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

Protein developability is an important, yet often overlooked, aspect of protein discovery campaigns that is a key driver of utility. Recent advances have improved developability screening capacity, making it an increasingly viable option in early-stage discovery. Here, we engineered one component of developability, stability, of two affibody proteins-one that targets death receptor 5 and another that targets tumor necrosis factor receptor 1-previously evolved to bind receptor and non-competitively inhibit signaling via conformational modulation. Starting from an error-prone PCR library of each affibody, variants were screened via yeast surface display binder selections, including depletion of non-specific binders, followed by developability assessment using the on-yeast protease and yeast display level assays. Multiplex deep sequencing identified variants for further evaluation. Purified variants exhibited elevated stability-8°C to 14°C increase in T(m,app)-with maintained 1-2 nM affinity for the TNFR1 affibody and 30-fold improvement in the DR5 affibody affinity to 0.8 nM.