68Ga-DOTA-PDL1P的设计合成及其在黑色素瘤小鼠模型中的应用研究

doi:10.19401/j.cnki.1007-3639.2023.04.005

中国癌症杂志 ›› 2023, Vol. 33 ›› Issue (4): 354-360.doi: 10.19401/j.cnki.1007-3639.2023.04.005

⁶⁸Ga-DOTA-PDL1P的设计合成及其在黑色素瘤小鼠模型中的应用研究

杨梓怡¹^,²^,³^,⁴^,⁵(), 李盼丽¹^,²^,³^,⁴^,⁵, 顾丙新¹^,²^,³^,⁴^,⁵, 刘成¹^,²^,³^,⁴^,⁵, 宋少莉¹^,²^,³^,⁴^,⁵, 许晓平¹^,²^,³^,⁴^,⁵()

1.复旦大学附属肿瘤医院核医学科，复旦大学上海医学院肿瘤学系，上海 200032
2.复旦大学生物医学影像研究中心，上海 200032
3.上海分子影像探针工程技术中心，上海 200032
4.上海市质子重离子医院核医学科，上海 201315
5.核物理与离子束应用教育部重点实验室，上海 200433

收稿日期:2022-09-05 修回日期:2023-03-08 出版日期:2023-04-30 发布日期:2023-05-15
通信作者: 许晓平（ORCID: 0000-0001-9656-6233），药师，副研究员。
作者简介:杨梓怡（ORCID: 0000-0002-8947-2623），住院医师。
基金资助:
国家自然科学基金(12275057);国家自然科学基金(81971648);国家自然科学基金重点项目(32030061);专利(CN 110981940 A)

The synthesis of a novel molecular imaging probe ⁶⁸Ga-DOTA-PDL1P and application in mouse model of melanoma

YANG Ziyi¹^,²^,³^,⁴^,⁵(), LI Panli¹^,²^,³^,⁴^,⁵, GU Bingxin¹^,²^,³^,⁴^,⁵, LIU Cheng¹^,²^,³^,⁴^,⁵, SONG Shaoli¹^,²^,³^,⁴^,⁵, XU Xiaoping¹^,²^,³^,⁴^,⁵()

1. Department of Nuclear Medicine, Fudan University Shanghai Cancer Center; Department of Oncology, Shanghai Medical College, Fudan University, Shanghai 200032, China
2. Center for Biomedical Imaging, Fudan University, Shanghai 200032, China
3. Shanghai Engineering Research Center of Molecular Imaging Probes, Shanghai 200032, China
4. Department of Nuclear Medicine, Shanghai Proton and Heavy Ion Center, Shanghai, 201321, China
5. Key Laboratory of Nuclear Physics and Ion-beam Application (MOE), Fudan University, Shanghai 200433, China

Received:2022-09-05 Revised:2023-03-08 Published:2023-04-30 Online:2023-05-15
Contact: XU Xiaoping

摘要/Abstract

摘要：

背景与目的：以程序性死亡[蛋白]-1（programmed death-1，PD-1）/程序性死亡[蛋白]配体-1（programmed death ligand-1，PD-L1）抑制剂为代表的免疫治疗药物在临床上获得巨大成功，但整体有效率差异很大。现阶段在肿瘤开始治疗前，检测患者PD-1/PD-L1的表达情况是临床用药的重要依据，但目前检测PD-L1表达的方法需要获取患者的标本，而获取标本存在创伤性。因此，迫切需要开发一种无创性活体内特异性检测PD-L1表达的方法。我们设计合成了一种新型靶向PD-L1多肽的正电子发射断层显像（positron emission tomography，PET）显像剂⁶⁸Ga-DOTA-PDL1P，本研究通过对⁶⁸Ga-DOTA-PDL1P的标记率、放射化学纯度及稳定性进行质量控制评估，并在黑色素瘤小鼠模型中进行应用评价，以期获得有转化前景的⁶⁸Ga-DOTA-PDL1P新型免疫PET探针。方法：利用镓-68（⁶⁸Ga）标记 DOTA-PDL1P，并对其放射性化学纯度和稳定性进行质量控制评估；使用鼠源性黑色素瘤B16-F10细胞系进行体外细胞摄取及阻断实验以评价其特异靶向性能；采用B16-F10荷瘤小鼠进行生物分布实验分析该PET显像剂在体内的代谢情况；通过PET/CT显像观察⁶⁸Ga-DOTA-PDL1P 在B16-F10荷瘤小鼠中的肿瘤摄取程度并进行半定量分析肿瘤靶本比（tumor/muscle，T/M）；另外对肿瘤样本进行放射自显影进一步分析肿瘤对⁶⁸Ga-DOTA-PDL1P探针的摄取情况。采用肿瘤细胞阳性比例分数（tumor cell proportion score，TPS）及联合阳性分数（combined positive score，CPS）指标分析免疫组织化学染色结果评价肿瘤组织的PD-L1表达情况。结果：⁶⁸Ga-DOTA-PDL1P的标记率及放射化学纯度均大于99%，并在磷酸缓冲盐溶液（phosphate-buffered saline，PBS）和胎牛血清（fetal bovine serum，FBS）中温育3 h后仍保持较好的稳定性（放化纯度均在90%以上）。细胞摄取结果示，⁶⁸Ga-DOTA-PDL1P可被B16-F10细胞特异性摄取，且该特异性摄取能够被PD-L1P多肽竞争结合阻断。生物分布实验结果示⁶⁸Ga-DOTA-PDL1P主要经泌尿系统排泄。荷瘤小鼠microPET/CT显像结果表明肿瘤组织高度摄取⁶⁸Ga-DOTA-PDL1P，T/M为6.7。放射自显影结果证实肿瘤⁶⁸Ga-DOTA-PDL1P高摄取部位与免疫组织化学染色PD-L1表达阳性区域一致，证明⁶⁸Ga-DOTA-PDL1P能够特异性与PD-L1阳性的肿瘤细胞结合。结论：本研究合成的⁶⁸Ga-DOTA-PDL1P新型PET显像剂稳定性好，能特异性靶向PD-L1阳性肿瘤细胞，是一种有转化前景的靶向PD-L1蛋白的新型免疫PET探针。

关键词: 细胞程序性死亡-配体1, 黑色素瘤, 镓放射性同位素, 正电子发射断层显像术

Abstract:

Background and purpose: The immunogenic drugs represented by programmed cell death 1 (PD-1) and programmed death ligand-1 (PD-L1) have achieved great success in clinic. However, with the development of clinical application, the overall effective rate varies greatly. At present, detection of the expression of PD-1/PD-L1 before the start of tumor treatment is an important basis for clinical medication. However, the current detection method of PD-L1 expression requires the acquisition of patient specimens, which is traumatic. Therefore, there is an urgent need to develop a noninvasive method to detect PD-L1 expression in vivo. This study innovatively designed and synthesized a new positron emission tomography (PET) imaging agent targeting PD-L1 polypeptide ⁶⁸Ga-DOTA-PDL1P. In this study, the labeling rate, radiochemical purity and stability of ⁶⁸Ga-DOTA-PDL1P were evaluated for quality control. And the application was evaluated in the mouse model of melanoma in order to obtain a promising ⁶⁸Ga-DOTA-PDL1P immune PET probe. Methods: Gallium-68 (⁶⁸Ga) was used to label DOTA-PDL1P, and its radiochemical purity and stability were evaluated by quality control. To evaluate its specific targeting performance, the cell uptake and blocking experiments were carried out in murine melanoma B16-F10 cells. The biodistribution experiment was performed in B16-F10 tumor-bearing mice to analyze the metabolism of the PET imaging agent in vivo. The Tumor uptake of ⁶⁸Ga-DOTA-PDL1P in B16-F10 tumor-bearing mice was observed by PET/CT imaging, and the tumor/muscle (T/M) ratio was semi-quantitatively analyzed. In addition, radioautography was performed to further analyze the tumor uptake of ⁶⁸Ga-DOTA-PDL1P probe. Tumor cell proportion score (TPS) and combined positive score (CPS) were used to analyze the immunohistochemical staining results to evaluate the expression of PD-L1 in tumor tissues. Results: The labeling efficiency and radiochemical purity of ⁶⁸Ga-DOTA-PDL1P were more than 99%, and the radiochemical purity of ⁶⁸Ga-DOTA-PDL1P was more than 90% after 3 h of incubation in PBS and fetal bovine serum. Cellular uptake results showed that ⁶⁸Ga-DOTA-PDL1P could be specifically taken up by B16-F10 cells, and the specific uptake could be blocked by PD-L1P peptide competitive binding. Biodistribution assay showed that ⁶⁸Ga-DOTA-PDL1P was mainly excreted through the urinary system. The results of microPET/CT imaging of tumor-bearing mice showed a high uptake of ⁶⁸Ga-DOTA-PDL1P in tumor tissues, and the T/M ratio was 6.7. The autoradiography results confirmed that ⁶⁸Ga-DOTA-PDL1P high uptake in the tumor was consistent with the PD-L1 positive area of immunohistochemical staining, which proved that ⁶⁸Ga-DOTA-PDL1P could specifically bind to PD-L1 positive malignant tumor cells. Conclusion: The novel ⁶⁸Ga-DOTA-PDL1P PET imaging agent synthesized in this study has good stability and can specifically target PD-L1 positive tumor cells, which is a promising new immune PET probe targeting PD-L1 protein.

Key words: PD-L1, Malignant melanoma, ⁶⁸Ga, PET/CT

中图分类号:

R730.44

杨梓怡, 李盼丽, 顾丙新, 刘成, 宋少莉, 许晓平. ⁶⁸Ga-DOTA-PDL1P的设计合成及其在黑色素瘤小鼠模型中的应用研究[J]. 中国癌症杂志, 2023, 33(4): 354-360.

YANG Ziyi, LI Panli, GU Bingxin, LIU Cheng, SONG Shaoli, XU Xiaoping. The synthesis of a novel molecular imaging probe ⁶⁸Ga-DOTA-PDL1P and application in mouse model of melanoma[J]. China Oncology, 2023, 33(4): 354-360.

图/表 5

参考文献 23

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⁶⁸Ga-DOTA-PDL1P的设计合成及其在黑色素瘤小鼠模型中的应用研究

The synthesis of a novel molecular imaging probe ⁶⁸Ga-DOTA-PDL1P and application in mouse model of melanoma

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