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

• Published in: Journal of Cancer Vol. 13; no. 4; pp. 1370 - 1384
• Main Authors: Li, Jingwen, Xiang, Lanxin, Wang, Qian, Ma, Xuqian, Chen, Xin, Zhu, Yuankui, Yang, Yaxi, Huang, Le, He, Huixia, Xu, Lilei, Liang, Xinjun, Dong, Shuang, Hu, Sheng, Li, Hanjie, Feng, Mingqian
• Format: Journal Article
• Language: English
• Published: Australia Ivyspring International Publisher 01.01.2022
• Subjects: Research Paper; GPC3; immunotoxin; monoclonal antibody; epitope mapping; hepatocellular carcinoma
• ISSN: 1837-9664; 1837-9664
• DOI: 10.7150/jca.66978

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 values in the range of 10 -10 M, and the highest affinity (K value of 0.0214 pM) was 40-fold higher than the previously generated high-affinity mAb YP7 (K value of 0.876 nM). Additionally, these mAbs exhibited excellent thermostability with T 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 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.