Glypican-3–Targeting F(ab′)2 for ⁸⁹Zr PET of Hepatocellular Carcinoma

Abstract

Hepatocellular carcinoma (HCC) is an increasingly lethal malignancy for which management is critically dependent on accurate imaging. Glypican-3 (GPC3) is a cell surface receptor overexpressed in most HCCs and provides a unique target for molecular diagnostics. The use of monoclonal antibodies (mAbs) that target GPC3 (αGPC3) in PET imaging has shown promise but comes with inherent limitations associated with mAbs such as long circulation times. This study used 89Zr-conjugated F(ab′)2 fragments directed against GPC3 (89Zr-αGPC3-F(ab′)2) to evaluate the feasibility of the fragments as a diagnostic immuno-PET imaging probe. Methods Immobilized ficin was used to digest αGPC3, creating αGPC3-F(ab′)2 fragments subsequently conjugated to 89Zr. In vivo biodistribution and PET studies were performed on GPC3-expressing HepG2 and GPC3-nonexpressing RH7777 orthotopic xenografts. Results Reliable αGPC3-F(ab′)2 production via immobilized ficin digestion was verified by high-performance liquid chromatography and sodium dodecyl sulfate polyacrylamide gel electrophoresis. 89Zr-αGPC3-F(ab′)2 demonstrated F(ab′)2-dependent, antigen-specific cell binding. HepG2 tumor uptake was higher than any other tissue, peaking at 100 ± 21 percentage injected dose per gram (%ID/g) 24 h after injection, a value 33- to 38-fold higher than GPC3-nonexpressing RH7777 tumors. The blood half-life of the 89Zr-αGPC3-F(ab′)2 conjugate was approximately 11 h, compared with approximately 115 h for historic mAb controls. This shorter half-life enabled clear tumor visualization on PET 4 h after administration, with a resultant peak tumor-to-liver contrast ratio of 23.3. Blocking antigen-expressing tumors with an excess of nonradiolabeled αGPC3 resulted in decreased tumor uptake similar to native liver. The kidneys exhibited high tissue uptake, peaking at 24 h with 83 ± 12 %ID/g. HepG2 tumors ranging from 1.5 to 7 mm were clearly visible on PET, whereas larger RH7777 tumors displayed signal lower than background liver tissue. Conclusion This study demonstrates the feasibility of using 89Zr-αGPC3-F(ab′) 2 for intrahepatic tumor localization with small-animal PET. Faster blood clearance and lower background liver uptake enable excellent signal-to-noise ratios at early time points. Increased renal uptake is similar to that as has been seen with clinical radioactive peptide imaging. 89Zr-αGPC3-F(ab′)2 addresses some of the shortcomings of whole-antibody immuno-PET probes. Further optimization is warranted to maximize probe sensitivity and specificity in the process of clinical translation.

Glypican‐3: A promising biomarker for hepatocellular carcinoma diagnosis and treatment

“…created the GPC3-F(ab ′ )2 fragment and conjugated it to89 Zr; the resultant GPC3-F(ab ′ )2-89 Zr probe showed specific HCC tumor-binding with a 11-h half-life, as compared with approximately 115 h for traditionally used mAb controls 115. This shorter half-life enabled clear tumor visualization in PET at 4 h after probe administration.…”

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).…”

“…GPC3-derived peptide vaccines, antibodies and their fragments targeting GPC3 have shown promising biological activity and target engagement in vivo, further underscoring the utility of GPC3 as a valuable target in HCC. However, most studies have used murine antibodies, limiting the potential for human translation (26)(27)(28)(29)(30). TAB-H14 is a full-length humanized IgG1 antibody with specificity to GPC3.…”

Galectin-3 Targeting in Thyroid Orthotopic Tumors Opens New Ways to Characterize Thyroid Cancer

“…Herein, we therefore performed the preclinical evaluation of gal-3 immunotargeting with 89 Zr-DFO-aGal3-F(ab9) 2 for detection of malignant thyroid nodules. F(ab) 2 fragments were used because of their faster blood clearance and lower liver uptake than monoclonal antibodies (6,19,25).…”