Purpose: This study aims to develop and evaluate a novel glypican-3 (GPC3)–targeted, single-chain variable fragment–based PET radiotracer for noninvasive assessment of GPC3 expression and precise diagnosis of hepatocellular carcinoma (HCC) in both preclinical models and a first-in-human clinical study. Experimental Design: The novel anti-GPC3 single-chain variable fragment, namely, XH-06, was labeled with gallium-68 to obtain [68Ga]Ga-XH-06. Cell uptake assays, small-animal PET imaging, and biodistribution studies were performed to evaluate its targeting ability. In the first-in-human study, eight patients with suspected HCC underwent [68Ga]Ga-XH-06 PET/MRI. Radiotracer uptake in tumors and normal tissues was quantified, and tumor-to-blood ratios and tumor-to-liver ratios were calculated. Results: [68Ga]Ga-XH-06 was synthesized with high radiochemical purity and exhibited specific uptake and efficient internalization in GPC3-positive cells. In subcutaneous and orthotopic animal models, [68Ga]Ga-XH-06 effectively visualized HCC tumors. Eight patients underwent [68Ga]Ga-XH-06 PET/MRI scans, and no adverse events were observed. The radiotracer successfully detected HCC lesions, including subcentimeter tumors, with high imaging contrast. Among seven patients with HCC, the median maximum standardized uptake value of the lesions was 16.9 (range, 8.2–51.8), the median tumor-to-liver ratio was 6.2 (range, 2.8–24.7), and the median tumor-to-blood ratio was 16.6 (range, 3.0–57.6) at 2.5 hours after injection. Conclusions: [68Ga]Ga-XH-06 is clinically promising for detecting GPC3-positive HCC with a favorable safety profile. Further investigation is warranted to validate the clinical value of GPC3-targeted PET imaging in HCC management. See related commentary by Seo and Flavell, p. 445

Supplementary Figure S4. Comparison of quantitative region-of-interest (ROI) data in [68Ga]Ga-XH-06 immunoPET imaging of Hep3B and SK-HEP-1 subcutaneous xenograft models.

Supplementary Table S2. Mean organ absorbed dose coefficients and whole-body effective dose for [68Ga]Ga-XH-06 in three healthy volunteers.

Purpose:This study aims to develop and evaluate a novel glypican-3 (GPC3)–targeted, single-chain variable fragment–based PET radiotracer for noninvasive assessment of GPC3 expression and precise diagnosis of hepatocellular carcinoma (HCC) in both preclinical models and a first-in-human clinical study.Experimental Design:The novel anti-GPC3 single-chain variable fragment, namely, XH-06, was labeled with gallium-68 to obtain [68Ga]Ga-XH-06. Cell uptake assays, small-animal PET imaging, and biodistribution studies were performed to evaluate its targeting ability. In the first-in-human study, eight patients with suspected HCC underwent [68Ga]Ga-XH-06 PET/MRI. Radiotracer uptake in tumors and normal tissues was quantified, and tumor-to-blood ratios and tumor-to-liver ratios were calculated.Results:[68Ga]Ga-XH-06 was synthesized with high radiochemical purity and exhibited specific uptake and efficient internalization in GPC3-positive cells. In subcutaneous and orthotopic animal models, [68Ga]Ga-XH-06 effectively visualized HCC tumors. Eight patients underwent [68Ga]Ga-XH-06 PET/MRI scans, and no adverse events were observed. The radiotracer successfully detected HCC lesions, including subcentimeter tumors, with high imaging contrast. Among seven patients with HCC, the median maximum standardized uptake value of the lesions was 16.9 (range, 8.2–51.8), the median tumor-to-liver ratio was 6.2 (range, 2.8–24.7), and the median tumor-to-blood ratio was 16.6 (range, 3.0–57.6) at 2.5 hours after injection.Conclusions:[68Ga]Ga-XH-06 is clinically promising for detecting GPC3-positive HCC with a favorable safety profile. Further investigation is warranted to validate the clinical value of GPC3-targeted PET imaging in HCC management.See related commentary by Seo and Flavell, p. 445

Background: B7-H3, a type I transmembrane glycoprotein belonging to the B7 superfamily, is an attractive target for antitumor therapies. B7-H3 demonstrates aberrant overexpression in various types of solid tumors while limited and low expression in normal human organs. Various types of treatment targeting B7-H3 have been reported. Among these treatments, antibody-drug conjugate (ADC) has shown potent activity and several clinical trials including DS7300a and MGC018 are currently ongoing. Methods: Here, we constructed CD276-8 ADC composed of anti-B7-H3 antibody CD276-8 with moderate affinity, enzymatically cleavable tetra-peptide–based linker and DXd. Characteristics including in vitro binding affinity and internalization activity was assessed by Bio-Layer Interferometry (BLI), flow cytometry and high content analysis (HCA). The cytotoxicity of CD276-8 ADC was evaluated by the cell-lines with B7-H3 expressed. Pharmacokinetics profiles and antitumor activity were evaluated in mice models in vivo. Finally, developability of CD276-8 ADC was assessed with plasma stability, accelerate stability and freeze-thawing studies using LC-MS and HPLC. Results: Characterization in vitro demonstrated the moderate affinity and acceptable internalization activity of CD276-8 ADC. In addition, CD276-8 ADC appeared potent antitumor activities in B7-H3-positive cell line-derived xenograft (CDX) models with acceptable pharmacokinetics profiles, although it showed less potent cytotoxicity in various cell lines in vitro, indicating acceptable developability. Conclusions: We developed CD276-8 ADC, a B7-H3-targeting ADC with moderate affinity, which delivers the TOP1 inhibitor DXd. This design enabled superior tumor penetration and favorable pharmacokinetics, resulting in potent antitumor efficacy in vivo. Our study highlights affinity optimization as a crucial strategy for enhancing ADC efficacy, positioning CD276-8 ADC as a promising therapeutic for B7-H3-expressing solid tumors.

Supplementary Figure S3. Quadrupole time-of-flight spectra of anti-GPC3 scFv (XH-06). The mass spectrum showed that the molecular weight of anti-GPC3 scFv was 26741 Da.