title: Evaluation of a HER2-targeting affibody molecule combining an N-terminal HEHEHE-tag with a GGGC chelator for 99mTc-labelling at the C terminus.
abstract: Affibody molecules are a class of small (ca.7 kDa) robust scaffold proteins with high potential as tracers for radionuclide molecular imaging in vivo. Incorporation of a cysteine-containing peptide-based chelator at the C terminus provides an opportunity for stable labelling with the radionuclide (99m)Tc. The use of a GGGC chelator at the C terminus has provided the lowest renal radioactivity retention of the previously investigated peptide-based chelators. Previously, it has also been demonstrated that replacement of the His(6)-tag with the negatively charged histidine-glutamate-histidine-glutamate-histidine-glutamate (HEHEHE)-tag permits purification of affibody molecules by immobilized metal ion affinity chromatography (IMAC) and provides low hepatic accumulation of radioactivity of conjugates site-specifically labelled at the C terminus using several different nuclides. We hypothesized that the combination of a HEHEHE-tag at the N terminus and a GGGC chelator at the C terminus of an affibody molecule would be a favourable format permitting IMAC purification and providing low uptake in excretory organs. To investigate this hypothesis, a (HE)(3)-Z(HER2:342)-GGGC affibody molecule was generated. It could be efficiently purified by IMAC and stably labelled with (99m)Tc. (99m)Tc-(HE)(3)-Z(HER2:342)-GGGC preserved specific binding to HER2-expressing cells. In NMRI mice, hepatic uptake of (99m)Tc-(HE)(3)-Z(HER2:342)-GGGC was lower than the uptake of the control affibody molecules, (99m)Tc-Z(HER2:2395)-VDC and (99m)Tc-Z(HER2:342)-GGGC. At 1 and 4 h after injection, the renal uptake of (99m)Tc-(HE)(3)-Z(HER2:342)-GGGC was 2-3-fold lower than uptake of (99m)Tc-Z(HER2:2395)-VDC, but it was substantially higher than uptake of (99m)Tc-Z(HER2:342)-GGGC. Further investigation indicated that a fraction of (99m)Tc was chelated by the HEHEHE-tag which caused a higher accumulation of radioactivity in the kidneys. Thus, a combination of a HEHEHE-tag and the GGGC chelator in targeting scaffold proteins was found to be undesirable in the case of (99m)Tc labelling due to a partial loss of site-specificity of nuclide chelation.
authors.affiliation: Division of Molecular Biotechnology, School of Biotechnology, AlbaNova University Center, Royal Institute of Technology, Stockholm, Sweden.
authors.forename: Hanna
authors.initials: H
authors.lastname: Lindberg
authors.forename: Camilla
authors.initials: C
authors.lastname: Hofström
authors.forename: Mohamed
authors.initials: M
authors.lastname: Altai
authors.forename: Hadis
authors.initials: H
authors.lastname: Honorvar
authors.forename: Helena
authors.initials: H
authors.lastname: Wållberg
authors.forename: Anna
authors.initials: A
authors.lastname: Orlova
authors.forename: Stefan
authors.initials: S
authors.lastname: Ståhl
authors.forename: Torbjörn
authors.initials: T
authors.lastname: Gräslund
authors.forename: Vladimir
authors.initials: V
authors.lastname: Tolmachev
mesh_terms: D000818:Animals; D000918:Antibody Specificity / Q000276:immunology; D002614:Chelating Agents / Q000737:chemistry; D051379:Mice; D057054:Molecular Imaging; D015609:Organotechnetium Compounds / Q000737:chemistry* / Q000378:metabolism / Q000493:pharmacokinetics*; D011485:Protein Binding; D055550:Protein Stability; D021381:Protein Transport; D019275:Radiopharmaceuticals / Q000737:chemistry* / Q000378:metabolism / Q000493:pharmacokinetics*; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000276:immunology* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000378:metabolism / Q000493:pharmacokinetics*; D013667:Technetium / Q000737:chemistry
chemical_list: D002614:Chelating Agents; D015609:Organotechnetium Compounds; D019275:Radiopharmaceuticals; D018719:Erb-b2 Receptor Tyrosine Kinases; D011993:Recombinant Fusion Proteins; D013667:Technetium; C573342:99mTc-(HE)3-Z(HER-342)-GGGC; C515865:Z(HER2.4)2 affibody