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

PURPOSE

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 (Mn, t = 5.6 d, β  = 29.6%, E(β) = 242 keV) is suitable for imaging at longer time points providing a complementary technique to Zirconium-89 (Zr, t = 3.3 d, β  = 22.7%, E(β) = 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 Mn under mild conditions using trastuzumab as a proof-of-concept.

PROCEDURES

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 [Mn]MnCl 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.

RESULTS

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 [Mn]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.

CONCLUSION

The bifunctional chelator p-SCN-Bn-Oxo-DO3A is promising for the development of Mn 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 [Mn]Mn-Oxo-DO3A-trastuzumab were obtained 14 d post-injection. This study illustrates the potential of [Mn]Mn-Oxo-DO3A for the evaluation of antibodies using PET imaging.