Identification of the Binding Epitope of an Anti-mouse CCR4 Monoclonal Antibody, C4Mab-1.

C-C chemokine receptor 4 (CCR4) is one of G protein-coupled receptors, and interacts with chemokines, CCL17 and CCL22. CCR4 is expressed on T cells such as helper T type 2 cells, regulatory T cells, and interleukin 17-producing T helper cells. CCR4 is associated with T cells trafficking into the tumor microenvironment, and is associated with tumor progression or metastasis. Therefore, CCR4 may be a potential therapeutic option for T cell malignancies. C₄Mab-1 is a novel anti-mouse CCR4 (mCCR4) monoclonal antibody produced by mCCR4 N-terminal peptide immunization. C₄Mab-1 is useful for flow cytometric analysis. In this study, we conducted the epitope mapping of C₄Mab-1 using enzyme-linked immunosorbent assay (ELISA) and peptide blocking assay. The result of ELISA indicated that Thr7, Asp8, and Gln11 of mCCR4 are the critical amino acids for the C₄Mab-1 binding. Furthermore, peptide blocking assay by flow cytometry showed that Thr7, Asp8, and Gln11 of mCCR4 are essential for C₄Mab-1 binding to mCCR4-overexpressed Chinese hamster ovary-K1 (CHO/mCCR4) cells, and Val6, Thr9, and Thr10 are involved in the C₄Mab-1 binding to CHO/mCCR4 cells. These results indicate that the critical binding epitope of C₄Mab-1 includes Thr7, Asp8, and Gln11 of mCCR4.