Nanobody-Based ImmunoPET for Hepatocellular Carcinoma

Abstract

HCC accounts for 75-90% of all primary liver cancers, the majority of which are treated with liver-directed therapy. Treatment response and recurrence are difficult to discern using conventional imaging with MR/CT. Tumor-selective PET imaging could help with clinical management in this setting. Here, we engineer HN3, a single-domain antibody (nanobody) specific to GPC3, a histopathologically-defining HCC marker, as an immunoPET agent. We compared both conventional and sortase-based site-specific modification methods for synthesizing HN3 immunoPET tracers. Stochastic lysine conjugation with deferoxamine (DFO-NCS) was done to synthesize nHN3-DFO. ssHN3-DFO was engineered utilizing sortase-mediated conjugation of HN3 containing an LPETG C-terminal tag and a triglycine-DFO chelator. Biolayer interferometry (BLI) and radioligand saturation assays were done to determine binding affinity pre- and post-Zirconium-89 labeling. Following, PET/CT with a terminal 3-hour biodistribution was done in mice inoculated with isogenic A431 and A431-GPC3+ xenografts to determine conjugate specificity for GPC3. Finally, conjugates were evaluated in a HepG2 liver cancer model via ex vivo biodistribution studies and a comparative PET/CT study in mice bearing HepG2 tumors that were imaged with both [18F]FDG and 89Zr-ssHN3. Both conjugates exhibited nanomolar binding affinity for GPC3 in vitro (11-30 nM for nHN3 and 10-15 nM for ssHN3). A431 and A431-GPC3+ PET/CT and biodistribution studies showed specificity to GPC3 by both probes, with more favorable tumor uptake by 89Zr-ssHN3 at 3 hours post-injection (14% IA/g vs. 7% IA/g for nHN3). Both tracers also displayed uptake in HepG2 (GPC3+) liver tumors, again with the site specifically conjugated probe having higher tumor accumulation and lower liver signal than the conventionally modified HN3 (7% IA/g vs. 5 % IA/g for tumor and 2% IA/g vs. 4% IA/g for liver at 1-hour post-injection). PET/CT studies in mice imaged with [18F]FDG and 89Zr-ssHN3 demonstrated more consistent tumor accumulation for the nanobody conjugate (4/4 mice had uptake by the tumor vs. 1/4 for FDG). We successfully designed, synthesized, and characterized novel GPC3-selective nanobody PET probes that can image liver tumors in vivo. The site-specifically conjugated tracer showed more favorable biodistribution and pharmacokinetic properties, resulting in a much higher tumor: liver signal compared to 89Zr-nHN3. We also show the superiority of the 89Zr-ssHN3 imaging over conventional [18F]FDG, highlighting a clear advantage in using targeted tumor imaging for this cancer type. Successful translation of the site-specifically conjugated nanobody may ultimately aid in characterizing lesions following liver-directed therapy and allow for more comprehensive screening, early diagnosis, and post-treatment surveillance of HCC.