Epitope Mapping of Anti-Mouse CCR1 Monoclonal Antibodies

The C-C motif chemokine receptor 1 (CCR1) plays key roles in guiding leukocyte movement during immune surveillance and inflammatory responses. Targeting CCR1 is a promising approach for treating autoimmune diseases and cancers. We previously developed anti-mouse CCR1 (mCCR1) monoclonal antibodies (clones C1Mab-2 and C1Mab-6) for use in flow cytometry and western blotting. However, the specific binding sites have not yet been identified. This study examined the binding epitope of C1Mab-2 and C1Mab-6. Analysis of mCCR1 mutants with altered extracellular domains showed that C1Mab-2 and C1Mab-6 bind to the N-terminal region of mCCR1. Additionally, PA-tag substitution experiments identified the epitope as comprising amino acids 1–13 of mCCR1. Further, alanine (or glycine) scanning within the N-terminal region (amino acids 2–13) was performed using flow cytometry and western blotting. The results demonstrated that Met1, Glu2, Asp5, and Phe6 are critical for recognition by C1Mab-2, while Met1, Glu2, Ile3, and Asp5 are essential for recognition by C1Mab-6. These findings enhance our under-standing of how mCCR1 interacts with C1Mabs.