(553g) Invited Talk: Engineering Covalent Antibodies on the Yeast Surface with an Expanded Genetic Code

Covalent drug discovery has reemerged as an important route to small-molecule therapeutic leads. However, extending these strategies to protein-based therapeutics is extremely challenging with the conventional genetic code. To overcome these limitations, we have utilized genetically encoded noncanonical amino acids (ncAAs) in combination with yeast display to efficiently prepare and characterize proteins containing either photocrosslinkable or spontaneously crosslinkable chemistries on the yeast surface. Using a combination of orthogonal translation systems for ncAA incorporation established by our lab and others, we have validated the presentation of four distinct crosslinkable chemistries in yeast-displayed proteins (3 photocrosslinkable chemistries and 1 spontaneously crosslinkable chemistry). Flow cytometry assays indicate translation efficiencies ranging from approximately 15 to 50 percent of wild-type protein translation. We used this ncAA-compatible yeast display platform to identify irreversible variants of single-domain antibodies (sdAbs), also called VHHs and nanobodies, targeting botulinum neurotoxin light chain A (LC/A). Starting from a series of structurally characterized sdAbs, we evaluated binding and crosslinking properties of antibodies substituted with photocrosslinkable or spontaneously crosslinkable ncAAs at various positions within four sdAbs. NcAA-substituted sdAbs generally retained binding function, and assays to identify covalent sdAbs on the yeast surface revealed numerous candidate photocrosslinkable variants (>15) and a more modest number of spontaneously crosslinkable variants (3) out of more than 20 variants of each crosslinking type tested. These results are consistent with the relative reactivities of the photocrosslinkable (high) and spontaneously crosslinkable (low) chemistries employed in these studies. Solution-based assays on selected clones revealed clear evidence for crosslinking and high to moderate target crosslinking selectivity in the presence of cell lysate. Overall, our findings highlight the power of yeast display in combination with noncanonical amino acids in the discovery of binding agents that covalently engage their targets. Ongoing work in the group and with our collaborators seeks to elucidate the molecular features that mediate crosslinking and to extend the tools available for covalent antibody discovery and engineering, with long-term goals to apply these unique antibodies in therapeutic settings.