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

Area of Science:

• Biotechnology
• Molecular Imaging
• Radiopharmaceutical Therapy

Background:

• Designed Ankyrin Repeat Proteins (DARPins) are small, high-affinity engineered scaffold proteins (ESPs) with potential in diagnostics and therapy.
• Current DARPin radioimmunodiagnostics face limitations due to high kidney accumulation, hindering therapeutic applications.
• Peptide nucleic acid (PNA)-based pretargeting has shown success with Affibody molecules, suggesting applicability to other ESPs.

Purpose of the Study:

• To investigate the feasibility of adapting a PNA-based pretargeting strategy for DARPins.
• To develop and evaluate a pretargeting system using the HER2-targeting DARPin G3.
• To compare the efficacy of DARPin-based pretargeting with direct targeting and Affibody-based pretargeting.

Main Methods:

• Site-specific conjugation of PNA to DARPin G3 via sortase A-mediated ligation, creating the primary agent G3-HP1.
• Labeling of G3-HP1 with ¹²⁵I and a complementary PNA probe (HP2) with ¹⁷⁷Lu (containing DOTA chelator).
• In vitro cell binding assays, in vivo biodistribution, and targeting specificity studies in HER2-positive and negative xenograft models.

Main Results:

• ¹²⁵I-G3-HP1 exhibited specific binding to HER2-expressing cells with picomolar affinity.
• ¹⁷⁷Lu-HP2 demonstrated HER2-specific, PNA-dependent binding to pretreated cells with subnanomolar affinity.
• Pretargeting increased tumor uptake 8-fold and significantly reduced off-target accumulation in kidneys, liver, and spleen compared to direct targeting.
• DARPin- and Affibody-based pretargeting showed comparable biodistribution and tumor uptake.

Conclusions:

• Sortase A-mediated coupling successfully established a PNA-based pretargeting system for DARPin G3.
• This approach expands the utility of pretargeting to another class of engineered scaffold proteins.
• DARPin-based pretargeting offers a promising strategy for improved tumor targeting in radioimmunodiagnostics and therapy.