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

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 have been 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, C 9 Mab-1 (IgG 1 , kappa), which can be used for flow cytometry, by immunizing mice with hCCR9-overexpressed Chinese hamster ovary-K1 cells. In this study, we examined the critical epitope of C 9 Mab-1, using enzyme-linked immunosorbent assay (ELISA) with synthesized peptides. First, we performed ELISA with deletion mutants, and C 9 Mab-1 reacted to the 1–20 amino acids sequence of hCCR9. Next, we analyzed the reaction to 20 point mutants, and C 9 Mab-1 did not recognize the alanine-substituted peptides of I10A, P11A, N12A, M13A, A14G, D16A, and Y17A. The results indicate that the binding epitope of C 9 Mab-1 includes Ile10, Pro11, Asn12, Met13, Ala14, Asp16, and Tyr17 of hCCR9.