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Background Hepatocellular carcinoma (HCC) remains a major cause of cancer-related mortality worldwide, highlighting the need for improved theranostic strategies. Glypican-3 (GPC3) is a membrane-anchored proteoglycan that is highly and selectively expressed in HCC but shows low expression in normal tissues, making it an attractive target for molecular imaging and targeted intervention. However, antibodies are constrained by their large molecular size, limited tumor penetration, and slow metabolism. Here, we report a GPC3-targeted radiotracer derived from dimeric cyclic peptides discovered de novo through a DNA-encoded library (DEL) screening platform, which exhibits robust tumor uptake and prolonged retention and has advanced to clinical imaging evaluation in HCC.Methods A DNA-encoded cyclic peptide library was synthesized and screened against GPC3 on magnetic beads, followed by PCR and next-generation sequencing. The top candidate was synthesized and evaluated by Surface plasmon resonance (SPR). Cy5-conjugated peptides were evaluated by confocal microscopy and flow cytometry. The DOTA-conjugated peptide was labelled with 68Ga ([68Ga]CTCN) for cellular uptake and PET/CT imaging in HepG2 xenografts. Finally, a first-in-human imaging study was performed.Results A DNA-encoded dimeric cyclic peptide library was constructed and screened against GPC3 ( IDDF2026-ABS-0481 Figure 1(A,B)). SPR confirmed a high-affinity binder (CTCN) of GPC3 (KD = 37 nM). A Cy5-conjugated peptide showed significantly higher uptake in GPC3-positive HepG2 cells than in GPC3-negative HT-1080 cells, as confirmed by flow cytometry and confocal imaging (IDDF2026-ABS-0481 Figure 1(C-E)). The 68Ga-labelled precursor ([68Ga]CTCN) was then assessed in vitro and in PET/CT, demonstrating high tumor uptake and prolonged retention in HepG2 xenografts, superior to a reported GPC3-targeting peptide and blockable by excess unlabeled peptide (IDDF2026-ABS-0481 Figure 1(F)). Preliminary clinical imaging showed positive uptake in a patient with a large diffuse lesion in the left hepatic lobe, indicating potential for HCC diagnosis.Conclusions This study identifies a novel GPC3-targeting cyclic peptide (CTCN) using a DNA-encoded dimeric peptide library, demonstrating high affinity, specificity, and favourable tumor uptake and retention in vitro and in vivo. Preliminary clinical imaging findings further support their potential for continued evaluation in HCC theranostics.Abstract IDDF2026-ABS-0481 Figure 1