Purpose

Enzymolysis clearance strategy, characterized by releasing the non-reabsorbable radioactive fragment under the specific cleavage of enzymes, is confirmed to be a safe and effective way to reduce the renal radioactivity accumulation in mice. However, the effectiveness of this strategy in humans remains unknown. Human epidermal growth factor receptor 2 (HER2) is overexpressed in various types of tumors, and radiolabeled HER2 Affibody is believed to be an attractive tool for HER2-targeted theranostics. However, its wide application is limited by the high and persistent renal uptake. In this study, we intend to validate the effectiveness of enzymolysis clearance strategy in reducing renal accumulation by using a modified HER2 Affibody.

Materials and methods

A new HER2 Affibody ligand, NOTA-MVK-Z_HER2:2891, containing a cleavable Met-Val-Lys (MVK) linker was synthesized and labeled with ⁶⁸Ga. The microPET imaging study was performed in SKOV-3 tumor mice to assess the uptakes of the control ligand and the MVK one in tumors and kidneys. Seven healthy volunteers were included for biodistribution and dosimetric studies with both the control and MVK ligands performed 1 week apart. Urine and blood samples from healthy volunteers were collected for in vivo metabolism study of the two ligands. Four HER2-positive and two HER2-negative patients were recruited for [⁶⁸Ga]Ga-NOTA-MVK-Z_HER2:2891 PET/CT imaging at 2 and 4 h post-injection (p.i.).

Results

[⁶⁸Ga]Ga-NOTA-MVK-Z_HER2:2891 was stable both in PBS and in mouse serum. MicroPET images showed that the tumor uptake of [⁶⁸Ga]Ga-NOTA-MVK-Z_HER2:2891 was comparable to that of [⁶⁸Ga]Ga-NOTA-Z_HER2:2891 at all the time points, while the kidney uptake was significantly reduced 40 min p.i. (P < 0.05). The biodistribution study in healthy volunteers showed that the kidney uptake of MVK ligand was significantly lower than that of the control ligand at 1 h p.i. (P < 0.05), with the SUV_mean of 34.3 and 45.8, respectively, while the uptakes of the two ligands in the other organs showed negligible difference. The effective doses of the MVK ligand and the control one were 26.1 and 28.7 µSv/MBq, respectively. The enzymolysis fragment of [⁶⁸Ga]Ga-NOTA-Met-OH was observed in the urine samples of healthy volunteers injected with the MVK ligand, indicating that the enzymolysis clearance strategy worked in humans. The PET/CT study of patients showed that the range of SUV_max of HER2-positive lesions was 9.4–21, while that of HER2-negative lesions was 2.7–6.2, which suggested that the MVK modification did not affect the ability of Z_HER2:2891 structure to bind with HER2.

Conclusion

We for the first time demonstrated that enzymolysis clearance strategy can effectively reduce renal radioactivity accumulation in humans. This strategy is expected to decrease renal radiation dose of peptide and small protein-based radiotracers, especially in the field of radionuclide therapy.

中文翻译：

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使用改良的 [68Ga]Ga-HER2 Affibody 对降低肾脏放射性的酶解清除策略进行首次人体验证

目的

酶解清除策略的特点是在酶的特异性裂解下释放不可重吸收的放射性片段，被证实是减少小鼠肾脏放射性蓄积的安全有效的方法。然而，这种策略对人类的有效性仍然未知。人表皮生长因子受体 2 (HER2) 在各种类型的肿瘤中过度表达，放射性标记的 HER2 Affibody 被认为是针对 HER2 靶向治疗诊断的有吸引力的工具。然而，其广泛应用受到肾脏高且持续摄取的限制。在本研究中，我们打算通过使用改良的 HER2 Affibody 来验证酶解清除策略在减少肾脏蓄积方面的有效性。

材料和方法

合成了一种新的 HER2 Affibody 配体 NOTA-MVK-Z _HER2:2891，含有可裂解的 Met-Val-Lys (MVK) 连接子，并用⁶⁸ Ga 进行标记。在 SKOV-3 肿瘤小鼠中进行 microPET 成像研究，以评估对照配体和 MVK 配体在肿瘤和肾脏中的摄取。七名健康志愿者被纳入生物分布和剂量测定研究，对照和 MVK 配体均间隔 1 周进行。收集健康志愿者的尿液和血液样本，用于两种配体的体内代谢研究。招募四名 HER2 阳性和两名 HER2 阴性患者在注射后 (pi) 2 小时和 4 小时进行 [ ⁶⁸ Ga]Ga-NOTA-MVK-Z _{HER2:2891 PET/CT 成像。}

结果

[ ⁶⁸ Ga]Ga-NOTA-MVK-Z _HER2:2891在 PBS 和小鼠血清中均稳定。^{MicroPET 图像显示，在所有时间点，[ 68} Ga]Ga-NOTA-MVK-Z _HER2:2891的肿瘤摄取与 [ ⁶⁸ Ga]Ga-NOTA-Z _HER2:2891相当，而肾脏摄取则为注射后 40 分钟显着降低（P  < 0.05）。健康志愿者的生物分布研究表明，感染后 1 h，MVK 配体的肾脏摄取显着低于对照配体（P  < 0.05），SUV_平均值分别为 34.3 和 45.8，而两者的摄取量其他器官中的配体显示出可忽略不计的差异。MVK配体和对照配体的有效剂量分别为26.1和28.7 µSv/MBq。^{在注射MVK配体的健康志愿者的尿液样本中观察到[ 68} Ga]Ga-NOTA-Met-OH的酶解片段，表明酶解清除策略在人类中有效。患者的PET/CT研究显示，HER2阳性病灶的SUV _{max范围为9.4~21，而HER2阴性病灶的SUV max范围为2.7~6.2，这表明MVK修饰并不影响Z HER2}的能力_:2891与 HER2 结合的结构。

结论

我们首次证明酶解清除策略可以有效减少人体肾脏放射性积累。该策略有望减少基于肽和小蛋白的放射性示踪剂的肾脏辐射剂量，特别是在放射性核素治疗领域。