Epitope Mapping of Anti-Mouse CCR3 Monoclonal Antibodies (CMab-6 and CMab-7).

One of G protein-coupled receptors, CC chemokine receptor 3 (CCR3), is expressed in eosinophils, basophils, a subset of Th2 lymphocytes, mast cells, and airway epithelial cells. CCR3 levels in the serum of colorectal cancer patients are significantly higher than in control groups. Moreover, CCR3 is essential for recruiting eosinophils into the lung. Therefore, CCR3 is considered both a therapeutic target for colorectal cancer and allergic diseases. Previously, we established anti-mouse CCR3 (mCCR3) monoclonal antibodies (mAbs), CMab-6 (rat IgG, kappa) and CMab-7 (rat IgG, kappa), by immunizing a rat with an N-terminal peptide of mCCR3. These mAbs can be used in flow cytometry and enzyme-linked immunosorbent assays. In this study, we performed the epitope mapping of CMab-6 and CMab-7 using alanine scanning. The reactivity between these mAbs and point mutants of mCCR3 were analyzed using flow cytometry. The results indicated that Phe3, Asn4, Thr5, Asp6, Glu7, Lys9, Thr10, and Glu13 of mCCR3 are essential for CMab-6 binding, whereas Phe15 and Glu16 are essential for CMab-7 binding.