Chemical Diversification of Simple Synthetic Antibodies

Antibodies possess properties that make them valuable as therapeutics, diagnostics, and basic research tools. However, antibody chemical reactivity and covalent antigen binding are constrained, or even prevented, by the narrow range of chemistries encoded in the canonical amino acids. In this work, we investigate strategies for leveraging an expanded range of chemical functionality to augment antibody binding properties. Using yeast displayed antibodies, we explored the presentation of noncanonical amino acids (ncAAs) in or near antibody complementarity determining regions (CDRs) and evaluated the properties of the resulting constructs. To enable systematic characterization of ncAA incorporation sites, we first investigated whether diversification of a single antibody loop would support isolation of binding clones. We constructed 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). Screens against a series of immunoglobulins from three species resulted in the isolation of antibodies exhibiting moderate affinities (double- to triple-digit nanomolar affinities) and, in several cases, single-species specificity. These findings confirmed that antibody specificity can be mediated by a single CDR. With this constrained diversity, we were able to utilize additional CDRs for the installation of chemically reactive and photo-crosslinkable ncAAs. Apparent binding affinities of ncAA-substituted synthetic antibodies on the yeast surface revealed that ncAA incorporation is generally well tolerated. However, changes in binding affinities did occur upon substitution, and varied based on factors including ncAA side chain identity, location of ncAA incorporation, and the ncAA incorporation machinery used. We further investigated chemical modifications facilitated by ncAA installation. Multiple azide-containing ncAAs supported both copper-catalyzed azide-alkyne cycloaddition (CuAAC) and strain-promoted azide-alkyne cycloaddition (SPAAC) without abrogation of binding function following the installation of bulky probes. Similarly, several alkyne substitutions facilitated CuAAC without apparent disruption of binding function. Finally, antibodies substituted with a photo-crosslinkable ncAA were evaluated for ultraviolet-mediated crosslinking on the yeast surface. Competition-based assays revealed position-dependent linkages that could not be displaced by excess soluble antigen, strongly suggesting successful crosslinking. Key findings regarding CuAAC reactions and photo-crosslinking on the yeast surface were confirmed using soluble forms of ncAA-substituted clones. These consistent behaviors between the yeast surface and in solution suggest that chemical diversification can be incorporated into yeast display screening approaches. Taken together, our results highlight the power of integrating the use of yeast display and ncAAs in search of proteins with “chemically augmented” binding functions. More specifically, our findings provide the means to productively integrate antibodies with ncAAs by leveraging simple synthetic antibodies. The efficient preparation and chemical diversification of antibodies on the yeast surface opens up new possibilities for discovering “drug-like” protein leads in high throughput.