Comparative Evaluation of Novel 177Lu-Labeled PNA Probes for Affibody-Mediated PNA-Based Pretargeting

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.

Advances in the Application of Radionuclide-Labeled HER2 Affibody for the Diagnosis and Treatment of Ovarian Cancer

“…To further improve the radioactive uptake of molecular probes by tumors and reduce the radioactive retention of normal organs such as the kidneys, the research team further optimized the molecular probes in a series of studies in which they conducted comparative evaluations in vivo and in vitro . The results showed that the molecular probe [ 177 Lu]Lu-HP16, containing 9 nucleic acid bases, has the highest tumor/kidney uptake ratio and is a promising molecular probe for targeted therapy of HER2-positive ovarian cancer ( 121 – 123 ).…”

Cucurbituril–Ferrocene: Host–Guest Based Pretargeted Positron Emission Tomography in a Xenograft Model

“…Furthermore, in clinical trials of the bsAb-hapten platform, immunogenicity of the pretargeting agents has been an issue when anti-inflammatory drugs are not used as part of the pretargeting scheme. − The other two platforms that have been reported are based on streptavidin–biotin binding or oligonucleotide hybridization as the pretargeting interaction. The clinical viability of the streptavidin–biotin methodology is hindered by problems with immunogenicity and the need to use clearing agents. , Finally, the nucleotide hybridization driven pretargeting platform, studied only in preclinical models, has suffered from poor tumor uptake (%ID/g) when full-length antibodies are used for targeting. − While full-length antibodies are currently the primary and most attractive targeting vectors for pretargeting, the nucleotide hybridization approach has shown encouraging results with affibody targeting vectors, suggesting they represent a potentially fruitful alternative. − …”

Explorations into Peptide Nucleic Acid Contrast Agents as Emerging Scaffolds for Breakthrough Solutions in Medical Imaging and Diagnosis

“…As an alternative to the use of PNA chimeras, PNAs may also be used as motifs for supramolecular recognition in pretargeting approaches. ,, A schematic representation can be found in Figure . Pretargeting approaches typically rely on either bioorthogonal click reactions in vitro or in vivo, such as the inverse electron-demand Diels–Alder (IEDDA) reaction between trans -cyclooctene and tetrazine, or on the supramolecular recognition of a host and a guest, such as the interaction between adamantane and cyclodextrin .…”

“…This alleviates the need for developing endosomal escape strategies and allows one to focus on other challenges, like the overall biodistribution and excretion of unbound probes. Several authors already successfully demonstrated the use of such a strategy involving PNAs. , …”