Comparative Preclinical Evaluation of HYNIC-Modified Designed Ankyrin Repeat Proteins G3 for the ^99mTc-Based Imaging of HER2-Expressing Malignant Tumors

HER2 status determination is a necessary step for the proper choice of therapy and selection of patients for the targeted treatment of cancer. Targeted radiotracers such as radiolabeled DARPins provide a noninvasive and effective way for the molecular imaging of HER2 expression. This study aimed to evaluate tumor-targeting properties of three Tc-99m-labeled DARPin G3 variants containing Gly-Gly-Gly-Cys (G(3)C), (Gly-Gly-Gly-Ser)(3)-Cys ((G(3)S)(3)C), or Glu-Glu-Glu-Cys (E3C) amino acid linkers at the C-terminus and conjugated to the HYNIC chelating agent, as well as to compare them with the clinically evaluated DARPin G3 labeled with Tc-99m(CO)(3) using the (HE)(3)-tag at the N-terminus. The labeling of DARPin G3-HYNIC variants provided radiochemical yields in the range of 50-80%. Labeled variants bound specifically to human HER2-expressing cancer cell lines with affinities in the range of 0.5-3 nM. There was no substantial influence of the linker and HYNIC chelator on the binding of Tc-99m-labeled DARPin G3 variants to HER2 in vitro; however, [Tc-99m]Tc-G3-(G(3)S)(3)C-HYNIC had the highest affinity. Comparative biodistribution of [Tc-99m]Tc-G3-G(3)C-HYNIC, [Tc-99m]Tc-G3-(G(3)S)(3)C-HYNIC, [Tc-99m]Tc-G3-E3C-HYNIC, and [Tc-99m]Tc-(HE)(3)-G3 in healthy CD1 mice showed that there was a strong influence of the linkers on uptake in normal tissues. [Tc-99m]Tc-G3-E3C-HYNIC had an increased retention of activity in the liver and the majority of other organs compared to the other conjugates. The tumor uptake of [Tc-99m]Tc-G3-(G(3)S)(3)C-HYNIC and [Tc-99m]Tc-(HE)(3)-G3 in Nu/j mice bearing SKOV-3 xenografts was similar. The specificity of tumor targeting in vivo was demonstrated for both tracers. [Tc-99m]Tc-G3-(G(3)S)(3)C-HYNIC provided comparable, although slightly lower tumor-to-lung, tumor-to spleen and tumor-to-liver ratios than [Tc-99m]Tc-(HE)(3)-G3. Radiolabeling of DARPin G3-HYNIC conjugates with Tc-99m provided the advantage of a single-step radiolabeling procedure; however, the studied HYNIC conjugates did not improve imaging contrast compared to the Tc-99m-tricarbonyl-labeled DARPin G3. At this stage, [Tc-99m]Tc-(HE)(3)-G3 remains the most promising candidate for the clinical imaging of HER2-overexpressing cancers.