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 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.
Highlights
Monoclonal antibodies are the current standard as biological recognition elements for in vitro and in vivo applications
We explore the tunability of 2 different protein catalyzed capture (PCC) for specific in vitro and in vivo applications
We developed an anti-vascular endothelial growth factor (VEGF) PCC as a positron emission tomography (PET) probe for in vivo imaging
Summary
Monoclonal antibodies (mAbs) are the current standard as biological recognition elements for in vitro and in vivo applications. Thermal and biological instabilities, batch-tobatch variability, and molecular size can limit the effectiveness of mAbs. For example, antibodies often lack the stability to serve as capture agents for environmentally demanding applications.[2]. The large size of mAbs can yield attractive pharmacokinetic (PK) properties for certain therapeutic tasks. Their large size can prevent the rapid in vivo clearance often required for in vivo imaging tasks.[3,4]. Some of these drawbacks are being actively addressed,[5] including the work by McConnell et al to produce stabilized IgG frameworks that can maintain >60% binding activity after 1 h at 908C.[6] Their large size can prevent the rapid in vivo clearance often required for in vivo imaging tasks.[3,4] Some of these drawbacks are being actively addressed,[5] including the work by McConnell et al to produce stabilized IgG frameworks that can maintain >60% binding activity after 1 h at 908C.[6]
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