Fab-PEG-Fab as a potential antibody mimetic

Area of Science:

• Biotechnology
• Immunology
• Protein Engineering

Background:

• Immunoglobulin G (IgG) antibodies possess flexible structures enabling epitope binding across diverse spatial ranges.
• The two fragment antigen-binding (Fab) regions in IgG are connected via a flexible linkage, mimicking a linear molecule.
• Polyethylene glycol (PEG) can serve as a scaffold to link two Fabs, creating novel Fab-PEG-Fab (FpF) constructs.

Purpose of the Study:

• To develop and characterize Fab-PEG-Fab (FpF) molecules as potential therapeutic agents.
• To investigate the structural and functional properties of FpFs compared to parent IgGs.
• To evaluate the therapeutic potential of FpFs in specific indications, particularly where the Fc region is not essential.

Main Methods:

• Fabs were obtained either directly or through proteolytic digestion of monoclonal IgGs.
• Site-specific conjugation was employed to attach Fabs to PEG scaffolds (6, 10, and 20 kDa) via bis-alkylation of cysteine thiols.
• Dynamic light scattering was used to assess the size of FpFs; binding affinities and functional activities (anti-angiogenesis) were evaluated in vitro.

Main Results:

• FpFs exhibited sizes comparable to IgG antibodies, smaller than expected based on PEGylation alone, suggesting inter-Fab interactions.
• Prepared FpFs targeting anti-VEGF and anti-Her2 demonstrated apparent affinities similar to their parent IgGs.
• Anti-VEGF FpFs showed slower dissociation rates and comparable or superior in vitro anti-angiogenic activity relative to bevacizumab.

Conclusions:

• Fab-PEG-Fab (FpF) molecules represent a promising novel format for antibody-based therapeutics.
• The FpF structure allows for functional antibody activity without the Fc region, potentially reducing immunogenicity or other Fc-mediated effects.
• Further investigation of FpFs is warranted for therapeutic applications where the Fc component is not required or may be disadvantageous.