Plug-and-play nucleic acid-mediated multimerization of biparatopic nanobodies for molecular imaging

• Published in: Molecular therapy. Nucleic acids 2024-09, Vol.35 (3), p.102305, Article 102305
• Main Authors: Teodori, Laura, Ochoa, Sarah K., Omer, Marjan, Andersen, Veronica L., Bech, Pernille, Su, Junyi, Bridoux, Jessica, Nielsen, Jesper S., Bertelsen, Mathias B., Hernot, Sophie, Gothelf, Kurt V., Kjems, Jørgen
• Format: Article
• Language: English
• Subjects: biparatopic; breast cancer; molecular imaging; MT: Oligonucleotides: Diagnostics and Biosensors; multimerization; nanobodies; nanostructure; nucleic acid; Original; single-domain antibodies; theranostic platform; tumor targeting
• ISSN: 2162-2531; 2162-2531
• DOI: 10.1016/j.omtn.2024.102305

In cancer molecular imaging, selecting binders with high specificity and affinity for biomarkers is paramount for achieving high-contrast imaging within clinical time frames. Nanobodies have emerged as potent candidates, surpassing antibodies in pre-clinical imaging due to their convenient production, rapid renal clearance, and deeper tissue penetration. Multimerization of nanobodies is a popular strategy to enhance their affinity and pharmacokinetics; however, traditional methods are laborious and may yield heterogeneous products. In this study, we employ a Holliday junction (HJ)-like nucleic acid-based scaffold to create homogeneous nanostructures with precise multivalent and multiparatopic nanobody displays. The plug-and-play assembly allowed the screening of several nanobody multimer configurations for the detection of the breast cancer biomarker, human epidermal growth factor receptor 2 (HER2). In vitro studies demonstrated significant improvements in binding avidity, particularly with the biparatopic construct exhibiting high sensitivity, surpassing that of traditional antibody-based cell binding. Furthermore, our HJ platform allowed for adaptation from fluorescence-based to nuclear imaging, as demonstrated in xenografted mice, thereby allowing for future in vivo applications. This work highlights the potential of nucleic acid-mediated multimerization to markedly enhance nanobody binding, by exploring synergistic combinations and offering versatility for both in vitro diagnostics and cancer molecular imaging with prospects for future theranostic applications. [Display omitted] Kjems and colleagues aim to enhance sensitivity and signal-to-noise ratio in molecular imaging applications by employing a modular plug-and-play approach to combine nanobodies with different affinities toward HER2 on a nucleic acid-based Holliday junction scaffold. Results show enhanced affinity and performance across various detection systems in vitro and in vivo.