Abstract 7026: Nanobody-PROTACs: Method development towards using single-domain antibodies to target undruggable proteins for degradation

Abstract Proteolysis targeting chimeras (PROTACs) are bifunctional molecules that can bring an E3 ligase (e.g. VHL) into the vicinity of a protein of interest (POI). The E3 ligase poly-ubiquitinates the POI, marking it for degradation via a proteasome. Uniquely, PROTACs have the ability to bind to any chosen amino acid region, meaning there is tailored targeting of the POI, followed by subsequent degradation. This means that PROTACs have the potential to degrade targets previously considered ‘undruggable’, such as the oncogene, KRAS, which is historically difficult to therapeutically target due to lack of deep binding pockets. Typically, PROTACs utilize small molecules to target the POI, however, our work aims to uncover the potential of nanobodies to replace these small molecules. The gold standard for protein targeting remains monoclonal antibodies, however, nanobodies possess the same antigen binding site specificity, whilst possessing a tertiary structure only a tenth of the size. Our work focuses on identifying whether the nanobody-PROTAC has the potential for cellular entry, and thus degradation of its POI. For proof of concept, we utilized an anti-GFP nanobody tagged with AZdye647, synthesized using the Michael addition reaction, followed by SDS-PAGE analysis to assess successful conjugation. This conjugate was then used to treat fixed and live cells expressing enhanced green fluorescent protein (EGFP), followed by fluorescent microscopy to confirm cellular entry and POI targeting. Initial SDS-PAGE analysis revealed the successful conjugation of the AZDye647 to the nanobody. Specific binding within fixed cells was confirmed using fluorescent microscopy, as co-localization of EGFP and the AZdye647-tagged anti-GFP nanobody was revealed. Our data also confirmed that liposomal encapsulation of the AZdye647-tagged anti-GFP nanobody yielded efficient delivery into live EGFP expressing cells. Together, this data provides confirmation that a nanobody-PROTAC would be capable of passing through the cellular membrane, maintaining its integrity and degrading its POI. Further understanding of whether the replacement of AZDye647 with an E3 ligase ligand could result in the degradation of ‘undruggable’ oncogenes such as KRAS, in vitro, will confirm whether this is a viable therapeutic approach to patients with KRAS-mutant cancers. Citation Format: Caroline Cope, Cristina Tufarelli, Robert Britton. Nanobody-PROTACs: Method development towards using single-domain antibodies to target undruggable proteins for degradation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 7026.