Radiolabeling of rituximab with 188Re and 99mTc using the tricarbonyl technology

IntroductionThe 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 the 99mTc- and 188Re-tricarbonyl core (IsoLink technology).MethodsThe native format of the antibody (RTXwt) as well as a reduced form (RTXred) was labeled with 99mTc/188Re(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.ResultsThe radiolabeling efficiency and kinetics of RTXred were superior to that of RTXwt (99mTc: 98% after 3 h for RTXred vs. 70% after 24 h for RTXwt). 99mTc(CO)3-RTXred was used without purification for in vitro and in vivo studies whereas 188Re(CO)3-RTXred was purified to eliminate free 188Re-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 of 99mTc(CO)3-RTXred but not with pre-purified 188Re(CO)3-RTXred. Both conjugates revealed high binding affinity to the CD20 antigen (Kd=5–6 nM). Tumor uptake of 188Re(CO)3-RTXred was 2.5 %ID/g and 0.8 %ID/g for 99mTc(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 for 99mTc(CO)3-RTXred and for 188Re(CO)3-RTXred 0.5 and 0.5 (24 h pi).ConclusionRituximab could be directly and stably labeled with the matched pair 99mTc/188Re 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.