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

Hanna Lindberg, Camilla Hofström, Mohamed Altai, Hadis Honorvar, Helena Wållberg, Anna Orlova, Stefan Ståhl, Torbjörn Gräslund, Vladimir Tolmachev

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16 Citations (Scopus)


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-glutamatehistidine-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 99mTc. 99mTc-(HE) 3-Z HER2:342-GGGC preserved specific binding to HER2-expressing cells. In NMRI mice, hepatic uptake of 99mTc-(HE) 3-Z HER2:342-GGGC was lower than the uptake of the control affibody molecules, 99mTc-ZHER2:2395-VDC and 99mTc-Z HER2:342-GGGC. At 1 and 4 h after injection, the renal uptake of 99mTc-(HE) 3-Z HER2:342-GGGC was 2-3-fold lower than uptake of 99mTc-ZHER2:2395-VDC, but it was substantially higher than uptake of 99mTc-Z HER2:342-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.

Original languageEnglish
Pages (from-to)641-651
Number of pages11
JournalTumor Biology
Issue number3
Publication statusPublished - Jun 2012
Externally publishedYes


  • Affibody molecules
  • Biodistribution
  • GGGC chelator
  • HEHEHE-tag
  • Radionuclide molecular imaging
  • Technetium-99m

ASJC Scopus subject areas

  • Cancer Research

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