Preclinical investigation of [¹⁴⁹Tb]Tb-DOTATATE and [¹⁴⁹Tb]Tb-DOTA-LM3 for tumor-targeted alpha therapy

Purpose: Terbium-149 is a short-lived α-particle emitter, potentially useful for tumor-targeted therapy. The aim of this study was to investigate terbium-149 in combination with the somatostatin receptor (SSTR) agonist DOTATATE and the SSTR antagonist DOTA-LM3. The radiopeptides were evaluated to compare their therapeutic efficacy in vitro and in vivo.

Methods: Terbium-149 was produced at ISOLDE/CERN and chemically purified at the Paul Scherrer Institute. Radiolabeling of somatostatin analogues with [¹⁴⁹Tb]TbCl₃ was performed under standard labeling conditions at pH 4.5. Cell viability (MTT) and survival assays (colony forming) assays were performed after 16-18 h exposure of SSTR-positive AR42J rat pancreatic tumor cells to various activity concentrations of [¹⁴⁹Tb]Tb-DOTATATE and [¹⁴⁹Tb]Tb-DOTA-LM3. DNA double-strand breaks were determined using immunofluorescence imaging of γ-H2A.X and 53BP1. Therapy studies were performed with AR42J tumor-bearing mice injected with 1 × 5 MBq or 2 × 5 MBq of the respective radiopeptide. The tolerability of up to 40 MBq [¹⁴⁹Tb]Tb-DOTATATE or 40 MBq [¹⁴⁹Tb]Tb-DOTA-LM3 was assessed with regard to undesired effects to the bone marrow and kidneys in immunocompetent mice without tumors.

Results: The radiolabeling of peptides was achieved at molar activities of up to 20 MBq/nmol at ≥ 98% radiochemical purity. AR42J cell viability was reduced in an activity-dependent manner, with [¹⁴⁹Tb]Tb-DOTA-LM3 being slightly more potent than [¹⁴⁹Tb]Tb-DOTATATE (EC₅₀: 0.5 vs. 1.2 kBq/mL). Both radiopeptides induced a similar number of γ-H2A.X and 53BP1 foci per nuclei, which indicated DNA damage in AR42J tumor cells. Injection of tumor-bearing mice with 1 × 5 MBq radiopeptide resulted in median survival times of 16.5 days and 19 days for [¹⁴⁹Tb]Tb-DOTATATE and [¹⁴⁹Tb]Tb-DOTA-LM3, respectively, as compared to only 8 days for untreated control mice. Application of 2 × 5 MBq of the radiopeptides further extended the median survival times to 30 days and 29 days, respectively. The blood cell counts and values for blood plasma biomarkers of treated mice without tumors were similar to those of untreated controls. Renal accumulation of [^99mTc]Tc-DMSA was similar in all mice, indicating normal kidney function.

Conclusion: ¹⁴⁹Tb-based radiopeptides effectively reduced the viability of tumor cells in vitro as well as the tumor growth in mice without causing relevant adverse events, irrespective of whether the SSTR agonist or antagonist was employed. These data encourage further preclinical application of terbium-149 to evaluate its potential in combination with other tumor-targeting agents.