title: Order of amino acids in C-terminal cysteine-containing peptide-based chelators influences cellular processing and biodistribution of 99mTc-labeled recombinant Affibody molecules.
abstract: Affibody molecules constitute a novel class of molecular display selected affinity proteins based on non-immunoglobulin scaffold. Preclinical investigations and pilot clinical data have demonstrated that Affibody molecules provide high contrast imaging of tumor-associated molecular targets shortly after injection. The use of cysteine-containing peptide-based chelators at the C-terminus of recombinant Affibody molecules enabled site-specific labeling with the radionuclide 99mTc. Earlier studies have demonstrated that position, composition and the order of amino acids in peptide-based chelators influence labeling stability, cellular processing and biodistribution of Affibody molecules. To investigate the influence of the amino acid order, a series of anti-HER2 Affibody molecules, containing GSGC, GEGC and GKGC chelators have been prepared and characterized. The affinity to HER2, cellular processing of 99mTc-labeled Affibody molecules and their biodistribution were investigated. These properties were compared with that of the previously studied 99mTc-labeled Affibody molecules containing GGSC, GGEC and GGKC chelators. All variants displayed picomolar affinities to HER2. The substitution of a single amino acid in the chelator had an appreciable influence on the cellular processing of 99mTc. The biodistribution of all 99mTc-labeled Affibody molecules was in general comparable, with the main difference in uptake and retention of radioactivity in excretory organs. The hepatic accumulation of radioactivity was higher for the lysine-containing chelators and the renal retention of 99mTc was significantly affected by the amino acid composition of chelators. The order of amino acids influenced renal uptake of some conjugates at 1 h after injection, but the difference decreased at later time points. Such information can be helpful for the development of other scaffold protein-based imaging and therapeutic radiolabeled conjugates.
authors.affiliation: Division of Biomedical Radiation Sciences, Department of Radiology, Oncology and Clinical Immunology, Rudbeck Laboratory, Uppsala University, 75185, Uppsala, Sweden.
authors.forename: Mohamed
authors.initials: M
authors.lastname: Altai
authors.forename: Helena
authors.initials: H
authors.lastname: Wållberg
authors.forename: Anna
authors.initials: A
authors.lastname: Orlova
authors.forename: Maria
authors.initials: M
authors.lastname: Rosestedt
authors.forename: Seyed Jalal
authors.initials: SJ
authors.lastname: Hosseinimehr
authors.forename: Vladimir
authors.initials: V
authors.lastname: Tolmachev
authors.forename: Stefan
authors.initials: S
authors.lastname: Ståhl
mesh_terms: D000596:Amino Acids / Q000737:chemistry*; D000818:Animals; D002614:Chelating Agents / Q000737:chemistry; D003545:Cysteine / Q000737:chemistry*; D006801:Humans; D007553:Isotope Labeling; D051379:Mice; D057054:Molecular Imaging; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry*; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000737:chemistry*; D011993:Recombinant Fusion Proteins / Q000737:chemistry*; D013667:Technetium / Q000737:chemistry; D014018:Tissue Distribution
chemical_list: D000596:Amino Acids; D002614:Chelating Agents; D003545:Cysteine; D010455:Peptides; D018719:Erb-b2 Receptor Tyrosine Kinases; D011993:Recombinant Fusion Proteins; D013667:Technetium; C508053:ERBB2 protein, human