Epitope Mapping of Anti-Mouse CCR3 Monoclonal Antibodies Using Flow Cytometry

The CC chemokine receptor 3 (CCR3) is a receptor for CC chemokines, including CCL5/RANTES, CCL7/MCP-3, and CCL11/eotaxin. CCR3 is expressed on the surface of eosinophils, basophils, a subset of Th2 lymphocytes, mast cells, and airway epithelial cells. CCR3 and its ligands are involved in airway hyperresponsiveness in allergic asthma, ocular allergies, and cancers. Therefore, CCR3 is an attractive target for those therapies. Previously, anti-mouse CCR3 (mCCR3) monoclonal antibodies (mAbs), C3Mab-3 (rat IgG2a, kappa), and C3Mab-4 (rat IgG2a, kappa) were developed using the Cell-Based Immunization and Screening (CBIS) method. In this study, the binding epitope of these mAbs was investigated using flow cytometry. A CCR3 extracellular domain-substituted mutant analysis showed that C3Mab-3, C3Mab-4, and a commercially available mAb (J073E5) recognized the N-terminal region (amino acids 1–38) of mCCR3. Next, alanine scanning was conducted in the N-terminal region. The results revealed that the Ala2, Phe3, Asn4, and Thr5 of mCCR3 are involved in C3Mab-3 binding, whereas Ala2, Phe3, and Thr5 are essential to C3Mab-4 binding, and Ala2 and Phe3 are crucial to J073E5 binding. These results reveal the involvement of the N-terminus of mCCR3 in the recognition of C3Mab-3, C3Mab-4, and J073E5.

In this study, the epitope mapping of anti-mCCR3 mAbs was conducted using flow cytometry, utilizing the extracellular region substitution and the alanine scanning methods to clarify the features of C 3 Mab-3 and C 3 Mab-4.

Determination of the C3Mab-3 Epitope Using Flow Cytometry and Alanine Scanning
Next, alanine scanning was conducted in the N-terminal region, except for Cys28. Thirty-six alanine substitution mutants of mCCR3 were constructed, and the mutant proteins were transiently expressed on the CHO-K1 cells. The reactivity against C3Mab-3, C3Mab-4, and J073E5 was assessed using flow cytometry. As shown in Figure 3A, C3Mab-3 did not react with four mutants (A2G, F3A, N4A, and T5A). In contrast, C3Mab-3 reacted with the other 32 mutants. These results show that four residues (Ala2, Phe3, Asn4, and Thr5) of mCCR3 are important for C3Mab-3 binding ( Figure 3B). C3Mab-4 did not react with three mutants (A2G, F3A, and T5A) but reacted with the others (Figure 4A), indicating that three residues (Ala2, Phe3, and Thr5) of mCCR3 are important for C3Mab-4 binding ( Figure 4B). J073E5 did not react with two mutants (A2G and F3A) but reacted with the others (Figure 5A), indicating that two residues (Ala2 and Phe3) of mCCR3 are important for J073E5 binding ( Figure 5B). The cell surface expression of the mCCR3 mutants on the CHO-K1 cells was confirmed using the anti-mCCR3 mAb, C3Mab-7. It has already been confirmed that Phe15 and Glu16 are essential for C3Mab-7 binding (manuscript submitted). We could confirm the cell surface expression of four mutants (A2G, F3A, N4A, and T5A) of mCCR3 using C3Mab-7 ( Figure 6).

Determination of the C 3 Mab-3 Epitope Using Flow Cytometry and Alanine Scanning
Next, alanine scanning was conducted in the N-terminal region, except for Cys28. Thirty-six alanine substitution mutants of mCCR3 were constructed, and the mutant proteins were transiently expressed on the CHO-K1 cells. The reactivity against C 3 Mab-3, C 3 Mab-4, and J073E5 was assessed using flow cytometry. As shown in Figure 3A, C 3 Mab-3 did not react with four mutants (A2G, F3A, N4A, and T5A). In contrast, C 3 Mab-3 reacted with the other 32 mutants. These results show that four residues (Ala2, Phe3, Asn4, and Thr5) of mCCR3 are important for C 3 Mab-3 binding ( Figure 3B). C 3 Mab-4 did not react with three mutants (A2G, F3A, and T5A) but reacted with the others (Figure 4A), indicating that three residues (Ala2, Phe3, and Thr5) of mCCR3 are important for C 3 Mab-4 binding ( Figure 4B). J073E5 did not react with two mutants (A2G and F3A) but reacted with the others (Figure 5A), indicating that two residues (Ala2 and Phe3) of mCCR3 are important for J073E5 binding ( Figure 5B). The cell surface expression of the mCCR3 mutants on the CHO-K1 cells was confirmed using the anti-mCCR3 mAb, C 3 Mab-7. It has already been confirmed that Phe15 and Glu16 are essential for C 3 Mab-7 binding (manuscript submitted). We could confirm the cell surface expression of four mutants (A2G, F3A, N4A, and T5A) of mCCR3 using C 3 Mab-7 ( Figure 6).

Discussion
We previously established various mAbs against membrane proteins using the CBIS method. Because mAbs sometimes recognize conformational epitopes, they can be applied to flow cytometry but not to Western blotting or ELISA. Two anti-mCCR3 mAbs examined in this study, C3Mab-3 and C3Mab-4, were established using the CBIS method [30]. We attempted to identify their epitopes using synthetic peptides and ELISA. However, they did not recognize the synthetic peptides, including the mCCR3 N-terminal region (aa [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19], which contains the epitope determined using flow cytometry (Figures 3  and 4). These results suggest that the residues participate in the formation of conformational epitopes and/or undergo post-translational modification on the cell surface. Furthermore, we could not exclude the possibility of the first Met as their epitopes. In the case of C3Mab-3 and C3Mab-4 epitopes, Asn4 and Thr5 are involved in recognition. Although Asn and Thr are known to be N-and O-glycosylated, respectively, there are no reports on the glycosylation of the Asn4 and Thr5 of mCCR3. Further studies are required to analyze the involvement of the post-translational modification of these residues in the recognition by C3Mab-3 and C3Mab-4.

Discussion
We previously established various mAbs against membrane proteins using the CBIS method. Because mAbs sometimes recognize conformational epitopes, they can be applied to flow cytometry but not to Western blotting or ELISA. Two anti-mCCR3 mAbs examined in this study, C 3 Mab-3 and C 3 Mab-4, were established using the CBIS method [30]. We attempted to identify their epitopes using synthetic peptides and ELISA. However, they did not recognize the synthetic peptides, including the mCCR3 N-terminal region (aa [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19], which contains the epitope determined using flow cytometry (Figures 3 and 4). These results suggest that the residues participate in the formation of conformational epitopes and/or undergo post-translational modification on the cell surface. Furthermore, we could not exclude the possibility of the first Met as their epitopes. In the case of C 3 Mab-3 and C 3 Mab-4 epitopes, Asn4 and Thr5 are involved in recognition. Although Asn and Thr are known to be Nand O-glycosylated, respectively, there are no reports on the glycosylation of the Asn4 and Thr5 of mCCR3. Further studies are required to analyze the involvement of the post-translational modification of these residues in the recognition by C 3 Mab-3 and C 3 Mab-4.
It has been reported that a CCR3 ligand, CCL11/eotaxin, binds to the N-terminal region of CCR3 [46,47]. Therefore, our established anti-mCCR3 mAbs could compete with ligand binding to mCCR3 and have neutralizing activity. Shen et al. reported that anti-CCR3 mAb could significantly suppress airway eosinophilia and mucus overproduction in asthmatic mice; therefore, the blockage of the CCR3 axis may be an attractive strategy for asthma therapy [48]. In future studies, we would like to examine the neutralizing activities of these anti-mCCR3 mAbs.