Project Summary Molecular imaging approaches that utilize monoclonal antibodies (mAbs) have shown great promise, but their extension into clinical practice is often difficult due to high radiation doses and inconvenient image acquisition intervals. These problems stem from the long-lived radioisotopes that are necessary to match the physiological properties of the mAbs themselves, namely a long in vivo half-life. Several pretargeting platforms, which aim to eliminate the dosimetry concerns by decoupling the radioisotope from the mAb, have been utilized successfully in preclinical models and with limited success in a clinical setting. However, to be widely applicable and translatable, pretargeting systems need to be robust, modular, and compatible for human use, and all of the platforms reported to date have not met those requirements. Broadly, the limitations of the predominant pretargeting platforms reported to date are non-modularity of the pretargeting components, intrinsic instability of the reactive ligands, or immunogenicity. Thus, we believe that taking advantage of the immense potential of pretargeting necessitates the development of a platform that is based on robust ligands that are amenable to a modular approach and are compatible with human use. We propose to develop a platform that meets these rigorous demands by utilizing the “host:guest” pair curcubit[7]uril (CB7) and adamantane (Adma). CB7 is known to rapidly form a strong, noncovalent interaction with Adma ligands, exhibiting similar kinetic and stability properties as biotin and streptavidin. We believe that this host:guest pair is ideal for development of a pretargeting platform for several reasons. First, CB7 may be easily functionalized for attachment to mAbs while Adma can be easily functionalized for radiolabeling with essentially any PET radioisotope, providing the requisite modularity. Additionally, neither functionality susceptible to racemization or prone to degradation, suggesting they are suitably robust. Finally, both CB7- and Adma-containing molecules have been reported as suitable for human use in various forms. Given the exceptional host:guest chemistry between CB7 and Adma-based compounds as well as their satisfaction of the above criteria for successful pretargeting platforms, we propose to develop and evaluate a pretargeted PET platform based on these compounds. The goal of the proposed project is to develop CB7-conjugated mAbs as well as Adma-containing ligands labeled with PET emitting radioisotopes. We will carry out a rigorous optimization of the pretargeting components and test our lead candidates using in vivo murine models of cancer. If successful, our pretargeting system could be the first to fully seize upon this concept of pretargeted PET imaging in a way that is widely applicable for human use. In doing so, we could alter the course of antibody-based PET imaging for precision medicine.