Evaluation of affibody molecule-based PNA-mediated radionuclide pretargeting: Development of an optimized conjugation...

We have previously developed a pretargeting approach for affibody-mediated cancer therapy based on PNA-PNA hybridization. In this article we have further developed this approach by optimizing the production of the primary agent, Z_HER2:342-SR-HP1, and labeling the secondary agent, HP2, with the therapeutic radionuclide ¹⁷⁷Lu. We also studied the biodistribution profile of ¹⁷⁷Lu-HP2 in mice, and evaluated pretargeting with ¹⁷⁷Lu-HP2 in vitro and in vivo.

The biodistribution profile of ¹⁷⁷Lu-HP2 was evaluated in NMRI mice and compared to the previously studied ¹¹¹In-HP2. Pretargeting using ¹⁷⁷Lu-HP2 was studied in vitro using the HER2-expressing cell lines BT-474 and SKOV-3, and in vivo in mice bearing SKOV-3 xenografts.

Using an optimized production protocol for Z_HER2:342-SR-HP1 the ligation time was reduced from 15h to 30min, and the yield increased from 45% to 70%. ¹⁷⁷Lu-labeled HP2 binds specifically in vitro to BT474 and SKOV-3 cells pre-treated with Z_HER2:342-SR-HP1. ¹⁷⁷Lu-HP2 was shown to have a more rapid blood clearance compared to ¹¹¹In-HP2 in NMRI mice, and the measured radioactivity in blood was 0.22±0.1 and 0.68±0.07%ID/g for ¹⁷⁷Lu- and ¹¹¹In-HP2, respectively, at 1h p.i. In contrast, no significant difference in kidney uptake was observed (4.47±1.17 and 3.94±0.58%ID/g for ¹⁷⁷Lu- and ¹¹¹In-HP2, respectively, at 1h p.i.). Co-injection with either Gelofusine or lysine significantly reduced the kidney uptake for ¹⁷⁷Lu-HP2 (1.0±0.1 and 1.6±0.2, respectively, vs. 2.97±0.87%ID/g in controls at 4h p.i.). ¹⁷⁷Lu-HP2 accumulated in SKOV-3 xenografts in BALB/C nu/nu mice when administered after injection of Z_HER2:342-SR-HP1. Without pre-injection of Z_HER2:342-SR-HP1, the uptake of ¹⁷⁷Lu-HP2 was about 90-fold lower in tumor (0.23±0.08 vs. 20.7±3.5%ID/g). The tumor-to-kidney radioactivity accumulation ratio was almost 5-fold higher in the group of mice pre-injected with Z_HER2:342-SR-HP1. In conclusion, ¹⁷⁷Lu-HP2 was shown to be a promising secondary agent for affibody-mediated tumor pretargeting in vivo.