MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume XX, Number XX, 2023
ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2023.0023
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Cx4Mab-1: A Novel Anti-Mouse CXCR4 Monoclonal
Antibody for Flow Cytometry

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

The CXC chemokine receptor 4 (CXCR4, CD184) is a member of the G protein-coupled receptor family that isexpressed in most leukocytes. Overexpression of CXCR4 is associated with poor prognosis in not only hematopoietic
malignancy but also solid tumors. Because CXCR4 is an attractive target for tumor therapy, reliable preclinicalmurine models using anti-CXCR4 monoclonal antibodies (mAbs) have been warranted. This study established anovel anti-mouse CXCR4 (mCXCR4) mAb using the Cell-Based Immunization and Screening method. Flowcytometric analysis showed that an anti-mCXCR4 mAb, Cx4Mab-1 (rat IgG2a, kappa), recognized mCXCR4overexpressed Chinese hamster ovary-K1 (CHO/mCXCR4) cells and endogenously mCXCR4-expressing mouse
myeloma P3X63Ag8U.1 (P3U1) cells. Furthermore, Cx4Mab-1 did not recognize mCXCR4-knockout P3U1 cells.
The dissociation constants of Cx4Mab-1 for CHO/mCXCR4 and P3U1 were determined as 6.4 · 10-9 M and
2.3 · 10-9 M, respectively, indicating that Cx4Mab-1 possesses a high affinity to both endogenous and exogenous
mCXCR4-expressing cells. These results indicate that Cx4Mab-1 could be a useful tool for preclinical mouse models.
Keywords: mouse CXCR4, monoclonal antibody, CBIS methodanimal experiment using a mouse colorectal cancer CT-26
cell line revealed that CXCR4 is important for metastasis tothe liver.17 Moreover, CXCR4 supports tumor growth byinducing angiogenesis.18,19 These findings showed thatCXCR4 is an attractive target for tumor therapy.
Mouse is a commonly used animal for preclinical studies topredict the efficiency and the safety of cancer therapies.20 For
assessment of cancer therapies, two models are established.
The first one is the transplantation of tumors into mice. The
other is the induction of tumors in mice by genetic modification and carcinogens. Both models with immunocompetentmice are used for the evaluation of cancer treatments in thepresence of host immunity, an important factor affecting theefficiency and the safety of cancer therapies.21 However, apreclinical model using anti-mouse CXCR4 (mCXCR4)
monoclonal antibodies (mAbs) has not been reported.
We have developed mAbs to mouse CCR322 and CCR8,23
members of chemokine receptors, by using the Cell-Based
Immunization and Screening (CBIS) method, which includes
immunization of antigen-overexpressing cell and highthroughput screening of hybridoma supernatants using flow
cytometry. In this report, we established a novel anti-mCXCR4
mAb using the CBIS method and evaluated its applications.

IntroductionC

XC chemokine receptor 4 (CXCR4, CD184) is amember of G-protein-coupled receptors for CXCL12(SDF-1).1,2 CXCR4 is expressed on most leukocytes.3–5
CXCR4 activation by CXCL12 induces cell migration, proliferation, and survival through G-proteins-dependent or-independent ( JAK/STAT) pathway.6,7 Furthermore, theCXCL12/CXCR4 axis plays a critical role in embryonic development.8 The mice lacking CXCR4 or CXCL12 die onembryonic day 18.5. Both mice exhibit abnormal development of the cerebellum, heart, and blood vessels in the gastrointestinal tract.9,10
Overexpression of CXCR4 is associated with poor prognosis in various types of cancers through promoting theproliferation and metastasis.6 CXCR4 is overexpressed inabout 60% of colorectal cancers,11 one of the most common
malignancies in the world. Many studies have reported thatpatients with high CXCR4-expressing colorectal cancers
showed frequent lymph node metastasis and distant metastasis.12–14 The metastasized colorectal cancer in the liver,which highly expresses CXCL12, exhibited elevatedexpression of CXCR4 compared to the primary site.15,16 An

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

12
Materials and Methods
Antibodies

Anti-mCXCR4 (CD184) mAbs (clone L276F12 and clone
2B11/CXCR4) were purchased from BioLegend (San Diego,CA) and BD Biosciences (Franklin Lakes, NJ), respectively.
Alexa Fluor 488-conjugated anti-rat immunoglobulin G(IgG) was purchased from Cell Signaling Technology, Inc.
(Danvers, MA).
Preparation of cell lines

subcloned into a pCAGzeo PAcH vector (PA tag added to
C-terminus of mCXCR4). The PA tag consists of 12 aminoacids (GVAMPGAEDDVV).24 The mCXCR4-PA plasmid
was transfected into LN229 and CHO-K1 cells, using a Neontransfection system (Thermo Fisher Scientific Inc., Waltham,MA). Stable transfectants were established by staining with
anti-mCXCR4 mAb (clone L276F12) and sorted 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 conducted. GeneArt
CRISPR nuclease vectors with orange fluorescent protein
plasmid, which target mCXCR4 (CGGCAATGGATTGGTGATCC), were purchased from Thermo Fisher ScientificInc., The knockout plasmid was transfected into P3U1 cells,
using a Neon transfection system. mCXCR4 knockout P3U1
(BINDS-56) was established by staining with anti-mCXCR4mAb and sorted using a cell sorter (SH800).

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LN229, Chinese hamster ovary (CHO)-K1 and P3X63Ag
8U.1 (P3U1) cells were obtained from the American Type
Culture Collection (Manassas, VA).
The synthesized DNA (Eurofins Genomics KK) encoding
mCXCR4 (Accession No.: NM_009911.3) was subsequently

OUCHIDA ET AL.

FIG. 1. The scheme of establishment of Cx4Mab-1 by the CBIS method. (A) LN229/mCXCR4 cells were immunized intoa Sprague–Dawley rat by intraperitoneal injections. (B) The splenocytes from the rat were fused with P3U1 cells. (C) The
culture supernatants were screened through flow cytometry to select anti-mCXCR4 mAb-producing hybridomas.
(D) Finally, Cx4Mab-1 was established by limiting dilution and some additional screenings. CBIS, Cell-Based Immunization and Screening; mAb, monoclonal antibody.

ANTI-MOUSE CXCR4 MAB

LN229 and mCXCR4-PA-overexpressed LN229 (LN229/
mCXCR4) 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 U/mL of penicillin, 100 lg/mL
streptomycin, and 0.25 lg/mL amphotericin B (Nacalai
Tesque, Inc.). CHO-K1, mCXCR4-PA-overexpressed CHOK1 (CHO/mCXCR4), P3U1, and BINDS-56 cells were cultured in a Roswell Park Memorial Institute (RPMI)-1640
medium (Nacalai Tesque, Inc.), supplemented with 10%
FBS, 100 U/mL of penicillin, 100 lg/mL of streptomycin,
and 0.25 lg/mL of amphotericin B (Nacalai Tesque, Inc.).
All cells were grown in a humidified incubator at 37C, in
an atmosphere of 5% carbon dioxide and 95% air.

3

plus Alhydrogel adjuvant 2% (InvivoGen, San Diego, CA). The
procedure included three additional injections every week
(1 · 109 cells), which were followed by a final booster intraperitoneal injection (1 · 109 cells), 2 days before harvesting
splenocytes. The harvested splenocytes were subsequently
fused with P3U1 cells, using PEG1500 (Roche Diagnostics,
Indianapolis, IN). For the hybridoma selection, cells were
cultured in the RPMI-1640 medium with 10% FBS, 100 U/mLof penicillin, 100 lg/mL of streptomycin, 0.25 lg/mL of amphotericin B, 5 lg/mL of Plasmocin, 5% Briclone (NICB,
Dublin, Ireland), and hypoxanthine, aminopterin and thymidine(Thermo Fisher Scientific, Inc.). The supernatants were subsequently screened by flow cytometry using CHO/mCXCR4.
Purification of Cx4Mab-1

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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 No.: 2022MdA-001) approved animal experiments.
To develop mAbs against mCXCR4, we intraperitoneallyimmunized a rat with LN229/mCXCR4 cells (1 · 109 cells)

The cultured supernatants of Cx4Mab-1-producing hybridomas were filtrated with Steritop (0.22 lm; Merck KGaA,
Darmstadt, Germany). The filtered supernatants were subsequently applied to 1 mL of Capto L (Cytiva, Marlborough,MA). After washing with phosphate-buffered saline (PBS),
bound antibodies were eluted with an IgG elution buffer(Thermo Fisher Scientific, Inc.), followed by immediate neutralization of eluates, using 1 M Tris-HCl (pH 8.0). Finally, theeluates were concentrated, after which PBS was replaced withthe elution buffer using Amicon Ultra (Merck KGaA).

FIG. 2. Flow cytometry of mCXCR4-overexpressed cells using anti-mCXCR4 mAbs. CHO/mCXCR4 cells were treatedwith 0.01–10 mg/mL of Cx4Mab-1, L276F12, or 2B11/CXCR4 followed by treatment with Alexa Fluor 488-conjugated antirat IgG. The red lines show the cells treated with each mAb. The black line shows the cells treated with blocking buffer andAlexa Fluor488-conjugated anti-rat IgG (negative control). CHO, Chinese hamster ovary; IgG, immunoglobulin G.

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FIG. 3. Flow cytometry of endogenously mCXCR4-expressed cells using anti-mCXCR4 mAbs. P3U1 (A) and mCXCR4knockout P3U1 (BINDS-56) (B) cells were treated with 0.01–10 mg/mL of Cx4Mab-1, L276F12, or 2B11/CXCR4 followed
by treatment with Alexa Fluor 488-conjugated anti-rat IgG. The red line shows the cells treated with each mAb. The blackline shows the cells treated with blocking buffer and Alexa Fluor 488-conjugated anti-rat IgG (negative control).
4

ANTI-MOUSE CXCR4 MAB
Flow cytometry

collected using the SA3800 Cell Analyzer (Sony Corporation, Tokyo, Japan).
To determine the dissociation constant (KD), anti-mCXCR4mAbs were serially diluted from 10 mg/mL to 0.61 ng/mL. The
geometric mean of fluorescence intensities of CHO/mCXCR4and P3U1 at each concentration was calculated by FlowJo
v10.8.1 (Becton, Dickinson & Company, Ashland, OR). The
KD was estimated by fitting saturation binding curves to thebuilt-in; one-site binding models in GraphPad PRISM 8(GraphPad Software, Inc., La Jolla, CA).

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CHO-K1 and CHO/mCXCR4 cells were harvested aftera brief exposure to 1 mM ethylenediaminetetraacetic acid
(Nacalai Tesque, Inc.). The cells were washed with 0.1%
bovine serum albumin in PBS and treated with 10 to
0.01 lg/mL anti-mCXCR4 mAbs (clone L276F12 andclone 2B11/CXCR4) and Cx4Mab-1 for 30 min at 4C.
The cells were treated with 2 lg/mL Alexa Fluor
488-conjugated anti-rat IgG. The fluorescence data were

5

FIG. 4. Kinetic analyses of anti-mCXCR4 mAbs against mCXCR4-expressed cells through flow cytometry. The determination of the binding affinity of Cx4Mab-1 (A), L276F12 (B), and 2B11/CXCR4 (C) against CHO/mCXCR4 or P3U1
cells by flow cytometry. The dots show the geometric mean of fluorescence intensity of CHO/mCXCR4 and P3U1 at eachconcentration. The solid lines are the fitting curve calculated by GraphPad PRISM 8.

6
Results
Establishment of a novel anti-mCXCR4 antibody

To establish anti-mCXCR4 mAbs, a rat was immunizedwith LN229/mCXCR4 cells (Fig. 1A). The hybridomas were
produced by the fusion of the splenocytes with P3U1 cells
using polyethylene glycol (Fig. 1B). The hybridomas, which
are producing CHO/mCXCR4-reactive and CHO-K1nonreactive mAbs, were selected by flow cytometry (Fig. 1C).
After limiting dilution, a clone Cx4Mab-1 (rat IgG2a, kappa)
was established (Fig. 1D).

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Specificity of Cx4Mab-1 in flow cytometry

We performed flow cytometry using Cx4Mab-1 and commercially available mAbs (clone L276F12 and clone 2B11/
CXCR4) against CHO/mCXCR4 and P3U1 cells. ThreemAbs reacted to CHO/mCXCR4 cells in a dose-dependent
manner (Fig. 2). Although Cx4Mab-1 and L276F12 did notbind to CHO-K1 cells even at 10 lg/mL, 2B11/CXCR4
reacted with CHO-K1 weakly (Supplementary Fig. S1).
Furthermore, three mAbs reacted to P3U1 cells in a dosedependent manner (Fig. 3A). In contrast, three mAbs did notbind to mCXCR4-knockout P3U1 (BINDS-56) cells even at
10 lg/mL (Fig. 3B). These results indicated that Cx4Mab-1
recognizes both endogenous and exogenous mCXCR4expressing cell lines.
Affinity of Cx4Mab-1 against
mCXCR4-expressing cells

To determine the KD of Cx4Mab-1 to mCXCR4, we performed kinetic analysis via flow cytometry using
CHO/mCXCR4 and P3U1 cells. The geometric mean of thefluorescence intensity of each concentration of Cx4Mab-1
was plotted. The KD values of Cx4Mab-1 for CHO/mCXCR4and P3U1 were determined as 6.4 · 10-9 M and 2.3 · 10-9 M,
respectively (Fig. 4A). The KD values of L276F12 for
CHO/mCXCR4 and P3U1 were determined as 1.2 · 10-8 M
and 1.5 · 10-9 M, respectively (Fig. 4B). The KD values of
2B11/CXCR4 for CHO/mCXCR4 and P3U1 were determined as 2.0 · 10-8 M and 2.5 · 10-9 M, respectively(Fig. 4C). These results indicated that Cx4Mab-1 possesses ahigh affinity to both endogenous and exogenous mCXCR4expressing cells.
Discussion

In this study, we compared the sensitivity, specificity, andaffinity of three anti-mCXCR4 mAbs (Cx4Mab-1, L276F12,
and 2B11/CXCR4) using flow cytometry. We found no difference in the sensitivity among three mAbs (Fig. 2). In
contrast, 2B11/CXCR4 reacted with CHO-K1 weakly (Supplementary Fig. S1), indicating that 2B11/CXCR4 mightcross-react with other molecules. Cx4Mab-1 possesses thebest affinity among three mAbs against CHO/mCXCR4(Fig. 4A). In contrast, L276F12 shows the best affinity among
three mAbs against P3U1 (Fig. 4B), indicating that Cx4Mab1 and L276F12 demonstrate better affinity than 2B11/
CXCR4. Overall, three mAbs possess high sensitivity, andaffinity against mCXCR4.
CXCL12-CXCR4 signaling contributes to tumor growthand metastasis in various types of cancer.6 Therefore, CXCR4-OUCHIDA ET AL.

targeted therapies have been developed. Ulocuplumab is afully human IgG4 anti-human CXCR4 (hCXCR4) mAb.25
Ulocuplumab induced apoptosis of leukemia cells fromchronic lymphocytic leukemia patients by blocking CXCL12
binding to hCXCR4. In addition, a phase Ib/II study in patientswith relapsed/refractory multiple myeloma reported that the
combination of ulocuplumab with lenalidomide and dexamethasone showed a high response rate (55.2%) and a clinicalbenefit rate (72.4%).26 PF-06747143, another humanized IgG1
anti-hCXCR4 mAb, also showed antitumor activities in multiple hematologic cancer models.27
A potential side effect of anti-CXCR4 therapy is the toxicity to normal leukocytes and hematopoietic stem cells(HSCs). The CXCL12/CXCR4 axis is essential for hematopoiesis in fetuses and adults. Deficiencies of CXCR4 or
CXCL12 exhibit hematopoietic defects in fetal mice.9,10
Studies using CXCR4 conditional knockout mice demonstrated that CXCL12/CXCR4 axis also plays critical roles inhematopoiesis in adult.28 In the bone marrow, CXCL12CXCR4 signaling tethers HSCs in the niches where the quiescent HSC pool is maintained by supplying the requisite
factors. AMD3100 (Plerixafor/Mozobil), an antagonist of
CXCR4, induces HSC mobilization from bone marrow toperipheral blood.29 Cx4Mab-1 could be a useful tool for developing anti-CXCR4 therapies in preclinical murine models.
Authors’ Contributions

T.O., H.S., 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 article. All authors
have read and agreed to the published version of the article.
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
Nos.: 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 (KAKENHI) Grant
Nos.: 23K19494 (to T.O.), 21K20789 (to T.T.), 22K06995(to H.S.), 21K07168 (to M.K.K.), and 22K07224 (to Y.K.).
Supplementary Material

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: November 07, 2023
Accepted: November 20, 2023