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 (T1/2 ) 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.
Subject(s)
Click Chemistry/methods , Peptide Library , Peptides/chemistry , Vascular Endothelial Growth Factor A/chemistry , Amino Acid Sequence , Animals , Antibodies/administration & dosage , Antibodies/chemistry , Antibodies/metabolism , Antigens, Bacterial/chemistry , Antigens, Bacterial/immunology , Antigens, Bacterial/metabolism , Bacterial Toxins/chemistry , Bacterial Toxins/immunology , Bacterial Toxins/metabolism , Calorimetry, Differential Scanning , Catalysis , Chromatography, High Pressure Liquid , Circular Dichroism , Colonic Neoplasms/diagnostic imaging , Colonic Neoplasms/metabolism , Female , HT29 Cells , Humans , Injections, Intraperitoneal , Injections, Intravenous , Ligands , Male , Mass Spectrometry , Mice , Microsomes, Liver/metabolism , Peptides/metabolism , Peptides/pharmacokinetics , Protein Binding , Transplantation, Heterologous , Vascular Endothelial Growth Factor A/metabolismABSTRACT
Concomitant advances made by the Human Genome Project and in the development of nucleic acid screening technologies are driving the expansion of pharmacogenomic research and molecular diagnostics. However, most current technologies are restrictive due to their complexity and/or cost, limiting the potential of personalized medicine. The invader assay, which can be used for genotyping as well as for gene expression monitoring without the need for intervening target amplification steps, presents an immediate solution that is accurate, simple to use, scaleable and cost-effective.
