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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 100°C, and > 81% binding activity for PA after heating at 90°C for 1 h. We discuss the potential of the PCC agent screening process for the discovery and enrichment of next generation antibody alternatives.

We describe a general synthetic strategy for developing high-affinity peptide binders against specific epitopes of challenging protein biomarkers. The epitope of interest is synthesized as a polypeptide, with a detection biotin tag and a strategically placed azide (or alkyne) presenting amino acid. This synthetic epitope (SynEp) is incubated with a library of complementary alkyne or azide presenting peptides. Library elements that bind the SynEp in the correct orientation undergo the Huisgen cycloaddition, and are covalently linked to the SynEp. Hit peptides are tested against the full-length protein to identify the best binder. We describe development of epitope-targeted linear or macrocycle peptide ligands against 12 different diagnostic or therapeutic analytes. The general epitope targeting capability for these low molecular weight synthetic ligands enables a range of therapeutic and diagnostic applications, similar to those of monoclonal antibodies.

Biopanning by bacterial display has many advantages over yeast and phage display, including the speed to discovery of affinity reagents and direct amplification of bound cells without the need to elute and reinfect. However, widespread use is limited, in part due to poor performance achieved using manual Magnetic-Activated Cell Sorting (MACS) methods, and an absence of widely-available, low cost, high-performance sorting alternatives. Here, we have developed a methodology for bacterial cell sorting using the semi-automated autoMACS® Pro Separator for the first time, and have produced a complete method for sorting of bacteria displaying 15-mer peptides on their cell surface using this device, including downstream bioinformatic analysis of candidates for binding to a target of interest. Two autoMACS® programs designed for isolation of target cells with low frequency were evaluated and adapted to bacterial biopanning, using protective antigen (PA) of Bacillus anthracis as the model system. In contrast to manual MACS, the bacterial display library was preferentially enriched by autoMACS® sorting, yielding several promising candidates after only three rounds of biopanning and bioinformatic analysis. Individual candidates were evaluated for relative binding to fluorescently-labeled PA target or streptavidin negative control using Fluorescence-Activated Cell Sorting (FACS). The top thirteen peptide candidates from the autoMACS® sort demonstrate binding to PA with low cross-reactivity to streptavidin, while only two of eighteen candidates from the manual sort showed binding to PA, and both demonstrated greater cross-reactivity to streptavidin. Overall, the autoMACS® platform quickly harvested higher affinity peptide candidates with demonstrated specificity to the PA target. Peptide candidates produced with this method contained the previously reported PA consensus WXCFTC, further validating this method and the commercially available autoMACS® platform as the first low cost, semi-automated biopanning approach for bacterial display that is widely accessible and more reliable than the MACS/FACS standard protocol.