Glypican-3 (GPC3) is a cell surface glypican that serves as a co-receptor for Wnt. Our laboratory has generated two high affinity antibodies targeting GPC3, HN3 and YP7. The HN3 human nanobody recognizes the N-lobe of GPC3, whereas the YP7 antibody recognizes the C-lobe of GPC3. Mutating residue F41 on the N-lobe of GPC3 inhibits binding of Wnt and the HN3 nanobody and inhibits activation of β-catenin. Both antibodies are fused to a fragment of Pseudomonas exotoxin A (PE38) to create recombinant immunotoxins. Interestingly, the HN3-derived immunotoxin (HN3-PE38) has superior antitumor activity as compared with the YP7-derived immunotoxin (YP7-PE38). Intravenous administration of HN3-PE38 causes regression of hepatocellular carcinoma (HCC) tumor xenografts in mice. Our study establishes GPC3 as a promising target for immunotoxin-based liver cancer therapy and demonstrates immunotoxin-induced tumor regression via dual mechanisms: inactivation of Wnt signaling via the HN3 nanobody and inhibition of protein synthesis via the PE bacteria toxin. However, immunogenicity and a short serum half-life may limit the ability of immunotoxins to transition to the clinic. To address these issues, we have recently engineered HN3-based immunotoxins to contain various deimmunized PE toxins. These new immunotoxins include HN3-T20, which is modified to remove domain II of the PE toxin and six T-cell epitopes. All of our immunotoxins display high affinity to human GPC3, with HN3-T20 having a KD value of 7.4 nM. A real-time cell growth inhibition assay demonstrates that a single dose of HN3-T20 at 1.6 nM is capable of inhibiting nearly all cell proliferation during the 10-day experiment. To enhance HN3-T20’s serum retention, we further engineer the HN3-T20 by adding a streptococcal albumin binding domain (ABD) and a llama single-domain antibody fragment (ALB1) specific for serum albumin. For the detection of immunotoxin in mouse serum, we develop a highly sensitive ELISA and find that HN3-ABD-T20 has a 45-fold higher serum half-life than HN3-T20 (326 min vs 7.3 minutes); consequently, HN3-ABD-T20 shows the best serum retention and remains detectible even after a 24-hour period in mice. Furthermore, addition of an albumin binding domain results in HN3-ABD-T20 mediated tumor regression in mice at a low dose (1 mg/kg). These data show that the albumin binding deimmunized immunotoxins are high potency therapeutics ready to be evaluated in clinical trials for the treatment of liver cancer.
Citation Format: Mitchell Ho, Bryan D Fleming, Tim F Greten, Ira Pastan. Immunotoxins targeting GPC3 for liver cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr CN07-02. doi:10.1158/1535-7163.TARG-19-CN07-02