RT Journal Article SR Electronic T1 Noninvasive evaluation of CD20 expression using 64Cu-labeled F(ab')2 fragments of obinutuzumab in lymphoma JF Journal of Nuclear Medicine JO J Nucl Med FD Society of Nuclear Medicine SP jnumed.120.246595 DO 10.2967/jnumed.120.246595 A1 Lei Kang A1 Cuicui Li A1 Zachary T. Rosenkrans A1 Jonathan W. Engle A1 Rongfu Wang A1 Dawei Jiang A1 Xiaojie Xu A1 Weibo Cai YR 2020 UL http://jnm.snmjournals.org/content/early/2020/08/21/jnumed.120.246595.abstract AB CD20 over-expressed non-Hodgkin lymphoma typically shows progressive malignancy. Obinutuzumab (obi) is a next-generation FDA-approved humanized monoclonal antibody that targets CD20. Previous studies with 89Zr-labeled obi has successfully imaged CD20 in vivo. However, delayed tumor uptake and increased radioactive exposure caused by long blood circulation limits its clinical translation. This study aims to develop 64Cu-labeled F(ab')2 fragments of obi for imaging CD20 in lymphoma xenograft tumor models. Methods F(ab’)2 fragments were produced from obi using an IdeS enzyme and purified with Protein A beads. SDS-PAGE and HPLC analysis 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. ELISA, 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-obi or 64Cu-NOTA-F(ab’)2-IgG. Results F(ab')2-obi and F(ab')2-IgG produced by the IdeS digestion system was confirmed with SDS-PAGE and HPLC analysis. 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 five lymphoma cell lines, Ramos showed the strongest expression of CD20 and CLL-155 showed the lowest as confirmed by ELISA, flow cytometry, and confocal imaging. PET imaging revealed rapid and sustained tumor uptake of 64Cu-NOTA-F(ab')2-obi in Ramos tumor-bearing mice. The peak tumor uptake (9.08 ± 1.67 %ID/g) in 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 p.i in the 64Cu-NOTA-F(ab')2-obi 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. Conclusions This study demonstrated that 64Cu-NOTA-F(ab')2-obi 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.