High-affinity antigen binding by chelating recombinant antibodies (CRAbs)

Area of Science:

• Biochemistry
• Immunology
• Molecular Biology

Background:

• Antibody fragments offer therapeutic potential but often lack sufficient binding affinity.
• The chelate effect, where multivalent binding enhances affinity, is a known phenomenon.
• Developing strategies to harness the chelate effect for antibody fragments is crucial for improving their efficacy.

Purpose of the Study:

• To develop a novel strategy for generating antibody fragments with enhanced binding affinities.
• To create a bispecific antibody fragment (CRAb) capable of binding adjacent epitopes simultaneously.
• To demonstrate the enhanced binding affinity of the CRAb compared to its individual components.

Main Methods:

• Computer graphic modeling was used to design a bispecific antibody fragment (CRAb) linking two single-chain antibody fragments (scFv) to adjacent epitopes.
• The CRAb gene was assembled and expressed in bacteria via secretion.
• Purified CRAb was analyzed for binding affinity using competition ELISA, bandshift assays, and fluorescence quench.

Main Results:

• Computer modeling predicted successful simultaneous binding of linked scFvs to adjacent epitopes on lysozyme.
• The expressed and purified CRAb demonstrated significantly higher binding affinity (Kd > 10^9 M^-1) compared to individual scFv fragments (Ka = 10^8 M^-1 and 10^6 M^-1).
• Affinity measurements confirmed a substantial increase, with Ka > 1.3 x 10^10 M^-1 observed via fluorescence quench.

Conclusions:

• The developed CRAb strategy effectively utilizes the chelate effect to achieve high-affinity antibody fragments.
• This approach offers a promising method for engineering potent antibody-based therapeutics.
• The CRAb represents a significant advancement in antibody engineering for enhanced antigen recognition and binding.