Incorporation of a Triglutamyl Spacer Improves the Biodistribution of Synthetic Affibody Molecules Radiofluorinated at the N-Terminus via Oxime Formation with 18F-4-Fluorobenzaldehyde

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 F-18 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 F-18-fluorobenzaldehyde (F-18-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 F-18-N-(4-fluorobenzylidine)oxime) (F-18-FBO)-labeled tracer. To verify this, we have produced two variants of the HER2-targeting Z(HER2:342) Affibody molecule by peptide synthesis: OA-PEP4313, where aminooxyacetic acid was conjugated directly to the N-terminal alanine, and OA-E-3-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 K-D = 49 pM to K-D = 180 pM. Radiolabeled F-18-FBO-E-3-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 F-18-FBO-E-3-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 F-18-FBA. It provides a tracer capable of producing high-contrast images of HER2-expressing tumors.