MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 40, Number 6, 2021
ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2021.0037

Original Articles
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Epitope Mapping of an Anti-Human CCR9 Monoclonal
Antibody (C9Mab-1) Using Enzyme-Linked
Immunosorbent Assay

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Junko Takei,1 Teizo Asano,1 Guanjie Li,2 Masaki Saito,2 Hiroyuki Suzuki,2Mika K. Kaneko,1 and Yukinari Kato1,2,i

One of G protein-coupled receptors, CCR9, is mainly expressed in the thymocytes and the small bowel. The
ligand of CCR9 is CCL25 (TECK), and the CCR9–CCL25 axis controls T cell maturation and intestinal
immune response. CCR9 is related to graft-versus-host disease and autoimmune diseases. Recent studies havebeen reported that CCR9 is also associated with tumor proliferation, apoptosis, migration, and drug resistance.
Therefore, CCR9-targeting therapy is receiving a lot of attention. Previously, we developed an anti-human
CCR9 (hCCR9) monoclonal antibody, C9Mab-1 (IgG1, kappa), which can be used for flow cytometry, by
immunizing mice with hCCR9-overexpressed Chinese hamster ovary-K1 cells. In this study, we examined thecritical epitope of C9Mab-1, using enzyme-linked immunosorbent assay (ELISA) with synthesized peptides.
First, we performed ELISA with deletion mutants, and C9Mab-1 reacted to the 1–20 amino acids sequence ofhCCR9. Next, we analyzed the reaction to 20 point mutants, and C9Mab-1 did not recognize the alaninesubstituted peptides of I10A, P11A, N12A, M13A, A14G, D16A, and Y17A. The results indicate that thebinding epitope of C9Mab-1 includes Ile10, Pro11, Asn12, Met13, Ala14, Asp16, and Tyr17 of hCCR9.
Keywords: human CCR9, C9Mab-1, epitope mapping, monoclonal antibody, enzyme-linked immunosorbent
assay

Introduction

C

hemokines are 8–14 kDa protein, which regulate
the development, homeostasis, and function of theimmune system. They are divided into four subfamiliesof C, CC, CXC, and CX3C based on the differences of
two cysteine positions at the N-terminus.(1,2) More than 50
chemokines and 20 chemokine receptors have been foundso far.(3) Chemokine receptors are G protein-coupled receptors with seven helical transmembrane regions. CCR9 is
one of the chemokine receptors with a molecular weight of*40,000.(4)CCR9 is mainly expressed on developing thymocytes andthe small intestine.(5,6) Although many chemokines recognize multiple receptors, and a single receptor can bind multiple chemokines, CCL25 (TECK) is the only ligand for

CCR9.(7,8) CCR9–CCL25 interaction plays important roles inproper T cell development and intestinal immune response
and inflammation.(7,9,10)
It has been reported that CCR9 expression is associatedwith various diseases. CCR9+ plasmacytoid dendritic cells
induce regulatory T cell function, resulting in suppression of
antigen-specific immune responses, including acute graftversus-host disease.(11) In patients with small bowel Crohn’sor celiac disease, but not in patients with purely colonic
Crohn’s disease, CCR9+ lymphocytes were elevated remarkably in peripheral blood lymphocytes. In contrast,
CCR9+ T cells in inflamed small intestine were significantly
lower than in normal small intestinal mucosa.(12) Moreover,in the past few years, it has been discovered that CCR9–
CCL25 interaction is related to tumor proliferation, metastasis, and drug resistance.

Departments of 1Antibody Drug Development and 2Molecular Pharmacology, Tohoku University Graduate School of Medicine,
Sendai, Japan.
i
ORCID ID (https://orcid.org/0000-0001-5385-8201).

239

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TAKEI ET AL.

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CCR9 is highly expressed in a variety of cancers, such as
T-lineage acute lymphoblastic leukemia, large B cell lymphoma, melanoma, ovarian cancer, and breast cancer.(4) Shenet al. reported that pancreatic cancer cells expressed CCR9, andits activation enhances proliferation of these cells.(13) Cutaneous
melanoma has a clear pattern of metastasis to the small intestine, which is caused by activation of CCR9.(14) CCR9–CCL25
interaction also mediates PI3K/AKT-dependent anti-apoptotic
signals in prostate cancer cells, resulting in low apoptosis andmodest chemotherapeutic response.(15) Therefore, CCR9 has
been expected to be a new target for cancer treatment.
In a previous study, we established an anti-human CCR9(hCCR9) monoclonal antibody (mAb), C9Mab-1 (mouse
IgG1, kappa), which is useful for flow cytometry by using aCell-Based Immunization and Screening (CBIS) method.(16)To clarify further characteristics of C9Mab-1, we herein
performed epitope mapping using enzyme-linked immunosorbent assay (ELISA).
Materials and Methods
hCCR9 peptides

hCCR9 peptides (accession No.: NM_031200), including
4 deletion mutants (Table 1) and 20 point mutants (Table 2),
were synthesized by utilizing PEPScreen (Sigma-AldrichCorp., St. Louis, MO). One cysteine residue was replaced byserine: C38S.
Enzyme-linked immunosorbent assay

Synthesized hCCR9 peptides (Tables 1 and 2) were
immobilized on Nunc Maxisorp 96-well immunoplates(Thermo Fisher Scientific, Inc., Waltham, MA) at a concentration of 10 lg/mL for 30 minutes at 37C. After washingwith phosphate-buffered saline containing 0.05% Tween20(PBST; Nacalai Tesque, Inc., Kyoto, Japan), wells were
blocked with 1% bovine serum albumin-containing PBST for
30 minutes at 37C. The plates were incubated with 1 lg/mL
of C9Mab-1, followed by a peroxidase-conjugated anti-mouse
immunoglobulins (1:2000 diluted; Agilent Technologies, Inc.,
Santa Clara, CA). 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

Table 2. Identification of C9Mab-1 Epitope Using
Point Mutants by Enzyme-Linked
Immunosorbent Assay
MutantpeptidesSequence

C9Mab-1

M01A
T02A
P03A
T04A
D05A
F06A
T07A
S08A
P09A
I10A
P11A
N12A
M13A
A14G
D15A
D16A
Y17A
G18A
S19A
E20A

ATPTDFTSPIPNMADDYGSE
MAPTDFTSPIPNMADDYGSE
MTATDFTSPIPNMADDYGSEMTPADFTSPIPNMADDYGSEMTPTAFTSPIPNMADDYGSEMTPTDATSPIPNMADDYGSEMTPTDFASPIPNMADDYGSEMTPTDFTAPIPNMADDYGSEMTPTDFTSAIPNMADDYGSEMTPTDFTSPAPNMADDYGSEMTPTDFTSPIANMADDYGSEMTPTDFTSPIPAMADDYGSEMTPTDFTSPIPNAADDYGSEMTPTDFTSPIPNMGDDYGSEMTPTDFTSPIPNMAADYGSEMTPTDFTSPIPNMADAYGSEMTPTDFTSPIPNMADDAGSEMTPTDFTSPIPNMADDYASE
MTPTDFTSPIPNMADDYGAE
MTPTDFTSPIPNMADDYGSA

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

+++, OD655 S0.6; +, 0.1& OD655 < 0.3; -, OD655 < 0.1.

method.(16) To reveal the binding epitope of C9Mab-1, we
synthesized 4 peptides (Table 1), which consist of 1–20
amino acids (aa), 11–30 aa, 21–40 aa, and 31–48 aa ofhCCR9, and performed ELISA. The results showed that
C9Mab-1 recognized the 1–20 aa (1-MTPTDFTSPIPNMADDYGSE-20) sequence of hCCR9 (Fig. 1A). The results
are summarized in Figure 1B.
Epitope mapping of C9Mab-1 using point mutants

Then, we synthesized 20 point mutant peptides of 1–20 aa
of hCCR9 (Table 2). C9Mab-1 exhibited reaction with
M01A, T02A, P03A, T04A, D05A, F06A, T07A, S08A,
P09A, D15A, G18A, S19A, E20A, and wild-type 1–20 aa(Fig. 2A). In contrast, C9Mab-1 did not react with I10A,
P11A, N12A, M13A, A14G, D16A, and Y17A (Fig. 2A),
indicating that Ile10, Pro11, Asn12, Met13, Ala14, Asp16,
and Tyr17 are included in the critical epitope of C9Mab-1.
The results are summarized in Figure 2B.

Epitope mapping of C9Mab-1 using deletion mutants

We previously established an anti-hCCR9 mAb (C9Mab1), which is useful for flow cytometry by the CBIS

Table 1. Identification of C9Mab-1 Epitope Using
Deletion Mutants by Enzyme-Linked
Immunosorbent Assay
PeptidesSequence

C9Mab-1

1–20
11–30
21–40
31–48

MTPTDFTSPIPNMADDYGSE
PNMADDYGSESTSSMEDYVNSTSSMEDYVNFNFTDFYSEKFNFTDFYSEKNNVRQFAS+++-+++, OD655 S0.6; -, OD655 < 0.1.

Discussion

CCR9 is a seven-transmembrane receptor with four extracellular domains. Among the four extracellular regions,the first one at N-terminus is the largest with 48 aa, and theothers are relatively small. So, we hypothesized that the
epitope of C9Mab-1 might be located at the extracellularregion of the N-terminus. ELISA revealed that Ile10, Pro11,
Asn12, Met13, Ala14, Asp16, and Tyr17 were critical aa,
which were included in the epitope of C9Mab-1.
Information about the binding epitope is important toelucidate the functional effect of mAbs. The ligand bindingsites of several chemokine receptors have been defined, andthe studies showed that the N-terminus of some receptors,
such as CCR2, CCR3, CCR5, and CXCR1, is important forDownloaded by Yukinari Kato from www.liebertpub.com at 12/27/21. For personal use only.

EPITOPE MAPPING OF ANTI-HCCR9

FIG. 1. Determination of the C9Mab-1 epitope for hCCR9
by ELISA using deletion mutants. (A) Synthesized peptides ofhCCR9 were immobilized on immunoplates. The plates wereincubated with C9Mab-1 (1 mg/mL), followed by incubation
with peroxidase-conjugated anti-mouse immunoglobulins.
(B) Schematic illustration of hCCR9 and the C9Mab-1 epitope. ELISA, enzyme-linked immunosorbent assay; hCCR9,
human CCR9.
ligand binding.(8) Some studies showed that the mAbs against
the N-terminus of CCR9 inhibited the interaction between
CCR9 and CCL25,(7,17) therefore, the N-terminus of CCR9
also appears to be important for ligand binding. Chamorro
et al. established an anti-hCCR9 mAb, clone 91R (mouse
IgG2b), which recognized the N-terminal region of CCR9 bygene gun immunization with full-length hCCR9 coding sequence inserted in a eukaryotic expression vector.
The clone 91R showed antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity against
acute T lymphoblastic leukemia MOLT-4 cells and significantly inhibited the tumor growth of xenografts.(17) Fromthese studies, it is expected that C9Mab-1 has an ability toblock CCR9–CCL25 binding and antitumor activities against
CCR9-expressing cells. We will examine these activities of
C9Mab-1 in a future study.
In this study, we investigated the binding epitope of
C9Mab-1 using synthesized peptides. Peptide scanning is
simple, relatively inexpensive, and useful for determining alinear epitope. However, peptide structure is unfolded anddifferent from native proteins, so, it is better to examine using
other ways together. X-ray crystallography is the most accurate method for epitope mapping; however, this method
requires difficult techniques and costs money and time.(18)241

FIG. 2. Determination of the C9Mab-1 epitope of hCCR9
by ELISA using point mutants. (A) Synthesized peptides ofhCCR9 were immobilized on immunoplates. The plates wereincubated with C9Mab-1 (1 mg/mL), followed by peroxidaseconjugated anti-mouse immunoglobulins. (B) Schematic illustration of hCCR9 and the C9Mab-1 epitope. The C9Mab-1
epitope of hCCR9 involves Ile10, Pro11, Asn12, Met13,
Ala14, Asp16, and Tyr17.
In a previous study, we developed a novel epitope mapping
method named RIEDL insertion for epitope mapping (REMAP)(19,20) using a RIEDL tag system.(21) This method isuseful for determining both linear and conformational epitopes. In a future study, we will also investigate the critical
epitope of C9Mab-1 using the REMAP method and compare
those results.
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) underGrant Nos. JP21am0401013 (to Y.K.) and JP21am0101078(to Y.K.), and by the Japan Society for the Promotion of
Science ( JSPS) grants-in-aid for scientific research (KAKENHI) Grant Nos. 21K15523 (to T.A.), 21K07168(to M.K.K.), and 19K07705 (to Y.K.).
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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
MiyagiJapan
E-mail: yukinarikato@med.tohoku.ac.jp
Received: August 30, 2021
Accepted: November 17, 2021