Evaluation of an Anti-HER2 Nanobody Labeled with 225AC for Targeted alpha-Particle Therapy of Cancer

Human epidermal growth factor receptor type 2 (HER2) is overexpressed in numerous carcinomas. Nano bodies (Nbs) are the smallest antibody-derived fragments with beneficial characteristics for molecular imaging and radionudide therapy. Therefore, HER2-targeting nanobodies could offer a valuable platform for radioimmunotherapy, especially when labeled with a-particle emitters, which provide highly lethal and localized radiation to targeted cells with minimal exposure to surrounding healthy tissues. In this study, the anti-HER2 2Rs15d-nanobody was conjugated with 2-(4-isothiocyanatobenzyl)-1,4,7,10-tetraazacydododecane-1,4,7,10-tetraacetic acid (p-SCN-Bn-DOTA) and radiolabeled with an alpha-emitter Ac-225 with a high yield (>90%) and a radiochemical purity above 95%. The Ac-225-DOTA-Nb binding affinity was 4.12 0.47 nM with an immunoreactive fraction above 80%. Binding to low HER2-expressing MDA-MB-231 cells was negligible, whereas HER2-overexpressing SKOV-3 cells could be blocked with an excess of unlabeled nanobody, confirming the specificity of binding.,Noncompeting binding to HER2 was observed in the presence of an excess of trastuzumab. The cell-associated fraction of Ac-225-DOTA-Nb was 34.72 +/- 16.66% over 24 h. In vitro, the radioconjugate was toxic in an HER2-mediated and dose-dependent manner, resulting in IC50 values of 10.2 and 322.1 kBq/mL for Ac-225-DOTA-Nb and the Ac-225-DOTA control, respectively, on SKOV-3 cells, and 282.2 kBq/mL for Ac-225-DOTA-Nb on MDA-MB-231 cells. Ex vivo biodistribution studies, performed in mice bearing subcutaneous HER2-overexpressing and low HER2-expressing tumors, showed a fast uptake in SKOV-3 tumors compared to MDA-MB-231 (4.01 +/- 1.58% ID/g vs 0.49 +/- 0.20% ID/g after 2 h), resulting also in high tumor-to-normal tissue ratios. In addition, coinjection of Ac-225-DOTA-Nb with Gelofusine reduced kidney retention by 70%. This study shows that Ac-225-DOTA-Nb is a promising new radioconjugate for targeted alpha-partide therapy and supports its further development.