Targeting of HER2-Expressing Tumors with a Site-Specifically 99mTc-Labeled Recombinant Affibody Molecule, ZHER2:2395, with C-Terminally Engineered Cysteine

The detection of human epidermal growth factor receptor type 2 (HER2) expression in malignant tumors provides important information influencing patient management. Radionuclide in vivo imaging of HER2 may permit the detection of HER2 in both primary tumors and metastases by a single noninvasive procedure. Small (7 kDa) high-affinity anti-HER2 Affibody molecules may be suitable tracers for SPECT visualization of HER2-expressing tumors. The use of generator-produced Tc-99m as a label would facilitate the prompt translation of anti-HER2 Affibody molecules into use in clinics. Methods: A C-terminal cysteine was introduced into the Affibody molecule Z(HER2:342) to enable site-specific labeling with Tc-99m. Two recombinant variants, His(6)-Z(HER2:342)-Cys (dissociation constant [K-D], 29 pM) and Z(HER2:2395)-Cys, lacking a His tag (K-D, 27 pM), were labeled with Tc-99m in yields exceeding 90%. The binding specificity and the cellular processing of Affibody molecules were studied in vitro. Biodistribution and gamma-camera imaging studies were performed in mice bearing HER2-expressing xenografts. Results: Tc-99m-His(6)-Z(HER2:342)-Cys was capable of targeting HER2-expressing SKOV-3 xenografts in SCID mice, but the liver radioactivity uptake was high. A series of comparative biodistribution experiments indicated that the presence of the His tag caused elevated accumulation in the liver. Tc-99m-Z(HER2:2395)-Cys, not containing a His tag, showed low uptake in the liver and high and specific uptake in HER2-expressing xenografts. Four hours after injection, the radioactivity uptake values (percentage of injected activity per gram of tissue [%IA/g]) were 6.9 +/- 2.5 (mean +/- SD) %IA/g in LS174T xenografts (moderate level of HER2 expression) and 15 +/- 3%IA/g in SKOV-3 xenografts (high level of HER2 expression). The corresponding tumor-to-blood ratios were 88 +/- 24 and 121 +/- 24, respectively. Both LS174T and SKOV-3 xenografts were clearly visualized with a clinical gamma-camera 1 h after injection of Tc-99m-Z(HER2:2395)-Cys. Conclusion: The Affibody molecule Tc-99m-Z(HER2:2395)-Cys is a promising tracer for SPECT visualization of HER2-expressing tumors.