Generation and Next-Generation Sequencing-Based Characterization of a Large Human Combinatorial Antibody Library

Area of Science:

• Biotechnology
• Immunology
• Molecular Biology

Background:

• Antibody phage display is crucial for developing target-specific monoclonal antibodies (mAbs).
• High-quality, diverse antibody libraries are essential for successful phage display applications.
• Existing libraries may not fully capture the breadth of human antibody repertoires.

Purpose of the Study:

• To construct a large and diverse human antibody library for phage display.
• To characterize the sequence diversity and uniqueness of the constructed library.
• To validate the library's utility for isolating high-affinity, cross-reactive mAbs.

Main Methods:

• Construction of a large human single-chain variable fragment (scFv) library (1.5 × 10^11 colonies) from stimulated peripheral blood mononuclear cells.
• Stimulation of B cells using R848 (Toll-like receptor 7/8 agonist) and interleukin-2.
• Next-generation sequencing (NGS) of heavy chain variable (VH) and kappa light chain variable (Vκ) domains to assess library diversity.

Main Results:

• The library comprises unique VH (approx. 94%) and Vκ (approx. 91%) sequences.
• NGS revealed greater diversity compared to germline sequences.
• Isolation of multiple high-affinity mAbs with broad cross-species reactivity against therapeutic targets.

Conclusions:

• The developed antibody library is highly diverse and contains unique antibody sequences.
• The library is suitable for isolating therapeutic and diagnostic monoclonal antibodies (mAbs).
• This resource facilitates the rapid development of recombinant human mAbs for various applications.