antibodies
Communication

Determination of the Binding Epitope of an Anti-Mouse CCR9
Monoclonal Antibody (C9Mab-24) Using the 1× Alanine and2× Alanine-Substitution Method
Hiyori Kobayashi 1,† , Teizo Asano 2,† , Tomohiro Tanaka 1 , Hiroyuki Suzuki 1, * , Mika K. Kaneko 2
and Yukinari Kato 1,2, *
1

2

*
†

Citation: Kobayashi, H.; Asano, T.;
Tanaka, T.; Suzuki, H.; Kaneko, M.K.;
Kato, Y. Determination of the Binding
Epitope of an Anti-Mouse CCR9
Monoclonal Antibody (C9 Mab-24)
Using the 1× Alanine and 2×

Department of Molecular Pharmacology, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi,
Aoba-ku, Sendai 980-8575, Japan
Department of Antibody Drug Development, Tohoku University Graduate School of Medicine,
2-1 Seiryo-machi, Aoba-ku, Sendai 980-8575, Japan
Correspondence: hiroyuki.suzuki.b4@tohoku.ac.jp (H.S.); yukinari.kato.e6@tohoku.ac.jp (Y.K.);
Tel.: +81-22-717-8207 (H.S. & Y.K.)
These authors contributed equally to this work.

Abstract: C-C chemokine receptor 9 (CCR9) is a receptor for C-C-chemokine ligand 25 (CCL25).
CCR9 is crucial in the chemotaxis of immune cells and inflammatory responses. Moreover, CCR9 ishighly expressed in tumors, including several solid tumors and T-cell acute lymphoblastic leukemia.
Several preclinical studies have shown that anti-CCR9 monoclonal antibodies (mAbs) exert antitumor
activity. Therefore, CCR9 is an attractive target for tumor therapy. In this study, we conducted the
epitope mapping of an anti-mouse CCR9 (mCCR9) mAb, C9 Mab-24 (rat IgG2a , kappa), using the 1×
alanine (1× Ala)- and 2× alanine (2× Ala)-substitution methods via enzyme-linked immunosorbent
assay. We first performed the 1× Ala-substitution method using one alanine-substituted peptides ofthe mCCR9 N-terminus (amino acids 1–19). C9 Mab-24 did not recognize two peptides (F14A andF17A), indicating that Phe14 and Phe17 are critical for C9 Mab-24-binding to mCCR9. Furthermore,
we conducted the 2× Ala-substitution method using two consecutive alanine-substituted peptides ofthe mCCR9 N-terminus, and showed that C9 Mab-24 did not react with four peptides (M13A–F14A,
F14A–D15A, D16A–F17A, and F17A–S18A), indicating that 13- MFDDFS-18 is involved in C9 Mab-24binding to mCCR9. Overall, combining, the 1× Ala- or 2× Ala-scanning methods could be useful forunderstanding for target–antibody interaction.

Alanine-Substitution Method.
Antibodies 2023, 12, 11.
https://doi.org/10.3390/

Keywords: mouse CCR9; monoclonal antibody; epitope mapping; alanine scanning; enzyme-linked
immunosorbent assay

antib12010011
Academic Editors: Buyong Ma andRui Gong
Received: 9 December 2022
Revised: 7 January 2023
Accepted: 25 January 2023
Published: 31 January 2023

Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms andconditions of the Creative Commons
Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

1. Introduction
C-C chemokine receptor 9 (CCR9) is a member of the G-protein coupled receptors
with seven transmembrane domains and four extracellular regions. Previous studies haveshown that CCR9 is expressed on the surface of immature T lymphocytes and intestinal
cells [1,2]. The C-C chemokine ligand 25 (CCL25), the only ligand for CCR9 [3,4], ismainly expressed in the thymus and intestinal epithelium [3,5–7]. CCL25 induces thechemotaxis of immature T cells into the thymus for their maturation [8]. Studies havedemonstrated that CCR9 and CCL25 play important roles in inflammatory diseases, suchas asthma [9], inflammatory bowel disease [10–12], and hepatitis [13,14]. Moreover, CCR9
is highly expressed in malignancies, such as lung cancer [15], breast cancer [16,17], ovariancancer [18], melanoma [19,20], and T-cell acute lymphoblastic leukemia (T-ALL) [21]. CCR9
is expressed in more than 70% cases of T-ALL. However, it is expressed in a small subset of
normal T cells [21]. The interaction of CCR9 and CCL25 activates the phosphatidylinositide
3-kinase (PI3K)/Akt signaling pathway, which is involved in the proliferation and survivalof tumor cells [18,22,23].

Antibodies 2023, 12, 11. https://doi.org/10.3390/antib12010011

https://www.mdpi.com/journal/antibodies

Antibodies 2023, 12, 11

2 of 9Several anti-CCR9 mAbs have been developed for therapeutic uses. Anti-human
CCR9 mAbs (clones 91R and 92R) exhibited cytotoxicity against CCR9-positive tumors
via antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity. These antibodies suppressed T-ALL proliferation in mouse xenograft models [24,25].
Moreover, Maciocia et al. developed a CCR9-specific mAb by gene-gun vaccination of rats
with a plasmid encoding human CCR9. They further produced the chimeric antigen receptor (CAR)-T cells, and showed a potent antitumor effect in cell lines and patient-derived
xenograft mouse models of T-ALL [21]. Therefore, CCR9 is considered as an attractive
target for tumor therapy [5,26].
Using the N-terminal peptide immunization, we developed anti-mouse chemokine
receptor mAbs against CCR2 [27], CCR3 [28], CCR4 [29], CCR6 [30], and CXCR6 [31].
Furthermore, we developed an anti-mouse CCR9 (mCCR9) mAb, C9 Mab-24 (rat IgG2a ,kappa), via peptide immunization of the mCCR9 N-terminus (amino acids 1–19) [32].
C9 Mab-24 could be applied to flow cytometry in mCCR9-overexpressed Chinese hamster
ovary-K1 cells and endogenously expressed RL2 cells [32]. In this study, we determined thebinding epitope of C9 Mab-24 using two different alanine scanning strategies via enzymelinked immunosorbent assay (ELISA).
2. Materials and Methods
2.1. Development of C9 Mab-24
Eurofins Genomics KK (Tokyo, Japan) synthesized a partial sequence of the N-terminal
extracellular region of mCCR9 (Accession No.: NP_001160097) with cysteine at its Cterminus (mCCR9p1-19C; sequence: MMPTELTSLIPGMFDDFSYC). Subsequently, the keyhole limpet hemocyanin (KLH) was conjugated at the C-terminus of the peptide (mCCR9p119C-KLH). Chinese hamster ovary (CHO)-K1 cells were obtained from the American Type
Culture Collection (Manassas, VA, USA). The expression plasmid of mCCR9 (pCMV6neomCCR9-Myc-DDK) was purchased from OriGene Technologies, Inc. (Rockville, MD, USA).
The mCCR9 plasmid was transfected into CHO-K1 cells, using a neon transfection system(Thermo Fisher Scientific Inc., Waltham, MA, USA). Stable transfectants were establishedthrough cell sorting using a cell sorter (SH800; Sony Corp., Tokyo, Japan) using Roswell
Park Memorial Institute (RPMI) 1640 medium (Nacalai Tesque, Inc.), containing 10%
heat-inactivated fetal bovine serum (FBS, Thermo Fisher Scientific Inc.), 100 units/mL of
penicillin, 100 µg/mL of streptomycin, and 0.25 µg/mL of amphotericin B (Nacalai Tesque,Inc.), and 0.5 mg/mL of G418 (Nacalai Tesque, Inc., Kyoto, Japan). CHO-K1, P3U1, mCCR9overexpressed CHO-K1 (CHO/mCCR9), and RL2 were cultured in RPMI 1640 medium
with 10% heat-inactivated FBS, 100 units/mL of penicillin, 100 µg/mL of streptomycin,
and 0.25 µg/mL of amphotericin B. Cells were grown in a humidified incubator at 37 ◦ C, at
an atmosphere of 5% CO2 and 95% air.
A five-week old Sprague–Dawley rat was purchased from 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
suffering and distress in the laboratory. The Animal Care and Use Committee of Tohoku
University (Permit number: 2019NiA-001) approved the animal experiments. The rat
was monitored daily for health during the full four-week duration of the experiment. A
reduction of more than 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 mCCR9,
one rat was immunized intraperitoneally, using 100 µg mCCR9p1-19C-KLH peptide with
Imject Alum (Thermo Fisher Scientific Inc.). The procedure included three additional
immunizations, which were followed by a final booster intraperitoneal injection, two days
before the harvest of spleen cells. Harvested spleen cells were subsequently fused with P3U1
cells, using PEG1500 (Roche Diagnostics, Indianapolis, IN, USA), after which hybridomas
were grown in an RPMI medium supplemented with hypoxanthine, aminopterin, andthymidine for the selection (Thermo Fisher Scientific Inc.). Supernatants were subsequently

Antibodies 2023, 12, 11

3 of 9screened with the mCCR9p1-19C peptide, using ELISA, following flow cytometry, using
CHO/mCCR9, CHO-K1 and RL2 cells.
2.2. ELISA
The mCCR9 peptides, such as wild type (WT), 19 of 1× alanine (1× Ala)-substituted
peptides (Table 1), and 18 of 2× alanine (2× Ala)-substituted peptides (Table 2), weresynthesized using PEPscreen (Sigma-Aldrich Corp., St. Louis, MO, USA). Each peptide
was immobilized on Nunc Maxisorp 96-well immuno plates (Thermo Fisher Scientific,Inc.) at a concentration of 1 µg/mL for 30 min at 37 ◦ C. As a negative control, no peptide
was immobilized on the immuno plates. After washing with phosphate-buffered saline
containing 0.05% Tween20 (PBST), the wells were blocked with 1% bovine serum albumin containing PBST for 30 min at 37 ◦ C. The plates were then incubated with C9 Mab-24(1 µg/mL), followed by a 1:20,000 dilution of peroxidase-conjugated anti-rat immunoglobulins (Sigma-Aldrich Corp.). Enzymatic reactions were performed using an ELISA POD
Substrate TMB Kit (Nacalai Tesque, Inc.). Optical density was detected at 655 nm using an
iMark microplate reader (Bio-Rad Laboratories, Inc., Berkeley, CA, USA).
Table 1. Identification of the C9 Mab-24 epitope using 1× Ala-substituted mCCR9 peptides.
PeptidesSequences

C9 Mab-24

WT
M1A
M2A
P3A
T4A
E5A
L6A
T7A
S8A
L9A
I10A
P11A
G12A
M13A
F14A
D15A
D16A
F17A
S18A
Y19A

MMPTELTSLIPGMFDDFSYAMPTELTSLIPGMFDDFSY
MAPTELTSLIPGMFDDFSY
MMATELTSLIPGMFDDFSY
MMPAELTSLIPGMFDDFSY
MMPTALTSLIPGMFDDFSY
MMPTEATSLIPGMFDDFSY
MMPTELASLIPGMFDDFSY
MMPTELTALIPGMFDDFSY
MMPTELTSAIPGMFDDFSY
MMPTELTSLAPGMFDDFSY
MMPTELTSLIAGMFDDFSY
MMPTELTSLIPAMFDDFSY
MMPTELTSLIPGAFDDFSY
MMPTELTSLIPGMADDFSY
MMPTELTSLIPGMFADFSYMMPTELTSLIPGMFDAFSYMMPTELTSLIPGMFDDASY
MMPTELTSLIPGMFDDFAY
MMPTELTSLIPGMFDDFSA

++
++
++
++
++
++
++
++
++
++
++
++
++
++
–
++
++
–
++
++

++, OD655 ≥ 0.3; –, OD655 < 0.1.

Table 2. Identification of the C9 Mab-24 epitope using 2× Ala-substituted mCCR9 peptides.
PeptidesWT
M1A–M2A
M2A–P3AP3A–T4A
T4A–E5A
E5A–L6A
L6A–T7A
T7A–S8A
S8A–L9A
L9A–I10A
I10A–P11A
P11A–G12A
G12A–M13A
M13A–F14A
F14A–D15A
D15A–D16A
D16A–F17A
F17A–S18A
S18A–Y19A++, OD655 ≥ 0.3; –, OD655 < 0.1.

Sequences

C9 Mab-24

MMPTELTSLIPGMFDDFSYAAPTELTSLIPGMFDDFSY
MAATELTSLIPGMFDDFSYMMAAELTSLIPGMFDDFSYMMPAALTSLIPGMFDDFSY
MMPTAATSLIPGMFDDFSY
MMPTEAASLIPGMFDDFSY
MMPTELAALIPGMFDDFSY
MMPTELTAAIPGMFDDFSY
MMPTELTSAAPGMFDDFSY
MMPTELTSLAAGMFDDFSY
MMPTELTSLIAAMFDDFSY
MMPTELTSLIPAAFDDFSY
MMPTELTSLIPGAADDFSY
MMPTELTSLIPGMAADFSYMMPTELTSLIPGMFAAFSY
MMPTELTSLIPGMFDAASYMMPTELTSLIPGMFDDAAY
MMPTELTSLIPGMFDDFAA

++
++
++
++
++
++
++
++
++
++
++
++
++
–
–
++
–
–
++

Antibodies 2023, 12, 11

4 of 9

3. Results
3.1. Epitope Determination Using 1× Ala-substituted mCCR9 Peptides
We previously developed an anti-mCCR9 mAb, C9 Mab-24 (rat IgG2a , kappa) using
peptide immunization of the mCCR9 N-terminus (amino acids 1–19) as shown in Materials
Antibodies 2022, 11, x FOR PEER REVIEW
4 of 9and Methods [32]. C9 Mab-24 is very useful for flow cytometry. Herein,
we determined thebinding epitope of C9 Mab-24 using two different alanine scanning strategies via ELISA.
We synthesized 19 different 1× Ala-substituted mCCR9 peptides (Table 1). According
G12A
MMPTELTSLIPAMFDDFSY
++
to
the
C9 Mab-24 reacted to 1× Ala-substituted
peptides, such as M1A, M2A,
M13A ELISA results,
MMPTELTSLIPGAFDDFSY++
P3A,
L6A, T7A, S8A, L9A, I10A, P11A, G12A,– M13A, D15A, D16A, S18A, andF14A T4A, E5A,
MMPTELTSLIPGMADDFSY
Y19A,
as WT (positive control) (Figure 1A). In contrast,
C9 Mab-24 did not react
D15A as well MMPTELTSLIPGMFADFSY++
D16A1× Ala-substituted
MMPTELTSLIPGMFDAFSY++ as well as a negative control
withpeptides, such as F14A and F17A
F17A 1A). TheseMMPTELTSLIPGMFDDASY– mCCR9 are the critical residues(Figureresults indicate that Phe14 and Phe17 of
S18A
MMPTELTSLIPGMFDDFAY++
for the C9 Mab-24 binding to mCCR9. Figure 1B summarizes the results. An alignment of
Y19A
MMPTELTSLIPGMFDDFSA++
rat,–, andCCR9 sequences (residues, 1–19) is shown in Figure 1C.
++,mouse,
OD655 ≥0.3;
OD655human<0.1.

9Mab-24
Figure1. Determinationof the Cof
for mCCR9
ELISA
using 1×Figure1. Determination
the Cepitope
epitopebyfor
mCCR9
byAla-substituted
ELISA using 1× Ala-substituted
9 Mab-24
peptides. (A) Synthesized 1× Ala-substituted peptides of mCCR9 were immobilized on immuno
peptides. (A) Synthesized 1× Ala-substituted peptides of mCCR9 were immobilized on immuno
plates. The plates were incubated with C9Mab-24 (1 μg/mL), followed by peroxidase-conjugatedplates.
The plates wereincubatedwith C9ofMab-24µg/mL),
followedby forperoxidase-conjugatedanti-rat
immunoglobulins.
(B) Schematic
illustration
mCCR9 (1andthe criticalamino acids
C9anti-rat
Mab-24 epitope.
(C) An alignment ofrat, and human
CCR9 sequences(residues,
immunoglobulins.
(B)mouse,
Schematic
illustrationof mCCR9
and 1–19).
the critical amino acids for

C9 Mab-24 epitope. (C) An alignment of mouse, rat, and human CCR9 sequences (residues, 1–19).

Antibodies 2023, 12, 11

5 of 93.2. Epitope Determination Using 2× Ala-substituted mCCR9 Peptides
The result of 1× Ala substitution showed that Phe14 and Phe17 of mCCR9 are themost critical for the C9 Mab-24-mCCR9 interaction, but did not show that only Phe14
and Phe17 of mCCR9 are enough for C9 Mab-24 binding to mCCR9. Therefore, we further
investigated the C9 Mab-24 epitope using 2× Ala-substituted mCCR9 peptides, as described
previously [33].
We synthesized 18 different 2× Ala-substituted mCCR9 peptides (Table 2). According
to the ELISA results, C9 Mab-24 reacted to 2× Ala-substituted peptides, such as M1A–M2A,
M2A–P3A, P3A–T4A, T4A–E5A, E5A–L6A, L6A–T7A, T7A–S8A, S8A–L9A, L9A–I10A,
I10A–P11A, P11A–G12A, G12A–M13A, and S18A–Y19A as well as WT (positive control)(Figure 2A). In contrast, C9 Mab-24 did not react with 2× Ala-substituted peptides, such as
M13A–F14A, F14A–D15A, D16A–F17A, and F17A–S18A along with the negative control(Figure2A). Moreover, the reactivity of C9 Mab-24 with D15A–D16A was weaker than that 6 of 9
Antibodies 2022, 11, x FOR PEER REVIEW
with WT (Figure 2A). These results indicate that 13- MFDDFS-18 is involved in C9 Mab-24binding to mCCR9. Figure 2B summarizes the results.

Figure 2.
2. Determination
Determination ofofthe
C9C
Mab-24
epitope for mCCR9 by ELISA using 2× Ala-substituted
Figurethe
9Mab-24 epitope for mCCR9 by ELISA using 2× Ala-substituted
peptides.
(A)
Synthesized2
×
Ala-substituted
peptidesof mCCR9
were were
immobilized
on immuno
peptides. (A) Synthesized 2× Ala-substituted
peptidesof mCCR9
immobilized
on immuno
plates.
The
plateswere
incubatedwithC
Mab-24(1µg/mL),
followedbyperoxidase-conjugatedplates. The plates were incubated with9 C9Mab-24 (1 μg/mL), followed by peroxidase-conjugatedanti-rat immunoglobulins.
immunoglobulins. (B)illustrationof mCCR9
and theC9theMab-24
epitopeepitope
region. region.
anti-rat(B)Schematic
Schematic
illustrationof mCCR9and
C9Mab-24

4. Discussion
The alanine-scanning mutagenesis method was first applied to antibody–antigen interaction in the human growth hormone (hGH) and 21 different anti-hGH mAbs using

Antibodies 2023, 12, 11

6 of 94. Discussion
The alanine-scanning mutagenesis method was first applied to antibody–antigen
interaction in the human growth hormone (hGH) and 21 different anti-hGH mAbs using ELISA [34]. Single alanine mutations were introduced at every residue within theside chains that have been suggested in mAbs recognition. Using the method, the highresolution “functional epitopes” could be mapped for each mAb [34].
We have established various anti-chemokine receptor mAbs against mouse CCR3 [35,36],
mouse CCR8 [37–39], and human CCR9 (hCCR9) (clone C9 Mab-1) [40] using the Cell-Based
Immunization and Screening (CBIS) method. We also identified the epitope of C9 Mab-1 in
the N-terminal region of hCCR9 [41]. Then, we immunized the peptide of the region, andestablished a clone, C9 Mab-11, which possesses a binding affinity comparable to C9 Mab-1 [42].
C9 Mab-1 possesses a wider epitope (10- IPNMA-14 and 16- DY-17 ) [41] than that of C9 Mab-11(11- PNMA-14 ) (manuscript submitted). In this study, we identified the epitope of the antimCCR9 mAb, C9 Mab-24, which was established by the N-terminal peptide immunization ofmCCR9 [32]. Using the 2× Ala- and 1× Ala-substitution methods, we found that 13- MFDDFS-18
is involved in C9 Mab-24-binding to mCCR9, and the phenylalanines at 14 and 17 are criticalamong the amino acids (Figures 1 and 2). The combination of 2× Ala- and 1× Ala-substitution
methods could contribute the determination of the region and center of the mAb epitope.
We previously applied the strategy to identify the epitope of an anti-mouse CXCR6mAb, Cx6 Mab-1 [33]. First, we could not identify the epitope of Cx6 Mab-1 by 1× Alasubstitution methods. Next, we performed the 2× Ala-substitution method, and could
determine the epitope of Cx6 Mab-1. The 2× Ala substitution could be another option
to determine the epitope of mAbs if the epitope was not determined by the 1× Alasubstitution method.
The N-terminal region of CCR9 is important for the CCL25 interaction. Anti-hCCR9
mAbs (91R and 92R) recognized the N-terminus of hCCR9, which was partially inhibited in
the presence of CCL25 [3,25]. The epitopes of 91R and 92R are located within 11–16 aminoacids of hCCR9 [24,25], which is almost identical to the epitopes of C9 Mab-1 and C9 Mab-11.
C9 Mab-24 also binds to the N-terminal region of mCCR9 (13- MFDDFS-18 ). Therefore, boththe neutralizing and biological activities of C9 Mab-24 should be investigated in the future.
The therapeutic success for refractory childhood leukemia relies on the development of
CAR-T targeting B-cell-specific antigen CD19 for B-ALL [43]. Although the therapy targets
the common B cell antigen, the background of the success is the ability to tolerate B-cellaplasia. Compared to B-cell aplasia, T-cell aplasia exhibits intolerable immunosuppression.
Therefore, a major hurdle for the development of CAR-T cell therapy for T-ALL is theinability to find T-ALL antigens that are not expressed in normal T-cells [44]. To overcome
this problem, CCR9 is expected for cell surface antigens unique to T-ALL cells. The CARCCR9, a CAR-T with a single-chain variable fragment (scFv) for hCCR9, demonstrated
cytotoxicity against CCR9-positive but not CCR9-negative T-ALL cells [21]. Although the
epitope of the scFv has not been reported, the investigation of a suitable epitope to exert
the CAR-T-mediated cytotoxicity is thought to be important for the future therapeutic
applications of our anti-CCR9 mAbs.
Author Contributions: H.K., T.T. and T.A. performed the experiments. M.K.K. and Y.K. designed
the experiments. H.K., T.A., H.S. and Y.K. analyzed the data. H.K., T.A., H.S. and Y.K. wrote themanuscript. All authors read and approved the final manuscript and agreed to be accountable
for all aspects of the research in ensuring that the accuracy or integrity of any part of the work is
appropriately investigated and resolved. All authors have read and agreed to the published versionof the manuscript.
Funding: This research was supported in part by the Japan Agency for Medical Research andDevelopment (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.), and by the Japan Society for the Promotion of Science (JSPS) Grants-in-Aid for Scientific Research (KAKENHI) grant nos.
21K20789 (to T.T.), 22K06995 (to H.S.), 22K15523 (to T.A.), 22K07168 (to M.K.K.), and 22K07224 (to Y.K.).

Antibodies 2023, 12, 11

7 of 9Institutional Review Board Statement: The animal study protocol was approved by the Animal
Care and Use Committee of Tohoku University (Permit number: 2019NiA-001) for studies involving animals.
Data Availability Statement: All related data and methods are presented in this paper. Additional
inquiries should be addressed to the corresponding authors.
Conflicts of Interest: The authors declare no conflict of interest involving this article.

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