In vivo evaluation of cysteine-based chelators for attachment of 99mTc to tumor-targeting affibody molecules

Thuy Tran, Torun Engfeldt, Anna Orlova, Charles Widström, Alexander Bruskin, Vladimir Tolmachev, Amelie Eriksson Karlström

Research output: Contribution to journalArticlepeer-review

57 Citations (Scopus)


Affibody molecules present a new class of affinity proteins, which utilizes a scaffold based on a 58-amino acid domain derived from protein A. The small (7 kDa) Affibody molecule can be selected to bind to cell-surface targets with high affinity. An Affibody molecule (ZHER2:342) with a dissociation constant (Kd) of 22 pM for binding to the HER2 receptor has been reported earlier. Preclinical and pilot clinical studies have demonstrated the utility of radiolabeled ZHER2:342 in imaging of HER2-expressing tumors. The small size and cysteine-free structure of Affibody molecules enable complete peptide synthesis and direct incorporation of radionuclide chelators. The goal of this study was to evaluate if incorporation of the natural peptide sequences cysteine-diglycine (CGG) and cysteine-triglycine (CGGG) sequences would enable labeling of Affibody molecules with 99mTc. In a model monomeric form, the chelating sequences were incorporated by peptide synthesis. The HER2-binding affinity was 280 and 250 pM for CGG-ZHER2:342 and CGGG-ZHER2:342, respectively. Conjugates were directly labeled with 99mTc with 90% efficiency and preserved the capacity to bind specifically to HER2-expressing cells. The biodistribution in normal mice showed a rapid clearance from the blood and the majority of organs (except kidneys). In the mice bearing SKOV-3 xenografts, tumor uptake of 99mTc-CGG- ZHER2:342 was HER2-specific and a tumor-to-blood ratio of 9.2 was obtained at 6 h postinjection. Gamma-camera imaging with 99mTc-CGG- ZHER2:342 clearly visualized tumors at 6 h postinjection. The results show that the use of a cysteine-based chelator enables 99mTc- labeling of Affibody molecules for imaging.

Original languageEnglish
Pages (from-to)549-558
Number of pages10
JournalBioconjugate Chemistry
Issue number2
Publication statusPublished - Mar 2007
Externally publishedYes

ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • Biomedical Engineering
  • Pharmacology
  • Pharmaceutical Science
  • Organic Chemistry

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