ImmunoPET imaging of Trop2 expression in triple-negative breast cancer using [⁶⁴Cu]Cu-NOTA-Trodelvy-F(ab')₂.

Abstract

The Trop2-targeting antibody-drug conjugate (ADC) sacituzumab govitecan (Trodelvy) has demonstrated remarkable efficacy in patients with metastatic triple-negative breast cancer (TNBC). ImmunoPET imaging offers a noninvasive method to visualize the expression and distribution of target antigens in vivo. In this study, we developed F(ab')₂ fragments of Trodelvy for immunoPET imaging to detect Trop2 expression in TNBC models, aiming to achieve a shorter imaging window. Trodelvy-F(ab')₂ was prepared using the IdeS protease kit and purified with Magne Protein A beads and MagneHis™ Ni Particles. The products were characterized by non-reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis and high-performance liquid chromatography. Trodelvy-F(ab')₂ was subsequently conjugated with p-SCN-Bn-NOTA (NOTA) for radiolabeling with ⁶⁴Cu. ImmunoPET imaging using [⁶⁴Cu]Cu-NOTA-Trodelvy-F(ab')₂ was conducted at multiple time points to assess its in vivo targeting capability. Immunohistochemical and immunofluorescence analyses were performed on tumor tissues obtained from tumor-bearing mice. The radiochemical yield of [⁶⁴Cu]Cu-NOTA-Trodelvy-F(ab')₂ exceeded 90%, with a radiochemical purity greater than 99%. High Trop2 expression was observed in MDA-MB-468 cells, whereas MDST8 cells exhibited low expression. The apparent dissociation constant (K_D) of [⁶⁴Cu]Cu-NOTA-Trodelvy-F(ab')₂ for MDA-MB-468 cells was determined to be 14.60 nM. ImmunoPET imaging revealed clear uptake of [⁶⁴Cu]Cu-NOTA-Trodelvy-F(ab')₂ in MDA-MB-468 tumors as early as 4 h post-injection (p.i.) (8.20 ± 0.98%ID/g), peaking at 12 h p.i. (11.13 ± 0.45%ID/g). Uptake was significantly higher compared to the MDST8 group (3.37 ± 0.45%ID/g at 4 h; 5.77 ± 0.74%ID/g at 12 h) and the blocking group (2.67 ± 0.21%ID/g at 4 h; 3.07 ± 0.37%ID/g at 12 h). [⁶⁴Cu]Cu-NOTA-Trodelvy-F(ab')₂ achieved significantly higher tumor-to-heart ratios in MDA-MB-468 tumors (3.87 ± 0.58 vs. 0.74 ± 0.19, P = 0.0019) at 12 h p.i., compared to [⁶⁴Cu]Cu-NOTA-Trodelvy, indicating superior tumor contrast. Our findings indicate that [⁶⁴Cu]Cu-NOTA-Trodelvy-F(ab')₂ exhibits rapid, specific, and sustained tumor accumulation in TNBC models, enabling precise and noninvasive monitoring of Trop2 expression.