Epitope Mapping of the Anti-CD44 Monoclonal Antibody (C₄₄Mab-46) Using the REMAP Method

CD44 functions as a major hyaluronan receptor on most cell types, with roles in cell adhesion, migration, proliferation, differentiation, and survival. The CD44 gene comprises 20 exons, with alternative splicing producing many different isoforms. CD44 variant isoforms exhibit tissue-specific expression patterns and have been studied as therapeutic targets for several cancers; therefore, anti-CD44 monoclonal antibodies (mAbs) are useful for investigating CD44 expression in various cancers. Previously, we established an anti-CD44 mAb, C₄₄Mab-46 (IgG₁, κ), by immunizing mice with the CD44v3-10 ectodomain. Although C₄₄Mab-46 recognized all CD44 isoforms, the binding epitope of C₄₄Mab-46 has not been determined. In this study, we first checked the reactivity of C₄₄Mab-46 to several CD44v3-10 deletion mutants such as dN79, dN124, dN147, and dN224. We found the N-terminus of the C₄₄Mab-46-binding epitope between residues 147 and 224 of CD44v3-10. We next investigated this epitope using a novel mapping system: RIEDL insertion for epitope mapping (REMAP) method. We constructed 31 CD44 standard (CD44s) mutants where the RIEDL tag was inserted into the expected epitope region in CD44s. We observed that the C₄₄Mab-46 epitope constituted five amino acids: ₁₇₄-TDDDV-₁₇₈ of CD44s. Thus, the REMAP method could be used to determine mAb binding epitopes for membrane proteins.