A novel heavy domain antibody library with functionally optimized complementarity determining regions.

Area of Science:

• Biotechnology
• Immunology
• Molecular Biology

Background:

• Synthetic antibody libraries are crucial for recombinant antibody selection.
• Library quality, defined by functional antibody content, dictates successful isolation.
• Existing libraries face challenges in folding, aggregation, and diversity.

Purpose of the Study:

• To construct a novel, high-quality synthetic single domain antibody library named Predator.
• To enhance library quality through specific mutations and optimized CDR3 design.
• To facilitate affinity maturation via a modular scaffold approach.

Main Methods:

• The HEL4 domain antibody scaffold was modified with mutations affecting folding and aggregation.
• CDR3 diversity was introduced using trinucleotide synthesis to prevent stop codons.
• A cycle-free elongation method converted single-stranded to double-stranded DNA.
• A modular scaffold with unique restriction sites enabled CDR shuffling for affinity maturation.

Main Results:

• The Predator library was successfully constructed with designed CDR3 diversity.
• Phage display selections yielded specific binders against purified antigens and eukaryotic cells.
• Characterization revealed that introduced mutations reduced the thermodynamic stability of selected clones.

Conclusions:

• The Predator library is a valuable tool for selecting functional recombinant antibodies.
• The library design, including CDR3 mimicry and modularity, shows promise.
• Further optimization may be needed to balance stability and binding affinity.