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

• Main Authors: Laura Teodori (Author), Sarah K. Ochoa (Author), Marjan Omer (Author), Veronica L. Andersen (Author), Pernille Bech (Author), Junyi Su (Author), Jessica Bridoux (Author), Jesper S. Nielsen (Author), Mathias B. Bertelsen (Author), Sophie Hernot (Author), Kurt V. Gothelf (Author), Jørgen Kjems (Author)
• Format: Book
• Published: Elsevier, 2024-09-01T00:00:00Z.
• Subjects: article
• Online Access: Connect to this object online.
• Notes: 2162-2531; 10.1016/j.omtn.2024.102305
• Keywords: MT: Oligonucleotides: Diagnostics and Biosensors; single-domain antibodies; nanobodies; nucleic acid; multimerization; nanostructure; Therapeutics. Pharmacology; RM1-950
• Source: Molecular Therapy: Nucleic Acids, Vol 35, Iss 3, Pp 102305- (2024)

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.