Noninvasive Evaluation of CD20 Expression Using 64Cu-Labeled F(ab′)2 Fragments of Obinutuzumab in Lymphoma
Noninvasive Evaluation of CD20 Expression Using 64Cu-Labeled F(ab′)2 Fragments of Obinutuzumab in Lymphoma
复制标题
使用 Obinutuzumab 的 64Cu 标记 F(ab-)2 片段对淋巴瘤中 CD20 表达进行无创评估
DOI：
10.2967/jnumed.120.246595
复制
发表时间：
2021-03-01
影响因子：
9.3
通讯作者：
Cai, Weibo
中科院分区：
文献类型：
--
作者：
Kang, Lei;Li, Cuicui;Cai, Weibo
CD 20过表达的非霍奇金淋巴瘤通常提示进行性恶性肿瘤。Obinutuzumab是美国食品和药物管理局批准的下一代靶向CD 20的人源化单克隆抗体。先前使用Zr-89标记的obinutuzumab的研究已经成功地在体内成像了CD 20。然而，由于长时间的血液循环导致的肿瘤摄取延迟和放射性暴露增加限制了其临床转化。本研究旨在开发Cu-64标记的Obinutuzumab的F（ab '）（2）片段，用于在淋巴瘤异种移植肿瘤模型中成像CD 20。研究方法：使用化脓性链球菌（Streptococcus pyogenes）的IgG降解酶（IdeS）酶从obinutuzumab产生F（ab '）（2）片段，并用蛋白A珠纯化。采用十二烷基硫酸钠聚丙烯酰胺凝胶电泳和高效液相色谱法对产品及其稳定性进行了评价。F（ab '）（2）产物与p-SCN-Bn-NOTA（NOTA）缀合用于Cu-64放射性标记。Western blotting检测淋巴瘤细胞CD 20表达水平。采用酶联免疫吸附试验、流式细胞术和共聚焦成像技术检测结合亲和力。使用Cu-64-NOTA-F（ab '）（2）-obinutuzumab或Cu-64-NOTA-F（ab'）（2）-IgG在皮下淋巴瘤荷瘤小鼠中进行了系列PET成像和生物分布研究。结果如下：通过十二烷基硫酸钠聚丙烯酰胺凝胶电泳和高效液相色谱法证实了由IdeS消化系统产生的F（ab '）（2）-obinutuzumab和F（ab'）（2）-IgG。Cu ~（64）标记F（ab '）（2）片段的放化纯度≥ 98%，比活度为56.3 ± 7.9MBq/mg（n = 6）。酶联免疫吸附试验、流式细胞术和共聚焦成像证实，在5种淋巴瘤细胞系中，拉莫斯显示出最强的CD 20表达，而CLL-155显示出最低。PET成像显示在荷拉莫斯肿瘤的小鼠中Cu-64-NOTA-F（ab '）（2）-obinutuzumab的快速和持续的肿瘤摄取。拉莫斯模型中的峰值肿瘤摄取（9.08 +/-1.67%注射剂量/克组织[%ID/g]）显著高于CCL-155模型（2.78 +/-0.62% ID/g）或Cu-64-NOTA-F（ab '）（2）-IgG对照（1.93 +/-0.26% ID/g，n = 4，P < 0.001）。在Cu-64-N 0 TA-F（ab '）（2）-obinutuzumab组中，在注射后48小时，肿瘤与血液和肿瘤与肌肉的比率分别为7.3 +/-1.6和21.9 +/-9.0。在测量的脱靶器官中，肾脏显示出最高的摄取。体外免疫荧光染色证实了拉莫斯和CCL-155肿瘤模型中的差异CD 20表达。结论：本研究表明，Cu-64-NOTA-F（ab '）（2）-obinutuzumab在具有高对比度的CD 20阳性淋巴瘤中具有快速和持续的肿瘤摄取，这可以在临床上无创地评估CD 20水平。
CD20-overexpressed non-Hodgkin lymphoma typically indicates progressive malignancy. Obinutuzumab is a next-generation Food and Drug Administration-approved humanized monoclonal antibody that targets CD20. Previous studies with Zr-89-labeled obinutuzumab have successfully imaged CD20 in vivo. However, delayed tumor uptake and increased radioactive exposure caused by long blood circulation limit its clinical translation. This study aimed to develop Cu-64-labeled F(ab')(2) fragments of obinutuzumab for imaging CD20 in lymphoma xenograft tumor models. Methods: F(ab')(2) fragments were produced from obinutuzumab using an IgG-degrading enzyme of Streptococcus pyogenes (IdeS) enzyme and purified with protein A beads. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and high-performance liquid chromatography were performed to evaluate the products and their stability. F(ab')(2) products were conjugated with p-SCN-Bn-NOTA (NOTA) for Cu-64 radio labeling. Western blotting was performed to screen the CD20 expression levels of lymphoma cells. Enzyme-linked immunosorbent assay, flow cytometry, and confocal imaging were used to test the binding affinity in vitro. Serial PET imaging and biodistribution studies in subcutaneous lymphoma-bearing mice were performed using Cu-64-NOTA-F(ab')(2)-obinutuzumab or Cu-64-NOTA-F(ab')(2)-IgG. Results: F(ab')(2)-obinutuzumab and F(ab')(2)-IgG produced by the IdeS digestion system were confirmed with sodium dodecyl sulfate polyacrylamide gel electrophoresis and high-performance liquid chromatography. The radiochemical purity of Cu-64-labeled F(ab')(2) fragments was no less than 98%, and the specific activity was 56.3 +/- 7.9 MBq/mg (n = 6). Among the 5 lymphoma cell lines, Ramos showed the strongest expression of CD20, and CLL-155 showed the lowest, as confirmed by enzyme-linked immunosorbent assay, flow cytometry, and confocal imaging. PET imaging revealed rapid and sustained tumor uptake of Cu-64-NOTA-F(ab')(2)-obinutuzumab in Ramos tumor-bearing mice. The peak tumor uptake (9.08 +/- 1.67 percentage injected dose per gram of tissue [%ID/g]) in the Ramos model was significantly higher than that in the CCL-155 model (2.78 +/- 0.62 %ID/g) or the Cu-64-NOTA-F(ab')(2)-IgG control (1.93 +/- 0.26 %ID/g, n = 4, P < 0.001). The tumor-to-blood and tumor-to-muscle ratios were 7.3 +/- 1.6 and 21.9 +/- 9.0, respectively, at 48 h after injection in the Cu-64-NOTA-F(ab')(2)-obinutuzumab group. Of the measured off target organs, the kidneys showed the highest uptake. Ex vivo immunofluorescent staining verified the differential CD20 expression in the Ramos and CCL-155 tumor models. Conclusion: This study demonstrated that Cu-64-NOTA-F(ab')(2)-obinutuzumab had a rapid and sustained tumor uptake in CD20-positive lymphoma with high contrast, which could enable noninvasive evaluation of CD20 levels in the clinic.