Epitope Mapping of the Anti-Human CCR2 Monoclonal Antibody C₂Mab-9

CC chemokine receptor type-2 (CCR2) belongs to the G protein-coupled receptors superfamily, localized on cell surface of some immune-related cells, including monocytes and macrophages. CCR2 and its ligand CCL2 are involved in the progression of various diseases such as cancers. Therefore, CCR2-targeted monoclonal antibodies (mAbs) are needed for treatment and diagnosis. Previously, we successfully developed an anti-human CCR2 (hCCR2) mAb, C₂Mab-9 (mouse IgG₁, kappa), which is applicable for flow cytometry and immunocytochemistry. In this study, we investigated the critical epitope of C₂Mab-9. We conducted enzyme-linked immunosorbent assay (ELISA) using several N-terminal peptides of hCCR2, and demonstrated that C₂Mab-9 recognizes 11-29 and 21-39 amino acids of hCCR2. We further performed ELISA using 20 peptides, which include alanine substitution of hCCR2. C₂Mab-9 lost the reaction to the alanine-substituted peptides of F23A, F24A, D25A, Y26A, and D27A. Among them, F23A, F24A, D25A, and Y26A did not block the C₂Mab-9 reaction with U937 cells in flow cytometry. These results indicate that the critical binding epitope of C₂Mab-9 includes Phe23, Phe24, Asp25, and Tyr26.