The Synthesis, Radiolabeling, and Characterization of 177Lu-Labeled Tetrazine Radioligands for Pretargeted Radioimmunotherapy

Objectives The synthesis, characterization, and optimization of ¹⁷⁷Lu-labeled tetrazine-bearing radioligands for use in pretargeted radioimmunotherapy.

Methods Current work in our group focuses on the pretargeted radiotherapy of pancreatic and colorectal cancer using the rapid and biorthogonal inverse electron demand Diels−Alder (IEDDA) reaction between tetrazine (Tz) and transcyclooctene (TCO). As part of an effort to develop a strategy for pretargeted radioimmunotherapy, Tz-PEG₇-DOTA and Tz-PEG₇-CHX-A˝-DTPA were synthesized modularly by standard chemical conjugation techniques. Structural characterizations were carried out by NMR and HRMS. Subsequently, ¹⁷⁷Lu-Tz-PEG₇-DOTA and ¹⁷⁷Lu-Tz-PEG₇-CHX-A”-DTPA were prepared via incubation of the ligand precursors with ¹⁷⁷LuCl₃ in ammonium acetate buffer. The stability of the radioligands was determined by agitation in PBS and human serum, followed by longitudinal analysis via instant thin layer chromatography (iTLC).

Results Tz-PEG₇-DOTA and Tz-PEG₇-CHX-A”-DTPA were synthesized with overall yields of ~34% and ~86%, respectively. Radiolabeling of both ligands resulted in the isolation of ¹⁷⁷Lu-Tz-PEG₇-DOTA and ¹⁷⁷Lu-Tz-PEG₇-CHX-A”-DTPA in >98% radioisotopic purity and specific activities of 311 μCi/μg and 307 μCi/μg respectively. Stability studies reveal that 93.6 ± 0.9% of the ¹⁷⁷Lu-Tz- PEG₇-DOTA remains intact after 48 hours compared to 81.7 ± 5% of ¹⁷⁷Lu-Tz-PEG₇-CHX-A”-DTPA. Biodistribution studies in athymic nude mice bearing SW1222 xenografts show that ¹⁷⁷Lu-Tz-PEG₇-DOTA and huA33-TCO form an effective pair for the pretargeting of colorectal cancer.

Conclusions We have successfully synthesized and radiolabeled Tz-PEG₇-DOTA and Tz-PEG₇-CHX-A"-DTPA for use in pretargeted radioimmunotherapy. ¹⁷⁷Lu-Tz-PEG₇-DOTA is an effective radioligand for in vivo pretargeting in a murine model of colorectal cancer. Pretargeted radiotherapy studies in murine models of colorectal and pancreatic cancer are forthcoming. $$graphic_B29CC900-A81E-45E2-A5DE-57B72BBD4FAD$$