Preclinical evaluation of [111In]-DOTA-trastuzumab for clinical trials

Context: Herceptin and its fragments have been radiolabeled and used in the imaging of human epidermal growth factor receptor 2 (HER2)/neu-positive tumors and development of diagnostic kits is of great importance in radiopharmacy. Aims: In this study, In-111-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-trastuzumab (In-111-DOTA-trastuzumab) was successively prepared and evaluated for ultimate use in the HER2 antigen imaging in oncology. Settings and Design: The conjugate was prepared, labeled and evaluated using in vitro (radioimmunoassay [RIA], enzyme-linked immunosorbent assay (ELISA), stability, binding, internalization)/in vivo (bio-distribution, single-photon emission computed tomography [SPECT]) experiments. Materials and Methods: In-111-DOTA-trastuzumab was prepared followed by determination of radiochemical purity (RCP), integrity of protein, immunoreactivity of radiolabeled antibody with HER2/neu antigen (by SkBr3 cell line binding and RIA methods) were determined followed by stability tests, internalization studies and the tissue bio-distribution determination in wild-type rats as well as SPECT imaging in SkBr3-bearing mice. Statistical Analysis Used: All values were expressed as mean +/- standard deviation (mean +/- SD) and the data were compared using Student's t-test. Statistical significance was defined as P < 0.05. Results: In-111-DOTA-trastuzumab was prepared (RCP >95 +/- 0.5%, S. A. 5.3 mu Ci/mu g) with the average number of chelators per antibody of 6: 1 showing significant immune-reactivity retention using ELISA. In vitro stability was >90% in phosphate buffered saline and 80 +/- 0.5% in serum over 48 h. Cell binding was significant (>0.79). In vitro internalization reached up to %12-13 in 10 h. Significant tumor uptake was observed. Conclusions: In vitro and in vivo/SPECT imaging in SkBr3-bearing mice demonstrated that In-111-DOTA-trastuzumab is a potential compound for molecular imaging of SPECT for diagnosis and follow-up of HER2 expression in oncology.