MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 41, Number 6, 2022
ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2021.0069
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Development of an Anti-Mouse CCR8 Monoclonal Antibody(C8Mab-1) for Flow Cytometry and Immunocytochemistry

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Masaki Saito,1 Hiroyuki Suzuki,1 Tomohiro Tanaka,2 Teizo Asano,2 Mika K. Kaneko,2 and Yukinari Kato1,2

It has been widely accepted that monoclonal antibody (mAb) is an effective tool for cancer immunotherapy.
The C-C motif chemokine receptor 8 (CCR8) is highly expressed in regulatory T cells and many cancers and isassociated with the progression of the cancers. However, its role in cancer progression remains unclear. Thus,
the development of mAbs for CCR8 leads to cancer immunotherapy and elucidation of unknown mechanismsof CCR8-dependent cancer progression. In this study, we have developed an anti-mouse CCR8 (mCCR8) mAb(clone C8Mab-1, rat IgG2a, kappa) using the Cell-Based Immunization and Screening (CBIS) method. We
showed that C8Mab-1 and its recombinant antibody (recC8Mab-1) bind to mCCR8-overexpressed Chinese
hamster ovary (CHO)-K1 cells (CHO/mCCR8), but not to the parental CHO-K1 cells, in flow cytometry andimmunofluorescence. Moreover, C8Mab-1 and recC8Mab-1 specifically reacted to P388 (a mouse lymphocytelike cells) and J774-1 (a mouse macrophage-like cells), which express endogenous mCCR8, in both applications. These results suggest that C8Mab-1, developed using the CBIS method, is useful for flow cytometry andimmunocytochemistry against exogenous and endogenous mCCR8.
Keywords: CCR8, monoclonal antibody, CBIS, flow cytometry, immunofluorescence
Introduction

I

mmunotherapy is a powerful approach for treating
many cancers. Notably, due to the effectiveness, monoclonal antibodies (mAbs) that target immune checkpoint
molecules have been developed, including cytotoxic
T lymphocyte antigen 4 (CTLA-4), programmed cell death 1(PD-1), and programmed death-ligand 1 (PD-L1).(1) CTLA-4
and PD-1 are highly expressed in regulatory T cells (Tregs)
that infiltrate into the tumor microenvironment and play asignificant role in the suppression of immune response in thetumor microenvironment.(2,3)
The development of various mAbs that target other molecules has been designed to find more effective strategies
for cancer immunotherapy. The tumor-infiltrating Tregs
highly express C-C motif chemokine receptor 8 (CCR8) on
the cell surface, in addition to CTLA-4 and PD-1.(4) CCR8
belongs to the C-C motif chemokine receptor family of
G protein-coupled receptor (GPCR) and is activated by
CCL1, CCL8, CCL16, and CCL18.(5–8) CCR8 is upregulated in many cancers, such as bladder cancer, breast cancer,

colorectal cancer, nonsmall cell lung cancer, and oral squamous cell carcinoma.(4,9–11) CCR8 is activated by CCL1
and promotes the activities of migration and antiapoptosis
in Tregs and lymphoma cells.(12–14)
Moreover, CCR8 is also highly expressed in bladder cancer cells, and the receptor promotes migration, invasion,and epithelial-mesenchymal transition through activation by
CCL18.(11) These studies demonstrate that CCR8 is associated with cancer progression, and CCR8 can be a target
molecule for cancer immunotherapy. In contrast, regulatory
mechanisms of cancer progression by CCR8 have remained
unclear.
mAbs that specifically bind to CCR8 would be usefulfor cancer immunotherapy and elucidation of the mechanisms of cancer progression. Despite the technical difficulty
in developing anti-GPCR mAbs,(15) we have developed ananti-GPCR mAbs using the Cell-Based Immunization andScreening (CBIS) method, including anti-mouse CCR3mAb (clone C3Mab-2),(16) anti-human CCR9 mAb (clone
C9Mab-1),(17) and anti-mouse CCR8 (mCCR8) mAb(clone C8Mab-2).(18) In this study, we developed another

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

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anti-mCCR8 mAb (clone C8Mab-1) and showed that C8Mab1 recognizes both exogenous and endogenous mCCR8 inflow cytometry and immunofluorescence.
Materials and Methods

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Cell lines

P3X63Ag8U.1 (P3U1) and Chinese hamster ovary (CHO)K1 were obtained from the American Type Culture Collection (ATCC, Manassas, VA). J774-1 and P388 were obtained
from the Cell Resource center for Biomedical ResearchInstitute of Development, Aging and Cancer, Tohoku University (Miyagi, Japan). mCCR8-overexpressed CHO-K1(CHO/mCCR8) was established in our previous study.(18) In
brief, 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).
P3U1, CHO-K1, P388, J774-1, and CHO/mCCR8 were
cultured in Roswell Park Memorial Institute (RPMI) 1640
medium (Nacalai Tesque, Inc., Kyoto, Japan), supplemented
with 10% heat-inactivated fetal bovine serum (Thermo Fisher
Scientific, Inc.), 100 units/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 5% CO2 and 95% air.
Animals

A female Sprague–Dawley (SD) rat (6-week-old) was
purchased from CLEA Japan (Tokyo, Japan). The animal washoused under specific pathogen-free conditions. All animal
experiments were conducted following the 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
health during the four full week’s duration of the experiment.
A decrease of more than 25% of the total body weight was
defined as a humane endpoint. The rat was euthanized bycervical dislocation, and death was verified by respiratory
and cardiac arrest.
Hybridoma production

To develop mAbs against mCCR8, the CBIS method (19–25)
was used. In brief, one SD rat was immunized with CHO/
mCCR8 cells (1 · 109) by the intraperitoneal route together
with the Imject Alum (Thermo Fisher Scientific, Inc.). The
procedure included three additional immunizations followed
by a final booster intraperitoneal injection, administered
2 days before spleen cells were harvested. Harvested spleencells were subsequently fused with P3U1 cells using PEG1500(Roche Diagnostics, Indianapolis, IN). The hybridomas weregrown in an RPMI 1640 medium supplemented with hypoxanthine, aminopterin, and thymidine for selection (Thermo
Fisher Scientific, Inc.). Culture supernatants were screenedusing flow cytometry.

SAITO ET AL.
Production of the recombinant antibody

To produce recombinant C8Mab-1 (recC8Mab-1), we subcloned variable (VH) and constant (CH) regions of heavy
chain cDNAs of C8Mab-1 into the pCAG-Neo vector alongwith variable (VL) and constant (CL) regions of light chain
cDNAs of C8Mab-1 into the pCAG-Ble vector (FUJIFILM
Wako Pure Chemical Corporation). We transfected C8Mab-1
vectors into ExpiCHO-S cells using the ExpiCHO Expression System (Thermo Fisher Scientific, Inc.). We purified theresulting mAb (recC8Mab-1) using Protein G-Sepharose(GE Healthcare Biosciences, Pittsburgh, PA).
Flow cytometry

Cells were harvested after brief exposure to 0.25% trypsin
and 1 mM of ethylenediaminetetraacetic acid (Nacalai Tesque, Inc.). They were washed with 0.1% bovine serum
albumin (BSA) in phosphate-buffered saline (PBS) andtreated with primary mAbs (10 lg/mL) for 30 minutes at 4C.
Then, the cells were treated with Alexa Fluor 488-conjugatedanti-rat IgG (1:2000; Cell Signaling Technology, Inc., Danvers, MA). Fluorescence data were collected using the EC800
Cell Analyzer (Sony Corp., Tokyo, Japan).
Determination of the binding affinity by flow cytometry

CHO/mCCR8 was suspended in 100 lL of serially diluted
anti-mCCR8 mAbs, and Alexa Fluor 488-conjugated anti-rat
IgG (1:200; Cell Signaling Technology, Inc.) was added.
Fluorescence data were collected using the EC800 Cell Analyzer. The dissociation constant (KD) was calculated by fitting
binding isotherms to built-in, one-site binding models in
GraphPad PRISM 8 (GraphPad Software, Inc., La Jolla, CA).
Immunocytochemistry

Subconfluent CHO-K1, CHO/mCCR8, P388, and J774-1
were fixed with 4% paraformaldehyde in PBS for 10 minutes and additional incubation with 50 mM of NH4Cl in PBS
supplemented with 0.2 mM of Ca2+ and 2 mM of Mg2+
(PBSc/m) for 10 minutes at room temperature. The cells were
blocked with a blocking buffer (PBSc/m supplemented with
0.5% BSA) for 30 minutes followed by incubation with primary mAbs (10 lg/mL) for 1 hour and Alexa Fluor 488conjugated anti-rat IgG (1:400; Cell Signaling Technology,Inc.) for 45 minutes at room temperature. The cell nuclei
were stained with 4¢,6-diamidino-2-phenylindole (Thermo
Fisher Scientific, Inc.). We acquired fluorescence images on a
BZ-X800 digital microscope (Keyence, Osaka, Japan) using
a 40 · objective.
Results
Establishment of anti-mCCR8 mAbs

To develop anti-mCCR8 mAbs, we used the CBIS method,using stable transfectants immunization and flow cytometrymediated hybridoma screening. A SD rat was immunizedwith CHO/mCCR8 cells. Hybridomas were seeded into
96-well plates, and CHO/mCCR8-positive and CHO-K1negative wells were selected. After limiting dilution, C8Mab1 (IgG2a, kappa) was finally established. We further produced
its recombinant antibody (recC8Mab-1).

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FIG. 1. Flow cytometry using anti-mCCR8 mAbs, C8Mab-1, and recC8Mab-1. CHO/mCCR8 (A), CHO-K1 (B), P388
(C), and J774-1 (D) were treated with 10 mg/mL C8Mab-1 or recC8Mab-1, followed by treatment with Alexa Fluor 488conjugated anti-rat IgG; red line, C8Mab-1 or recC8Mab-1 treated cells; black line, negative control. CHO, Chinese hamster
ovary; mAb, monoclonal antibody; mCCR8, mouse C-C motif chemokine receptor 8.
335

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SAITO ET AL.

Flow cytometry

We performed flow cytometry using C8Mab-1 andrecC8Mab-1 against CHO/mCCR8 and CHO-K1. Both
C8Mab-1 and recC8Mab-1 recognized CHO/mCCR8 (Fig. 1A),
but not CHO-K1 (Fig. 1B). Both C8Mab-1 and recC8Mab-1
also reacted with P388 (Fig. 1C) and J774-1 (Fig. 1D),
expressing endogenous mCCR8.
Determination of the binding affinity of C8Mab-1

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C8Mab-1 might detect the conformational epitope of
CCR8; therefore, we could not determine the binding affinity
using Surface Plasmon Resonance. In this study, we assessed
the binding affinity of C8Mab-1 and recC8Mab-1 with CHO/
mCCR8 by flow cytometry. The KD of C8Mab-1 andrecC8Mab-1 for CHO/mCCR8 was 6.7 · 10-7 M and 1.1 ·10-7 M, respectively (Supplementary Fig. S1), indicating that
C8Mab-1 possesses a low affinity for CHO/mCCR8 cells.
Immunocytochemistry using C8Mab-1 and recC8Mab-1

We applied C8Mab-1 and recC8Mab-1 to immunocytochemistry against exogenously expressing mCCR8. We
found that C8Mab-1 and recC8Mab-1 bound to CHO/mCCR8(Fig. 2A), but not CHO-K1 (Fig. 2B), indicating that both
antibodies recognize exogenous mCCR8. We also used
C8Mab-1 and recC8Mab-1 for immunocytochemistry against
endogenously expressing mCCR8 in P388 and J774-1 cells.
We found that C8Mab-1 and recC8Mab-1 specifically recognize endogenous mCCR8 in both cells (Fig. 2C, D)
Note that the intensities of the fluorescent signals wereweak because the expression level of endogenous mCCR8 is
expected to be low in both cells.
Discussion

In this study, we developed a novel mAb for mCCR8,
C8Mab-1, using the CBIS method. Both C8Mab-1 andrecC8Mab-1 bound to mCCR8 with low affinity (Supplementary Fig. S1), but they specifically recognized exogenousand endogenous mCCR8 in both flow cytometry (Fig. 1) andimmunocytochemistry (Fig. 2).
These mAbs would be useful for elucidation of themechanism of mCCR8 in cancer progression, such as
analysis of the expression level and intracellular distribution of mCCR8 in target cells, and isolation of CCR8+
cells by combining with a cellular sorting technique.
Confirming other applications of C8Mab-1 and recC8Mab1, including western blotting, immunohistochemistry, andimmunoprecipitation, is required to promote basic research in the future. In contrast, flow cytometric analysis
revealed that C8Mab-1 and recC8Mab-1 bind to mCCR8
with low affinity. We previously developed another
mCCR8 mAb (C8Mab-2), which binds to mCCR8 withmoderate affinity in flow cytometry.(18) To uncover aminoacid sequences of the epitopes of C8Mab-1 and C8Mab-2
would be advantageous for developing novel mAbs thatpossess higher affinity.
Recent studies have revealed that anti-CCR8 mAbs anda CCR8-specific nanobody display antitumor activity inbladder cancer, colon cancer, and nonsmall cell lung cancer.(26–28) A mechanism of the antitumor activity is thedepletion of CCR8+ Tregs at the tumor microenviron-

FIG. 2. Immunocytochemistry using C8Mab-1 andrecC8Mab-1. CHO/mCCR8 (A), CHO-K1 cells (B), P388
(C), and J774-1 (D) were treated with buffer control,
10 mg/mL C8Mab-1, or recC8Mab-1, followed by treatment
with Alexa Fluor 488-conjugated anti-rat IgG. DAPI wasused for nuclear staining. Scale bars, 20 mm. DAPI, 4¢,6diamidino-2-phenylindole.

ment.(27,29) In contrast, we have not evaluated the antitumor activity of C8Mab-1 and recC8Mab-1. It is important toinvestigate their antitumor activity by Tregs depletion using
in vivo mice tumor models. Furthermore, since CCR8 ishighly expressed in cancer cells, the evaluation of their antitumor activity via antibody-dependent cellular cytotoxicity
and complement-dependent cytotoxicity is also thought to beimportant.
CCR8 plays an important role in other physiological
functions. The receptor is involved in the promotion of theonset of allergy and inflammatory diseases by recruiting
Tregs, T helper 2 (TH2) cells, and eosinophils into the
inflammation site.(30–33) CCR8 expressed in neurons causes

ANTI-MOUSE CCR8 FOR FLOW CYTOMETRY AND IMMUNOCYTOCHEMISTRY

diabetic neuropathy via stimulation by CCL1.(34) Thus, it isexpected that C8Mab-1 and recC8Mab-1 are also effective
for treating these diseases.
In summary, we developed a novel mAb for mCCR8,
C8Mab-1, in this study. C8Mab-1 and recC8Mab-1 are applicable to flow cytometry and immunocytochemistry. ThesemAbs would be valuable for elucidating the roles of CCR8 incancer.
Author Disclosure Statement

No competing financial interests exist.

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Funding Information

This research was supported, in part, by Japan Agency
for Medical Research and Development (AMED) under
grant numbers: JP22ama121008 (to Yukinari Kato),
JP21am0401013 (to Yukinari Kato), and JP21am0101078 (toYukinari Kato), and by the Japan Society for the Promotionof Science ( JSPS) Grants-in-Aid for Scientific Research(KAKENHI) grant nos. 21K06059 (to Masaki Saito)
21K15523 (to Teizo Asano), 21K20789 (to Tomohiro Tanaka), 21K07168 (to Mika K. Kaneko), and 19K07705(to Yukinari Kato).
Supplementary Material

Supplementary Figure S1
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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
JapanE-mail: yukinarikato@med.tohoku.ac.jp
Received: December 27, 2021
Accepted: March 2, 2022