Abstract A069: Generation of llama-derived phage display library specific to hepatocellular carcinoma tumor targets
Authors: Nambiar Divya , Esparza Thomas J , Choyke Peter L , Escorcia Freddy E 
Published: December 2023

Abstract
Background: Primary liver cancer is the third leading cause of cancer-related deaths worldwide, with its incidence and mortality continuing to increase. Hepatocellular carcinoma (HCC) accounts for approximately 90% of these cases, and outcomes for most patients are poor. Unlike many cancer types, liver cancer cannot reliably be detected by 18F-fluorodeoxyglucose positron emission tomography (18F-FDG-PET) due to its limited tumor uptake. Curiously, despite the advent of precision oncology, no HCC-selective agents are clinically approved for the diagnosis or treatment of HCC in the United States. There are multiple tumor targets, including Glypican-3(GPC3) and Basigin (BSG) which are overexpressed on HCC tumors. GPC3 is a heparin sulfate proteoglycan expressed in 75% of HCC tumors, and rarely presented on other tissue types, making it a promising HCC-specific target. Similarly, BSG is a transmembrane glycoprotein that is expressed in 60% of HCC tumors, and is closely associated with tumor invasion, metastasis, and angiogenesis. Accordingly, radiopharmaceutical agents specific to GPC3 or BSG could allow for HCC-selective imaging and therapy.
Methods:Using standard methods, a young, naïve llama was immunized four times with human and murine GPC3, along with BSG . Nanobody cDNA sequences were isolated from the B cells of the llama, amplified by PCR, cloned into phagemids, and transformed into E. Coli cells to create a nanobody library with over 10⁶ clones. Phage, displaying the nanobody protein, are then produced and used for immunopanning to enrich for specific nanobodies with affinity against each antigen. Using antigen-coated high binding tubes, phage particles displaying a specific nanobody will bind to the antigen and be eluted following stringent wash conditions, while non-specific phage particles are discarded. The eluted phage is then amplified, and a repeat round of phage display is performed. Enriched clones will then be screened by ELISA to identify high affinity binders. Lead candidate nanobodies are then sequenced and unique clones expressed to determine yield, stability, and purity. Once specific nanobodies are identified, additional biophysical characterization will be performed.
Results: An antibody titer was performed by ELISA, demonstrating a successful immune response to all antigens. A phage display library was successfully created that is specific to human GPC3, murine GPC3 and BSG, with over 10⁶ clones. It is anticipated that after a few rounds of immunopanning, specific nanobodies will be identified.
Conclusion: Thus far, a phage display library was created that is specific to mGPC3, hGPC3 and BSG. Immunopanning is currently underway to select out clones that are specific to the targeted antigens. Once enriched clones are isolated, orthogonal in vitro binding affinity assays, such as ELISA, flow cytometry and biolayer interferometry, will be used to measure specificity and strength of antigen binding. Subsequently, assessment of in vivo target binding will be performed using biodistribution studies and PET imaging.
Citation Format: Divya Nambiar, Thomas J Esparza, Peter L Choyke, Freddy E Escorcia. Generation of llama-derived phage display library specific to hepatocellular carcinoma tumor targets [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 A069.