Effectiveness of [⁶⁷Cu]Cu-trastuzumab as a theranostic against HER2-positive breast cancer

Purpose To evaluate the imaging and therapeutic properties (theranostic) of Cu-67-labeled anti-human epidermal growth factor receptor II (HER2) monoclonal antibody trastuzumab against HER2-positive breast cancer (BC). Methods We conjugated trastuzumab with p-SCN-Bn-NOTA, 3p-C-NETA-NCS, or p-SCN-Bn-DOTA, and radiolabeled with [Cu-67]CuCl2. Immunoconjugate internalization was evaluated in BT-474, JIMT-1 and MCF-7 BC cells. In vitro stability was studied in human serum (HS) and Phosphate Buffered Saline (PBS). Flow cytometry, radioligand binding and immunoreactive fraction assays were carried out. ImmunoSPECT imaging of [Cu-67]Cu-NOTA-trastuzumab was done in mice bearing BT-474, JIMT-1 and MCF-7 xenografts. Pharmacokinetic was studied in healthy Balb/c mice while dosimetry was done in both healthy Balb/c and in athymic nude mice bearing JIMT-1 xenograft. The therapeutic effectiveness of [Cu-67]Cu-NOTA-trastuzumab was evaluated in mice bearing BT-474 and JIMT-1 xenografts after a single intravenous (i.v.) injection of similar to 16.8 MBq. Results Pure immunoconjugates and radioimmunoconjugates (> 95%) were obtained. Internalization was HER2 density-dependent with highest internalization observed with NOTA-trastuzumab. After 5 days, in vitro stabilities were 97 +/- 1.7%, 31 +/- 6.2%, and 28 +/- 4% in HS, and 79 +/- 3.5%, 94 +/- 1.2%, and 86 +/- 2.3% in PBS for [Cu-67]Cu-NOTA-trastuzumab, [Cu-67]Cu-3p-C-NETA-trastuzumab and [Cu-67]Cu-DOTA-trastuzumab, respectively. [Cu-67]Cu-NOTA-trastuzumab was chosen for further evaluation. BT-474 flow cytometry showed low K-D, 8.2 +/- 0.2 nM for trastuzumab vs 26.5 +/- 1.6 nM for NOTA-trastuzumab. There were 2.9 NOTA molecules per trastuzumab molecule. Radioligand binding assay showed a low K-D of 2.1 +/- 0.4 nM and immunoreactive fraction of 69.3 +/- 0.9. Highest uptake of [Cu-67]Cu-NOTA-trastuzumab was observed in JIMT-1 (33.9 +/- 5.5% IA/g) and BT-474 (33.1 +/- 10.6% IA/g) xenograft at 120 h post injection (p.i.). Effectiveness of the radioimmunoconjugate was also expressed as percent tumor growth inhibition (%TGI). [Cu-67]Cu-NOTA-trastuzumab was more effective than trastuzumab against BT-474 xenografts (78% vs 54% TGI after 28 days), and JIMT-1 xenografts (90% vs 23% TGI after 19 days). Mean survival of [Cu-67]Cu-NOTA-trastuzumab, trastuzumab and saline treated groups were > 90, 77 and 72 days for BT-474 xenografts, while that of JIMT-1 were 78, 24, and 20 days, respectively. Conclusion [Cu-67]Cu-NOTA-trastuzumab is a promising theranostic agent against HER2-positive BC.