Development, High-Throughput Profiling, and Biopanning of a Large Phage Display Single-Domain Antibody Library

Area of Science:

• Biotechnology
• Immunology
• Molecular Biology

Background:

• Immunoglobulin G-based monoclonal antibodies (mAbs) show limitations in tissue penetration and efficacy for multifactorial diseases due to their large size.
• Alternative antibody formats are needed to overcome these limitations.

Purpose of the Study:

• To construct and characterize a large-scale phage display library of single-domain antibodies (sdAbs, also known as VHHs) derived from alpacas.
• To evaluate the potential of this library for developing novel diagnostic and therapeutic agents.

Main Methods:

• Construction of a phage display library using 1.6 × 10^9 lymphocytes from 20 alpacas, yielding approximately 7.16 × 10^10 colonies.
• High-throughput profiling using next-generation sequencing of 5.65 × 10^6 full-length VHH sequences to assess library diversity and uniqueness (92% uniqueness observed).
• Systematic characterization to identify sdAbs with high affinity for therapeutically relevant antigens.

Main Results:

• A highly diverse alpaca sdAb phage display library was successfully constructed.
• The library contains a significant repertoire of VHH sequences, with high uniqueness.
• Multiple sdAbs demonstrating high affinity for three key therapeutic antigens were identified.

Conclusions:

• The developed alpaca sdAb phage display library is a valuable resource for both diagnostic and therapeutic applications.
• The natural VHH antibody repertoire provides a foundation for creating humanized synthetic sdAb libraries.
• This advancement holds potential for further development of sdAb-based therapeutics.