Shark IgNAR antibody mimotopes target a murine immunoglobulin through extended CDR3 loop structures

Area of Science:

• Immunology
• Structural Biology
• Biotechnology

Background:

• Mimotopes mimic antigen epitopes, recognized by antibodies with high affinity.
• Peptides and anti-idiotypic antibodies are known mimotope classes.
• Single domain IgNAR antibodies possess long CDR3 loops, suitable for mimotope development.

Purpose of the Study:

• To develop and characterize novel mimotopes using single domain IgNAR antibodies.
• To investigate the binding affinity and structural basis of IgNAR-derived mimotopes.
• To explore the potential of IgNAR variable domains (V(NAR)s) as constrained peptide libraries.

Main Methods:

• Screening an in vitro phage-display library of IgNAR variable domains against a target antibody (MAb5G8).
• Characterizing four selected V(NAR) mimotopes for binding specificity and affinity using competition studies.
• Conducting crystallographic studies of V(NAR) proteins to determine structural features of mimotope presentation.

Main Results:

• Four V(NAR) mimotopes were identified, binding specifically to the MAb5G8 antibody's paratope.
• V(NAR) mimotopes exhibited enhanced binding affinities (3-46 nM) compared to the parental antigen (175 nM).
• Crystallography revealed SYP motifs on CDR3 loops, stabilized by various interactions, positioned for antibody contact.

Conclusions:

• Single domain IgNAR antibodies can generate high-affinity mimotopes targeting specific epitopes.
• The structural features of IgNAR CDR loops, including stabilization, contribute to enhanced binding.
• Phage-display selection from constrained IgNAR libraries offers a powerful method for discovering novel mimotopes with potentially superior topologies.