Evaluation of affibody molecule-based PNA-mediated radionuclide pretargeting: Development of an optimized conjugation protocol and 177Lu labeling.

INTRODUCTION: 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, ZHER2:342-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. METHODS: 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. RESULTS AND CONCLUSION: Using an optimized production protocol for ZHER2:342-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 ZHER2:342-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 ZHER2:342-SR-HP1. Without pre-injection of ZHER2:342-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 ZHER2:342-SR-HP1. In conclusion, 177Lu-HP2 was shown to be a promising secondary agent for affibody-mediated tumor pretargeting in vivo.

Influence of the N-Terminal Composition on Targeting Properties of Radiometal-Labeled Anti-HER2 Scaffold Protein ADAPT6.

Radionuclide-imaging-based stratification of patients to targeted therapies makes cancer treatment more personalized and therefore more efficient. Albumin-binding domain derived affinity proteins (ADAPTs) constitute a novel group of imaging probes based on the scaffold of an albumin-binding domain (ABD). To evaluate how different compositions of the N-terminal sequence of ADAPTs influence their biodistribution, a series of human epidermal growth factor receptor type 2 (HER2)-binding ADAPT6 derivatives with different N-terminal sequences were created: GCH6DANS (2), GC(HE)3DANS (3), GCDEAVDANS (4), and GCVDANS(5). These were compared with the parental variant: GCSS(HE)3DEAVDANS (1). All variants were site-specifically conjugated with a maleimido-derivative of a DOTA chelator and labeled with 111In. Binding to HER2-expressing cells in vitro, in vivo biodistribution as well as targeting properties of the new variants were compared with properties of the 111In-labeled parental ADAPT variant 1 (111In-DOTA-1). The composition of the N-terminal sequence had an apparent influence on biodistribution of ADAPT6 in mice. The use of a hexahistidine tag in 111In-DOTA-2 was associated with elevated hepatic uptake compared to the (HE)3-containing counterpart, 111In-DOTA-3. All new variants without a hexahistidine tag demonstrated lower uptake in blood, lung, spleen, and muscle compared to uptake in the parental variant. The best new variants, 111In-DOTA-3 and 111In-DOTA-5, provided tumor uptakes of 14.6 ± 2.4 and 12.5 ± 1.3% ID/g at 4 h after injection, respectively. The tumor uptake of 111In-DOTA-3 was significantly higher than the uptake of the parental 111In-DOTA-1 (9.1 ± 2.0% ID/g). The tumor-to-blood ratios of 395 ± 75 and 419 ± 91 at 4 h after injection were obtained for 111In-DOTA-5 and 111In-DOTA-3, respectively. In conclusion, the N-terminal sequence composition affects the biodistribution and targeting properties of ADAPT-based imaging probes, and its optimization may improve imaging contrast.