Development of a CD9-Targeted Radiopharmaceutical for Imaging and Radionuclide Therapy in CD9-Positive Glioma

High expression of CD9 (cluster of differentiation 9) is closely associated with the poor prognosis of glioma, and it is necessary to develop targeted radiopharmaceuticals for diagnosis and treatment. The therapeutic efficacy of peptide-based drugs is often limited by their rapid metabolism. This study aims to develop an integrated theranostic radiopharmaceutical capable of in vivo CD9 targeting by modifying peptides with maleimide. The CD9-binding molecule DOTA-M-P was synthesized and labeled with 68Ga and 177Lu by using an indirect labeling method. Construction of a CD9-overexpressing cell line (U87-CD9) to simulate an in vivo and in vitro model of the invasive glioma subtype before the affinity of DOTA-M-P for the CD9 protein was assessed through cellular assays. In vivo small animal PET/CT and SPECT/CT imaging and biodistribution studies were conducted to verify pharmacokinetics and tumor-targeting retention capabilities. DNA damage assays and Western blot analyses were employed to explore the therapeutic mechanisms. Radioligand therapy studies were performed to evaluate the therapeutic efficacy. Then OLINDA/EXM was employed to estimate the effective dose to human organs from 177Lu-DOTA-M-P for assessing its dose safety. In vitro cellular assays demonstrated that DOTA-M-P exhibits a moderate affinity for CD9. In vivo imaging studies demonstrated the modest targeting and retention capabilities of DOTA-M-P. Biodistribution experiments indicated that DOTA-M-P is primarily metabolized via the kidneys. Mechanistic studies suggested that 177Lu-DOTA-M-P induces DNA damage, thereby activating the mitochondrial apoptotic pathway. Targeted radioligand therapy results revealed that a single dose of 18.5 MBq of 177Lu-DOTA-M-P significantly inhibited U87-CD9 tumor growth. The effective doses for all human organs were estimated to be below the single-dose limit (21 CFR 361.1) established by U.S. regulatory standards, demonstrating a favorable safety profile for clinical translation. We developed a CD9-targeted peptide precursor, DOTA-M-P, enabling dual-functional radionuclide imaging and therapy for glioma, with human dose estimates supporting personalized regimens and theranostic applications.