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.

Graphical Abstract

中文翻译：

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[149Tb]Tb-DOTATATE 和 [149Tb]Tb-DOTA-LM3 用于肿瘤靶向 α 治疗的临床前研究

 目的

Terbium-149 是一种短寿命的 α 粒子发射器，可能可用于肿瘤靶向治疗。本研究的目的是研究铽 149 与生长抑素受体 （SSTR） 激动剂 DOTATATE 和 SSTR 拮抗剂 DOTA-LM3 的联合使用。对放射性肽进行评估，以比较它们在体外和体内的治疗效果。

 方法

铽-149 由 ISOLDE/CERN 生产，并在 Paul Scherrer 研究所进行化学纯化。在 pH 4.5 的标准标记条件下，用 [¹⁴⁹Tb]TbCl₃ 对生长抑素类似物进行放射性标记。在 SSTR 阳性 AR42J 大鼠胰腺肿瘤细胞暴露于各种活性浓度的 [¹⁴⁹Tb]Tb-DOTATATE 和 [¹⁴⁹Tb]Tb-DOTA-LM3 16-18 小时后，进行细胞活力 （MTT） 和存活测定（集落形成）测定。使用 γ-H2A.X 和 53BP1 的免疫荧光成像确定 DNA 双链断裂。用注射 1 × 5 MBq 或 2 × 5 MBq 相应放射性肽的 AR42J 荷瘤小鼠进行治疗研究。评估高达 40 MBq [¹⁴⁹Tb]Tb-DOTATATE 或 40 MBq [¹⁴⁹Tb]Tb-DOTA-LM3 的耐受性，以评估对无肿瘤免疫功能正常小鼠骨髓和肾脏的不良影响。

 结果

在放射化学纯度≥高达 20 MBq/nmol 的摩尔活性下，实现了肽的放射性标记。AR42J 细胞活力以活性依赖性方式降低，[¹⁴⁹Tb]Tb-DOTA-LM3 比 [¹⁴⁹Tb]Tb-DOTATATE 略强（EC₅₀：0.5 vs. 1.2 kBq/mL）。两种放射性肽诱导每个细胞核相似数量的 γ-H2A.X 和 53BP1 病灶，这表明 AR42J 肿瘤细胞中的 DNA 损伤。注射 1 × 5 MBq 放射性肽的荷瘤小鼠导致 [¹⁴⁹Tb]Tb-DOTATATE 和 [¹⁴⁹Tb]Tb-DOTA-LM3 的中位生存时间分别为 16.5 天和 19 天，而未治疗的对照小鼠仅为 8 天。应用 2 × 5 MBq 的放射性肽进一步将中位生存时间分别延长至 30 天和 29 天。无肿瘤治疗小鼠的血细胞计数和血浆生物标志物值与未治疗对照组相似。所有小鼠的 [^99mTc] Tc-DMSA 的肾脏积累相似，表明肾功能正常。

 结论

¹⁴⁹ 元基于 TB 的放射性肽有效降低了体外肿瘤细胞的活力以及小鼠的肿瘤生长，而不会引起相关不良事件，无论是否使用 SSTR 激动剂或拮抗剂。这些数据鼓励铽 149 的进一步临床前应用，以评估其与其他肿瘤靶向药物联合使用的潜力。

 图形摘要