ImmunoPET as Stoichiometric Sensor for Glypican-3 in Models of Hepatocellular Carcinoma

Abstract

Background: Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide. While conventional imaging approaches like ultrasound, CT, and MRI play critical roles in the diagnosis and surveillance of HCC, improved methods for detection and assessment of treatment response are needed. One promising approach is the use of radiolabeled antibodies for positron emission tomography (immunoPET) imaging. Glypican-3 (GPC3) is a proteoglycan that is highly expressed in the majority of HCC tumors. GPC3-specific antibodies are used to diagnose HCC histopathologically, and have been proposed as a treatment of HCC. Here, we design, synthesize and demonstrate that our humanized immunoPET agent, [ 89 Zr]Zr-DFO-TAB-H14, can stoichiometrically bind to models of human liver cancer with varied GPC3 expression. Methods: The GPC3-specific monoclonal humanized IgG1, TAB-H14, was used as a scaffold for engineering our immunoPET agent. Fluorescent and deferroxamine (DFO) chelate conjugates of TAB-H14 were characterized using mass spectrometry. Binding affinity of TAB-H14 and conjugates for GPC3 was determined in cell-free biolayer interferometry, and cell-based radioimmunoassays. GPC3-expression was assessed by flow cytometry and immunofluorescence using commercially available anti-GPC3 antibodies and TAB-H14 in GPC3 -(A431) and GPC3 + cell lines including an engineered line (A431-GPC3 + , G1) and liver cancer lines (HepG2, Hep3B, and Huh7). DFO-TAB-H14, was radiolabeled with Zr-89. Mice were subcutaneously engrafted with the aforementioned cell lines and in vivo target engagement of the immunoPET agent [ 89 Zr]Zr-DFO-TAB-H14 was determined using PET/CT, quantitative biodistribution, and autoradiography. Results: TAB-H14 demonstrated subnanomolar to nanomolar affinity for human GPC3. Fluorescently tagged TAB-H14 was able to bind to GPC3 on cell membranes of GPC3-expressing lines by flow cytometry. These results were confirmed by immunofluorescence staining of A431, G1 HepG2, Hep3B, and Huh7 tumor sections. ImmunoPET imaging with [ 89 Zr]Zr-DFO-TAB-H14 showed stoichiometric tumor uptake corresponding to the cell surface expression levels. Autoradiography and immunostaining confirmed in vivo findings. Conclusion: We systematically demonstrate that the humanized immnoPET agent [ 89 Zr]Zr-DFO-TAB-H14 specifically and stoichiometrically binds to GPC3 in several models of human liver cancer, serving as a promising in vivo GPC3 sensor. This agent may provide utility in HCC diagnosis and surveillance, and the selection of candidates for GPC3-directed therapies.

Site-Specifically Conjugated Single-Domain Antibody Successfully Identifies Glypican-3–Expressing Liver Cancer by Immuno-PET

“…The GPC3-specific imaging capabilities of 89 Zr-ssHN3 place it among a select few radiotracers with clinical potential for liver tumor PET imaging. Although GPC3-targeted antibody conjugates have demonstrated specific tumor accumulation, these agents suffer from slow tumor uptake kinetics and are generally unsuitable for same-day imaging (11)(12)(13). Single-domain antibodies represent an alternative targeting biomolecule with excellent specificity and rapid clearance.…”

“…Radiolabeling of DFO-conjugated single-domain antibodies was conducted using a modification of established methods (12,29). Gentisic acid (10 mL, 10 mg/mL) was added to a solution of 89 Zr(oxalate) 2 (40 mL, 92.5 MBq, cyclotron at the National Institutes of Health Clinical Center) in oxalic acid.…”

“…Analysis of the original single-domain antibodies and resulting DFO conjugates by size-exclusion high-performance liquid chromatography and high-resolution mass spectrometry confirmed their purity and identity (Supplemental Figs. [3][4][5][6][7][8][9][10][11][12][13][14]. Synthetic schema for nHN3 and ssHN3 are presented in Figure 1.…”

“…Therefore, it is an attractive HCC-selective target that, if leveraged for molecular imaging, may help characterize postablation lesions and allow for more comprehensive screening, early diagnosis, and posttreatment surveillance. Full-length antibodies specific to GPC3 have been evaluated for immuno-PET imaging in preclinical (11,12) and clinical (13) studies. However, despite good imaging characteristics, the long blood half-life of full-length antibodies precludes sameday imaging, compromising existing patient workflows in the clinic.…”