Original Articles

MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 41, Number 6, 2022
ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2022.0027

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Development of a Sensitive Anti-Human CCR9 Monoclonal
Antibody (C9Mab-11) by N-Terminal Peptide Immunization

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Tomohiro Tanaka,1 Hiroyuki Suzuki,2 Yu Isoda,1 Teizo Asano,1 Takuro Nakamura,1 Miyuki Yanaka,1
Saori Handa,1 Nozomi Takahashi,1 Saori Okuno,1 Takeo Yoshikawa,3 Guanjie Li,2 Ren Nanamiya,1Nohara Goto,2 Nami Tateyama,1 Yuki Okada,2 Hiyori Kobayashi,2 Mika K. Kaneko,1 and Yukinari Kato1–3

The C-C chemokine receptor 9 (CCR9) belongs to the G-protein-coupled receptor superfamily, and is highly
expressed on the T cells and intestinal cells. CCR9 regulates various immune responses by binding to the C-C
chemokine ligand, CCL25, and is involved in inflammatory diseases and tumors. Therefore, the development ofsensitive monoclonal antibodies (mAbs) for CCR9 is necessary for treatment and diagnosis. In this study, weestablished a specific anti-human CCR9 (hCCR9) mAb; C9Mab-11 (mouse IgG2a, kappa), using the synthetic
peptide immunization method. C9Mab-11 reacted with hCCR9-overexpressed Chinese hamster ovary-K1(CHO/hCCR9) and hCCR9-endogenously expressed MOLT-4 (human T-lymphoblastic leukemia) cells in flow
cytometry. The dissociation constant (KD) of C9Mab-11 for CHO/hCCR9 and MOLT-4 cells were determined
to be 1.2 · 10-9 M and 4.9 · 10-10 M, respectively, indicating that C9Mab-11 possesses a high affinity for bothexogenously and endogenously hCCR9-expressing cells. Furthermore, C9Mab-11 clearly detected hCCR9protein in CHO/hCCR9 cells using western blot analysis. In summary, C9Mab-11 can be a useful tool for
analyzing hCCR9-related biological responses.
Keywords: human CCR9, monoclonal antibody, flow cytometry, western blot

Introduction

C

hemokines are 8- to 12-kDa chemotactic cytokinesthat regulate cell migration by binding to seventransmembrane G-protein-coupled receptors (GPCRs).
Chemokines play pivotal roles during development, inflammation, and pathological processes, including cancer progression.1 They can be divided into four categories: CC,
CXC, CX3C, and XC subfamilies, depending on the number
and position of N-terminal cysteine residue.
The C-C chemokine receptor 9 (CCR9), a member of
GPCRs, is highly expressed in gut-homing T cells, thymocytes,
B cells, dendritic cells, and intestinal cells.2 CCR9 on immature
T cells contributes to T cell activation and infiltration bybinding to its dedicated ligand, CCL25/thymus-expressedchemokine.3 CCL25 is mainly secreted from epithelial cellsof the thymus and small intestine, and inducts the T cells into
intestinal tissues.4–6 CCR9/CCL25 has been reported to be
involved in various inflammatory diseases.7 CCR9 expres-sion is increased in mice myocardial infarction model, andabrogation of CCR9 improved the mice survival.8
In hepatitis, CCR9+ macrophages induced acute liver
damage by interacting with helper T1 (Th1) cells.9 In dextran
sulfate sodium-induced mice colitis, a model of inflammatory
bowel disease (IBD), both CCR9 and CCL25 expression
were elevated. In the colitis model, CCR9-knockout mice
exhibited higher IBD score and mortality.10 Therefore, thetargeting CCR9/CCL25 could be an attractive therapeuticstrategy because of its involvement with inflammationassociated diseases. A clinical trial of the CCR9 antagonist
CCX282-B against IBD had been conducted.11,12 CCR9 was
also found to be expressed in T-acute lymphoblastic leukemia (T-ALL), which contributes to the progression of
T-ALL.13 CD4+CCR9+T cells have been found to express
large amounts of interleukin 21, inducible T cell costimulators, and the transcription factors, including Bcl-6.
Furthermore, the CD4+CCR9+T cells support antibody production from B cells.14

1
Department of Antibody Drug Development, 2Department of Molecular Pharmacology, and 3Department of Pharmacology, Tohoku
University Graduate School of Medicine, Sendai, Japan.

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Previously, we have developed various anti-GPCR
monoclonal antibodies (mAbs) against mouse CCR315–17 andmouse CCR818–20 by using the Cell-Based Immunization andScreening (CBIS) method. Furthermore, anti-human CCR9(hCCR9) mAb (clone C9Mab-1) was also established using
the CBIS method.21 We also identified the epitope of
C9Mab-1 on the N-terminal region of the hCCR9 protein.22
The aim of this study is to obtain more sensitive anti-hCCR9
mAbs by the synthetic peptide immunization method using
the epitope region of C9Mab-1.
Materials and Methods

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Cell lines

Chinese hamster ovary (CHO)-K1 and P3X63Ag8U.1 (P3U1)
cells were obtained from the American Type Culture Collection(ATCC; Manassas, VA). MOLT-4 (a human T-lymphoblastic
leukemia cell line) was provided from the Japanese Collection of
Research Bioresources (JCRB) Cell Bank (Osaka, Japan). The
expression plasmid of hCCR9 (pCMV6neoCCR9-Myc-DDK)
was purchased from OriGene Technologies, Inc. (Rockvile, MD)
and was transfected into CHO-K1 cells using Lipofectamine
LTX with Plus Reagent (Thermo Fisher Scientific, Inc., Waltham, MA). Stable transfectants were selected by limiting dilution and cultivation in a medium containing 0.5 mg/mL of G418
(Nacalai Tesque, Inc., Kyoto, Japan).
CHO-K1, P3U1, CHO/hCCR9, and MOLT-4 were cultured in a Roswell Park Memorial Institute (RPMI) 1640
medium (Nacalai Tesque, Inc.) that was supplemented with
10% heat-inactivated fetal bovine serum (Thermo Fisher
Scientific, Inc.), 100 U/mL of penicillin, 100 lg/mL of
streptomycin, and 0.25 lg/mL of amphotericin B (Nacalai
Tesque, Inc.). Then, cells were grown in a humidified incubator, which was supplied with 5% CO2 and 95% air at 37C.
Hybridoma production

Two 6-week-old female BALB/c mice were purchasedfrom CLEA Japan (Tokyo, Japan). Mice were housed under
specific pathogen-free conditions. Then, animal experiments
were conducted, following relevant guidelines and regulations to minimize animal suffering and distress in the laboratory. The Animal Care and Use Committee of Tohoku
University approved animal experiments (permit number:
2019NiA-001). The mouse health was monitored daily during the entire 4-week duration of the experiment, and a reduction of more than 25% of the total body weight was
denoted as a humane endpoint. Subsequently, mice wereeuthanized through cervical dislocation, after which respiratory and cardiac arrest were used to verify death.
We immunized two BALB/c mice with keyhole limpet hemocyanin-conjugated hCCR9 peptide (100 lg):
4-TDFTSPIPNMADDYGSEST-22 + C-terminal cysteine.
The administration was conducted through the intraperitoneal route with an Imject Alum (Thermo Fisher Scientific,Inc.). The procedure included three additional immunization
procedures (100 lg of peptide), followed by a final booster
intraperitoneal injection (100 lg of peptide) 2 days before its
spleen cells were harvested.
The harvested spleen cells were subsequently fused with
P3U1 cells, using polyethylene glycol 1500 (PEG1500;
Roche Diagnostics, Indianapolis, IN). Then, hybridomas

TANAKA ET AL.

were grown in an RPMI 1640 medium supplemented withhypoxanthine, aminopterin, and thymidine for selection(Thermo Fisher Scientific, Inc.). Cultured supernatants were
finally screened using enzyme-linked immunosorbent assay(ELISA) and flow cytometric analysis.
Enzyme-linked immunosorbent assay

A synthesized hCCR9 peptide (4-TDFTSPIPNMADDYGSEST-22 + C-terminal cysteine) was immobilized on
Nunc Maxisorp 96-well immunoplates (Thermo Fisher Scientific, Inc.) at a concentration of 1 lg/mL peptide at 37C for
30 minutes. After washing with phosphate-buffered saline(PBS) containing 0.05% Tween20 (PBST; Nacalai Tesque,Inc.), wells were blocked with 1% bovine serum albumin(BSA)-containing PBST for 30 minutes at 37C. Plates werethen incubated with culture supernatants, followed byperoxidase-conjugated anti-mouse immunoglobulins (1:2000
diluted; Agilent Technologies, Inc., Santa Clara, CA). Enzymatic reactions were conducted using the ELISA POD substrate TMB kit (Nacalai Tesque, Inc.), followed by themeasurement of the optical density at 655 nm using an iMark
microplate reader (Bio-Rad Laboratories, Inc., Berkeley, CA).
Flow cytometry

Cells were washed with 0.1% BSA in PBS and treated with
C9Mab-11 or recombinant C9Mab-1 (recC9Mab-1)23 for 30
minutes at 4C. Then, cells were treated with Alexa Fluor
488-conjugated anti-mouse IgG (1:1000; Cell Signaling
Technology, Inc., Danvers, MA), followed by the collectionof fluorescence data, using SA3800 Cell Analyzer (SonyCorp., Tokyo, Japan).
Determination of the binding affinity by flow cytometry

CHO/hCCR9 and MOLT-4 cells were suspended in 100 lL
serially diluted anti-hCCR9 mAbs, after which Alexa Fluor
488-conjugated anti-mouse IgG (1:200; Cell Signaling Technology, Inc.) was added. Fluorescence data were subsequently
collected, using BD FACSLyric (BD Biosciences), followed by
the calculation of the dissociation constant (KD) by fitting thebinding isotherms into the built-in one-site binding model in
GraphPad PRISM 6 (GraphPad Software, Inc., La Jolla, CA).
Western blot analysis

Cell lysates (10 lg) were boiled in sodium dodecyl sulfate
sample buffer (Nacalai Tesque, Inc.). Proteins were electrophoresed on 5%–20% polyacrylamide gels (FUJIFILM Wako
Pure Chemical Corporation, Osaka, Japan) and transferred onto
polyvinylidene difluoride (PVDF) membranes (Merck KGaA,
Darmstadt, Germany). After blocking with 4% nonfat milk
(Nacalai Tesque, Inc.), PVDF membranes were incubated with
1 lg/mL of C9Mab-11 and 1 lg/mL of an anti-DYKDDDDK
mAb (clone 1E6; FUJIFILM Wako Pure Chemical Corporation), followed by incubation with peroxidase-conjugated antimouse IgG (1:2000; Agilent Technologies, Inc.); or 1 lg/mL ofan anti-isocitrate dehydrogenase 1 (IDH1) mAb (clone RcMab1),24,25 followed by incubation with peroxidase-conjugated antirat IgG (1:10,000; Sigma-Aldrich Corp., St. Louis, MO). Blots
were developed using Pierce ECL Plus Western Blotting
Substrate (Thermo Fisher Scientific, Inc.) and imaged with a
Sayaca-Imager (DRC Co. Ltd., Tokyo, Japan).

DEVELOPMENT OF ANTI-HCCR9 MAB
Results
Establishment of anti-hCCR9 mAbs

(6.58%), and finally established C9Mab-11 (mouse IgG2a,kappa) after cloning by the limiting dilution.
Flow cytometry

Flow cytometry was performed using C9Mab-11 andrecC9Mab-1 against exogenously hCCR9-overexpressed
CHO/hCCR9 and endogenously hCCR9-expressed MOLT-4
cells. Results showed that C9Mab-11 recognized

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We first immunized mice with the hCCR9 peptide (Fig. 1).
Hybridomas were seeded into 96-well plates, after which
ELISA was used to select reactive wells for hCCR9 peptide,followed by the selection of CHO/hCCR9 and MOLT-4
cells-reactive supernatants of hybridoma using flow cytometry. We obtained reactive supernatants in 63 of 958 wells

305

FIG. 1. A schematic illustration about the production of anti-hCCR9 mAbs. The mice were intraperitoneally immunizedwith the hCCR9 peptide. Screening of hybridoma was then conducted by ELISA, followed by flow cytometry using hCCR9expressing cells. ELISA, enzyme-linked immunosorbent assay; mAbs, monoclonal antibodies; hCCR9, human CCR9.

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FIG. 2. Flow cytometry using anti-hCCR9 mAbs. CHO/hCCR9 cells (A), CHO-K1 cells (B), and MOLT-4 cells (C) weretreated with 0.1, 1, 10 mg/mL of C9Mab-11 and recC9Mab-1, followed by treatment with Alexa Fluor 488-conjugated antimouse IgG. Black line represents the negative control. CHO, Chinese hamster ovary; recC9Mab-1, recombinant C9Mab-1.
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DEVELOPMENT OF ANTI-HCCR9 MAB

CHO/hCCR9 in a dose-dependent manner (Fig. 2A) but did
not react with parental CHO-K1 cells (Fig. 2B). C9Mab-11
reacted with MOLT-4 cells in a dose-dependent manner(Fig. 2C). Although recC9Mab-1 reacted with both
CHO/hCCR9 (Fig. 2A) and MOLT-4 (Fig. 2C) in the similar
way with C9Mab-11, the reactivity of C9Mab-11 is moresensitive than recC9Mab-1 at a concentration of 1 lg/mL.

DYKDDDDK mAb (clone 1E6) was used as a positive
control and could also detect a band of the same position in
CHO/hCCR9 cell lysates. An anti-IDH1 mAb (clone RcMab1) was used for internal control. These results indicate that
C9Mab-11 can detect hCCR9 in western blot analyses.

Determination of the binding affinity of C9Mab-11

Using the CBIS method, we have developed many mAbs,
which could recognize the biological structure and modification, including folding and glycosylation in the membraneproteins.16,18,20,21,23,26–32 We also developed an anti-hCCR9mAb (clone C9Mab-1) by using CBIS method.21 By evaluating the mAb epitope, we could find a better peptide immunogen for the same target protein. We, therefore,
identified the epitope of C9Mab-1 as Ile10, Pro11, Asn12,
Met13, Ala14, Asp16, and Tyr17 of hCCR9 by ELISA.22 In
this study, we immunized mice with the N-terminal peptideof hCCR9, including these amino acids, and obtained ahighly sensitive mAb (clone C9Mab-11).
The development of anti-GPCR antibodies has been reported to be difficult due to the complexity of its folded
three-dimensional structure, the small-exposed area of extracellular epitopes, and the difficulty of purifying a functional protein as an antigen.33 The design of antigenic epitopeis important for antibody production by peptide immunization method. Thus, the combination with CBIS and peptide
immunization method considered to be an effective method
for efficiently obtaining highly sensitive mAbs. We successfully established anti-GPCRs mAbs against the mCCR315–17
and mCCR818,19 by CBIS method.
In the future study, we will try to obtain more highly
sensitive anti-GPCR mAbs by using the same strategy of
C9Mab-11 development. In our previous study, numerous
mAbs against membrane proteins, such as EGFR,34 HER2,35
HER3,36 CD20,26 CD44,32 CD133,37 EpCAM,28
TROP2,29,30 and podoplanin27,31,38–40 have been produced
by using CBIS method and the epitopes of those mAbs havebeen already identified.41–44 It is expected that more sensitive
and useful mAbs will be obtained against these proteins byimmunizing the identified epitopes.
The N-terminus of several GPCRs, including CCR2,
CCR3, CCR5, and CXCR1 was defined as the ligand-binding
region.45 The N-terminus of CCR9 is also thought to beimportant for the ligand binding, because the mAb, whichbinds to N-terminus of CCR9, was reported to block the interaction between CCR9 and CCL25.46,47 The CCR9/CCL25
expression is increased in various inflammatory diseases,
such as myocardial infarction, rheumatoid arthritis, IBD,
asthma, and tumors8–10,13; therefore, the CCR9/CCL25 interaction is expected as an ideal therapeutic target.
The C9Mab-11 may have therapeutic effects for the
aforementioned diseases. In the immune system, CCR9 plays
a vital role in the regulation of T cells. T cells recruitment to
the intestine and colon is triggered by CCR9 stimulation.48,49
A tumor acidity-responsive nanoparticle delivery system(NP-siCD47/CCL25) can release CCL25 protein and CD47
small interfering RNA in tumor. The released CCL25 promoted the infiltration of CCR9+CD8+ T cells in tumor, which
resulted in inhibition of tumor growth and metastasis througha T cell-dependent immunity.50 Therefore, CCR9+ T cells
have positive effect for cancer treatment, through activation

The binding affinity of C9Mab-11 was assessed with
CHO/hCCR9 and MOLT-4 cells using flow cytometry. Results showed that the KD values of C9Mab-11 for
CHO/hCCR9 and MOLT-4 cells were 1.2 · 10-9 M and
4.9 · 10-10 M, respectively (Fig. 3A, B). These results indicated that C9Mab-11 possesses high affinity for CHO/hCCR9and MOLT-4 cells.
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307

Western blot analyses

We investigated whether C9Mab-11 can be used forwestern blot analysis by analyzing CHO-K1 andCHO/hCCR9 cell lysates. As shown in Figure 4, C9Mab-11
could clearly detect hCCR9 as around 48-kDa band (hCCR9+ Myc-DDK tag) in CHO/hCCR9 cell lysates, whereas noband was detected in parental CHO-K1 cells. An anti-

FIG. 3. The determination of the binding affinity of
C9Mab-11. CHO/hCCR9 (A) or MOLT-4 (B) cells weresuspended in 100 mL serially diluted C9Mab-11 (10–0.0006 mg/mL for both cells). Then, cells were treated with
Alexa Fluor 488-conjugated anti-mouse IgG. Fluorescencedata were subsequently collected using BD FACSLyric,after the calculation of the dissociation constant (KD) by
GraphPad PRISM 6.

Discussion

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TANAKA ET AL.

FIG. 4. The detection of hCCR9 by western blot analysis. Cell lysates of CHO-K1 and CHO/hCCR9 (10 mg) were
electrophoresed and transferred onto PVDF membranes. The membranes were incubated with 1 mg/mL of C9Mab-11,
1 mg/mL of an anti-DYKDDDDK mAb (clone 1E6), and 1 mg/mL of an anti-IDH1 mAb (clone RcMab-1) and subsequently
with peroxidase-conjugated anti-mouse or anti-rat immunoglobulins. IDH1, isocitrate dehydrogenase 1; PVDF, polyvinylidene difluoride.

of tumor immunity. Recently, it has shown that chimericantigen receptor T cells targeting CCR9 possess antileukemic
activity against T-ALL in vitro and in vivo.51 C9Mab-11 is
expected to be effective in cancer immunotherapy.
C9Mab-11 is applicable to various experiments, such as
flow cytometry, ELISA, and western blot analysis. Therefore,
C9Mab-11 will also contribute to further research related to
CCR9 functions.
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 grantnos. JP22ama121008 (to Y.K.), JP22am0401013 (to Y.K.),
JP22bm1004001 (to Y.K.), JP22ck0106730 (to Y.K.), andJP21am0101078 (to Y.K.).
References

1. Nagarsheth N, Wicha MS, Zou W. Chemokines in thecancer microenvironment and their relevance in cancer
immunotherapy. Nat Rev Immunol 2017;17:559–572; doi:
10.1038/nri.2017.49
2. Ozga AJ, Chow MT, Luster AD. Chemokines and the immune response to cancer. Immunity 2021;54:859–874; doi:
10.1016/j.immuni.2021.01.012
3. Tu Z, Xiao R, Xiong J, et al. CCR9 in cancer: Oncogenicrole and therapeutic targeting. J Hematol Oncol 2016;9:10;
doi: 10.1186/s13045-016-0236-7

4. Wurbel MA, Malissen B, Campbell JJ. Complex regulationof CCR9 at multiple discrete stages of T cell development.
Eur J Immunol 2006;36:73–81; doi: 10.1002/eji.200535203
5. Wurbel MA, Philippe JM, Nguyen C, et al. The chemokine
TECK is expressed by thymic and intestinal epithelial cells
and attracts double- and single-positive thymocytes expressing the TECK receptor CCR9. Eur J Immunol 2000;
30:262–271; doi: 10.1002/1521-4141(200001)30:1 <262::
Aid-immu262>3.0.Co;2-0
6. Campbell DJ, Butcher EC. Intestinal attraction: CCL25
functions in effector lymphocyte recruitment to the small
intestine. J Clin Invest 2002;110:1079–1081; doi: 10.1172/
jci16946
7. Wu X, Sun M, Yang Z, et al. The roles of CCR9/CCL25 in
inflammation and inflammation-associated diseases. Front
Cell Dev Biol 2021;9:686548; doi: 10.3389/fcell.2021.686548
8. Huang Y, Wang D, Wang X, et al. Abrogation of CC
chemokine receptor 9 ameliorates ventricular remodeling inmice after myocardial infarction. Sci Rep 2016;6:32660;
doi: 10.1038/srep32660
9. Nakamoto N, Ebinuma H, Kanai T, et al. CCR9+ macrophages are required for acute liver inflammation in mousemodels of hepatitis. Gastroenterology 2012;142:366–376;
doi: 10.1053/j.gastro.2011.10.039
10. Wurbel MA, McIntire MG, Dwyer P, et al. CCL25/CCR9interactions regulate large intestinal inflammation in amurine model of acute colitis. PLoS One 2011;6:e16442;
doi: 10.1371/journal.pone.0016442
11. Walters MJ, Wang Y, Lai N, et al. Characterization of
CCX282-B, an orally bioavailable antagonist of the CCR9chemokine receptor, for treatment of inflammatory bowel
disease. J Pharmacol Exp Ther 2010;335:61–69; doi:
10.1124/jpet.110.169714

Downloaded by 210.167.28.137 from www.liebertpub.com at 12/28/22. For personal use only.

DEVELOPMENT OF ANTI-HCCR9 MAB

12. Zhang J, Romero J, Chan A, et al. Biarylsulfonamide CCR9
inhibitors for inflammatory bowel disease. Bioorg Med
Chem Lett 2015;25:3661–3664; doi: 10.1016/j.bmcl.2015.06.046
13. Qiuping Z, Qun L, Chunsong H, et al. Selectively increased
expression and functions of chemokine receptor CCR9 on
CD4+ T cells from patients with T-cell lineage acutelymphocytic leukemia. Cancer Res 2003;63:6469–6477.
14. McGuire HM, Vogelzang A, Ma CS, et al. A subset ofinterleukin-21+ chemokine receptor CCR9+ T helper cells
target accessory organs of the digestive system in autoimmunity. Immunity 2011;34:602–615; doi: 10.1016/j.immuni.2011.01.021
15. Asano T, Suzuki H, Goto N, et al. Establishment of novelanti-mouse CCR3 monoclonal antibodies (C(3)Mab-6 andC(3)Mab-7) by N-terminal peptide immunization. Monoclon Antib Immunodiagn Immunother 2022;41:94–100;
doi: 10.1089/mab.2021.0065
16. Asano T, Suzuki H, Tanaka T, et al. C(3)Mab-3: A
monoclonal antibody for mouse CC chemokine receptor 3
for flow cytometry. Monoclon Antib Immunodiagn Immunother 2022;41:74–79; doi: 10.1089/mab.2021.0062
17. Saito M, Harigae Y, Li G, et al. C(3)Mab-2: An anti-mouse
CCR3 monoclonal antibody for immunocytochemistry.
Monoclon Antib Immunodiagn Immunother 2022;41:45–
49; doi: 10.1089/mab.2021.0050
18. Saito M, Suzuki H, Tanaka T, et al. Development of ananti-mouse CCR8 monoclonal antibody (C(8)Mab-1) forflow cytometry and immunocytochemistry. Monoclon Antib Immunodiagn Immunother 2022; doi: 10.1089/mab.2021.0069
19. Saito M, Tanaka T, Asano T, et al. C(8)Mab-2: An anti-mouse
C-C motif chemokine receptor 8 monoclonal antibody for
immunocytochemistry. Monoclon Antib Immunodiagn Immunother 2022;41:115–119; doi: 10.1089/mab.2021.0045
20. Suzuki H, Saito M, Asano T, et al. C(8)Mab-3: An antimouse CCR8 monoclonal antibody for immunocytochemistry. Monoclon Antib Immunodiagn Immunother 2022;41:
110–114; doi: 10.1089/mab.2022.0002
21. Nanamiya R, Takei J, Asano T, et al. Development of antihuman CC chemokine receptor 9 monoclonal antibodies forflow cytometry. Monoclon Antib Immunodiagn Immunother 2021;40:101–106; doi: 10.1089/mab.2021.0007
22. Takei J, Asano T, Li G, et al. Epitope mapping of an antihuman CCR9 monoclonal antibody (C(9)Mab-1) using
enzyme-linked immunosorbent assay. Monoclon AntibImmunodiagn Immunother 2021;40:239–242; doi: 10.1089/
mab.2021.0037
23. Saito M, Suzuki H, Harigae Y, et al. C(9)Mab-1: An antimouse CCR9 monoclonal antibody for immunocytochemistry. Monoclon Antib Immunodiagn Immunother 2022;41:
120–124; doi: 10.1089/mab.2021.0052
24. Kato Y. Specific monoclonal antibodies against IDH1/2
mutations as diagnostic tools for gliomas. Brain Tumor
Pathol 2015;32:3–11; doi: 10.1007/s10014-014-0202-4
25. Ikota H, Nobusawa S, Arai H, et al. Evaluation of IDH1
status in diffusely infiltrating gliomas by immunohistochemistry using anti-mutant and wild type IDH1 antibodies. Brain Tumor Pathol 2015;32:237–244; doi:
10.1007/s10014-015-0222-8
26. Furusawa Y, Kaneko MK, Kato Y. Establishment ofC(20)Mab-11, a novel anti-CD20 monoclonal antibody, forthe detection of B cells. Oncol Lett 2020;20:1961–1967;
doi: 10.3892/ol.2020.11753

309

27. Hosono H, Asano T, Takei J, et al. Development of an antielephant podoplanin monoclonal antibody PMab-265 forflow cytometry. Monoclon Antib Immunodiagn Immunother 2021;40:141–145; doi: 10.1089/mab.2021.0015
28. Li G, Suzuki H, Asano T, et al. Development of a novelanti-EpCAM monoclonal antibody for various applications.
Antibodies (Basel) 2022;11; doi: 10.3390/antib11020041
29. Sayama Y, Kaneko MK, Kato Y. Development and characterization of TrMab-6, a novel anti-TROP2 monoclonal
antibody for antigen detection in breast cancer. Mol Med
Rep 2021;23; doi: 10.3892/mmr.2020.11731
30. Sayama Y, Kaneko MK, Takei J, et al. Establishment of anovel anti-TROP2 monoclonal antibody TrMab-29 for
immunohistochemical analysis. Biochem Biophys Rep
2021;25:100902; doi: 10.1016/j.bbrep.2020.100902
31. Tanaka T, Asano T, Sano M, et al. Development of
monoclonal antibody PMab-269 against California Sea
Lion Podoplanin. Monoclon Antib Immunodiagn Immunother 2021;40:124–133; doi: 10.1089/mab.2021.0011
32. Yamada S, Itai S, Nakamura T, et al. Detection of highCD44 expression in oral cancers using the novel monoclonal antibody, C(44)Mab-5. Biochem Biophys Rep 2018;
14:64–68; doi: 10.1016/j.bbrep.2018.03.007
33. Jo M, Jung ST. Engineering therapeutic antibodies targeting G-protein-coupled receptors. Exp Mol Med 2016;48:
e207; doi: 10.1038/emm.2015.105
34. Itai S, Kaneko MK, Fujii Y, et al. Development of EMab-51,
a sensitive and specific anti-epidermal growth factor receptor
monoclonal antibody in flow cytometry, western blot, andimmunohistochemistry. Monoclon Antib Immunodiagn Immunother 2017;36:214–219; doi: 10.1089/mab.2017.0028
35. Kaneko MK, Yamada S, Itai S, et al. Development of ananti-HER2 monoclonal antibody H2Mab-139 against coloncancer. Monoclon Antib Immunodiagn Immunother 2018;37:59–62; doi: 10.1089/mab.2017.0052
36. Asano T, Ohishi T, Takei J, et al. Anti-HER3 monoclonal
antibody exerts antitumor activity in a mouse model ofcolorectal adenocarcinoma. Oncol Rep 2021;46; doi:
10.3892/or.2021.8124
37. Itai S, Fujii Y, Nakamura T, et al. Establishment of CMab-43,
a sensitive and specific anti-CD133 monoclonal antibody, for
immunohistochemistry. Monoclon Antib Immunodiagn Immunother 2017;36:231–235; doi: 10.1089/mab.2017.0031
38. Yamada S, Ogasawara S, Kaneko MK, et al. LpMab-23: A
cancer-specific monoclonal antibody against human podoplanin. Monoclon Antib Immunodiagn Immunother 2017;36:72–76; doi: 10.1089/mab.2017.0001
39. Yamada S, Itai S, Nakamura T, et al. PMab-52: Specific andsensitive monoclonal antibody against cat podoplanin for
immunohistochemistry. Monoclon Antib Immunodiagn Immunother 2017;36:224–230; doi: 10.1089/mab.2017.0027
40. Furusawa Y, Takei J, Sayama Y, et al. Development of ananti-bear podoplanin monoclonal antibody PMab-247 forimmunohistochemical analysis. Biochem Biophys Rep
2019;18:100644; doi: 10.1016/j.bbrep.2019.100644
41. Takei J, Asano T, Suzuki H, et al. Epitope mapping of theanti-CD44 monoclonal antibody (C(44)Mab-46) using
alanine-scanning mutagenesis and surface plasmon resonance. Monoclon Antib Immunodiagn Immunother 2021;
40:219–226; doi: 10.1089/mab.2021.0028
42. Kaneko MK, Oki H, Hozumi Y, et al. Monoclonal antibody
LpMab-9 recognizes O-glycosylated N-terminus of human
podoplanin. Monoclon Antib Immunodiagn Immunother2015;34:310–317; doi: 10.1089/mab.2015.0022

Downloaded by 210.167.28.137 from www.liebertpub.com at 12/28/22. For personal use only.

310

43. Tanaka T, Asano T, Sano M, et al. Epitope mapping of theanti-California Sea Lion podoplanin monoclonal antibody
PMab-269 using alanine-scanning mutagenesis and ELISA.
Monoclon Antib Immunodiagn Immunother 2021;40:196–
200; doi: 10.1089/mab.2021.0017
44. Asano T, Takei J, Suzuki H, et al. Epitope mapping of ananti-HER2 monoclonal antibody (H(2)Mab-181) using
enzyme-linked immunosorbent assay. Monoclon AntibImmunodiagn Immunother 2021;40:255–260; doi: 10.1089/
mab.2021.0029
45. Allen SJ, Crown SE, Handel TM. Chemokine: Receptor
structure, interactions, and antagonism. Annu Rev Immunol
2007;25:787–820;
doi:
10.1146/annurev.immunol.24.021605.090529
46. Zabel BA, Agace WW, Campbell JJ, et al. Human G proteincoupled receptor GPR-9-6/CC chemokine receptor 9 is selectively expressed on intestinal homing T lymphocytes,
mucosal lymphocytes, and thymocytes and is required for
thymus-expressed chemokine-mediated chemotaxis. J Exp
Med 1999;190:1241–1256; doi: 10.1084/jem.190.9.1241
47. Chamorro S, Vela M, Franco-Villanueva A, et al. Antitumor effects of a monoclonal antibody to human CCR9 in
leukemia cell xenografts. MAbs 2014;6:1000–1012; doi:
10.4161/mabs.29063
48. Tubo NJ, Wurbel MA, Charvat TT, et al. A systemicallyadministered small molecule antagonist of CCR9 acts as atissue-selective inhibitor of lymphocyte trafficking. PLoS
One 2012;7:e50498; doi: 10.1371/journal.pone.0050498

TANAKA ET AL.

49. Greis C, Rasuly Z, Janosi RA, et al. Intestinal T lymphocyte homing is associated with gastric emptying and epithelial barrier function in critically ill: A prospective
observational study. Crit Care 2017;21:70; doi: 10.1186/
s13054-017-1654-9
50. Chen H, Cong X, Wu C, et al. Intratumoral delivery of
CCL25 enhances immunotherapy against triple-negative
breast cancer by recruiting CCR9(+) T cells. Sci Adv 2020;
6:eaax4690; doi: 10.1126/sciadv.aax4690
51. Maciocia PM, Wawrzyniecka PA, Maciocia NC, et al.
Anti-CCR9 chimeric antigen receptor T cells for T-cell
acute lymphoblastic leukemia. Blood 2022;140:25–37; doi:
10.1182/blood.2021013648

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: August 4, 2022
Accepted: September 30, 2022