MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 41, Number 4, 2022
ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2022.0001
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Development of an Anti-human CCR2 Monoclonal
Antibody (C2Mab-9) by N-Terminal Peptide Immunization

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

The CC chemokine receptor type-2 (CCR2) is one of the members of the G protein-coupled receptor superfamily, which are expressed on the cell surface of immune and tumor cells. CCR2 binds to the C-C motif
chemokine ligand 2 (CCL2)/monocyte chemoattractant protein-1 (MCP-1), which is produced by various cells,
including tumor and immune-related cells. Therefore, the development of sensitive monoclonal antibodies(mAbs) for CCR2 has been desired for treatment and diagnosis. In this study, we established a specific antihuman CCR2 (hCCR2) mAb, C2Mab-9 (mouse IgG1, kappa), using the synthetic peptide immunization method.
Flow cytometric and immunocytochemical results showed that C2Mab-9 reacted with hCCR2-expressing U937
(human histiocytic lymphoma) and natural killer cells. Furthermore, C2Mab-9 showed the moderate binding
affinity for both cells. Conclusively, C2Mab-9 can be a useful tool for analyzing hCCR2-related biologicalresponses.
Keywords: CCR2, monoclonal antibody, flow cytometry

Introduction

G

protein-coupled receptor (GPCR) is a seventransmembrane chemokine receptor, which can trigger
intracellular signals by binding its cognate chemokine ligand
to influence various cellular functions.(1–3) Chemokines play
important roles in immune responses, such as infiltration andmigration of immune-related cells.(3,4) Depending on thenumber and position of the N-terminal cysteine residues,
chemokines are further divided into four different subfamilies, namely CC, CXC, CX3C, and XC.(1,5)
The C-C motif chemokine ligand 2 (CCL2)/monocyte
chemoattractant protein-1 (MCP-1) is one of the C-C motif
chemokine ligands. CCL2 is produced by fibroblasts, endothelial, epithelial, myeloid, and tumor cells.(5,6) In addition,
CCL2 plays a crucial role in attracting monocytes, T lymphocytes, and natural killer (NK) cells.(7,8) Furthermore,
CCL2 can activate several GPCRs, including the CC chemokine receptor type-2 (CCR2), CCR4, and CCR5.(9)Notably, the primary receptor of CCL2 is CCR2, whose
expression has been observed in multiple cells, including
monocytes, macrophages, dendritic cells, and epithelial

cells.(10) The CCL2-attracted monocytes to the lung control
the SARS-CoV-2 burden and the inflammatory response
during infection.(11)The CCL2–CCR2 axis has been reported in many diseases,
such as immune disorders and cancers.(12,13) In cancers, highCCL2 expression influences cancer progression, angiogenesis, and metastasis.(7,14,15) CCL2 has been reported to behighly upregulated in bone tumors(15) and inflammatory
breast cancer.(16) Furthermore, the invasive lesions of breast
ductal carcinoma show high CCR2 and CCL2 expression.(17)Therefore, various CCR2-expressing cells are involved indisease pathogenesis by interacting with CCL2.
We have previously developed various monoclonal antibodies (mAbs) against membrane proteins, such as HER3,(18)
EpCAM,(19) KLRG1,(20) TIGIT,(21) TROP2,(22,23) programmed cell death ligand 1 (PD-L1),(24) CD19,(25) CD20,(26,27)
CD44,(28) CD133,(29) and podoplanin(30–37) by using the
Cell-Based Immunization and Screening (CBIS) method.
Moreover, we have also developed anti-GPCR mAbs againstmouse CCR3,(38) mouse CCR8,(39) and human CCR9(40)using the CBIS method. In contrast, we have not established sensitive anti-GPCR mAbs, using synthetic peptide

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

188

ANTI-HUMAN CCR2 MONOCLONAL ANTIBODY

immunization. In this study, we attempted to develop
anti-human CCR2 (hCCR2) mAbs using synthetic peptide
immunization.
Materials and Methods
Antibodies

The anti-hCCR2 mAb (clone K036C2) was purchased from
BioLegend (San Diego, CA). The peroxidase-conjugatedanti-mouse immunoglobulins was purchased from Agilent
Technologies, Inc. (Santa Clara, CA). The Alexa Fluor 488conjugated anti-mouse IgG was purchased from Cell Signaling Technology, Inc. (Danvers, MA).

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Cell lines

P3X63Ag8U.1 (P3U1) was obtained from the American
Type Culture Collection (Manassas, VA). U937 (The humanhistiocytic lymphoma) was obtained from the JapaneseCollection of Research Bioresources Cell Bank (Osaka,
Japan). The human NK cells (donor lot. 4022602, purity >70%)
were purchased from Takara Bio (Shiga, Japan).
P3U1 and U937 cells were cultured in a Roswell Park
Memorial Institute (RPMI) 1640 medium (Nacalai Tesque,Inc., Kyoto, Japan) that was supplemented with 10% heatinactivated fetal bovine serum (Thermo Fisher Scientific,
Inc., Waltham, MA), 100 units/mL penicillin, 100 lg/mL
streptomycin, and 0.25 lg/mL amphotericin B (Nacalai
Tesque, Inc.). Subsequently, cells were grown in a humidified
incubator, which was supplied with 5% CO2 and 95% air at
37C.

189

Roche Diagnostics, Indianapolis, IN). Thereafter, hybridomas were grown in an RPMI medium supplemented withhypoxanthine, aminopterin, and thymidine for selection(Thermo Fisher Scientific, Inc.). Cultured supernatants were
finally screened using enzyme-linked immunosorbent assay(ELISA) and flow cytometric analysis.
ELISA

A mixture of synthesized hCCR2 peptides, including
11-RNTNESGEEVTTF
FDYDYG-29, and 21-TTFFDYDYGAPSHKFDVKQ-39 +C-terminal cysteine, was immobilized on Nunc Maxisorp
96-well immunoplates (Thermo Fisher Scientific, Inc.) at aconcentration of 1 lg/mL for each peptide and a temperatureof 37C for 30 minutes. After washing with phosphatebuffered saline (PBS) containing 0.05% Tween20 (PBST;

1-MLSTSRSRFIRNTNESGEE-19,

Animals

Two 6-week-old female BALB/c mice were purchasedfrom CLEA (Tokyo, Japan). Mice were housed under specificpathogen-free conditions. Afterward, animal experiments
were conducted, following relevant guidelines and regulations to minimize animal suffering and distress in the laboratory. The animal care and use committee of Tohoku
University approved animal experiments (Permit number:
2019NiA-001). The mouse health was monitored daily during the entire 4-week duration of the experiment and areduction of >25% of the total body weight was denoted asa humane endpoint. Subsequently, mice were euthanizedthrough cervical dislocation, after which respiratory and cardiac arrest was used to verify death.
Hybridoma production

Two BALB/c mice were immunized using three keyhole
limpet hemocyanin (KLH)-conjugated hCCR2 peptides(100 lg of each peptide), including 1-MLSTSRSRFIRNTNE
SGEE-19, 11-RNTNESGEEVTTFFDYDYG-29, and 21-TTFF
DYDYGAPSHKFDVKQ-39 (the 32nd Cys was changed to
Ser in the 3rd peptide) + C-terminal cysteine. The administration was conducted through the intraperitoneal route withan Imject Alum Adjuvant (Thermo Fisher Scientific, Inc.).
The procedure included three additional immunization procedures (100 lg of each peptide), followed by a final booster
intraperitoneal injection (100 lg of each peptide) 2 days
before their spleen cells were harvested.
The harvested spleen cells were subsequently fused with
P3U1 cells, using polyethylene glycol 1500 (PEG1500;

FIG. 1. A diagram showing the production of anti-hCCR2
mAbs. The mice were intraperitoneally immunized with
hCCR2 peptides. Screening of hybridoma was then conducted by ELISA, followed by flow cytometry using hCCR2expressing cells. ELISA, enzyme-linked immunosorbent
assay; hCCR2, human CC chemokine receptor type-2;
mAbs, monoclonal antibodies.

190

Nacalai Tesque, Inc.), wells were blocked with 1% bovine
serum albumin (BSA)-containing PBST for 30 minutes at
37C.
Subsequently, plates were incubated with culture supernatants, followed by peroxidase-conjugated anti-mouse
immunoglobulins (1:2000 diluted). Enzymatic reactions
were conducted using the ELISA POD substrate TMB kit(Nacalai Tesque, Inc.), followed by the measurement of the
optical density at 655 nm using an iMark microplate reader(Bio-Rad Laboratories, Inc., Berkeley, CA).

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Flow cytometry

U937 and human NK cells were washed with 0.1% BSA
in PBS and treated with primary mAbs for 30 minutes at
4C. Thereafter, cells were treated with Alexa Fluor 488conjugated anti-mouse IgG (1:1000), followed by the collection of fluorescence data using an SA3800 cell analyzer(Sony Corp., Tokyo, Japan).
Determination of the binding affinity by flow cytometry

U937 and human NK cells were suspended in 100 lL
serially diluted anti-hCCR2 mAbs, after which Alexa Fluor
488-conjugated anti-mouse IgG (1:200) was added. Fluorescence data were subsequently collected using BD FACSLyric (BD Biosciences), followed by the calculation of the
dissociation constant (KD) through fitting binding isotherms
to the built-in one-site binding models in GraphPad PRISM 8(GraphPad Software, Inc., La Jolla, CA).

TANAKA ET AL.
ImmunocytochemistryU937 and human NK cells were centrifuged at 270 · g for
5 minutes and the cell pellets were suspended with 4% paraformaldehyde in PBS for 10 minutes before the quenching in
50 mM NH4Cl in PBSc/m (PBS supplemented with 0.2 mM
Ca2+ and 2 mM Mg2+) for 10 minutes. The cells were further
incubated in the blocking buffer (0.5% BSA in PBSc/m) for 30
minutes, then in the primary mAbs (10 mg/mL) for 2 hours, andsubsequently in an additional Alexa Fluor 488-conjugated antimouse IgG (1:400) and 4¢,6-diamidino-2-phenylindole (DAPI;
Thermo Fisher Scientific, Inc.) for 45 minutes. Lastly, the cells
were mounted using ProLong Glass Antifade Mountant. The
fluorescent images were acquired using a digital microscope(BZ-X800; Keyence, Osaka, Japan) with a 40 · objective.
Results
Establishment of anti-hCCR2 mAbs

We first immunized mice with the hCCR2 peptides(Fig. 1). Hybridomas were seeded into 96-well plates, after
which ELISA was used to select positive wells for hCCR2
peptides, followed by the selection of U937 and NK cellsreactive supernatants using flow cytometry. We obtained
reactive supernatants in 16 out of the 956 wells (1.67%) andfinally established C2Mab-9 (mouse IgG1, kappa) aftercloning by the limiting dilution.
Flow cytometry

Flow cytometry was performed using C2Mab-9 against
U937 and NK cells. Results showed that C2Mab-9 recognized

FIG. 2. Flow cytometry results using anti-hCCR2 mAbs. U937 (human histiocytic lymphoma) cells (A) and human NK
cells (B) were treated with 0.01–10 mg/mL of C2Mab-9 and K036C2, followed by treatment with Alexa Fluor 488conjugated anti-mouse IgG. The black line represents the negative control. NK, natural killer.

ANTI-HUMAN CCR2 MONOCLONAL ANTIBODY

U937 (Fig. 2A) and NK cells (Fig. 2B) in a dose-dependent
manner. Another anti-hCCR2 mAb (clone K036C2) also
recognized U937 (Fig. 2A) and NK cells (Fig. 2B) dosedependently.
Determination of the binding affinity of C2Mab-9

The binding affinity of C2Mab-9 was assessed with U937
and NK cells based on flow cytometry. Results showed that
the KD of C2Mab-9 for U937 and NK cells was 2.9 · 10-8 M
and 7.9 · 10-8 M, respectively (Supplementary Fig. S1).
These results indicated that C2Mab-9 possesses moderateaffinities for U937 and NK cells.

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Immunocytochemistry

Immunocytochemistry was performed using C2Mab-9
against U937 and NK cells. Results showed that C2Mab-9
and anti-hCCR2 mAb (clone K036C2), but not buffer control, were bound to U937 and NK cells (Fig. 3), indicatingthat C2Mab-9 specifically recognizes endogenous hCCR2
in immunocytochemistry.

191
Discussion

The GPCRs possess various functions in normal and pathological conditions and they have been focused on as pivotal
drug targets.(41) Although it is scientifically well known to bedifficult to develop anti-GPCR mAbs,(42) we have successfully established various anti-GPCR mAbs, including antimouse CCR3 mAbs,(38) mouse CCR8 mAbs,(39) and human
CCR9 mAbs(40) using the CBIS method. In this study, wedeveloped a novel anti-hCCR2 mAb (C2Mab-9) throughpeptide immunization. The KD of C2Mab-9 for U937 and NK
cells was 2.9 · 10-8 M and 7.9 · 10-8 M, respectively, indicating that C2Mab-9 possesses a moderate affinity against
endogenous hCCR2-expressing cells.
In the tumor microenvironment (TME), CCR2 has animportant function as an immunoregulatory molecule.(9,13,43)
CCL2 expression was associated with PD-1–related gene signatures in patients with esophageal squamous cell carcinoma.(43) The tumor-secreted CCL2 recruits CCR2-expressing
monocytes into the tumor, where they differentiate into M2
macrophages, known as tumor-associated macrophages

FIG. 3. Immunocytochemistry using anti-hCCR2 mAbs. U937 cells (A) and NK cells (B) were treated with control,
K036C2 (10 mg/mL), and C2Mab-9 (10 mg/mL), followed by treatment with Alexa Fluor 488-conjugated anti-mouse IgG
and DAPI. Scale bars: 20 mm. DAPI, 4¢,6-diamidino-2-phenylindole.

192(TAMs). TAMs express cytokines and chemokines that can
suppress antitumor immunity and promote tumor progression.(5) In brain tumors, CCL2–CCR2 signaling triggers theinfiltration of TAM and regulatory T cells, which contributes
the formation of immunosuppressive TME.(44–46)
In addition, interactions between CCL2–CCR2 have beenshown to recruit immunosuppressive cells such as CCR2+
myeloid-derived suppressor cells(47) and metastasis-promoting
monocytes.(48) In anti-PD-1–resistant glioma-bearing micemodel, CCR2 deficiency unmasked an anti-PD-1 effect andenhanced the survival. Furthermore, the CCR2 antagonist(CCX872) enhanced the effect of anti-PD-1 therapy in theglioma.(49) Therefore, it is important to evaluate the neutralizing activity of C2Mab-9 in the future.
Author Disclosure Statement

Downloaded by Yukinari Kato from www.liebertpub.com at 08/26/22. For personal use only.

No competing financial interests exist.
Funding Information

This research was supported in part by Japan Agency for
Medical Research and Development (AMED) under Grant
Nos JP22ama121008 (to Y.K.), JP22am0401013 (to Y.K.),
JP22bm1004001 (to Y.K.), JP22ck0106730 (to Y.K.), andJP21am0101078 (to Y.K.).
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
Miyagi 980-8575
JapanE-mail: yukinari.kato.e6@tohoku.ac.jp
Received: January 9, 2022
Accepted: July 11, 2022