MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 41, Number 3, 2022
ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2022.0010
Open camera or QR reader andscan code to access this article
and other resources online.

Cx6Mab-1: A Novel Anti-Mouse CXCR6 Monoclonal
Antibody Established by N-Terminal Peptide Immunization

Downloaded by Yukinari Kato from www.liebertpub.com at 06/23/22. For personal use only.

Kaishi Kitamura,1,* Hiroyuki Suzuki,1,* Mika K. Kaneko,2 and Yukinari Kato1,2

The CXC chemokine receptor 6 (CXCR6) is a member of the G protein-coupled receptor family that is highly
expressed in helper T type 1 cells, natural killer cells, cytotoxic T lymphocytes, and various type of cells intumor microenvironment (TME). CXCR6 has been proposed as a therapeutic target against tumors throughregulation of the tumor TME. In this study, we developed specific and sensitive monoclonal antibodies(mAbs) for mouse CXCR6 (mCXCR6), which are useful for flow cytometry and Western blotting by
N-terminal peptide immunization into rat. The established anti-mCXCR6 mAb, Cx6Mab-1 (rat IgG1,
kappa), reacted with not only mCXCR6-overexpressed Chinese hamster ovary-K1 (CHO/mCXCR6) but
also mCXCR6-endogenously expressed cell lines, such as P388 (mouse lymphoid neoplasm) and J774-1(mouse macrophage-like) through flow cytometry. Kinetic analyses using flow cytometry indicated that the
dissociation constants (KD) of Cx6Mab-1 for CHO/mCXCR6, P388, and J774-1 cells were 1.7 · 10-9 M,
3.4 · 10-7 M, and 3.8 · 10-7 M, respectively. Furthermore, Cx6Mab-1 could detect endogenous mCXCR6 in
P388 and J774-1 cells by Western blotting. These results indicated that Cx6Mab-1 is useful for detecting
mCXCR6 by flow cytometry and Western blotting, and provides a possibility for targeting CXCR6-expressing
cells in vivo experiments.
Keywords: mouse CXCR6, monoclonal antibody, peptide immunization

IntroductionT

he CXC chemokine receptor 6 (CXCR6) is a chemokine receptor for CXCL16. CXCR6 is primarily expressed in T cells, including cytotoxic T lymphocytes (CTLs),
helper T type 1 (Th1) cells, natural killer (NK) cells, andNK T cells. Upon the chemokines binding to CXCR6, thechemokines can stimulate intracellular signaling pathway
in CXCR6-expressing cells, which mediate various cellularfunctions, including the infiltration into target tissues.(1)CXCR6 has been reported to play a critical role in lymphocyte homeostasis in the liver, based on the constitutive
secretion of CXCL16 in liver sinusoids. CXCL16 supports
the maintenance of CXCR6-expressing NK, NK T, and CD8+
memory T cells in liver.(2)CXCL16 is the largest ligand compared with the other
CXC chemokines. CXCL16 has been found in soluble form

(sCXCL16) and transmembrane form (TM-CXCL16).(3)Therefore, the forward and reverse signaling pathways aretransduced through TM-CXCL16 and CXCR6 interaction.
In forward signaling, sCXCL16 or TM-CXCL16 binds to
CXCR6 that promotes signal transduction from CXCR6.
In reverse signaling, CXCR6-bound TM-CXCL16 mediates
the signal transduction.(4)The expression of CXCL16 and CXCR6 has been observed in numerous human cancers, including pancreaticcancer, thyroid carcinoma, and breast cancer, and influences
both tumor and the tumor microenvironment (TME).(4) The
growing number of evidence has shown the relationship between chronic inflammation and tumor development.(5) CXCL16–CXCR6 axis plays critical roles in not
only lymphocyte recruitment to tumor sites, but also in tumor development through activation of tumor invasivenessand cancer-associated fibroblasts (CAFs).(4)Departments of 1Molecular Pharmacology and 2Antibody Drug Development, Tohoku University Graduate School of Medicine, Sendai,
Japan.
*Both these authors contributed equally to this study.

133

134

Using the Cell-Based Immunization and Screening (CBIS)
method, we have developed many monoclonal antibodies(mAbs) against membrane proteins, such as anti-CD19,(6) antiCD20,(7,8) anti-CD44,(9) anti-CD133,(10) anti-EpCAM,(11,12) antiHER3,(13) anti-KLRG1,(14) anti-TIGIT,(15) anti-TROP2,(16,17)
anti-programmed cell death ligand 1 (PD-L1),(18) andanti-podoplanin(19–30) mAbs. The CBIS method includes theimmunization of antigen-overexpressing cells and the highthroughput hybridoma screening using flow cytometry.
Anti-chemokine receptor mAbs, including anti-mouse
CCR3,(31) anti-mouse CCR8,(32) and anti-human CCR9(33)mAbs, were also successfully developed using the CBIS
method. Furthermore, we have developed several antichemokine receptor mAbs by peptide immunization.(34) This
study developed anti-mouse CXCR6 (mCXCR6) mAbs using N-terminal peptide immunization.

Downloaded by Yukinari Kato from www.liebertpub.com at 06/23/22. For personal use only.

Materials and Methods
Preparation of cell lines

Chinese hamster ovary (CHO)-K1 and P3X63Ag8U.1(P3U1) cells were obtained from the American Type Culture
Collection (Manassas, VA). P388 (mouse lymphoid neoplasm) and J774-1 (mouse macrophage-like) cells wereobtained from the Cell Resource Center for Biomedical
Research Institute of Development, Aging and Cancer
Tohoku University (Miyagi, Japan). The synthesized DNA(Eurofins Genomics KK, Tokyo, Japan) encoding mCXCR6(Accession No. NM_030712) was subsequently subcloned
into a pCAG-Ble vector (FUJIFILM Wako Pure Chemical
Corporation, Osaka, Japan).
Then, the mCXCR6 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), after which cultivation in a
medium, containing 0.5 mg/mL of Zeocin (InvivoGen, San
Diego, CA), was conducted.
CHO-K1, mCXCR6-overexpressed CHO-K1 (CHO/
mCXCR6), P3U1, P388, and J774-1 were cultured in a
Roswell Park Memorial Institute (RPMI) 1640 medium(Nacalai Tesque, Inc., Kyoto, Japan), with 10% heatinactivated fetal bovine serum (FBS; Thermo Fisher Scientific, Inc.), 100 units/mL of penicillin, 100 lg/mL of
streptomycin, and 0.25 lg/mL of amphotericin B (Nacalai
Tesque, Inc.). Cells were grown in a humidified incubator at
37C, at an atmosphere of 5% CO2 and 95% air.
Antibodies

An anti-mCXCR6 mAb (clone SA051D1) was purchasedfrom BioLegend (San Diego, CA). A secondary Alexa Fluor
488-conjugated anti-rat IgG was purchased from Cell Signaling Technology, Inc. (Danvers, MA).
PeptidesEurofins Genomics KK (Tokyo, Japan) synthesized a partial
sequence of the N-terminal extracellular region of mCXCR6(1-MDDGHQESALYDGHYEGDF-19) plus C-terminal cysteine. Subsequently, the keyhole limpet hemocyanin (KLH)
was conjugated at the C-terminus of the peptide.

KITAMURA ET AL.
Production of hybridomas

A Sprague–Dawley rat (5 weeks old) were purchasedfrom CLEA Japan (Tokyo, Japan). Animals were housed
under specific pathogen-free conditions. All animal experiments were also conducted according to relevant
guidelines and regulations to minimize animal sufferingand distress in the laboratory. The Animal Care and Use
Committee of Tohoku University (Permit No. 2019NiA001) approved animal experiments. Rats were monitored
daily for health during the full 4-week duration of the experiment. A reduction of >25% of the total body weight was
defined as a humane endpoint. During sacrifice, the rat was
euthanized through cervical dislocation, after which death
was verified through respiratory and cardiac arrest.
To develop mAbs against mCXCR6, we intraperitoneallyimmunized one rat with 100 lg of the KLH-conjugated
mCXCR6 peptide plus Imject Alum (Thermo Fisher Scientific, Inc.). The procedure included three additional immunization every week (100 lg/rat), which was followed bya final booster intraperitoneal injection (100 lg/rat), 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 RPMI medium with 10% FBS, 100 units/mLof penicillin, 100 lg/mL of streptomycin, and 0.25 lg/mLof amphotericin B.
For the hybridoma selection, hypoxanthine, aminopterin,
and thymidine (HAT; Thermo Fisher Scientific, Inc.) wereadded into the medium. Supernatants were subsequently
screened using enzyme-linked immunosorbent assay (ELISA)
with the mCXCR6 peptide, followed by flow cytometry,using CHO/mCXCR6, CHO-K1, P388, and J774-1 cells.
Enzyme-linked immunosorbent assay

The synthesized peptide (MDDGHQESALYDGHYE
GDFC) was immobilized on Nunc Maxisorp 96-well immunoplates (Thermo Fisher Scientific, Inc.) at a concentrationof 1 lg/mL for 30 minutes at 37C. After washing withphosphate-buffered saline (PBS) containing 0.05% Tween 20(PBST; Nacalai Tesque, Inc.), wells were blocked with 1%
bovine serum albumin (BSA)-containing PBST for 30 minutes at 37C. Plates were then incubated with supernatantsof hybridomas, followed by peroxidase-conjugated anti-rat
immunoglobulins (1:20,000 diluted; Sigma-Aldrich Corp.,
St. Louis, MO). Next, enzymatic reactions were conducted,using ELISA POD Substrate TMB Kit (Nacalai Tesque, Inc.),
followed by measurement of the optical density at 655 nm,
using iMark microplate reader (Bio-Rad Laboratories, Inc.,
Berkeley, CA).
Purification of mAbs

The cultured supernatants of Cx6Mab-1–producing hybridomas were collected through centrifugation at 2330 · g for
5 minutes, followed by filtration using Steritop (0.22 mm;
Merck KGaA, Darmstadt, Germany). Filtered supernatants
were subsequently applied to 1 mL of Ab-Capcher ExTra(ProteNova Co., Ltd., Kagawa, Japan). After washing with
PBS, bound antibodies were eluted with IgG Elution Buffer(Cat. No.: 21009, Thermo Fisher Scientific, Inc.), followed
by immediate neutralization of eluates, using 1 M Tris-HCl

ANTIMOUSE CXCR6 MONOCLONAL ANTIBODY135(pH 8.0). Finally, eluates were concentrated, after which PBS
was used to replace with the elution buffer, using Amicon
Ultra (Merck KGaA).
Flow cytometric analyses

CHO-K1 and CHO/mCXCR6 were harvested after a brief
exposure to 0.25% trypsin and 1 mM ethylenediaminetetraacetic acid (EDTA; Nacalai Tesque, Inc.). Cells were
subsequently washed with 0.1% BSA 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 488conjugated anti-rat IgG (1:2000; Cell Signaling Technology,Inc.), after which fluorescence data were collected using theSA3800 Cell Analyzer (Sony Biotechnology Corp.).

Downloaded by Yukinari Kato from www.liebertpub.com at 06/23/22. For personal use only.

Determination of dissociation constant (KD)through flow cytometry

CHO/mCXCR6, P388, and J774-1 were suspended in
100 lL serially diluted anti-mCXCR6 mAbs, after which
50 lL Alexa Fluor 488-conjugated anti-rat IgG (1:200; Cell
Signaling Technology, Inc.) was added. Then, fluorescencedata were collected, using BD FACSLyric (BD Biosciences,Franklin Lakes, NJ). The KD was subsequently calculated byfitting saturation binding curves to the built-in one-sitebinding models in GraphPad PRISM 8 (GraphPad Software,Inc., La Jolla, CA).
Western blot analysis

Cell lysates were prepared by hypotonic cell lysis buffer(20 mM HEPES–KOH, pH 7.5, 10 mM KCl, 1.5 mM MgCl2,
0.2 mM EDTA, 0.1% Triton-X100), and were boiled in
sodium dodecyl sulfate (SDS) sample buffer (Nacalai Tesque, Inc.). Proteins (10 lg for CHO-K1 and CHO/mCXCR6
or 15 lg for P388 and J774-1) were separated on 5%–20%
polyacrylamide gels (FUJIFILM Wako Pure Chemical Corporation) and transferred onto polyvinylidene difluoride(PVDF) membranes (Merck KGaA).
After blocking with 4% skim milk (Nacalai Tesque, Inc.)in PBST, membranes were incubated with 10 lg/mL ofCx6Mab-1, 1 lg/mL of an anti-isocitrate dehydrogenase 1(IDH1; RcMab-1)(35,36) or 1 lg/mL of an anti-b-actin mAb(clone AC-15; Sigma-Aldrich Corp.). Membranes werethen incubated with either peroxidase-conjugated anti-rat
and anti-mouse immunoglobulins (diluted 1:1000; Agilent
Technologies, Inc., Santa Clara, CA). 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-mCXCR6 mAbs
by peptide immunization

To develop anti-mCXCR6 mAbs, one rat was immunizedwith the KLH-conjugated mCXCR6 peptide (Fig. 1A).
Spleen was then excised from the rat, after which splenocytes
were fused with myeloma P3U1 cells (Fig. 1B). Developed
hybridomas were subsequently seeded into twenty 96-wellplates and cultivated for 6 days. Then, positive wells forthe naked mCXCR6 peptide were selected using ELISA,

FIG. 1. A schematic illustration showing the productionof anti-mCXCR6 mAbs. (A) The mCXCR6p1-19C-KLH
peptide was immunized into an SD rat, using an intraperitoneal injection. (B) Spleen cells were then fused with P3U1
cells. (C) Subsequently, culture supernatants were screened
through ELISA and flow cytometry to select anti-mCXCR6mAb-producing hybridomas. (D) After limiting dilution
and some additional screenings, anti-mCXCR6 mAbs were
finally established. CXCR6, CXC chemokine receptor 6;
ELISA, enzyme-linked immunosorbent assay; KLH, keyhole limpet hemocyanin; mAbs, monoclonal antibodies;
mCXCR6, mouse CXCR6; SD, Sprague–Dawley.
followed by the selection of mCXCR6-expressing cellreactive and CHO-K1 nonreactive supernatants, using flow
cytometry (Fig. 1C). The ELISA screening identified 26
out of 1914 wells (1.36%), which strongly reacted with the
naked mCXCR6 peptide.
The flow cytometric screening identified 12 out of the
26 wells (46.2%), which exhibited strong signals to CHO/
mCXCR6 cells, but not to CHO-K1 cells. In addition, positive signals were also obtained in endogenous mCXCR6expressing P388 and J774-1 cells in 5 out of the 12 wells(41.7%). After the limiting dilution and several additional
screenings, an anti-mCXCR6 mAb (clone Cx6Mab-1; rat
IgG1, kappa) was finally established (Fig. 1D).
Flow cytometric analysis

We conducted flow cytometry using two anti-mCXCR6
mAbs: Cx6Mab-1 and SA051D1 against CHO/mCXCR6,
P388, and J774-1 cell lines. Both Cx6Mab-1 and SA051D1

136

Kinetic analysis of the interaction between Cx6Mab-1
and mCXCR6-expressing cells through flow cytometry

To determine the KD of Cx6Mab-1 with mCXCR6expressing cells, we conducted kinetic analysis of the interaction between Cx6Mab-1 and CHO/mCXCR6, P388, andJ774-1 cell lines using flow cytometry. The geometric meanof the fluorescence intensity was then plotted versus theconcentration of Cx6Mab-1. The KD of Cx6Mab-1 for CHO/
mCXCR6, P388, and J774-1 cells was determined as
1.7 · 10-9 M, 3.4 · 10-7 M, and 3.8 · 10-7 M, respectively

Downloaded by Yukinari Kato from www.liebertpub.com at 06/23/22. For personal use only.

recognized CHO/mCXCR6 cells dose-dependently at 10, 1,
and 0.1 lg/mL. Cx6Mab-1 could recognize CHO/mCXCR6
cells at low concentration of 0.01 lg/mL; in contrast, SA051D1
needed >0.1 lg/mL for the detection of CHO/mCXCR6(Fig. 2A). Parental CHO-K1 cells were not recognizedeven at 10 lg/mL of both mAbs (Fig. 2B). Cx6Mab-1 andSA051D1 reacted with P388 and J774-1 cells at >1 lg/mL(Fig. 3A, B). These results suggested that Cx6Mab-1 specifically recognizes mCXCR6, and is also useful for detecting
endogenous mCXCR6 by flow cytometry.

KITAMURA ET AL.

FIG. 2. Flow cytometry to mCXCR6 overexpressing cells using anti-mCXCR6 mAbs. CHO/mCXCR6 (A) and CHO-K1(B) cells were treated with 0.01–10 mg/mL of Cx6Mab-1 and SA051D1, followed by treatment with Alexa Fluor 488conjugated anti-rat IgG. CHO, Chinese hamster ovary.

137

Downloaded by Yukinari Kato from www.liebertpub.com at 06/23/22. For personal use only.

ANTIMOUSE CXCR6 MONOCLONAL ANTIBODY

FIG. 3. Flow cytometry to endogenously mCXCR6-expressing cells using anti-mCXCR6 mAbs. P388 (A) and J-774-1(B) cells were treated with 0.01–10 mg/mL of Cx6Mab-1 and SA051D1, followed by treatment with Alexa Fluor 488conjugated anti-rat IgG.
(Fig. 4). These results indicate that Cx6Mab-1 possesses thehigh affinity for CHO/mCXCR6 and the moderate affinity forP388 and J774-1 cells.
Western blot analysis

Western blotting was performed to further assess thesensitivity of Cx6Mab-1. Lysates of CHO-K1 and CHO/
mCXCR6 cells were probed. As shown in Figure 5A, Cx6Mab-1
detected mCXCR6 as a *40-kDa main band. However,Cx6Mab-1 did not detect any bands from lysates of CHO-K1
cells. Next, we examined the detection of endogenous mCXCR6

using lysates from P388 and J774-1 cells. As shown in
Figure 5B, Cx6Mab-1 could detect mCXCR6 as a *40-kDa
main band, and double bands between 75 and 100 kDa fromlysates of P388 and J774-1 cells. Similar molecular weightbands were also detected in lysates of CHO/mCXCR6 cells(Fig. 5A). These results suggest that Cx6Mab-1 can detect bothexogenous and endogenous mCXCR6.
Discussion

In this study, we developed novel anti-mCXCR6 mAbs using
N-terminal peptide immunization methods, and investigatedDownloaded by Yukinari Kato from www.liebertpub.com at 06/23/22. For personal use only.

138

KITAMURA ET AL.

FIG. 5. Western blotting using Cx6Mab-1. (A) Cell lysates of CHO-K1 and CHO/mCXCR6 were electrophoresed
and transferred onto PVDF membranes. The membranes
were incubated with 10 mg/mL of Cx6Mab-1 and 1 mg/mLof anti-IDH mAb (RcMab-1) and subsequently withperoxidase-conjugated anti-rat immunoglobulins. (B) Cell
lysates of P388 and J-774-1 were electrophoresed andtransferred onto PVDF membranes. The membranes wereincubated with 10 mg/mL of Cx6Mab-1 and 1 mg/mL of antib-actin mAb and subsequently with peroxidase-conjugatedanti-rat and mouse immunoglobulins, respectively. Arrows
indicate the predicted size of mCXCR6 (*40 kDa). IDH,
isocitrate dehydrogenase; PVDF, polyvinylidene difluoride.
FIG. 4. The determination of the binding affinity of
Cx6Mab-1. CHO/mCXCR6 (A), P388 (B), and J-774-1(C) cells were suspended in 100 mL serially diluted Cx6Mab1 (100 to 0.006 mg/mL for CHO/mCXCR6 and 400 to
0.025 mg/mL for P388 and J-774-1). Then, cells were treatedwith Alexa Fluor 488-conjugated anti-rat IgG. Fluorescencedata were subsequently collected using a BD FACSLyric,after the calculation of the dissociation constant (KD) by
GraphPad PRISM 8.
the usefulness for flow cytometry and Western blotting fordetecting the endogenous mCXCR6. The synthesized peptide
immunization is a simple strategy and possesses several advantages to generate anti-CXCR mAbs since the N-terminal
regions of CXCR families are largely and linearly exposed on
the cell surface.(37) The neutralizing activity could be expected because the N-terminus of CXCR families is known tobe the ligand-binding domain.(37)

Although the three-dimensional structure of CXCR6and its ligand CXCL16 has not been solved experimentally, ahomology model of CXCR6–CXCL16 was reported throughcoarse-grained molecular dynamics simulations. According
to the simulations, the N-terminal region of CXCR6 incomplex with CXCL16 is positioned in the middle of extracellular loops, triggers the system’s evolution, rotations,movements, and transmembrane interactions, and changes
the inactive state to an active state.(38) Since the CXCL16CXCR6 interaction mediates the forward and reverse signaling pathways,(3,4) further investigation is required toevaluate the effect of Cx6Mab-1 to these pathways.
CXCL16–CXCR6 axis has been reported to possess thecomplex roles in TME. It mediates tumor progression
directly through promoting survival, proliferation, and metastasis of tumor cells or indirectly through enhancing lymphocytes, macrophages, and mesenchymal stem cells (MSCs)

Downloaded by Yukinari Kato from www.liebertpub.com at 06/23/22. For personal use only.

ANTIMOUSE CXCR6 MONOCLONAL ANTIBODY

infiltration.(4) The signaling of CXCR6 in recruited MSCs
leads to the conversion into CAFs.(39) Furthermore, therecruitment of tumor-promoting M2 macrophages is also promoted.(40) These events play critical roles in tumor progression
through activating the metastatic programs and angiogenesis.
In our previous studies, we changed the isotype of mAbs
into mouse IgG2a to retain antibody-dependent cellularcytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), which caused high antitumor activities in mice
xenograft models.(41–43) Since the subclass of Cx6Mab-1 is
rat IgG1, it does not possess ADCC and CDC. Therefore, infurther studies, the subclass of Cx6Mab-1 will be converted
into mouse IgG2a to evaluate the effect to CXCR6-expressing
tumor cells, CAFs, and macrophages in mice tumor models.
CXCR6 mediates the conversion of stem-like memory
cells into effector-like CTLs. In TME, CCR7+ dendritic cells
exist in perivascular niche, and secrete CXCL16 and transpresent the survival cytokine interleukin-15 (IL-15). CXCR6
expression and IL-15 trans-presentation are critical for thesurvival and local expansion of effector-like CTLs in TME
to potentiate their antitumor response.(44) These observations
indicate a critical function of CXCR6 for sustaining antitumor immune responses of CD8+ CTLs.
In tumor immune therapy, the infiltration of CTLs into
tumor is important factors to predict the efficacy.(45,46) The
PD-1+ effector-like CTLs uniformly express CXCR6 at thehighest level.(44) Therefore, the diagnostic use of Cx6Mab-1
to examine the CXCR6 expression in tumor-infiltrating CTLs
could provide the important information for combination
therapy of immune checkpoint inhibitor with CXCR6 agonists developed in the future.
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 JP22ama121008 (to Y.K.), JP22am0401013 (toY.K.), JP22bm1004001 (to Y.K.), JP22ck0106730 (to Y.K.),
and JP21am0101078 (to Y.K.).
References

1. Urbantat RM, Vajkoczy P, and Brandenburg S: Advancesin chemokine signaling pathways as therapeutic targets inglioblastoma. Cancers (Basel) 2021;13:2983.
2. Tse SW, Radtke AJ, Espinosa DA, Cockburn IA, andZavala F: The chemokine receptor CXCR6 is required forthe maintenance of liver memory CD8+ T cells specific for
infectious pathogens. J Infect Dis 2014;210:1508–1516.
3. Abel S, Hundhausen C, Mentlein R, Schulte A, Berkhout
TA, Broadway N, Hartmann D, Sedlacek R, Dietrich S,
Muetze B, Schuster B, Kallen KJ, Saftig P, Rose-John S,
and Ludwig A: The transmembrane CXC-chemokine
ligand 16 is induced by IFN-gamma and TNF-alpha andshed by the activity of the disintegrin-like metalloproteinase ADAM10. J Immunol 2004;172:6362–6372.
4. Gowhari Shabgah A, Qasim MT, Mojtaba Mostafavi S,
Olegovna Zekiy A, Ezzatifar F, Ahmadi M, Mohammadian
Haftcheshmeh S, and Gholizadeh Navashenaq J: CXC

139

chemokine ligand 16: a Swiss army knife chemokine incancer. Expert Rev Mol Med 2021;23:e4.
5. Greten FR, and Grivennikov SI: Inflammation and cancer:
triggers, mechanisms, and consequences. Immunity 2019;51:27–41.
6. Yamada S, Kaneko MK, Sayama Y, Asano T, Sano M,
Yanaka M, Nakamura T, Okamoto S, Handa S, Komatsu Y,
Nakamura Y, Furusawa Y, Takei J, and Kato Y: Development of novel mouse monoclonal antibodies against
human CD19. Monoclon Antib Immunodiagn Immunother2020;39:45–50.
7. Furusawa Y, Kaneko MK, and Kato Y: Establishment ofC(20)Mab-11, a novel anti-CD20 monoclonal antibody, forthe detection of B cells. Oncol Lett 2020;20:1961–1967.
8. Furusawa Y, Kaneko MK, and Kato Y: Establishment of ananti-CD20 monoclonal antibody (C(20)Mab-60) for immunohistochemical analyses. Monoclon Antib Immunodiagn
Immunother 2020;39:112–116.
9. Yamada S, Itai S, Nakamura T, Yanaka M, Kaneko MK,
and Kato Y: Detection of high CD44 expression in oral
cancers using the novel monoclonal antibody, C(44)Mab-5.
Biochem Biophys Rep 2018;14:64–68.
10. Itai S, Fujii Y, Nakamura T, Chang YW, Yanaka M, Saidoh
N, Handa S, Suzuki H, Harada H, Yamada S, Kaneko MK,
and Kato Y: Establishment of CMab-43, a sensitive andspecific anti-CD133 monoclonal antibody, for immunohistochemistry. Monoclon Antib Immunodiagn Immunother2017;36:231–235.
11. Hosono H, Ohishi T, Takei J, Asano T, Sayama Y, Kawada
M, Kaneko MK, and Kato Y: The anti-epithelial celladhesion molecule (EpCAM) monoclonal antibody
EpMab-16 exerts antitumor activity in a mouse model ofcolorectal adenocarcinoma. Oncol Lett 2020;20:383.
12. Kaneko MK, Ohishi T, Takei J, Sano M, Nakamura T,
Hosono H, Yanaka M, Asano T, Sayama Y, Harada H,
Kawada M, and Kato Y: AntiEpCAM monoclonal antibody
exerts antitumor activity against oral squamous cell carcinomas. Oncol Rep 2020;44:2517–2526.
13. Asano T, Ohishi T, Takei J, Nakamura T, Nanamiya R,
Hosono H, Tanaka T, Sano M, Harada H, Kawada M,
Kaneko MK, and Kato Y: Anti-HER3 monoclonal antibody
exerts antitumor activity in a mouse model of colorectal
adenocarcinoma. Oncol Rep 2021;46:173.
14. Asano T, Nanamiya R, Tanaka T, Kaneko MK, and Kato
Y: Development of antihuman killer cell lectin-like receptor subfamily G member 1 monoclonal antibodies for flow
cytometry. Monoclon Antib Immunodiagn Immunother2021;40:76–80.
15. Takei J, Asano T, Nanamiya R, Nakamura T, Yanaka M,
Hosono H, Tanaka T, Sano M, Kaneko MK, Harada H, andKato Y: Development of anti-human T cell immunoreceptor with Ig and ITIM domains (TIGIT) monoclonal
antibodies for flow cytometry. Monoclon Antib Immunodiagn Immunother 2021;40:71–75.
16. Sayama Y, Kaneko MK, Takei J, Hosono H, Sano M,
Asano T, and Kato Y: Establishment of a novel antiTROP2 monoclonal antibody TrMab-29 for immunohistochemical analysis. Biochem Biophys Rep 2021;25:
100902.
17. Tanaka T, Ohishi T, Asano T, Takei J, Nanamiya R,
Hosono H, Sano M, Harada H, Kawada M, Kaneko MK,
and Kato Y: An anti-TROP2 monoclonal antibody TrMab-6
exerts antitumor activity in breast cancer mouse xenograftmodels. Oncol Rep 2021;46:132.

Downloaded by Yukinari Kato from www.liebertpub.com at 06/23/22. For personal use only.

140

18. Yamada S, Itai S, Nakamura T, Yanaka M, Chang YW,
Suzuki H, Kaneko MK, and Kato Y: Monoclonal antibody
L(1)Mab-13 detected human PD-L1 in lung cancers.
Monoclon Antib Immunodiagn Immunother 2018;37:110–
115.
19. Yamada S, Itai S, Nakamura T, Yanaka M, Saidoh N,
Chang YW, Handa S, Harada H, Kagawa Y, Ichii O,
Konnai S, Kaneko MK, and Kato Y: PMab-52: specific andsensitive monoclonal antibody against cat podoplanin for
immunohistochemistry. Monoclon Antib Immunodiagn
Immunother 2017;36:224–230.
20. Furusawa Y, Kaneko MK, Nakamura T, Itai S, Fukui M,
Harada H, Yamada S, and Kato Y: Establishment of a
monoclonal antibody PMab-231 for tiger podoplanin.
Monoclon Antib Immunodiagn Immunother 2019;38:
89–95.
21. Furusawa Y, Takei J, Sayama Y, Yamada S, Kaneko MK,
and Kato Y: Development of an anti-bear podoplanin
monoclonal antibody PMab-247 for immunohistochemical
analysis. Biochem Biophys Rep 2019;18:100644.
22. Furusawa Y, Yamada S, Itai S, Nakamura T, Takei J,
Sano M, Harada H, Fukui M, Kaneko MK, and Kato Y:
Establishment of a monoclonal antibody PMab-233 forimmunohistochemical analysis against Tasmanian devil
podoplanin. Biochem Biophys Rep 2019;18:100631.
23. Goto N, Suzuki H, Tanaka T, Asano T, Kaneko MK, andKato Y: Development of a monoclonal antibody PMab-292
against ferret podoplanin. Monoclon Antib Immunodiagn
Immunother 2022;41:101–109.
24. Furusawa Y, Yamada S, Itai S, Sano M, Nakamura T,
Yanaka M, Handa S, Mizuno T, Maeda K, Fukui M,
Harada H, Kaneko MK, and Kato Y: Establishment of
monoclonal antibody PMab-202 against horse podoplanin.
Monoclon Antib Immunodiagn Immunother 2018;37:233–
237.
25. Kato Y, Yamada S, Furusawa Y, Itai S, Nakamura T,
Yanaka M, Sano M, Harada H, Fukui M, and Kaneko MK:
PMab-213: a monoclonal antibody for immunohistochemical analysis against pig podoplanin. Monoclon AntibImmunodiagn Immunother 2019;38:18–24.
26. Furusawa Y, Yamada S, Nakamura T, Sano M, Sayama Y,
Itai S, Takei J, Harada H, Fukui M, Kaneko MK, and Kato
Y: PMab-235: a monoclonal antibody for immunohistochemical analysis against goat podoplanin. Heliyon 2019;5:e02063.
27. Kato Y, Furusawa Y, Yamada S, Itai S, Takei J, Sano M,
and Kaneko MK: Establishment of a monoclonal antibody
PMab-225 against alpaca podoplanin for immunohistochemical analyses. Biochem Biophys Rep 2019;18:100633.
28. Kato Y, Furusawa Y, Itai S, Takei J, Nakamura T, Sano M,
Harada H, Yamada S, and Kaneko MK: Establishment ofan anticetacean podoplanin monoclonal antibody PMab237 for immunohistochemical analysis. Monoclon AntibImmunodiagn Immunother 2019;38:108–113.
29. Kato Y, Furusawa Y, Sano M, Takei J, Nakamura T,
Yanaka M, Okamoto S, Handa S, Komatsu Y, Asano T,
Sayama Y, and Kaneko MK: Development of an anti-sheep
podoplanin monoclonal antibody PMab-256 for immunohistochemical analysis of lymphatic endothelial cells.
Monoclon Antib Immunodiagn Immunother 2020;39:
82–90.
30. Tanaka T, Asano T, Sano M, Takei J, Hosono H, Nanamiya
R, Nakamura T, Yanaka M, Harada H, Fukui M, Suzuki H,
Uchida K, Nakagawa T, Kato Y, and Kaneko MK:

KITAMURA ET AL.

Development of monoclonal antibody PMab-269 against
California sea lion podoplanin. Monoclon Antib Immunodiagn Immunother 2021;40:124–133.
31. Asano T, Nanamiya R, Takei J, Nakamura T, Yanaka M,
Hosono H, Tanaka T, Sano M, Kaneko MK, and Kato Y:
Development of anti-mouse CC chemokine receptor 3
monoclonal antibodies for flow cytometry. Monoclon AntibImmunodiagn Immunother 2021;40:107–112.
32. Tanaka T, Nanamiya R, Takei J, Nakamura T, Yanaka M,
Hosono H, Sano M, Asano T, Kaneko MK, and Kato Y:
Development of anti-mouse CC chemokine receptor 8
monoclonal antibodies for flow cytometry. Monoclon AntibImmunodiagn Immunother 2021;40:65–70.
33. Nanamiya R, Takei J, Asano T, Tanaka T, Sano M,
Nakamura T, Yanaka M, Hosono H, Kaneko MK, and Kato
Y: Development of anti-human CC chemokine receptor 9
monoclonal antibodies for flow cytometry. Monoclon AntibImmunodiagn Immunother 2021;40:101–106.
34. Tanaka T, Li G, Asano T, Saito M, Kaneko MK, Suzuki H,
and Kato Y: Development of a novel anti-mouse CCR2
monoclonal antibody (C2Mab-6) by N-terminal peptide
immunization. Monoclon Antib Immunodiagn Immunother2022;41:80–86.
35. Kato Y: Specific monoclonal antibodies against IDH1/2
mutations as diagnostic tools for gliomas. Brain Tumor
Pathol 2015;32:3–11.
36. Ikota H, Nobusawa S, Arai H, Kato Y, Ishizawa K, Hirose
T, and Yokoo H: Evaluation of IDH1 status in diffusely
infiltrating gliomas by immunohistochemistry using antimutant and wild type IDH1 antibodies. Brain Tumor Pathol
2015;32:237–244.
37. Chain B, Arnold J, Akthar S, Brandt M, Davis D,
Noursadeghi M, Lapp T, Ji C, Sankuratri S, Zhang Y,
Govada L, Saridakis E, and Chayen N: A linear epitope in
the N-terminal domain of CCR5 and its interaction with
antibody. PLoS One 2015;10:e0128381.
38. Aguilera-Durán G, and Romo-Mancillas A: Behavior ofchemokine receptor 6 (CXCR6) in complex with CXCL16
soluble form chemokine by molecular dynamic simulations: general protein–ligand interaction model and 3DQSAR studies of synthetic antagonists. Life (Basel) 2021;
11:346.
39. Jung Y, Kim JK, Shiozawa Y, Wang J, Mishra A, Joseph J,
Berry JE, McGee S, Lee E, Sun H, Wang J, Jin T, Zhang H,
Dai J, Krebsbach PH, Keller ET, Pienta KJ, and Taichman
RS: Recruitment of mesenchymal stem cells into prostate
tumours promotes metastasis. Nat Commun 2013;4:1795.
40. Krawczyk KM, Nilsson H, Allaoui R, Lindgren D,
Arvidsson M, Leandersson K, and Johansson ME: Papillary
renal cell carcinoma-derived chemerin, IL-8, and CXCL16
promote monocyte recruitment and differentiation into
foam-cell macrophages. Lab Invest 2017;97:1296–1305.
41. Takei J, Kaneko MK, Ohishi T, Hosono H, Nakamura T,
Yanaka M, Sano M, Asano T, Sayama Y, Kawada M,
Harada H, and Kato Y: A defucosylated antiCD44 monoclonal antibody 5mG2af exerts antitumor effects in mouse
xenograft models of oral squamous cell carcinoma. Oncol
Rep 2020;44:1949–1960.
42. Itai S, Ohishi T, Kaneko MK, Yamada S, Abe S, Nakamura
T, Yanaka M, Chang YW, Ohba SI, Nishioka Y, Kawada
M, Harada H, and Kato Y: Anti-podocalyxin antibody
exerts antitumor effects via antibody-dependent cellularcytotoxicity in mouse xenograft models of oral squamouscell carcinoma. Oncotarget 2018;9:22480–22497.

ANTIMOUSE CXCR6 MONOCLONAL ANTIBODY

Downloaded by Yukinari Kato from www.liebertpub.com at 06/23/22. For personal use only.

43. Hosono H, Takei J, Ohishi T, Sano M, Asano T, Sayama Y,
Nakamura T, Yanaka M, Kawada M, Harada H, Kaneko
MK, and Kato Y: AntiEGFR monoclonal antibody
134mG2a exerts antitumor effects in mouse xenograftmodels of oral squamous cell carcinoma. Int J Mol Med
2020;46:1443–1452.
44. Di Pilato M, Kfuri-Rubens R, Pruessmann JN, Ozga AJ,
Messemaker M, Cadilha BL, Sivakumar R, Cianciaruso
C, Warner RD, Marangoni F, Carrizosa E, Lesch S,
Billingsley J, Perez-Ramos D, Zavala F, Rheinbay E,
Luster AD, Gerner MY, Kobold S, Pittet MJ, and Mempel
TR: CXCR6 positions cytotoxic T cells to receive criticalsurvival signals in the tumor microenvironment. Cell 2021;
184:4512.e22–4530.e22.
45. Galon J, and Bruni D: Approaches to treat immune hot,altered and cold tumours with combination immunotherapies. Nat Rev Drug Discov 2019;18:197–218.

141

46. Kraehenbuehl L, Weng CH, Eghbali S, Wolchok JD, andMerghoub T: Enhancing immunotherapy in cancer by targeting emerging immunomodulatory pathways. Nat Rev
Clin Oncol 2022;19:37–50.

Address correspondence to:
Yukinari Kato
Department of Molecular Pharmacology
Tohoku University Graduate School of Medicine
2-1, Seiryo-machi, Aoba-ku
Sendai, Miyagi 980-8575
JapanE-mail: yukinarikato@med.tohoku.ac.jp
Received: February 14, 2022
Accepted: April 19, 2022