Designed Ankyrin Repeat Protein–Mediated Peptide Nucleic Acid–Based Pretargeting: A Proof-of-Principle Study

Pretargeting Proof of concept Ankyrin repeat
DOI: 10.2967/jnumed.125.269533 Publication Date: 2025-05-15T16:05:13Z
ABSTRACT
Designed ankyrin repeat proteins (DARPins) are a class of engineered scaffold (ESPs) with molecular weight approximately 15 kDa and picomolar affinity for tumor antigen targets. Proof-of-concept studies have demonstrated the potential DARPin radioimmunodiagnostics in humans. However, high accumulation activity kidneys limits their use conventional radionuclide therapy. A peptide nucleic acid (PNA)-based pretargeted approach was successfully applied to Affibody molecules, another ESP. We hypothesized that this method could also enable controlled conversion DARPins into pretargeting probes. In proof-of-principle study, we tested hypothesis using human epidermal growth factor receptor type 2 (HER2)-targeting G3 as model. Methods: performed site-specific coupling PNA sortase A-mediated ligation. The modified at C-terminus recognition sequence. GGG-modified hybridization probe (HP1) containing 15-base sequence attached A, creating primary agent G3-HP1. To evaluate cell binding specificity biodistribution, G3-HP1 labeled 125I. complementary PNA-based secondary HP2 DOTA chelator 177Lu. vitro were HER2-expressing lines. Biodistribution vivo targeting evaluated mice HER2-positive SKOV-3 HER2-negative Ramos xenografts. [177Lu]Lu-HP2 head-to-head compared molecule-mediated ZHER2:342-HP1 direct [177Lu]Lu-DOTA-G3. Results: [125I]I-G3-HP1 specific cells affinity. showed HER2-specific PNA-dependent G3-HP1-pretreated subnanomolar confirmed uptake [125I]I-G3-HP1. xenografts HER2-dependent PNA-mediated case preinjection. increased 8-fold Pretargeting substantially decreased (∼9-fold), liver (∼370-fold), spleen (6.5-fold). biodistribution strikingly similar cases Affibody- DARPin-based pretargeting. Conclusion: Sortase enables development system G3, expanding application ESPs.
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