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

Abstract

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.