title: Influence of an aliphatic linker between DOTA and synthetic Z(HER2:342) Affibody molecule on targeting properties of the (111)In-labeled conjugate.
abstract: BACKGROUND
  Affibody molecules are small (∼6.5 kDa) scaffold proteins suitable for radionuclide imaging of tumor-associated molecular targets. Site-specific labeling of Affibody molecules made by peptide synthesis can be achieved by coupling a chelator to N-terminus in the last synthesis step. The goal of this study was to evaluate the influence of a 6-aminohexanoic linker between DOTA and Z(HER2:342) on targeting properties of (111)In-labeled conjugate.
  
  
  METHODS
  A DOTA-conjugated 6-aminohexanoic linker-containing variant of Z(HER2:342) (ABY-003) was produced by peptide synthesis, and the in vitro binding affinity, specificity and cellular processing were evaluated. The biodistribution of (111)In-ABY-003 in normal mice was compared to (111)In-ABY-002 (DOTA-Z(HER2:342-pep2)) lacking the linker. Tumor-targeting properties of (111)In-ABY-003 were evaluated in mice bearing HER2-expressing xenografts.
  
  
  RESULTS
  The dissociation constant of ABY-003 was in the low picomolar range, slightly higher than for ABY-002. (111)In-ABY-003 bound specifically to HER2-expressing cells in vitro. The cellular retention was efficient but slightly worse than for (111)In-ABY-002. In normal mice, the clearance of (111)In-ABY-003 from blood and other tissues was slightly but significantly faster compared to (111)In-ABY-002. Targeting of HER2-expressing xenografts by (111)In-ABY-003 was receptor-specific. Due to faster clearance, the tumor-to-blood ratio for (111)In-ABY-003 at 4 h postinjection was improved compared to (111)In-ABY-002. The capacity of (111)In-ABY-003 to visualize HER2-expressing tumors was confirmed by gamma camera imaging.
  
  
  CONCLUSIONS
  A 6-aminohexanoic linker between the DOTA chelator and N-terminus of synthetic Z(HER2:342) had a measurable effect on affinity, cellular retention of radioactivity and blood clearance. The linker might be used for modulation of targeting properties of Affibody molecules.
authors: Division of Biomedical Radiation Sciences, Department of Radiology, Oncology and Clinical Immunology, Rudbeck Laboratory, Uppsala University, SE-751 85 Uppsala, Sweden. vladimir.tolmachev@bms.uu.se
authors: Vladimir
authors: V
authors: Tolmachev
authors: Joachim
authors: J
authors: Feldwisch
authors: Malin
authors: M
authors: Lindborg
authors: Barbro
authors: B
authors: Baastrup
authors: Mattias
authors: M
authors: Sandström
authors: Anna
authors: A
authors: Orlova
mesh_terms: D000595:Amino Acid Sequence; D015119:Aminocaproic Acid / Q000737:chemistry; D000818:Animals; D045744:Cell Line, Tumor; D002471:Cell Transformation, Neoplastic; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D006573:Heterocyclic Compounds, 1-Ring / Q000737:chemistry*; D006801:Humans; D007205:Indium Radioisotopes; D007553:Isotope Labeling; D051379:Mice; D008969:Molecular Sequence Data; D010051:Ovarian Neoplasms / Q000000981:diagnostic imaging / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D010455:Peptides / Q000737:chemistry; D011877:Radionuclide Imaging; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000378:metabolism* / Q000493:pharmacokinetics
chemical_list: D006573:Heterocyclic Compounds, 1-Ring; D007205:Indium Radioisotopes; D010455:Peptides; D011993:Recombinant Fusion Proteins; C533351:affibody (ZHER2-342)2; C071349:1,4,7,10-tetraazacyclododecane- 1,4,7,10-tetraacetic acid; D015119:Aminocaproic Acid