Radiolabeling of rituximab with188 Re and99m Tc using the tricarbonyl technology

Abstract Introduction The most successful clinical studies of immunotherapy in patients with non-Hodgkin's lymphoma (NHL) use the antibody rituximab (RTX) targeting CD20+ B-cell tumors. Rituximab radiolabeled with β− emitters could potentiate the therapeutic efficacy of the antibody by virtue of the particle radiation. Here, we report on a direct radiolabeling approach of rituximab with the99m Tc- and188 Re-tricarbonyl core (IsoLink technology). Methods The native format of the antibody (RTXwt ) as well as a reduced form (RTXred ) was labeled with99m Tc/188 Re(CO)3 . The partial reduction of the disulfide bonds to produce free sulfhydryl groups (–SH) was achieved with 2-mercaptoethanol. Radiolabeling efficiency, in vitro human plasma stability as well as transchelation toward cysteine and histidine was investigated. The immunoreactivity and binding affinity were determined on Ramos and/or Raji cells expressing CD20. Biodistribution was performed in mice bearing subcutaneous Ramos lymphoma xenografts. Results The radiolabeling efficiency and kinetics of RTXred were superior to that of RTXwt (99m Tc: 98% after 3 h for RTXred vs. 70% after 24 h for RTXwt ).99m Tc(CO)3 -RTXred was used without purification for in vitro and in vivo studies whereas188 Re(CO)3 -RTXred was purified to eliminate free188 Re-precursor. Both radioimmunoconjugates were stable in human plasma for 24 h at 37°C. In contrast, displacement experiments with excess cysteine/histidine showed significant transchelation in the case of99m Tc(CO)3 -RTXred but not with pre-purified188 Re(CO)3 -RTXred . Both conjugates revealed high binding affinity to the CD20 antigen ( Kd =5–6 nM). Tumor uptake of188 Re(CO)3 -RTXred was 2.5 %ID/g and 0.8 %ID/g for99m Tc(CO)3 -RTXred 48 h after injection. The values for other organs and tissues were similar for both compounds, for example the tumor-to-blood and tumor-to-liver ratios were 0.4 and 0.3 for99m Tc(CO)3 -RTXred and for188 Re(CO)3 -RTXred 0.5 and 0.5 (24 h pi). Conclusion Rituximab could be directly and stably labeled with the matched pair99m Tc/188 Re using the IsoLink technology under retention of the biological activity. Labeling kinetics and yields need further improvement for potential routine application in radioimmunodiagnosis and therapy.