MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 43, Number 4, 2024
ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2024.0004

Short Communication
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Development of a Sensitive Anti-Mouse CCR5 Monoclonal
Antibody for Flow Cytometry

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Hiroyuki Suzuki, Tomohiro Tanaka, Guanjie Li, Tsunenori Ouchida, Mika K. Kaneko, and Yukinari Kato

C-C chemokine receptor 5 (CCR5), a member of the G protein-coupled receptor family, is the most common coreceptor for the human immunodeﬁciency virus type 1. CCR5 is also involved in the pathogenesis of tumors andinﬂammatory diseases. The CCR5 antagonists including monoclonal antibodies (mAbs) have been developed andevaluated in clinical trials. In this study, we developed novel mAbs for mouse CCR5 (mCCR5) using the CellBased Immunization and Screening (CBIS) method. One of the established anti-mCCR5 mAbs, C5Mab-2 (rat
IgG2b, kappa), reacted with mCCR5-overexpressed Chinese hamster ovary-K1 (CHO/mCCR5) and an endogenously mCCR5-expressing cell line (L1210) by ﬂow cytometry. Using ﬂow cytometry, the dissociation constant(KD) of C5Mab-2 for CHO/mCCR5 was determined as 4.3 · 10-8 M. These results indicated that C5Mab-2 isuseful for the detection of mCCR5 in ﬂow cytometry and may be applicable to obtain the proof of concept in preclinical studies.
Keywords: mouse CCR5, monoclonal antibody, Cell-Based Immunization and Screening, CBIS
proinﬂammatory cytokines.6 These functions mediate theprogression of tumors7,8 and inﬂammatory diseases.6
Unique functions of CCR5 in the brain have been reported.
CCR5 is expressed in cortical neurons after stroke.9 In a mousemodel of traumatic brain injury, CCR5 knockdown reduces
learning deﬁcits and improves cognitive function. Furthermore, the FDA-approved CCR5 antagonist maraviroc promotes recovery of function in stroke and traumatic brain
injury.9 These results suggest that CCR5 is a promising
molecular target for the recovery from stroke and traumaticbrain injury.
In recent years, the CCR5 antagonists including monoclonal
antibodies (mAbs) have been developed and evaluated for thetreatment of tumors and inﬂammatory diseases in clinical trials.6,10 We have developed anti-mouse GPCR mAbs against
CCR1 (clone C1Mab-6),11 CCR3 (clones C3Mab-2, C3Mab-3,
and C3Mab-4),12–14 CCR8 (clones C8Mab-1, C8Mab-2, andC8Mab-3),15–17 CXCR1 (clone Cx1Mab-1),18 CXCR3 (clone
Cx3Mab-4),19 and CXCR4 (clone Cx4Mab-1)20 using the CellBased Immunization and Screening (CBIS) method. The CBIS
method includes the immunization of antigen-overexpressed
cells and high-throughput hybridoma screening using ﬂow
cytometry. In this study, a novel anti-mCCR5 mAb wasdeveloped by the CBIS method.

Introduction-C chemokine receptor 5 (CCR5) is a cell membraneprotein of G protein-coupled receptor (GPCR) family,
which is an important regulator for lymphocyte migration
and activation.1 In the 1980s, studies showed that CCR5 is
the most common coreceptor for the human immunodeﬁciency virus type 1 (HIV-1).2 The lack of CCR5 offers protection against HIV infection.3 Therefore, CCR5 antagonists
were developed as anti-HIV drugs.4 A CCR5 antagonist,maraviroc, was developed for acquired immunodeﬁciency
syndrome (AIDS) therapy and is the only U.S. Food andDrug Administration (FDA)-approved drug for marketing.5
CCR5 possesses the typical structure of seven transmembrane GPCR.1 CCR5 is expressed on macrophages, dendritic
cells, and natural killer cells, which are cells of the innate
immune response, as well as on T and B cells of the adaptive
immune response.4 CCR5 serves as a receptor for inﬂammatory b-chemokines including regulated on activation, normal
T-expressed and secreted [RANTES; C-C chemokine ligand
5 (CCL5)], macrophage-inﬂammatory protein-1a (MIP-1a;
CCL3), and MIP-1b (CCL4).4 Upon binding to these ligands,
CCR5 activates downstream signaling, which plays criticalroles in the innate and adaptive immune response through thepromotion of lymphocyte migration and the secretion ofC

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

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ANTI-MOUSE CCR5 MAB
Materials and Methods

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Preparation of cell lines

L1210 (mouse lymphocytic leukemia) was obtained from the
Cell Resource Center for Biomedical Research Institute of Development, Aging and Cancer, Tohoku University (Miyagi, Japan).
LN229, Chinese hamster ovary (CHO)-K1, and P3X63Ag8U.1(P3U1) were obtained from the American Type Culture Collection (Manassas, VA).
The synthesized DNA (Euroﬁns Genomics KK, Tokyo,
Japan) encoding mCCR5 (Accession No.: NM_009917) was
subsequently subcloned into pCAGzeo and pCAGzeo-cPA
vectors (FUJIFILM Wako Pure Chemical Corporation, Osaka,
Japan). The PA tag can be detected by an anti-human podoplanin mAb (clone NZ-1).21 The mCCR5 plasmids were transfected into CHO-K1 and LN229 cells, using a Neontransfection system (Thermo Fisher Scientiﬁc Inc., Waltham,MA). Stable transfectants were established through cell sorting
using a cell sorter (SH800; Sony Corp., Tokyo, Japan), after
which cultivation in a medium containing 0.5 mg/mL of Zeocin (InvivoGen, San Diego, CA) was performed.
L1210, CHO-K1, mCCR5-overexpressed CHO-K1 (CHO/
mCCR5), and P3U1 were cultured in a Roswell Park Memorial
Institute (RPMI)-1640 medium (Nacalai Tesque, Inc., Kyoto,
Japan), with 10% heat-inactivated fetal bovine serum (FBS;
Thermo Fisher Scientiﬁc Inc.), 100 units/mL of penicillin,
100 lg/mL of streptomycin, and 0.25 lg/mL of amphotericin
B (Nacalai Tesque, Inc.). LN229 and mCCR5-overexpressed
LN229 (LN229/mCCR5 or LN229/mCCR5-PA) were cultured
in Dulbecco’s modiﬁed Eagle medium (DMEM; Nacalai Tesque, Inc.), supplemented with 10% FBS, 100 units/mL of penicillin, 100 lg/mL streptomycin, and 0.25 lg/mL amphotericin
B. All cells were grown in a humidiﬁed incubator at 37C, in
an atmosphere of 5% CO2 and 95% air.
Production of hybridomas

A 5-week-old Sprague–Dawley rat was purchased from
CLEA Japan (Tokyo, Japan). The animal was housed under
speciﬁc pathogen-free conditions. All animal experiments
were performed according to the relevant guidelines and regulations to minimize animal suffering and distress in the laboratory. The Animal Care and Use Committee of Tohoku
University (Permit number: 2022MdA-001) approved animal
experiments. The rat was monitored daily for health during
the complete 4-week duration of the experiment. A reductionof >25% of the total body weight was deﬁned as a humane
endpoint. During the sacriﬁce, the rat was euthanized throughcervical dislocation, after which death was veriﬁed throughrespiratory and cardiac arrest.
To develop mAbs against mCCR5, we intraperitoneallyimmunized one rat with CHO/mCCR5 (1 · 109 cells) plus
Alhydrogel adjuvant 2% (InvivoGen). The procedure included
three additional injections every week (1 · 109 cells/rat),
which were followed by a ﬁnal booster intraperitoneal injection (1 · 109 cells/rat), 2 days before harvesting spleen cells.
The harvested spleen cells were subsequently fused with
P3U1 cells, using PEG1500 (Roche Diagnostics, Indianapolis,
IN), after which hybridomas were grown in the RPMI-1640
medium with 10% FBS, 5% Briclone (NICB, Dublin, Ireland), 100 units/mL of penicillin, 100 lg/mL of streptomycin,

97

and 0.25 lg/mL of amphotericin B. For the hybridoma selection, hypoxanthine, aminopterin, and thymidine (HAT;
Thermo Fisher Scientiﬁc Inc.) were added into the medium.
The supernatants were subsequently screened using ﬂow
cytometry using LN229/mCCR5-PA and LN229.
Antibodies

An anti-mCCR5 mAb (clone HM-CCR5, Armenian hamster
IgG) conjugated with Alexa Fluor 488 was purchased from
BioLegend (San Diego, CA). Alexa Fluor 488-conjugated antirat IgG was purchased from Cell Signaling Technology, Inc.
(Danvers, MA).
The cultured supernatant of C5Mab-2 hybridomas wasapplied to 1 mL of Ab-Capcher (ProteNova, Kagawa, Japan).
After washing with phosphate-buffered saline (PBS), the antibodies were eluted with an IgG elution buffer (Thermo Fisher
Scientiﬁc Inc.). Finally, the eluates were concentrated, andthe elution buffer was replaced with PBS using Amicon Ultra(Merck KGaA, Darmstadt, Germany).
Flow cytometric analysis

Cells were harvested after a brief exposure to 0.25% trypsin and 1 mM ethylenediaminetetraacetic acid (EDTA,
Nacalai Tesque, Inc.). The cells were subsequently washedwith 0.1% bovine serum albumin (BSA) in PBS (blocking
buffer) and treated with 0.01, 0.1, 1, and 10 lg/mL of primary mAbs for 30 minutes at 4C. The C5Mab-2-treatedcells were further incubated with Alexa Fluor 488-conjugatedanti-rat IgG (1:2,000). The ﬂuorescence data were collectedusing the SA3800 Cell Analyzer (Sony Corp.).
Determination of dissociation constant (KD) by flow cytometry

CHO/mCCR5 cells were suspended in serially diluted
C5Mab-2 for 30 minutes at 4C. The cells were treated with
Alexa Fluor 488-conjugated anti-rat IgG (1:200). CHO/
mCCR5 cells were suspended in serially diluted HM-CCR5
conjugated with Alexa Fluor 488. The ﬂuorescence data
were collected, using the SA3800 Cell Analyzer. The KD
was subsequently calculated by ﬁtting saturation binding
curves to the built-in, one-site binding models in GraphPad
PRISM 6 (GraphPad Software, Inc., La Jolla, CA).
Results
Development of anti-mCCR5 mAbs using the CBIS method

To develop anti-mCCR5 mAb, one rat was immunized with
CHO/mCCR5 cells (Fig. 1A). The spleen was then excisedfrom the rat, and splenocytes were fused with myeloma P3U1
cells (Fig. 1B). The developed hybridomas were subsequently
seeded into 96-well plates and cultivated for 6 days. The positive wells were screened by the selection of LN229/mCCR5PA-reactive and LN229-nonreactive supernatants using ﬂow
cytometry (Fig. 1C). After the limiting dilution and several
additional screenings, an anti-mCCR5 mAb, C5Mab-2 (rat
IgG2b, kappa), was ﬁnally established (Fig. 1D).
Flow cytometric analysis using C5Mab-2

We conducted ﬂow cytometry using two anti-mCCR5
mAbs: C5Mab-2 and HM-CCR5 (from BioLegend) against

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SUZUKI ET AL.

and 1.6 · 10-7 M, respectively (Fig. 3). These results indicatethat C5Mab-2 possesses the superior afﬁnity to CHO/mCCR5
compared with HM-CCR5.

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Discussion

In this study, we developed a novel anti-mCCR5 mAb,
C5Mab-2 using the CBIS method and showed the usefulness
for ﬂow cytometry to detect the exogenous and endogenous
mCCR5 (Fig. 2). The understanding of the structural-based
CCR activation is important for the development of therapeutic
agents. Among the CCR family members, CCR5 has been
structurally solved in both inactive and active states.22,23
Recently, 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.24 We previously determined the
epitope of C3Mab-3 and C3Mab-4 (anti-mouse CCR3 mAbs)12
and C8Mab-2 (an anti-mouse CCR8 mAb)25 using the chimeric proteins and the alanine scanning in ﬂow cytometry. We
should determine the epitope and antagonistic activity of
C5Mab-2 in future studies.
CCR5 is the principal HIV coreceptor2 and is involved in the
disease progression in tumors7 and inﬂammatory diseases.26
CCR5 has been implicated in the inﬂammatory complications

FIG. 1. The production of an anti-mCCR5 mAb, C5Mab-2.
(A) CHO/mCCR5 cells were immunized into a Sprague–
Dawley rat. (B) The spleen cells were fused with P3U1 cells.
(C) To select anti-mCCR5 mAb-producing hybridomas, thesupernatants were screened by ﬂow cytometry using LN229
and LN229/mCCR5-PA cells. (D) After limiting dilution, ananti-mCCR5 mAb, C5Mab-2 was ﬁnally established.
CHO/mCCR5, CHO-K1, and L1210 cells. C5Mab-2 recognized CHO/mCCR5 cells dose-dependently at 10, 1, 0.1, and0.01 lg/mL (Fig. 2A). In contrast, HM-CCR5 needed >0.1 lg/
mL for the detection of CHO/mCCR5 clearly (Fig. 2A). Parental CHO-K1 cells were not recognized even at 10 lg/mL of all
mAbs (Fig. 2B). The superior reactivity of C5Mab-2 comparedwith HM-CCR5 was also observed in LN229/mCCR5 cells(Supplementary Fig. S1).
We next investigated the reactivity of C5Mab-2 against an
endogenously mCCR5-expressing cell line, L1210. C5Mab-2
reacted with L1210 at >0.1 lg/mL (Fig. 2C). In contrast, HMCCR5 could react with L1210 at >1 lg/mL, but the reaction
was weak at 0.1 lg/mL. These results suggested that C5Mab-2
speciﬁcally recognizes mCCR5 and is also useful for detecting
endogenous mCCR5 by ﬂow cytometry.
Determination of the binding affinity of C5Mab-2 using
flow cytometry

To determine the KD of anti-mCCR5 mAbs against CHO/
mCCR5, we conducted ﬂow cytometry, and the geometricmean of the ﬂuorescence intensity was plotted versus the concentration of C5Mab-2. The KD values of C5Mab-2 and HMCCR5 for CHO/mCCR5 were determined as 4.3 · 10-8 M

FIG. 2. Flow cytometry of mCCR5-expressed cells using
C5Mab-2 and HM-CCR5. CHO/mCCR5 (A), CHO-K1 (B),and L1210 (C) cells were treated with 0.01–10 lg/mL of
C5Mab-2 or HM-CCR5 conjugated with Alexa Fluor 488(black line). The C5Mab-2-treated cells were further incubatedwith anti-rat IgG conjugated with Alexa Fluor 488. The ﬁlled
gray represents the negative control (blocking buffer).

ANTI-MOUSE CCR5 MAB

99

therapies have been applied to neural disorders such as Alzheimer’s disease.31 CCR5 plays an important role in modulating Ab1–42-induced learning and memory deﬁcits in a mousemodel, which suggests that CCR5 antagonists are a potential
treatment to improve cognitive deﬁcits associated with Alzheimer’s disease.32 Therefore, C5Mab-2 could contribute to
the treatment of memory deﬁcits in mouse preclinical studies.
Authors’ Contribution

H.S., T.T., G.L., and T.O. performed the experiments.
M.K.K. and Y.K. designed the experiments. H.S. and M.K.K.
analyzed the data. H.S. wrote the article. All authors have readand agreed to the published version of the article.
Author Disclosure Statement

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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 (to Y.K.),
JP23bm1123027 (to Y.K.), and JP23ck0106730 (to Y.K.) andby the Japan Society for the Promotion of Science (JSPS)
Grants-in-Aid for Scientiﬁc Research (KAKENHI) grant numbers 22K06995 (to H.S.), 21K20789 (to T.T.), 23K19494 (toT.O.), 21K07168 (to M.K.K.), and 22K07224 (to Y.K.).
Supplementary Material

FIG. 3. The binding afﬁnity of C5Mab-2. (A) CHO/
mCCR5 cells were suspended in serially diluted C5Mab-2.
The cells were treated with anti-rat IgG conjugated with
Alexa Fluor 488. (B) CHO/mCCR5 cells were suspended
in serially diluted HM-CCR5 conjugated with Alexa Fluor
488. The ﬂuorescence data were subsequently collectedusing the SA3800 Cell Analyzer, followed by the calculation of the KD using GraphPad PRISM 6.
of coronavirus disease 2019 (COVID-19).27 Therefore, CCR5
antagonists have been tested in various clinical researches.28
Furthermore, an anti-CCR5 mAb (PRO 140) has been developed and investigated for HIV, breast cancer, and COVID-19
in phase II or III trials.6,10
Using mouse models, CCR5 has been implicated to have anovel function in neural systems. CCR5 was ﬁrst identiﬁed to
be involved in plasticity and memory in a reverse genetic
screening. In total, 148 transgenic and knockout mutant mousestrains were screened for contextual memory phenotypes. The
CCR5 knockout resulted in memory enhancements for contextual conditioning when tested 24 hours after training.29 In addition, decreasing the function of CCR5 enhances the long-term
potentiation and hippocampus-dependent memory, whereas
neuronal CCR5 overexpression caused memory deﬁcits inmice.29 These results indicated that CCR5 is a suppressor forcortical plasticity and hippocampal learning and memory. Furthermore, an age-related increase in CCL5-CCR5 expression inneurons leads to impairments in memory linking in middleaged mice that can be reversed by the FDA-approved CCR5
inhibitor, maraviroc.30 These results suggested that the CCL5CCR5 axis is a potential target for memory deﬁcits. MAb

Supplementary Figure S1

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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: February 9, 2024
Accepted: February 24, 2024