AFM-Based Single-Molecule Force Spectroscopy of PEG-Anti-PEG Antibody Interactions

Area of Science:

• Biochemistry
• Immunology
• Materials Science

Background:

• Poly(ethylene glycol) (PEG) is a crucial stealth polymer in drug delivery, enhancing stability and circulation by evading immune detection.
• Increasing reports of anti-PEG antibodies in humans necessitate a deeper understanding of PEG-antibody interactions to mitigate adverse effects like accelerated drug clearance.

Purpose of the Study:

• To investigate the molecular-scale mechanics of PEG-antibody binding using single-molecule force spectroscopy.
• To elucidate how PEG terminal chemistry and antibody maturation influence binding affinity and interaction dynamics.

Main Methods:

• Atomic force microscopy-based single-molecule force spectroscopy (AFM-SMFS) was employed to measure binding forces and rupture distances.
• Quartz crystal microbalance with dissipation (QCM-D) and Fourier-transform infrared (FTIR) spectroscopy were used for complementary binding and structural analysis.
• Methoxy-terminated PEG (m-PEG) and hydroxy-terminated PEG (HO-PEG) were tested against Fv-clasps from naïve (M9) and affinity-matured (M11) anti-PEG IgMs.

Main Results:

• The affinity-matured IgM (M11) demonstrated stronger binding to PEG at shorter rupture distances compared to the naïve IgM (M9).
• The M11 and m-PEG pair exhibited the most intense binding signatures.
• PEG's hydrated structure significantly influences binding: HO-PEG forms extended layers, while m-PEG adopts compact conformations, affecting antibody accessibility.

Conclusions:

• Antibody maturation and PEG hydration state are critical determinants of PEG-antibody binding mechanics.
• These findings offer molecular-level insights for designing PEGylated therapeutics with improved performance and reduced immunogenicity.