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 Kd values in the range of 10-9-10-14 M, and the highest affinity (Kd value of 0.0214 pM) was 40-fold higher than the previously generated high-affinity mAb YP7 (Kd value of 0.876 nM). Additionally, these mAbs exhibited excellent thermostability with Tm 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.