Incorporation of a triglutamyl spacer improves the biodistribution of synthetic affibody molecules radiofluorinated at the n-terminus via oxime formation with 18F-4-fluorobenzaldehyde

Daniel Rosik, Alf Thibblin, Gunnar Antoni, Hadis Honarvar, Joanna Strand, Ram Kumar Selvaraju, Mohamed Altai, Anna Orlova, Amelie Eriksson Karlström, Vladimir Tolmachev

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25 Citations (Scopus)


Affibody molecules are a class of affinity agents for molecular imaging based on a non-immunoglobulin protein scaffold. Previous studies have demonstrated high contrast for in vivo imaging of cancer-associated molecular abnormalities using Affibody molecules. Using the radionuclide 18F for labeling and PET as the imaging modality, the sensitivity of molecular imaging using Affibody molecules can be further increased. The use of oxime formation between an aminooxy-functionalized peptide and 18F- fluorobenzaldehyde (18F-FBA) is a promising way of radiolabeling of targeting peptides. However, previous studies demonstrated that application of this method to Affibody molecules is associated with high liver uptake. We hypothesized that incorporation of a triglutamyl spacer between the aminooxy moiety and the N-terminus of a synthetic Affibody molecule would decrease the hepatic uptake of the 18F-N-(4-fluorobenzylidine)oxime) ( 18F-FBO)-labeled tracer. To verify this, we have produced two variants of the HER2-targeting ZHER2:342 Affibody molecule by peptide synthesis: OA-PEP4313, where aminooxyacetic acid was conjugated directly to the N-terminal alanine, and OA-E3-PEP4313, where a triglutamyl spacer was introduced between the aminooxy moiety and the N-terminus. We have found that the use of the spacer is associated with a minor decrease of affinity, from KD = 49 pM to KD = 180 pM. Radiolabeled 18F-FBO-E3-PEP4313 demonstrated specific binding to HER2-expressing ovarian carcinoma SKOV-3 cells and slow internalization. Biodistribution studies in mice demonstrated that the use of a triglutamyl linker decreased uptake of radioactivity in liver 2.7-fold at 2 h after injection. Interestingly, radioactivity uptake in kidneys was also reduced (2.4-fold). Experiments in BALB/C nu/nu mice bearing SKOV-3 xenografts demonstrated HER2-specific uptake of 18F-FBO-E3-PEP4313 in tumors. At 2 h pi, the tumor uptake (20 ± 2% ID/g) exceeded uptake in liver 5-fold and uptake in kidneys 3.6-fold. The tumor-to-blood ratio was 21 ± 3. The microPET/CT imaging experiment confirmed the biodistribution data. In conclusion, the use of a triglutamyl spacer is a convenient way to improve the biodistribution profile of Affibody molecules labeled at the N-terminus using 18F-FBA. It provides a tracer capable of producing high-contrast images of HER2-expressing tumors.

Original languageEnglish
Pages (from-to)82-92
Number of pages11
JournalBioconjugate Chemistry
Issue number1
Publication statusPublished - 15 Jan 2014
Externally publishedYes

ASJC Scopus subject areas

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

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