Yttrium-90-Labeled Anti-Glypican 3 Radioimmunotherapy Halts Tumor Growth in an Orthotopic Xenograft Model of Hepatocellular Carcinoma

Abstract

Hepatocellular carcinoma (HCC) is the second most lethal malignancy globally and is increasing in incidence in the United States. Unfortunately, there are few effective systemic treatment options, particularly for disseminated disease. Glypican-3 (GPC3) is a proteoglycan cell surface receptor overexpressed in most HCCs and provides a unique target for molecular therapies. We have previously demonstrated that PET imaging using a ⁸⁹Zr-conjugated monoclonal anti-GPC3 antibody (αGPC3) can bind to minute tumors and allow imaging with high sensitivity and specificity in an orthotopic xenograft mouse model of HCC and that serum alpha-fetoprotein (AFP) levels are highly correlated with tumor size in this model. In the present study, we conjugated ⁹⁰Y, a high-energy beta-particle-emitting radionuclide, to our αGPC3 antibody to develop a novel antibody-directed radiotherapeutic approach for HCC. Luciferase-expressing HepG2 human hepatoblastoma cells were orthotopically implanted in the livers of athymic nude mice, and tumor establishment was verified at 6 weeks after implantation by bioluminescent imaging and serum AFP concentration. Tumor burden by bioluminescence and serum AFP concentration was highly correlated in our model. Yttrium-90 was conjugated to αGPC3 using the chelating agent 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) and injected via the tail vein into the experimental mice at a dose of 200 μCi/mouse or 300 μCi/mouse. Control mice received DOTA-αGPC3 without radionuclide. At 30 days after a single dose of the radioimmunotherapy agent, mean serum AFP levels in control animals increased dramatically, while animals treated with 200 μCi only experienced a minor increase, indicating cessation of tumor growth, and animals treated with 300 μCi experienced a reduction in serum AFP concentration, indicating tumor shrinkage. Mean tumor-bearing liver weight in control animals was also significantly greater than that in animals that received either dose of ⁹⁰Y-αGPC3. These results were achieved without significant toxicity as measured by body condition scoring and body weight. The results of this preclinical pilot demonstrate that GPC3 can be used as a target for radioimmunotherapy in an orthotopic mouse model of HCC and may be a target of clinical significance, particularly for disseminated HCC.

Figure 1

DOTA conjugation maintains αGPC3 binding in vitro. Flow cytometry on fixed HepG2 cells demonstrates a significant increase in mean fluorescence of both unconjugated and DOTA-conjugated αGPC3 compared to unstained cells and isotype-matched primary antibody control samples (^∗p < 0.001). Error bars represent standard deviation of triplicate measurements for >10,000 cell counts per sample.

Figure 2

Plot of serum AFP concentration compared to tumor bioluminescence by IVIS imaging in tumor-bearing mice. Serum AFP concentration in mice 6 weeks after orthotopic HepG2-Red-FLuc xenograft implantation is highly correlated with tumor bioluminescence by IVIS imaging, indicating that AFP excretion by HepG2 orthotopic xenografts is dependent on tumor size.

Figure 3

Mean serum AFP concentration of tumor-bearing mice at specified time points after administration of radioimmunotherapy. Serum AFP steadily increases over time in control animals (n = 7), while AFP concentration in animals treated with 200 μCi (n = 9) or 300 μCi (n = 9) of the ⁹⁰Y-αGPC3 conjugate remained at pretreatment levels and was significantly lower than that in control animals by 30 days (^∗p < 0.05). Error bars represent standard error of the mean concentration.

Figure 4

Mean weight of tumor-bearing livers at the conclusion of the 30-day radioimmunotherapy trial. The weight of tumor-bearing livers resected en bloc is increased in control animals compared to those treated with either 200 μCi or 300 μCi ⁹⁰Y-αGPC3 conjugate (^∗p=0.05; ⁺p < 0.05). Error bars represent standard error of the mean organ weight.