Shark Attack: high affinity binding proteins derived from shark vNAR domains by stepwise in vitro affinity maturation

Area of Science:

• Biotechnology
• Molecular Biology
• Immunology

Background:

• Shark vNAR domains offer unique therapeutic potential due to their small size and high stability.
• Developing efficient in vitro methods for generating high-affinity vNAR binders is crucial for therapeutic applications.

Purpose of the Study:

• To establish a novel stepwise in vitro affinity maturation method for antigen-specific shark vNAR domains.
• To generate high-affinity vNAR binders against therapeutically relevant antigens using semi-synthetic repertoires.

Main Methods:

• Utilized yeast surface display for screening a CDR3-randomized vNAR library from non-immunized bamboo sharks.
• Performed affinity maturation by diversifying CDR1 of target-enriched vNAR populations.
• Characterized selected vNAR molecules using thermal shift assays and biolayer interferometry.

Main Results:

• Successfully isolated antigen-binding vNAR domains against EpCAM, EphA2, and HTRA1.
• Achieved rapid selection of nanomolar binders for EpCAM and HTRA1 through affinity maturation.
• Demonstrated that high-affinity vNAR binders can be generated without compromising scaffold thermostability.

Conclusions:

• The described method provides an efficient platform for in vitro affinity maturation of vNAR domains.
• This approach facilitates the development of novel therapeutic antibodies with high affinity and stability.