title: 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 (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.
authors.affiliation: Sensors and Electron Devices Directorate, U.S. Army Research Laboratory, Adelphi, MD, 20783.
authors.forename: Matthew B
authors.identifier: http://orcid.org/0000-0001-9305-3846
authors.initials: MB
authors.lastname: Coppock
authors.affiliation: Excet, Springfield, VA 22151 supporting USA Edgewood Chemical Biological Center, Aberdeen Proving Ground, MD, 21010.
authors.forename: Candice R
authors.initials: CR
authors.lastname: Warner
authors.affiliation: Federal Staffing Resources, Annapolis, MD supporting U.S. Army Research Laboratory, Adelphi, MD, 20783.
authors.forename: Brandi
authors.initials: B
authors.lastname: Dorsey
authors.affiliation: Weapons and Materials Research Directorate, U.S. Army Research Laboratory, Aberdeen Proving Ground, MD, 21005.
authors.forename: Joshua A
authors.initials: JA
authors.lastname: Orlicki
authors.affiliation: Sensors and Electron Devices Directorate, U.S. Army Research Laboratory, Adelphi, MD, 20783.
authors.forename: Deborah A
authors.initials: DA
authors.lastname: Sarkes
authors.affiliation: Indi Molecular, 6162 Bristol Parkway, Culver City, CA, 90230.
authors.forename: Bert T
authors.initials: BT
authors.lastname: Lai
authors.affiliation: Indi Molecular, 6162 Bristol Parkway, Culver City, CA, 90230.
authors.forename: Suresh M
authors.initials: SM
authors.lastname: Pitram
authors.affiliation: Indi Molecular, 6162 Bristol Parkway, Culver City, CA, 90230.
authors.forename: Rosemary D
authors.initials: RD
authors.lastname: Rohde
authors.affiliation: Indi Molecular, 6162 Bristol Parkway, Culver City, CA, 90230.
authors.forename: Jacquie
authors.initials: J
authors.lastname: Malette
authors.affiliation: Indi Molecular, 6162 Bristol Parkway, Culver City, CA, 90230.
authors.forename: Jeré A
authors.initials: JA
authors.lastname: Wilson
authors.affiliation: Integrated Diagnostics, Seattle, WA, 98109.
authors.forename: Paul
authors.initials: P
authors.lastname: Kearney
authors.affiliation: Integrated Diagnostics, Seattle, WA, 98109.
authors.forename: Kenneth C
authors.initials: KC
authors.lastname: Fang
authors.affiliation: Integrated Diagnostics, Seattle, WA, 98109.
authors.forename: Scott M
authors.initials: SM
authors.lastname: Law
authors.affiliation: Integrated Diagnostics, Seattle, WA, 98109.
authors.forename: Sherri L
authors.initials: SL
authors.lastname: Candelario
authors.affiliation: Division of Chemistry and Chemical Engineering, California Institute of Technology, 1200 East California Boulevard, Pasadena, CA, 91125.
authors.forename: Blake
authors.initials: B
authors.lastname: Farrow
authors.affiliation: Sensors and Electron Devices Directorate, U.S. Army Research Laboratory, Adelphi, MD, 20783.
authors.forename: Amethist S
authors.initials: AS
authors.lastname: Finch
authors.affiliation: Indi Molecular, 6162 Bristol Parkway, Culver City, CA, 90230.
authors.forename: Heather D
authors.initials: HD
authors.lastname: Agnew
authors.affiliation: Division of Chemistry and Chemical Engineering, California Institute of Technology, 1200 East California Boulevard, Pasadena, CA, 91125.
authors.forename: James R
authors.initials: JR
authors.lastname: Heath
authors.affiliation: Sensors and Electron Devices Directorate, U.S. Army Research Laboratory, Adelphi, MD, 20783.
authors.forename: Dimitra N
authors.initials: DN
authors.lastname: Stratis-Cullum
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000008:administration & dosage / Q000737:chemistry / Q000378:metabolism; D000942:Antigens, Bacterial / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D001427:Bacterial Toxins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D002152:Calorimetry, Differential Scanning; D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D057930:Click Chemistry / Q000379:methods*; D003110:Colonic Neoplasms / Q000000981:diagnostic imaging / Q000378:metabolism; D005260:Female; D019073:HT29 Cells; D006801:Humans; D007274:Injections, Intraperitoneal; D007275:Injections, Intravenous; D008024:Ligands; D008297:Male; D013058:Mass Spectrometry; D051379:Mice; D008862:Microsomes, Liver / Q000378:metabolism; D019151:Peptide Library; D010455:Peptides / Q000737:chemistry* / Q000378:metabolism / Q000493:pharmacokinetics; D011485:Protein Binding; D014183:Transplantation, Heterologous; D042461:Vascular Endothelial Growth Factor A / Q000737:chemistry* / Q000378:metabolism
chemical_list: D000906:Antibodies; D000942:Antigens, Bacterial; D001427:Bacterial Toxins; D008024:Ligands; D019151:Peptide Library; D010455:Peptides; D042461:Vascular Endothelial Growth Factor A; C030325:anthrax toxin
keywords: biological stability; peptide; protective antigen; protein catalyzed capture agent; synthetic antibody; thermal stability; vascular endothelial growth factor