG2 cells. The KD
of C6Mab-19 for HuH-7 and HepG2 was determined to be
1.8 · 10-10 M and 6.9 · 10-10 M, respectively (Fig. 3C, D).
Therefore, C6Mab-19 possesses a high affinity for exogenously and endogenously hCCR6-expressing cells.
Immunohistochemical analysis using C6Mab-19
against non-Hodgkin lymphoma tissues

We stained an FFPE lymph node tissue from patients with
non-Hodgkin lymphoma using C6Mab-19 to investigate theuse of C6Mab-19 for immunohistochemical analysis. C6Mab19 strongly stained non-Hodgkin lymphoma cells (Fig. 4A, B).
In contrast, no staining was observed without primary mAb(Fig. 4C, D). Therefore, C6Mab-19 is useful for immunohistochemical staining of FFPE sections.
Discussion

We have previously established various mAbs against
membrane proteins using the Cell-Based Immunization andScreening (CBIS) method. The CBIS method is a valid
technique to develop mAbs against complex structured antigens, including GPCRs. Using the CBIS method, weTANAKA ET AL.

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FIG. 1. A schematic illustration of the production flow of anti-hCCR6 mAbs. (A) The BALB/c mice were immunizedwith the hCCR6 peptide via the intraperitoneal route. (B) Hybridoma was produced by the fusion of P3U1 and mice
splenocytes cells. (C) The first screening of hybridomas was conducted by ELISA against hCCR6p1–19C peptide, followedby flow cytometric secondary screening using hCCR6-expressing cells and parental cells. (D) Anti-hCCR6 mAbs were
established after cloning via limiting dilution and additional analysis. ELISA, enzyme-linked immunosorbent assay;
hCCR6, human CC chemokine receptor 6; mAbs, monoclonal antibodies.
successfully developed an anti-human CCR9 mAb (C9Mab-1)
and confirmed that its epitope is located at the N-terminus ofhuman CCR9.30,33 By identifying the epitope of C9Mab-1,
we found that the N-terminal region of chemokine receptors
is highly antigenic and have successfully developed mAbs
against various mouse or human chemokine receptors, including CCR2,19,29 CCR3,20,23 CCR4,24 CCR6,25 CCR9,27,31
and CXCR6,28 by using peptides in the N-terminal region as
immunogens. Likewise, we successfully established a novelmAb against hCCR6 (clone C6Mab-19), which is able torecognize exogenous and endogenous hCCR6 by flow cytometry and immunohistochemistry.
The N-terminus of GPCRs, including CCR6, CCR9, andCXCR6, is identified as the ligand-binding domain.34–37 An
anti-hCCR6 mAb (clone 1C6) can block the CCL20-CCR6
interaction and inhibit hCCR6-expressing cells’ chemotaxis.36
The epitope of 1C6 was determined as the N-terminus of
hCCR6.36 CCL20 binds to all three extracellular domainsof CCR6 and N-terminal residues from Tyr27 to Leu38.7
Conversely, N-terminal residues before Tyr27 in CCR6 mayDownloaded by Yukinari Kato from www.liebertpub.com at 08/25/23. For personal use only.

FIG. 2. The analysis of reactivity of C6Mab-19 by flow cytometry. CHO/hCCR6 (A) or CHO-K1 (B) cells were treatedwith 0.001, 0.01, 0.1, and 1 mg/mL of C6Mab-19, followed by treatment with Alexa Fluor 488-conjugated anti-mouse IgG(red line). (C) CHO/hCCR6 cells were treated with 0.001, 0.01, 0.1, and 1 mg/mL of G034E3, which is a commerciallyavailable anti-hCCR6 mAb from BioLegend, followed by treatment with Alexa Fluor 488-conjugated anti-mouse IgG (redline). HepG2 (D) or HuH-7 (E) cells were treated with 0.001, 0.01, 0.1, and 1 mg/mL of C6Mab-19, followed by treatment
with Alexa Fluor 488-conjugated anti-mouse IgG (red line). The black line shows the negative control (blocking buffer).
CHO, Chinese hamster ovary.

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FIG. 3. The determination of the KD of anti-hCCR6 mAbs against hCCR6-expressing cells. CHO/hCCR6 (A, B), HuH-7(C), and HepG2 (D) cells were suspended in serially diluted C6Mab-19 or G034E3 at the indicated concentrations. Then,cells were treated with Alexa Fluor 488-conjugated anti-mouse IgG. Fluorescence data were subsequently obtained using
BD FACSLyric, followed by the calculation of the dissociation constant (KD) by GraphPad Prism 6.

FIG. 4. The immunohistochemical staining using C6Mab-19 against non-Hodgkin lymphoma tissues. After antigen retrieval, serial tissue sections of non-Hodgkin lymphoma (Catalog number: T2235161-1; BioChain) were incubated with
0.3 mg/mL of C6Mab-19 (A, B), or without primary antibody (C, D) followed by treatment with the Envision+ kit. The color
development was performed using DAB, and the sections were counterstained with hematoxylin. The scale bar indicated
100 mm. DAB, 3,3¢-diaminobenzidine tetrahydrochloride.
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ESTABLISHMENT OF ANTI-HCCR6 MAB

not contribute to the interaction with CCL20.7 The second
extracellular domain and N-terminal residues from Tyr27 toLeu38 in CCR6 are the central sites of interaction in CCR6.
The epitope of C6Mab-19 is presented in the 1st to 19th aminoacids of hCCR6; therefore, C6Mab-19 might not possess theneutralizing activity for CCL20. We try to establish the mAbs,
which have the neutralizing activity for CCL20 by applying the
peptide immunization method, in the future studies.
CCL20/CCR6 has been reported to be involved in many
inflammatory and autoimmune diseases, and the CCL20/
CCR6 axis has been a therapeutic target.4 Small molecule
inhibitors for CCL20/CCR6, such as compound 1b andMR120 are expected to ameliorate symptoms of chronic
colitis in preclinical models.10,38 Interestingly, a recent report
revealed that the high prevalence of CCR6 in patients withacute myocardial infarction makes it an independent biomarker and correlates with the severity.39 Lipid-loweringdrugs ameliorate psoriasis by suppressing inflammatory responses that also involve CCL20/CCR6.40
Furthermore, oral squamous cell carcinoma patients withdrinking and smoking habits significantly increased the percentage of CD4+ T cells, which express CD45RO and CCR6
in the blood and tumor microenvironment compared with thepatients who do not drink or smoke.41 Hence, CCR6 is apotent immunoregulatory factor in the formation of pathologies. Therefore, CCR6 is an attractive target to help patientswith various inflammatory diseases. C6Mab-19 can be used inflow cytometry (Fig. 2) and immunohistochemistry (Fig. 4);
thus, it may contribute to the diagnosis of such diseases.
Additionally, C6Mab-19 may treat such inflammatory diseases, although further validation is needed.
CCL20, which is secreted into tumor tissues, recruits immunosuppressive CCR6-expressing cells, including Treg
cells.13 CCL20/CCR6-guided circulating Tregs infiltrate into
tumors, resulting in tumor progression and poor prognosis inpatients with hepatocellular carcinoma.14 Hence, cancer
treatment strategies using CCR6-expressing CAR-T cells
were designed.18,42 We could detect CCR6-expressing CAR-T
cells in blood or tissues using flow cytometry or immunohistochemistry because C6Mab-19 possesses extremely highbinding affinity against hCCR6.
Author Disclosure Statement

The authors have no conflict of interest.
Funding Information

This research was supported in part by Japan Agency for
Medical Research and Development (AMED) underGrant Numbers: JP22ama121008 (to Y.K.), JP22am0401013(to Y.K.), JP22bm1004001 (to Y.K.), JP22ck0106730(to Y.K.), and JP21am0101078 (to Y.K.), and by Japan Society for the Promotion of Science ( JSPS) Grants-in-Aid for
Scientific Research (KAKENHI) grant nos. 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 Antibody Drug Development
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: March 25, 2023
Accepted: June 17, 2023