Optimal composition and position of histidine-containing tags improves biodistribution of (99m)Tc-labeled DARPin G3

Radionuclide molecular imaging of HER2 expression in disseminated cancer enables stratification of patients for HER2-targeted therapies. DARPin G3, a small (14 kDa) engineered scaffold protein, is a promising probe for imaging of HER2. We hypothesized that position (C- or N-terminus) and composition (hexahistidine or (HE)(3)) of histidine-containing tags would influence the biodistribution of [(99m)Tc]Tc(CO)(3)-labeled DARPin G3. To test the hypothesis, G3 variants containing tags at N-terminus (H(6)-G3 and (HE)(3)-G3) or at C-terminus (G3-H(6) and G3-(HE)(3)) were labeled with [(99m)Tc]Tc(CO)(3). Labeling yield, label stability, specificity and affinity of the binding to HER2, biodistribution and tumor targeting properties of these variants were compared side-by-side. There was no substantial influence of position and composition of the tags on binding of [(99m)Tc]Tc(CO)(3)-labeled variants to HER2. The specificity of HER2 targeting in vivo was confirmed. The tumor uptake in BALB/c nu/nu mice bearing SKOV3 xenografts was similar for all variants. On the opposite, there was a strong influence of the tags on uptake in normal tissues. The tumor-to-liver ratio for [(99m)Tc]Tc(CO)(3)-(HE)(3)-G3 was three-fold higher compared to the hexahistidine-tag containing variants. Overall, [(99m)Tc]Tc(CO)(3)-(HE)(3)-G3 variant provided the highest tumor-to-lung, tumor-to-liver, tumor-to-bone and tumor-to-muscle ratios, which should improve sensitivity of HER2 imaging in these common metastatic sites.