Synthetic single-framework antibody library integrated with rapid affinity maturation by VL shuffling

Area of Science:

• Immunotechnology
• Molecular Biology
• Biochemistry

Background:

• Antibody affinity maturation is crucial for enhancing antibody properties from in vitro libraries.
• Existing methods can be limited in diversity and efficiency.

Purpose of the Study:

• To develop a rapid affinity maturation strategy for antibodies using a synthetic human scFv library.
• To improve antibody binding affinities against lysozyme.

Main Methods:

• Constructed a synthetic human scFv antibody library in a single framework for rapid affinity maturation.
• Employed updated Kunkel's mutagenesis to generate diversity, focusing on V(H) domains.
• Utilized phage display and panning techniques for library selection and enrichment.
• Incorporated enriched V(H) genes into a mature library for further V(L) domain variant selection.

Main Results:

• Generated a phage-displayed library with 3 × 10(10) unique members, emphasizing V(H) diversity.
• Identified several unique anti-lysozyme antibodies with dissociation constants (K(d)) between 0.8-10 nM after affinity maturation.
• Achieved significantly higher binding affinities compared to antibodies selected from the primary universal library (K(d) <20 nM).

Conclusions:

• The single-framework strategy enables efficient transfer of V(H) domain diversity for antibody affinity maturation.
• This method leads to a diverse set of antibodies with high binding affinities in the low nanomolar range.
• The approach offers a streamlined process for generating potent antibodies without complex ligation steps.