Yeast Display Enables Identification of Covalent Single Domain Antibodies Against Botulinum Neurotoxin Light Chain A

Abstract

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, a high-throughput protein discovery platform, 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. This underscores the utility of high-throughput platforms both to identify crosslinkable antibodies and to inform future rational and computational designs of such antibodies. 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.

Systematic evaluation of protein-small molecule hybrids on the yeast surface

“…High-throughput experimentation on the yeast surface enables the rapid discovery of inhibitors from large collections while simultaneously yielding data to better inform future hybrid design efforts. The ncAAmediated production of clickable conjugates described here complements and extends prior work from our group to "chemically enhance" binding proteins by introducing photocrosslinkable and proximity-induced crosslinkable groups in binding proteins 23,24 .…”

“…20 Here, we used noncanonical amino acid (ncAA) substitutions and copper-catalyzed azide-alkyne cycloaddition (CuAAC) click chemistry to combinatorially vary the attachment site and hCA-targeting warhead. [21][22][23][24] To increase the throughput of hybrid generation, we also established a 96-well plate-based CuAAC protocol that facilitates rapid assembly and evaluation of hybrid construction and properties in parallel. These investigations allowed us to identify hybrids that retain hCA binding and inhibition while differing substantially from the previously described "parent" hybrids in terms of conjugated amino acid, conjugation chemistry, and even the CA-targeting warhead (with some loss of isoform selectivity noted).…”

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

“…Yeast display serves as a powerful platform to engineer proteins and enzymes and supports assays for quantitatively evaluating protein properties including affinity, stability, activity, and specificity (Cherf and Cochran, 2015). Previously, we have shown that chemically expanding yeast-displayed antibodies with ncAAs results in constructs with new properties including bio-orthogonal reactivity, photocrosslinkability, and spontaneous crosslinkability (Alcala-Torano et al, 2022; Islam et al, 2021). Incorporation of a second noncanonical functionality within chemically expanded, yeast-displayed proteins is expected to usher in a broader range of applications such as the preparation of doubly labeled proteins, parallel introduction of two distinct chemical properties such as crosslinkable groups and recognition motifs (for example, post-translational modifications), or additional chemical moieties of fundamental importance or commercial value (Oller-Salvia and Chin, 2019; Zheng et al, 2018b).…”

“…Efficient routes to doubly labeled proteins on the yeast surface are expected to enable applications such as FRET to study protein conformation and dynamics; doubly labeled proteins have previously facilitated FRET studies in vitro and in vivo, including on the surfaces of live mammalian cells (Cui et al, 2017;Maurel et al, 2008;Wang et al, 2014;Wu et al, 2012). Beyond bioconjugation strategies, proteins with dual ncAAs can support applications including intramolecular protein stapling, tethering strategies for small molecules or other drug modalities, and simultaneous presentation of two distinct chemistries such as crosslinking chemistries and posttranslational modifications (Alcala-Torano et al, 2022;Bednar et al, 2021;Islam et al, 2021;Meineke et al, 2020;Ren et al;Zheng et al, 2018b). Thus, the emerging dual ncAA incorporation strategies in this work along with the expanding collection of OTSs supporting diverse ncAA incorporation in yeast will streamline the realization of these intricate protein manipulations in the near future.…”

Protein Engineering and High‐Throughput Screening by Yeast Surface Display: Survey of Current Methods

“…[65] This concept was extended via incorporation of photoreactive (azido-phenylalanine) and proximity-reactive (O-(2-bromoethyl)-tyrosine) ncAAs adjacent to the binding site in single-domain antibodies to create covalent inhibitors of botulinum neurotoxin light chain A. [66] The utility of ncAAs reaches beyond engineered ligands. Expansion of the chemical repertoire of enzymes can enable catalysis of otherwise intractable chemical reactions.…”