MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 41, Number 5, 2022
ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2022.0019

Short Communications
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Epitope Mapping of an Anti-Mouse CXCR6 Monoclonal
Antibody (Cx6Mab-1) Using the 2 · Alanine Scanning Method

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

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, cytotoxic T lymphocytes (CTLs), and natural killer cells. CXCR6 plays
critical roles in local expansion of effector-like CTLs in tumor microenvironment to potentiate the antitumor
response. Therefore, the development of anti-CXCR6 monoclonal antibodies (mAbs) is essential to evaluate theimmune microenvironment of tumors. Using N-terminal peptide immunization, we previously developed ananti-mouse CXCR6 (mCXCR6) mAb, Cx6Mab-1 (rat IgG1, kappa) , which is useful for flow cytometry andwestern blotting. In this study, we determined the critical epitope of Cx6Mab-1 by enzyme-linked immunosorbent assay (ELISA) using the 1 · alanine scanning (1 · Ala-scan) method or the 2 · alanine scanning(2 · Ala-scan) method. Although we first performed ELISA by 1 · Ala-scan using one alanine-substituted
peptides of mCXCR6 N-terminal domain (amino acids 1–20), we could not identify the Cx6Mab-1 epitope. We
next performed ELISA by 2 · Ala-scan using two alanine (or glycine) residues-substituted peptides of
mCXCR6 N-terminal domain, and found that Cx6Mab-1 did not recognize S8A–A9G, A9G–L10A, L10A–
Y11A, and G13A–H14A of the mCXCR6 N-terminal peptide. The results indicate that the binding epitope of
Cx6Mab-1 includes Ser8, Ala9, Leu10, Tyr11, Gly13, and His14 of mCXCR6. Therefore, we could demonstratethat the 2 · Ala scan method is useful for determining the critical epitope of mAbs.
Keywords: mouse CXCR6, epitope mapping, monoclonal antibody, enzyme-linked immunosorbent assay,
2 · Ala scanning
IntroductionT

he CXC chemokine receptor 6 (CXCR6) is primarily expressed in T cells, including cytotoxic T lymphocytes (CTLs), helper T type 1 (Th1) cells, natural killer(NK) cells, and NK T cells. Upon the ligand (CXCL16) binding to CXCR6, it stimulates intracellular signaling pathway
and mediates various cellular functions, including the infiltration into target tissues.1 CXCL16 is constitutively secreted
by liver sinusoids, which plays critical roles in the maintenance of NK, NK T, and CD8+ memory T cells and lymphocyte homeostasis in the liver.2
CXCL16-CXCR6 axis also plays complex roles in tumor
microenvironment (TME).3 It promotes tumor progression directly through enhancing survival, proliferation, andmetastasis of tumor cells. Furthermore, it potentiates
tumor-promoting M2 macrophages infiltration,4 and stimulates the conversion from mesenchymal stem cells into
cancer-associated fibroblasts.5 In contrast, CXCR6 mediates the differentiation into effector-like CTLs. In TME,CXCL16 is secreted by CCR7+ dendritic cells, which alsotrans-present the survival cytokine interleukin-15 (IL-15).
CXCR6 expression and IL-15 trans-presentation toeffector-like CTLs are critical for the survival and local
expansion in TME to enhance their antitumor immune
response.6 These results indicate a critical function of
CXCR6 for potentiating CD8+ CTLs-mediated antitumor
immune responses.
We have developed monoclonal antibodies (mAbs) against
chemokine receptors, including anti-mouse CCR2,7 mouse

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

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ISODA ET AL.

CCR3,8–10 mouse CCR4,11 mouse CCR8,12–14 and human
CCR9,15 and also determined the binding epitope.16 We
also established an anti-mouse CXCR6 (mCXCR6) mAb
Cx6Mab-1 (rat IgG1, kappa) by N-terminal peptide immunization.17 To clarify further characteristics of Cx6Mab-1,
we performed epitope mapping by enzyme-linked immunosorbent assay (ELISA) using the 1 · alanine scanning(1 · Ala-scan) method or the 2 · alanine scanning (2 · Alascan) method.
Materials and Methods

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PeptidesThe mCXCR6 (Accession No. NM_030712) peptide(1-MDDGHQESALYDGHYEGDFW-20) and one alanine(or glycine) residue-substituted peptides (Supplementary
Table S1) and two alanine (or glycine) residues-substituted
peptides (Table 1) were synthesized by utilizing PEPscreen(Sigma-Aldrich Corp., St. Louis, MO).
Enzyme-linked immunosorbent assay

Synthesized mCXCR6 peptides were immobilized on
Nunc Maxisorp 96-well immunoplates (Thermo Fisher Scientific, Inc., Waltham, MA) at a concentration of 1 lg/mL
for 30 minutes at 37C. After washing with phosphatebuffered saline (PBS) containing 0.05% Tween20 (PBST;
Nacalai Tesque, Inc., Kyoto, Japan), wells were blockedwith 1% bovine serum albumin-containing PBST for 30 minutes at 37C. The plates were incubated with 1 lg/mL ofCx6Mab-1, followed by a peroxidase-conjugated anti-rat
immunoglobulins (1:10000 diluted; Sigma-Aldrich Corp.).
Enzymatic reactions were performed using the ELISA POD
Substrate TMB Kit (Nacalai Tesque, Inc.). Optical density
was measured at 655 nm using an iMark microplate reader(Bio-Rad Laboratories, Inc., Berkeley, CA).

Results
Epitope mapping of Cx6Mab-1 with alanine-substituted
mCXCR6 peptides

We previously established an anti-mCXCR6 mAb, Cx6Mab-1(rat IgG1, kappa), by immunizing rats with KLH-conjugated
mCXCR6 N-terminal domain (1-MDDGHQESALYDGHYEG
DF-19)+C-terminal cysteine residue.17 To reveal the binding
epitope of Cx6Mab-1, we first synthesized one-alanine (orglycine)-substituted peptides of mCXCR6, which is called as
1 · Ala-scan method (Supplementary Table S1). However,Cx6Mab-1 reacted with all one-alanine (or glycine)-substituted
peptides and wild-type (WT) (Supplementary Fig. S1).
We next synthesized mutant peptides that sequential two
amino acids were substituted to two alanine (or glycine)residues, which is called as 2 · Ala-scan method. For
instance, a peptide (M1A–D2A) indicates the alanine substitution of first Met and second Asp of mCXCR6 peptide(Table 1). As shown in Figure 1A, Cx6Mab-1 exhibited
reaction with M1A–D2A, D2A–D3A, D3A–G4A, G4A–
H5A, H5A–Q6A, Q6A–E7A, E7A–S8A, Y11A–D12A,
D12A–G13A, H14A–Y15A, Y15A–E16A, E16A–G17A,
G17A–D18A, D18A–F19A, F19A–W20A, and WT. In
contrast, Cx6Mab-1 did not react with S8A–A9G, A9G–
L10A, L10A–Y11A, and G13A–H14A (Fig. 1), indicatingthat Ser8, Ala9, Leu10, Tyr11, Gly13, and His14 are included in the critical epitope of Cx6Mab-1. The results are
summarized in Table 1. Figure 1B shows the schematic illustration of mCXCR6 and the critical epitope of Cx6Mab-1.
Discussion

A strategy, called alanine-scanning mutagenesis, was firstused to identify specific residues in human growth hormone(hGH) that participate in the binding to the hGH receptor.
Single alanine mutations were introduced at every residue
within the regions that have been suggested in receptor

Table 1. Identification of the Cx6Mab-1 Epitope Using 2 · Alanine-Substituted mCXCR6 Peptides
PeptidesM1A–D2A
D2A–D3A
D3A–G4A
G4A–H5A
H5A–Q6A
Q6A–E7A
E7A–S8A
S8A–A9G
A9G–L10A
L10A–Y11A
Y11A–D12A
D12A–G13A
G13A–H14A
H14A–Y15A
Y15A–E16A
E16A–G17A
G17A–D18A
D18A–F19A
F19A–W20A+++, OD655 S 0.3;-, OD655 < 0.1.

Sequences

Cx6Mab-1

AADGHQESALYDGHYEGDFWMAAGHQESALYDGHYEGDFWMDAAHQESALYDGHYEGDFWMDDAAQESALYDGHYEGDFWMDDGAAESALYDGHYEGDFWMDDGHAASALYDGHYEGDFWMDDGHQAAALYDGHYEGDFWMDDGHQEAGLYDGHYEGDFWMDDGHQESGAYDGHYEGDFWMDDGHQESAAADGHYEGDFWMDDGHQESALAAGHYEGDFWMDDGHQESALYAAHYEGDFWMDDGHQESALYDAAYEGDFWMDDGHQESALYDGAAEGDFWMDDGHQESALYDGHAAGDFWMDDGHQESALYDGHYAADFWMDDGHQESALYDGHYEAAFWMDDGHQESALYDGHYEGAAW
MDDGHQESALYDGHYEGDAA

+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++

EPITOPE MAPPING USING 2 · ALA-SCAN

277

CXCR6 plays a critical function for sustaining antitumor
immune responses of CD8+ CTLs through the conversion ofstem-like memory cells into effector-like CTLs in TME.6 In
tumor immune therapy, the infiltration of CTLs into tumor
is important factors to predict the efficacy.23,24 Cx6Mab-1
could be useful for investigating the expression of mCXCR6
in tumor infiltrating CTLs and use of combination therapy
with immune checkpoint inhibitors in mice experiments.
Author Disclosure Statement

No competing financial interests exist.

Downloaded by Yukinari Kato from www.liebertpub.com at 10/28/22. For personal use only.

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.).
Supplementary Material

Supplementary Figure S1
Supplementary Table S1References

FIG. 1. Determination of the Cx6Mab-1 epitope of
mCXCR6 by ELISA using 2 · Ala-scan method. (A) The
2 · alanine-substituted mCXCR6 peptides were immobilized
on immunoplates. The plates were incubated with Cx6Mab1 (1 mg/mL), followed by peroxidase-conjugated anti-rat
immunoglobulins. (B) Schematic illustration of mCXCR6and the Cx6Mab-1 epitope. The Cx6Mab-1 epitope involves
Ser8, Ala9, Leu10, Tyr11, Gly13, and His14 of mCXCR6.
ELISA, enzyme-linked immunosorbent assay; 2 · Ala-scan,
2 · alanine scanning; mCXCR6, mouse CXC chemokine
receptor 6.

recognition.18 The strategy was also applied to antibody–antigen interaction to analyze the functional epitope of hGH
important for the binding to 21 different anti-hGH mAbs
using ELISA.19 In this study, we could not identify the epitope of Cx6Mab-1 by 1 · Ala-scan (Supplementary Fig. S1).
However, the introduction of sequential amino acids substitution (2 · Ala-scan) could determine the epitope of Cx6Mab1 (Fig. 1). This 2 · Ala-scan method could be another option
to determine the epitope of mAbs.
The N-terminus of chemokine receptors is known to be theligand-binding domain.20 The 3D structure of CXCR6 and its
ligand CXCL16 has not been solved. In contrast, a molecular
dynamics simulation of CXCR6-CXCL16 was reported.
According to the simulation, acidic residues of humanCXCR6, including E8, D9, and D17, interact with basic
residues of human CXCL16.21 In mCXCR6, these aminoacids are thought to be conserved in and out of Cx6Mab-1
epitope. Therefore, further investigation is required to evaluate the agonistic or antagonistic effects of Cx6Mab-1
through the investigation of intracellular signaling pathways
and cellular responses.3,22

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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: May 7, 2022
Accepted: September 26, 2022