Engineered Anti‐GPC3 Immunotoxin, HN3‐ABD‐T20, Produces Regression in Mouse Liver Cancer Xenografts Through Prolonged Serum Retention

Abstract

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. Conclusion 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.

Development of Glypican-3 Targeting Immunotoxins for the Treatment of Liver Cancer: An Update

“…We also tested the ability of our HN3-based immunotoxin to display cross-species binding to mouse GPC3. HN3 immunotoxin displayed a high affinity for both human (4.9 nM) and mouse (5.8 nM) GPC3 when tested with an Octet system [27]. This suggests that the Wnt binding domain we have identified on the N-lobe of GPC3 is highly conserved between both human and mouse GPC3.…”

“…HN3 staining showed little to no binding on healthy adult liver tissues [27]. Importantly, vital organs such as the heart, brain, lungs and kidney were also found to be negative for HN3 staining [27]. These results suggest that HN3-based immunotoxins will show low off-target binding in patients.…”

“…To help anticipate any off-target side effects of HN3, we utilized a cryopreserved human tissue microarray that retained the conformational epitope needed for HN3 binding. HN3 staining showed little to no binding on healthy adult liver tissues [27]. Importantly, vital organs such as the heart, brain, lungs and kidney were also found to be negative for HN3 staining [27].…”

“…We and others have demonstrated that GPC3 is a suitable target for antibody-based therapeutics for several reasons [18]. First, as an oncofetal proteoglycan, GPC3 is primarily expressed in embryonic tissues and has virtually no expression in healthy human tissues [15][16][17][22][23][24][25][26][27][28]. Consequently, GPC3 has a high tumor specificity due to its upregulation in hepatocellular carcinoma and lack of expression in healthy human tissues.…”

ImmunoPET as Stoichiometric Sensor for Glypican-3 in Models of Hepatocellular Carcinoma

“…Since only about half of HCCs take up 18 FDG PET, the standard functional tracer, immunoPET may serve as an informative imaging biomarker in such circumstances and could allow more timely local or systemic interventions (5). GPC3 is a known marker of HCC and several GPC3-directed agents, including vaccines, monoclonal antibodies, bispecific T-cell engagers, single-domain antibody conjugates, targeted peptides, and chimeric antigen receptor T-cells (26,28,30,(35)(36)(37)(38)(39). Accordingly, identifying tumors that express GPC3 with immunoPET could help better enrich for populations that may most benefit from these treatments (32).…”