Engineering a β-Sheet Enables Bispecific Binding in Single VHH Domains

Area of Science:

• Biotechnology
• Protein Engineering
• Immunology

Background:

• Bispecific protein binding offers therapeutic advantages but is often limited by the large size and complexity of traditional antibody assemblies.
• Developing smaller, more versatile bispecific agents is crucial for advancing therapeutic applications.

Purpose of the Study:

• To engineer a novel bispecific binding platform within a single VHH nanobody domain.
• To demonstrate the feasibility of creating dual binders for therapeutic targets like EGFR/PD-L1 and HER2/TfR.

Main Methods:

• Engineered VHH nanobodies with an additional binding paratope in the β-sheet, independent of the native CDR paratope.
• Created dual binders for EGFR/PD-L1 and HER2/TfR target combinations.
• Assessed binding affinities and demonstrated modularity by grafting binding sites into different scaffolds and cell surface receptors.

Main Results:

• Developed a VHH nanobody platform enabling bispecific binding within a single domain.
• Achieved high-affinity dual binding (nM range) for EGFR/PD-L1 and HER2/TfR targets.
• Demonstrated the modularity and versatility of the engineered binding sites and VHH scaffolds.

Conclusions:

• Established a novel method for engineering bivalency into single immunoglobulin scaffold domains.
• Highlighted the potential of β-sheet paratopes for high-affinity bispecific binding.
• This platform offers a promising alternative to conventional bispecific antibodies for therapeutic development.