Increasing the developability of lambda chain antibodies: engineering to confer protein L binding activity and its...

Area of Science:

• Biotechnology and Protein Engineering
• Immunology and Antibody Therapeutics

Background:

• Protein L (PpL) is crucial for antibody purification, binding to the variable light chain.
• A limitation of Protein L is its inability to bind lambda chain antibodies, hindering their developability.
• Engineering lambda antibodies is essential for expanding their utility in biotechnology.

Purpose of the Study:

• To engineer lambda chain antibodies with Protein L binding capabilities.
• To improve the developability of lambda antibodies using the anti-Acanthamoeba IgG mAb3 as a template.
• To enhance thermal stability and maintain antigen recognition of engineered antibody fragments.

Main Methods:

• Construction and expression of recombinant single-chain variable fragments (scFvs) derived from mAb3.
• Assessment of Protein L binding using Protein L-affinity purification, ELISA, and western blotting.
• Evaluation of thermal stability via melting temperature measurements and antigen reactivity profiling.

Main Results:

• Four functional mAb3-derived scFvs were produced, with two demonstrating Protein L binding.
• One Protein L-binding scFv exhibited enhanced thermal stability, increasing melting temperature by 8.7°C to 67.2°C, with high production yields.
• Engineered scFvs and derived minibody and scFv-Fc fragments retained antigen binding and Protein L interaction profiles.

Conclusions:

• Lambda chain antibodies can be engineered for Protein L binding through targeted mutations.
• This engineering approach significantly enhances antibody developability for therapeutic and diagnostic purposes.
• The study provides a viable strategy for optimizing lambda antibodies for biotechnological applications.