Optimized preparation and preliminary evaluation of [ 64 Cu]–DOTA–trastuzumab for targeting ErbB2/Neu expression Behrooz Alirezapour • Amir Reza Jalilian • Mohammad Javad Rasaee • Saeed Rajabifar • Kamal Yavari • Mohsen Kamalidehghan • Fatemeh Bolourinovin • Gholamreza Aslani Received: 5 June 2012 Ó Akade ´miai Kiado ´, Budapest, Hungary 2012 Abstract Breast cancer radioimmunoscintigraphy tar- geting 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 [ 64 Cu]CuCl 2 after conjugation with DOTA- NHS-ester. The conjugate was purified by molecular fil- tration, the average number of DOTA conjugated per mAb was calculated and total concentration was determined by spectrophotometric method. DOTA–trastuzumab was labeled with 64 Cu produced by 68 Zn(p,an) 64 Cu nuclear reaction (30 MeV protons at 180 lA). Radiochemical purity, integrity of protein after radiolabeling and immu- noreactivity 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 inter- nalization studies were performed with the SkBr3 cell line and the tissue biodistribution of the 64 Cu–DOTA–trast- uzumab was evaluated in wild-type rat (90 ± 5.5 lCi, 2, 6, 12, 24 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 lCi/lg. 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 electropho- resis. The 64 Cu–DOTA–trastuzumab showed high immu- noreactivity 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 64 Cu–DOTA–trastuzumab showed that up to 11.5 % of the radioimmunoconjugate internalized after 10 h. The accu- mulation of the radiolabeled mAb in liver, skin, intestine, lung, spleen, kidney and other tissues demonstrates a similar pattern to the other radiolabeled anti-HER2 im- munoconjugates. 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 64 Cu  Trastuzumab  Radiolabeling  Conjugation  Biodistribution Introduction The HER2/neu antigen is a transmembrane receptor [1] overexpressed in 25–30 % of breast cancers [2]. The overexpression has been implicated in the carcinogenesis of breast cancer and is an independent prognostic indicator of survival in patients [3]. Trastuzumab is a humanized IgG1 monoclonal antibody (mAb) recognizing an epitope in the extracellular domain of the receptor and is used for immunotherapy for HER2/neu-positive tumors [4]. Breast cancer radioimmunoscintigraphy targeting HER2/ neu expression has been proposed by different research groups and could allow direct assessment of the receptor status of primary and metastatic lesions suggesting the B. Alirezapour  M. J. Rasaee Department of Clinical Biochemistry, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran A. R. Jalilian (&)  S. Rajabifar  K. Yavari  M. Kamalidehghan  F. Bolourinovin  G. Aslani Nuclear Science and Technology Research Institute (NSTRI), Tehran, Iran e-mail: jalili@aeoi.org.ir 123 J Radioanal Nucl Chem DOI 10.1007/s10967-012-1939-z