Protein catalyzed capture agents with tailored performance for in vitro and in vivo applications

Abstract

We report on peptide-based ligands matured through the protein catalyzed capture (PCC) agent method to tailor molecular binders for in vitro sensing/diagnostics and in vivo pharmacokinetics parameters. A vascular endothelial growth factor (VEGF) binding peptide and a peptide against the protective antigen (PA) protein of Bacillus anthracis discovered through phage and bacterial display panning technologies, respectively, were modified with click handles and subjected to iterative in situ click chemistry screens using synthetic peptide libraries. Each azide-alkyne cycloaddition iteration, promoted by the respective target proteins, yielded improvements in metrics for the application of interest. The anti-VEGF PCC was explored as a stable in vivo imaging probe. It exhibited excellent stability against proteases and a mean elimination in vivo half-life (T 1/2 ) of 36 min. Intraperitoneal injection of the reagent results in slow clearance from the peritoneal cavity and kidney retention at extended times, while intravenous injection translates to rapid renal clearance. The ligand competed with the commercial antibody for binding to VEGF in vivo. The anti-PA ligand was developed for detection assays that perform in demanding physical environments. The matured anti-PA PCC exhibited no solution aggregation, no fragmentation when heated to 1008C, and > 81% binding activity for PA after heating at 908C for 1 h. We discuss the potential of the PCC agent screening process for the discovery and enrichment of next generation antibody alternatives.

Protein-Catalyzed Capture Agents

“…These PCCs represent scientific demonstrations of diagnostic reagents 16 , allosteric 19 and direct 12 inhibitors, in-cell protein inhibitors via degradation tags 126 , and in vivo imaging. 26 Certain metrics of these PCCs are listed in Table 3.…”

Unraveling the Roots of Selectivity of Peptide Affinity Reagents for Structurally Similar Ribosomal Inactivating Protein Derivatives

“…Combined with semi-automated Magnetic Activated Cell Sorting (autoMACS) methods, this technology has been employed for the rapid discovery of robust recognition elements with affinity towards potential biological threats and food toxins, such as Bacillus anthracis and Staphococcal enterotoxin B (SEB) [7,9,11,14]. After the initial rapid biopanning procedure to enrich for peptide capture candidates against the target of interest, the peptide ligands can be synthesized off-cell for further testing and immediate use or successfully matured to more robust, higher affinity synthetic peptide capture reagents using Protein Catalyzed Capture Agent (PCC Agent) technology [19,20,21] since these peptides are an alternative precursor for PCC strategies which otherwise require structural and sequence information for the target of interest [22,23]. Additionally, discovery in a bacterial peptide display system allows for direct use of peptide recognition elements while displayed on the cell surface of Escherichia coli ( E. coli ) bacteria.…”

Epitope Targeting of Tertiary Protein Structure Enables Target‐Guided Synthesis of a Potent In‐Cell Inhibitor of Botulinum Neurotoxin

“…They utilized the active site of an enzyme as a highly selective promoter of the 1,3-dipolar cycloaddition reaction to assemble a divalent inhibitor from two small molecule libraries – one presenting and azide, the other an acetylene. Over the last several years we have applied in situ click to the discovery of peptides targeting a variety of proteins and post-translational modifications [9] . To build a BoNT inhibitor, we sought to further generalize the target-guided synthesis approach by exploiting the tertiary structure of the BoNT LC as a landscape for assembling a potent inhibitor (Scheme 1).…”