Antibodies that recognize amyloid-forming proteins associated with neurodegenerative disorders (e.g., Alzheimer’s disease) are valuable for therapeutic and diagnostic applications. We have developed a novel approach for designing antibody fragments specific for amyloidogenic proteins that is inspired by the molecular interactions that mediate protein aggregation. We find that grafting hydrophobic peptides from amyloidogenic polypeptides into the complementarity-determining regions of antibody fragments generates Grafted AMyloid-Motif AntiBODIES (gammabodies) that recognize aggregated proteins with high nanomolar binding affinities (Perchiacca et al., <i>PNAS</i>, 2012). Our recent work has focused on improving single (V<sub>H</sub>) and multidomain (scFv) gammabodies to achieve low nanomolar binding affinities. Toward this aim, we have used targeted mutagenesis and yeast surface display to select for antibody fragments with improved affinity. We find that variants with mutations at the edges of the grafted binding loops can be readily selected with increased binding affinity and minimal change in antibody stability and structure. In addition, we also find that co-selection for antibody binding to conformational ligands (such as Protein A) and antigen at the same time leads to identification of antibody variants that are well folded and possess specific binding activity. Finally, we find that the use of specific types of stringent screening solutions significantly improves the quality of the selected variants. We expect that these findings will improve antibody selections using yeast surface display and increase the likelihood of identifying high-quality antibody fragments.

Directed evolution methods for improving the affinity and stability of Alzheimer’s antibody fragments

251st National Meeting (2016)

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