Antibody light chain variable domains and their biophysically improved versions for human immunotherapy

Area of Science:

• Biotechnology
• Immunology
• Protein Engineering

Background:

• Antibody variable domains are crucial for therapeutic applications.
• Identifying stable and non-aggregating domains is essential for drug development.
• Light chain variable domains (VLs) have potential as immunotherapeutics.

Purpose of the Study:

• To investigate the potential of human antibody light chain variable domains (VLs) as immunotherapeutics.
• To assess the biophysical properties of VLs, including aggregation, stability, and expression.
• To evaluate the impact of engineered disulfide bonds on VL properties.

Main Methods:

• Generated a naive human VL phage display library.
• Selected non-aggregating VL domains by panning against protein L.
• Characterized VL domains for aggregation, protein L binding, thermal stability, and protease resistance.
• Engineered a non-canonical disulfide linkage in VLs and re-evaluated properties.

Main Results:

• Isolated diverse non-aggregating VL domains with high yields and protein L binding.
• VLs exhibited favorable thermal stability and protease resistance, comparable to heavy chain variable domains (VHs).
• Engineered disulfide bonds significantly increased melting temperatures (Tm) and pepsin resistance, with minimal impact on aggregation and binding.

Conclusions:

• Human VL repertoires are a rich source of non-aggregating domains with desirable biophysical characteristics for therapeutics.
• Engineered VLs with enhanced stability present a promising format for oral immunotherapies.
• The study highlights the therapeutic potential of VL domains, particularly engineered variants, due to their stability and favorable properties.