In vitro cytotoxicity of Auger electron-emitting [(67)Ga]Ga-trastuzumab

INTRODUCTION: Molecular radiotherapy exploiting short-range Auger electron-emitting radionuclides has potential for targeted cancer treatment and, in particular, is an attractive option for managing micrometastatic disease. Here, an approach using chelator-trastuzumab conjugates to target radioactivity to breast cancer cells was evaluated as a proof-of-concept to assess the suitability of (67)Ga as a therapeutic radionuclide. METHODS: THP-trastuzumab and DOTA-trastuzumab were synthesised and radiolabelled with Auger electron-emitters (67)Ga and (111)In, respectively. Radiopharmaceuticals were tested for HER2-specific binding and internalisation, and their effects on viability (dye exclusion) and clonogenicity of HER2-positive HCC1954 and HER2–negative MDA-MB-231 cell lines was measured. Labelled cell populations were studied by microautoradiography. RESULTS: Labelling efficiencies for [(67)Ga]Ga-THP-trastuzumab and [(111)In]In-DOTA-trastuzumab were 90% and 98%, respectively, giving specific activities 0.52 ± 0.16 and 0.61 ± 0.11 MBq/μg (78–92 GBq/μmol). At 4 nM total antibody concentration and 200 × 10(3) cells/mL, [(67)Ga]Ga-THP-trastuzumab showed higher percentage of cell association (10.7 ± 1.3%) than [(111)In]In-DOTA-trastuzumab (6.2 ± 1.6%; p = 0.01). The proportion of bound activity that was internalised did not differ significantly for the two tracers (62.1 ± 1.4% and 60.8 ± 15.5%, respectively). At 100 nM, percentage cell binding of both radiopharmaceuticals was greatly reduced compared to 4 nM and did not differ significantly between the two (1.2 ± 1.0% [(67)Ga]Ga-THP-trastuzumab and 0.8 ± 0.9% for [(111)In]In-DOTA-trastuzumab). Viability and clonogenicity of HER2-positive cells decreased when each radionuclide was incorporated into cells by conjugation with trastuzumab, but not when the same level of radioactivity was confined to the medium by omitting the antibody conjugation, suggesting that (67)Ga needs to be cell-bound or internalised for a therapeutic effect. Microautoradiography showed that radioactivity bound to individual cells varied considerably within the population. CONCLUSIONS: [(67)Ga]Ga-THP-trastuzumab reduced cell viability and clonogenicity only when cell-bound, suggesting (67)Ga holds promise as a therapeutic radionuclide as part of a targeted radiopharmaceutical. The causes and consequences of non-homogeneous uptake among the cell population should be explored.