MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 43, Number 4, 2024
ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2024.0009
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Development of Sensitive Anti-Mouse CCR5 Monoclonal
Antibodies Using the N-Terminal Peptide Immunization

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Rena Ubukata,* Hiroyuki Suzuki,* Tomohiro Tanaka, Guanjie Li, Mika K. Kaneko, and Yukinari Kato

One of the G protein-coupled receptors, C-C chemokine receptor 5 (CCR5), is an important regulator for the activation of T and B lymphocytes, dendritic cells, natural killer cells, and macrophages. Upon binding to its ligands,
CCR5 activates downstream signaling, which is an important regulator in the innate and adaptive immune response
through the promotion of lymphocyte migration and the secretion of proinﬂammatory cytokines. Anti-CCR5
monoclonal antibodies (mAbs) have been developed and evaluated in clinical trials for tumors and inﬂammatory
diseases. In this study, we developed novel mAbs for mouse CCR5 (mCCR5) using the N-terminal peptide immunization. Among the established anti-mCCR5 mAbs, C5Mab-4 (rat IgG2a, kappa) and C5Mab-8 (rat IgG1, kappa),
recognized mCCR5-overexpressing Chinese hamster ovary-K1 (CHO/mCCR5) and an endogenously mCCR5expressing cell line (L1210) by ﬂow cytometry. The dissociation constant (KD) values of C5Mab-4 and C5Mab-8
for CHO/mCCR5 were determined as 3.5 · 10-8 M and 7.3 · 10-9 M, respectively. Furthermore, both C5Mab-4
and C5Mab-8 could detect mCCR5 by western blotting. These results indicated that C5Mab-4 and C5Mab-8 are
useful for detecting mCCR5 by ﬂow cytometry and western blotting and provide a possibility to obtain the proof
of concept in preclinical studies.
Keywords: mouse CCR5, monoclonal antibody, peptide immunization, ﬂow cytometry, western blotting
Introduction-C chemokine receptor 5 (CCR5) is a G proteincoupled receptor (GPCR) that plays an important role
in regulating lymphocyte migration and activation.1 CCR5 isexpressed on dendritic cells, macrophages, and natural killer
cells, which are cells of the innate immune response, as wellas on T and B cells of the adaptive immune response.2 CCR5
serves as a receptor for macrophage-inﬂammatory protein-1a(MIP-1a); also known as C-C chemokine ligand 5 (CCL5),
MIP-1b; also known as C-C chemokine ligand 4 (CCL4),and inﬂammatory b-chemokines including regulated on activation, normal T-expressed and secreted (RANTES); also
known as CCL5.2 CCR5 binds to these ligands and activatesdownstream signaling, which mediates the innate and adaptive immune response through the promotion of lymphocyte
migration and the secretion of proinﬂammatory cytokines.3
These functions mediate the progression of tumors4,5 andinﬂammatory diseases.3
CCR5 is identiﬁed as the principal coreceptor for humanimmunodeﬁciency virus type 1 (HIV-1),6 and is involved inCdisease progression in tumors4 and inﬂammatory diseases.7
CCR5 has also been implicated in the inﬂammatory complications of coronavirus disease 2019 (COVID-19).8 Therefore, a CCR5 antagonist, maraviroc, was ﬁrst developed
for acquired immunodeﬁciency syndrome (AIDS) therapy
and is the only U.S. Food and Drug Administration (FDA)approved drug for marketing.9 Furthermore, CCR5 antagonists, including monoclonal antibodies (mAbs), have beentested in various clinical trials.10 An anti-CCR5 mAb (PRO140) has been developed and investigated for HIV, breast
cancer, and COVID-19 in phase II or III trials.3,11
We have developed anti-mouse chemokine receptor mAbs
against CCR1 (clone C1Mab-6),12 CCR3 (clones C3Mab-2,
C3Mab-3, and C3Mab-4),13–15 CCR5 (clone C5Mab-2),16 CCR8
(clones C8Mab-1, C8Mab-2, and C8Mab-3),17–19 CXCR1 (clone
Cx1Mab-1),20 CXCR3 (clone Cx3Mab-4),21 and CXCR4 (clone
Cx4Mab-1)22 using the Cell-Based Immunization and Screening(CBIS) method. The CBIS method includes the immunizationof antigen-overexpressing cells and high-throughput hybridoma
screening using ﬂow cytometry. Furthermore, we establishedanti-mouse chemokine receptor mAbs against CCR2 (clone

Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, Sendai, Japan.
*These authors contributed equally to this work.

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ANTI-MOUSE CCR5 MABS

C2Mab-6),23 CCR3 (clones C3Mab-6 and C3Mab-7),24 CCR4
(clone C4Mab-1),25 CCR9 (clone C9Mab-24),26 and CXCR6(clone Cx6Mab-1)27 using the N-terminal peptide immunization.
In this study, novel anti-mCCR5 mAbs were developedby the N-terminal peptide immunization.
Materials and Methods

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Cell lines

The L1210 cell line (mouse lymphocytic leukemia) was
obtained from the Cell Resource Center for Biomedical
Research, Institute of Development, Aging, and Cancer
Tohoku University (Miyagi, Japan). The LN229, Chinese
hamster ovary (CHO)-K1, and P3X63Ag8U.1 (P3U1) celllines 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).28 The mCCR5 plasmids
were transfected into CHO-K1 and LN229, using a Neontransfection system (Thermo Fisher Scientiﬁc Inc., Waltham,MA). Stable transfectants were established by cell sorting
using a cell sorter (SH800; Sony Corp., Tokyo, Japan) andwere cultivated in a medium containing 0.5 mg/mL of Zeocin (InvivoGen, San Diego, CA).
GeneArt CRISPR nuclease vectors with OFP plasmid,
which target mCCR5 (TrueGuide Synthetic sgRNA,CRISPR59005_SGM), were purchased from Thermo Fisher
Scientiﬁc Inc. The knockout plasmid was transfected into
L1210 cells using a Neon transfection system. mCCR5
knockout L1210 (BINDS-57) was established by staining
with anti-mCCR5 mAb (clone HM-CCR5, BioLegend, San
Diego, CA) and sorted using a cell sorter (SH800).
CHO-K1, mCCR5-overexpressing CHO-K1 (CHO/mCCR5),mCCR5-PA-overexpressing CHO-K1 (CHO/mCCR5-PA),
L1210, BINDS-57, and P3U1 were cultured in a (RPMI)-1640
medium (Nacalai Tesque, Inc., Kyoto, Japan) with 10% heatinactivated fetal bovine serum (FBS; Thermo Fisher ScientiﬁcInc.), 100 units/mL of penicillin, 100 lg/mL of streptomycin, and0.25 lg/mL of amphotericin B (Nacalai Tesque, Inc.). LN229
and mCCR5-overexpressing LN229 (LN229/mCCR5) were cultured in Dulbecco’s modiﬁed Eagle’s 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.
PeptidesEuroﬁns Genomics KK (Tokyo, Japan) synthesized a partial sequence of the N-terminal extracellular region ofmCCR5 (1-MDFQGSVPTYSYDIDYGMS-19) with a C-terminal cysteine. Subsequently, keyhole limpet hemocyanin(KLH) was conjugated to the C-terminus of the peptide.
Production of hybridomas

A ﬁve-week-old Sprague–Dawley rat was purchased from
CLEA Japan (Tokyo, Japan). The animal was housed under113

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 theanimal experiments. The rat was monitored daily for healthduring the complete four-week duration of the experiment.
A reduction of more than 25% of the total body weight wasdeﬁned as a humane endpoint. During the sacriﬁce, the rat
was euthanized through cervical dislocation, after which
death was veriﬁed through respiratory and cardiac arrest.
To develop mAbs against mCCR5, we intraperitoneallyimmunized one rat with 100 mg of the KLH-conjugated mCCR5
peptide (mCCR5-KLH) with Alhydrogel adjuvant 2% (InvivoGen). The procedure included three additional immunizations
every week (100 mg/rat), followed by a ﬁnal booster intraperitoneal injection (100 mg/rat) two days before the harvest of spleencells. The harvested spleen cells were subsequently fused with
P3U1 cells using PEG1500 (Roche Diagnostics, Indianapolis,
IN), after which hybridomas were grown in RPMI-1640
medium with 10% FBS, 5% Briclone (NICB, Dublin, Ireland),
100 units/mL of penicillin, 100 lg/mL of streptomycin, and0.25 lg/mL of amphotericin B. For the hybridoma selection,
hypoxanthine, aminopterin, and thymidine (HAT; Thermo
Fisher Scientiﬁc Inc.) were added to the medium. The supernatants were subsequently screened using enzyme-linked immunosorbent assay (ELISA) with the mCCR5 peptide, followed by
ﬂow cytometry using CHO/mCCR5 and CHO-K1.
Antibodies

We previously developed an anti-mCCR5 mAb (C5Mab-2)
using the immunization of CHO/mCCR5 cells.16 Alexa Fluor
488-conjugated anti-rat IgG and peroxidase-conjugated antirat IgG (A9542) were purchased from Cell Signaling Technology, Inc. (Danvers, MA) and Sigma-Aldrich Corp. (St.
Louis, MO), respectively.
The cultured supernatants of C5Mab-4 and C5Mab-8producing hybridomas were applied to 1 mL of Protein G
Sepharose 4 Fast Flow (GE Healthcare, Chicago, IL) and
Ab-Capcher (ProteNova, Kagawa, Japan), respectively.
After washing with phosphate-buffered saline (PBS), themAbs were eluted with an IgG elution buffer (Thermo
Fisher Scientiﬁc Inc.). Finally, the eluates were concentrated, and the elution buffer was replaced with PBS using
Amicon Ultra (Merck KGaA, Darmstadt, Germany).
ELISA

The synthesized mCCR5 peptide (MDFQGSVPTYSYDIDYGMSC) was immobilized on Nunc Maxisorp 96-wellimmunoplates (Thermo Fisher Scientiﬁc Inc.) at a concentration of 1 mg/mL for 30 minutes at 37C. After washing with
PBS containing 0.05% Tween 20 (PBST; Nacalai Tesque,Inc.), the wells were blocked with 1% bovine serum albumin(BSA)-containing PBST for 30 minutes at 37C. Plates werethen incubated with supernatants of hybridomas, followedby peroxidase-conjugated anti-rat IgG (1:20,000 diluted).
Next, enzymatic reactions were conducted using the ELISAPOD Substrate TMB Kit (Nacalai Tesque, Inc.), followed bymeasurement of the optical density at 655 nm using an iMark

114

UBUKATA ET AL.

microplate reader (Bio-Rad Laboratories, Inc., Berkeley,
CA).

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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 washed with 0.1%
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. For the
peptide inhibition assay, C5Mab-4 and C5Mab-8 (1 lg/mL)
were preincubated with 20 lg/mL of mCCR5 peptide ordimethyl sulfoxide (DMSO) for 15 minutes at 4C, and thenincubated with the cells for 30 minutes at 4C. The cells weretreated with Alexa Fluor 488-conjugated anti-rat IgG (1:2000).
The ﬂuorescence data were collected using the SA3800 Cell
Analyzer (Sony Corp.). An anti-isocitrate dehydrogenase 1(IDH1) mAb (RcMab-1)29,30 or an anti-mouse TERT mAb(TmMab-1, http://www.med-tohoku-antibody.com/topics/001_
paper_antibody_PDIS.htm) were used as isotype control rat
IgG2a and rat IgG1, respectively.
Determination of dissociation constant (KD) by flow cytometry

CHO/mCCR5 cells were suspended in serially-diluted primary mAbs for 30 minutes at 4C. The cells were treatedwith Alexa Fluor 488-conjugated anti-rat IgG (1:200). 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).
Western blotting

Cell lysates from CHO-K1 and CHO/mCCR5-PA wereboiled in sodium dodecyl sulfate (SDS) sample buffer (Nacalai Tesque, Inc.). Proteins (10 mg) were separated on 5%–20%
polyacrylamide gels (FUJIFILM Wako Pure Chemical Corporation) and transferred onto polyvinylidene diﬂuoride membranes (Merck KGaA). After blocking with 4% skim milk
(Nacalai Tesque, Inc.) in PBST, membranes were incubatedwith 1 lg/mL of C5Mab-4, C5Mab-8, RcMab-1, or 0.1 lg/mL
of NZ-1. Membranes were then incubated with peroxidaseconjugated anti-rat IgG (diluted 1:10,000). Finally, protein
bands were detected with ImmunoStar LD (FUJIFILM Wako
Pure Chemical Corporation) using a Sayaca-Imager (DRC Co.
Ltd., Tokyo, Japan).
Results
Development of anti-mCCR5 mAbs using N-terminal
peptide immunization

To develop anti-mCCR5 mAbs, one rat was immunizedwith mCCR5-KLH (Fig. 1A). Spleen was then excised fromthe rat, after which splenocytes were fused with myeloma
P3U1 cells. Developed hybridomas were subsequently seeded
into ﬁfteen 96-well plates and cultivated for six days. Then,positive wells for the naked mCCR5 peptide were selectedusing ELISA, followed by the selection of CHO/mCCR5reactive and CHO-K1-nonreactive supernatants using ﬂow
cytometry (Fig. 1B). The ELISA screening identiﬁed 68 out

FIG. 1. The production of anti-mCCR5 mAbs. (A) CCR5
N-terminal peptide conjugated with KLH (mCCR5-KLH)
was immunized into a Sprague–Dawley rat. The spleen cells
were fused with P3U1 cells. (B) To select anti-mCCR5
mAb-producing hybridomas, the supernatants were screenedby ELISA and ﬂow cytometry using CHO-K1 and CHO/
mCCR5 cells. (C) After limiting dilution, anti-mCCR5
mAbs, C5Mab-4 and C5Mab-8 were ﬁnally established.
ELISA, enzyme-linked immunosorbent assay. mAb, monoclonal antibody.
of 1438 wells (4.7%), which strongly reacted with the naked
mCCR5 peptide. The ﬂow cytometric screening identiﬁed 18
out of the 68 wells (26.5%), which exhibited strong signals to
CHO/mCCR5 cells, but not to CHO-K1 cells. After the limiting dilution and several additional screenings, C5Mab-4 (rat
IgG2a, kappa) and C5Mab-8 (rat IgG1, kappa) were ﬁnallyestablished (Fig. 1C).
Flow cytometric analysis using C5Mab-4 and C5Mab-8

We conducted ﬂow cytometry using three anti-mCCR5
mAbs: C5Mab-2, C5Mab-4, and C5Mab-8 against CHO/
mCCR5 and CHO-K1 cells. C5Mab-2 was previously established by the CBIS method.16 Both C5Mab-4 and C5Mab-8
recognized CHO/mCCR5 cells dose-dependently at 10, 1,
0.1, and 0.01 lg/mL (Fig. 2A). C5Mab-8 exhibited a superior
reactivity against CHO/mCCR5 cells at 0.1 and 0.01 lg/mL
compared to C5Mab-2 and C5Mab-4 (Fig. 2A). Parental CHOK1 cells were not recognized by any mAbs even at 10 lg/mL(Fig. 2B). The superior reactivity of C5Mab-8 was also observedin LN229/mCCR5 cells (Supplementary Fig. S1).
A commercially available anti-mCCR5 mAb (clone
HM-CCR5) reacted with mCCR5-expressing cell line,
L1210 cells (Fig. 3A). Furthermore, mCCR5-knockout
L1210 (BINDS-57) cells were not recognized by HMCCR5, indicating that L1210 cells express mCCR5(Fig. 3A). We next investigated the reactivity of C5Mab-4
and C5Mab-8 against an endogenously mCCR5-expressingDownloaded by Yukinari Kato from www.liebertpub.com at 08/13/24. For personal use only.

ANTI-MOUSE CCR5 MABS

FIG. 2. Flow cytometry of mCCR5-overexpressed CHOK1 cells using anti-mCCR5 mAbs. CHO/mCCR5 (A) andCHO-K1 (B) cells were treated with 0.01–10 mg/mL of
C5Mab-4, C5Mab-8, or C5Mab-2 (black line). The mAbstreated cells were further incubated with anti-rat IgG conjugated with Alexa Fluor 488. The ﬁlled gray represents
the negative control (blocking buffer). mAb, monoclonal
antibody.
cell line, L1210. C5Mab-4 reacted with L1210 at more than
1 lg/mL (Fig. 3B). C 5Mab-8 and C5 Mab-2 could reactwith L1210 at more than 0.1 lg/mL. We did not detect
the reactivity of isotype control rat IgG 2a and rat IgG 1
against CHO/mCCR5 and L1210 cells (Supplementary
Fig. S2).
We next performed a peptide-blocking assay. As shownin Figure 4, C5Mab-4 and C5Mab-8 reacted with the CHO/
mCCR5 and L1210 cells. These reactions were completely
neutralized by mCCR5 peptide (Fig. 4). These results indicated that the reaction by C5Mab-4 and C5Mab-8 wasmediated by the recognition of mCCR5.

115

FIG. 3. Flow cytometry of endogenous mCCR5-expressing
cells using anti-mCCR5 mAbs. (A) L1210 cells and BINDS57 cells were treated with 1 mg/mL of HM-CCR5 (blackline). The ﬁlled gray represents the negative control (blocking
buffer). (B) L1210 cells were treated with 0.01–10 mg/mL of
C5Mab-4, C5Mab-8, or C5Mab-2 (black line). The mAbstreated cells were further incubated with anti-rat IgG
conjugated with Alexa Fluor 488. The ﬁlled gray represents the negative control (blocking buffer). mAb,
monoclonal antibody.
Detection of mCCR5 using C5Mab-4 and C5Mab-8 byWestern blotting

Western blotting was performed to assess the reactivityof C5Mab-4 and C5Mab-8. Lysates of CHO-K1 and CHO/
mCCR5-PA cells were probed. C5Mab-4 (Fig. 6A) andC5Mab-8 (Fig. 6B) detected mCCR5 as a *40-kDa band. In
contrast, C5Mab-4 and C5Mab-8 did not show any bands fromthe lysates of CHO-K1 cells. C5Mab-4 exhibited a superior
reactivity against CHO/mCCR5 cell lysate compared to
C5Mab-8 (Fig. 6A and B, the same exposure time). In contrast,
C5Mab-2 could not detect the CHO/mCCR5 cell lysate(Fig. 6C). A similar molecular weight band was also
detected by NZ-1 (Fig. 6D). An anti-IDH1 mAb, RcMab1, was used as an internal control (Fig. 6E). These results
suggest that C5Mab-4 and C5Mab-8 are useful for detecting mCCR5 by western blotting.
Discussion

Determination of the binding affinity of C5Mab-4 and C5Mab-8
using flow cytometry

To determine the KD values of C5Mab-4 and C5Mab-8
against CHO/mCCR5, we conducted ﬂow cytometry. The
geometric mean of the ﬂuorescence intensity was plotted versus the concentration. The KD values of C5Mab-4 andC5Mab-8 for CHO/mCCR5 were determined to be 3.5 · 10-8
M and 7.3 · 10-9 M, respectively (Fig. 5). These results indicate that C5Mab-4 and C5Mab-8 possess superior afﬁnity to
CHO/mCCR5 compared to C5Mab-2 (4.3 · 10-8).16

In this study, we developed novel anti-mCCR5 mAbs
(C5Mab-4 and C5Mab-8) using the N-terminal peptide immunization and showed their usefulness for ﬂow cytometry(Figs. 2 and 3) and western blotting (Fig. 6) to detect
mCCR5. C5Mab-4 and C5Mab-8 possess superior afﬁnities(3.5 · 10-8 M and 7.3 · 10-9 M, respectively, Fig. 5) compared to that of a commercially available anti-mCCR5 mAb(clone HM-CCR5, 1.6 · 10-7 M).16 As described in the
results section, not all ELISA-positive supernatants recognized CHO/mCCR5 in ﬂow cytometry. One possibility is a
modiﬁcation of the CCR5 N-terminal region. The tyrosine

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116

FIG. 4. A peptide-blocking assay using C5Mab-4 andC5Mab-8 with mCCR5 peptide. C5Mab-4 or C5Mab-8(1 mg/mL) plus mCCR5 peptide (20 lg/mL) or control(2% DMSO in blocking buffer) were reacted with CHO/
mCCR5 (A) or L1210 (B) for 30 minutes at 4C, followed
by treatment with Alexa Fluor 488-conjugated anti-rat
IgG. The ﬁlled gray represents the negative control(blocking buffer). DMSO, dimethyl sulfoxide.
sulfation of the human CCR5 N-terminal region enhances thebinding afﬁnity of ligands and HIV envelope glycoprotein.31,32 The human CCR5 N-terminal region possesses four
potentially sulfated tyrosine residues, three of which are conserved in mCCR5 and included in the immunogen.33 Therefore, the epitope of C5Mab-4 and C5Mab-8 is thought toexclude the potentially sulfated tyrosine residues. We previously determined the epitope of Cx6Mab-1 (an anti-mouse
CXCR6 mAb) using 1· and 2· alanine scanning methods.34
We should determine the epitope C5Mab-4 and C5Mab-8 infuture studies.
An understanding of the structure-based activation of
CCR is important for the development of therapeutic agents.
Among the CCR family members, human CCR5 has been
structurally solved in both inactive and active states.35,36
Based on the nuclear magnetic resonance structure of CCL5
in complex with an N-terminal fragment (residues 1 to 27)
of human CCR5 sulfated at residues Y10 and Y14, the sulfation at Y10 and Y14, which are conserved in mCCR5, was

UBUKATA ET AL.

FIG. 5. The binding afﬁnity of anti-mCCR5 mAbs.
CHO/mCCR5 cells were suspended in serially diluted
C5Mab-4 (A) or C5Mab-8 (B). The cells were treated with
anti-rat IgG conjugated with Alexa Fluor 488. The ﬂuorescence data were subsequently collected using theSA3800 Cell Analyzer, followed by the calculation of the
KD using GraphPad PRISM 6. mAb, monoclonal antibody.
also revealed to be important for chemokine afﬁnity.37 Furthermore, the structures of CCR8 in complex with either the
antagonistic mAb or the endogenous ligand CCL1 have been
determined, which provide insight into the speciﬁc activation
mechanism by CCL1 and inhibition by mAb.38 We should
determine the antagonistic activity of anti-mCCR5 mAbs,
which could contribute to the understanding of the inactive
state of mCCR5.
Unique functions of CCR5 in the neural systems have beenreported using mouse models.39 By screening 148 transgenic
and knockout mutant mouse strains for contextual memory phenotypes, CCR5 was ﬁrst identiﬁed as being involved in plasticity
and memory. The CCR5-knockout mice exhibited a phenotype
of memory enhancements for contextual conditioning after training.39 Furthermore, decreased CCR5 function enhances longterm potentiation and hippocampus-dependent memory, while
overexpression of CCR5 in neurons caused memory deﬁcits inmice.39 These results indicate that CCR5 is a suppressor forcortical plasticity and hippocampus-dependent memory.
In addition, an age-related increase in CCL5–CCR5
expression in neurons leads to impairments in memory linking in middle-aged mice that can be reversed by the FDAapproved CCR5 inhibitor, maraviroc.40 These results suggest

ANTI-MOUSE CCR5 MABS

117
Supplementary Material

Supplementary Figure S1
Supplementary Figure S2

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References

FIG. 6. Western blotting using anti-mCCR5 mAbs. The
lysates of CHO-K1 and CHO/mCCR5-PA cells were electrophoresed and transferred onto polyvinylidene ﬂuoride membranes. The membranes were incubated with 1 mg/mL of
C5Mab-4 (A), 1 mg/mL of C5Mab-8 (B), 1 mg/mL of C5Mab-2(C), 0.1 lg/mL of NZ-1 (an anti-PA tag mAb) (D), or
1 mg/mL of RcMab-1 (an anti-IDH1 mAb) (E). The
membranes were subsequently incubated with peroxidase-conjugated anti-rat IgG. Note that the exposure
time of C5Mab-4 and C5Mab-8 blotting was the same.
IDH1, isocitrate dehydrogenase 1; mAb, monoclonal
antibody.
that the CCL5–CCR5 axis is a potential target for addressing
memory deﬁcits. MAb therapies have been applied to neuraldisorders such as Alzheimer’s disease. The accumulation ofsoluble and insoluble aggregated amyloid-beta (Ab) isthought to initiate or progress pathological processes.41 CCR5
plays an important role in modulating Ab1–42-induced learning and memory deﬁcits in a mouse model, which suggeststhat CCR5 antagonists are a potential treatment to improve
cognitive deﬁcits associated with Alzheimer’s disease.42
Therefore, C5Mab-4 and C5Mab-8 could contribute to mousepreclinical studies aimed at developing treatments for memory deﬁcits.
Authors’ Contributions

R.U., H.S., T.T., and G.L. 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 read and agreed to the published version of the article.
Author Disclosure Statement

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: JP24ama121008 (to Y.K.), JP23am0401013(to Y.K.), JP24bm1123027 (to Y.K.), and JP24ck0106730(to Y.K.), and by the Japan Society for the Promotion of
Science (JSPS) Grants-in-Aid for Scientiﬁc Research(KAKENHI) grant nos. 22K06995 (to H.S.), 21K20789(to T.T.), 21K07168 (to M.K.K.), and 22K07224 (toY.K.).

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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: March 31, 2024
Accepted: May 3, 2024