Design, Construction, and Validation of a Yeast-Displayed Chemically Expanded Antibody Library.

In vitro display technologies, exemplified by phage and yeast display, have emerged as powerful platforms for antibody discovery and engineering. However, the identification of antibodies that disrupt target functions beyond binding remains a challenge. In particular, there are very few strategies that support identification and engineering of either protein-based irreversible binders or inhibitory enzyme binders. Expanding the range of chemistries in antibody libraries has the potential to lead to efficient discovery of function-disrupting antibodies. In this work, we describe a yeast display-based platform for the discovery of chemically diversified antibodies. We constructed a billion-member antibody library that supports the presentation of a range of chemistries within antibody variable domains via noncanonical amino acid (ncAA) incorporation and subsequent bioorthogonal click chemistry conjugations. Use of a polyspecific orthogonal translation system enables introduction of chemical groups with various properties, including photo-reactive, proximity-reactive, and click chemistry-enabled functional groups for library screening. We established conjugation conditions that facilitate modification of the full library, demonstrating the feasibility of sorting the full billion-member library in "protein-small molecule hybrid" format in future work. Here, we conducted initial library screens after introducing O-(2-bromoethyl)tyrosine (OBeY), a weakly electrophilic ncAA capable of undergoing proximity-induced crosslinking to a target. Enrichments against donkey IgG and protein tyrosine phosphatase 1B (PTP1B) each led to the identification of several OBeY-substituted clones that bind to the targets of interest. Flow cytometry analysis on the yeast surface confirmed higher retention of binding for OBeY-substituted clones compared to clones substituted with ncAAs lacking electrophilic side chains after denaturation. However, subsequent crosslinking experiments in solution with ncAA-substituted clones yielded inconclusive results, suggesting that weakly reactive OBeY side chain is not sufficient to drive robust crosslinking in the clones isolated here. Nonetheless, this work establishes a multi-modal, chemically expanded antibody library and demonstrates the feasibility of conducting discovery campaigns in chemically expanded format. This versatile platform offers new opportunities for identifying and characterizing antibodies with properties beyond what is accessible with the canonical amino acids, potentially enabling discovery of new classes of reagents, diagnostics, and even therapeutic leads.

Genome-Wide Screen for Enhanced Noncanonical Amino Acid Incorporation in Yeast.

Expanding the genetic code beyond the 20 canonical amino acids enables access to a wide range of chemical functionality that is inaccessible within conventionally biosynthesized proteins. The vast majority of efforts to expand the genetic code have focused on the orthogonal translation systems required to achieve the genetically encoded addition of noncanonical amino acids (ncAAs) into proteins. There remain tremendous opportunities for identifying genetic and genomic factors that enhance ncAA incorporation. Here we describe genome-wide screening strategies to identify factors that enable more efficient addition of ncAAs to biosynthesized proteins. These unbiased screens can reveal previously unknown genes or mutations that can enhance ncAA incorporation and deepen our understanding of the translation apparatus.

Systematic Evaluation of Protein-Small Molecule Hybrids on the Yeast Surface.

Protein-small molecule hybrids are structures that have the potential to combine the inhibitory properties of small molecules and the specificities of binding proteins. However, achieving such synergies is a substantial engineering challenge with fundamental principles yet to be elucidated. Recent work has demonstrated the power of the yeast display-based discovery of hybrids using a combination of fibronectin-binding domains and thiol-mediated conjugations to introduce small-molecule warheads. Here, we systematically study the effects of expanding the chemical diversity of these hybrids on the yeast surface by investigating a combinatorial set of fibronectins, noncanonical amino acid (ncAA) substitutions, and small-molecule pharmacophores. Our results show that previously discovered thiol-fibronectin hybrids are generally tolerant of a range of ncAA substitutions and retain binding functions to carbonic anhydrases following click chemistry-mediated assembly of hybrids with diverse linker structures. Most surprisingly, we identified several cases where replacement of a potent acetazolamide warhead with a substantially weaker benzenesulfonamide warhead still resulted in the assembly of multiple functional hybrids. In addition to these unexpected findings, we expanded the throughput of our system by validating a 96-well plate-based format to produce yeast-displayed hybrid conjugates in parallel. These efficient explorations of hybrid chemical diversity demonstrate that there are abundant opportunities to expand the functions of protein-small molecule hybrids and elucidate principles that dictate their efficient discovery and design.

Dual Noncanonical Amino Acid Incorporation Enabling Chemoselective Protein Modification at Two Distinct Sites in Yeast.

Incorporation of more than one noncanonical amino acid (ncAA) within a single protein endows the resulting construct with multiple useful features such as augmented molecular recognition or covalent cross-linking capabilities. Herein, for the first time, we demonstrate the incorporation of two chemically distinct ncAAs into proteins biosynthesized in Saccharomyces cerevisiae. To complement ncAA incorporation in response to the amber (TAG) stop codon in yeast, we evaluated opal (TGA) stop codon suppression using three distinct orthogonal translation systems. We observed selective TGA readthrough without detectable cross-reactivity from host translation components. Readthrough efficiency at TGA was modulated by factors including the local nucleotide environment, gene deletions related to the translation process, and the identity of the suppressor tRNA. These observations facilitated systematic investigation of dual ncAA incorporation in both intracellular and yeast-displayed protein constructs, where we observed efficiencies up to 6% of wild-type protein controls. The successful display of doubly substituted proteins enabled the exploration of two critical applications on the yeast surface─(A) antigen binding functionality and (B) chemoselective modification with two distinct chemical probes through sequential application of two bioorthogonal click chemistry reactions. Lastly, by utilizing a soluble form of a doubly substituted construct, we validated the dual incorporation system using mass spectrometry and demonstrated the feasibility of conducting selective labeling of the two ncAAs sequentially using a "single-pot" approach. Overall, our work facilitates the addition of a 22nd amino acid to the genetic code of yeast and expands the scope of applications of ncAAs for basic biological research and drug discovery.

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.

Flow cytometric evaluation of yeast-bacterial cell-cell interactions.

Synthetic cell-cell interaction systems can be useful for understanding multicellular communities or for screening binding molecules. We adapt a previously characterized set of synthetic cognate nanobody-antigen pairs to a yeast-bacteria coincubation format and use flow cytometry to evaluate cell-cell interactions mediated by binding between surface-displayed molecules. We further use fluorescence-activated cell sorting to enrich a specific yeast-displayed nanobody within a mixed yeast-display population. Finally, we demonstrate that this system supports the characterization of a therapeutically relevant nanobody-antigen interaction: a previously discovered nanobody that binds to the intimin protein expressed on the surface of enterohemorrhagic Escherichia coli. Overall, our findings indicate that the yeast-bacteria format supports efficient evaluation of ligand-target interactions. With further development, this format may facilitate systematic characterization and high-throughput discovery of bacterial surface-binding molecules.

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.

Genome-Wide Screen for Enhanced Noncanonical Amino Acid Incorporation in Yeast.

Numerous applications of noncanonical amino acids (ncAAs) in basic biology and therapeutic development require efficient protein biosynthesis using an expanded genetic code. However, achieving such incorporation at repurposed stop codons in cells is generally inefficient and limited by complex cellular processes that preserve the fidelity of protein synthesis. A more comprehensive understanding of the processes that contribute to ncAA incorporation would aid in the development of genomic engineering strategies for augmenting genetic code manipulation. In this work, we used a series of fluorescent reporters to screen a pooled Saccharomyces cerevisiae molecular barcoded yeast knockout (YKO) collection. Fluorescence-activated cell sorting enabled isolation of strains encoding single-gene deletions exhibiting improved ncAA incorporation efficiency in response to the amber (TAG) stop codon; 55 unique candidate deletions were identified. The deleted genes encoded for proteins that participate in diverse cellular processes, including many genes that have no known connection with protein translation. We then verified that two knockouts, yil014c-aΔ and alo1Δ, exhibited improved ncAA incorporation efficiency starting from independently acquired strains possessing the knockouts. Using additional orthogonal translation systems and ncAAs, we determined that yil014c-aΔ and alo1Δ enhance ncAA incorporation efficiency without loss of fidelity over a wide range of conditions. Our findings highlight opportunities for further modulating gene expression with genetic, genomic, and synthetic biology approaches to improve ncAA incorporation efficiency. In addition, these discoveries have the potential to enhance our fundamental understanding of protein translation. Ultimately, cells that efficiently biosynthesize ncAA-containing proteins will streamline the realization of applications utilizing expanded genetic codes ranging from basic biology to drug discovery.

High-Throughput Aminoacyl-tRNA Synthetase Engineering for Genetic Code Expansion in Yeast.

Protein expression with genetically encoded noncanonical amino acids (ncAAs) benefits a broad range of applications, from the discovery of biological therapeutics to fundamental biological studies. A major factor limiting the use of ncAAs is the lack of orthogonal translation systems (OTSs) that support efficient genetic code expansion at repurposed stop codons. Aminoacyl-tRNA synthetases (aaRSs) have been extensively evolved in Escherichia coli but are not always orthogonal in eukaryotes. In this work, we use a yeast display-based ncAA incorporation reporter platform with fluorescence-activated cell sorting to screen libraries of aaRSs in high throughput for (1) the incorporation of ncAAs not previously encoded in yeast; (2) the improvement of the performance of an existing aaRS; (3) highly selective OTSs capable of discriminating between closely related ncAA analogues; and (4) OTSs exhibiting enhanced polyspecificity to support translation with structurally diverse sets of ncAAs. The number of previously undiscovered aaRS variants we report in this work more than doubles the total number of translationally active aaRSs available for genetic code manipulation in yeast. The success of myriad screening strategies has important implications related to the fundamental properties and evolvability of aaRSs. Furthermore, access to OTSs with diverse activities and specific or polyspecific properties is invaluable for a range of applications within chemical biology, synthetic biology, and protein engineering.

Engineering Proteins Containing Noncanonical Amino Acids on the Yeast Surface.

Yeast display has been used to advance many critical research areas, including the discovery of unique protein binders and biological therapeutics. In parallel, noncanonical amino acids (ncAAs) have been used to tailor antibody-drug conjugates and enable discovery of therapeutic leads. Together, these two technologies have allowed for generation of synthetic antibody libraries, where the introduction of ncAAs in yeast-displayed proteins allows for library screening for therapeutically relevant targets. The combination of yeast display with genetically encoded ncAAs increases the available chemistry in proteins and advances applications that require high-throughput strategies. In this chapter, we discuss methods for displaying proteins containing ncAAs on the yeast surface, generating and screening libraries of proteins containing ncAAs, preparing bioconjugates on the yeast surface in large scale, generating and screening libraries of aminoacyl-tRNA synthetases (aaRSs) for encoding ncAAs by using reporter constructs, and characterizing ncAA-containing proteins secreted from yeast. The experimental designs laid out in this chapter are generalizable for discovery of protein binders to a variety of targets and aaRS evolution to continue expanding the genetic code beyond what is currently available in yeast.

Exploration of Methanomethylophilus alvus Pyrrolysyl-tRNA Synthetase Activity in Yeast.

Archaeal pyrrolysyl-tRNA synthetases (PylRSs) have been used to genetically encode over 200 distinct noncanonical amino acids (ncAAs) in proteins in Escherichia coli and mammalian cells. This vastly expands the range of chemical functionality accessible within proteins produced in these organisms. Despite these clear successes, explorations of PylRS function in yeast remain limited. In this work, we demonstrate that the Methanomethylophilus alvus PylRS (MaPylRS) and its cognate tRNACUAMaPyl support the incorporation of ncAAs into proteins produced in Saccharomyces cerevisiae using stop codon suppression methodologies. Additionally, we prepared three MaPylRS mutants originally engineered in E. coli and determined that all three were active with one or more ncAAs, although with low efficiencies of ncAA incorporation in comparison to the parent MaPylRS. Alongside MaPylRS variants, we evaluated the activity of previously reported Methanosarcina mazei, Methanosarcina barkeri, and chimeric M. mazei and M. barkeri PylRSs. Using S. cerevisiae RJY100 and pairing these PylRSs with the M. mazei tRNACUA, we did not observe any detectable stop codon suppression activity under the same conditions that produced moderately efficient ncAA incorporation with MaPylRS. The addition of MaPylRS/tRNACUAMaPyl to the orthogonal translation machinery toolkit in S. cerevisiae potentially opens the door to hundreds of ncAAs that have not previously been genetically encodable using other aminoacyl-tRNA synthetase/tRNA pairs. Extending the scope of ncAA incorporation in yeast could powerfully advance chemical and biological research for applications ranging from basic biological discovery to enzyme engineering and therapeutic protein lead discovery.

Incorporating, Quantifying, and Leveraging Noncanonical Amino Acids in Yeast.

Genetic code expansion has allowed for extraordinary advances in enhancing protein chemical diversity and functionality, but there remains a critical need for understanding and engineering genetic code expansion systems for improved efficiency. Incorporation of noncanonical amino acids (ncAAs) at stop codons provides a site-specific method for introducing unique chemistry into proteins, though often at reduced yields compared to wild-type proteins. A powerful platform for ncAA incorporation supports both the expression and evaluation of chemically diverse proteins for a broad range of applications. In yeast, ncAAs have been used to study dynamic cellular processes such as protein-protein interactions and also allow for exploration of eukaryotic-specific biology such as epigenetics. Furthermore, yeast display is an advantageous technology for engineering and screening the properties of proteins in high throughput. The protocols presented in this chapter describe detailed methods for the yeast-based genetic encoding of ncAAs in proteins intracellularly or on the yeast surface. In addition, methods are presented for modifying proteins on the yeast surface using bioorthogonal chemical reactions and evaluating reaction efficiency. Finally, protocols are included for the preparation of libraries that involve genetic code expansion. Libraries of proteins that contain ncAAs or libraries of the cellular machinery required to encode ncAAs can be constructed and screened in high throughput for many biological and chemical applications. Efficient incorporation of ncAAs facilitates elucidation of fundamental eukaryotic biology and advances tools for enzyme and genome engineering to evolve host cells that are better able to accommodate alternative genetic codes.

Intracellular Delivery of Antibodies for Selective Cell Signaling Interference.

Many intracellular signaling events remain poorly characterized due to a general lack of tools to interfere with "undruggable" targets. Antibodies have the potential to elucidate intracellular mechanisms via targeted disruption of cell signaling cascades because of their ability to bind to a target with high specificity and affinity. However, due to their size and chemical composition, antibodies cannot innately cross the cell membrane, and thus access to the cytosol with these macromolecules has been limited. Herein we describe strategies for accessing the intracellular space with recombinant antibodies mediated by cationic lipid nanoparticles to selectively disrupt intracellular signaling events. Together, our results demonstrate the use of recombinantly produced antibodies, delivered at concentrations of 10 nM, to selectively interfere with signaling driven by a single posttranslational modification. Efficient intracellular delivery of engineered antibodies opens up possibilities for modulation of previously "undruggable" targets, including for potential therapeutic applications.

Broadening the Toolkit for Quantitatively Evaluating Noncanonical Amino Acid Incorporation in Yeast.

Genetic code expansion is a powerful approach for advancing critical fields such as biological therapeutic discovery. However, the machinery for genetically encoding noncanonical amino acids (ncAAs) is only available in limited plasmid formats, constraining potential applications. In extreme cases, the introduction of two separate plasmids, one containing an orthogonal translation system (OTS) to facilitate ncAA incorporation and a second for expressing a ncAA-containing protein of interest, is not possible due to a lack of the available selection markers. One strategy to circumvent this challenge is to express the OTS and protein of interest from a single vector. For what we believe is the first time in yeast, we describe here several sets of single plasmid systems (SPSs) for performing genetic code manipulation and compare the ncAA incorporation capabilities of these plasmids against the capabilities of previously described dual plasmid systems (DPSs). For both dual fluorescent protein reporters and yeast display reporters tested with multiple OTSs and ncAAs, measured ncAA incorporation efficiencies with SPSs were determined to be equal to efficiencies determined with DPSs. Click chemistry on yeast cells displaying ncAA-containing proteins was also shown to be feasible in both formats, although differences in reactivity between formats suggest the need for caution when using such approaches. Additionally, we investigated whether these reporters would support the separation of yeast strains known to exhibit distinct ncAA incorporation efficiencies. Model sorts conducted with mixtures of two strains transformed with the same SPS or DPS both led to the enrichment of a strain known to support a higher efficiency ncAA incorporation, suggesting that these reporters will be suitable for conducting screens for strains exhibiting enhanced ncAA incorporation efficiencies. Overall, our results confirm that SPSs are well behaved in yeast and provide a convenient alternative to DPSs. SPSs are expected to be invaluable for conducting high-throughput investigations of the effects of genetic or genomic changes on ncAA incorporation efficiency and, more fundamentally, the eukaryotic translation apparatus.

Engineering Selectively Targeting Antimicrobial Peptides.

The rise of antibiotic-resistant strains of bacterial pathogens has necessitated the development of new therapeutics. Antimicrobial peptides (AMPs) are a class of compounds with potentially attractive therapeutic properties, including the ability to target specific groups of bacteria. In nature, AMPs exhibit remarkable structural and functional diversity, which may be further enhanced through genetic engineering, high-throughput screening, and chemical modification strategies. In this review, we discuss the molecular mechanisms underlying AMP selectivity and highlight recent computational and experimental efforts to design selectively targeting AMPs. While there has been an extensive effort to find broadly active and highly potent AMPs, it remains challenging to design targeting peptides to discriminate between different bacteria on the basis of physicochemical properties. We also review approaches for measuring AMP activity, point out the challenges faced in assaying for selectivity, and discuss the potential for increasing AMP diversity through chemical modifications.

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.

ASAP-SML: An antibody sequence analysis pipeline using statistical testing and machine learning.

Antibodies are capable of potently and specifically binding individual antigens and, in some cases, disrupting their functions. The key challenge in generating antibody-based inhibitors is the lack of fundamental information relating sequences of antibodies to their unique properties as inhibitors. We develop a pipeline, Antibody Sequence Analysis Pipeline using Statistical testing and Machine Learning (ASAP-SML), to identify features that distinguish one set of antibody sequences from antibody sequences in a reference set. The pipeline extracts feature fingerprints from sequences. The fingerprints represent germline, CDR canonical structure, isoelectric point and frequent positional motifs. Machine learning and statistical significance testing techniques are applied to antibody sequences and extracted feature fingerprints to identify distinguishing feature values and combinations thereof. To demonstrate how it works, we applied the pipeline on sets of antibody sequences known to bind or inhibit the activities of matrix metalloproteinases (MMPs), a family of zinc-dependent enzymes that promote cancer progression and undesired inflammation under pathological conditions, against reference datasets that do not bind or inhibit MMPs. ASAP-SML identifies features and combinations of feature values found in the MMP-targeting sets that are distinct from those in the reference sets.