MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 41, Number 2, 2022
ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2021.0063
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and other resources online.

Development of a Novel Anti-Mouse CCR2 Monoclonal
Antibody (C2Mab-6) by N-Terminal Peptide Immunization

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

The CC chemokine receptor type-2 (CCR2) belongs to the G-protein-coupled receptor superfamily, expressedon the cell surface of immune cells and tumors. CCR2 binds to the CC motif chemokine 2/monocyte chemoattractant protein-1, a CC chemokine, which is produced by various cells, including immune-related cells
and tumors. Therefore, the development of sensitive monoclonal antibodies (mAbs) for CCR2 has been desired
for treatment and diagnosis. This study established a novel, specific, and sensitive anti-mouse CCR2 (mCCR2)mAb; C2Mab-6 (rat IgG1, kappa), using the mCCR2 synthetic peptide immunization method. C2Mab-6 reactedwith mCCR2-overexpressed Chinese hamster ovary-K1 cells and L1210 (murine leukemia) cells, which express
endogenous mCCR2 in flow cytometry. Furthermore, C2Mab-6 showed a high binding affinity for both cells.
Hence, C2Mab-6 can be a useful tool for analyzing mCCR2-related biological responses, using flow cytometry.
Keywords: CCR2, monoclonal antibody, flow cytometry

cells, including monocytes, dendritic cells, macrophages, andepithelial cells. It has also been reported that the CCL2-CCR2
axis correlates with many diseases, such as immune disorders
and cancers.(12–14)
In viral and bacterial infections, CCL2 and CCR2expressing immune cells, such as macrophages and neutrophils, contribute to innate immune responses in respiratoryorgans.(15) Alternatively, in tumors, CCL2 influences cancer
growth, angiogenesis, and metastasis.(9,10,16) It has also beenreported as a highly upregulated chemokine in bone tumors.(17) Therefore, various cells expressing CCR2 are involved in disease pathogenesis by interacting with CCL2.
Using the Cell-Based Immunization and Screening (CBIS)
method, we have developed many monoclonal antibodies (mAbs) against membrane proteins, such as antiCD19,(18) anti-CD20,(19,20) anti-CD44,(21) anti-CD133,(22)anti-EpCAM,(23,24) anti-HER3,(25) anti-KLRG1,(26) antiTIGIT,(27) anti-TROP2,(28,29) anti-programmed cell deathligand 1 (PD-L1),(30) and anti-podoplanin(31–41) mAbs. We
have also successfully developed anti-GPCR mAbs, including anti-mouse CCR3,(42) anti-mouse CCR8,(43) and antihuman CCR9(44) mAbs, using the CBIS method. However,Introduction

C

hemokines play important roles in immune responses, such as infiltration and migration,(1–4) aresmall-molecule proteins, which belong to the largest subfamily of cytokines.(3) They can be classified into four different subfamilies: CC, CXC, CX3C, and XC, depending on
the number and position of cysteine residues in theirN-terminus.(5) By binding to thier receptors, which are seventransmembrane G-protein-coupled receptors (GPCRs), chemokines can trigger an intracellular signal, which influences
various cellular functions.(6)The CC motif chemokine 2 (CCL2)/monocyte chemoattractant protein-1 is one of the CC motifs of chemokine ligands, with a molecular weight of 13,000. Various cells
produce CCL2, including tumor cells, endothelial cells, epithelial cells, myeloid cells, and fibroblasts.(7,8) CCL2 also
plays a crucial role in attracting monocytes, T lymphocytes,
and natural killer cells.(9) Moreover, several GPCRs bind
CCL2, including the CC chemokine receptor type-2 (CCR2),
CCR4, and CCR5.(10) Notably, CCR2 is a primary receptor of
CCL2,(11) whose expression has been observed in multiple

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

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ANTI-MOUSE CCR2 MONOCLONAL ANTIBODY

we have not established anti-GPCR mAbs, using the synthetic peptide immunization method yet. In this study, wedeveloped anti-mouse CCR2 (mCCR2) mAbs using thesynthetic peptide immunization method.
Materials and Methods

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Cell lines

Chinese hamster ovary (CHO)-K1 and P3X63Ag8U.1(P3U1) cells were obtained from the American Type Culture
Collection (Manassas, VA). The L1210 (murine leukemia)
cell line was obtained from the Cell Resource Center for
Biomedical Research Institute of Development, Aging andCancer, Tohoku University (Miyagi, Japan). Synthesized
DNA (Eurofins Genomics KK, Tokyo, Japan) encoding
mCCR2 (Accession No. NM_009915) was subcloned into a
pCAG-Ble vector (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan), after which the mCCR2 plasmid wastransfected using a Neon transfection system (Thermo Fisher
Scientific, Inc., Waltham, MA). Subsequently, CHO/mCCR2
was established using a cell sorter (SH800; Sony Corp.,
Tokyo, Japan), following cultivation in a medium containing
0.5 mg/mL Zeocin (InvivoGen, San Diego, CA).
CHO-K1, P3U1, CHO/mCCR2, and L1210 were cultured
in a Roswell Park Memorial Institute (RPMI) 1640 medium(Nacalai Tesque, Inc., Kyoto, Japan) that was 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.). Then, cells were grown in a humidified incubator,
which was supplied with 5% CO2 and 95% air at 37C.

81

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 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 weregrown in an RPMI 1640 medium supplemented with hypoxanthine, aminopterin, and thymidine for selection (Thermo
Fisher Scientific, Inc.). Cultured supernatants were finally
screened using enzyme-linked immunosorbent assay (ELISA)
and flow cytometric analysis.

Antibodies

The anti-mCCR2 mAb (clone EPR20844) was purchasedfrom Abcam (Cambridge, United Kingdom). The secondary
Alexa Fluor 488-conjugated anti-rat IgG and Alexa Fluor
488-conjugated anti-rabbit IgG were purchased from Cell
Signaling Technology, Inc. (Danvers, MA).
Hybridoma production

A 6-week-old female Sprague–Dawley (SD) rat was purchased from CLEA, Japan (Tokyo, Japan). The animal washoused under specific pathogen-free conditions. Then, animal experiments were conducted, following relevant guidelines and regulations to minimize animal suffering anddistress in the laboratory. The Animal Care and Use Committee of Tohoku University approved animal experiments(Permit No. 2019NiA-001). The rat health was monitored
daily during the entire 4-week duration of the experiment,
and a reduction of more than 25% of the total body weightwas 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 mCCR2 peptide immunization method was used todevelop mAbs against mCCR2. In brief, one SD rat was immunized, using three keyhole limpet hemocyanin-conjugated
mCCR2 peptides (100 lg of each peptide), including
1-MEDNNMLPQFIHGILSTSH-19, 11-IHGILSTSHSLFTRSI
QEL-29, and 21-LFTRSIQELDEGATTPYDY-39+C-terminal
cysteine. The administration was conducted through the intraperitoneal route with an Imject Alum (Thermo Fisher

FIG. 1. A schematic procedure of anti-mCCR2 mAbs
production. The rat was intraperitoneally immunized with
mCCR2 peptides. Screening was then conducted by ELISA,
using mCCR2 peptides, and flow cytometry using parental
cells and mCCR2-overexpressed CHO-K1 cells. CHO,
Chinese hamster ovary; ELISA, enzyme-linked immunosorbent assay; mAbs, monoclonal antibodies; mCCR2,mouse CC chemokine receptor type-2.

82
ELISA

wells were blocked with 1% bovine serum albumin (BSA)containing PBST for 30 minutes at 37C. Plates werethen incubated with culture supernatants, followed byperoxidase-conjugated anti-rat immunoglobulins (1:2000
diluted; Agilent Technologies, Inc., Santa Clara, CA).
Afterward, enzymatic reactions were conducted, using the
ELISA POD substrate TMB kit (Nacalai Tesque, Inc.),
following the measurement of the optical density at 655 nm,
using an iMark microplate reader (Bio-Rad Laboratories,
Inc., Berkeley, CA).

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A mixture of synthesized mCCR2 peptides, including-MEDNNMLPQFIHGILSTSH1
19, 11-IHGILSTSHSLFTRSI
QEL-29, and 21-LFTRSIQELDEGATTPYDY-39+C-terminal
cysteine was immobilized on Nunc Maxisorp 96-well immunoplates (Thermo Fisher Scientific, Inc.) at a concentration of 1 lg/mL for each peptide for 30 minutes at 37C.
After washing with phosphate-buffered saline (PBS) containing 0.05% Tween20 (PBST; Nacalai Tesque, Inc.),

TANAKA ET AL.

FIG. 2. Flow cytometry using anti-mCCR2 mAbs. CHO/mCCR2 cells (A), CHO-K1 cells (B), and L1210 cells (C) weretreated with 0.01–10 mg/mL of C2Mab-6 and EPR20844, followed by treatment with Alexa Fluor 488-conjugated anti-rat
IgG and Alexa Fluor 488-conjugated anti-rabbit IgG, respectively. Black line represents the negative control.

ANTI-MOUSE CCR2 MONOCLONAL ANTIBODY

83

Flow cytometry

CHO-K1 and CHO/mCCR2 cells were harvested after
brief exposure to 1 mM ethylenediaminetetraacetic acid
(Nacalai Tesque, Inc.). CHO-K1, CHO/mCCR2, and L1210
cells were washed with 0.1% BSA in PBS and treated withprimary mAbs for 30 minutes at 4C. Afterward, cells weretreated with Alexa Fluor 488-conjugated anti-rat IgG(1:1000) or Alexa Fluor 488-conjugated anti-rabbit IgG(1:1000) as well, following the collection of fluorescencedata, using a SA3800 Cell Analyzer (Sony Corp., Tokyo,
Japan).

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Determination of the binding affinity by flow cytometry

CHO/mCCR2 and L1210 were suspended in 100 lL serially diluted anti-mCCR2 mAbs, after which Alexa Fluor 488conjugated anti-rat IgG (1:200) or Alexa Fluor 488conjugated anti-rabbit IgG (1:200) were added. Fluorescencedata were subsequently collected, using BD FACSLyric(BD Biosciences), following calculation of the dissociation
constant (KD) by fitting the binding isotherms into the builtin; one-site binding model in GraphPad PRISM 8 (GraphPad
Software, Inc., La Jolla, CA).
Results
Establishment of anti-mCCR2 mAbs

To develop anti-mCCR2 mAbs, we used the mCCR2
peptide immunization method. Hybridoma screening was
conducted using ELISA and flow cytometry (Fig. 1). A rat
was immunized with three kinds of mCCR2 synthetic peptides. Subsequently, hybridomas were seeded into 96-wellplates, after which ELISA was used to extract positive wells
for mCCR2 peptides, followed by the selection of CHO/
mCCR2-reactive and CHO-K1-nonreactive supernatants
using flow cytometry. Afterward, we obtained CHO/mCCR2reactive supernatants in 3 of 1916 wells (0.16%), following
the establishment of C2Mab-6 (rat IgG1, kappa) after cloningby limiting dilution.
Flow cytometry

Flow cytometry was conducted using C2Mab-6 against
CHO/mCCR2, CHO-K1, and L1210. Results showed that
C2Mab-6 dose-dependently recognized CHO/mCCR2(Fig. 2A), but not CHO-K1 (Fig. 2B). Another anti-mCCR2
mAb (clone EPR20844 from Abcam) also dose-dependently
recognized CHO/mCCR2 (Fig. 2A), but not CHO-K1(Fig. 2B). However, both C2Mab-6 and EPR20844 reactedwith CHO/mCCR2, even at 0.01 lg/mL concentration(Fig. 2A). Both C2Mab-6 and EPR20844 also reacted dosedependently with endogenous mCCR2-expressing L1210(Fig. 2C).
Determination of the binding affinity of C2Mab-6

The binding affinity of C2Mab-6 was assessed with
CHO/mCCR2 and L1210, using flow cytometry. Results
showed that the KD of C2Mab-6 for CHO/mCCR2 and L1210
were 1.6 · 10-9 and 1.8 · 10-10 M, respectively (Fig. 3). In
contrast, the KD of the commercially available anti-mCCR2
mAb (clone EPR20844) for CHO/mCCR2 and L1210 were

FIG. 3. The determination of the binding affinity of C2Mab6. CHO/mCCR2 (A) or L1210 (B) cells were suspended in
100 mL serially diluted C2Mab-6 (25–0.0015 mg/mL for
CHO/mCCR2 and 2–0.0001 mg/mL for L1210). Then, cells
were treated with Alexa Fluor 488-conjugated anti-rat IgG.
Fluorescence data were subsequently collected using a BD
FACSLyric, following the calculation of the dissociation
constant (KD) by GraphPad PRISM 8.
7.6 · 10-9 and 3.3 · 10-9 M, respectively (Fig. 4). These results therefore indicate that C2Mab-6 possesses a high affinity
for CHO/mCCR2 and L1210 cells.
Discussion

It has been reported that the development of therapeutic
agents targeting the CCL2-CCR2 axis can be effective fortreating or managing various diseases.(45) Nonetheless, antiGPCR mAbs are difficult to develop, due to problems withfolded-protein structures, small epitope regions, and purification of functional antigens.(45) Therefore, we have trieddeveloping various anti-GPCR mAbs, including anti-mouse
CCR3,(42) anti-mouse CCR8,(43) and anti-human CCR9(44)mAbs, using the CBIS method. However, we have not established the production of anti-GPCR mAbs, using synthetic
peptide immunization. Thus, in this study, we developed anovel anti-mCCR2 mAb (C2Mab-6) through peptide immunization. The KD of C2Mab-6 for CHO/mCCR2 and L1210
were 1.6 · 10-9 and 1.8 · 10-10 M, respectively, indicatingthat C2Mab-6 possesses a high affinity against mCCR2expressing cells. We demonstrated peptide immunization of
the GPCR N-terminal region as the powerful method forestablishing high-affinity mAbs.

84

TANAKA ET AL.

In our previous studies, we changed the isotype of mAbs
into mouse IgG2a to retain the antibody-dependent cellularcytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), which led to high antitumor activities in mouse
xenograft models.(52–54) However, since the subclass of
C2Mab-6 was rat IgG1, it did not possess ADCC and CDC.
Therefore, in further studies, we will convert the subclass of
C2Mab-6 into mouse IgG2a to evaluate whether the depletionof CCR2-expressing immunosuppressive cells can enhance
antitumor activities in mouse syngeneic and xenograftmodels of human cancers.
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: JP21am0401013 (to Yukinari Kato) and JP21am0101078 (to Yukinari Kato).
References

FIG. 4. The determination of the binding affinity of
EPR20844. CHO/mCCR2 (A) or L1210 (B) cells weresuspended in 100 mL serially diluted EPR20844 (10–0.0006 mg/mL). Cells were then treated with Alexa Fluor
488-conjugated anti-rabbit IgG. Fluorescence data were
subsequently collected using a BD FACSLyric, following
the calculation of the dissociation constant (KD) by GraphPad PRISM 8.

In the tumor microenvironment (TME), CCR2 has been
focused on as an immunoregulatory molecule. Tumorsecreted CCL2 recruits CCR2-expressing monocytes into thetumor, where it differentiates into M2 macrophages, known
as tumor-associated macrophages (TAM).(10,13) Previously,
in an esophageal squamous cell carcinoma (ESCC) cohort
study, CCL2 expression and TAM accumulation becamepoor prognostic factors.(46) In breast and pancreatic cancers,
high serum CCL2 levels were associated with malignancy.(47,48) In addition, studies have shown that the CCL2CCR2 axis contributes to the construction of TMEs for
immune evasion by recruiting immunosuppressive cells, such
as CCR2+ myeloid-derived suppressor cells, Th2 cells, andregulatory T cells. Besides, deleting CCR2 suppresses PD-1
expression in cytotoxic T lymphocytes.
CCL2 expression was associated with PD-1-related genesignatures in patients with ESCC.(46) Moreover, as a therapeutic approach, the CCR2 antagonist enhanced the effect ofanti-PD-1 therapy on bladder cancer(49) and glioma.(50) Thus,
it is of interest to evaluate the neutralizing activity of C2Mab6 for clinical applications in immune therapy. Recently,
evaluating the tumor and tumor immune microenvironment
was proposed to be essential for cancer diagnosis.(51)Nevertheless, further evaluation should apply C2Mab-6 toimmunohistochemistry for cancer diagnosis.

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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 9, 2021
Accepted: January 25, 2022