Bioorthogonal Diels–Alder Click Chemistry-Based Pretargeted PET Imaging Strategy for Monitoring Programmed Death-Ligand 1 Expression

The development of antibody tracers for positron emission tomography (PET) imaging enables real-time monitoring of tumor expression of programmed cell death ligand 1 (PD-L1) <i>in vivo</i>, aiming to facilitate the selection of immunotherapy responders. However, the slow pharmacokinetics of the antibodies in vivo limits their applications in PET imaging with commonly used radionuclides with short half-lives. In this study, we developed a pretargeted PET imaging strategy based on Diels-Alder (IEDDA) click chemistry to overcome these limitations. Atezolizumab and durvalumab, the most commonly used PD-L1 antibodies in cancer immunotherapy, were selected and compared in the development of the pretargeted PET imaging strategy. Fluorine-18-labeled derivatives of methyl tetrazine ([¹⁸F]Tz, [¹⁸F]PEG₆-Tz, and [¹⁸F]PEG₁₂-Tz) were tested in biodistribution and PET imaging of A549-PDL1 xenografts (PD-L1 positive) pretargeted with the trans-cyclooctene (TCO)-functionalized atezolizumab/durvalumab. The biodistribution and imaging results indicated that atezolizumab-TCO/[¹⁸F]PEG₁₂-Tz was more suitable for pretargeted PET imaging strategy, and the optimal interval time was 48 h after atezolizumab-TCO administration, where the atezolizumab-TCO/[¹⁸F]PEG₁₂-Tz pretargeted approach clearly delineated the A549-PDL1 tumor with a tumor-to-muscle ratio of 5.33, while the ratios are 3.39 and 2.39 for durvalumab/[¹⁸F]PEG₁₂-Tz and mock-pretargeting controls, respectively. In conclusion, a pretargeted ¹⁸F-immuno-PET imaging technology was successfully established on atezolizumab. The high-contrast PET images of the A549-PDL1 tumor models demonstrate that the pretargeting strategy incorporating short-lived fluorine-18 is viable in identifying tumors with high PD-L1 expression, marking this strategy as a potential candidate for further clinical translation.