A phage display approach to identify highly selective covalent binders

Chen, Shiyu; Bogyo, Matthew

doi:10.1101/791533

Cited by 6 publications

(5 citation statements)

References 58 publications

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“…52,123 In this regard, the simple synthetic antibodies and characterization strategies elucidated in this work could facilitate sideby-side evaluations of multiple chemical modification strategies. Finally, while many routes to prepare chemically modified peptides in display formats are now available, 29,[35][36][124][125][126][127][128][129][130][131] very few analogous approaches for the chemical diversification of proteins have been described. 53,60 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.…”

Section: Discussionmentioning

confidence: 99%

Chemical Diversification of Simple Synthetic Antibodies

et al. 2021

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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...

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Section: Discussionmentioning

confidence: 99%

Chemical Diversification of Simple Synthetic Antibodies

et al. 2021

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show abstract

Section: Discussionmentioning

confidence: 99%

Chemical Diversification of Simple Synthetic Antibodies

Islam¹,

Kehoe²,

Lissoos³

et al. 2020

Preprint

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<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>

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“…1k) can provide the pegylated and lipidated ligands and reduce the number of steps for optimization of leads. The "late" nature of 1,3-diketone ligation makes it an interesting candidate for ligation of reactive warheads for genetically-encoded discovery of covalent 26 or reversible covalent inhibitors 6 . Combination of this modification with silent encoding opens new opportunities in encoding of diverse number of macrocycles with pharmacophores, warheads and functionalities that would be difficult to introduce by other methods.…”

Section: Discussionmentioning

confidence: 99%