antibodies
Communication

Identification of the Binding Epitope of an Anti-Mouse CCR6
Monoclonal Antibody (C6Mab-13) Using 1× Alanine Scanning
Tomohiro Tanaka 1 , Mayuki Tawara 1 , Hiroyuki Suzuki 1, * , Mika K. Kaneko 1,2
12*

Citation: Tanaka, T.; Tawara, M.;
Suzuki, H.; Kaneko, M.K.; Kato, Y.
Identification of the Binding Epitopeof an Anti-Mouse CCR6 Monoclonal

and Yukinari Kato 1,2, *Department of Molecular Pharmacology, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi,
Aoba-ku, Sendai 980-8575, Japan; tomohiro.tanaka.b5@tohoku.ac.jp (T.T.);
tawara.mayuki.p8@dc.tohoku.ac.jp (M.T.); k.mika@med.tohoku.ac.jp (M.K.K.)
Department of Antibody Drug Development, Tohoku University Graduate School of Medicine,
2-1 Seiryo-machi, Aoba-ku, Sendai 980-8575, Japan
Correspondence: hiroyuki.suzuki.b4@tohoku.ac.jp (H.S.); yukinari.kato.e6@tohoku.ac.jp (Y.K.);
Tel.: +81-22-717-8207 (H.S. & Y.K.)

Abstract: CC chemokine receptor 6 (CCR6) is one of the members of the G-protein-coupled receptor(GPCR) family that is upregulated in many immune-related cells, such as B lymphocytes, effector
and memory T cells, regulatory T cells, and immature dendritic cells. The coordination between
CCR6 and its ligand CC motif chemokine ligand 20 (CCL20) is deeply involved in the pathogenesisof various diseases, such as cancer, psoriasis, and autoimmune diseases. Thus, CCR6 is an attractive
target for therapy and is being investigated as a diagnostic marker for various diseases. In a previous
study, we developed an anti-mouse CCR6 (mCCR6) monoclonal antibody (mAb), C6 Mab-13 (rat
IgG1 , kappa), that was applicable for flow cytometry by immunizing a rat with the N-terminal
peptide of mCCR6. In this study, we investigated the binding epitope of C6 Mab-13 using an enzymelinked immunosorbent assay (ELISA) and the surface plasmon resonance (SPR) method, which wereconducted with respect to the synthesized point-mutated-peptides within the 1–20 amino acid regionof mCCR6. In the ELISA results, C6 Mab-13 lost its ability to react to the alanine-substituted peptideof mCCR6 at Asp11, thereby identifying Asp11 as the epitope of C6 Mab-13. In our SPR analysis, thedissociation constants (KD ) could not be calculated for the G9A and D11A mutants due to the lackof binding. The SPR analysis demonstrated that the C6 Mab-13 epitope comprises Gly9 and Asp11.
Taken together, the key binding epitope of C6 Mab-13 was determined to be located around Asp11 on
mCCR6. Based on the epitope information, C6 Mab-13 could be useful for further functional analysis
of mCCR6 in future studies.

Antibody (C6 Mab-13) Using 1×
Alanine Scanning. Antibodies 2023, 12,

Keywords: mouse CCR6; monoclonal antibody; epitope; ELISA; SPR

32. https://doi.org/10.3390/
antib12020032
Academic Editor: Anthony Rees
Received: 16 March 2023
Revised: 28 March 2023
Accepted: 17 April 2023
Published: 28 April 2023

Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms andconditions of the Creative Commons
Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

1. Introduction
The CC chemokine receptor 6 (CCR6) is a seven-transmembrane chemokine receptor
belonging to the G-protein-coupled receptor (GPCR) family [1–5]. CCR6 was identified asa specific receptor for the CC motif chemokine ligand 20 (CCL20) in 1997 [6]. It is reportedly associated with various diseases, such as cancer [7–9], autoimmune diseases [10–13],
psoriasis [14–17], and inflammatory bowel disease (IBD) [18–22]. The expression of CCR6
is found in B cells or T cells [23], such as effector memory T cells [24], immature dendritic
cells [25], Th17 cells [26], and regulatory T (Treg) cells [27], and thus affects the activity anddirectionality of immune cells [23,24,26]. Mice lacking CCR6 exhibited impaired leukocyte
homeostasis, which results in severe contact hypersensitivity and defects in delayed-type
hypersensitivity responses. These results suggested that CCR6 plays a critical role in theregulation of leukocyte homeostasis [28].
The chemokine ligand CCL20, also known as macrophage inflammatory protein-3α(MIP-3α) [29], liver- and activation-regulated chemokine (LARC) [30], or Exodus-1 [31], isa crucial CCR6 ligand. The binding of CCL20 to CCR6 can activate a variety of intracellular

Antibodies 2023, 12, 32. https://doi.org/10.3390/antib12020032

https://www.mdpi.com/journal/antibodies

Antibodies 2023, 12, 32

2 of 9signaling pathways, including the calcium signaling, PI3K-Akt, MEK-ERK, STAT3, and
NF-κB pathways [32]. These pathways play essential roles in the differentiation, migration,and plasticity of CD4+ T lymphcytes [33–36]. These findings suggest that CCR6-CCL20
signaling could provoke cross-talk with the signalling of T-cell receptors and cytokines toregulate the mitigation of CD4+ T lymphcytes in the inflammatory microenvironment.
CCL20 is secreted by various immune-related cells, such as B cells [23], Th17 cells [37],dendritic cells [38], and natural killer cells [39]. Although various CC chemokine receptor–
ligand pairs exist, the CCR6/CCL20-regulated immune response has currently become afocus of immunological research with respect to disease development [10,38,40,41]. The
expression of CCR6 and CCL20 has been found to be dysregulated in the colonic mucosa
and serum from IBD patients [20,22]. CCR6+ T lymphcytes are involved in an imiquimodinduced psoriasis model [42]. Furthermore, the tumor-promoting effects of CCR6/CCL20
within the tumor microenvironment have been reported in many cancer types, such as renal
cell carcinoma [43], gastric cancer [44], cervical cancer [45], and lung cancer [46,47]. Treg
cells in peripheral blood (~60%) express CCR6, presenting increased suppressive activity
and higher FOXP3 expression in patients with oral squamous cell carcinoma [48]. Thesefindings have made the CCL20/CCR6 axis an attractive therapeutic target for various
diseases., and inhibitors targeting the CCR6/CCL20 axis are also being actively developed [10].
Previously, we developed various monoclonal antibodies (mAbs) against chemokine
receptors, including mouse CCR2 [49], mouse CCR3 [50], mouse CCR4 [51], mouse CCR6(mCCR6) [52], mouse CCR9 [53], and mouse CXCR6 [54]. The N-terminus of GPCRs, including CCR6, CCR9, and CXCR6, has been identified as a ligand-binding domain [55–58].
Interestingly, the binding between CCL20 and CCR6 has been elucidated [59]. CCR6 andCCL20 have shallow binding modes on the receptor surface, which induce allosteric conformational changes and are considered to trigger binding to intracellular G-proteins [59].
Analysis of the ligand-binding mode and the characterization of antibody epitopes areimportant for predicting neutralizing activity and assessing efficacy against antigens.
In this study, we performed an epitope identification of a rat anti-mCCR6 mAb (C6 Mab13; IgG1 , kappa) using enzyme-linked immunosorbent assay (ELISA) and surface plasmon
resonance (SPR) analysis against the alanine-substituted N-terminal peptides of mCCR6.
2. Materials and Methods
2.1. Antibodies
The rat anti-mCCR6 mAb (clone C6 Mab-13) used herein was previously developed [52].
In summary, one rat was intraperitoneally immunized with a keyhole-limpet-hemocyanin(KLH)-conjugated N-terminal peptide of mCCR6 (1–19 amino acids (aa) + C-terminal
cysteine). Subsequently, the hybridoma supernatants were screened with the mCCR6p119C peptide using ELISA following flow cytometry using mCCR6-overexpressed CHO-K1
cells and endogenously mCCR6-expressed P388 (mouse lymphoid neoplasma) and J774-1(mouse macrophage-like) cells [52].
We purchased secondary peroxidase-conjugated anti-rat immunoglobulins from SigmaAldrich Corp. (St. Louis, MO, USA).
2.2. Peptides
The mCCR6 (Accession No.: NM_001190333.1) peptide (1 -MNSTESYFGTDDYDNTEYYS)
and
1× alanine residue-substituted peptides (Table 1) were synthesized utilizing PEP20
screen (Sigma-Aldrich Corp.).

Antibodies 2023, 12, 32

3 of 9

Table 1. Identification of C6 Mab-13 epitope using point mutant peptides of mCCR6 via enzymelinked immunosorbent assay.
PeptidesSequences

C6 Mab-13 Reactivity

p1–20 (WT)
M1A
N2A
S3A
T4A
E5A
S6A
Y7A
F8A
G9A
T10A
D11A
D12A
Y13A
D14A
N15A
T16A
E17A
Y18A
Y19A
S20A

MNSTESYFGTDDYDNTEYYSANSTESYFGTDDYDNTEYYSMASTESYFGTDDYDNTEYYSMNATESYFGTDDYDNTEYYSMNSAESYFGTDDYDNTEYYSMNSTASYFGTDDYDNTEYYSMNSTEAYFGTDDYDNTEYYSMNSTESAFGTDDYDNTEYYSMNSTESYAGTDDYDNTEYYSMNSTESYFATDDYDNTEYYSMNSTESYFGADDYDNTEYYSMNSTESYFGTADYDNTEYYSMNSTESYFGTDAYDNTEYYSMNSTESYFGTDDADNTEYYSMNSTESYFGTDDYANTEYYSMNSTESYFGTDDYDATEYYSMNSTESYFGTDDYDNAEYYSMNSTESYFGTDDYDNTAYYSMNSTESYFGTDDYDNTEAYSMNSTESYFGTDDYDNTEYASMNSTESYFGTDDYDNTEYYA

+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++

+++, OD655 = 0.3; -, OD655 < 0.1.

2.3. ELISA
Synthesized mCCR6 peptides were immobilized on Nunc Maxisorp 96-well immunoplates (Thermo Fisher Scientific Inc., Waltham, MA, USA) at 10 µg/mL for 30 min at 37 ◦ C.
After being washed with phosphate-buffered saline (PBS) containing 0.05% Tween20 (PBST;
Nacalai Tesque, Inc., Kyoto, Japan), the wells were blocked with 1% bovine serum albumin(BSA)-containing PBST for 30 min at 37 ◦ C. The plates were incubated with 10 µg/mL of
C6 Mab-13 for 30 min at 37 ◦ C followed by peroxidase-conjugated anti-rat immunoglobulins(1:20,000 diluted; Sigma-Aldrich Corp.) for 30 min at 37 ◦ C. Enzymatic reactions wereconducted at room temperature using the ELISA POD Substrate TMB Kit (Nacalai Tesque,Inc.). Optical density was measured at 655 nm using an iMark microplate reader (Bio-Rad
Laboratories, Inc., Berkeley, CA, USA).
2.4. Measurement of Dissociation Constants Using Surface Plasmon Resonance (SPR)
The dissociation constants (KD ) between C6 Mab-13 and the epitope region peptides
were measured using SPR. C6 Mab-13 was immobilized on the CM5 sensor chip according to
the manufacturer’s protocol (Cytiva, Marlborough, MA, USA). In summary, C6 Mab-13 wasdiluted to 10 µg/mL by an acetate buffer (pH 4.0; Cytiva) and immobilized using an aminecoupling reaction. The surface of flow cell 2 of the CM5 sensor chip was treated with 1-ethyl3-(3-dimethylaminopropyl)-carbodiimide and N-hydroxysuccinimide (NHS), followed byan injection of C6 Mab-13. The unreacted NHS-ester was blocked with ethanolamine after
C6 Mab-13 immobilization. The KD between C6 Mab-13 and mCCR6 peptides (50, 25, 12.5,
6.25, and 3.13 µM) were measured using Biacore X100 (Cytiva) at 25 ◦ C. The buffer was
filtrated with PBS containing 0.05% (v/v) Tween 20 and 0.24% (v/v) dimethyl sulfoxide(FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan). The single-cycle kinetics
method was used to measure the binding signals. The data were analyzed using 1:1
binding kinetics to determine the association rate constant (ka ), dissociation rate constant(kd ), and KD using BIAevaluation software (Cytiva).

Antibodies 2023, 12, x FOR PEER REVIEW

Antibodies 2023, 12, 32

4 of 9analyzed using 1:1 binding kinetics to determine the association rate constant (ka), disso4 of 9
ciation rate constant (kd), and KD using BIAevaluation software (Cytiva).
3. Results
3. Results
3.1. Epitope
Epitope Identification
IdentificationofofCC6Mab-13
Mab-13by
byELISAELISAUsing
Using1×
1×Alanine-Substituted
Alanine-SubstitutedmCCR6
mCCR6Peptides
Pep3.1.
6tides
We previously
previously developed
developed an
an anti-mCCR6
anti-mCCR6 mAb,
mAb, CC66Mab-13 (rat IgG
IgG11, kappa), by imWe
munizing
a
rat
with
a
KLH-conjugated
mCCR6
N-terminal
peptide[52].
C6Mab-13
is apmunizing rat with a KLH-conjugated mCCR6 N-terminal peptide [52].
C6 Mab-13
isplicable to to
ELISA
and
is usefulforfordetecting
mCCR6-expressing
cells
viaviaflow
cytometry
applicable
ELISA
and
is usefuldetecting
mCCR6-expressing
cells
flow
cytom[52].[52].
To characterize
the binding
epitopeof C6Mab-13,
we synthesized
20 different
1× alaetry
To characterize
the binding
epitopeof C6 Mab-13,
we synthesized
20 different
mCCR6
peptidesbetween
Met1 toSer20.
The sequences
are listed
Ta1nine-substituted
× alanine-substituted
mCCR6
peptidesbetween
Met1
to Ser20.
The sequences
are inlistedbleTable
1. Theresults
of of
ELISA
using
in
1. Theresults
ELISA
usingalanine-substituted
alanine-substitutedpeptides
peptidesand
and C
C66Mab-13
Mab-13 demonstrated
strated thatthat CC66Mab-13 bound to point mutants, such as M1A, N2A, S3A, T4A, E5A, S6A,
Y7A,
Y7A, F8A,
F8A, G9A,
G9A, T10A,
T10A, D12A,
D12A, Y13A,
Y13A, D14A,
D14A, N15A,
N15A, T16A, E17A, Y18A, Y19A, and S20A,
as
as wellwell asas thethe 1–20
1–20 aa
aa wild-typewild-type (WT)
(WT) sequence
sequence (positive control) (Figure 1A). In contrast,C
C66Mab-13 did not react with the D11A
D11A peptide
peptide (Figure(Figure 1A).
1A). These
These results
results indicate
indicate that
that
Asp11
the criticalcriticalaa,
aa,whichwhichisis
included
C6 Mab-13
epitope.
The resultsareAsp11 was theincluded
in in
thetheC6Mab-13
epitope.
The results
are sumsummarized
schematicallyin
Figure1B.
marized schematically in Figure 1B.

Figure 1.
1. Determination
Determination ofof thethe CC66Mab-13
Mab-13 epitope
epitope byby ELISAELISA using
using alanine-substituted
alanine-substituted peptides
peptides ofof
FiguremCCR6.
(A)
Synthesizedpeptides
of
mCCR6
(10
µ
g/mL)
were
immobilizedon
immunoplates
for
mCCR6. (A) Synthesized peptides of mCCR6 (10 µg/mL) were immobilized on immunoplates for
30 minmin atat 37
37 ◦°C.
The plates
plates were
were incubatedincubatedwith
with1010µg/mLµ g/mL ofof C
C6Mab-13,
followed by treatment with
30
C. The6 Mab-13, followed by treatment withperoxidase-conjugated anti-rat immunoglobulins. Optical density was measured at 655 nm (OD655)
peroxidase-conjugated anti-rat immunoglobulins. Optical density was measured at 655 nm (OD655)
using a microplate reader. (B) The schematic illustration of mCCR6 and the C6Mab-13 epitope. The
using a microplate reader. (B) The schematic illustration of mCCR6 and the C6 Mab-13 epitope. The
C6Mab-13 epitope of mCCR6 comprises Asp11 from ELISA experiments.
C6 Mab-13 epitope of mCCR6 comprises Asp11 from ELISA experiments.

3.2. Epitope Identification of C6 Mab-13 by SPR Using 1× Alanine-Substituted mCCR6 Peptides
To confirm the C6 Mab-13 epitope, we measured the binding affinity between C6 Mab-13
and the synthesized peptides, including 20 point mutants and the WT of mCCR6, using Biacore

Antibodies 2023, 12, 32

5 of 9

X100. The peptides’ sequences are presented in Table 1, and the measured values are summarized in Table 2. The ka , kd , and KD of G9A and D11A were not determined. These results
demonstrated that Gly9 and Asp11 were the critical amino acids of the C6 Mab-13 epitope.
Table 2. The KD between C6 Mab-13 and 1× alanine-substituted peptides determined by surface
plasmon resonance.
Peptideska (/ms)kd (/s)KD (M)

p1_20 (WT)
M1A
N2A
S3A
T4A
E5A
S6A
Y7A
F8A
G9A
T10A
D11A
D12A
Y13A
D14A
N15A
T16A
E17A
Y18A
Y19A
S20A

6.84 × 103

3.77 × 10−3

6.94 × 103

4.15 × 10−3

7.86 × 103
7.62 × 103
7.92 × 103
8.20 × 103
9.05 × 103
8.16 × 103
1.43 × 103
ND
1.31 × 104
ND
7.43 × 103
1.43 × 103
6.87 × 103
6.19 × 103
6.23 × 103
6.38 × 103
5.23 × 103
5.75 × 103
4.68 × 103

4.23 × 10−3
4.53 × 10−3
4.55 × 10−3
4.64 × 10−3
5.25 × 10−3
3.45 × 10−3
1.23 × 10−2
ND
3.15 × 10−2
ND
7.09 × 10−3
1.30 × 10−2
1.05 × 10−2
5.61 × 10−3
5.17 × 10−3
6.67 × 10−3
5.56 × 10−3
6.02 × 10−3
5.96 × 10−3

5.52 × 10−7
5.99 × 10−7
5.38 × 10−7
5.94 × 10−7
5.75 × 10−7
5.65 × 10−7
5.81 × 10−7
4.23 × 10−7
8.55 × 10−6ND
2.40 × 10−6
ND
9.55 × 10−7
9.12 × 10−6
1.53 × 10−6
9.06 × 10−7
8.30 × 10−7
1.05 × 10−6
1.06 × 10−6
1.05 × 10−6
1.27 × 10−6

WT, wild-type; ND, not determined.

Mutant peptides of F8A, T10A, Y13A, and D14A increased the KD values by 15.5-, 4.4-,
16.5-, and 2.8-fold, respectively (Table 2), indicating that Phe8, Thr10, Tyr13, and Asp14
may contribute to the binding of C6 Mab-13 to mCCR6.
4. Discussion
This study examined the binding epitope of C6 Mab-13 through a 1× alanine-substitutedpeptide-scanning method using ELISA and SPR. We concluded that Asp11 is a pivotal
epitope aa in ELISA, while Gly9 and Asp11 are critical in SPR. This epitope is locatedoutside the region of all three extracellular domains of CCR6 and N-terminal residues fromTyr27 to Leu38, to which the chemokine ligand CCL20 binds [59,60]. There is a possibility
that structural changes might occur upon C6 Mab-130 s binding to CCR6, which leads toallosteric effects on CCL20 binding. Therefore, we will investigate the neutralizing activityof C6 Mab-13 between CCL20 and CCR6 in the future study.
A recent report showed that low rather than high affinity of mAb to a target provokes
elevated activity through inducing the clustering of receptors. These findings provide
new insights for antibody-mediated receptor signaling [61]. Since CCR6 is involved in
intracellular signaling [62], the relationship between antibody affinity and the effect ofcellular signaling should be investigated in future studies.
The epitope-mapping results obtained using ELISA (Figure 1) and SPR (Table 2) indicated a similar region of mCCR6 as the binding epitope. However, Gly9 was only identified
as the critical aa by via SPR analysis (Table 2). The experimental system differs between
both experiments, as follows: (i) for ELISA, the synthesized peptides were immobilized
on immunoplates, while C6 Mab-13 was immobilized on a CM5 sensor chip in the SPR
analysis; (ii) the reaction times between the antigen and the antibody were different; andAntibodies 2023, 12, 32

6 of 9(iii) the secondary antibody was only used for ELISA. These different conditions may haveprecipitated the inconsistent results of both experiments in this study.
In the SPR analysis, mutant peptides of F8A, T10A, Y13A, and D14A increased the KD
values by 15.5-, 4.4-, 16.5-, and 2.8-fold, respectively (Table 2). These results indicate that
Phe8, Thr10, Tyr13, and Asp14 may contribute to C6 Mab-130 s binding to mCCR6. In the
future, we will adopt the cell-based alanine- or 2× alanine-scanning methods for a detailed
epitope analysis of C6 Mab-13, as we have clarified the epitope of mAb [63].
When CCL20 is secreted in tumor tissues [64], it attracts CCR6-expressing Treg
cells [65], which are involved in tumor progression and poor prognosis [66,67]. Therefore, novel cancer treatment strategies using CCR6-expressing chimeric antigen receptor-T(CAR-T) cells have been designed [68,69]. Furthermore, removing immunosuppressive
cells, such as CCR6+ Treg cells, may enhance antitumor efficacy [70]. In this study, wedemonstrated that C6 Mab-13 possesses high binding affinity against mCCR6, which was
expressed in Chinese hamster ovary-K1 cells (KD : 2.8 × 10−9 M according to flow cytometric analysis) [52]. Therefore, C6 Mab-13 is expected to be useful for antitumor evaluations
considering the depletion of CCR6-expressing Treg cells in mouse models.
Author Contributions: T.T. and M.T. performed the experiments. M.K.K. and Y.K. designed theexperiments. T.T. and H.S. analyzed the data. T.T., H.S. and Y.K. wrote the manuscript. All authors
have read and agreed to the published version of the manuscript.
Funding: This research was supported in part by Japan Agency for Medical Research and Development (AMED) under Grant Numbers: JP22ama121008 (to Y.K.), JP22am0401013 (to Y.K.),
JP22bm1004001 (to Y.K.), JP22ck0106730 (to Y.K.), and JP21am0101078 (to Y.K.), and by the Japan
Society for the Promotion of Science (JSPS) Grants-in-Aid for Scientific Research (KAKENHI) grantnos. 21K20789 (to T.T.), 22K06995 (to H.S.), 21K07168 (to M.K.K.), and 22K07224 (to Y.K.).
Institutional Review Board Statement: Not applicable for studies not involving humans or animals.
Informed Consent Statement: Not applicable.
Data Availability Statement: All related data and methods are presented in this paper. Additional
inquiries should be addressed to the corresponding authors.
Conflicts of Interest: The authors declare no conflict of interest.

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