Fibronectin type III domain based monobody with high avidity

Area of Science:

• Biochemistry
• Molecular Biology
• Protein Engineering

Background:

• Affinity molecules are crucial in biomedical research and clinical applications.
• Optimizing protein binding affinity, like antibody fragments, via affinity maturation is often time-consuming and limited by protein properties.
• Fibronectin type III domain (FN3) monobodies offer an alternative scaffold for developing targeted therapeutics.

Purpose of the Study:

• To develop a multivalent strategy for engineering FN3 monobodies with enhanced binding strength and stability.
• To create a pentameric FN3 monobody targeting the alphavbeta3 integrin, a tumor-related biomarker.
• To assess the binding affinity, specificity, and stability of the multivalent FN3 monobody.

Main Methods:

• A multivalent strategy was developed by fusing an alphavbeta3-binding FN3 monobody with a COMP pentamerization domain via a linker.
• The fusion protein was expressed in Escherichia coli and purified.
• The self-assembled pentameric monobody was characterized and compared to its monomeric counterpart.

Main Results:

• The fusion protein expressed well in the soluble fraction of E. coli and self-assembled into a stable pentamer.
• The pentameric monobody exhibited significantly tighter binding to alphavbeta3 integrin compared to the monomeric form.
• The pentameric monobody demonstrated a much slower off-rate and maintained excellent specificity for alphavbeta3.

Conclusions:

• A multivalent strategy using a COMP pentamerization domain can significantly enhance the binding strength and stability of FN3 monobodies.
• This approach provides a robust method for developing high-affinity binders for biomedical research and potential clinical applications.
• The described strategy is broadly applicable for engineering other FN3 monobodies to improve their targeting capabilities.