MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 43, Number 3, 2024
ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2023.0024

Short Communication
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Cx3Mab-4: A Novel Anti-Mouse CXCR3 Monoclonal
Antibody for Flow Cytometry

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Tsunenori Ouchida,* Yu Isoda,* Tomohiro Tanaka, Mika K. Kaneko, Hiroyuki Suzuki, and Yukinari Kato

C-X-C motif chemokine receptor 3 (CXCR3, CD183) is a G-protein-coupled receptor for CXCL9, CXCL10,and CXCL11. CXCR3 induces chemotaxis of immune cells and promotes inflammation. Various mouse models
have been developed to mimic the pathogenesis of diseases and used in the evaluation of therapeutics for these
diseases. Although CXCR3 is an attractive target to suppress inflammation, anti-CXCR3 therapeutic agents
have not been approved. In this study, we established a novel anti-mouse CXCR3 (mCXCR3) monoclonal
antibody, Cx3Mab-4 (rat IgG1, kappa), using the Cell-Based Immunization and Screening method. Flow cytometric analysis demonstrated that Cx3Mab-4 bound to mCXCR3-overexpressed Chinese hamster ovary-K1(CHO/mCXCR3) cells, but did not react to parental CHO-K1 cells. The dissociation constant of Cx3Mab-4 wasdetermined as 1.3 · 10-9 M, indicating that Cx3Mab-4 possesses a high affinity to mCXCR3-expressing cells.
Cx3Mab-4 could be useful for targeting CXCR3-expressing cells in preclinical mouse models.
Keywords: mouse CXCR3, monoclonal antibody, CBIS methodcolon did not increase after the challenge. Moreover, the elevations of proinflammatory cytokines, such as IL-6, TNF-a,and IFN-g by DSS-challenge reduced in CXCR3-deficientmice.9 These facts indicate that CXCR3 plays a pivotal rolein the development of IBD.
To understand pathogenesis and develop therapeutics for
inflammatory diseases, various mouse models have beenreported.8,10–12 Although CXCR3 is an attractive target for
anti-inflammatory therapies, few preclinical models using
anti-mouse CXCR3 (mCXCR3) monoclonal antibodies(mAbs) have been developed. We have established mAbs
against mouse CCR313 and CCR814 using the Cell-Based
Immunization and Screening (CBIS) method. In this report,
we developed a novel anti-mCXCR3 mAb using the CBIS
method and evaluated its applications.

IntroductionC-X-C motif chemokine receptor 3 (CXCR3, CD183)
is a G-protein-coupled receptor expressed on T cells andnatural killer cells.1 CXCR3 signaling contributes to the accumulation of these cells to inflammation sites. Interferons(IFNs), cytokines secreted in inflammation sites, regulate
CXCR3 signaling and expression. IFN-a, -b, and -g elevate
the expression of CXCL9, CXCL10, and CXCL11, which
induce CXCR3-mediated chemotaxis in various types of cells
including macrophages, dendritic cells (DCs), fibrocytes, andendothelial cells.2,3 Additionally, IFN-g is essential for
CXCR3 expression induced by T cell receptor stimulation in
T cells.4 CXCR3 activation leads to actin polymerization andmigration of T cells to inflammation sites through G-protein
and phospholipase C-dependent pathways.5
CXCR3 is involved in inflammatory diseases.6,7 Dextran
Sulfate Sodium (DSS)-induced colitis is a commonly usedmouse model for inflammatory bowel disease (IBD).8 In
CXCR3-deficient mice challenged with 2.5% DSS, bodyweight loss, a sign of the severity of colitis, was lower thanthat of wild type mice.9 The number of leukocytes in the

Materials and Methods
Antibodies, cell lines, and plasmids

An anti-mCXCR3 mAb (clone CXCR3-173) was purchasedfrom BioLegend (San Diego, CA). Alexa Fluor 488-conjugatedDepartment of Antibody Drug Development, Tohoku University Graduate School of Medicine, Sendai, Japan.
This paper was previously published in preprint.org (doi: 10.20944/preprints202311.0805.v1).
*These authors contributed equally to this work.

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ANTI-MOUSE CXCR3 MAB

anti-rat IgG (4416) and anti-Armenian hamster IgG (ab173003)
were purchased from Cell Signaling Technology, Inc., (Danvers,MA) and Abcam (Cambridge, UK), respectively.
LN229, Chinese hamster ovary (CHO)-K1, andP3X63Ag8U.1 (P3U1) cells were obtained from the American Type Culture Collection (Manassas, VA).
The synthesized DNA (Eurofins Genomics KK) encoding
mCXCR3 (Accession No.: NM_009910.3) was subcloned
into a pCAGzeo_PAcH vector (PA tag15 added to the
C-terminus of mCXCR3). The pCAGzeo_mCXCR3-PA wastransfected into LN229 and CHO-K1 cells using a Neontransfection system (Thermo Fisher Scientific, Inc., Waltham, MA). Clones stably expressing mCXCR3-PA were
established using a cell sorter (SH800; Sony Corp., Tokyo,
Japan) after culturing in a medium, containing 0.5 mg/mL of
Zeocin (InvivoGen, San Diego, CA).
LN229 and mCXCR3-PA-overexpressed LN229 (LN229/
mCXCR3-PA) cells were cultured in Dulbecco’s Modified
Eagle Medium (Nacalai Tesque, Inc., Kyoto, Japan), supplemented with 10% heat-inactivated fetal bovine serum(FBS; Thermo Fisher Scientific, Inc.), 100 lg/mL streptomycin, 100 U/mL of penicillin, and 0.25 lg/mL amphotericin
B (Nacalai Tesque, Inc.). CHO-K1 and mCXCR3-PAoverexpressed CHO-K1 (CHO/mCXCR3-PA) cells weremaintained in a Roswell Park Memorial Institute (RPMI)1640 medium (Nacalai Tesque, Inc.), supplemented with
10% FBS, 100 lg/mL of streptomycin, 100 U/mL of penicillin, and 0.25 lg/mL of amphotericin B. All cells weregrown in a humidified incubator at 37C, in an atmosphere of
5% CO2 and 95% air.
Production of hybridomas

A five-week-old Sprague–Dawley rat was purchasedfrom CLEA Japan (Tokyo, Japan). The animal was housed
under specific pathogen-free conditions. All animal experiments were approved by the Animal Care and Use
Committee of Tohoku University (Permit number:
2022MdA-001).
To develop mAbs against mCXCR3, we intraperitoneallyimmunized one rat with CHO/mCXCR3-PA cells (1 · 109
cells) plus Alhydrogel adjuvant 2% (InvivoGen). After four
additional injections every week (1 · 109 cells), the splenocytes were harvested. The splenocytes were fused with
P3U1 cells using PEG1500 (Roche Diagnostics, Indianapolis, IN). Hybridomas were selected by cultivationin the RPMI-1640 medium with 10% FBS, 100 lg/mLof streptomycin, 100 U/mL of penicillin, 0.25 lg/mL ofamphotericin B, 5 lg/mL of Plasmocin, 5% Briclone(NICB, Dublin, Ireland), and hypoxanthine/aminopterin/
thymidine (HAT; Thermo Fisher Scientific, Inc.). The supernatants were screened using flow cytometry using LN229/
mCXCR3-PA.
Purification of mAbs

The cultured supernatants of anti-mCXCR3 mAbsproducing hybridomas were filtrated with Steritop (0.22 lm;
Merck KGaA, Darmstadt, Germany). After filtration, thesupernatants were applied to 1 mL of Ab-Capcher ExTra(ProteNova Co., Ltd., Kagawa, Japan). Bound Cx3Mab-4 waseluted with an IgG elution buffer (Thermo Fisher Scientific,Inc.), followed by immediate neutralization of eluates, using

1 M Tris-HCl (pH 8.0). The buffer of eluates was replacedwith phosphate-buffered saline (PBS) using Amicon Ultra(Merck KGaA).
Flow cytometry

CHO-K1 and CHO/mCXCR3-PA cells were harvested
after a brief exposure to 0.25% trypsin in 1 mM ethylenediaminetetraacetic acid (EDTA; Nacalai Tesque, Inc.). The
cells were washed with 0.1% bovine serum albumin in PBS
and treated with anti-mCXCR3 mAbs for 30 min at 4C.
After washing, the cells were treated with Alexa Fluor 488conjugated secondary antibodies. Flow cytometric analysis
was performed using the SA3800 Cell Analyzer (Sony Corp.).
To determine the dissociation constant (KD), antimCXCR3 mAbs were diluted serially from 100 mg/mL to
6 ng/mL. The geometric mean of fluorescence intensities of
CHO/mCXCR3-PA at each concentration was calculatedby FlowJo v10.8.1 (BD Biosciences, Franklin Lakes, NJ).
The KD was estimated by fitting saturation binding curves tothe built-in; one-site binding models in GraphPad PRISM 8(GraphPad Software, Inc., La Jolla, CA).
Results
Development of anti-mCXCR3 mAbs

We developed anti-mCXCR3 mAbs using the CBIS
method. The CBIS method is composed of four steps (Fig. 1).
First, a rat was immunized with CHO/mCXCR3-PA cells(Fig. 1A). The splenocytes were collected and fused with
P3U1 cells (Fig. 1B). The reactivities to LN229/mCXCR3-PA
cells of each supernatant were observed by flow cytometry(Fig. 1C). Finally, Cx3Mab-4 (rat IgG1, kappa) was developed (Fig. 1D).
Flow cytometry

To check the specificity of Cx3Mab-4 and CXCR3-173(a commercially available anti-mCXCR3 mAb), we performed flow cytometry against CHO/mCXCR3-PA and
CHO-K1. Both Cx3Mab-4 and CXCR3-173 reacted to CHO/
mCXCR3-PA cells in a dose-dependent manner (Fig. 2A). In
contrast, Cx3Mab-4 and CXCR3-173 did not bind to CHOK1 cells even at 10 lg/mL (Fig. 2B). No difference was observed between two anti-mCXCR3 mAbs.
Determination of dissociation constantof anti-mCXCR3 mAbs against CHO/
mCXCR3-PA cells

We determined the apparent dissociation constant (KD) of
Cx3Mab-4 and CXCR3-173 against mCXCR3 by flow cytometry. The geometric mean of the fluorescence intensity of
CHO/mCXCR3-PA at each concentration of Cx3Mab-4 andCXCR3-173 was plotted. By fitting one-site binding models,
both KD values of Cx3Mab-4 and CXCR3-173 for CHO/
mCXCR3-PA were determined as 1.3 · 10-9 M (Fig. 3), indicating that both Cx3Mab-4 and CXCR3-173 possess highaffinity for CHO/mCXCR3-PA cells.
Discussion

The CXCR3 ligands, CXCL9, CXCL10, and CXCL11, areabundantly expressed in the intestinal mucosa of IBD

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OUCHIDA ET AL.

FIG. 1. The workflow of development of Cx3Mab-4 by CBIS method. (A) CHO/mCXCR3-PA cells were injected
intraperitoneally into a Sprague–Dawley rat. (B) After five immunizations, the splenocytes from the rat were fused with
P3U1 cells. Hybridomas were seeded in 96-well plates. (C) The culture supernatants of each well were screened throughflow cytometry to distinguish anti-mCXCR3 mAb-producing hybridomas. (D) Cx3Mab-4 was established through limiting
dilution and some additional screenings. CBIS, Cell-Based Immunization and Screening; mAb, monoclonal antibody;
mCXCR3, mouse CXCR3.

patients.16–18 IBD is characterized by chronic, uncontrolledinflammation in the intestinal mucosa.19 The chemokines
induce cellular trafficking and enhance inflammation
through binding to CXCR3 expressed on proinflammatory
lymphocytes. The inhibition of infiltration has been shownto attenuate inflammation.19 Eldelumab (MDX-1100, BMS936557) is a fully human IgG1 mAb to CXCL10 and havebeen developed for IBD treatment. Unfortunately, in thephase II study, the eldelumab exposure-remission was not
sufficient in patients with IBD.20,21 Since not only CXCL10
but also CXCL9 and CXCL11 are elevated in IBD, antiCXCR3 therapy is also expected for IBD treatment. In thisstudy, we developed a novel anti-mCXCR3 mAb, Cx3Mab4 which is useful for flow cytometry (Figs. 2 and 3). To
apply preclinical mouse models of IBD, we should determine the epitope of Cx3Mab-4 and investigate the neutralizing effect against the ligands. We have already determinedthe epitopes of anti-mouse chemokine receptor mAbs
against CCR2 (C2Mab-6),22 CCR3 (C3Mab-3, 4, 6, and7),23,24 CCR6 (C6Mab-13),25 CCR9 (C9Mab-24),26 andCXCR6 (Cx6Mab-1).27,28
Infiltration of regulatory T (Treg) cells into solid tumors
represents a barrier to cancer immunotherapy.29 Chemokine
receptors including CXCR3 play a critical role in Treg cells

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ANTI-MOUSE CXCR3 MAB

FIG. 2. Flow cytometry of mCXCR3-overexpressed cells using Cx3Mab-4. CHO/mCXCR3-PA (A) and CHO-K1(B) cells were treated with 0.1–10 mg/mL of Cx3Mab-4 or CXCR3-173, followed by treatment with Alexa Fluor 488conjugated secondary antibodies. The red lines show the cells treated with each mAbs. The black lines show the cells
treated with blocking buffer and Alexa Fluor 488-conjugated secondary antibodies (negative control). CHO, Chinese
hamster ovary.
recruitment into inflamed tumor tissue, and are importanttherapeutic targets.30 CXCR3+ Treg cells increase in multiple
tumor models and are activated through interaction with
DCs.30 CXCR3 ablation in Treg cells disrupted the DC-Treg
cells interaction and increased DC-CD8+ T cell interactions,which resulted in increased CD8+ T cell priming and activation in tumors.30 These results show that CXCR3 is criticalfor Treg cells accumulation and immune suppression in tumors. Therefore, depletion of CXCR3+ Treg cells in tumors is

expected to potentiate antitumor immunity. In our previous
studies, we changed the isotype of mAbs into mouse IgG2a toretain antibody-dependent cellular cytotoxicity (ADCC) andcomplement-dependent cytotoxicity (CDC).31 Since thesubclass of Cx3Mab-4 is rat IgG1, it does not possess ADCC
and CDC. Therefore, in further studies, the subclass of
Cx3Mab-4 will be converted into mouse IgG2a to evaluate theeffect of depletion of mCXCR3+ Treg cells in preclinicalmouse models.

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(KAKENHI) grant nos. 23K19494 (to T.O.), 21K20789(to T.T.), 22K06995 (to H.S.), 21K07168 (to M.K.K.), and22K07224 (to Y.K.).

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References

FIG. 3. Determination of dissociation constant of Cx3Mab4 against mCXCR3-overexpressed cells. The binding affinity of Cx3Mab-4 (A) and CXCR3-173 (B) against CHO/
mCXCR3-PA cells was determined by flow cytometry. The
dots show the geometric mean of fluorescence intensity of
CHO/mCXCR3-PA at each concentration. The solid lines
are the fitting curve calculated by GraphPad PRISM 8.

Authors’ Contributions

T.O., Y.I., and T.T. performed the experiments. M.K.K.
and Y.K. designed the experiments. H.S. and M.K.K. analyzed the data. T.O. and Y.K. wrote the manuscript. All authors have read and agreed to the published version of themanuscript.
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 Grant
Numbers: JP23ama121008 (to Y.K.), JP23am0401013(to Y.K.), 23bm1123027h0001 (to Y.K.), and JP23ck0106730(to Y.K.), and by the Japan Society for the Promotion of
Science ( JSPS) Grants-in-Aid for Scientific Research

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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 980-8575
MiyagiJapan
E-mail: yukinari.kato.e6@tohoku.ac.jp
Received: November 13, 2023
Accepted: January 17, 2024