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 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.

CD38‐Targeted Theranostics of Lymphoma with 89Zr/177Lu‐Labeled Daratumumab

“…[ 12 ] CD38 has been found to be highly expressed for some lymphoma such as MM. [ 25 ] The first anti‐CD38‐humanized mAb daratumumab was subsequently approved for the treatment of relapsed MM. In our study, 89 Zr‐labeled dara showed a high tumor uptake at around 20 %ID g −1 and low uptake in other nontarget organs for CD38‐positive models, providing excellent tumor contrast.…”

“…Its stability was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and radio thin-layer chromatography (TLC) analysis as previously reported. [25] PET Imaging of 89 Zr-Labeled Antibodies: PET imaging was performed using an Inveon PET/CT scanner (Siemens) after intravenously injecting 89 Zr-labeled dara (≈7.4 MBq). At each time point, mice were anesthetized and scanned in a prone position.…”

“…For direct method, N‐Hydroxysuccinimide –fluorescein (NHS–FITC, ThermoFisher)‐conjugated antibody was prepared and purified. [ 25 ] Then, FITC‐conjugated DTPA– or Df–dara were tested and processed using a LSRFortessa cell analyzer (BD Biosciences) and FlowJo software (Tree Star) for the mean fluorescence intensities.…”

“…Its stability was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) and radio thin‐layer chromatography (TLC) analysis as previously reported. [ 25 ]…”

High-performance renal imaging with a radiolabeled, non-excretable chimeric fusion protein

“…Additionally, passive tracer excretion via the kidneys may hinder active observation of renal dysfunction, scaling down necessary imaging contrast needed for confident diagnosis. Low molecular weight antibody fragments in the forms of Fab, single-chain Fv fragment (scFv), diabody, and nanobody, have all been found to excrete from the kidneys 1 , 13 . Going through lysosomal proteolysis, glomerular filtration, and subsequent tubular reabsorption, these radioactive renal metabolites display rapid renal excretion and steady kidney retention, adding difficulties to kidney function interpretation.…”