Background and Aims
Treatment of hepatocellular carcinomas using our glypican‐3 (GPC3)‐targeting human nanobody (HN3) immunotoxins causes potent tumor regression by blocking protein synthesis and down‐regulating the Wnt signaling pathway. However, immunogenicity and a short serum half‐life may limit the ability of immunotoxins to transition to the clinic.
Approach and Results
To address these concerns, we engineered HN3‐based immunotoxins to contain various deimmunized Pseudomonas exotoxin (PE) domains. This included HN3‐T20, which was modified to remove T‐cell epitopes and contains a PE domain II truncation. We compared them to our previously reported B‐cell deimmunized immunotoxin (HN3‐mPE24) and our original HN3‐immunotoxin with a wild‐type PE domain (HN3‐PE38). All of our immunotoxins displayed high affinity to human GPC3, with HN3‐T20 having a KD value of 7.4 nM. HN3‐T20 retained 73% enzymatic activity when compared with the wild‐type immunotoxin in an adenosine diphosphate–ribosylation assay. Interestingly, a real‐time cell growth inhibition assay demonstrated that a single dose of HN3‐T20 at 62.5 ng/mL (1.6 nM) was capable of inhibiting nearly all cell proliferation during the 10‐day experiment. To enhance HN3‐T20’s serum retention, we tested the effect of adding a streptococcal albumin‐binding domain (ABD) and a llama single‐domain antibody fragment specific for mouse and human serum albumin. For the detection of immunotoxin in mouse serum, we developed a highly sensitive enzyme‐linked immunosorbent assay and found that HN3‐ABD‐T20 had a 45‐fold higher serum half‐life than HN3‐T20 (326 minutes vs. 7.3 minutes); consequently, addition of an ABD resulted in HN3‐ABD‐T20–mediated tumor regression at 1 mg/kg.
These data indicate that ABD‐containing deimmunized HN3‐T20 immunotoxins are high‐potency therapeutics ready to be evaluated in clinical trials for the treatment of liver cancer.