Development of ⁵²Mn Labeled Trastuzumab for Extended Time Point PET Imaging of HER2

Due to their long circulation time in the blood, monoclonal antibodies (mAbs) such as trastuzumab, are usually radiolabeled with long-lived positron emitters for the development of agents for Positron Emission Tomography (PET) imaging. Manganese-52 (⁵²Mn, t_1/2 = 5.6 d, β⁺  = 29.6%, E(β_ave) = 242 keV) is suitable for imaging at longer time points providing a complementary technique to Zirconium-89 (⁸⁹Zr, t_1/2 = 3.3 d, β⁺  = 22.7%, E(β_ave) = 396 keV)) because of its long half-life and low positron energy. To exploit these properties, we aimed to investigate suitable bifunctional chelators that could be readily conjugated to antibodies and labeled with ⁵²Mn under mild conditions using trastuzumab as a proof-of-concept.

Trastuzumab was incubated with S-2-(4-isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane tetraacetic acid (p-SCN-Bn-DOTA), 1-Oxa-4,7,10-tetraazacyclododecane-5-S-(4-isothiocyantobenzyl)-4,7,10-triacetic acid (p-SCN-Bn-Oxo-DO3A), and 3,6,9,15-tetraazabicyclo[9.3.1] pentadeca-1(15),11,13-triene-4-S-(4-isothiocyanatobenzyl)-3,6,9-triacetic acid (p-SCN-Bn-PCTA) at a tenfold molar excess. The immunoconjugates were purified, combined with [⁵²Mn]MnCl₂ at different ratios, and the labeling efficiency was assessed by iTLC. The immunoreactive fraction of the radiocomplex was determined through a Lindmo assay. Cell studies were conducted in HER2 + (BT474) and HER2- (MDA-MB-468) cell lines followed by in vivo studies.

Trastuzumab-Oxo-DO3A was labeled within 30 min at 37 °C with a radiochemical yield (RCY) of 90 ± 1.5% and with the highest specific activity of the chelators investigated of 16.64 MBq/nmol. The labeled compound was purified with a resulting radiochemical purity of > 98% and retained a 67 ± 1.2% immunoreactivity. DOTA and PCTA immunoconjugates resulted in < 50 ± 2.5% (RCY) with similar specific activity. Mouse serum stability studies of [⁵²Mn]Mn-Oxo-DO3A-trastuzumab showed 95% intact complex for over 5 days. Cell uptake studies showed higher uptake in HER2 + (12.51 ± 0.83% /mg) cells compared to HER2- (0.85 ± 0.10%/mg) cells. PET images of mice bearing BT474 tumors showed high tumor uptake that was consistent with the biodistribution (42.02 ± 2.16%ID/g, 14 d) compared to MDA-MB-468 tumors (2.20 ± 0.80%ID/g, 14 d). Additionally, both models exhibited low bone uptake of < 1% ID/g.

The bifunctional chelator p-SCN-Bn-Oxo-DO3A is promising for the development of ⁵²Mn radiopharmaceuticals as it was easily conjugated, radiolabeled at mild conditions, and illustrated stability for a prolonged duration both in vitro and in vivo. High-quality PET/CT images of [⁵²Mn]Mn-Oxo-DO3A-trastuzumab were obtained 14 d post-injection. This study illustrates the potential of [⁵²Mn]Mn-Oxo-DO3A for the evaluation of antibodies using PET imaging.