Bioorthogonal Diels–Alder\nClick Chemistry-Based\nPretargeted PET Imaging Strategy for Monitoring Programmed Death-Ligand\n1 Expression

The development of antibody tracers for positron emission\ntomography\n(PET) imaging enables real-time monitoring of tumor expression of\nprogrammed cell death ligand 1 (PD-L1) <i>in vivo</i>, aiming\nto facilitate the selection of immunotherapy responders. However,\nthe slow pharmacokinetics of the antibodies in vivo limits their applications\nin PET imaging with commonly used radionuclides with short half-lives.\nIn this study, we developed a pretargeted PET imaging strategy based\non Diels–Alder (IEDDA) click chemistry to overcome these limitations.\nAtezolizumab and durvalumab, the most commonly used PD-L1 antibodies\nin cancer immunotherapy, were selected and compared in the development\nof the pretargeted PET imaging strategy. Fluorine-18-labeled derivatives\nof methyl tetrazine ([<sup>18</sup>F]­Tz, [<sup>18</sup>F]­PEG<sub>6</sub>-Tz, and [<sup>18</sup>F]­PEG<sub>12</sub>-Tz) were tested in biodistribution\nand PET imaging of A549-PDL1 xenografts (PD-L1 positive) pretargeted\nwith the trans-cyclooctene (TCO)-functionalized atezolizumab/durvalumab.\nThe biodistribution and imaging results indicated that atezolizumab-TCO/[<sup>18</sup>F]­PEG<sub>12</sub>-Tz was more suitable for pretargeted PET\nimaging strategy, and the optimal interval time was 48 h after atezolizumab-TCO\nadministration, where the atezolizumab-TCO/[<sup>18</sup>F]­PEG<sub>12</sub>-Tz pretargeted approach clearly delineated the A549-PDL1\ntumor with a tumor-to-muscle ratio of 5.33, while the ratios are 3.39\nand 2.39 for durvalumab/[<sup>18</sup>F]­PEG<sub>12</sub>-Tz and mock-pretargeting\ncontrols, respectively. In conclusion, a pretargeted <sup>18</sup>F-immuno-PET imaging technology was successfully established on atezolizumab.\nThe high-contrast PET images of the A549-PDL1 tumor models demonstrate\nthat the pretargeting strategy incorporating short-lived fluorine-18\nis viable in identifying tumors with high PD-L1 expression, marking\nthis strategy as a potential candidate for further clinical translation.