Products Cited in Publications Worldwide Shorter Peptide Nucleic Acid Probes Improve Affibody-Mediated Peptide Nucleic Acid-Based Pretargeting

Shorter Peptide Nucleic Acid Probes Improve Affibody-Mediated Peptide Nucleic Acid-Based Pretargeting

Kristina Westerlund; Maryam Oroujeni; Maxime Gestin; Jacob Clinton; Alia Hani Rosly; Hanna Tano; Anzhelika Vorobyeva; Anna Orlova; Amelie Eriksson Karlström; Vladimir Tolmachev

DOI:

Abstract

Affibody-mediated PNA-based pretargeting shows promise for HER2-expressing tumor radiotherapy. In our recent study, a 15-mer ZHER2:342-HP15 affibody-PNA conjugate, in combination with a shorter 9-mer [177Lu]Lu-HP16 effector , emerged as the most effective pretargeting strategy. It offered a superior tumor-to-kidney uptake ratio and more efficient tumor targeting compared to longer radiolabeled effector probes containing 12 or 15 complementary bases. To enhance the production efficiency of our pretargeting system, we here introduce even shorter 6-, 7-, and 8-mer secondary probes, designated as HP19, HP21, and HP20, respectively. We also explore the replacement of the original 15-mer Z-HP15 primary with shorter 12-mer Z-HP12 and 9-mer Z-HP9 alternatives. This extended panel of shorter PNA-based probes was synthesized using automated microwave-assisted methods and biophysically screened in vitro to identify shorter combinations with the most effective binding properties. In a mouse xenograft model, we evaluated the biodistribution of these probes, comparing them to the Z-HP15:[177Lu]Lu-HP16 combination. Tumor-to-kidney ratios at 4 and 144 h postinjection of the secondary showed no significant differences among the Z-HP9:[177Lu]Lu-HP16, Z-HP9:[177Lu]Lu-HP20, and the Z-HP15:[177Lu]Lu-HP16 pairs. Importantly, tumor uptake significantly exceeded, by several hundred-fold, that of most normal tissues, with kidney uptake being the critical organ for radiation therapy. This suggests that using a shorter 9-mer primary , Z-HP9, in combination with 9-mer HP16 or 8-mer HP20 secondary probes effectively targets tumors while minimizing the dose-limiting kidney uptake of radionuclide. In conclusion, the Z-HP9:HP16 and Z-HP9:HP20 combinations offer good prospects for both cost-effective production and efficient in vivo pretargeting of HER2-expressing tumors.

Keywords

affibody ; pretargeting ; nucleic acid ; radiotherapy

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