Arginine mutations in antibody complementarity-determining regions display context-dependent affinity/specificity...

Area of Science:

• Immunology
• Biochemistry
• Structural Biology

Background:

• Antibodies mature through accumulating charged mutations in complementarity-determining regions (CDRs) to enhance electrostatic interactions and improve affinity.
• However, charged mutations, particularly positively charged ones, can lead to antibody polyspecificity and reduced binding specificity.
• The precise balance between enhancing antibody affinity and maintaining specificity through CDR mutations remains incompletely understood.

Purpose of the Study:

• To investigate the trade-offs between antibody affinity and specificity concerning charged mutations in CDRs.
• To explore the role of arginine mutations in CDRs and their impact on binding to the Alzheimer's amyloid β peptide.
• To elucidate the structural and environmental factors governing the specificity of antibody variants.

Main Methods:

• Selection of single-chain antibody fragments against the Alzheimer's amyloid β peptide using varying stringency.
• Alanine-scanning mutagenesis to assess the contribution of specific CDR mutations to antibody affinity.
• Structural modeling and molecular simulations to analyze the microenvironments of CDR mutations.

Main Results:

• Antibody variants selected under stringent conditions showed improved specificity despite enrichment in arginine CDR mutations.
• Antibody affinity was highly dependent on arginine mutations in less specific variants, but less so in highly specific variants.
• Structural analysis revealed that arginine mutations in hydrophobic CDR regions correlated with higher specificity, while those in hydrophilic regions correlated with lower specificity.

Conclusions:

• Arginine mutations in antibody CDRs have context-dependent effects on specificity.
• Over-reliance on arginine for affinity can compromise antibody specificity.
• The relative contribution of arginine residues to overall antibody affinity, influenced by their local environment, governs the affinity-specificity balance.