title: Increasing the Net Negative Charge by Replacement of DOTA Chelator with DOTAGA Improves the Biodistribution of Radiolabeled Second-Generation Synthetic Affibody Molecules.
abstract: A promising strategy to enable patient stratification for targeted therapies is to monitor the target expression in a tumor by radionuclide molecular imaging. Affibody molecules (7 kDa) are nonimmunoglobulin scaffold proteins with a 25-fold smaller size than intact antibodies. They have shown an apparent potential as molecular imaging probes both in preclinical and clinical studies. Earlier, we found that hepatic uptake can be reduced by the incorporation of negatively charged purification tags at the N-terminus of Affibody molecules. We hypothesized that liver uptake might similarly be reduced by positioning the chelator at the N-terminus, where the chelator-radionuclide complex will provide negative charges. To test this hypothesis, a second generation synthetic anti-HER2 ZHER2:2891 Affibody molecule was synthesized and labeled with (111)In and (68)Ga using DOTAGA and DOTA chelators. The chelators were manually coupled to the N-terminus of ZHER2:2891 forming an amide bond. Labeling DOTAGA-ZHER2:2891 and DOTA-ZHER2:2891 with (68)Ga and (111)In resulted in stable radioconjugates. The tumor-targeting and biodistribution properties of the (111)In- and (68)Ga-labeled conjugates were compared in SKOV-3 tumor-bearing nude mice at 2 h postinjection. The HER2-specific binding of the radioconjugates was verified both in vitro and in vivo. Using the DOTAGA chelator gave significantly lower radioactivity in liver and blood for both radionuclides. The (111)In-labeled conjugates showed more rapid blood clearance than the (68)Ga-labeled conjugates. The most pronounced influence of the chelators was found when they were labeled with (68)Ga. The DOTAGA chelator gave significantly higher tumor-to-blood (61 ± 6 vs 23 ± 5, p < 0.05) and tumor-to-liver (10.4 ± 0.6 vs 4.5 ± 0.5, p < 0.05) ratios than the DOTA chelator. This study demonstrated that chelators may be used to alter the uptake of Affibody molecules, and most likely other scaffold-based imaging probes, for improvement of imaging contrast.
authors.affiliation: Division of Protein Technology, School of Biotechnology, KTH Royal Institute of Technology, AlbaNova University Center , 106 91 Stockholm, Sweden.
authors.forename: Kristina
authors.initials: K
authors.lastname: Westerlund
authors.affiliation: Institute for Immunology, Genetics and Pathology, Uppsala University , 751 05 Uppsala, Sweden.
authors.forename: Hadis
authors.initials: H
authors.lastname: Honarvar
authors.affiliation: Division of Protein Technology, School of Biotechnology, KTH Royal Institute of Technology, AlbaNova University Center , 106 91 Stockholm, Sweden.
authors.forename: Emily
authors.initials: E
authors.lastname: Norrström
authors.affiliation: Institute for Immunology, Genetics and Pathology, Uppsala University , 751 05 Uppsala, Sweden.
authors.forename: Joanna
authors.initials: J
authors.lastname: Strand
authors.affiliation: Preclinical PET Platform, Department of Medicinal Chemistry, Uppsala University , 751 05 Uppsala, Sweden.
authors.forename: Bogdan
authors.initials: B
authors.lastname: Mitran
authors.affiliation: Preclinical PET Platform, Department of Medicinal Chemistry, Uppsala University , 751 05 Uppsala, Sweden.
authors.forename: Anna
authors.initials: A
authors.lastname: Orlova
authors.affiliation: Division of Protein Technology, School of Biotechnology, KTH Royal Institute of Technology, AlbaNova University Center , 106 91 Stockholm, Sweden.
authors.forename: Amelie
authors.initials: A
authors.lastname: Eriksson Karlström
authors.affiliation: Institute for Immunology, Genetics and Pathology, Uppsala University , 751 05 Uppsala, Sweden.
authors.forename: Vladimir
authors.initials: V
authors.lastname: Tolmachev
mesh_terms: D000577:Amides / Q000737:chemistry; D000818:Animals; D045744:Cell Line, Tumor; D002614:Chelating Agents / Q000737:chemistry*; D005260:Female; D005710:Gallium Radioisotopes / Q000737:chemistry; D006801:Humans; D007205:Indium Radioisotopes / Q000737:chemistry; D007553:Isotope Labeling / Q000379:methods; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D057054:Molecular Imaging / Q000379:methods; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism*; D011868:Radioisotopes / Q000737:chemistry; D019275:Radiopharmaceuticals / Q000737:chemistry*; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000378:metabolism; D014018:Tissue Distribution
chemical_list: D000577:Amides; D002614:Chelating Agents; D005710:Gallium Radioisotopes; D007205:Indium Radioisotopes; D011506:Proteins; D011868:Radioisotopes; D019275:Radiopharmaceuticals; D018719:Erb-b2 Receptor Tyrosine Kinases
keywords: Affibody molecule; DOTAGA; HER2; molecular imaging; radiolabeling