[WS9] Synergistic development of protein science and antibody engineering

[WS9-01] Development of a novel epitope-mapping method using a peptide-tag system

^○Yukinari Kato, Teizo Asano, Mika Kaneko (Tohoku Univ. Grad. Sch. Med.)

Background: To clarify the binding region of monoclonal antibodies (mAbs) to target molecules is very critical for understanding the pharmacological function of each mAb. We often experience the difficulty of determining the mAb epitope against membrane proteins using point mutants. Purpose: We aimed to develop a novel method to determine the binding region of mAbs using the epitope tag system. Methods: We first checked the reactivity of an anti-CD44 mAb (C44Mab-5) to several deletion mutants of CD44. We then employed the RIEDL tag system ("RIEDL" peptide and LpMab-7 mAb). We inserted the "RIEDL" peptide into CD44. Produced transfectants were stained by LpMab-7 and C44Mab-5 in flow cytometry. Results: C44Mab-5 did not react with the 30-361 amino acid (aa) deletion mutant of CD44. Further, the reaction of C44Mab-5 to RIEDL tag-inserted CD44 from 25 aa to 36 aa was lost although LpMab-7 detected RIEDL tag-inserted CD44 from 22 aa to 41 aa. Conclusions: The epitope of C44Mab-5 for CD44 was determined to be the peptide from 25-36 aa of CD44 using RIEDL insertion for epitope mapping (REMAP) method. REMAP method could be useful for determining the critical epitope of functional mAbs against many target molecules.