Epitope Mapping of an Anti-Mouse CCR2 Monoclonal Antibody (C₂Mab-6) Using Enzyme-Linked Immunosorbent Assay

CC chemokine receptor type-2 (CCR2) is a member of the G protein-coupled receptors, and is mainly expressed on cell surface of immune cells. CCR2 binds to its ligand, C-C motif chemokine 2 (also named as monocyte chemoattractant protein-1), which involves in the tumor progression by modulating the tumor microenvironment. Therefore, the monoclonal antibody (mAb) targeting CCR2 could be one of the strategies for cancer treatment. In this study, we investigated the critical epitope of C₂Mab-6, an anti-mouse CCR2 (mCCR2) mAb developed by N-terminal peptides immunization. We first performed enzyme-linked immunosorbent assay (ELISA) using N-terminal peptides of mCCR2 and demonstrated that C₂Mab-6 recognizes 1-19 amino acids of mCCR2. We further performed ELISA using 20 alanine-substituted peptides of mCCR2. C₂Mab-6 lost the reaction to the alanine-substituted peptides of D3A, N4A, M6A, P8A, Q9A, and F10A. These results indicate that the binding epitope of C₂Mab-6 includes Asp3, Asn4, Met6, Pro8, Gln9, and Phe10 of mCCR2.