(627d) Laying the Foundation for Performing Drug Discovery on the Yeast Surface

Antibodies exhibit a versatile range of molecular recognition properties that make them cornerstones of therapeutics, diagnostics, and basic research tools. However, some antibody properties, including chemical reactivity and covalent antigen binding, are constrained by the narrow range of chemistries encoded in the canonical set of amino acids. In this work, we investigate strategies for leveraging an expanded range of chemical functionality to augment antibody binding properties. We explore this area using the combination of yeast display and noncanonical amino acids. The efforts we describe to augment antibody binding functions with additional chemistries are distinct from efforts to prepare antibody-drug conjugates, where antibody binding and chemical modification occur in very distinct portions of the antibody structure. Enhancing binding function with additional chemistries requires the presentation of ncAAs in or near antibody complementarity determining regions (CDRs). To enable systematic exploration of ncAA presentation sites, we first investigated whether diversification of a single antibody loop would support isolation of binding clones. We prepared a billion-member library containing canonical amino acid diversity and loop length diversity only within the 3rd complementarity determining region of the heavy chain (CDR H3). Antibodies isolated from this library against a series of immunoglobulins from different species exhibit single-species specificity and moderate affinities (double- to triple-digit nanomolar affinities) despite possessing only changes in CDR H3. Because of the narrow location of amino acid diversity in the library, this enabled us to rapidly explore where within antibody structures to place noncanonical amino acids (ncAAs) containing a series of chemically reactive and photocrosslinkable side chains using ncAA-compatible yeast display. Apparent binding affinities on the yeast surface of ncAA-substituted synthetic antibodies vary based on factors including ncAA side chain identity, location of ncAA incorporation in or near antibody CDRs, and ncAA incorporation machinery used. However, numerous substitutions result in clones that retain binding function, confirming that several positions within antibodies support ncAA incorporation despite their proximity to the presumed binding site in CDR H3. We further investigated whether ncAA-containing antibodies displayed on yeast could be chemically modified with bioorthogonal chemistries and what effects these modifications might have on antibody binding. Multiple azide-containing ncAAs supported both copper-catalyzed azide-alkyne cycloaddition (CuAAC) and strain-promoted azide-alkyne cycloaddition without apparent changes in binding function compared to unmodified controls. Alkyne-containing antibodies also appear to support CuAAC without apparent changes in binding function. Finally, we investigated whether antibodies substituted with a photocrosslinkable ncAA would support ultraviolet-mediated crosslinking on the yeast surface. We observed position-dependent photocrosslinking events on the yeast surface consistent with our findings in control solution-based photocrosslinking experiments with the same clones. Taken together, our results highlight the power of integrating the use of yeast display and ncAAs in search of proteins with “chemically augmented” binding function. More specifically, our findings provide one route to productively integrate antibodies with ncAAs by leveraging the use of simple synthetic clones. We believe that these findings are important foundational elements paving the way to performing drug discovery on the yeast surface.