Detection of HER2-overexpression in
tumors and
metastases is important for the selection of patients who will benefit from
trastuzumab treatment. Earlier investigations showed successful imaging of HER2-positive
tumors in patients using
indium- or
gallium-labeled Affibody molecules. The goal of this study was to evaluate the use of (99m)Tc-labeled Affibody molecules for the detection of HER2 expression. The Affibody molecule Z(HER2:342) with the
chelator sequences
mercaptoacetyl-Gly-Glu-Gly (maGEG) and
mercaptoacetyl-Glu-Glu-Glu (
maEEE) was synthesized by
peptide synthesis and labeled with technetium-99m. Binding specificity, cellular retention, and in vitro stability were investigated. The biodistribution of (99m)Tc-maGEG-Z(HER2:342) and (99m)Tc-
maEEE-Z(HER2:342) was compared with (99m)Tc-maGGG-Z(HER2:342) in normal mice, and the
tumor targeting properties of (99m)Tc-
maEEE-Z(HER2:342) were determined in SKOV-3 xenografted nude mice. The results showed that the Affibody molecules were efficiently labeled with technetium-99m. The labeled conjugates were highly stable in vitro with preserved HER2-binding capacity. The use of
glutamic acid in the
chelator sequences for (99m)Tc-labeling of Z(HER2:342) reduced the hepatobiliary excretion 3-fold with a single Gly-to-Glu substitution and 10-fold with three Gly-to-Glu substitutions. (99m)Tc-
maEEE-Z(HER2:342) showed a receptor-specific
tumor uptake of 7.9 +/- 1.0 %IA/g and a
tumor-to-blood ratio of 38 at 4 h pi.
Gamma-camera imaging with (99m)Tc-
maEEE-Z(HER2:342) could detect HER2-expressing
tumors in xenografts already at 1 h pi. It was concluded that
peptide synthesis for the coupling of
chelator sequences to Affibody molecules for (99m)Tc labeling is an efficient way to modify the in vivo kinetics. Increased hydrophilicity, combined with improved stability of the mercaptoacetyl-triglutamyl
chelator, resulted in favorable biodistribution, making (99m)Tc-
maEEE-Z(HER2:342) a promising tracer for clinical imaging of HER2 overexpression in
tumors.