MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 43, Number 4, 2024
ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2024.0002

Original Articles
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Epitope Mapping of an Anti-Mouse CCR8 Monoclonal
Antibody C8Mab-2 Using Flow Cytometry

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Hiyori Kobayashi, Hiroyuki Suzuki, Tomohiro Tanaka, Mika K. Kaneko, and Yukinari Kato

The C-C motif chemokine receptor 8 (CCR8) is highly and selectively expressed in regulatory T (Treg) cells
and is associated with tumor progression. The massive accumulation of Treg cells into tumors suppresses theeffector function of CD8+ cells against tumor cells. Therefore, selective depletion of Treg cells using antiCCR8 monoclonal antibodies (mAbs) reinvigorates antitumor immune responses and improves responses tocancer immunotherapy. Previously, we developed an anti-mouse CCR8 (mCCR8) mAb, C8Mab-2, using the
Cell-Based Immunization and Screening method. In this study, the binding epitope of C8Mab-2 was investigated using ﬂow cytometry. The mCCR8 extracellular domain-substituted mutant analysis showed that
C8Mab-2 recognizes the N-terminal region (1–33 amino acids) of mCCR8. Next, 1·alanine (or glycine) scanning and 2·alanine (or glycine) scanning were conducted in the N-terminal region. The results revealed thatthe 17-DFFTAP-22 sequence is important for the recognition by C8Mab-2, and Thr20 is a central amino acidof the epitope. These results revealed the involvement of the N-terminus of mCCR8 in the recognition by
C8Mab-2.
Keywords: mouse CCR8, monoclonal antibody, epitope mapping, alanine scanning, ﬂow cytometry
Introduction

he C-C motif chemokine receptor 8 (CCR8) is amember of G protein-coupled receptors (GPCRs)
family. The C-C motif chemokine ligands (CCLs),
including CCL1, CCL8, CCL16, and CCL18, are known
as ligands for human CCR8. CCL1 is the only ligand for
CCR8,1 which is produced by CD11b+CD14+ myeloid
cells during the inﬁltration of regulatory T (Treg) cells
into tumor. 2 Upon binding of CCL1 to CCR8, the
FOXP3 is upregulated by the STAT3 pathway, and theactivated CCR8+ Treg cells potently suppress antitumor
immunity through secretion of granzyme B and IL-10.3
Increased expression of CCR8 is observed in Treg cells,
especially in cancer patients.4 Patients with high levelsof Treg cells exhibit poor prognoses and clinical outcomes in several cancers.5 Therefore, it has been proposed that depletion of tumor-inﬁltrated Treg cells could
restore antitumor immunity and improve responses totumor immunotherapy. 6 Recent preclinical mouse models have revealed that depletion of Treg cells using ananti-mouse CCR8 (mCCR8) monoclonal antibody (mAb)
exhibited strong antitumor responses through dramaticTchanges of the intratumor CD8+ T-cell proﬁle7 or enhanced
the antitumor effects of antiprogrammed cell death 1 (PD-1)
therapy.8
The understanding of the structural-based CCR activation
is important for the development of therapeutic agents.
Among the CCR family members, CCR2 and CCR5 havebeen structurally solved in both inactive and active states,9–12
while inactive state of CCR7 and CCR9 and active state of
CCR1 and CCR6 structures are also characterized.13–16 Furthermore, the structures of CCR8 in complex with either the
antagonistic mAb or the endogenous ligand CCL1 were
determined, which provides the speciﬁc activation mechanism by CCL1 and inhibition by mAb.17
We have developed anti-mouse GPCR mAbs against CCR1
(clone C1Mab-6),18 CCR3 (clones C3Mab-2, C3Mab-3, andC3Mab-4),19–21 CCR8 (clones C8Mab-1, C8Mab-2, andC8Mab-3),22–24 CXCR1 (clone Cx1Mab-1),25 CXCR3 (clone
Cx3Mab-4),26 and CXCR4 (clone Cx4Mab-1)27 using the
Cell-Based Immunization and Screening (CBIS) method.
For the determination of the epitopes, we have faced difﬁcultyusing conventional methods such as enzyme-linked immunosorbent assay. In this study, epitope mapping of an anti-mCCR8
mAb was conducted by ﬂow cytometry-based approaches.

Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, Sendai, Japan.

101

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Materials and Methods
Cell lines

System (Thermo Fisher Scientiﬁc Inc.). Stable transfectants were selected using SH800. Cells were cultured in
Roswell Park Memorial Institute-1640 medium (Nacalai
Tesque, Inc., Kyoto, Japan) supplemented with 10% heatinactivated fetal bovine serum (Thermo Fisher ScientiﬁcInc.), 100 units/mL of penicillin, 100 lg/mL streptomycin,
and 0.25 lg/mL amphotericin B (Nacalai Tesque, Inc.) at
37C in a humidiﬁed atmosphere containing 5% CO2. The
stable transfectants were cultivated in a medium containing
0.5 mg/mL of Zeocin.
Plasmid construction

Synthesized DNAs (Euroﬁns Genomics KK) encoding
mouse CCR3 (mCCR3; Accession No.: NM_009914.4)
and mCCR8 were subcloned into a pCAG-Ble vector. Chimeric mutants including mCCR3 (mCCR8p1–33), mCCR3

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Chinese hamster ovary (CHO)-K1 cell was obtained from theAmerica Type Culture Collection (ATCC, Manassas, VA).
Stable transfectants of CHO/mCCR8 were established in
our previous study.24 Brieﬂy, synthesized DNA (Euroﬁns
Genomics KK, Tokyo, Japan) encoding mCCR8 (Accession
No.: NM_007720.2) was subcloned into a pCAG-Ble vector(FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan).
mCCR8 plasmid was transfected using a Neon Transfection
System (Thermo Fisher Scientiﬁc Inc., Waltham, MA). CHO/
mCCR8 cells were sorted using a cell sorter (SH800; Sony
Corp., Tokyo, Japan) and cultivated in a medium containing 0.5
mg/mL of Zeocin (InvivoGen, San Diego, CA).
The chimeric and the point mutant plasmids were transfected into CHO-K1 cells using the Neon Transfection

KOBAYASHI ET AL.

FIG. 1. Schematic illustration of chimeric proteins. The four extracellular regions of mCCR8, including the N-terminal
region (1–33 aa), ECL1 (92–105 aa), ECL2 (170–200 aa), and ECL3 (262–278 aa), were substituted into the corresponding
regions of mCCR3. aa, amino acids; mCCR, mouse C-C motif chemokine receptor; ECL, extracellular loop.

EPITOPE MAPPING OF ANTI-MOUSE CCR8 MAB(mCCR8p92–105), mCCR3 (mCCR8p170–200), and mCCR3(mCCR8p262–278) were produced with a PA16 tag at theirN-terminus using the HotStar HiFidelity polymerase kit (Qiagen Inc., Hilden, Germany). Alanine (or glycine) substitutionsin the mCCR8 N-terminal region were conducted using QuikChange Lightning Site-Directed Mutagenesis Kits (Agilent
Technologies Inc., Santa Clara, CA, USA). PCR fragments
bearing the desired mutations were inserted into the pCAG-Ble
vector (FUJIFILM Wako Pure Chemical Corporation) using
the In-Fusion HD Cloning Kit (Takara Bio, Inc., Shiga, Japan).
Antibodies

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C8Mab-2 was established by the CBIS method.23 NZ-1 (ananti-PA16 tag mAb) was described previously.28 A secondary
Alexa Fluor 488-conjugated anti-rat immunoglobulin G (IgG) was
purchased from Cell Signaling Technology, Inc. (Danvers, MA).
Flow cytometry

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 in
phosphate-buffered saline, cells were treated with C8Mab-2(10 lg/mL) or NZ-1 (1 lg/mL) for 30 minutes at 4C andsubsequently with Alexa Fluor 488-conjugated anti-rat IgG(1:2000; Cell Signaling Technology, Inc.). Fluorescence data
were obtained using the SA3800 Cell Analyzer (Sony Corp.).
Results
Determination of the epitope of an anti-mCCR8 mAb by flow
cytometry using chimeric proteins

We employed the CBIS method to develop an antimCCR8 mAb (clone C8Mab-2).23 C8Mab-2 is applicable for103

ﬂow cytometry and immunocytochemistry. To investigate thebinding epitope of C8Mab-2, we focused on four extracellular regions of mCCR8, including the N-terminal region[1–33 amino acids (aa)], extracellular loop 1 (ECL1; 92–
105 aa), ECL2 (170–200 aa), and ECL3 (262–278 aa). The
four extracellular regions of mCCR8 were substituted into
the corresponding regions of mCCR3, which possesses asimilar structure to mCCR8. As shown in Figure 1,
mCCR3 (mCCR8p1–33), mCCR3 (mCCR8p92–105),mCCR3 (mCCR8p170–200), and mCCR3 (mCCR8p262–
278) were generated. The chimeric proteins were transiently expressed on CHO-K1 cells, and the reactivities to
C8Mab-2 were analyzed using ﬂow cytometry (Fig. 2A).
C8Mab-2 reacted with mCCR3 (mCCR8p1–33) andCHO/mCCR8 cells, but not with other chimeric proteins(Fig. 2A). The cell surface expression of each mutant was
conﬁrmed by an anti-PA16 tag mAb, NZ-1 (Fig. 2B).
These results indicated that the N-terminal region ofmCCR8 is recognized by C8Mab-2.
Determination of the C8Mab-2 epitope by flow cytometry
using 1·alanine scanning

Next, 1·alanine scanning was conducted in the N-terminal region except for Cys23. Thirty-two 1·alanine (or glycine) substitution mutants of mCCR8 were constructed,
and the mutant proteins were stably expressed on CHO-K1
cells. The reactivity against C8Mab-2 was assessed using
ﬂow cytometry. As shown in Figure 3A, C8Mab-2 did notreact with a mutant (T20A). In contrast, C8Mab-2 reactedwith the other 31 mutants. The cell surface expression ofeach mutant was conﬁrmed by NZ-1 (Fig. 3B). These
results showed that Thr20 of mCCR8 is important for
C8Mab-2 binding.

FIG. 2. Determination of the epitope of an anti-mCCR8 mAb by ﬂow cytometry using chimeric proteins. C8Mab-2(10 lg/mL) (A) and an anti-PA16 tag mAb, NZ-1 (1 lg/mL) (B) were treated with CHO-K1 cells which were transiently expressed the chimeric proteins, CHO/mCCR8 or CHO-K1 cells for 30 minutes at 4C, followed by the additionof Alexa Fluor 488-conjugated anti-rat IgG. Red lines show the cells with C8Mab-2 or NZ-1 treatment, and black lines
show cells treated with a blocking buffer as a negative control. CHO-K1 cell, Chinese hamster ovary-K1 cell; mCCR,
mouse C-C motif chemokine receptor; IgG, Immunoglobulin G.

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104

KOBAYASHI ET AL.

FIG. 3. Determination of the C8Mab-2 epitope by ﬂow cytometry using 1·alanine scanning. C8Mab-2 (10 lg/mL) (A) andNZ-1 (1 lg/mL) (B) were treated with CHO-K1 cells which were stably expressed mutant proteins or CHO-K1 cells for 30
minutes at 4C, followed by the addition of Alexa Fluor 488-conjugated anti-rat IgG. Red lines show the cells with C8Mab-2
or NZ-1 treatment, and black lines show cells treated with a blocking buffer as a negative control. CHO-K1 cell, Chinese hamster ovary-K1 cell; IgG, Immunoglobulin G.
Determination of the C8Mab-2 epitope by flow cytometry
using 2·alanine scanning

We also examined the reactivity of C8Mab-2 against
2·alanine (or glycine)-substituted mCCR8. We constructedthirty 2·alanine (or glycine)-substituted mutants in the Nterminal region of mCCR8 except for Cys23. The mutant
proteins were transiently expressed on CHO-K1 cells. The
reactivity against C8Mab-2 was assessed using ﬂow cytometry. As shown in Figure 4A, C8Mab-2 did not react with thefour mutants (D17A-F18A, F19A-T20A, T20A-A21G, and
A21G-P22A). In contrast, C8Mab-2 reacted with the other26 mutants. The cell surface expression of each mutant was
conﬁrmed by NZ-1 (Fig. 4B). These results showed that a
motif from Asp17 to Pro22 in mCCR8 is important for
C8Mab-2 recognition.

Discussion

In this study, we performed the ﬂow cytometry-based epitope
mapping of an anti-mouse CCR8 mAb (C8Mab-2) using the chimeric proteins (Figs. 1 and 2). Furthermore, we determined thatthe 17-DFFTAP-22 is important for the recognition by C8Mab-2
in the 2·alanine scanning (Fig. 4), and Thr20 is a central aminoacid of the epitope by the 1·alanine scanning (Fig. 3). Figure 5
summarizes the epitope of C8Mab-2. In the epitope mapping ofan anti-human CCR8 mAb (clone mAb1), CCR8 chimeras, inwhich the N-terminus, ECL1, ECL2, or ECL3 were replacedwith the corresponding sequences from human CCR5, wereused.17 The mAb1 did not recognize ECL1-replaced CCR8 andECL2-replaced CCR8, suggesting that both ECL1 and ECL2
are required for mAb1 binding.17 Because we could not determine the binding epitope of our other anti-mCCR8 mAbs

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EPITOPE MAPPING OF ANTI-MOUSE CCR8 MAB

FIG. 4. Determination of the C8Mab-2 epitope by ﬂow cytometry using 2·alanine scanning. C8Mab-2 (10 lg/mL)(A) and NZ-1 (1 lg/mL) (B) were treated with CHO-K1 cells which were transiently expressed mutant proteins or
CHO-K1 cells for 30 minutes at 4C, followed by the addition of Alexa Fluor 488-conjugated anti-rat IgG. Red lines
show the cells with C8Mab-2 or NZ-1 treatment, and black lines show cells treated with a blocking buffer as a negative
control. CHO-K1 cell, Chinese hamster ovary-K1 cell; IgG, Immunoglobulin G.
(C8Mab-1 and C8Mab-3) in this study (data not shown), thesubstitution of two ECLs may be required for the identiﬁcationof the epitopes of those anti-mCCR8 mAbs.
The “hot” tumor is characterized by the massive inﬁltrationand enrichment with CD8+ effector T cells, which is important
for the antitumor immune responses. Immune checkpoint

inhibitors such as anti-PD-1 mAbs are effective in hot tumors.
However, the response rate of immune checkpoint inhibitors isstill low due to the lack of CD8+ effector T-cell inﬁltration or
accumulation of Treg cells suppressing the effector activities,
which is characterized as “cold tumors.”29,30 Since CCR8
expression is increased in tumor-inﬁltrated Treg cells,

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KOBAYASHI ET AL.

FIG. 5. Schematic illustration of the C8Mab-2 epitope, which was identiﬁed by 1·alanine scanning and 2·alanine
scanning. 17-DFFTAP-22 is important for the recognition by C8Mab-2 in the 2·alanine scanning (underlined). Thr20(red) is determined to be a central amino acid of the epitope by the 1·alanine scanning.
CCR8 is one of the promising target for depleting of Treg
cells selectively in tumors.4 Anti-mCCR8 mAbs have beenused to suppress cancer growth in several cancer models.7,8,31 Furthermore, an anti-human CCR8 mAb (S-531011)
was developed.32 S-531011 has antibody-dependent cellularcytotoxicity activity against tumor-inﬁltrating CCR8+ Treg
cells and neutralization activity against the CCR8 signaling.32
Meanwhile, there is no information about the relationshipbetween the Treg cells-depleting activity and epitope of themAbs. Our strategy for epitope identiﬁcation would contributenot only to the understanding of mAb-epitope interaction but
also to the improvement of those therapeutic mAbs.
Author’s Contribution

H.K. and T.T. performed the experiments. M.K.K. and
Y.K. designed the experiments. H.S. and M.K.K. analyzed
the data. H.S. and Y.K. wrote the article. All authors haveread and agreed to the published version of the article.
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) under Grant
Numbers: JP23ama121008 (to Y.K.), JP23am0401013 (toY.K.), JP23bm1123027 (to Y.K.), and JP23ck0106730 (toY.K.), and by the 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.), and 22K07224 (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, Miyagi 980-8575
JapanE-mail: yukinari.kato.e6@tohoku.ac.jp
Received: January 23, 2024
Accepted: February 24, 2024