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

Abstract

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.

Keywords

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

Notes

Acknowledgments

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

References

  1. Ahlgren S, Tolmachev V (2010) Radionuclide molecular imaging using Affibody molecules. Curr Pharm Biotechnol 11:581–589PubMedCrossRefGoogle Scholar
  2. Ahlgren S, Orlova A, Rosik D, Sandström M, Sjöberg A, Baastrup B, Widmark O, Fant G, Feldwisch J, Tolmachev V (2008) Evaluation of maleimide derivative of DOTA for site-specific labeling of recombinant Affibody molecules. Bioconjug Chem 19:235–243PubMedCrossRefGoogle Scholar
  3. Ahlgren S, Wållberg H, Tran TA, Widström C, Hjertman M, Abrahmsén L, Berndorff D, Dinkelborg LM, Cyr JE, Feldwisch J, Orlova A, Tolmachev V (2009) Targeting of HER2-expressing tumors using a site-specifically 99mTc-labeled recombinant Affibody molecule ZHER2:2395 with C-terminal engineered cysteine. J Nucl Med 50:781–789PubMedCrossRefGoogle Scholar
  4. Ahlgren S, Orlova A, Wållberg H, Hansson M, Sandström M, Lewsley R, Wennborg A, Abrahmsén L, Tolmachev V, Feldwisch J (2010a) Targeting of HER2-expressing tumors using 111In-ABY-025, a second generation Affibody molecule with a fundamentally re-engineered scaffold. J Nucl Med 51:1131–1138PubMedCrossRefGoogle Scholar
  5. Ahlgren S, Andersson K, Tolmachev V (2010b) Kit formulation for 99mTc-labeling of recombinant anti-HER2 Affibody molecules with a C-terminally engineered cysteine. Nucl Med Biol 37:539–546PubMedCrossRefGoogle Scholar
  6. Baum RP, Prasad V, Müller D, Schuchardt C, Orlova A, Wennborg A, Tolmachev V, Feldwisch J (2010) Molecular imaging of HER2-expressing malignant tumors in breast cancer patients using synthetic 111In- or 68Ga-labeled Affibody. J Nucl Med 51:892–897PubMedCrossRefGoogle Scholar
  7. Britz-Cunningham SH, Adelstein SJ (2003) Molecular targeting with radionuclides: state of the science. J Nucl Med 44:1945–1961PubMedGoogle Scholar
  8. Cameron DA, Stein S (2008) Drug insight: intracellular inhibitors of HER2—clinical development of lapatinib in breast cancer. Nat Clin Pract Oncol 5:512–520PubMedCrossRefGoogle Scholar
  9. Chang HR (2010) Trastuzumab-based neoadjuvant therapy in patients with HER2-positive breast cancer. Cancer 116:2856–2867PubMedCrossRefGoogle Scholar
  10. Citri A, Yarden Y (2006) EGF-ERBB signalling: towards the systems level. Nat Rev Mol Cell Biol 7:505–516PubMedCrossRefGoogle Scholar
  11. Ekblad T, Tran T, Orlova A et al (2008) Development and preclinical characterisation of 99mTc-labelled Affibody molecules with reduced renal uptake. Eur J Nucl Med Mol Imaging 35:2245–2255PubMedCrossRefGoogle Scholar
  12. Ekblad T, Orlova A, Feldwisch J, Wennborg A, Eriksson Karlström A, Tolmachev V (2009) Positioning of 99mTc-chelators influences radiolabeling, stability and biodistribution of Affibody molecules. Bioorg Med Chem Lett 19:3912–3914PubMedCrossRefGoogle Scholar
  13. Engfeldt T, Orlova A, Tran T et al (2007a) Imaging of HER2-expressing tumours using a synthetic Affibody molecule containing the 99mTc-chelating mercaptoacetyl-glycyl-glycyl-glycyl (MAG3) sequence. Eur J Nucl Med Mol Imaging 34:722–733PubMedCrossRefGoogle Scholar
  14. Engfeldt T, Tran T, Orlova A, Widström Ch, Eriksson Karlström A, Tolmachev V (2007b) 99mTc-chelator engineering to improve tumour targeting properties of a HER2-specific Affibody molecule. Eur J Nucl Med Mol Imaging 34:1843–1853PubMedCrossRefGoogle Scholar
  15. Hynes NE, MacDonald G (2009) ErbB receptors and signaling pathways in cancer. Curr Opin Cell Biol 21:177–184PubMedCrossRefGoogle Scholar
  16. Löfblom J, Feldwisch J, Tolmachev V, Carlsson J, Ståhl S, Frejd FY (2010) Affibody molecules: engineered proteins for therapeutic, diagnostic and biotechnological applications. FEBS Lett 584:2670–2680PubMedCrossRefGoogle Scholar
  17. Mankoff DA (2009) Molecular imaging to select cancer therapy and evaluate treatment response. Q J Nucl Med Mol Imaging 53:181–192PubMedGoogle Scholar
  18. Miao Z, Levi J, Cheng Z (2010) Protein scaffold-based molecular probes for cancer molecular imaging. Amino Acids. doi: 10.1007/s00726-010-0503-9
  19. Nygren PA (2008) Alternative binding proteins: affibody binding proteins developed from a small three-helix bundle scaffold. FEBS J 275:2668–2676PubMedCrossRefGoogle Scholar
  20. Orlova A, Tolmachev V, Pehrson R, Lindborg M, Tran T, Sandström M, Nilsson FY, Wennborg A, Abrahmsén L, Feldwisch J (2007) Synthetic affibody molecules: a novel class of affinity ligands for molecular imaging of HER2-expressing malignant tumors. Cancer Res 67:2178–2186PubMedCrossRefGoogle Scholar
  21. Orlova A, Magnusson M, Eriksson T, Nilsson M, Larsson B, Höiden-Guthenberg I, Widström C, Carlsson J, Tolmachev V, Ståhl S, Nilsson F (2006) Tumor imaging using a picomolar affinity HER2 binding Affibody molecule. Cancer Res 66:4339–4348CrossRefGoogle Scholar
  22. Tolmachev V (2008) Imaging of HER-2 overexpression in tumors for guiding therapy. Curr Pharm Des 14:2999–3011PubMedCrossRefGoogle Scholar
  23. Tolmachev V, Nilsson FY, Widström C, Andersson K, Rosik D, Gedda L, Wennborg A, Orlova A (2006) 111In-benzyl-DTPA-ZHER2:342, an affibody-based conjugate for in vivo imaging of HER2 expression in malignant tumors. J Nucl Med 47:846–853PubMedGoogle Scholar
  24. Tolmachev V, Stone-Elander S, Orlova A (2010a) Current approaches to the use of radiolabeled tyrosine kinase-targeting drugs for patient stratification and treatment response monitoring: prospects and pitfalls. Lancet Oncol 11:992–1000PubMedCrossRefGoogle Scholar
  25. Tolmachev V, Hofström C, Malmberg J et al (2010b) HEHEHE-tagged affibody molecule may be purified by IMAC, is conveniently labeled with [99(m)Tc(CO)3](+), and shows improved biodistribution with reduced hepatic radioactivity accumulation. Bioconjug Chem 21:2013–2022PubMedCrossRefGoogle Scholar
  26. Tolmachev V, Velikyan I, Sandström M, Orlova A (2010c) A HER2-binding Affibody molecule labelled with 68Ga for PET imaging: direct in vivo comparison with the 111In-labelled analogue. Eur J Nucl Med Mol Imaging 37:1356–1367PubMedCrossRefGoogle Scholar
  27. Tran T, Engfeldt T, Orlova A et al (2007a) In vivo evaluation of cysteine-based chelators for attachment of 99mTc to tumor-targeting Affibody molecules. Biocojug Chem 18:549–558CrossRefGoogle Scholar
  28. Tran T, Engfeldt T, Orlova A et al (2007b) 99mTc-maEEE-ZHER2:342, an Affibody molecule-based tracer for detection of HER2-expression in malignant tumors. Bioconjug Chem 18:1956–1964PubMedCrossRefGoogle Scholar
  29. Tran T, Ekblad T, Orlova A et al (2008) Effects of Lysine-containing mercaptoacetyl-based Chelators on the Biodistribution of 99mTc-labeled anti-HER2. Bioconjug Chem 19:2568–2576PubMedCrossRefGoogle Scholar
  30. Tran TA, Rosik D, Abrahmsén L et al (2009) Design, synthesis and biological evaluation of a HER2-specific affibody molecule for molecular imaging. Eur J Nucl Med Mol Imaging 36:1864–1873PubMedCrossRefGoogle Scholar
  31. Wållberg H, Orlova A (2008) Slow internalization of anti-HER2 synthetic affibody monomer 111In-DOTA-ZHER2:342-pep2: implications for development of labeled tracers. Cancer Biother Radiopharm 23:435–442PubMedCrossRefGoogle Scholar
  32. Wållberg H, Ahlgren S, Widström C, Orlova A (2010) Evaluation of the radiocobalt-labeled [MMA-DOTA-Cys61]-Z HER2:2395(-Cys) affibody molecule for targeting of HER2-expressing tumors. Mol Imaging Biol 12:54–62PubMedCrossRefGoogle Scholar
  33. Wållberg H, Orlova A, Altai M, Widström C, Hosseinimehr SJ, Malmberg J, Ståhl S, Tolmachev V (2011a) Molecular design and optimization of 99mTc-labeled recombinant affibody molecules improves their biodistribution and imaging properties. J Nucl Med 52:461–469Google Scholar
  34. Wållberg H, Löfdahl PÅ, Tschapalda K, Uhlén M, Tolmachev V, Nygren PÅ, Ståhl S (2011b) Affinity recovery of eight HER2-binding affibody variants using an anti-idiotypic affibody ligand. Protein Exp Purif 76:127–135CrossRefGoogle Scholar

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

Personalised recommendations