Engineering Affibody Binders to Death Receptor 5 and Tumor Necrosis Factor Receptor 1 With Improved Stability

Area of Science:

• Biotechnology
• Protein Engineering
• Drug Discovery

Background:

• Protein developability, particularly stability, is crucial for therapeutic utility but often overlooked in early discovery.
• Affibody proteins targeting death receptor 5 (DR5) and tumor necrosis factor receptor 1 (TNFR1) were previously evolved for signaling inhibition.

Purpose of the Study:

• To engineer enhanced stability in DR5 and TNFR1 affibody proteins.
• To assess the impact of stability engineering on binding affinity and other developability aspects.

Main Methods:

• Error-prone PCR libraries were created for each affibody.
• Yeast surface display and binder selections were employed, including depletion of non-specific binders.
• On-yeast protease and yeast display level assays were used for developability assessment.
• Multiplex deep sequencing identified promising variants for purification and further analysis.

Main Results:

• Engineered affibody variants demonstrated significantly increased stability, with an 8°C to 14°C rise in apparent melting temperature (Tm,app).
• The TNFR1 affibody variants maintained their high binding affinity (1-2 nM).
• The DR5 affibody variants showed a 30-fold improvement in binding affinity, reaching 0.8 nM.

Conclusions:

• Protein stability can be effectively engineered in affibody scaffolds using yeast display selection methods.
• Enhanced stability can be achieved without compromising, and in some cases improving, binding affinity.
• This approach advances the development of more robust and potentially more effective protein-based therapeutics.