Site-Specifically Conjugated Single-Domain Antibody Successfully Identifies Glypican-3–Expressing Liver Cancer by Immuno-PET
Stanley Fayn, Anna King, Nicholas T. Gutsche, Zhijian Duan, Jesse Buffington, Colleen P. Olkowski, Ying Fu, Jessica Hong, Deepak Sail, Kwamena E. Baidoo, Rolf E. Swenson, Ross W. Cheloha, Mitchell Ho, Peter L. Choyke, Freddy E. Escorcia
Abstract
Primary liver cancer is the third leading cause of cancer-related deaths, and its incidence and mortality are increasing worldwide. Hepatocellular carcinoma (HCC) accounts for 80% of primary liver cancer cases. Glypican-3 (GPC3) is a heparan sulfate proteoglycan that histopathologically defines HCC and represents an attractive tumor-selective marker for radiopharmaceutical imaging and therapy for this disease. Single-domain antibodies are a promising scaffold for imaging because of their favorable pharmacokinetic properties, good tumor penetration, and renal clearance. Although conventional lysine-directed bioconjugation can be used to yield conjugates for radiolabeling full-length antibodies, this stochastic approach risks negatively affecting target binding of the smaller single-domain antibodies. To address this challenge, site-specific approaches have been explored. Here, we used conventional and sortase-based site-specific conjugation methods to engineer GPC3-specific human single-domain antibody (HN3) PET probes. <b>Methods:</b> Bifunctional deferoxamine (DFO) isothiocyanate was used to synthesize native HN3 (nHN3)-DFO. Site-specifically modified HN3 (ssHN3)-DFO was engineered using sortase-mediated conjugation of triglycine-DFO chelator and HN3 containing an LPETG C-terminal tag. Both conjugates were radiolabeled with <sup>89</sup>Zr, and their binding affinity in vitro and target engagement of GPC3-positive (GPC3<sup>+</sup>) tumors in vivo were determined. <b>Results:</b> Both <sup>89</sup>Zr-ssHN3 and <sup>89</sup>Zr-nHN3 displayed nanomolar affinity for GPC3 in vitro. Biodistribution and PET/CT image analysis in mice bearing isogenic A431 and A431-GPC3<sup>+</sup> xenografts, as well as in HepG2 liver cancer xenografts, showed that both conjugates specifically identify GPC3<sup>+</sup> tumors. <sup>89</sup>Zr-ssHN3 exhibited more favorable biodistribution and pharmacokinetic properties, including higher tumor uptake and lower liver accumulation. Comparative PET/CT studies on mice imaged with both <sup>18</sup>F-FDG and <sup>89</sup>Zr-ssHN3 showed more consistent tumor accumulation for the single-domain antibody conjugate, further establishing its potential for PET imaging. <b>Conclusion:</b><sup>89</sup>Zr-ssHN3 showed clear advantages in tumor uptake and tumor-to-liver signal ratio over the conventionally modified <sup>89</sup>Zr-nHN3 in xenograft models. Our results establish the potential of HN3-based single-domain antibody probes for GPC3-directed PET imaging of liver cancers.