Co-Evolution of Affinity and Stability for Domain Antibodies That Recognize Hydrophobic Antigens

Phage and yeast surface display are invaluable methods for evolving and tailoring the properties of biomolecules ranging from peptides and small proteins to multidomain enzymes and full-length antibodies. These methods have been used in many innovative ways for improving not only biomolecular activity but also stability as well. We have sought to use yeast surface display to evolve the affinity and stability of single-domain (V_H and V_L) antibodies specific for hydrophobic peptides and proteins linked to Alzheimer’s and Parkinson’s diseases. Interestingly, we find that the binding affinity of selected antibody mutants is anti-correlated with folding stability. Multiple rounds of mutagenesis and selection result in high-affinity domain antibodies that are unfolded on the surface of yeast and as isolated domains when produced in bacteria. The unfolded antibodies are soluble and well-expressed in bacteria, suggesting that the yeast secretory pathway removes aggregation-prone variants. To improve selection of stable domain antibodies, we have replaced sequence-specific antibodies used for detecting yeast display levels with conformational probes specific for folded antibodies. This leads to significant improvements in co-evolution of antibody affinity and stability. We are currently employing this approach for optimizing domain antibodies specific for soluble and aggregated forms of several hydrophobic (amyloid-forming) proteins, as well as evaluating these evolved antibodies for their ability to detect and inhibit toxic protein aggregation.