Design and preclinical evaluation of lipophilicity-enhanced bicyclic peptide probes targeting Nectin4 Labeled with 68Ga/18F

Antibody-drug conjugates (ADCs) targeting Nectin4 have shown substantial clinical efficacy in the treatment of progressive or metastatic urothelial carcinoma (mUC). Accumulating evidence indicates that therapeutic responsiveness to ADCs is closely associated with the level of Nectin4 expression, which can be noninvasively evaluated using positron emission tomography (PET). However, our previously developed molecular probe, DN68, has displayed predominant renal excretion, a pharmacokinetic feature that may compromise its ability to reliably visualize primary mUC lesions. In the present study, we sought to develop novel molecular probes with reduced renal retention to improve PET imaging of mUC. To enhance hepatic metabolic processing, a lipid-soluble linker was introduced into the molecular probe precursor NOTA-DN96, thereby modulating its lipid-water partition coefficient and altering its in vivo clearance profile. The targeting characteristics of the novel precursor, radiolabeled with either [⁶⁸Ga]Ga or [¹⁸F]AlF, were systematically assessed through in vitro cellular uptake assays and in vivo imaging studies using Nectin4-positive (Nectin4+) tumor-bearing models. Based on overall imaging performance, the optimal probe was subsequently selected for further validation in mUC models. Radiolabeling of [⁶⁸Ga]Ga-NOTA-DN96 and [¹⁸F]AlF-NOTA-DN96 achieved efficiencies exceeding 95%, with radiochemical purity consistently greater than 99%. Cellular uptake assays demonstrated markedly higher accumulation of both tracers in MC38-Nectin4 cells (Nectin4+) compared with MC38 cells (Nectin4-negative, Nectin4-). In agreement with these results, biodistribution studies and PET imaging revealed pronounced and specific tracer accumulation in MC38-Nectin4 tumor models, whereas uptake in MC38 tumors remained substantially lower. The tumor-to-muscle (T/M) ratio increased progressively over time and reached its maximum at 120 min post-injection (p.i.). Moreover, tumor uptake of [⁶⁸Ga]Ga-NOTA-DN96 was consistently higher than that of [¹⁸F]AlF-NOTA-DN96 across all tumor models examined. Notably, both tracers exhibited increased hepatic uptake accompanied by reduced renal accumulation, indicating a shift toward enhanced hepatobiliary clearance. Collectively, [⁶⁸Ga]Ga-/[¹⁸F]AlF-NOTA-DN96 demonstrated excellent stability, high target specificity, and partial hepatobiliary elimination, highlighting their potential as promising PET probes for imaging Nectin4-expressing tumors. Their distinct pharmacokinetic characteristics also provided valuable insights for future probe design and optimization. These findings, therefore, established a foundation for the development of Nectin4-targeted PET imaging strategies in precision oncology.