GPC3-targeted immunoPET imaging of hepatocellular carcinomas.

Abstract

Early detection of hepatocellular carcinoma (HCC) remains a clinical challenge. Glypican 3 (GPC3) is a proteoglycan highly specific for HCC and is a potential diagnostic and therapeutic target for HCC. This work aims to develop GPC3-targeted immuno-positron emission tomography (immunoPET) imaging strategies and to assess the diagnostic values in preclinical HCC models. Flow cytometry was used to screen GPC3-positive HCC cell lines. The expression of GPC3 in HCCs was detected by immunohistochemistry on tissue microarray. A novel GPC3-specific single domain antibody (sdAb) was produced and labeled with gallium-68 (⁶⁸Ga, T_1/2 = 1.1 h) and fluorine-18 (¹⁸F, T_1/2 = 1.8 h) to develop radiotracers with different half-lives. The diagnostic efficacies of the developed probes (i.e., [⁶⁸Ga]Ga-NOTA-G2, [¹⁸F]F-G2, and [⁶⁸Ga]Ga-NOTA-ABDG2) were interrogated in preclinical HCC models bearing varying GPC3 levels. GPC3 was strongly expressed on HCC cell lines and patients with poorly differentiated HCC. [⁶⁸Ga]Ga-NOTA-G2 immunoPET imaging specifically delineated the subcutaneous HCC lesions, outperforming the traditional ¹⁸F-fluorodeoxyglucose PET and the nonspecific [⁶⁸Ga]Ga-NOTA-NbGFP immunoPET. ImmunoPET imaging with [¹⁸F]F-G2 also efficiently diagnosed the tumors with clarity. Moreover, the fusion of G2 to an albumin-binding domain (ABD) significantly increased the tumor uptake and decreased kidney accumulation of the radiotracer when compared to [⁶⁸Ga]Ga-NOTA-G2. In the work, we successfully developed sdAb-derived GPC3-targeted immunoPET imaging strategies and characterized the superior diagnostic accuracies in preclinical HCC models. Furthermore, we synthesized a fusion protein ABDG2 with improved targeting and pharmacokinetic properties, serving as a promising candidate for developing radioimmunotherapy agents.