title: Design, Preparation, and Characterization of PNA-Based Hybridization Probes for Affibody-Molecule-Mediated Pretargeting.
abstract: In radioimmunotherapy, the contrast between tumor and normal tissue can be improved by using a pretargeting strategy with a primary targeting agent, which is conjugated to a recognition tag, and a secondary radiolabeled molecule binding specifically to the recognition tag. The secondary molecule is injected after the targeting agent has accumulated in the tumor and is designed to have a favorable biodistribution profile, with fast clearance from blood and low uptake in normal tissues. In this study, we have designed and evaluated two complementary peptide nucleic acid (PNA)-based probes for specific and high-affinity association in vivo. An anti-HER2 Affibody-PNA chimera, Z(HER2:342)-SR-HP1, was produced by a semisynthetic approach using sortase A catalyzed ligation of a recombinantly produced Affibody molecule to a PNA-based HP1-probe assembled using solid-phase chemistry. A complementary HP2 probe carrying a DOTA chelator and a tyrosine for dual radiolabeling was prepared by solid-phase synthesis. Circular dichroism (CD) spectroscopy and UV thermal melts showed that the probes can hybridize to form a structured duplex with a very high melting temperature (T(m)), both in HP1:HP2 and in Z(HER2:342)-SR-HP1:HP2 (T(m) = 86-88 °C), and the high binding affinity between Z(HER2:342)-SR-HP1 and HP2 was confirmed in a surface plasmon resonance (SPR)-based binding study. Following a moderately fast association (1.7 × 10(5) M(-1) s(-1)), the dissociation of the probes was extremely slow and <5% dissociation was observed after 17 h. The equilibrium dissociation constant (K(D)) for Z(HER2:342)-SR-HP1:HP2 binding to HER2 was estimated by SPR to be 212 pM, suggesting that the conjugation to PNA does not impair Affibody binding to HER2. The biodistribution profiles of (111)In- and (125)I-labeled HP2 were measured in NMRI mice, showing very fast blood clearance rates and low accumulation of radioactivity in kidneys and other organs. The measured radioactivity in blood was 0.63 ± 0.15 and 0.41 ± 0.15%ID/g for (125)I- and (111)In-HP2, respectively, at 1 h p.i., and at 4 h p.i., the kidney accumulation of radioactivity was 0.17 ± 0.04%ID/g for (125)I-HP2 and 3.83 ± 0.39%ID/g for (111)In-HP2. Taken together, the results suggest that a PNA-based system has suitable biophysical and in vivo properties and is a promising approach for pretargeting of Affibody molecules.
authors.affiliation: †School of Biotechnology, Division of Protein Technology, KTH Royal Institute of Technology, 106 91 Stockholm, Sweden.
authors.forename: Kristina
authors.initials: K
authors.lastname: Westerlund
authors.affiliation: ‡Department of Immunology, Genetics and Pathology, Uppsala University, 751 05 Uppsala, Sweden.
authors.forename: Hadis
authors.initials: H
authors.lastname: Honarvar
authors.affiliation: ‡Department of Immunology, Genetics and Pathology, Uppsala University, 751 05 Uppsala, Sweden.
authors.forename: Vladimir
authors.initials: V
authors.lastname: Tolmachev
authors.affiliation: †School of Biotechnology, Division of Protein Technology, KTH Royal Institute of Technology, 106 91 Stockholm, Sweden.
authors.forename: Amelie
authors.initials: A
authors.lastname: Eriksson Karlström
mesh_terms: D000818:Animals; D002614:Chelating Agents / Q000737:chemistry; D015195:Drug Design; D005260:Female; D007205:Indium Radioisotopes / Q000493:pharmacokinetics; D007457:Iodine Radioisotopes / Q000493:pharmacokinetics; D007668:Kidney / Q000378:metabolism*; D051379:Mice; D009693:Nucleic Acid Hybridization; D015341:Nucleic Acid Probes / Q000737:chemistry* / Q000493:pharmacokinetics*; D020135:Peptide Nucleic Acids / Q000737:chemistry*; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000276:immunology / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000493:pharmacokinetics*; D060327:Solid-Phase Synthesis Techniques; D020349:Surface Plasmon Resonance; D014018:Tissue Distribution
chemical_list: D002614:Chelating Agents; D007205:Indium Radioisotopes; D007457:Iodine Radioisotopes; D015341:Nucleic Acid Probes; D020135:Peptide Nucleic Acids; D018719:Erb-b2 Receptor Tyrosine Kinases; D011993:Recombinant Fusion Proteins; C533351:affibody (ZHER2-342)2