Stability convergence in natural antibodies with ultra-long hypervariable loops.

Area of Science:

• Immunology
• Structural Biology
• Biochemistry

Background:

• Antibodies utilize complementarity-determining regions (CDRs) for antigen binding.
• Bovine antibodies possess a unique subset with ultra-long CDRs (ulCDRs), featuring a distinct stalk-and-knob structure.
• The structural and functional principles governing bovine ulCDRs remain largely unexplored.

Purpose of the Study:

• To investigate the impact of diverse natural ulCDRs on antibody structure, stability, and function.
• To explore the feasibility of grafting ulCDRs onto a common antibody scaffold.

Main Methods:

• Swapping various natural ulCDRs onto a single antibody scaffold.
• Analysis of secondary structure fingerprints.
• Thermal stability assays.
• Antigen specificity and binding affinity measurements.
• Hydrogen-deuterium exchange experiments.
• Molecular dynamics simulations.

Main Results:

• All ulCDR-swap variants displayed highly similar secondary structure profiles.
• Remarkably consistent thermal stability was observed across variants.
• Antibody specificity and high-affinity antigen binding were preserved.
• Molecular simulations indicated subtle variations due to stalk-scaffold interactions.

Conclusions:

• Natural ulCDRs can be effectively grafted onto a common Fab scaffold while retaining essential antibody characteristics.
• The study elucidates key principles for ulCDR integration, offering insights for antibody engineering.
• Findings have significant implications for developing advanced antibody-based therapeutics and diagnostics.