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Development of a Novel Epitope Mapping System: RIEDL Insertion for Epitope Mapping Method.

Teizo Asano1, Mika K Kaneko1, Yukinari Kato1,2

  • 1Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, Sendai, Japan.

Monoclonal Antibodies in Immunodiagnosis and Immunotherapy
|August 23, 2021
PubMed
Summary

A new RIEDL insertion for epitope mapping (REMAP) method precisely identifies monoclonal antibody (mAb) binding regions on membrane proteins like CD44. This technique overcomes challenges with traditional epitope mapping, enabling better understanding of mAb function.

Keywords:
C44Mab-5CD44RIEDL tagepitope mappingmonoclonal antibody

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Area of Science:

  • Immunology
  • Molecular Biology
  • Biochemistry

Background:

  • Determining monoclonal antibody (mAb) binding regions is crucial for understanding their pharmacological functions.
  • Traditional epitope mapping methods like deletion and point mutants face challenges, especially with membrane proteins.
  • A novel approach is needed to accurately map mAb epitopes on complex protein targets.

Purpose of the Study:

  • To develop and validate a novel epitope tag system for determining mAb binding regions.
  • To specifically map the epitope of an anti-CD44 mAb (C44Mab-5) using the new method.
  • To assess the utility of the RIEDL tag system for epitope mapping of functional mAbs.

Main Methods:

  • Utilized deletion mutants of CD44 to assess reactivity with an anti-CD44 mAb (C44Mab-5).
  • Employed the RIEDL tag system, inserting the "RIEDL" peptide into CD44 between amino acids 21-41.
  • Analyzed mAb binding using flow cytometry on transfectants expressing modified CD44, including RIEDL tag insertion.

Main Results:

  • C44Mab-5 showed no reactivity with a CD44 deletion mutant from amino acids 30-361.
  • The reactivity of C44Mab-5 was lost when the RIEDL tag was inserted between amino acids 25-36 of CD44.
  • LpMab-7, an antibody against the RIEDL tag, detected most RIEDL tag-inserted CD44 constructs (AA 21-41).

Conclusions:

  • The RIEDL insertion for epitope mapping (REMAP) method successfully determined the C44Mab-5 epitope on CD44 to be amino acids 25-36.
  • The REMAP method provides a valuable tool for mapping critical epitopes of functional mAbs against various target molecules.
  • This novel technique offers a solution for epitope determination challenges, particularly for membrane proteins.