Comparative Evaluation of Novel 177Lu-Labeled PNA Probes for Affibody-Mediated PNA-Based Pretargeting
Hanna Tano1, Maryam Oroujeni2, Anzhelika Vorobyeva2,3
1Department of Protein Science, School of Engineering Sciences in Chemistry, Biotechnology and Health, KTH Royal Institute of Technology, 106 91 Stockholm, Sweden.
Abstract:
Affibody-mediated PNA-based pretargeting is a promising approach to radionuclide therapy of HER2-expressing tumors. In this study, we test the hypothesis that shortening the PNA pretargeting probes would increase the tumor-to-kidney dose ratio. The primary probe ZHER2:342-SR-HP15 and the complementary secondary probes HP16, HP17, and HP18, containing 9, 12, and 15 nucleobases, respectively, and carrying a 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) chelator were designed, synthesized, characterized in vitro, and labeled with 177Lu. In vitro pretargeting was studied in HER2-expressing SKOV3 and BT474 cell lines. The biodistribution of these novel probes was evaluated in immunodeficient mice bearing SKOV3 xenografts and compared to the previously studied [177Lu]Lu-HP2. Characterization confirmed the formation of high-affinity duplexes between HP15 and the secondary probes, with the affinity correlating with the length of the complementary PNA sequences. All the PNA-based probes were bound specifically to HER2-expressing cells in vitro. In vivo studies demonstrated HER2-specific uptake of all 177Lu-labeled probes in xenografts in a pretargeting setting. The ratio of cumulated radioactivity in the tumor to the radioactivity in kidneys was dependent on the secondary probe's size and decreased with an increased number of nucleobases. The shortest PNA probe, [177Lu]Lu-HP16, showed the highest tumor-to-kidney ratio. [177Lu]Lu-HP16 is the most promising secondary probe for affibody-mediated tumor pretargeting.
Insights
Shortening peptide nucleic acid (PNA) probes enhances radionuclide therapy for HER2-expressing tumors. The shortest PNA probe, [177Lu]Lu-HP16, demonstrated the highest tumor-to-kidney ratio, indicating its promise for affibody-mediated pretargeting.
Area of Science:
- Oncology
- Radiochemistry
- Molecular Biology
Background:
- Affibody-mediated peptide nucleic acid (PNA)-based pretargeting is a developing strategy for radionuclide therapy targeting human epidermal growth factor receptor 2 (HER2)-expressing tumors.
- Optimizing pretargeting probe design is crucial for improving therapeutic efficacy and minimizing off-target toxicity.
Purpose of the Study:
- To investigate the impact of PNA probe length on the tumor-to-kidney dose ratio in HER2-targeted radionuclide therapy.
- To evaluate novel PNA-based secondary probes for affibody-mediated pretargeting in vitro and in vivo.
Main Methods:
- Designed, synthesized, and characterized three PNA-based secondary probes (HP16, HP17, HP18) of varying lengths (9, 12, 15 nucleobases) conjugated to a DOTA chelator.
- Labeled probes with Lutetium-177 ([177Lu]) and assessed in vitro binding to HER2-expressing cell lines (SKOV3, BT474).
- Evaluated biodistribution and tumor-to-kidney ratios in mice bearing HER2-expressing xenografts.
Main Results:
- High-affinity duplex formation was confirmed between the primary probe (ZHER2:342-SR-HP15) and secondary probes, with affinity correlating to PNA length.
- [177Lu]-labeled PNA probes showed specific binding to HER2-expressing cells in vitro and HER2-specific uptake in xenografts in vivo.
- The tumor-to-kidney ratio was dependent on secondary probe size, decreasing with increased nucleobase count; the shortest probe, [177Lu]Lu-HP16, yielded the highest ratio.
Conclusions:
- Shorter PNA sequences in secondary probes improve the tumor-to-kidney ratio for affibody-mediated pretargeting.
- [177Lu]Lu-HP16 is identified as the most promising secondary probe for enhancing HER2-targeted radionuclide therapy efficacy.
- This study supports the optimization of PNA probe design for improved therapeutic outcomes in HER2-positive cancers.
