Strategies for enriching and characterizing proteins with inhibitory properties on the yeast surface.

Display technologies are powerful tools for discovering binding proteins against a broad range of biological targets. However, it remains challenging to adapt display technologies for the discovery of proteins that inhibit the enzymatic activities of targets. Here, we investigate approaches for discovering and characterizing inhibitory antibodies in yeast display format using a well-defined series of constructs and the target matrix metalloproteinase-9. Three previously reported antibodies were used to create model libraries consisting of inhibitory, non-inhibitory, and non-binding constructs. Conditions that preferentially enrich for inhibitory clones were identified for both magnetic bead-based enrichments and fluorescence-activated cell sorting. Half maximal inhibitory concentration (IC50) was obtained through yeast titration assays. The IC50 of the inhibitory antibody obtained in yeast display format falls within the confidence interval of the IC50 value determined in soluble form. Overall, this study identifies strategies for the discovery and characterization of inhibitory clones directly in yeast display format.

Yeast Display Enables Identification of Covalent Single-Domain Antibodies against Botulinum Neurotoxin Light Chain A.

While covalent drug discovery is reemerging as an important route to small-molecule therapeutic leads, strategies for the discovery and engineering of protein-based irreversible binding agents remain limited. Here, we describe the use of yeast display in combination with noncanonical amino acids (ncAAs) 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 previously described, structurally characterized sdAbs, we evaluated the properties of antibodies substituted with reactive ncAAs capable of forming covalent bonds with nearby groups after UV irradiation (when using 4-azido-l-phenylalanine) or spontaneously (when using O-(2-bromoethyl)-l-tyrosine). Systematic evaluations in yeast display format of more than 40 ncAA-substituted variants revealed numerous clones that retain binding function while gaining either UV-mediated or spontaneous crosslinking capabilities. Solution-based analyses indicate that ncAA-substituted clones exhibit site-dependent target specificity and crosslinking capabilities uniquely conferred by ncAAs. Interestingly, not all ncAA substitution sites resulted in crosslinking events, and our data showed no apparent correlation between detected crosslinking levels and distances between sdAbs and LC/A residues. Our findings highlight the power of yeast display in combination with genetic code expansion in the discovery of binding agents that covalently engage their targets. This platform streamlines the discovery and characterization of antibodies with therapeutically relevant properties that cannot be accessed in the conventional genetic code.

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 canonical amino acids. In this work, we investigate strategies for leveraging an expanded range of chemical functionality using yeast displayed antibodies containing noncanonical amino acids (ncAAs) in or near antibody complementarity determining regions (CDRs). To enable systematic characterization of the effects of ncAA incorporation on antibody function, we first investigated whether diversification of a single antibody loop would support the isolation of binding clones against immunoglobulins from three species. We constructed and screened a billion-member library containing canonical amino acid diversity and loop length diversity only within the third complementarity determining region of the heavy chain (CDR-H3). Isolated clones exhibited moderate affinities (double- to triple-digit nanomolar affinities) and, in several cases, single-species specificity, confirming that antibody specificity can be mediated by a single CDR. This constrained diversity enabled the utilization of additional CDRs for the installation of chemically reactive and photo-cross-linkable ncAAs. Binding studies of ncAA-substituted antibodies revealed that ncAA incorporation is reasonably well tolerated, with observed changes in affinity occurring as a function of ncAA side chain identity, substitution site, and the ncAA incorporation machinery used. Multiple azide-containing ncAAs supported copper-catalyzed azide-alkyne cycloaddition (CuAAC) and strain-promoted azide-alkyne cycloaddition (SPAAC) without the abrogation of binding function. Similarly, several alkyne substitutions facilitated CuAAC without the apparent disruption of binding. Finally, antibodies substituted with a photo-cross-linkable ncAA were evaluated for ultraviolet-mediated cross-linking on the yeast surface. Competition-based assays revealed position-dependent covalent linkages, strongly suggesting successful cross-linking. Key findings regarding CuAAC reactions and photo-cross-linking on the yeast surface were confirmed using soluble forms of ncAA-substituted clones. The consistency of findings on 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. This includes strategies for systematically introducing small molecule functionality within binding protein structures and evaluating protein-based covalent target binding. The efficient preparation and chemical diversification of antibodies on the yeast surface open up new possibilities for discovering "drug-like" protein leads in high throughput.

Future prospects for noncanonical amino acids in biological therapeutics.

There is growing evidence that noncanonical amino acids (ncAAs) can be utilized in the creation of biological therapeutics ranging from protein conjugates to cell-based therapies. However, when does genetically encoding ncAAs yield biologics with unique properties compared to other approaches? In this review, we attempt to answer this question in the broader context of therapeutic development, emphasizing advances within the past two years. In several areas, ncAAs add valuable routes to therapeutically relevant entities, but application-specific needs ultimately determine whether ncAA-mediated or alternative solutions are preferred. Looking forward, using ncAAs to perform 'protein medicinal chemistry,' in which atomic-level changes to proteins dramatically enhance therapeutic properties, is a promising emerging area. Further upgrades to the performance of ncAA incorporation technologies will be essential to realizing the full potential of ncAAs in biological therapeutics.