Developing drug-like single-domain antibodies (VHH) from in vitro libraries

Area of Science:

• Biotechnology
• Immunology
• Protein Engineering

Background:

• Variable heavy chain (VHH) domains offer therapeutic potential but require optimization for developability.
• Existing VHH libraries often lack sufficient humanness, stability, or facile purification methods.

Purpose of the Study:

• To create a novel VHH library optimized for therapeutic discovery.
• To enhance VHH humanness, stability, affinity, diversity, and developability.
• To establish a method for facile purification using Protein A without an Fc domain.

Main Methods:

• Humanized VHH scaffolds were engineered with human CDR1, CDR2, and CDR3 sequences.
• CDR1 and CDR2 libraries were generated from next-generation sequencing data and filtered for Protein A binding via yeast display.
• CDR3 sequences were amplified from human B cells and combined with CDR1-2 libraries, followed by optimization for stability and diversity.

Main Results:

• A diverse VHH library was successfully constructed, incorporating optimized human CDR sequences.
• High-affinity (100 pM–10 nM) and developable VHH binders were directly isolated.
• The study presents the first systematic developability assessment of isolated VHH molecules without Fc fusion.

Conclusions:

• The developed VHH library provides a powerful platform for discovering therapeutic candidates with improved properties.
• Optimized VHHs demonstrate high affinity and developability, suitable for therapeutic applications.
• This work establishes a new standard for evaluating VHH developability independently of Fc fusion.