MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 42, Number 2, 2023
ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2022.0035
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Epitope Mapping of the Novel Anti-Human CCR9 Monoclonal
Antibody (C9Mab-11) by 2 · Alanine Scanning

Downloaded by Yukinari Kato from www.liebertpub.com at 04/27/23. For personal use only.

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

We recently developed a novel anti-human C-C chemokine receptor 9 (hCCR9) monoclonal antibody (mAb),
C9Mab-11, which is applicable to flow cytometry, western blotting, and enzyme-linked immunosorbent assay(ELISA). This study aims to identify the binding epitope of C9Mab-11 by using 1 · and 2 · alanine (or glycine)substituted-hCCR9 peptides (1 · and 2 · Ala-scan) by ELISA. According to the 1 · Ala-scan analysis, theresponse of C9Mab-11 was diminished against M13A of the hCCR9 peptide, but was not eliminated. In the2 · Ala-scan analysis, the reactions were abolished in the substitution of P11A-N12A, N12A-M13A, andM13A-A14G of hCCR9 N-terminal peptides. The results indicate that the binding epitope of C9Mab-11 includes Pro11, Asn12, Met13, and Ala14 of hCCR9, with the region around Met13 being particularly important.
The successful identification of the C9Mab-11 epitope might be useful for the future pathophysiological analysisof hCCR9.
Keywords: human CCR9, monoclonal antibody, epitope, ELISA, 2 · Ala-scantrigger acute liver damage by collaborating with helper T1(Th1) cells.9 However, CCR9 axis inhibition avoided pDCs
capture in the small intestine, enhanced pDCs infiltration into
the liver, and protected against liver injury.10 Therefore,
managing the CCR9/CCL25 axis could be a target for
immunotherapy in numerous inflammation-associateddisorders.
CCR9 is highly expressed in T cell acute lymphoblastic
leukemia (T-ALL), contributing to the progression ofT-ALL.11 Furthermore, CCR9 expression has been confirmedwith high probability in melanomas that have metastasized tothe intestine.12,13 Thus, CCR9 is considered a therapeutictarget for cancer. In the T-ALL-bearing mouse model, an antihuman CCR9 monoclonal antibody (hCCR9 mAb) (92R) andhumanized 92R (Srb1) inhibited tumor growth and prolonged
the survival of mice.14,15 A clinical study of the CCR9 antagonist CCX282-B against intestinal bowel disease wasconducted.16,17 However, there are still no approved drugs
despite CCR9 being an attractive target.
Previously, we developed various anti-mouse and antihuman GPCR mAbs,18–25 including an anti-hCCR9 mAb(clone C9Mab-11).26 We further characterized the binding

IntroductionT

he C-C chemokine receptor 9 (CCR9) is a member of
the G protein-coupled receptors (GPCRs), which arehighly expressed in T cells, thymocytes, B cells, plasmacytoid dendritic cells (pDCs), and intestinal cells.1 Chemokine
ligand 25 (CCL25), formerly thymus-expressed chemokine(TECK), regulates the activation and infiltration of immature
T cells by binding to CCR9.2,3 CCL25 is the only known
ligand for CCR9 and is mainly secreted from the epithelial
cells of the thymus and small intestine. In addition, CCR9+ Tcells are gut-homing T cells because the CCR9 and CCL25
interactions potently recruit T cells to intestinal tissues.4–6
CCR9 on immune cells guides the spatial organization andcellular communications of immune cells within tissues.
The CCR9/CCL25 axis is involved in the development ofvarious inflammatory diseases. The increase in CCR9+monocytes was confirmed in the synovium of patients withrheumatoid arthritis.7 In the ovalbumin-induced allergic inflammation model, the recruitment of eosinophils and T cells
was impaired at the peribronchial and perivenular levels inCCR9-deficient mice.8 In hepatitis, CCR9+ macrophages

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

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ISODA ET AL.

epitopes of these mAbs.27–29 The binding epitope ofCx6Mab-1, an anti-mouse CXCR6 mAb, was identified byenzyme-linked immunosorbent assay (ELISA) using the twoalanine scanning method (2 · Ala-scan).29 We demonstrated
that the 2 · Ala-scan was an effective method for epitope
mapping of mAbs.
In this study, we determined the epitope of C9Mab-11
using the 1 · and 2 · Ala-scan strategies.
Materials and Methods
PeptidesDownloaded by Yukinari Kato from www.liebertpub.com at 04/27/23. For personal use only.

The hCCR9 (Accession No. NM_031200) peptide (4TDFTSPIPNMADDYGSEST-22), one-alanine (or glycine)residue-substituted peptide (Supplementary Table S1), andtwo alanine (or glycine) residue-substituted peptides(Table 1) were synthesized using PEPScreen (Sigma-AldrichCorp., St. Louis, MO).
Enzyme-linked immunosorbent assay

Synthesized hCCR9 peptides were immobilized on Nunc
Maxisorp 96-well immunoplates (Thermo Fisher Scientific,Inc., Waltham, MA) at a 1 lg/mL concentration for 30 minutes at 37C. After washing with phosphate-buffered saline
containing 0.05% Tween20 (PBST; Nacalai Tesque, Inc.,Kyoto, Japan), the 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-11, followed byperoxidase-conjugated anti-mouse immunoglobulin (1:2000
diluted; Agilent Technologies Inc., Santa Clara, CA). Enzymatic reactions were performed using the ELISA POD
Substrate TMB Kit (Nacalai Tesque, Inc.). The optical density was measured at 655 nm using an iMark microplate
reader (Bio-Rad Laboratories, Inc., Berkeley, CA).

Table 1. Identification of the C9Mab-11 Epitope
Using 2 · Alanine-Substituted hCCR9 Peptides
PeptidesSequences

WTTDFTSPIPNMADDYGSEST
T4A–D5AAAFTSPIPNMADDYGSEST
D5A–F6ATAATSPIPNMADDYGSEST
F6A–T7A
TDAASPIPNMADDYGSEST
T7A–S8A
TDFAAPIPNMADDYGSEST
S8A–P9ATDFTAAIPNMADDYGSEST
P9A–I10ATDFTSAAPNMADDYGSEST
I10A–P11A
TDFTSPAANMADDYGSEST
P11A–N12ATDFTSPIAAMADDYGSEST
N12A–M13A TDFTSPIPAAADDYGSEST
M13A–A14G TDFTSPIPNAGDDYGSEST
A14G–D15A TDFTSPIPNMGADYGSEST
D15A–D16A TDFTSPIPNMAAAYGSEST
D16A–Y17A TDFTSPIPNMADAAGSEST
Y17A–G18A TDFTSPIPNMADDAASEST
G18A–S19ATDFTSPIPNMADDYAAEST
S19A–E20ATDFTSPIPNMADDYGAAST
E20A–S21ATDFTSPIPNMADDYGSAATS21A–T22ATDFTSPIPNMADDYGSEAA

C9Mab-11+++++++++++++++++++++++++++++++++++++++++++++++++++, OD655S0.3; -, OD655 < 0.1.
hCCR9, human C-C chemokine receptor 9; WT, wild-type.

Results and Discussion
Epitope mapping of C9Mab-11 with alanine-substituted
hCCR9 peptides

We previously established an anti-hCCR9 mAb (cloneC9Mab-11) by immunizing the hCCR9 N-terminal peptide(4-TDFTSPIPNMADDYGSEST-22) + C-terminal cysteine
residue with keyhole limpet hemocyanin.26 To investigate
the binding epitope of C9Mab-11, we first synthesized
one-alanine- (or glycine-) substituted peptides of hCCR9,
referred to as the 1 · Ala-scan method (SupplementaryTable S1). C9Mab-11 reacted with all one-alanine- (or glycine-) substituted and wild-type (WT) hCCR9 peptides.
However, compared with other substituted peptides, thereaction of C9Mab-11 against M13A was attenuated (Supplementary Fig. S1).
Furthermore, we synthesized 18 peptides, substituting twoamino acid sequences with two alanine (or glycine) residues(2 · Ala-scan method). For instance, a peptide (T4A–D5A)
indicates the alanine substitution of the fourth Thr and thefifth Asp of the hCCR9 peptide (Table 1). As shown inFigure 1A, C9Mab-11 exhibited reactions with T4A–D5A,
D5A–F6A, F6A–T7A, T7A–S8A, S8A–P9A, P9A–I10A,
I10A–P11A, A14G–D15A, D15A–D16A, D16A–Y17A,
Y17A–G18A, G18A–S19A, S19A–E20A, E20A–S21A,S21A–T22A, and WT of hCCR9 peptides. However, C9Mab11 did not react with P11A–N12A, N12A–M13A, andM13A–A14G (Fig. 1A), indicating that Pro11, Asn12,
Met13, and Ala14 are included in the critical epitope ofC9Mab-11. The results are summarized in Table 1. Figure 1B
describes a schematic illustration of hCCR9 and the estimated binding epitope of C9Mab-11.
The 2 · Ala-scan is a useful method for identifying theepitope of mAbs. Considering the results of the 1 · Alascan (Supplementary Fig. S1), we speculate that aminoacids in the region centered on Met13 of hCCR9 are pivotal
epitope for C9Mab-11 binding. Anti-hCCR9 mAbs, 91R(mouse IgG2b), and 92R (mouse IgG2a) generated by immunization of plasmid bearing-hCCR9 cDNA using thegene gun method have been reported to possess antitumor
effects in mouse models.14,15,30 The hotspot of epitopes of91R and 92R are 11-PNMADD-16 of hCCR9,14 consistent
with the epitope of C9Mab-11 in this study (Fig. 1). Since
the subclass of C9Mab-11 is also mouse IgG2a,26 C9Mab11 is expected to have an antitumor effect on CCR9expressing cancers. In addition to antitumor activity, chimeric antigen receptor-T cells that target hCCR9 have highantitumor activity against T-ALL.31 C9Mab-11 could
contribute to the development of novel immunotherapy
modalities.
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 grantnos. JP22ama121008 (to Y.K.), JP22am0401013 (to Y.K.),
JP22bm1004001 (to Y.K.), JP22ck0106730 (to Y.K.), andJP21am0101078 (to Y.K.), and by the Japan Society for thePromotion of Science ( JSPS) Grants-in-Aid for ScientificDownloaded by Yukinari Kato from www.liebertpub.com at 04/27/23. For personal use only.

IDENTIFICATION OF C9MAB-11 EPITOPE

75

FIG. 1. Epitope identification of the C9Mab-11 by ELISA using the 2 · Ala-scan method. (A) The 2 · alanine-substitutedand WT of hCCR9 peptides were immobilized on immunoplates. The plates were incubated with C9Mab-11 (1 mg/mL),followed by peroxidase-conjugated anti-mouse immunoglobulins. (B) Schematic illustration of hCCR9 and the critical
epitope of C9Mab-11. The C9Mab-11 epitope involves Pro11, Asn12, Met13, and Ala14 of hCCR9. Star describes theestimated epitope from Supplementary Figure S1. 2 · Ala-scan, 2 · alanine scanning; ELISA, enzyme-linked immunosorbent assay; hCCR9, human C-C chemokine receptor 9; WT, wild-type.

Research (KAKENHI) grant nos. 21K20789 (to T.T.),
22K06995 (to H.S.), 21K07168 (to M.K.K.), and 22K07224(to Y.K.).
Supplementary Material

Supplementary Table S1
Supplementary Figure S1
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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: October 2, 2022
Accepted: March 21, 2023