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 KD 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.

Keywords: glypican-3 or GPC3; hepatocellular carcinoma; mouse xenograft model; recombinant immunotoxin; single-domain antibody fragment.

Figure 1. Construction of mPE24-based immunotoxins

(A) Schematic of HN3-PE38, HN3-mPE24 and HN3-HN3-mPE24 immunotoxins. (B) SDS-PAGE to show the purified immunotoxins. Four micrograms of protein for each sample was loaded.

Figure 2. Binding properties of new immunotoxins

(A) ELISA result showing the binding affinities of indicated immunotoxins on GPC3. (B) Representative flow cytometry result showed the binding affinity of indicated immunotoxins on G1 cells.

Figure 3. Cytotoxicity of mPE24-based immunotoxins in HCC cell culture

(A) Inhibition of cell proliferation of A431 cells and G1 cells by indicated immunotoxins, determined by WST-8 assay. A431 was used as an antigen negative cell line. The dashed line indicates the value of IC₅₀. Values represent mean ± SD. (B) Cytotoxicity of indicated immunotoxins on the designated HCC cells, determined by a WST-8 assay. SK-hep1 was used as an antigen negative cell line. The dash line represents 50% live cells.

Figure 4. Comparison of anti-tumor activity of immunotoxins at a low dose in mice

(A) Anti-tumor activity indicated by immunotoxins. Nude mice were s.c. inoculated with 8 × 10⁶ Hep3B cells. When tumors reached an average volume of 100 mm³, mice were administered indicated doses of immunotoxins intravenously (0.6 mg kg⁻¹) every other day for ten injections. Arrows indicate individual injections. Control and HN3-HN3-mPE24: n = 8, HN3-PE38 and HN3-mPE24: n = 9. Values represent mean ± SE. (B) Body weight of the mice treated in A. Arrows indicate individual injections. Values represent mean ± SE.

Figure 5. Toxicity determination of HN3-HN3-mPE24 and HN3-mPE24 in mice

Nude mice were treated with the indicated dose of the HN3-HN3-mPE24 (A) or HN3-mPE24 (B) immunotoxin intravenously every other day for a total three injections. Arrows indicated individual injections. n = 3/group.

Figure 6. Anti-tumor activity comparison of anti-GPC3 immunotoxins at their tolerated doses in mice

(A) Anti-tumor activity of HN3-PE38, HN3-HN3-mPE24 and HN3-mPE24 at their tolerated dose. Nude mice were s.c. inoculated with 8 × 10⁶ Hep3B cells. When tumors reached an average volume of 100 mm³, mice were administered HN3-PE38 (0.6 mg kg⁻¹), HN3-HN3-mPE24 (2.5 mg kg⁻¹) and HN3-mPE24 (5 mg kg⁻¹) intravenously every other day for ten injections. Right panel shows amplified curves below 300 mm³. Arrows indicated individual injections. (B) Body weight of the mice treated in A. Arrow indicated individual injections. Values represent mean ± SE. (C) Representative photographs of treated mice at day 38. (D) Mouse serum AFP levels at day 38. Values represent mean ± SD. P* < 0.05. (E) H&E and immunohistochemistry staining of GPC3 on control and HN3-mPE24 treated tumor at day 38. Scale bar: 50 μm. (F) Survival curve for mice treated in A. *p < 0.05, ***p < 0.001 and ****p < 0.0001.