PT - JOURNAL ARTICLE AU - Lei Kang AU - Cuicui Li AU - Zachary T. Rosenkrans AU - Jonathan W. Engle AU - Rongfu Wang AU - Dawei Jiang AU - Xiaojie Xu AU - Weibo Cai TI - Noninvasive evaluation of CD20 expression using <sup>64</sup>Cu-labeled F(ab')2 fragments of obinutuzumab in lymphoma AID - 10.2967/jnumed.120.246595 DP - 2020 Aug 01 TA - Journal of Nuclear Medicine PG - jnumed.120.246595 4099 - http://jnm.snmjournals.org/content/early/2020/08/21/jnumed.120.246595.short 4100 - http://jnm.snmjournals.org/content/early/2020/08/21/jnumed.120.246595.full 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&lt;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.