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.