An engineered human IgG1 CH2 domain with decreased aggregation and nonspecific binding

Area of Science:

• Biotechnology
• Protein Engineering
• Immunology

Background:

• The immunoglobulin (Ig) CH2 domain is a valuable scaffold for therapeutic development.
• Previous work enhanced CH2 stability via disulfide bonds and N-terminal truncation (m01s).
• Both isolated CH2 and m01s exhibit aggregation due to identified aggregation-prone regions (APRs).

Purpose of the Study:

• To reduce aggregation in CH2-based scaffolds.
• To improve the developability of CH2-based therapeutics.
• To identify specific CH2 mutants with reduced aggregation and nonspecific binding.

Main Methods:

• Utilized computational methods to identify aggregation-prone regions (APRs) in CH2 domains.
• Generated a phage display library of CH2 mutants.
• Employed high-temperature incubation and panning with a specific monoclonal antibody to select for stable, correctly folded mutants.
• Performed additional mutagenesis and characterized selected clones using turbidity assays and dynamic light scattering.

Main Results:

• Identified m01s5 with smaller APRs and m01s5.4 with significantly reduced aggregation compared to m01s.
• m01s5.4 demonstrated substantially lower nonspecific binding.
• Engineering of an m01s-based tumor antigen binder resulted in reduced aggregation without compromising binding affinity.

Conclusions:

• Developed a novel approach combining computational analysis and phage display to minimize protein aggregation.
• Successfully generated CH2 mutants (e.g., m01s5.4) with significantly reduced aggregation.
• These engineered CH2 scaffolds offer improved developability for potential therapeutics.