Epitope Mapping of Anti-Mouse C-C Motif Chemokine Receptor 1 Monoclonal Antibodies Developed by the Cell-Based Immunization and Screening Method

Abstract

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 monoclonal antibodies against mouse CCR1 (mCCR1) (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 using flow cytometry. 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 1st Met and amino acids 2–13 of mCCR1. Further alanine (or glycine) scanning within the N-terminal region (amino acids 2–13) demonstrated that Glu2, Asp5, and Phe6 are essential for recognition by C₁Mab-2, and Glu2, Ile3, and Asp5 are crucial for recognition by C₁Mab-6 in flow cytometry and Western blotting. These findings contribute to the understanding of mCCR1 and C₁Mab interaction.