Noninvasive Evaluation of CD20 Expression Using ⁶⁴Cu-Labeled F(ab′)₂ Fragments of Obinutuzumab in Lymphoma

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 ⁸⁹Zr-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 ⁶⁴Cu-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 ⁶⁴Cu 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 ⁶⁴Cu-NOTA-F(ab′)₂-obinutuzumab or ⁶⁴Cu-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 ⁶⁴Cu-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 ⁶⁴Cu-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 ⁶⁴Cu-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 ⁶⁴Cu-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 ⁶⁴Cu-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.