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 89 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 64 Cu-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 64 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 64 Cu-NOTA-F(ab′) 2 -obinutuzumab or 64 Cu-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 64 Cu-labeled F(ab′) 2 fragments was no less than 98%, and the specific activity was 56.3 ± 7.9 MBq/mg (n 5 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 64 Cu-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 64 Cu-NOTA-F(ab′) 2 -IgG control (1.93 ± 0.26 %ID/ g, n 5 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 64 Cu-NOTA-F(ab′) 2 -obinutuzumab group. Of the measured offtarget 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 64 Cu-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.

Objectives. To explore the diagnostic value of 18F-FDG PET/CT bone marrow uptake pattern (BMUP) in detecting bone marrow involvement (BMI) in pediatric neuroblastoma (NB) patients. Methods. Ninety-eight NB patients were enrolled in BMI analysis. Four patterns of bone marrow uptake were categorized based on pretreatment cF-FDG PET/CT images. Some crucial inspection indexes and 18F-FDG PET/CT metabolic parameters were analyzed. The BMUP was divided into BMUP1, BMUP2, BMUP3, and BMUP4. Paired-like homeobox 2b (PHOX2B) of bone marrow and blood, bone marrow biopsy (BMB) result, and 18F-FDG PET/CT were compared to detect BMI. All patients were followed up for at least six months. Results. BMUP had excellent consistency among different physicians. Kappa coefficients of two residents and two attending physicians and between the resident and attending physician, were 0.857, 0.891, and 0.845, respectively. The optimal cut-off value of SUVmax-Bone/Liver was 2.08 to diagnose BMI for BMUP3 patients, and the area under curve (AUC) was 0.873. AUC of PHOX2B of bone marrow (PHOX2B of BM), PHOX2B of blood, BMB, and 18F-FDG PET/CT were 0.916, 0.811, 0.806, and 0.904, respectively. There was no significant difference between PHOX2B of BM and PET/CT. Positive predictive value, negative predictive value, sensitivity, and specificity in diagnosis of BMI were 92.9%, 92.9%, 97.0%, and 83.9% for PET/CT and 96.7%, 80.6%, 89.6%, and 93.5% for PHOX2B of BM, respectively. Conclusions. BMUP of pretreatment 18F-FDG PET/CT is a simple and practical method, which has a relatively high diagnostic efficiency in detecting BMI and might decrease unnecessary invasive inspections in some pediatric NB patients.

No abstract