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Optimizing peptide nucleic acid-based pretargeting for enhanced targeted radionuclide therapy

Altai, Mohamed LU ; Nagy, Ábel ; Granit, Pauline ; Zedan, Wahed LU ; Cerezo-Magaña, Myriam LU orcid ; Park, Julie ; Lückerath, Katharina ; Geres, Susanne LU ; Sydoff, Marie LU and Thorek, Daniel L.J. , et al. (2025) In Journal of Controlled Release 381.
Abstract

Radiolabeled targeting agents have emerged as valuable tools for the treatment of disseminated cancer. Monoclonal antibodies (mAbs) are widely employed as carriers for diagnostic and therapeutic radionuclides due to their exceptional specificity and affinity. However, their prolonged circulatory half-life can diminish diagnostic efficacy and increase radiation exposure to non-target tissues in therapeutic applications, resulting in dose-limiting toxicities. To overcome this limitation, pretargeting technologies emerge as promising strategies to enhance tumor-to-background ratio and reduce radiation exposure of healthy tissues. Our previous work introduced a pretargeting concept leveraging the specific interaction between two peptide... (More)

Radiolabeled targeting agents have emerged as valuable tools for the treatment of disseminated cancer. Monoclonal antibodies (mAbs) are widely employed as carriers for diagnostic and therapeutic radionuclides due to their exceptional specificity and affinity. However, their prolonged circulatory half-life can diminish diagnostic efficacy and increase radiation exposure to non-target tissues in therapeutic applications, resulting in dose-limiting toxicities. To overcome this limitation, pretargeting technologies emerge as promising strategies to enhance tumor-to-background ratio and reduce radiation exposure of healthy tissues. Our previous work introduced a pretargeting concept leveraging the specific interaction between two peptide nucleic acid (PNA) probes, HP1 and HP2, as the recognition mechanism. This early iteration of the PNA-based concept showed limited efficacy when used with mAb-based vectors. To improve its performance, we re-engineered the primary and secondary targeting agents by incorporating newly designed PNA-probes. As the primary targeting agent, we functionalized trastuzumab (T), a well-characterized human epidermal growth factor receptor 2 (HER2)-targeting IgG1 mAb, with a 9-mer PNA probe (HP9). Both FcIII-based covalent UV-light crosslinking and enzyme-mediated glyco-engineering click-chemistry methods were applied to generate trastuzumab-PNA conjugates T-FcIII-HP9 and T-gly-HP9, respectively. As a radionuclide-carrying secondary agent, we utilized a 9-mer complementary PNA probe, HP16, which forms a stable duplex with HP9 as well as displaying favorable in vivo kinetics. Biacore and flow cytometry assessment of the HP9-conjugated trastuzumab agents demonstrated retained HER2-binding properties. The secondary HP16 probe, labeled with either a dye or a radionuclide, showed cell surface accumulation contingent on the presence of HP9 on the primary HER2-targeting agents. In vivo, T-gly-HP9 exhibited significantly longer blood circulation half-life and superior tumor uptake compared to T-FcIII-HP9. Further, therapeutic dosing with [177Lu]-HP16 of trastuzumab-HP9 pretargeted HER2+ tumor models resulted in significantly delayed disease progression and extended survival compared to untreated subjects. Furthermore, pretargeted [177Lu]-HP16 exhibited comparable efficacy to [177Lu]-trastuzumab in both delaying disease progression and prolonging survival. In conclusion, the optimization of our PNA-based pretargeting system has resulted in exceptional in vivo targeting characteristics and therapeutic efficacy, validating the potential of this novel approach.

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organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
Antibody, Peptide nucleic acid, Pretargeting, Radiotherapy, Site-specific labeling
in
Journal of Controlled Release
volume
381
article number
113551
publisher
Elsevier
external identifiers
  • scopus:85218886381
  • pmid:39986477
ISSN
0168-3659
DOI
10.1016/j.jconrel.2025.02.047
language
English
LU publication?
yes
id
91d593f1-34fc-44fc-8b48-4f4fcb27631b
date added to LUP
2025-06-18 13:27:37
date last changed
2025-07-30 17:15:22
@article{91d593f1-34fc-44fc-8b48-4f4fcb27631b,
  abstract     = {{<p>Radiolabeled targeting agents have emerged as valuable tools for the treatment of disseminated cancer. Monoclonal antibodies (mAbs) are widely employed as carriers for diagnostic and therapeutic radionuclides due to their exceptional specificity and affinity. However, their prolonged circulatory half-life can diminish diagnostic efficacy and increase radiation exposure to non-target tissues in therapeutic applications, resulting in dose-limiting toxicities. To overcome this limitation, pretargeting technologies emerge as promising strategies to enhance tumor-to-background ratio and reduce radiation exposure of healthy tissues. Our previous work introduced a pretargeting concept leveraging the specific interaction between two peptide nucleic acid (PNA) probes, HP1 and HP2, as the recognition mechanism. This early iteration of the PNA-based concept showed limited efficacy when used with mAb-based vectors. To improve its performance, we re-engineered the primary and secondary targeting agents by incorporating newly designed PNA-probes. As the primary targeting agent, we functionalized trastuzumab (T), a well-characterized human epidermal growth factor receptor 2 (HER2)-targeting IgG<sub>1</sub> mAb, with a 9-mer PNA probe (HP9). Both FcIII-based covalent UV-light crosslinking and enzyme-mediated glyco-engineering click-chemistry methods were applied to generate trastuzumab-PNA conjugates T-FcIII-HP9 and T-gly-HP9, respectively. As a radionuclide-carrying secondary agent, we utilized a 9-mer complementary PNA probe, HP16, which forms a stable duplex with HP9 as well as displaying favorable in vivo kinetics. Biacore and flow cytometry assessment of the HP9-conjugated trastuzumab agents demonstrated retained HER2-binding properties. The secondary HP16 probe, labeled with either a dye or a radionuclide, showed cell surface accumulation contingent on the presence of HP9 on the primary HER2-targeting agents. In vivo, T-gly-HP9 exhibited significantly longer blood circulation half-life and superior tumor uptake compared to T-FcIII-HP9. Further, therapeutic dosing with [<sup>177</sup>Lu]-HP16 of trastuzumab-HP9 pretargeted HER2+ tumor models resulted in significantly delayed disease progression and extended survival compared to untreated subjects. Furthermore, pretargeted [<sup>177</sup>Lu]-HP16 exhibited comparable efficacy to [<sup>177</sup>Lu]-trastuzumab in both delaying disease progression and prolonging survival. In conclusion, the optimization of our PNA-based pretargeting system has resulted in exceptional in vivo targeting characteristics and therapeutic efficacy, validating the potential of this novel approach.</p>}},
  author       = {{Altai, Mohamed and Nagy, Ábel and Granit, Pauline and Zedan, Wahed and Cerezo-Magaña, Myriam and Park, Julie and Lückerath, Katharina and Geres, Susanne and Sydoff, Marie and Thorek, Daniel L.J. and Westerlund, Kristina and Ulmert, David and Karlström, Amelie Eriksson}},
  issn         = {{0168-3659}},
  keywords     = {{Antibody; Peptide nucleic acid; Pretargeting; Radiotherapy; Site-specific labeling}},
  language     = {{eng}},
  publisher    = {{Elsevier}},
  series       = {{Journal of Controlled Release}},
  title        = {{Optimizing peptide nucleic acid-based pretargeting for enhanced targeted radionuclide therapy}},
  url          = {{http://dx.doi.org/10.1016/j.jconrel.2025.02.047}},
  doi          = {{10.1016/j.jconrel.2025.02.047}},
  volume       = {{381}},
  year         = {{2025}},
}