Highly Potent Immunotoxins Targeting the Membrane-distal N-lobe of GPC3 for Immunotherapy of Hepatocellular Carcinoma

Glypican-3 (GPC3) has become a compelling target for immunotherapy of hepatocellular carcinoma, including antibody-drug conjugate (ADC), and ADC-like immunotoxin. To investigate the impact of epitopes on the potency of ADCs, current study generated a large panel of chicken monoclonal antibodies (mAbs) that targeted 12 different and over-lapping epitopes on GPC3. These mAbs demonstrated a very high affinity with K(d) values in the range of 10(-9)-10(-14) M, and the highest affinity (K(d) value of 0.0214 pM) was 40-fold higher than the previously generated high-affinity mAb YP7 (K(d) value of 0.876 nM). Additionally, these mAbs exhibited excellent thermostability with T(m) values in the range of 45-82 °C. As a proof-of-concept study for ADC, we made immunotoxins (scFv fused with PE24, the 24-kDa cytotoxic domain of Pseudomonas exotoxin A) based on these mAbs, and we found that immunotoxins targeting the N-lobe of GPC3 were overall much more potent than those targeting the C-lobe and other locations. One representative N-lobe-targeting immunotoxin J80A-PE24 demonstrated 3 to 13-fold more potency than the hitherto best immunotoxin HN3-PE24 that was previously developed. J80A-PE24 could suppress tumor growth much greater than HN3-PE24 in a xenograft mouse model. Combination of J80A-PE24 with an angiogenesis inhibitor FGF401 showed additive effect, which dramatically shrank tumor growth. Our work demonstrated that, due to high affinity, excellent thermostability and potency, chicken mAbs targeting the N-lobe of GPC3 are appealing candidates to develop potent ADCs for immunotherapy of liver cancer.