Mutational and biophysical robustness in a prestabilized monobody

Area of Science:

• Protein engineering
• Biotechnology
• Immunology

Background:

• Fibronectin type III (FN3) monobodies offer a non-antibody scaffold with simpler architecture and binding loops.
• Previous development of FN3Con resulted in a hyperstable monobody with therapeutic and diagnostic potential.
• Prestabilization of protein scaffolds can mitigate stability-function trade-offs during biological activity evolution.

Purpose of the Study:

• To assess if the FN3Con monobody scaffold can achieve antibody-like binding to therapeutic targets while maintaining extreme stability.
• To investigate the strategy of loop grafting onto a prestabilized scaffold for developing novel protein domains.
• To evaluate the stability and binding affinity of a new FN3Con-based construct targeting VEGFR2.

Main Methods:

• Grafting binding loops from Adnectin-anti-VEGFR2 (CT-322) onto the prestabilized FN3Con scaffold.
• Characterizing the binding affinity of the resulting FN3Con-anti-VEGFR2 construct to VEGFR2.
• Assessing the thermostability and long-term storage stability of the engineered monobody.
• Investigating the impact of buffer excipients on monobody stability through accelerated trials.

Main Results:

• The FN3Con-anti-VEGFR2 construct successfully bound to the therapeutic target VEGFR2 with high affinity.
• The engineered monobody maintained high thermostability and remarkable long-term stability, retaining activity after two years at 36 °C.
• Buffer excipient investigations doubled the monomeric monobody presence in accelerated stability trials.
• Loop grafting onto a prestabilized scaffold proved to be a viable strategy for developing protein domains with desirable biophysical characteristics.

Conclusions:

• The FN3Con scaffold is well-suited for developing stable, antibody-like binding domains.
• This approach enables the creation of monobodies with high affinity and exceptional stability for therapeutic and diagnostic applications.
• The developed FN3Con-anti-VEGFR2 construct demonstrates significant potential for shelf-stable diagnostics and therapeutics.