Optimized preparation and preliminary evaluation of [[sup.64]Cu]-DOTA-trastuzumab for targeting ErbB2/Neu expression

Breast cancer radioimmunoscintigraphy targeting HER2/neu expression is a growing field of work in nuclear medicine research. Trastuzumab is a monoclonal antibody that binds with high affinity to HER2/neu, which is over expressed on breast and other tumors. Developing new tracers for the detection of this cancer is of great interest. In this study, trastuzumab was successively labeled with [[sup.64]Cu]Cu[Cl.sub.2] after conjugation with DOTA-NHS-ester. The conjugate was purified by molecular filtration, the average number of DOTA conjugated per mAb was calculated and total concentration was determined by spectrophotometric method. DOTA-trastuzumab was labeled with [sup.64]Cu produced by Zn(p,αn) Cu nuclear reaction (30 MeV protons at 180 µA). Radiochemical purity, integrity of protein after radiolabeling and immunoreactivity of radiolabeled mAb trastuzumab with HER2/ neu antigen and SkBr3 cell line were performed by RIA. In vitro stability of radiolabeled mAb in human serum was determined by thin layer chromatography. In vitro internalization studies were performed with the SkBr3 cell line and the tissue biodistribution of the [sup.64]Cu-DOTA-trastuzumab was evaluated in wild-type rat (90 ± 5.5 µCi, 2, 6, 12, 2 h p.i.). The radioimmunoconjugate was prepared with a radiochemical purity of higher than 96 ± 0.5 % (ITLC) and specific activity as high as 5.3 µCi/µg. The average number of chelators per antibody for the conjugate used in this study was 5.8/1. The sample was showed to have similar patterns of migration in the gel electrophoresis. The [sup.64]Cu-DOTA-trastuzumab showed high immunoreactivity towards HER2/neu antigen and SkBr3 cell line. In vitro stability of the labeled product was found to be more than 94 % in PBS and 82 ± 0.5 % in human serum over 48 h. In vitro internalization studies of the [sup.64]Cu-DOTA-trastuzumab showed that up to 11.5 % of the radioimmunoconjugate internalized after 10 h. The accumulation of the radiolabeled mAb in liver, skin, intestine, lung, spleen, kidney and other tissues demonstrates a similar pattern to the other radiolabeled anti-HER2 immunoconjugates. [sup.64]Cu-DOTA-trastuzumab is a potential compound for molecular imaging of PET for diagnosis and treatment studies and follow-up of HER2 expression in oncology. Keywords [sup.64]Cu * Trastuzumab * Radiolabeling * Conjugation * Biodistribution