Epitope Mapping of the Anti-CD44 Monoclonal Antibody (CMab-46) Using Alanine-Scanning Mutagenesis and Surface Plasmon Resonance.

CD44 is a type I transmembrane protein expressed in various kinds of normal cancer cells, including pancreatic, breast, and oral cancers. CD44 is associated with cancer progression, metastases, and treatment resistance. CD44 consists of 20 exons, and various isoforms exist due to alternative splicing of the central 10 exons. Some splicing variants show cancer-specific expression patterns and are related to prognosis of patients with cancer. Therefore, CD44 targeting therapy has been attracting attention. In a previous study, we established an anti-CD44 monoclonal antibody, CMab-46 (IgG, kappa), useful for flow cytometry, Western blotting, and immunohistochemistry by immunizing mice with CD44v3-10 ectodomain. This study investigated the binding epitope of CMab-46 using enzyme-linked immunosorbent assay (ELISA) and the surface plasmon resonance (SPR) with the synthesized peptide. ELISA results using deletion mutants showed that CMab-46 reacted with the amino acids (aa) of 161-180 aa of CD44. Further examination of the CMab-46 epitope using ELISA with point mutants in 161-180 aa of CD44 demonstrates that the CMab-46 epitope comprised Thr174, Asp177, and Val178. The SPR with point mutants in 161-180 aa of CD44 demonstrated that the CMab-46 epitope comprises Thr174, Asp175, Asp176, Asp177, and Val178. Together, the CMab-46 epitope was determined to be located in exon 5 of CD44.