Click Chemistry–Enabled Pretargeted Alpha Therapy with Astatine-211

Abstract

This thesis aimed to develop a pretargeted radioimmunotherapy (PRIT) system based on the inverse electron-demand Diels–Alder (IEDDA) click reaction between tetrazine (Tz) and trans-cyclooctene (TCO), enabling targeted delivery of the alpha-emitter astatine-211 for systemic tumor treatment. To achieve this, poly-L-lysine-(PL)-based effector molecules and clearing agents (CAs) were designed, synthesized, and evaluated alongside the pretargeting agents (PRIT agents). The work was conducted through a series of studies that progressively developed and assessed the system. Initially, astatinated effectors based on PL scaffolds of two sizes (10 and 21 kDa) were designed and functionalized with Tz. Their performance was evaluated in vitro within the click chemistry PRIT system and in vivo through biodistribution studies in tumor-free mouse models. Subsequently, Tz-functionalized CAs based on the same scaffold and size variants were developed and compared to a biotin-based CA used in a streptavidin–biotin PRIT system. In the final stage, a smaller effector construct (4.2 kDa) was introduced, and in vivo studies in tumor-bearing mice compared the performance of the 4.2 and 10 kDa effectors alongside evaluation of the TCO-based PRIT agent. Overall, the results demonstrate that PL is a versatile scaffold for constructing both effectors and CAs. The effectors achieved tumor targeting with a notably homogeneous intratumoral distribution of radioactivity, representing an important advantage for alpha therapy of solid tumors. While the system shows promise, additional studies are needed to better understand the in vivo behavior of the effector–PRIT agent complex and to enhance therapeutic efficacy. The CAs effectively removed circulating PRIT agents, particularly for the larger polymer variant, underscoring the importance of size. Collectively, these findings support the feasibility of the PL approach using click chemistry and provide a foundation for continued optimization.