161Tb-Based Anti-L1CAM Radioimmunotherapy Shows Superior Efficacy in Eliminating Ovarian Cancer Stem Cells Compared with177Lu in Preclinical Models of Ovarian Cancer

Cancer stem cells (CSCs) are highly tumorigenic, self-renewable cells with a key role in tumor relapse, metastasis, and therapy resistance. Effective CSC-targeted therapies remain an unmet clinical need, strongly dependent on the selection of suitable targets and thorough validation of therapeutic agents. L1 cell adhesion molecule (L1CAM) is a targetable CSC-associated biomarker aberrantly expressed in various malignancies, including ovarian cancer (OC). 161Tb is attractive for clinical application because of its substantial emission of conversion electrons/Auger electrons as well as β- emission. Leveraging the high cytotoxicity of conversion electrons/Auger electrons, 161Tb is promising for radioimmunotherapy against radioresistant tumor cells such as CSCs. The aim of this study was to confirm the presence of L1CAM+/CD133+ ovarian CSCs in patient samples and preclinically investigate, in a tumor prevention mouse model, 161Tb-based anti-L1CAM radioimmunotherapy as a new therapeutic modality against CSCs compared with 177Lu-based anti-L1CAM radioimmunotherapy. Methods: L1CAM+/CD133+ CSCs were examined in OC samples by immunofluorescence. After radiolabeling anti-L1CAM DOTA-chCE7 with 177Lu or 161Tb and purification, we assessed radioimmunoconjugate quality by determining the radiochemical purity and the immunoreactive fraction. The internalized and membrane-bound fractions and the radiocytotoxicity of radiolabeled DOTA-chCE7 were evaluated with cell uptake and cell proliferation assays. Ovarian L1CAM+/CD133+ CSCs were sorted via fluorescence-activated cell sorting from OVCAR8 and SKOV3ip cells and inoculated into immunocompromised mice, who then received treatment with [177Lu]Lu-DOTA-chCE7 or [161Tb]Tb-DOTA-chCE7. Results: L1CAM+/CD133+ CSCs (0.3%-21%) were confirmed in samples from patients who were chemotherapy-naïve or had relapsed OC. [177Lu]Lu-DOTA-chCE7 and [161Tb]Tb-DOTA-chCE7 were produced with high radiochemical purity and retained 76%-96% immunoreactivity. Cell uptake after 15 h ranged from 50% to 75% for both radioimmunoconjugates. [161Tb]Tb-DOTA-chCE7 showed significantly increased cytotoxicity, eliminating all ovarian CSCs and tumor cells differentiated from the CSCs in vivo, compared with [177Lu]Lu-DOTA-chCE7 (3 tumors in OVCAR8 group and 1 tumor in SKOV3ip group). Follow-up tumor analysis confirmed that sorted ovarian L1CAM+/CD133+ CSCs regenerated the tumor heterogeneity in vivo. Conclusion: This work addresses the critical need for CSC-specific therapies in the clinics by establishing 161Tb-based anti-L1CAM radioimmunotherapy as a novel therapeutic modality against CSCs. We found that 161Tb-based anti-L1CAM radioimmunotherapy eliminated ovarian CSCs more efficiently than 177Lu-based anti-L1CAM radioimmunotherapy, emphasizing its promising therapeutic potential.