Elsevier

Biomaterials

Volume 35, Issue 25, August 2014, Pages 6964-6971
Biomaterials

Imaging of hepatocellular carcinoma patient-derived xenografts using 89Zr-labeled anti-glypican-3 monoclonal antibody

https://doi.org/10.1016/j.biomaterials.2014.04.089Get rights and content

Abstract

Imaging probes for early detection of hepatocellular carcinoma (HCC) are highly desired to overcome current diagnostic limitations which lead to poor prognosis. The membrane protein glypican-3 (GPC3) is a potential molecular target for early HCC detection as it is over-expressed in >50% of HCCs, and is associated with early hepatocarcinogenesis. We synthesized the positron emission tomography (PET) probe 89Zr-DFO-1G12 by bioconjugating and radiolabeling the anti-GPC3 monoclonal antibody (clone 1G12) with 89Zr, and evaluated its tumor-targeting capacity. In vitro, 89Zr-DFO-1G12 was specifically taken up into GPC3-positive HCC cells only, but not in the GPC3-negative prostate cancer cell line (PC3). In vivo, 89Zr-DFO-1G12 specifically accumulated in subcutaneous GPC3-positive HCC xenografts only, but not in PC3 xenografts. Importantly, 89Zr-DFO-1G12 delineated orthotopic HCC xenografts from surrounding normal liver, with tumor/liver (T/L) ratios of 6.65 ± 1.33 for HepG2, and 4.29 ± 0.52 for Hep3B xenografts. It also delineated orthotopic xenografts derived from three GPC3-positive HCC patient specimens, with T/L ratios of 4.21 ± 0.64, 2.78 ± 0.26, and 2.31 ± 0.38 at 168 h p.i. Thus, 89Zr-DFO-1G12 is a highly translatable probe for the specific and high contrast imaging of GPC3-positive HCCs, which may aid early detection of HCC to allow timely intervention.

Introduction

Hepatocellular carcinoma (HCC) is the third leading cause of cancer deaths worldwide [1], [2], [3]. Early detection of HCC is crucial and may significantly improve its outcome, since its typically late diagnosis is associated with limited treatment options and lower chances of survival [4], [5], [6]. Current diagnostic imaging of HCC based on ultrasonography, computed tomography, and magnetic resonance imaging [7] is limited as they are often incapable of detecting HCC lesions <2 cm, and of differentiating between HCC lesions and other benign liver lesions, leading to false-positive diagnoses [8]. Thus, it is imperative to develop new molecular imaging techniques which can improve the sensitivity and specificity of HCC detection.

Molecular imaging of cancer can potentially improve early diagnosis and clinical management of cancer patients [9], [10]. Positron emission tomography (PET) using tumor-targeting radiolabeled-molecules has gained wide acceptance in oncology, allowing improved diagnosis and clinical management of cancer patients [9]. A variety of molecules, including monoclonal antibodies (mAbs), antibody fragments, small proteins, peptides, and small molecules can be used as tumor-targeting molecules with different levels of tumor accessibility and specificity [11], [12], [13]. The widespread availability of highly specific antibodies has led to rapid advances in antibody-based probe development for PET imaging [9], [10], [14]. Generally, intact antibody molecules have relatively slow pharmacokinetics, which require multiple days to reach their optimal biodistribution within the body [15], [16]; therefore, PET radioisotopes with a long half-life such as 89Zr (78.4 h) or 124I (100.3 h) are particularly suitable for intact antibodies. However, since the liver is largely responsible for antibody clearance, the applicability of immuno-PET for HCC imaging remains unclear, with the major hurdle being high normal liver uptake and resulting poor tumor-to-liver ratio.

The successful early detection of HCC lesions will require the combined selection of highly specific targets and effective approaches to decrease non-specific liver uptake of the imaging probe. Although other HCC associated biomarkers such as epidermal growth factor receptor (EGFR) has been used as an imaging target for HCC imaging, these approaches were typically associated with high liver background and unfavorable tumor-to-liver ratios [17], [18]. In HCC, the heparin sulfate proteoglycan glypican-3 (GPC3) is a rational molecular target for HCC diagnostic imaging because it is: (i). a cell-membrane receptor that is readily accessible for antibody-mediated targeting and binding [19]; (ii). expressed in more than 50% of HCC patients [20], [21], [22]; (iii). capable of distinguishing malignant HCCs from normal liver, and pre-neoplastic and benign liver lesions [8], [22], [23], [24]; (iv). expressed at higher levels in small HCCs than in cirrhosis and other types of small focal lesions, suggesting that the transition from premalignant lesions to small HCC is usually associated with elevated GPC3 [20], [22], [24], [25], [26]. Thus, detection of HCC based on GPC3 expression may aid early diagnosis.

We therefore hypothesized that an immuno-PET probe based on 89Zr-radiolabeled, anti-GPC3 monoclonal antibody (mAb) may offer the potential to accurately identify GPC3-positive HCC cells. We tested this probe for its specificity for detecting HCC cells in vitro and in vivo, and further determined its suitability for clinical translation using orthotopic HCC patient-derived xenografts.

Section snippets

Bioconjugation and radiolabeling

The anti-human GPC3 mAb (Clone 1G12, BioMosaics Inc., Burlington, VT) or non-targeting mouse IgG (Jackson ImmunoResearch, Inc.) were conjugated with desferrioxamine (DFO) and radiolabeled with 89Zr (University of Wisconsin, Madison, WI) as previously described [27], [28]. The radiolabeled products, 89Zr-DFO-1G12 or 89Zr-DFO-IgG respectively, were eluted by phosphate-buffered saline (PBS, pH 7.4) and passed through a 0.22-μm Millipore filter into a sterile vial for in vitro and animal

Affinity and specificity of anti-GPC3-mAb in vitro

We first demonstrated that the mouse anti-GPC3 mAb (clone 1G12) has high binding affinity (mean KD value = 0.41 ± 0.05 nm; Fig. 1A) to recombinant human GPC3 protein using an ELISA-based procedure [30]. Using the same mAb for Western blotting and immunofluorescence, we observed varying levels of GPC3 protein expression in a panel of HCC cell lines (HepG2, Hep3B, Huh 7, PLC/PRF/5 and SNU449) and the prostate cancer cell line (PC3), with highest levels in HepG2 cells, and undetectable levels in

Discussion

We successfully synthesized the 89Zr-DFO-1G12 immuno-PET probe for imaging of HCC based on GPC3 expression, and demonstrated its ability to specifically identify GPC3-expressing HCC cells in vitro and in vivo. Importantly, 89Zr-DFO-1G12 delineated GPC3-expressing orthotopic HCC patient-derived xenografts from surrounding normal liver tissue, suggesting its potential for clinical translation.

Despite recent advances in positron emission tomography imaging using tumor-seeking

Conclusions

We have demonstrated that immuno-PET imaging of HCC based on GPC3 is a feasible and highly translatable approach. The specificity provided by the anti-GPC3 antibody, coupled with the high tumor-to-liver ratios provided by the 89Zr-mAb, makes 89Zr-DFO-1G12 the most specific immuno-PET probe for HCC to date that can achieve high resolution imaging of GPC3-expressing HCCs.

Acknowledgments

This work was supported by the T.S. Kwok Liver Research Foundation, the C.J. Huang Foundation (to the Asian Liver Center, Stanford University), and the Stanford Cancer Center (DCRA). The authors thank Dr. Tim Doyle from the Stanford Center for Innovation in in vivo Imaging for his expert knowledge and technical advice for PET/CT imaging.

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