Noninvasive Evaluation of Trop2 Expression Using 64Cu-NOTA-Trodelvy-F(ab')2 in Gastric and Pancreatic Cancer.

This study aimed to develop and evaluate a 64Cu-labeled NOTA-conjugated F(ab')2 fragment of Trodelvy for ImmunoPET imaging of Trop2 expression in gastric and pancreatic cancer models. Trodelvy was enzymatically digested using IdeS protease to generate F(ab')2 fragments, which were subsequently conjugated to a NOTA chelator and radiolabeled with 64Cu. In vitro binding affinity and cellular uptake were assessed in Trop2-positive and Trop2-negative cancer cell lines using flow cytometry, immunofluorescence, and cell binding assays. In vivo ImmunoPET imaging and biodistribution studies were conducted in subcutaneous xenograft models. In NCI-N87 xenografts, 64Cu-NOTA-Trodelvy-F(ab')2 showed peak tumor uptake at 12 h postinjection (10.83 ± 2.76%ID/g), significantly higher than in HGC-27 (3.33 ± 1.12%ID/g, p = 0.0110). In BxPC3 xenografts, uptake reached 10.00 ± 0.89%ID/g at 12 h, compared to 3.13 ± 0.96%ID/g in AsPC1 (p < 0.0001). Tumor-to-heart ratios were significantly improved in Trop2-positive tumors: NCI-N87 (3.53 ± 0.22 vs 0.83 ± 0.34 for intact Trodelvy, p = 0.0003) and BxPC3 (3.77 ± 0.07 vs 0.77 ± 0.19, p < 0.0001). Ex vivo biodistribution at 48 h confirmed high tumor retention (NCI-N87: 4.14 ± 0.44%ID/g; BxPC3: 4.10 ± 0.31%ID/g) and significantly lower uptake in control and IgG-F(ab')2 groups (p < 0.01). Although renal uptake was elevated due to the clearance of antibody fragments, histological analyses showed no signs of off-target toxicity. The rapid tumor targeting and favorable pharmacokinetics of 64Cu-NOTA-Trodelvy-F(ab')2 support its utility as a same-day ImmunoPET imaging agent. This practical and sensitive platform is highly valuable for patient stratification and therapeutic monitoring in Trop2-targeted cancer treatment.