Amino Acids

, Volume 42, Issue 5, pp 1975–1985 | Cite as

Order of amino acids in C-terminal cysteine-containing peptide-based chelators influences cellular processing and biodistribution of 99mTc-labeled recombinant Affibody molecules

  • Mohamed Altai
  • Helena Wållberg
  • Anna Orlova
  • Maria Rosestedt
  • Seyed Jalal Hosseinimehr
  • Vladimir Tolmachev
  • Stefan Ståhl
Original Article


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.


Affibody molecule Technetium-99m Molecular imaging HER2 C-terminal cysteine Peptide-based chelator 



This research was financially supported by grants from the Swedish Cancer Society (Cancerfonden) and the Swedish Research Council (Vetenskapsrådet).


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Copyright information

© Springer-Verlag 2011

Authors and Affiliations

  • Mohamed Altai
    • 1
  • Helena Wållberg
    • 2
  • Anna Orlova
    • 1
  • Maria Rosestedt
    • 1
  • Seyed Jalal Hosseinimehr
    • 1
    • 3
  • Vladimir Tolmachev
    • 1
  • Stefan Ståhl
    • 2
  1. 1.Division of Biomedical Radiation Sciences, Department of Radiology, Oncology and Clinical Immunology, Rudbeck LaboratoryUppsala UniversityUppsalaSweden
  2. 2.Division of Molecular Biotechnology, School of Biotechnology, AlbaNova University CenterRoyal Institute of TechnologyStockholmSweden
  3. 3.Department of Radiopharmacy, Faculty of PharmacyMazandaran University of Medical SciencesSariIran

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