MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 41, Number 2, 2022
ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2021.0064
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Development of a Novel Anti-Mouse CCR4 Monoclonal
Antibody (C4Mab-1) by N-Terminal Peptide Immunization

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

The CC chemokine receptor type-4 (CCR4) belongs to the G-protein-coupled receptor superfamily, expressedon the cell surface of T cells and its malignancy. Two CCR4 ligands (CCL17 and CCL22) bind to CCR4 thatmediate physiological and pathological functions of T cell immune responses. Anti-CCR4 monoclonal antibody(mAb) mogamulizumab is approved for adult T cell leukemia/lymphoma and cutaneous T cell lymphomas. In
addition, mogamulizumab can deplete regulatory T cells, implying the application to solid tumors as an immunomodulator. Therefore, the development of sensitive mAbs for CCR4 has been desired for basic research,
diagnosis, and therapy. In this study, a specific, and sensitive anti-mouse CCR4 (mCCR4) mAb, C4Mab-1 (rat
IgG1, kappa), was established using N-terminal peptide immunization. C4Mab-1 reacted with mCCR4overexpressed Chinese hamster ovary (CHO)-K1 cells, P388 (mouse lymphoid neoplasm), and J774-1 (mouse
macrophage-like) cells in flow cytometry. Kinetic analyses using flow cytometry showed that KDs of C4Mab-1
for CHO/mCCR4, P388, and J774-1 cells were 4.2 · 10-9 M, 5.4 · 10-7 M, and 1.1 · 10-6 M, respectively.
C4Mab-1 could be a valuable tool for elucidating mCCR4-related biological responses.
Keywords: CCR4, monoclonal antibody, flow cytometry

malignancies. Mogamulizumab (Poteligeo) is a defucosylated humanized monoclonal antibody (mAb) targeting CCR4.
Mogamulizumab binds to Fcg receptor IIIa on natural killer
cells and exerts potent antibody-dependent cellular cytotoxicity (ADCC) against the CCR4+ cells.(9,10) Mogamulizumab
was first approved in Japan for ATLL therapy.(11,12)
Clinical studies of mogamulizumab in CTCLs were conducted. Mogamulizumab efficiently decreased levels of CCR4+
malignant T cells and CCR4+ Tregs in CTCL patients.(13,14)
Mogamulizumab significantly improved the progression-freesurvival and overall response rate compared with vorinostat, a histone deacetylase inhibitor approved for treating
CTCLs.(15,16) From these results, the US Food and Drug
Administration approved mogamulizumab in 2018 for treating adult patients with relapsed or refractory mycosis fungoides (MF) and Sézary syndrome (SS), which are the mostcommon types of CTCLs.(17)In solid tumors, Treg cells play a critical role in suppressing tumor immunity.(18) Treg cells abundantly infiltrate into
tumor tissues, associated with poor prognosis in cancer patients. Removal of Treg cells is thought to increase theIntroduction

C

C chemokine receptor 4 (CCR4) is a chemokine
receptor for two CC chemokine ligands, CCL17 andCCL22. CCR4 is primarily expressed in T cells, including
helper T type 2 (Th2), regulatory T (Treg), and Th17-cells.(1)Upon the chemokines binding to CCR4, the chemokines
can trigger intracellular signaling of CCR4 expressing cells,
which mediates various cellular functions, including theinfiltration into target tissues.(2)In addition, elevated serum levels of CCL17 and CCL22
have been reported in patients with atopic dermatitis andcorrelate with disease severity and treatment response.(3–5)
Accordingly, the serum CCL17 test was approved by theJapanese Pharmaceutical and Medical Devices Agency and is
now regarded as an excellent biomarker for disease severity
and therapeutic response in the disease.(6)In addition, CCR4 is strongly expressed in T cell malignancies, including adult T cell leukemia/lymphoma (ATLL)(7)and cutaneous T cell lymphomas (CTCLs).(8) Therefore, CCR4
has been regarded as a possible therapeutic target for these

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

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88

antitumor immune responses and potentiate the efficacy of
immune checkpoint inhibitors. Since mogamulizumab efficiently depletes Treg cells, clinical trials in advanced solid
tumors are currently conducted singly or combined withimmune checkpoint inhibitors.(19,20)
Using the cell-based immunization and screening (CBIS)
method, many mAbs have been developed against membraneproteins, such as anti-CD19,(21) anti-CD20,(22,23) antiCD44,(24) anti-CD133,(25) anti-EpCAM,(26,27) anti-HER3,(28)anti-KLRG1,(29) anti-TIGIT,(30) anti-TROP2,(31,32) antiprogrammed cell death ligand 1,(33) and anti-podoplanin(34–44)
mAbs. CBIS method includes immunizing antigenoverexpressing cells and the high-throughput hybridoma
screening using flow cytometry. Anti-chemokine receptors
mAbs, including anti-mouse CCR3,(45) anti-mouse CCR8,(46)and anti-human CCR9(47) mAbs, were also successfully
developed using the CBIS method. This study developed
anti-mouse CCR4 (mCCR4) mAbs using N-terminal peptide
immunization.
Materials and Methods
Cell lines

Chinese hamster ovary (CHO)-K1 and P3X63Ag8U.1(P3U1) cells were obtained from the American Type Culture
Collection (Manassas, VA). P388 (mouse lymphoid neoplasma) and J774-1 (mouse macrophage-like) cells wereobtained 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 mCCR4(accession no. NM_009916) was subcloned into a pCAG-Ble
vector (FUJIFILM Wako Pure Chemical Corporation, Osaka,
Japan).
mCCR4 plasmid was transfected into CHO-K1 cells
using the Neon transfection system (Thermo Fisher Scientific, Inc., Waltham, MA). Stable transfectants were developed through cell sorting using a cell sorter (SH800; Sony
Corp., Tokyo, Japan) and cultivation in a medium containing
0.5 mg/mL Zeocin (InvivoGen, San Diego, CA).
CHO-K1, P3U1, mCCR4-overexpressed CHO-K1 (CHO/
mCCR4), P388, and J774-1 were cultured in a Roswell Park
Memorial Institute (RPMI) 1640 medium (Nacalai Tesque,Inc., Kyoto, Japan), supplemented with 10% heat-inactivatedfetal 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 an atmosphere of 5% CO2
and 95% air.
Antibodies

The anti-mCCR4 mAb (clone 2G12) was bought from
BioLegend (San Diego, CA). The secondary Alexa Fluor 488conjugated anti-rat IgG and anti-Armenian hamster IgG wasbought from Cell Signaling Technology, Inc. (Danvers, MA).
Hybridoma production

A 6-week-old female Sprague–Dawley (SD) rat wasbought from CLEA, Japan (Tokyo, Japan). The animal washoused under specific pathogen-free conditions. Then, animal experiments were performed, following relevant guide-

TAKEI ET AL.

lines and regulations to reduce animal suffering and distressin the laboratory. The Animal Care and Use Committee of
Tohoku University approved the animal experiments (Permit
no. 2019NiA-001). Rat health was monitored daily during
the entire 4-week duration of the experiment, and a declineof >25% of the total body weight was denoted as a humane
endpoint. Subsequently, the rat was euthanized through cervical dislocation, after which respiratory and cardiac arrest
was used to verify death.
The mCCR4 peptide immunization method was used todevelop mAbs against mCCR4. In brief, one SD rat was
immunized with the keyhole limpet hemocyanin-conjugated
mCCR4 peptide (100 lg): 1-MNATEVTDTTQDETVYNSY-19+ C-terminal cysteine. The administration was performed
through the intraperitoneal route with an Imject Alum (Thermo
Fisher Scientific, Inc.).
The procedure involved three additional immunization
procedures (100 lg), followed by a final booster intraperitoneal injection (100 lg) 2 days before its spleen cells were
harvested. The harvested spleen cells were subsequently
fused with P3U1 cells using polyethylene glycol 1500
(PEG1500; Roche Diagnostics, Indianapolis, IN). Then,hybridomas were cultured in an RPMI 1640 medium supplemented with hypoxanthine, aminopterin, and thymidine
for selection (Thermo Fisher Scientific, Inc.). Cultured supernatants were finally screened by enzyme-linked immunosorbent assay (ELISA) and flow cytometric analysis.
ELISA

The synthesized mCCR4 peptide: 1-MNATEVTDTTQDETVYNSY-19 + C-terminal cysteine was immobilized on
Nunc Maxisorp 96-well Immuno plates (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.
Plates were then incubated with culture supernatants, followed by peroxidase-conjugated anti-rat immunoglobulins(1:2000 diluted; Agilent Technologies, Inc., Santa Clara, CA).
Afterward, enzymatic reactions were performed using the
ELISA POD substrate TMB kit (Nacalai Tesque, Inc.), followed by the optical density measurement at 655-nm, using
an iMark microplate reader (Bio-Rad Laboratories, Inc.,
Berkeley, CA).
Flow cytometry

CHO-K1 and CHO/mCCR4 cells were harvested after brief
exposure to 1 mM ethylenediaminetetraacetic acid (Nacalai
Tesque, Inc.). CHO-K1, CHO/mCCR4, P388, and J774-1 cells
were washed using 0.1% BSA in PBS and treated with primary mAbs for 30 minutes at 4C. Afterward, cells weretreated using Alexa Fluor 488-conjugated anti-rat IgG or antiArmenian hamster IgG (1:1000) also, followed by the collection of fluorescence data, using the EC800 Cell Analyzer(Sony Corp., Tokyo, Japan).
Determination of the binding affinity by flow cytometry

CHO-K1, CHO/mCCR4, P388, and J774-1 cells weresuspended in 100 lL serially diluted anti-mCCR4 mAbs,

ANTI-MOUSE CCR4 MONOCLONAL ANTIBODY

after which Alexa Fluor 488-conjugated anti-rat IgG antiArmenian hamster IgG (1:200) was added. Fluorescence data
were subsequently obtained, using the BD FACSLyric(BD Biosciences, Franklin Lakes, NJ), followed by the calculation of the dissociation constant (KD) by fitting thebinding isotherms into the built-in one-site binding model in
GraphPad PRISM 8 (GraphPad Software, Inc., La Jolla, CA).
Results

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Establishment of anti-mCCR4 mAbs

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Flow cytometry

Flow cytometry was performed using C4Mab-1 against
CHO-K1, CHO/mCCR4, P388, and J774-1. Results indicatedthat C4Mab-1 dose-dependently recognized CHO/mCCR4,
but not CHO-K1 (Fig. 2A). Another anti-mCCR4 mAb(clone 2G12 from BioLegend) also dose-dependently recognized CHO/mCCR4, but not CHO-K1 (Fig. 2B). In addition,
C4Mab-1 and 2G12 reacted with CHO/mCCR4, even at 0.01lg/mL concentration (Fig. 2A, B). C4Mab-1 and 2G12
also reacted dose-dependently with endogenous mCCR4expressing P388 and J774-1 (Fig. 2A, B).

To develop anti-mCCR4 mAbs, the mCCR4 peptide
immunization method was employed. Hybridoma screening
was performed using ELISA and flow cytometry (Fig. 1).
A rat was immunized with the CCR4 synthetic peptide. Subsequently, hybridomas were seeded into 96-well plates, after
which ELISA was used to obtain positive wells for mCCR4
peptides, followed by selecting CHO/mCCR4-reactive and
CHO-K1-nonreactive supernatants using flow cytometry.
Finally, C4Mab-1 (rat IgG1, kappa) was established after
limiting dilution.

FIG. 1. A schematic procedure of anti-mCCR4 mAbs
production. The rat was intraperitoneally immunized withthe mCCR4 peptide. The screening was then conducted by
ELISA, using the mCCR4 peptide, flow cytometry using
parental cells and mCCR4-overexpressed CHO-K1 cells.
CHO-K1, Chinese hamster ovary-K1; ELISA, enzymelinked immunosorbent assay; mAbs, monoclonal antibodies;
mCCR4, mouse CCR4.

FIG. 2. Flow cytometry using anti-mCCR4 mAbs. CHOK1, CHO/mCCR4, P388, and J774-1 were treated with
0.01–10 mg/mL of C4Mab-1 (A) and 2G12 (B), followed bytreatment with Alexa Fluor 488-conjugated anti-rat IgG oranti-Armenian hamster IgG.

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Determination of the binding affinity of C4Mab-1

The binding affinity of C4Mab-1 was analyzed with CHO/
mCCR4, P388, and J774-1, using flow cytometry. Results
indicated that the KD of C4Mab-1 for CHO/mCCR4, P388,
and J774-1 was 4.2 · 10-9 M, 5.4 · 10-7 M, and 1.1 · 10-6 M,
respectively (Fig. 3A). In contrast, the KD of 2G12 for CHO/
mCCR4, P388, and J774-1 was 3.6 · 10-9 M, 1.2 · 10-6 M,
and 1.4 · 10-6 M, respectively (Fig. 3B). These results, therefore, show that C4Mab-1 has a high affinity for CHO/mCCR4
and a moderate affinity for P388 and J774-1 cells.
Discussion

tors (GPCRs). The generation of anti-GPCR mAbs is difficult because of the problems in protein folding, small
epitope regions, and purification of functional antigens.(48)Therefore, there were attempts to develop different antiGPCR mAbs, including anti-mouse CCR3,(45) anti-mouse
CCR8,(46) and anti-human CCR9(47) mAbs, using the CBIS
method.
In this study, a novel anti-mCCR4 mAb (C4Mab-1)
through peptide immunization was developed. The KDs
of C4Mab-1 for CHO/mCCR4, P388, and J774-1 cells
were 4.2 · 10-9 M, 5.4 · 10-7 M, and 1.1 · 10-6 M, respectively, showing that C4Mab-1 possessed a high tomoderate affinity against mCCR4-expressing cells. The
epitope of mogamulizumab was shown at positions 12–29
of the N-terminal residues (DESIYSNYYLYESIPKPC)

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Chemokine receptors, such as CCR4, are a family of
the seven-transmembrane domain G protein-coupled recep-

TAKEI ET AL.

FIG. 3. The determination of the binding affinity of C4Mab-1. CHO/mCCR4, P388, and J774-1 cells were suspended in
100 mL serially diluted C4Mab-1 (A) and 2G12 (B). The concentration of each mAb is as follows: C4Mab-1 for CHO/
mCCR4, 12.5–0.006 mg/mL; C4Mab-1 for P388, 100–0.006 mg/mL; C4Mab-1 for J774-1, 100–0.006 mg/mL; 2G12 for
CHO/mCCR4, 12.5–0.006 mg/mL; 2G12 for P388, 100–0.006 mg/mL; 2G12 for J774-1, 250–0.02 mg/mL. Then, cells weretreated with Alexa Fluor 488-conjugated anti-rat IgG or anti-Armenian hamster IgG.

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ANTI-MOUSE CCR4 MONOCLONAL ANTIBODY

of human CCR4.(49) Therefore, in humans and mice,N-terminal CCR4 is an essential region to generate highaffinity mAbs.
CTCLs are a heterogeneous group of non-Hodgkin skinlymphomas obtained from skin-homing T cells. The skinhoming effector/memory T cells express cutaneous lymphocyte antigen (CLA) and CCR4.(5) In contrast, malignant
T cells of MF/SS are characterized as CLA+CCR4+CD4+
T cells, while skin lesions express high levels of CCR4 ligands, CCL17 and CCL22.(50) Therefore, the neutralizing
activity of C4Mab-1 is also important to suppress the T cellhoming to target lesions. Further investigation is needed toevaluate the neutralizing activity of C4Mab-1 for CCR4CCL17/CCL22-mediated homing and chemoattraction.
Foxp3-expressing Treg cells are indispensable for preventing autoimmunity. Treg cells also abundantly infiltrate
into tumor tissues and suppress antitumor immunity. Therefore, the removal of Treg cells improves antitumor immunity
but may also trigger autoimmunity.(18) Therefore, an essential point in developing Treg-depleting cancer immunotherapy is how to specifically deplete Treg cells infiltrating into
tumors without affecting tumor-reactive effector T cells,
while suppressing autoimmunity.
These marker proteins include T cell accessory molecules,
including CD25, CTLA-4, GITR, 4-1BB, OX-40, LAG3,TIGIT, CCR8, and CCR4.(18) In our previous studies, wechanged the isotype of mAbs into mouse IgG2a to retain
ADCC and complement-dependent cytotoxicity (CDC),
which caused high antitumor activities in mouse xenograftmodels.(51–53) Since the subclass of C4Mab-1 was rat IgG1,
it did not possess ADCC and CDC. Therefore, in further studies, the subclass of C4Mab-1 will be converted into
mouse IgG2a to evaluate the effect of depletion of CCR4+
Treg cells in cancer immunotherapy.
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 grantnos. JP22ama121008 (to Y.K.), JP21am0401013 (to Y.K.), andJP21am0101078 (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 980-8575
JapanE-mail: yukinarikato@med.tohoku.ac.jp
Received: December 15, 2021
Accepted: February 14, 2022