Noninvasive Evaluation of CD20 Expression Using <sup>64</sup>Cu-Labeled F(ab′)<sub>2</sub> Fragments of Obinutuzumab in Lymphoma
Lei Kang, Cuicui Li, Zachary T. Rosenkrans, Jonathan W. Engle, Rongfu Wang, Dawei Jiang, Xiaojie Xu, Weibo Cai
Abstract
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 <sup>89</sup>Zr-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 <sup>64</sup>Cu-labeled F(ab′)<sub>2</sub> fragments of obinutuzumab for imaging CD20 in lymphoma xenograft tumor models. <b>Methods:</b> F(ab′)<sub>2</sub> fragments were produced from obinutuzumab using an IgG-degrading enzyme of <i>Streptococcus pyogenes</i> (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′)<sub>2</sub> products were conjugated with p-SCN-Bn-NOTA (NOTA) for <sup>64</sup>Cu radiolabeling. 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 <sup>64</sup>Cu-NOTA-F(ab′)<sub>2</sub>-obinutuzumab or <sup>64</sup>Cu-NOTA-F(ab′)<sub>2</sub>-IgG. <b>Results:</b> F(ab′)<sub>2</sub>-obinutuzumab and F(ab′)<sub>2</sub>-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 <sup>64</sup>Cu-labeled F(ab′)<sub>2</sub> fragments was no less than 98%, and the specific activity was 56.3 ± 7.9 MBq/mg (<i>n</i> = 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 <sup>64</sup>Cu-NOTA-F(ab′)<sub>2</sub>-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 <sup>64</sup>Cu-NOTA-F(ab′)<sub>2</sub>-IgG control (1.93 ± 0.26 %ID/g, <i>n</i> = 4, <i>P</i> < 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 <sup>64</sup>Cu-NOTA-F(ab′)<sub>2</sub>-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. <b>Conclusion:</b> This study demonstrated that <sup>64</sup>Cu-NOTA-F(ab′)<sub>2</sub>-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.