MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 42, Number 1, 2023
ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2022.0029

Short Communications
Open camera or QR reader andscan code to access this article
and other resources online.

Epitope Mapping Using the Cell-Based 2 · Alanine
Substitution Method About the Anti-mouse CXCR6Monoclonal Antibody, Cx6Mab-1

Downloaded by Yukinari Kato from www.liebertpub.com at 02/28/23. For personal use only.

Yu Isoda,1,* Tomohiro Tanaka,1,* Hiroyuki Suzuki,2 Teizo Asano,1 Takeo Yoshikawa,3 Kaishi Kitamura,2Yuma Kudo,2 Ryo Ejima,2 Kazuki Ozawa,2 Mika K. Kaneko,1 and Yukinari Kato1–3

An anti-mouse CXC chemokine receptor 6 (mCXCR6) monoclonal antibody (mAb), Cx6Mab-1, was developed
recently. Cx6Mab-1 is applicable for flow cytometry, Western blotting, and enzyme-linked immunosorbent
assay. The purpose of this study is to determine the binding epitope of Cx6Mab-1 using 2 · alanine mutated
mCXCR6. Analysis of flow cytometry revealed that Cx6Mab-1 did not recognize S8A-A9G, L10A-Y11A,
D12A-G13A, and H14A-Y15A mutants of mCXCR6. The results clearly indicate that the binding epitope of
Cx6Mab-1 includes Ser8, Ala9, Leu10, Tyr11, Asp12, Gly13, His14, and Tyr15 of mCXCR6. The successful
determination of the Cx6Mab-1 epitope might contribute to the pathophysiological investigation of mCXCR6.
Keywords: mouse CXCR6, monoclonal antibody, epitope, flow cytometry, 2 · Ala scanning

deficiency virus type 2.9 During acute hepatitis B virus
infection, liver-resident CXCR3+CXCR6+ gd T cells work aprotective role by producing high level of IFN-g.10 CXCR6+
NK cells contribute to immune memory against antigens suchas viruses and haptens.11 CXCR6-expressing T cells play
pivotal roles in infection responses.
In tumors, CXCL16/CXCR6 expression has been
confirmed in various cancers.12 Tumor-infiltrating
CXCR6+CD8+ T cells exert antitumor effect in immunotherapy. Anti-programmed cell death-1 antibodies are lesseffective in CXCR6 knockout mice.1 In tumor microenvironment, CXCR6 in CTLs and trans-presented interleukin15 promote the survival and local expansion of CTLs,
maximizing subsequent antitumor effects.13 In contrast,
upregulation of CXCL16 supports cancer progression andresults in poor prognosis.12 High expression of CXCL16/CXCR6 has both positive and negative prognostic effects incancer, requiring further study. The functional antibodies
against CXCR6 and CXCL16 are required for therapy anddiagnosis.

IntroductionT

he CXC chemokine receptor 6 (CXCR6), also known
as CD186, is a G protein-coupled receptor (GPCR) withseven transmembrane regions. CXCR6 is preferentially expressed on T cells, including memory T cells, cytotoxic T
lymphocytes (CTLs), helper T type 1 cells, and natural killer(NK) cells.1–3 In hepatic CXCR6+ NK cells, the survival of
the T cells depends on the signaling of CXCR6.4
T cells that participate in numerous immune responses arerecruited through the interaction with a chemokine, CXC
chemokine ligand 16 (CXCL16).5,6 CXCL16 is a specificchemokine ligand for CXCR6, which is expressed constitutively on liver sinusoidal endothelial cells, spleen, intestine,
lungs, and skin, and is known to have transmembrane andsoluble forms.7,8
The roles of CXCR6 and CXCL16 have been actively
investigated in their association with diseases including
cancer and viral infections. CXCR6 was found as a coreceptor on CD4+ and CD8+ T cells for the human immuno-

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

22

Downloaded by Yukinari Kato from www.liebertpub.com at 02/28/23. For personal use only.

FIG. 1. Epitope determination of Cx6Mab-1 using 2 · Ala (or Gly) substituted mutants by flow cytometry. (A) CHO-K1
cells, which transiently overexpressed WT or two amino acids-substituted mutants (D2A-D3A, G4A-H5A, Q6A-E7A, S8AA9G, L10A-Y11A, D12A-G13A, H14A-Y15A, E16A-G17A, D18A-F19A, and W20A-L21A) of mCXCR6, were incubatedwith NZ-1 (anti-PA tag mAb; red line), or buffer control (black line) for 30 minutes at 4C, followed by treatment withsecondary antibody. (B) CHO-K1 cells, which transiently expressed WT or two amino acids-substituted mutants (D2AD3A, G4A-H5A, Q6A-E7A, S8A-A9G, L10A-Y11A, D12A-G13A, H14A-Y15A, E16A-G17A, D18A-F19A, and W20AL21A) of mCXCR6, were incubated with Cx6Mab-1 (anti-mCXCR6 mAb; red line), or buffer control (black line) for 30
minutes at 4C, followed by treatment with secondary antibody. (C) Schematic illustration of mCXCR6 and the Cx6Mab-1
epitope. The Cx6Mab-1 epitope involves Ser8, Ala9, Leu10, Tyr11, Asp12, Gly13, His14, and Tyr15 of mCXCR6, shown
underline. mCXCR6, mouse CXC chemokine receptor 6; WT, wild type.
23

24

We have successfully developed various anti-GPCR
monoclonal antibodies (mAbs), including anti-mouse
CCR2,14 human CCR2,15 mouse CCR3,16–18 mouse CCR4,19
mouse CCR8,20 human CCR9,21 and mouse CXCR6(mCXCR6).22 Furthermore, we performed epitope mapping
about these mAbs.23–25 The estimated epitope of Cx6Mab-1
was also determined by enzyme-linked immunosorbent assay(ELISA) experiments using two alanine scanning methods.26
In this study, we perform epitope analysis using two sequential alanine (or glycine) substitution strategies in a system of mutated mCXCR6-overexpressed cells that maintainstructural features by flow cytometry.
Materials and Methods

Downloaded by Yukinari Kato from www.liebertpub.com at 02/28/23. For personal use only.

Cell lines

Chinese hamster ovary-K1 (CHO-K1) cells were purchased from the American Type Culture Collection (Manassas, VA). The mCXCR6 mutation plasmids, pCAG-Ble
vector (FUJIFILM Wako Pure Chemical Corporation, Osaka,
Japan), containing an N-terminal PA tag,27–32 were transfected into CHO-K1 cells using a Neon Transfection System(Thermo Fisher Scientific, Inc., Waltham, MA). The PA tag
comprises 12 amino acids, GVAMPGAEDDVV, and isrecognized by NZ-1 mAb.27,33
Cells transiently transfected with wild type (WT) or two
alanine (or glycine) residues-substituted mCXCR6 mutants
were cultured in a Roswell Park Memorial Institute (RPMI)
1640 medium (Nacalai Tesque, Inc., Kyoto, Japan), supplemented with 10% heat-inactivated fetal bovine serum (FBS)(Thermo Fisher Scientific, Inc.), 100 U/mL penicillin,
100 lg/mL streptomycin, and 0.25 lg/mL amphotericin B(Nacalai Tesque, Inc.), and 0.5 mg/mL of Zeocin (InvivoGen,
San Diego, CA). Then, cells were grown in a humidified
incubator, which was supplied with 5% CO2 and 95% air at
37C.
Production of mCXCR6 2 · alanine mutants

Synthesized DNA (Eurofins Genomics KK, Tokyo, Japan)
encoding mCXCR6 (Accession No. NM_030712) with
N-terminal PA tag was subcloned into a pCAG-Ble vector.
Plasmids were transfected using a neon transfection system.
Substitutions of consecutive amino acids with two alanine(or glycine) in the mCXCR6 sequence were produced by
QuikChange Lightning Site Directed Mutagenesis Kits
(Agilent Technologies, Inc., Santa Clara, CA). Polymerase
chain reaction fragments-bearing desired mutations wereinserted into the pCAG-Ble vector using KAPA HiFi HotStart ReadyMix (NIPPON Genetics Co., Ltd., Tokyo, Japan).
Flow cytometry

Transiently transfected CHO-K1 cells were detached by
0.25% trypsin in 1 mM ethylenediaminetetraacetic acid
(EDTA; Nacalai Tesque, Inc.) and collected using 10% FBS
containing RPMI 1640 medium. After washing with 0.1%
bovine serum albumin (BSA; Nacalai Tesque, Inc.) in
phosphate-buffered saline (PBS; Nacalai Tesque, Inc.), cells
were treated with anti-mCXCR6 mAb (Cx6Mab-1; 1 lg/mL)
or NZ-1 (anti-PA tag; 1 lg/mL) for 30 minutes at 4C, followed by incubation with Alexa Fluor 488-conjugated anti-rat
IgG (1:1000; product no. 4416; Cell Signaling Technology,

ISODA ET AL.

Inc.). Fluorescence data were collected using the SA3800
Cell Analyzer (Sony Biotechnology Corp., Tokyo, Japan).
Results and Discussion
Epitope mapping of Cx6Mab-1 with alanine-substituted
mCXCR6 in CHO-K1

We previously developed an anti-mCXCR6 mAb(Cx6Mab-1) by N-terminal peptide immunization ofmCXCR6.22 To identify the binding epitope of Cx6Mab-1,
we produced 10 mutants of mCXCR6, in which sequential
two amino acids were replaced to two alanine (or glycine)residues. This method was named as ‘‘2 · Ala-scan method.’’
Based on past ELISA analysis, we could not determine theepitope with 1 · Ala-scan method26; therefore, we introduced
2 · Ala-scan method using cells in this study. For instance, amutant (D2A-D3A) indicates the alanine substitution of second
Asp and third Asp of mCXCR6 protein. As shown in Figure 1A,
all 2 · Ala mutants and WT mCXCR6 containing the N-terminal
PA tag were recognized by NZ-1 (an anti-PA tag mAb), indicating that the expression level of each construct was sufficient.
Cx6Mab-1 recognized D2A-D3A, G4A-H5A, Q6A-E7A,
E16A-G17A, D18A-F19A, and W20A-L21A, but not S8AA9G, L10A-Y11A, D12A-G13A, and H14A-Y15A (Fig. 1B).
These results suggest that Ser8, Ala9, Leu10, Tyr11,
Asp12, Gly13, His14, and Tyr15 are included in the critical
epitope of Cx6Mab-1. The results are shown as a schematic
illustration in Figure 1C. Previous results of ELISA experiments indicated that Ser8, Ala9, Leu10, Tyr11, Gly13, andHis14 were the critical epitopes of Cx6Mab-1, we speculatethat this discrepancy is due to the structural differences between peptides and proteins. This 2 · Ala-scanning strategy
could be another option to identify the epitope of mAbs even
if the conventional alanine scanning is not effective.
Several GPCRs, including CCR2, CCR3, CCR5, CCR9,and CXCR1, interact with ligands at the N-terminal region.34,35 A molecular dynamics simulation about docking of
CXCL16–CXCR6 was reported.36 Cx6Mab-1 may block
CXCL16 binding, although further approach is needed.
CXCL16 is highly expressed by tumor cells, including pancreatic cancer, and tumor-infiltrating immune cells. Since
CXCR6-armed T cells enhance the recognition and lysis oftumor,37 Cx6Mab-1 is expected to be one of the immunotherapy strategies by applying modalities such as bispecificantibodies against tumor antigen.
Author Disclosure Statement

No competing financial interests exist.
Funding Information

This research was supported in part by Japan Agency for
Medical Research and Development (AMED) under Grant
Numbers JP22ama121008 (to Y.K.), JP22am0401013(to Y.K.), JP22bm1004001 (to Y.K.), JP22ck0106730(to Y.K.), and JP21am0101078 (to Y.K.).
References

1. Wang B, Wang Y, Sun X, et al. CXCR6 is required for antitumor efficacy of intratumoral CD8(+) T cell. J Immunother
Cancer 2021;9:e003100. doi: 10.1136/jitc-2021-003100

Downloaded by Yukinari Kato from www.liebertpub.com at 02/28/23. For personal use only.

IDENTIFICATION OF CX6MAB-1 EPITOPE USING FLOW CYTOMETRY

2. Aviles-Padilla K, Angelo LS, Fan D, et al. CXCR6(+) andNKG2C(+) natural killer cells are distinct with unique
phenotypic and functional attributes following bone marrow transplantation. Front Immunol 2022;13:886835; doi:
10.3389/fimmu.2022.886835
3. Tse SW, Radtke AJ, Espinosa DA, et al. The chemokine
receptor CXCR6 is required for the maintenance of liver
memory CD8+ T cells specific for infectious pathogens.
J Infect Dis 2014;210:1508–1516; doi: 10.1093/infdis/jiu281
4. Angelo LS, Hogg GD, Abeynaike S, et al. Phenotypic andfunctional plasticity of CXCR6(+) peripheral blood NK
cells. Front Immunol 2021;12:810080; doi: 10.3389/fimmu.2021.810080
5. Lehrke M, Millington SC, Lefterova M, et al. CXCL16 is amarker of inflammation, atherosclerosis, and acute coronary syndromes in humans. J Am Coll Cardiol 2007;49:
442–449; doi: 10.1016/j.jacc.2006.09.034
6. Dhoke NR, Kaushik K, Das A. Cxcr6-based mesenchymal
stem cell gene therapy potentiates skin regeneration inmurine diabetic wounds. Mol Ther 2020;28:1314–1326;
doi: 10.1016/j.ymthe.2020.02.014
7. Abel S, Hundhausen C, Mentlein R, et al. The transmembrane CXC-chemokine ligand 16 is induced by IFN-gamma
and TNF-alpha and shed by the activity of the disintegrinlike metalloproteinase ADAM10. J Immunol 2004;172:
6362–6372; doi: 10.4049/jimmunol.172.10.6362
8. Korbecki J, Bajdak-Rusinek K, Kupnicka P, et al. The roleof CXCL16 in the pathogenesis of cancer and other diseases. Int J Mol Sci 2021;22:3490; doi: 10.3390/ijms220
73490
9. Blaak H, Boers PH, Gruters RA, et al. CCR5, GPR15, andCXCR6 are major coreceptors of human immunodeficiency
virus type 2 variants isolated from individuals with andwithout plasma viremia. J Virol 2005;79:1686–1700; doi:
10.1128/jvi.79.3.1686-1700.2005
10. Wang Y, Guan Y, Hu Y, et al. Murine CXCR3(+)CXCR6(+)gdT cells reside in the liver and provide protection against HBV infection. Front Immunol 2021;12:757379; doi: 10.3389/fimmu.2021.757379
11. Paust S, Gill HS, Wang BZ, et al. Critical role for thechemokine receptor CXCR6 in NK cell-mediated antigenspecific memory of haptens and viruses. Nat Immunol
2010;11:1127–1135; doi: 10.1038/ni.1953
12. Gowhari Shabgah A, Qasim MT, Mojtaba Mostafavi S,
et al. CXC chemokine ligand 16: A Swiss army knife
chemokine in cancer. Expert Rev Mol Med 2021;23:e4;
doi: 10.1017/erm.2021.7
13. Di Pilato M, Kfuri-Rubens R, Pruessmann JN, et al.
CXCR6 positions cytotoxic T cells to receive critical survival signals in the tumor microenvironment. Cell 2021;
184:4512–4530.e4522; doi: 10.1016/j.cell.2021.07.015
14. Tanaka T, Li G, Asano T, et al. Development of a novelanti-mouse CCR2 monoclonal antibody (C(2)Mab-6) by
N-terminal peptide immunization. Monoclon Antib Immunodiagn Immunother 2022;41:80–86; doi: 10.1089/
mab.2021.0063
15. Tanaka T, Li G, Saito M, et al. Development of an antihuman CCR2 monoclonal antibody (C(2)Mab-9) by
N-terminal peptide immunization. Monoclon Antib Immunodiagn Immunother 2022;41:188–193; doi: 10.1089/mab.2022.0001
16. Asano T, Nanamiya R, Takei J, et al. Development of antimouse CC chemokine receptor 3 monoclonal antibodies for25

flow cytometry. Monoclon Antib Immunodiagn Immunother 2021;40:107–112; doi: 10.1089/mab.2021.0009
17. 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
18. 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
19. Takei J, Suzuki H, Asano T, et al. Development of a novelanti-mouse CCR4 monoclonal antibody (C(4)Mab-1) by
N-terminal peptide immunization. Monoclon Antib Immunodiagn Immunother 2022;41:87–93; doi: 10.1089/
mab.2021.0064
20. Tanaka T, Nanamiya R, Takei J, et al. Development of antimouse CC chemokine receptor 8 monoclonal antibodies forflow cytometry. Monoclon Antib Immunodiagn Immunother 2021;40:65–70; doi: 10.1089/mab.2021.0005
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. Kitamura K, Suzuki H, Kaneko MK, et al. Cx(6)Mab-1: A
novel anti-mouse CXCR6 monoclonal antibody establishedby N-terminal peptide immunization. Monoclon AntibImmunodiagn Immunother 2022;41:133–141; doi: 10.1089/
mab.2022.0010
23. 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
24. Asano T, Suzuki H, Tanaka T, et al. Identification of thebinding epitope of an anti-mouse CCR4 monoclonal antibody, C(4)Mab-1. Monoclon Antib Immunodiagn Immunother 2022; doi: 10.1089/mab.2022.0015
25. Tanaka T, Li G, Asano T, et al. Epitope mapping of theanti-human CCR2 monoclonal antibody c(2)Mab-9.
Monoclon Antib Immunodiagn Immunother 2022;41:150–
156; doi: 10.1089/mab.2022.0012
26. Isoda Y, Tanaka T, Suzuki H, et al. Epitope mapping of ananti-mouse CXCR6 monoclonal antibody (Cx6Mab-1) using the 2 · alanine scanning method. Monoclon Antib Immunodiagn Immunother 2022;41:275–278; doi: 10.1089/mab.2022.0019
27. Fujii Y, Kaneko M, Neyazaki M, et al. PA tag: A versatile
protein tagging system using a super high affinity antibody
against a dodecapeptide derived from human podoplanin.
Protein Expr Purif 2014;95:240–247; doi: 10.1016/j.pep.2014.01.009
28. Fujii Y, Matsunaga Y, Arimori T, et al. Tailored placementof a turn-forming PA tag into the structured domain of aprotein to probe its conformational state. J Cell Sci 2016;
129:1512–1522; doi: 10.1242/jcs.176685
29. Tamura R, Oi R, Akashi S, et al. Application of the NZ-1 Fab
as a crystallization chaperone for PA tag-inserted target proteins. Protein Sci 2019;28:823–836; doi: 10.1002/pro.3580
30. Tabata S, Kitago Y, Fujii Y, et al. An anti-peptide monoclonal antibody recognizing the tobacco etch virus
protease-cleavage sequence and its application to a tandem
tagging system. Protein Expr Purif 2018;147:94–99; doi:
10.1016/j.pep.2018.03.004

Downloaded by Yukinari Kato from www.liebertpub.com at 02/28/23. For personal use only.

26

31. Tamura-Sakaguchi R, Aruga R, Hirose M, et al. Moving
toward generalizable NZ-1 labeling for 3D structure determination with optimized epitope-tag insertion. Acta
Crystallogr D Struct Biol 2021;77:645–662; doi:
10.1107/S2059798321002527
32. Imaizumi Y, Takanuki K, Miyake T, et al. Mechanistic
insights into intramembrane proteolysis by E. coli site-2
protease homolog RseP. Sci Adv 2022;8:eabp9011; doi:
10.1126/sciadv.abp9011
33. Kato Y, Kaneko MK, Kuno A, et al. Inhibition of tumor
cell-induced platelet aggregation using a novel antipodoplanin antibody reacting with its platelet-aggregationstimulating domain. Biochem Biophys Res Commun 2006;
349:1301–1307; doi: 10.1016/j.bbrc.2006.08.171
34. 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
35. 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
36. Aguilera-Durán G, Romo-Mancillas A. Behavior of chemokine receptor 6 (CXCR6) in complex with CXCL16ISODA ET AL.

soluble form chemokine by molecular dynamic simulations: General protein–ligand interaction model and 3DQSAR studies of synthetic antagonists. Life (Basel) 2021;
11:346; doi: 10.3390/life11040346
37. Lesch S, Blumenberg V, Stoiber S, et al. T cells armed with
C-X-C chemokine receptor type 6 enhance adoptive celltherapy for pancreatic tumours. Nat Biomed Eng 2021;5:1246–1260; doi: 10.1038/s41551-021-00737-6

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: yukinari.kato.e6@tohoku.ac.jp
Received: August 27, 2022
Accepted: September 30, 2022