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 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.

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

“…The ncAA-mediated production of clickable conjugates described here complements and extends prior work from our group to "chemically enhance" binding proteins by introducing photo-cross-linkable and proximity-induced cross-linkable groups in binding proteins. 23,24 This growing toolkit and numerous related strategies combining proteins and small molecules 36−39 provide exciting opportunities to combinatorially evaluate hybrids for properties not accessible in either proteins or small molecules alone.…”

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

“…Next, we extended our dual ncAA incorporation system to yeast-displayed proteins, as yeast display is a powerful platform to engineer proteins and supports myriad assays for evaluating properties like affinity, stability, activity, and specificity. ,, We began by identifying two sites in a previously reported synthetic antibody fragment (Donkey1.1 scFv)L93 (located in complementarity determining region L3; CDR-L3) and H54 (located in CDR-H2) for encoding ncAAs in response to TAG and TGA (Figure A) . Both of these sites have been previously demonstrated to tolerate different ncAAs inserted in response to the TAG codon.…”

“…16,82−84 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 cross-linking chemistries or post-translational modifications. 3,12,13,18,72,85 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.…”

“…Sequential SPAAC and CuAAC reactions led to doubly functionalized antibodies in both yeast display and soluble formats, which has not previously been achieved in yeast-based ncAA incorporation systems. Other promising biorthogonal conjugation chemistries for double labeling explorations on the yeast surface and in yeast-produced proteins include strain-promoted inverse electron-demand Diels–Alder cycloaddition (SPIEDAC) and the chemoselective rapid azo-coupling reaction (CRACR). − ,,, Doubly labeled proteins on the yeast surface are expected to enable applications such as FRET to study protein conformation and dynamics, as evidenced by prior work in mammalian cells. ,− 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 cross-linking chemistries or post-translational modifications. ,,,,, 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 ]…”