Determination of the Binding Epitope of an Anti-Mouse CCR9 Monoclonal Antibody (C9Mab-24) Using the 1× Alanine and 2× Alanine-Substitution Method

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 is highly 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, C9Mab-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 of the mCCR9 N-terminus (amino acids 1–19). C9Mab-24 did not recognize two peptides (F14A and F17A), indicating that Phe14 and Phe17 are critical for C9Mab-24-binding to mCCR9. Furthermore, we conducted the 2× Ala-substitution method using two consecutive alanine-substituted peptides of the mCCR9 N-terminus, and showed that C9Mab-24 did not react with four peptides (M13A–F14A, F14A–D15A, D16A–F17A, and F17A–S18A), indicating that 13-MFDDFS-18 is involved in C9Mab-24-binding to mCCR9. Overall, combining, the 1× Ala- or 2× Ala-scanning methods could be useful for understanding for target–antibody interaction.


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 have shown 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], is mainly expressed in the thymus and intestinal epithelium [3,[5][6][7]. CCL25 induces the chemotaxis of immature T cells into the thymus for their maturation [8]. Studies have demonstrated that CCR9 and CCL25 play important roles in inflammatory diseases, such as asthma [9], inflammatory bowel disease [10][11][12], and hepatitis [13,14]. Moreover, CCR9 is highly expressed in malignancies, such as lung cancer [15], breast cancer [16,17], ovarian cancer [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 survival of tumor cells [18,22,23].
Several 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].
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, and thymidine for the selection (Thermo Fisher Scientific Inc.). Supernatants were subsequently screened with the mCCR9p1-19C peptide, using ELISA, following flow cytometry, using CHO/mCCR9, CHO-K1 and RL2 cells.

Epitope Determination Using 2× Ala-substituted mCCR9 Peptides
The result of 1× Ala substitution showed that Phe14 and Phe17 of mCCR9 are the most critical for the C 9 Mab-24-mCCR9 interaction, but did not show that only Phe14 and Phe17 of mCCR9 are enough for C 9 Mab-24 binding to mCCR9. Therefore, we further investigated the C 9 Mab-24 epitope using 2× Ala-substituted mCCR9 peptides, as described previously [33].

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 Figure 2. Determination of the C 9 Mab-24 epitope for mCCR9 by ELISA using 2× Ala-substituted peptides. (A) Synthesized 2× Ala-substituted peptides of mCCR9 were immobilized on immuno plates. The plates were incubated with C 9 Mab-24 (1 µg/mL), followed by peroxidase-conjugated anti-rat immunoglobulins. (B) Schematic illustration of mCCR9 and the C 9 Mab-24 epitope region.

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 the side chains that have been suggested in mAbs recognition. Using the method, the highresolution "functional epitopes" could be mapped for each mAb [34].
We previously applied the strategy to identify the epitope of an anti-mouse CXCR6 mAb, Cx 6 Mab-1 [33]. First, we could not identify the epitope of Cx 6 Mab-1 by 1× Alasubstitution methods. Next, we performed the 2× Ala-substitution method, and could determine the epitope of Cx 6 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 amino acids of hCCR9 [24,25], which is almost identical to the epitopes of C 9 Mab-1 and C 9 Mab-11. C 9 Mab-24 also binds to the N-terminal region of mCCR9 ( 13-MFDDFS -18 ). Therefore, both the neutralizing and biological activities of C 9 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-cell aplasia. 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 the inability 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 CAR-CCR9, 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.

Institutional 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.