Engineering protein translation machinery to incorporate noncanonical amino acids (ncAAs) into proteins has advanced applications ranging from proteomics to single-molecule studies. As applications of ncAAs emerge, efficient ncAA incorporation is crucial to exploiting unique chemistries. We have established a quantitative reporter platform to evaluate ncAA incorporation in response to the TAG (amber) codon in yeast. This yeast display-based reporter utilizes an antibody fragment containing an amber codon at which a ncAA is incorporated when the appropriate orthogonal translation system (OTS) is present. Epitope tags at both termini allow for flow cytometry-based end point readouts of OTS efficiency and fidelity. Using this reporter, we evaluated several factors that influence amber suppression, including the amber codon position and different aminoacyl-tRNA synthetase/tRNA (aaRS/tRNA) pairs. Interestingly, previously described aaRSs that evolved from different parent enzymes to incorporate O-methyl-l-tyrosine exhibit vastly different behavior. Escherichia coli leucyl-tRNA synthetase variants demonstrated efficient incorporation of a range of ncAAs, and we discovered unreported activities of several variants. Compared to a plate reader-based reporter, our assay yields more precise bulk-level measurements while also supporting single-cell readouts compatible with cell sorting. This platform is expected to allow quantitative elucidation of principles dictating efficient stop codon suppression and evolution of next-generation stop codon suppression systems to further enhance genetic code manipulation in eukaryotes. These efforts will improve our understanding of how the genetic code can be further evolved while expanding the range of chemical diversity available in proteins for applications ranging from fundamental epigenetics studies to engineering new classes of therapeutics.

The combination of protein display technologies and noncanonical amino acids (ncAAs) offers unprecedented opportunities for the high throughput discovery and characterization of molecules suitable for addressing fundamental and applied problems in biological systems. Here we demonstrate that ncAA-compatible yeast display facilitates evaluations of conjugation chemistry and stability that would be challenging or impossible to perform with existing mRNA, phage, or E. coli platforms. Our approach enables site-specific introduction of ncAAs into displayed proteins, robust modification at azide-containing residues, and quantitative evaluation of conjugates directly on the yeast surface. Moreover, screening allows for the selective enrichment of chemically modified constructs while maintaining a genotype-phenotype linkage with encoded azide functionalities. Thus, this platform is suitable for the high throughput characterization and screening of libraries of chemically modified polypeptides for therapeutic lead discovery and other biological applications.

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, o...

<p>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 <a>copper-catalyzed azide-alkyne cycloaddition (CuAAC) and strain-promoted azide-alkyne cycloaddition </a>(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.</p>

Although cancer‐promoting activities of extracellular proteases and peptidases in the tumor microenvironment are well known, the disruption of individual enzymes in this environment remains a fundamental challenge. Individual matrix metalloproteinases (MMPs), in particular, have proven difficult to selectively inhibit due to their structural similarities. To better understand the roles of these enzymes in cancer progression, and to potentially begin to target them in vivo, selective inhibitors of these enzymes are needed. We are developing a protein‐small molecule “hybrid” approach to meet this need by conjugating inhibitory small molecules to single‐chain variable antibody fragments (scFvs) that selectively bind a unique target.To this end, we have established a platform to introduce a versatile range of chemical functionalities into scFvs on the yeast surface. Using amber suppression, we can introduce noncanonical amino acids (ncAAs) at specific sites in displayed scFvs that are predicted to be close to the antigen binding site. In our proof‐of‐concept model, we have been investigating the possibility of adding enzyme active site‐targeting chemical groups to scFvs that bind to MMP‐9. Flow cytometry results show that constructs containing ncAAs with an azide side chain retain MMP‐9 binding. Importantly, introducing the azide functionality enables us to conjugate MMP‐targeting small molecules to displayed scFvs using bioorthogonal copper‐catalyzed azide‐alkyne cycloadditions (“click chemistry”). We have investigated installation of MMP‐targeting small molecules in different antibodies and at various scFv positions. In some cases, we see almost complete reaction, but the extent of reaction across antibodies and sites is dependent on the small molecule used and the site of ncAA incorporation. Systematic exploration of reaction conditions including time and click chemistry ligand has led to conditions that enhance the extent of reaction of small molecules at different sites. Work to evaluate enhancements to binding and inhibition of MMP‐9 with the resulting antibody‐small molecule “hybrid” conjugates is ongoing. We anticipate that adding more chemical functionality into proteins will facilitate selective targeting of broad classes of enzymes active in the tumor microenvironment and in other complex diseases.Support or Funding InformationTufts Summer Scholars, Tufts Faculty Research FundThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

<p>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 <a>copper-catalyzed azide-alkyne cycloaddition (CuAAC) and strain-promoted azide-alkyne cycloaddition </a>(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.</p>