Abstract A068: Site-specific modification of nanobodies for ImmunoPET of Liver Cancer utilizing Self-labeling nanobody-tag pair (SLANT) technology
Authors: Fayn Stanley , Cabalteja Chino C , Lee Woonghee , Nambiar Divya , Baidoo Kwamena E , Escorcia Freddy E , Cheloha Ross W 
Published: December 2023

Abstract
Visualization of tumor cells is critical in diagnosing, monitoring, and treating patients with cancer. Liver cancer has an 18 percent 5-year survival rate and is the third leading cause of cancer-related deaths worldwide. Hepatocellular carcinoma (HCC), the most common type of liver cancer, only takes up 18F-fluorodeoxyglucose (18F-FDG), the most commonly used clinical positron emission tomography (PET) tracer, 50% of the time. As such, developing an HCC-selective imaging agent could improve our ability for earlier diagnosis, post-treatment surveillance, and ability to increase patient longevity and quality of life. Single domain antibodies (sdAbs), or nanobodies (Nb), are attractive scaffolds for radiopharmaceutical development because of their high target affinity and specificity, ready availability from recombinant expression in bacteria, and small size for rapid clearance, better tumor penetration, and same-day imaging. However, conventional conjugation chemistry that exploits cysteines or lysines found on the native protein can abrogate the binding site of the smaller Nbs. We have previously used sortase A-based site-specific conjugation to address these challenges; however, improvements were still possible. Here, we developed a novel self-labeling Nb-tag pair (SLANT) that allows fast and specific covalent labeling of a Nb based on peptide epitope binding. We used SLANT to engineer a heterodimeric Nb construct consisting of a Nb (HN3) specific for Glypican-3, a heparin sulfate proteoglycan that is overexpressed in 75-90% of all HCCs and our self-labeling Nb. This design allows for site-specific labeling of the heterodimeric (GPC3-specific) construct in a manner that avoids modification of the tumor antigen-specific protomer. Labeling is achieved via exposure of the fusion protein to a crosslinking peptide functionalized with a chelator (DOTA). This methodology produces labeled protein at over 90 percent yields within minutes compared to less than 30 percent yields utilizing enzymatic methods. Biolayer interferometry and radioligand saturation studies were done to determine the binding affinity of the conjugate for GPC3 pre- and post-Indium-111 labeling. SPECT/CT and biodistribution studies were done at 1h, 3hrs, and 24hrs with HepG2 and HepG2-GPC3- xenografts to assess conjugate specificity and ability to accumulate in GPC3+ liver tumors. Our conjugate exhibited a nanomolar affinity for GPC3 in vitro (17-36 nM). SPECT/CT and biodistribution studies showed specificity for GPC3 in HepG2 liver cancer xenografts (~5.5% IA/g at 1-hour post-injection (p.i.)) and minimal uptake in HepG2 GPC3- tumors (~0.62% IA/g at 1-hour p.i.). Our results highlight the development of a new labeling paradigm applied to GPC3+ liver cancer. This approach can be used for same-day imaging and the rapid development of SPECT and PET imaging tracers for different cellular markers on cancer cells, addressing known tumor heterogeneity.
Citation Format: Stanley Fayn, Chino C Cabalteja, Woonghee Lee, Divya Nambiar, Kwamena E Baidoo, Freddy E Escorcia, Ross W Cheloha. Site-specific modification of nanobodies for ImmunoPET of Liver Cancer utilizing Self-labeling nanobody-tag pair (SLANT) technology [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A068.