An antibody loop replacement design feasibility study and a loop-swapped dimer structure

Area of Science:

• Antibody engineering
• Protein structure analysis
• Rational drug design

Background:

• Complementarity determining region (CDR) loops are key to antibody-antigen interactions.
• Current antibody design strategies, like mutation-based approaches, have limitations in expanding conformational space.
• Crystal structure diversity offers opportunities for structure-based antibody design targeting CDR loops.

Purpose of the Study:

• To investigate the feasibility of replacing single CDR antibody loops as a rational antibody design strategy.
• To explore if CDR loop replacement can expand conformational space beyond simple mutation strategies.
• To analyze the structural and functional consequences of CDR loop replacement in an anti-VLA1 antibody.

Main Methods:

• Computational design and structural analysis of CDR loop replacements.
• X-ray crystallography to determine the structure of designed antibody variants.
• Circular dichroism and tryptophan fluorescence spectroscopy to assess protein stability and unfolding.
• Affinity measurements to evaluate the functional impact of designed modifications.

Main Results:

• CDR loop replacement by design was confirmed as feasible, with designed loops adopting desired conformations.
• Initial L1 loop replacement in the anti-VLA1 antibody did not improve binding affinity.
• Structural analysis revealed unintended domain swapping in one design, linked to structural instability and affinity loss.
• A second round of design successfully eliminated domain swapping through targeted mutation.

Conclusions:

• Replacing single CDR loops is a viable strategy for antibody engineering, offering conformational control.
• Structural instability and unintended oligomerization can arise from CDR loop modifications, impacting antibody function.
• Iterative design and structural analysis are crucial for overcoming challenges and achieving desired antibody properties.