Construction of an immunotoxin, HN3-mPE24, targeting glypican-3 for liver cancer therapy

Abstract

Glypican-3 (GPC3) is overexpressed in hepatocellular carcinoma (HCC). We constructed a recombinant immunotoxin, HN3-mPE24, which contains a truncated form of Pseudomonas exotoxin A. The toxin portion lacks most of domain II and has seven point mutations in domain III to remove the B-cell epitopes thought to be responsible for causing off-target side effects and immunogenicity. We also fused a bivalent HN3 to mPE24. We tested these two molecules for GPC3 binding and cytotoxicity in HCC cell models. The K_D values of HN3-mPE24 and HN3-HN3-mPE24 for GPC3-expressing tumor cells were 12 nM and 1.4 nM, respectively. The IC₅₀ values of HN3-mPE24 and HN3-HN3-mPE24 for HCC cells were 0.2 nM and 0.4 nM, respectively. We also evaluated their toxicity and anti-tumor efficacy in mice. The maximum tolerated doses of HN3-mPE24 and HN3-HN3-mPE24 were 7 mg kg^-1 and 3.6 mg kg^-1, respectively. We treated mice with 5 mg kg^-1 of HN3-mPE24 intravenously every other day for ten injections. The alpha-fetoprotein level of HN3-mPE24 treated group was approximately 700 fold less than that of the untreated group (1.1 μg ml^-1 vs. 692.1 μg ml^-1). In addition, 25% of the mice treated with HN3-mPE24 survived to the end of this study, which was 105 days after HCC tumor implantation. In conclusion, the HN3-mPE24 immunotoxin caused liver tumor regressions and extended survival with no significant side effects in mice. It is a promising candidate for the treatment of liver cancer that may be readily translated to humans.