Immunotoxin targeting glypican-3 regresses liver cancer via dual inhibition of Wnt signalling and protein synthesis

Abstract

Glypican-3 is a cell surface glycoprotein that associates with Wnt in liver cancer. We develop two antibodies targeting glypican-3, HN3 and YP7. The first antibody recognizes a functional epitope and inhibits Wnt signalling, whereas the second antibody recognizes a C-terminal epitope but does not inhibit Wnt signalling. Both are fused to a fragment of Pseudomonas exotoxin A (PE38) to create immunotoxins. Interestingly, the immunotoxin based on HN3 (HN3-PE38) has superior antitumor activity as compared with YP7 (YP7-PE38) both in vitro and in vivo. Intravenous administration of HN3-PE38 alone, or in combination with chemotherapy, induces regression of Hep3B and HepG2 liver tumour xenografts in mice. This study establishes glypican-3 as a promising candidate for immunotoxin-based liver cancer therapy. Our results demonstrate immunotoxin-induced tumour regression via dual mechanisms: inactivation of cancer signalling via the antibody and inhibition of protein synthesis via the toxin.

Figure 1. GPC3 expression and internalization in HCC cells

(a) Flow cytometry results, detecting cell surface GPC3 expression in the GPC3 positive cell lines HepG2, Hep3B, Huh-1, and Huh-7. Cell line A431 does not express GPC3, and is shown as a negative control. G1 cells, which over-express GPC3 in the A431 cell line is shown as a positive control. Shaded gray peaks represent the cell surface staining with isotype control; white peaks represent the cell surface staining of GPC3. (b) Western blot analysis of endogenous GPC3 expression in HCC and other cell lines. For each sample, 20μg total protein was loaded for detection. (c) Quantitation of GPC3 sites per HCC cell using QuantiBrite PE beads. (d) Flow cytometry detection of internalization rates of GPC3. Each western blot and flow cytometry results representative of at least three independent experiments.

Figure 2. Affinity and cytotoxicity of HN3-PE38 and YP7-PE38

(a) ELISA and flow cytometry analysis of HN3-PE38 and YP7-PE38 binding affinities for GPC3 protein and G1 cells. The results are representative of at least three independent experiments. (b) Inhibition of cell proliferation on different cell lines by HN3-PE38 and YP7-PE38 immunotoxins, determined by a WST-8 assay. SK-Hep-1 was showed as an antigen negative cell line. Dashed line indicated the value of IC₅₀. IT: immunotoxin. Values represent mean ± s.d. (c) Cytotoxicity of HN3-PE38 on wild type or GPC3 knocked down Hep3B and HepG2 cells. Western blot showed knock down efficiency. wt: wide type, kd: knockdown. IT: immunotoxin. Dashed line indicated the value of IC₅₀. Values represent mean ± s.d.

Figure 3. Construction and analysis of inactive anti-GPC3 immunotoxins

(a) Flow cytometry analysis of active and inactive HN3-PE38 and YP7-PE38 binding affinities for G1 cells. The K_D values for G1 cells were based on mean fluorescence intensity (MFI). (b) [³H] Leucine incorporation assay to detect protein synthesis on Hep3B cells treated with active or inactive anti-GPC3 immunotoxins. Dashed line indicated the value of IC₅₀. Values represent mean ± s.d. (c) Cytotoxicity of active and inactive HN3-PE38 and YP7-PE38 on Hep3B cells, determined by a WST-8 assay. Dashed line indicated the value of IC₅₀. Values represent mean ± s.d.

Figure 4. Inhibition of Wnt3a-induced β-catenin and Yap signaling by inactive HN3-PE38

(a) Topflash activity of HEK293Topflash cells treated with 0.5μg ml⁻¹ inactive HN3-PE38 and YP7-PE38 in the presence of Wnt3a. Inactive HS20-PE38 and active irrelevant IT were set up as a positive and negative control, respectively. IT: immunotoxin. Values represent mean ± s.d. P**<0.01 compared with Wnt3a treated group, Student’s t-test. (b) Active β-catenin expression on HEK293Topflash cells treated as described in (a) for 12h. (c) Yap/TEAD activity of Hep3B cells treated with 10μg ml⁻¹ inactive HN3-PE38 and YP7-PE38 in the presence of Wnt3a for 16h. Values represent mean ± s.d. P**<0.01 compared with Wnt3a treated group, Student’s t-test. (d) Cytotoxicity of HN3-PE38 and YP7-PE38 on wild type and Yap knockdown Hep3B cells. Data was presented as fold change of IC50 (wt vs kd). Western blot showed knock down efficiency. Values represent mean ± s.d., P*<0.05 and P**<0.01 compared with HN3-PE38 treated group, Student’s t-test.

Figure 5. In vivo anti-tumor activities of HN3-PE38 and YP7-PE38

(a) Toxicity detection of HN3-PE38 and YP7-PE38 in vivo. BALB/c nu/nu mice were treated with indicated dose of immunotoxins intravenously every other day for a total of six injections. Arrow indicated individual injection. n=5/group. (b) Anti-tumor activity of HN3-PE38 and YP7-PE38. BALB/c nu/nu mice were subcutaneously inoculated with 5 ×10⁶ Hep3B cells. When tumors reached an average volume of 100 mm³, mice were administered indicated doses of immunotoxins intravenously every other day for six injections. Right panel showed amplified curves below 400mm³. Arrow indicated individual injection. n=5/group. Values represent mean ± s.e.m., P***<0.001, paired Student’s t-test. (c) Body weight of the mice treated in (b). Arrow indicated individual injection. n=5/group. Values represent mean ± s.e.m. (d) Survival curve for mice treated in (b). n=5/group. P*<0.05, Log-rank (Mantel-Cox) test.

Figure 6. Immunohistochemistry analysis of HN3-PE38 and YP7-PE38 treated tumors

β-catenin staining and phosphorylated Yap staining on HN3-PE38 and YP7-PE38 treated Hep3B tumors. Scale bar: 50μm. Arrow indicates nuclear staining of β-catenin.

Figure 7. Combination of HN3-PE38 immunotoxin with chemotherapeutic drugs

(a) Combination of HN3-PE38 with sorafenib on Hep3B model. BALB/c nu/nu mice with Hep3B tumor were treated with 100mg kg⁻¹sorafenib and 0.4mg kg⁻¹HN3-PE38 every other day for six injections when tumors reached an average volume of 120 mm³. Right panel showed amplified curves below 500mm³. Arrow: HN3-PE38 injection; Arrow head: sorafenib delivery. n=4/group. Values represent mean ± s.e.m. (b) Combination of HN3-PE38 with irinotecan on Hep3B model. BALB/c nu/nu mice were treated with 100mg kg⁻¹ irinotecan once and 0.4mg kg⁻¹ HN3-PE38 every other day when tumors reached an average volume of 170 mm³. Arrow: HN3-PE38 injection; Arrow head: irinotecan injection. n=5/group. Values represent mean ± s.e.m. (c) Combination of HN3-PE38 with irinotecan on HepG2 model. BALB/c nu/nu mice were treated with 100mg kg⁻¹ irinotecan once and 0.6mg kg⁻¹ HN3-PE38 every other day for a total of six injections. Arrow: HN3-PE38 injection; Arrow head: irinotecan injection. n=7/group. Values represent mean ± s.e.m. Dashed line indicated the detection limit. (d) Tumor weight of (c) on day 51. Values represent mean ± s.e.m. P*<0.05; P**<0.01 and P***<0.001, Student’s t-test. n=7/group. (e) Serum AFP levels of the mice treated in (c) on day 51. Values represent mean ± s.d. P*<0.05 and P***<0.001, Student’s t-test. n=7/group. (f) The correlation between AFP and tumor size. Correlation was measured by GraphPad Prism 6.0.