Noninvasive Evaluation of Trop2 Expression Using ⁶⁴ Cu-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. <i>In vitro</i> binding affinity and cellular uptake were assessed in Trop2-positive and Trop2-negative cancer cell lines using flow cytometry, immunofluorescence, and cell binding assays. <i>In vivo</i> 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, <i>p</i> = 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 (<i>p</i> < 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, <i>p</i> = 0.0003) and BxPC3 (3.77 ± 0.07 vs 0.77 ± 0.19, <i>p</i> < 0.0001). <i>Ex vivo</i> 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 (<i>p</i> < 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.