Modulation of single-chain antibody affinity with temperature-responsive elastin-like polypeptide linkers

Area of Science:

• Biotechnology
• Protein Engineering
• Immunology

Background:

• Single-chain antibodies (scFv) are engineered proteins comprising variable heavy (VH) and variable light (VL) domains.
• Traditional scFv utilize flexible peptide linkers, such as (GGGGS)3, for structural integrity.
• Stimulus-responsive peptides offer potential for dynamic control over protein function.

Purpose of the Study:

• To investigate the impact of incorporating elastin-like polypeptide (ELP) linkers into anti-fluorescein single-chain antibodies.
• To evaluate how linker length and composition influence antibody binding and ligand release characteristics.
• To explore the potential of stimulus-responsive linkers for modulating antibody affinity.

Main Methods:

• Genetic modification of anti-fluorescein single-chain antibodies to include (VPGXG)n ELP linkers (n=1.2-3, X=Val or His).
• Production and characterization of engineered single-chain antibodies.
• Assessment of equilibrium affinity (KD) and binding kinetics at varying temperatures.

Main Results:

• Engineered scFv with ELP linkers demonstrated equilibrium affinity comparable to wild-type scFv at room temperature.
• Increased temperature led to altered binding kinetics and accelerated ligand release.
• Observed changes correlate with the known temperature-induced contraction and molecular ordering of ELPs.

Conclusions:

• Stimulus-responsive ELP linkers can modulate the binding and release properties of single-chain antibodies.
• Temperature-triggered affinity changes in scFv are achievable through linker engineering.
• This approach holds promise for applications in biosensors, drug delivery, and bioseparations.