Radiolabeled small-molecule DOTA-haptens can be combined with antitumor/anti-DOTA bispecific antibodies (BsAbs) for pretargeted radioimmunotherapy (PRIT). For optimized delivery of the theranostic γ- and β-emitting isotope ¹⁷⁷Lu with DOTA-based PRIT (DOTA-PRIT), bivalent Gemini (DOTA-Bn-thiourea-PEG4-thiourea-Bn-DOTA, aka (3,6,9,12-tetraoxatetradecane-1,14-diyl)bis(DOTA-benzyl thiourea)) was developed. Methods: Gemini was synthesized by linking 2 S-2-(4-isothiocyanatobenzyl)-DOTA molecules together via a 1,14-diamino-PEG4 linker. [¹⁷⁷Lu]Lu-Gemini was prepared with no-carrier-added ¹⁷⁷LuCl₃ to a molar-specific activity of 123 GBq/μmol and radiochemical purity of more than 99%. The specificity of BsAb-¹⁷⁷Lu-Gemini was verified in vitro. Subsequently, we evaluated biodistribution and whole-body clearance for [¹⁷⁷Lu]Lu-Gemini and, for comparison, our gold-standard monovalent [¹⁷⁷Lu]Lu-S-2-(4-aminobenzyl)-DOTA ([¹⁷⁷Lu]Lu-DOTA-Bn) in naïve (tumor-free) athymic nude mice. For our proof-of-concept system, a 3-step pretargeting approach was performed with an established DOTA-PRIT regimen (anti-GPA33/anti-DOTA IgG-scFv BsAb, a clearing agent, and [¹⁷⁷Lu]Lu-Gemini) in mouse models. Results: Initial in vivo studies showed that [¹⁷⁷Lu]Lu-Gemini behaved similarly to [¹⁷⁷Lu]Lu-DOTA-Bn, with almost identical blood and whole-body clearance kinetics, as well as biodistribution and mouse kidney dosimetry. Pretargeting [¹⁷⁷Lu]Lu-Gemini to GPA33-expressing SW1222 human colorectal xenografts was highly effective, leading to absorbed doses of [¹⁷⁷Lu]Lu-Gemini for blood, tumor, liver, spleen, and kidneys of 3.99, 455, 6.93, 5.36, and 14.0 cGy/MBq, respectively. Tumor–to–normal tissue absorbed-dose ratios (i.e., therapeutic indices [TIs]) for the blood and kidneys were 114 and 33, respectively. In addition, we demonstrate that the use of bivalent [¹⁷⁷Lu]Lu-Gemini in DOTA-PRIT leads to improved TIs and augmented [¹⁷⁷Lu]Lu-Gemini tumor uptake and retention in comparison to monovalent [¹⁷⁷Lu]Lu-DOTA-Bn. Finally, we established efficacy in SW1222 tumor-bearing mice, demonstrating that a single injection of anti-GPA33 DOTA-PRIT with 44 MBq (1.2 mCi) of [¹⁷⁷Lu]Lu-Gemini (estimated tumor-absorbed dose, 200 Gy) induced complete responses in 5 of 5 animals and a histologic cure in 2 of 5 (40%) animals. Moreover, a significant increase in survival compared with nontreated controls was noted (maximum tolerated dose not reached). Conclusion: We have developed a bivalent DOTA-radiohapten, [¹⁷⁷Lu]Lu-Gemini, that showed improved radiopharmacology for DOTA-PRIT application. The use of bivalent [¹⁷⁷Lu]Lu-Gemini in DOTA-PRIT, as opposed to monovalent [¹⁷⁷Lu]Lu-DOTA-Bn, allows curative treatments with considerably less administered ¹⁷⁷Lu activity while still achieving high TIs for both the blood (>100) and the kidneys (>30).

放射性标记的小分子 DOTA 半抗原可与抗肿瘤/抗 DOTA 双特异性抗体 (BsAb) 组合用于预靶向放射免疫治疗 (PRIT)。用于使用基于 DOTA 的 PRIT (DOTA-PRIT)、二价 Gemini (DOTA-Bn-硫脲-PEG4-硫脲-Bn-DOTA，又名 (3,6,9) 优化治疗诊断 γ 和 β 发射同位素¹⁷⁷ Lu 的递送开发了 ,12-四氧四烷-1,14-二基)双(DOTA-苄基硫脲))。方法： Gemini 是通过 1,14-二氨基-PEG4 连接体将 2 个S -2-(4-异硫氰酸苄基)-DOTA 分子连接在一起合成的。采用不添加载体的¹⁷⁷ LuCl ₃制备了[ ¹⁷⁷ Lu]Lu-Gemini，其摩尔比活度为123 GBq/μmol，放射化学纯度大于99%。 BsAb- ¹⁷⁷ Lu-Gemini 的特异性已在体外得到验证。随后，我们评估了 [ ¹⁷⁷ Lu]Lu-Gemini 的生物分布和全身清除率，并且为了进行比较，我们的金标准单价 [ ¹⁷⁷ Lu]Lu- S -2-(4-氨基苄基)-DOTA ([ ¹⁷⁷ Lu] Lu-DOTA-Bn）在幼稚（无肿瘤）无胸腺裸鼠中。对于我们的概念验证系统，使用已建立的 DOTA-PRIT 方案（抗 GPA33/抗 DOTA IgG-scFv BsAb、清除剂和 [ ¹⁷⁷ Lu]Lu-Gemini）执行 3 步预靶向方法在小鼠模型中。结果：初步体内研究表明，[ ¹⁷⁷ Lu]Lu-Gemini 的行为与[ ¹⁷⁷ Lu]Lu-DOTA-Bn 相似，具有几乎相同的血液和全身清除动力学，以及生物分布和小鼠肾脏剂量测定。 [ ¹⁷⁷ Lu]Lu-Gemini 预靶向表达 GPA33 的 SW1222 人结肠直肠异种移植物非常有效，导致血液、肿瘤、肝脏、脾脏和肾脏的 [ ¹⁷⁷ Lu]Lu-Gemini 吸收剂量为 3.99、455、6.93、分别为 5.36 和 14.0 cGy/MBq。血液和肾脏的肿瘤与正常组织吸收剂量比（即治疗指数 [TI]）分别为 114 和 33。此外，我们证明，与单价[ ^177Lu ]Lu-DOTA相比，在DOTA-PRIT中使用二价[ ^177Lu ]Lu-Gemini可改善TI并增强[ ^177Lu ]Lu-Gemini肿瘤摄取和保留布恩。最后，我们在 SW1222 荷瘤小鼠中建立了功效，证明单次注射抗 GPA33 DOTA-PRIT 和 44 MBq (1.2 mCi) 的 [ ¹⁷⁷ Lu]Lu-Gemini（估计肿瘤吸收剂量，200 Gy）可诱导5 只动物中有 5 只完全缓解，5 只动物中有 2 只 (40%) 组织学治愈。此外，与未治疗的对照相比，存活率显着增加（未达到最大耐受剂量）。结论：我们开发了一种二价 DOTA-放射性半抗原，[ ¹⁷⁷ Lu]Lu-Gemini，其在 DOTA-PRIT 应用中显示出改进的放射性药理学。与单价 [ ¹⁷⁷ Lu]Lu-DOTA-Bn 相比，在 DOTA-PRIT 中使用二价 [ ¹⁷⁷ Lu]Lu-Gemini 可以在显着减少¹⁷⁷ Lu 活性的情况下进行治疗，同时仍然实现血液的高 TI（ >100) 和肾脏 (>30)。