MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 42, Number 1, 2023
ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2022.0032
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Establishment of a Sensitive Monoclonal Antibody
Against Mouse CCR9 (C9Mab-24) for Flow Cytometry

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Hiyori Kobayashi,1,* Teizo Asano,2,* Hiroyuki Suzuki,1 Tomohiro Tanaka,2 Takeo Yoshikawa,3Mika K. Kaneko,2 and Yukinari Kato1–3

The CC chemokine receptor 9 (CCR9), also known as CD199, is one of chemokine receptors. The CC
chemokine ligand 25 (CCL25) is known to be the only ligand for CCR9. The CCR9-CCL25 interaction plays
important roles in chemotaxis of lymphocytes and tumor cell migration. Therefore, CCR9-CCL25 axis is apromising target for tumor therapy and diagnosis. In this study, we established a sensitive and specificmonoclonal antibody (mAb) against mouse CCR9 (mCCR9) using N-terminal peptide immunization method.
The established anti-mCCR9 mAb, C9Mab-24 (rat immunoglobulin [IgG]2a, kappa), reacted with mCCR9overexpressed Chinese hamster ovary-K1 (CHO/mCCR9) and mCCR9-endogenously expressed cell line, RL2,
through flow cytometry. Kinetic analyses using flow cytometry showed that the dissociation constants (KD) of
C9Mab-24 for CHO/mCCR9 and RL2 cell lines were 6.0 · 10-9 M and 4.7 · 10-10 M, respectively. Results
indicated that C9Mab-24 is useful for detecting mCCR9 through flow cytometry, thereby providing a possibility
for targeting mCCR9-expressing cells in vivo experiments.
Keywords: mouse CCR9, monoclonal antibody, flow cytometry, dissociation constantfor the CCR9. The CCR9-CCL25 mediates chemotaxis oflymphocytes.(9–11) Furthermore, the CCR9-CCL25 axis plays
important roles in the inhibition of apoptosis,(12,13) proliferation,(14–16) invasion,(17) and metastasis(18–20) during tumor progression.(21–24) CCR9 is highly expressed in T cell lineage acutelymphoblastic leukemia (T-ALL) cells,(25) lung adenocarcinoma tissues,(26) breast cancer cell line (MDA-MB-231),(27) andovarian cancer cell lines (OVCAR-3 and CAOV-3).(28)The elevated expression of CCL25 is observed in
T-ALL,(29) prostate,(30) breast,(28) and ovarian cancers.(28,31)
The CCR9–CCL25 interaction mediates PI3K/AKTdependent antiapoptotic signals, which results in low apoptosis and modest chemotherapeutic response.(30) Therefore,
the CCR9-CCL25 interaction will be a promising target forcancer treatment and diagnosis.
In this study, we established a sensitive and specificmonoclonal antibody (mAb) against mouse CCR9 (mCCR9)
using N-terminal peptide immunization method and determined the dissociation constants (KD) using mCCR9expressed cell lines by flow cytometry.

Introduction

C

hemokines are 8–14 kDa proteins, which are known tobe ligand for G protein-coupled receptors (GPCRs).
Chemokine-GPCR pathway regulates cellular trafficking oflymphocytes and plays an important role in the immune
system.(1,2) Chemokines are divided into four subfamilies,
CC, CXC, XC, and CX3C chemokines, based on the differences of cysteine positions at their N-terminus.(3,4) Chemokine receptors are also classified into four families that
correspond to their ligand subfamilies: CC chemokine receptor (CCR), CXC chemokine receptor (CXCR), CX3C
chemokine receptor (CX3CR), and XC chemokine receptor(XCR).(5) Chemokines involve in the pathogenesis of several
diseases, including allergic inflammatory disease, humanimmunodeficiency virus-associated diseases, and cancer.(6–8)
CCR9, also known as CD199, is a member of GPCR family
consisting of an extracellular N-terminus, seven membranespanning regions, and a cytoplasmic C-terminus.(9–11) CC
chemokine ligand 25 (CCL25) is known to be the only ligand

Departments of 1Molecular Pharmacology, 2Antibody Drug Development, and 3Pharmacology, Tohoku University Graduate Schoolof Medicine, Sendai, Japan.
*These authors contributed equally in this study.

15

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Materials and Methods
PeptidesEurofins Genomics KK (Tokyo, Japan) synthesized apartial sequence of the N-terminal extracellular region ofmCCR9 (accession no. NP_001160097) with cysteine at
its C-terminus (mCCR9p1-19C; sequence: MMPTELTSLIPGMFDDFSYC). Subsequently, the keyhole limpet hemocyanin (KLH) was conjugated at the C-terminus of the peptide (mCCR9p1-19C-KLH).

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Preparation of cell lines

Chinese hamster ovary (CHO)-K1 and P3X63Ag8U.1(P3U1) cells were obtained from the American Type Culture
Collection (Manassas, VA). RL2 cells (mouse leukemia
cells) were obtained from the Cell Resource Center for
Biomedical Research Institute of Development, Aging andCancer Tohoku University (Miyagi, Japan). The expression
plasmid of mCCR9 (pCMV6neo-mCCR9-Myc-DDK) was
purchased from OriGene Technologies, Inc. (Rockville,
MD). The mCCR9 plasmid was transfected into CHO-K1
cells, using a Neon transfection system (Thermo Fisher Scientific, Inc., Waltham, MA). Stable transfectants were established through cell sorting using a cell sorter (SH800;
Sony Corp., Tokyo, Japan) using Roswell Park Memorial
Institute (RPMI) 1640 medium (Nacalai Tesque, Inc., Kyoto,
Japan), containing 10% heat-inactivated fetal bovine serum(FBS; Thermo Fisher Scientific, Inc.), 100 U/mL of penicillin, 100 lg/mL of streptomycin, 0.25 lg/mL of amphotericin
B (Nacalai Tesque, Inc.), and 0.5 mg/mL of G418 (Nacalai
Tesque, Inc.).
CHO-K1, P3U1, mCCR9-overexpressed CHO-K1(CHO/mCCR9), and RL2 were cultured in RPMI 1640 medium with 10% heat-inactivated FBS, 100 U/mL of penicillin, 100 lg/mL of streptomycin, and 0.25 lg/mL ofamphotericin B. Cells were grown in a humidified incubator
at 37C, at an atmosphere of 5% CO2 and 95% air.
Antibodies

Anti-mCCR9 mAbs (clone 9B1 and CW-1.2) were purchased from BioLegend (San Diego, CA). A secondary Alexa
Fluor 488-conjugated anti-rat immunoglobulin (IgG) was
purchased from Cell Signaling Technology, Inc. (Danvers,
MA).
Production of hybridomas

A 5-week-old Sprague–Dawley rat was purchased from
CLEA Japan (Tokyo, Japan). Animals were housed under
specific pathogen-free conditions. All animal experiments
were also conducted according to relevant guidelines andregulations to minimize animal suffering and distress in thelaboratory. The Animal Care and Use Committee of Tohoku
University (permit no. 2019NiA-001) approved animal experiments. The rat was monitored daily for health during thefull 4-week duration of the experiment. A reduction of morethan 25% of the total body weight was defined as a humane
endpoint. During sacrifice, the rat was euthanized throughcervical dislocation, after which death was verified throughrespiratory and cardiac arrest.

KOBAYASHI ET AL.

To develop mAbs against mCCR9, one rat was immunizedintraperitoneally, using 100 lg mCCR9p1-19C-KLH peptide
with Imject Alum (Thermo Fisher Scientific, Inc.). The procedure included three additional immunizations, which were
followed by a final booster intraperitoneal injection, 2 days
before the harvest of spleen cells. Harvested spleen cells were
subsequently fused with P3U1 cells, using PEG1500 (Roche
Diagnostics, Indianapolis, IN), after which hybridomas weregrown in an RPMI1640 medium supplemented with hypoxanthine, aminopterin, and thymidine for the selection(Thermo Fisher Scientific, Inc.). Supernatants were subsequently screened with the mCCR9p1-19C peptide, using
enzyme-linked immunosorbent assay (ELISA), after flow
cytometry, using CHO/mCCR9 and CHO-K1 cells.
Enzyme-linked immunosorbent assay

The synthesized peptide, mCCR9p1-19C, was immobilized on Nunc Maxisorp 96-well immunoplates (Thermo Fisher Scientific, Inc.) at a concentration of 1 lg/mL for
30 minutes at 37C. After washing with phosphate-bufferedsaline (PBS) containing 0.05% Tween20 (PBST; Nacalai
Tesque, Inc.), wells were blocked with 1% bovine serum
albumin-containing PBST for 30 minutes at 37C. Plates
were then incubated with supernatants of hybridomas, followed by a peroxidase-conjugated anti-rat IgG (1:20000 diluted; Sigma-Aldrich Corp., St. Louis, MO). Next, enzymatic
reactions were conducted, using an ELISA POD Substrate
TMB Kit (Nacalai Tesque, Inc.), followed by the measurement of the optical density at 655 nm, using an iMark microplate reader (Bio-Rad Laboratories, Inc., Berkeley, CA).
Purification of mAbs

The cultured supernatants of C9Mab-24-expressing hybridomas were collected through centrifugation at 2330 · g
for 5 minutes, followed by filtration using Steritop (0.22 lm;
Merck KGaA, Darmstadt, Germany). Filtered supernatants
were subsequently applied to 1 mL Protein G-Sepharose(Cytiva, Marlborough, MA). After washing with PBS, boundantibodies were eluted with an IgG elution buffer (Thermo
Fisher Scientific, Inc.), followed by an immediate neutralization of eluates, using 1 M Tris-HCl. Finally, eluates wereconcentrated, after which PBS was used to replace the elution
buffer, using Amicon Ultra (Merck KGaA).
Flow cytometric analyses

CHO-K1 and CHO/mCCR9 cells were harvested after an
exposure to 1 mM ethylenediaminetetraacetic acid (EDTA;
Nacalai Tesque, Inc.). CHO-K1, CHO/mCCR9, and RL2cells were washed with 0.1% bovine serum albumin in PBS
and treated with 0.01, 0.1, 1, and 10 lg/mL primary mAbs for
30 minutes at 4C. Then, cells were treated with Alexa Fluor
488-conjugated anti-rat IgG (1:2000 diluted), after which
fluorescence data were collected, using the BD FACSLyric(BD Biosciences, Franklin Lakes, NJ).
Determination of dissociation constants (KD)through flow cytometry

CHO/mCCR9 and RL2 cells were suspended in 100 lL
serially diluted anti-mCCR9 mAbs, after which 50 lL Alexa
Fluor 488-conjugated anti-rat IgG (1:200 diluted) was added.

ESTABLISHMENT OF ANTI-MOUSE CCR9 MONOCLONAL ANTIBODY

Afterward, fluorescence data were collected, using BD
FACSLyric. The KD were subsequently calculated by fitting
saturation binding curves to the built-in one-site binding
models in GraphPad PRISM 8 (GraphPad Software, Inc., La
Jolla, CA).
Results
Development of anti-mCCR9 mAbs
by peptide immunization

To develop anti-mCCR9 mAbs, one rat was immunizedwith an mCCR9p1-19C-KLH peptide (Fig. 1A). Spleen wasthen excised from the rat, after which splenocytes were fusedwith myeloma P3U1 cells using PEG1500 (Fig. 1B). De-

17veloped hybridomas were subsequently seeded into 20 of 96well plates and cultivated for 7 days. Then, wells in which
cultured supernatants were positive for the mCCR9p1-19C
peptide through ELISA were selected (Fig. 1C).
The ELISA screening approach identified strong signals
from mCCR9p1-19C peptide-immunized wells, no signal
was detected from control wells in 152 of 1916 wells(7.93%). Afterward, ELISA-positive wells were screenedusing flow cytometry for the selection of mCCR9-expressing
cell-reactive and CHO-K1-nonreactive supernatants(Fig. 1C). The flow cytometric screening approach identified
strong signals from CHO/mCCR9 cells, and a weak or no
signal from CHO-K1 cells in 37/152 (24.3%). After limiting
dilution and several additional screenings, anti-mCCR9
mAbs, C9Mab-24 (rat IgG2a, kappa) was finally established(Fig. 1D).
Flow cytometric analysis

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We conducted flow cytometric analysis using three antimCCR9 mAbs, C9Mab-24 and the commercially available
anti-mCCR9 mAbs (clone 9B1 and CW-1.2 from BioLegend), against mCCR9-expressed cell lines CHO/mCCR9and RL2. All three mAbs recognized CHO/mCCR9 cells
dose-dependently (Fig. 2A), whereas parental CHO-K1 cells
were not recognized by them except for 10 lg/mL of CW-1.2(Fig. 2B). Although 9B1 did not react with CHO/mCCR9cells at 0.01 lg/mL, C9Mab-24 and CW-1.2 reacted with thecells even at 0.01 lg/mL (Fig. 2A). All three mAbs also reacted with RL2 cells dose-dependently, but 9B1 did not reactwith RL2 cells at 0.1 and 0.01 lg/mL (Fig. 2C). These results
suggested that C9Mab-24 is specific for mCCR9, and is usefulfor detecting exogenous and endogenous mCCR9 throughflow cytometry.
Kinetic analysis of the interaction of anti-mCCR9 mAbs
with mCCR9-expressed cells through flow cytometry

FIG. 1. Schematic illustration of the production of antimCCR9 mAbs. (A) mCCR9p1-19C-KLH peptide was immunized into one SD rat using an intraperitoneal injection.
(B) Spleen was excised and spleen cells were fused with P3U1
cells. (C) Culture supernatants were screened by ELISA andthen flow cytometry to select hybridomas that are producing
anti-mCCR9 mAb. (D) After limiting dilution and some additional screenings, mCCR9-specific mAb was finally established.
ELISA, enzyme-linked immunosorbent assay; KLH, keyhole
limpet hemocyanin; P3U1, P3X63Ag8U.1; mAb, monoclonal
antibody; mCCR9, mouse CCR9; SD, Sprague–Dawley.

To determine the apparent KD of anti-mCCR9 mAbs with
mCCR9-expressed cells, we conducted kinetic analysis of
the interaction of C9Mab-24, 9B1, and CW-1.2 with
CHO/mCCR9 and RL2 cells using flow cytometry. The
geometric means of the fluorescence intensity were thenplotted versus the concentration of anti-mCCR9 mAbs, following fitting through one-site binding models in GraphPad
PRISM 8. The KD of C9Mab-24 for CHO/mCCR9 and RL2cells were determined as 6.0 · 10-9 M (Fig. 3A) and
4.7 · 10-10 M (Fig. 3B), respectively. The KD of 9B1 for
CHO/mCCR9 and RL2 cells were determined as 2.7 · 10-8
M (Fig. 3C) and 8.7 · 10-8 M (Fig. 3D), respectively. The KD
of CW-1.2 for CHO/mCCR9 and RL2 cells were determined
as 4.3 · 10-9 M (Fig. 3E) and 2.9 · 10-10 M (Fig. 3F), respectively. These results indicate that C9Mab-24 and CW-1.2
possess a high affinity for both CHO/mCCR9 cells and RL2cells.
Discussion

Previously, we have succeeded in development of mAbs
against chemokine receptors, including mouse CCR2,(32)human CCR2,(33) mouse CCR3,(34–36) mouse CCR4,(37)mouse CCR8,(38–40) human CCR9,(41) and mouse
CXCR6.(42) In this study, we developed a novel anti-mCCR9

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FIG. 2. Flow cytometry using anti-mCCR9 mAbs. CHO/mCCR9 (A), CHO-K1 (B), and RL2 (C) cells were treated with
0.01, 0.1, 1, and 10 mg/mL of C9Mab-24 (upper panels), 9B1 (middle panels), or CW-1.2 (lower panels), followed bytreatment with Alexa Fluor 488-conjugated anti-rat IgG. Filled, without primary Ab as negative controls. CHO, Chinese
hamster ovary; IgG, immunoglobulin.
18

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ESTABLISHMENT OF ANTI-MOUSE CCR9 MONOCLONAL ANTIBODY

19

FIG. 3. Determination of the KD of anti-mCCR9 mAbs against mCCR9-expressing cells. CHO/mCCR9 (A, C, E) and RL2 (B, D, F) were suspended in 100 mL serially diluted C9Mab-24 (0.0006–10 mg/mL, A, B), 9B1 (0.006–100 mg/mL, C, D) or
CW-1.2 (0.0006–10 mg/mL, E, F). Alexa Fluor 488-conjugated anti-rat IgG was then added. Fluorescence data wereobtained using BD FACSLyric. KD were calculated by fitting saturation binding curves to the built-in one-site binding
models in GraphPad PRISM 8.
mAb, C9Mab-24 (rat IgG2a, kappa), through immunization
with the N-terminal peptide of mCCR9. C9Mab-24 can detectboth exogenous and endogenous mCCR9 in flow cytometricanalysis (Fig. 2).
The specific and sensitive antibodies are essential to develop therapeutic antibodies. Therapeutic antibodies possess
several mechanisms of action, including neutralization,(43)antibody-dependent cellular cytotoxicity (ADCC), andcomplement-dependent cytotoxicity (CDC).(44) Furthermore,therapeutic antibodies can be utilized as a drug delivery
carrier,(45) Sharma et al. reported that blocking of the CCR9CCL25 interaction by anti-CCR9 mAb abrogates the CCL25mediated resistance to a chemotherapeutic agent, etoposidein prostate cancer.(30) It has been also reported that anti-

CCR9 mAb inhibited the ovarian cancer migration and invasiveness toward chemotactic gradients of CCL25.(28)
Therefore, CCR9 is an attractive therapeutic target and antiCCR9 mAbs are useful for cancer treatment.
In this study, we showed the specificity (Fig. 2) and highaffinities (Fig. 3A, B) of C9Mab-24. It has been reported that
GPCRs, including CCR9, CCR2, CCR3, CCR5, andCXCR1, interact with their ligands at the N-terminal
region.(11,46–48) Since C9Mab-24 was developed by immunizing N-terminal peptide of mCCR9, C9Mab-24 is expected to inhibit the binding of CCL25 to CCR9. Moreover,rat IgG2a possesses ADCC and CDC activities. Therefore,
C9Mab-24 could be used for the elimination of mCCR9positive cells in vivo.

20

KOBAYASHI ET AL.

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) to Y.K. under
grant nos. JP22ama121008, JP22am0401013, JP22bm1004001,
JP22ck0106730, and JP21am0101078.

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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: yukinari.kato.e6@tohoku.ac.jp
Received: September 3, 2022
Accepted: October 10, 2022