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Evaluation of a HER2-targeting affibody molecule combining an N-terminal HEHEHE-tag with a GGGC chelator for 99mTc-labelling at the C terminus

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Tumor Biology

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 99mTc. 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 His6-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-ZHER2:342-GGGC affibody molecule was generated. It could be efficiently purified by IMAC and stably labelled with 99mTc. 99mTc-(HE)3-ZHER2:342-GGGC preserved specific binding to HER2-expressing cells. In NMRI mice, hepatic uptake of 99mTc-(HE)3-ZHER2:342-GGGC was lower than the uptake of the control affibody molecules, 99mTc-ZHER2:2395-VDC and 99mTc-ZHER2:342-GGGC. At 1 and 4 h after injection, the renal uptake of 99mTc-(HE)3-ZHER2:342-GGGC was 2–3-fold lower than uptake of 99mTc-ZHER2:2395-VDC, but it was substantially higher than uptake of 99mTc-ZHER2:342-GGGC. Further investigation indicated that a fraction of 99mTc 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 99mTc labelling due to a partial loss of site-specificity of nuclide chelation.

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Acknowledgements

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

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Authors and Affiliations

  1. Division of Molecular Biotechnology, School of Biotechnology, AlbaNova University Center, Royal Institute of Technology, Stockholm, Sweden

    Hanna Lindberg, Camilla Hofström, Helena Wållberg, Stefan Ståhl & Torbjörn Gräslund

  2. Biomedical Radiation Sciences, Rudbeck Laboratory, Uppsala University, 751 85, Uppsala, Sweden

    Mohamed Altai, Hadis Honorvar & Vladimir Tolmachev

  3. Department of Medicinal Chemistry, Preclinical PET Platform, Uppsala University, Uppsala, Sweden

    Anna Orlova

Authors
  1. Hanna Lindberg
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  2. Camilla Hofström
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  3. Mohamed Altai
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  4. Hadis Honorvar
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  5. Helena Wållberg
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  6. Anna Orlova
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  7. Stefan Ståhl
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  8. Torbjörn Gräslund
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  9. Vladimir Tolmachev
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Corresponding author

Correspondence to Vladimir Tolmachev.

Additional information

Hanna Lindberg and Camilla Hofström contributed equally to this study.

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Lindberg, H., Hofström, C., Altai, M. et al. Evaluation of a HER2-targeting affibody molecule combining an N-terminal HEHEHE-tag with a GGGC chelator for 99mTc-labelling at the C terminus. Tumor Biol. 33, 641–651 (2012). https://doi.org/10.1007/s13277-011-0305-z

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  • DOI: https://doi.org/10.1007/s13277-011-0305-z

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