Evaluation of a Maleimido Derivative of NOTA for Site-Specific Labeling of Affibody Molecules

Radionuclide molecular imaging has the potential to improve cancer treatment by selection of patients for targeted therapy. Affibody molecules are a class of small (7 kDa) high-affinity targeting proteins with appreciable potential as molecular imaging probes. The NOTA chelator forms stable complexes with a number of radionuclides suitable for SPECT or PET imaging. A maleimidoethylmonoamide NOTA (MMA-NOTA) has been prepared for site-specific labeling of Affibody molecules having a unique C-terminal cysteine. Coupling of the MMA-NOTA to the anti-HER2 Affibody molecule Z HER2:2395 resulted in a conjugate with an affinity (dissociation constant) to HER2 of 72 pM. Labeling of [MMA-NOTA-Cys 61 ]-Z HER2:2395 with 111 In gave a yield of >95% after 20 min at 60 °C. In vitro cell tests demonstrated specific binding of [ 111 In-MMA-NOTA-Cys 61 ]-Z HER2:2395 to HER2-expressing cell lines. In mice bearing prostate cancer DU-145 xenografts, the tumor uptake of [ 111 In-MMA-NOTA-Cys 61 ]-Z HER2:2395 was 8.2 ± 0.9% IA/g and the tumor-to-blood ratio was 31 ± 1 (4 h postinjection). DU-145 xenografts were clearly visualized by a gamma camera. Direct in vivo comparison of [ 111 In-MMA-NOTA-Cys 61 ]-Z HER2:2395 and [ 111 In-MMA-DOTA-Cys 61 ]-Z HER2:2395 demonstrated that both conjugates provided equal radioactivity uptake in tumors, but the tumor-to-organ ratios were better for [ 111 In-MMA-NOTA-Cys 61 ]-Z HER2:2395 due to more efficient clearance from normal tissues. In conclusion, coupling of MMA-NOTA to a cysteine-containing Affibody molecule resulted in a site-specifically labeled conjugate, which retains high affinity, can be efficiently labeled, and allows for high-contrast imaging.