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. 2019 Aug;70(2):563-576.
doi: 10.1002/hep.30326. Epub 2019 Feb 19.

Glypican-3-Specific Antibody Drug Conjugates Targeting Hepatocellular Carcinoma

Ying Fu  1 , Daniel J Urban  2 , Roger R Nani  3 , Yi-Fan Zhang  1 , Nan Li  1 , Haiying Fu  1 , Hamzah Shah  1 , Alexander P Gorka  3 , Rajarshi Guha  2 , Lu Chen  2 , Matthew D Hall  2 , Martin J Schnermann  3 , Mitchell Ho  1
Affiliations

Glypican-3-Specific Antibody Drug Conjugates Targeting Hepatocellular Carcinoma

Ying Fu et al. Hepatology. 2019 Aug.

Abstract

Hepatocellular carcinoma (HCC) is the second most common cause of cancer-related death in the world. Therapeutic outcomes of HCC remain unsatisfactory, and novel treatments are urgently needed. GPC3 (glypican-3) is an emerging target for HCC, given the findings that 1) GPC3 is highly expressed in more than 70% of HCC; (2) elevated GPC3 expression is linked with poor HCC prognosis; and (3) GPC3-specific therapeutics, including immunotoxin, bispecific antibody and chimeric antigen receptor T cells. have shown promising results. Here, we postulate that GPC3 is a potential target of antibody-drug conjugates (ADCs) for treating liver cancer. To determine the payload for ADCs against liver cancer, we screened three large drug libraries (> 9,000 compounds) against HCC cell lines and found that the most potent drugs are DNA-damaging agents. Duocarmycin SA and pyrrolobenzodiazepine dimer were chosen as the payloads to construct two GPC3-specific ADCs: hYP7-DC and hYP7-PC. Both ADCs showed potency at picomolar concentrations against a panel of GPC3-positive cancer cell lines, but not GPC3 negative cell lines. To improve potency, we investigated the synergetic effect of hYP7-DC with approved drugs. Gemcitabine showed a synergetic effect with hYP7-DC in vitro and in vivo. Furthermore, single treatment of hYP7-PC induced tumor regression in multiple mouse models. Conclusion: We provide an example of an ADC targeting GPC3, suggesting a strategy for liver cancer therapy.

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Conflict of interest statement

CONFLICTS OF INTEREST

The National Cancer Institute (NCI) holds patent rights to anti-GPC3 antibodies in many jurisdictions, including the United States [e.g., U.S. Patent 9,409,994, U.S. Patent 9,206,257, U.S Patent 9,304,364, and U.S. Patent 9,932,406], China, Japan, South Korea, Singapore and Europe. Claims cover the antibodies themselves, as well as conjugates that utilize the antibodies, such as recombinant immunotoxins (RITs), antibody drug conjugates (ADCs), bispecific antibodies and modified T cell receptors (TCRs)/chimeric antigen receptors (CARs), and vectors expressing these constructs. Anyone interested in licensing these antibodies can contact the NCI Technology Transfer Office or the corresponding author of this report (M.H.) for additional information.

Figures

Figure 1.
Figure 1.
(A) Screening of three libraries of payloads against liver cancer cell lines. (B) Examples of payloads with highest potency against Hep3B cell line. (C) Structure of hYP7-DC and hYP7-PC. (D) Binding assay of hYP7-DC and hYP7-PC against A431-GPC3 and A431. (E) Internalization rate of hYP7-PC and hYP7-DC (both at 100ng/ml, 4 h incubation) against A431-GPC3 cells. (F) Cytotoxicity curve of hYP7-DC. Error bars denote SD. (G) Western blot for cleaved poly ADP-ribose polymerase (cleaved PARP) and cleaved caspase-9 after treatment of ADCs in Hep3B cells for 4 days. (H-I) Toxicity of hYP7-DC (H) and hYP7-PC (I) after cathepsin B digestion against A431 (GPC3 negative) cells.
Figure 2.
Figure 2.
Internalization and trafficking of hYP7-Alexa647, hYP7-DC and hYP7-PC. (A) Time-dependent images of Alexa labeled hYP7 (hYP7-Alexa647) in A431-GPC3 cells. (B-C) GPC+ (A431-GPC3) and GPC− (A431) cells were incubated with hYP7-DC or hYP7-PC for 4h at 37°C. Cells were fixed, permeabilized, and hYP7-DC or hYP7-PC visualized using a fluorophore labeled secondary antibody (red). Nuclei and lysosomes were stained with anti-Lamp1 (green) and Hoechst 33342 (blue), respectively. Lysosome (green circle) around hYP7-DC (red) or hYP7-PC (red).
Figure 3.
Figure 3.
Synergistic effect of hYP7-DC and Gemcitabine in vitro and in vivo. (A) Combination screen of hYP7-DC with different small molecule anticancer agents. (B) Summary of hYP7-DC matrix synergy and antagonism. (C) Example of synergistic effect of hYP7-DC with gemcitabine. (D) Tumor volume (left), bodyweight change (middle) and Kaplan-Meier survival curve (right) for each group from the Hep3B xenograft model. Error bars denote SD. *p<0.05, **p<0.01, ***p<0.001.
Figure 4.
Figure 4.
Anticancer efficacy of hYP7-PC in the HepG2-Luc and Hep3B-Luc xenograft models. (A) Treatment scheme. HepG2-Luc or Hep3B-Luc cells were injected i.p., single-dose treatment of hYP7-PC (2.5 mg/kg) was given 7 days after disease establishment (n=5 per group). (B) Bioluminescence imaging (BLI) of liver burden change before and after treatment with PBS (control) or hYP7-PC (2.5 mg/kg). (C). Spider plot of individual tumor burden from the HepG2-Luc model. (D) Quantification of tumor burden from the HepG2-Luc model. (E). Spider plot of individual tumor burden from the Hep3B-Luc model. (F) Quantification of tumor burden from the Hep3B-Luc model. (G-H) Kaplan-Meier survival curve for each group (n=5 per group) and bodyweight change from the HepG2-Luc model. (I-J) Kaplan-Meier survival curve for each group (n=5 per group) and bodyweight change from the Hep3B-Luc model. Error bars denote SD. *p<0.05, **p<0.01, ***p<0.001.
Figure 4.
Figure 4.
Anticancer efficacy of hYP7-PC in the HepG2-Luc and Hep3B-Luc xenograft models. (A) Treatment scheme. HepG2-Luc or Hep3B-Luc cells were injected i.p., single-dose treatment of hYP7-PC (2.5 mg/kg) was given 7 days after disease establishment (n=5 per group). (B) Bioluminescence imaging (BLI) of liver burden change before and after treatment with PBS (control) or hYP7-PC (2.5 mg/kg). (C). Spider plot of individual tumor burden from the HepG2-Luc model. (D) Quantification of tumor burden from the HepG2-Luc model. (E). Spider plot of individual tumor burden from the Hep3B-Luc model. (F) Quantification of tumor burden from the Hep3B-Luc model. (G-H) Kaplan-Meier survival curve for each group (n=5 per group) and bodyweight change from the HepG2-Luc model. (I-J) Kaplan-Meier survival curve for each group (n=5 per group) and bodyweight change from the Hep3B-Luc model. Error bars denote SD. *p<0.05, **p<0.01, ***p<0.001.
Figure 5.
Figure 5.
(A-C) Single-dose antitumor activity of hYP7-DC in xenograft model of A431-GPC3 cells. Localized tumor models were developed in nude mice with A431-GPC3 cells, hYP7-DC was injected when tumor volume reached 160 mm3. Tumor volume (A) and bodyweight (C) were measured daily. (C) Kaplan-Meier survival curve for each group (n=7 per group). (D-F) Singe-dose antitumor activity of hYP7-DC in xenograft model of HCC. Localized tumor models were developed in nude mice with Hep3B cells, different doses of hYP7-DC were injected when tumor volume reached 100 mm3. Animals were monitored for 2 weeks. Tumor volume (D) and bodyweight (F) were measured daily. (E) Kaplan-Meier survival curve for each group (n=8 per group). Error bars denote SD. *p<0.05, **p<0.01, ***p<0.001.
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