Isolation of a Cross-reactive Glypican-3 Nanobody from a Llama-Derived Phage Display Library

Introduction: 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. Glypican-3 (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. Accordingly, radiopharmaceutical agents specific to GPC3 could allow for HCC-selective imaging and therapy.

While full-length antibodies can be engineered for use as scaffolds to build radiopharmaceutical imaging or therapeutic agents, their long blood half-life makes them suboptimal because of poor tumor:blood or hematopoietic toxicity, respectively. Llamas, along with other camelids, produce a subclass of IgGs that have an unpaired heavy-chain variable domain, which has been shown to function like a conventional IgG. These heavy-chain domains can be expressed as a single domain known as a VHH region, or nanobody. Nanobodies are an attractive option, compared to a full-length antibody, for targeting GPC3 because their smaller size leads to superior kinetic properties, clearance rate, and tissue penetration. Here, we aimed to design an immunized phage display library to isolate specific, high-affinity nanobodies against GPC3.

Methods: Using standard methods, an adult llama was immunized five times with human and murine GPC3. Nanobody cDNA sequences were isolated from the B cells of the llama, amplified by PCR, cloned into phagemids, and transfected into E. Coli cells to create a phage display library with over 10⁸ clones. Phage were isolated from the library and immunopanning was performed. Using a GPC3-coated high binding tube, phage containing a specific nanobody bound to the surface and were eluted, while non-specific phage were discarded. Candidate clones were screened and identified using ELISA. Candidate phage clones were eluted, and the subsequent nanobodies were isolated and sequenced.

Results: After multiple rounds of immunopanning, two nanobodies, DN1 and DN2, specifically bound to human and murine GPC3. These nanobodies possess two distinct amino acid differences: two isoleucine residues in DN1 become leucine and valine in DN2. DN1 and DN2 also displayed similar nanomolar affinity for GPC3 by quantitative ELISA.

Conclusions: Thus far, two nanobodies have been identified to specifically bind to GPC3 by ELISA. Biolayer interferometry and flow cytometry studies are currently underway, and if promising will be followed by assessment of in vivo target binding by biodistribution studies and PET imaging.