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.