Epitope Mapping of the Anti-Human CC Chemokine Receptor Type-2 Monoclonal Antibody (K036C2)

CC chemokine receptor type-2 (CCR2) belongs to the G protein-coupled receptors superfamily, and is localized on cell surface of tumor cells and some immune cells, including monocytes and macrophages. CCR2 is a receptor for monocyte chemoattractant protein-1/C-C motif chemokine 2, and is involved in the progression of various diseases such as cancers. Therefore, the development of CCR2-targeted monoclonal antibody (mAb) is desired. Its characterization, including epitope of mAb, is very important for antibody applications. In this study, we investigated the critical epitope of K036C2, which is a commercially available anti-human CCR2 (hCCR2) mAb. We conducted enzyme-linked immunosorbent assay (ELISA) using three N-terminal peptides of hCCR2 and demonstrated that K036C2 recognizes 11-29 and 21-39 amino acids of hCCR2. We further performed ELISA using 20 peptides, which include alanine substitution of hCCR2. K036C2 lost the reaction to the alanine-substituted peptides of D25A, Y26A, D27A, G29A, and A30G. These results indicate that the critical binding epitope of K036C2 includes Asp25, Tyr26, Asp27, Gly29, and Ala30 of hCCR2.