Radiolabeling of rituximab with (188)Re and (99m)Tc using the tricarbonyl technology.

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 ß(-) 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 (99m)Tc- and (188)Re-tricarbonyl core (IsoLink technology). METHODS: The native format of the antibody (RTX(wt)) as well as a reduced form (RTX(red)) was labeled with (99m)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 RTX(red) were superior to that of RTX(wt) ((99m)Tc: 98% after 3 h for RTX(red) vs. 70% after 24 h for RTX(wt)). (99m)Tc(CO)(3)-RTX(red) was used without purification for in vitro and in vivo studies whereas (188)Re(CO)(3)-RTX(red) was purified to eliminate free (188)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 of (99m)Tc(CO)(3)-RTX(red) but not with pre-purified (188)Re(CO)(3)-RTX(red). Both conjugates revealed high binding affinity to the CD20 antigen (K(d) = 5-6 nM). Tumor uptake of (188)Re(CO)(3)-RTX(red) was 2.5 %ID/g and 0.8 %ID/g for (99m)Tc(CO)(3)-RTX(red) 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 (99m)Tc(CO)(3)-RTX(red) and for (188)Re(CO)(3)-RTX(red) 0.5 and 0.5 (24 h pi). CONCLUSION: Rituximab could be directly and stably labeled with the matched pair (99m)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.

Efficient tumor targeting with high-affinity designed ankyrin repeat proteins: effects of affinity and molecular size.

Slow-clearing, tumor-targeting proteins such as monoclonal antibodies typically exhibit high tumor accumulation but low tissue contrast, whereas intermediate-sized proteins such as scFvs show faster clearance but only moderate tumor accumulation. For both, tumor targeting does not seem to improve further above an optimal affinity. We show here that with very small high-affinity proteins such as designed ankyrin repeat proteins (DARPins), these limits can be overcome. We have systematically investigated the influence of molecular mass and affinity on tumor accumulation with DARPins with specificity for HER2 in SK-OV-3.ip nude mouse xenografts. DARPins with a mass of 14.5 kDa and affinities between 270 nmol/L and 90 pmol/L showed a strong correlation of tumor accumulation with affinity to HER2, with the highest affinity DARPin reaching 8% ID/g after 24 hours and 6.5% ID/g after 48 hours (tumor-to-blood ratio >60). Tumor autoradiographs showed good penetration throughout the tumor mass. Genetic fusion of two DARPins (30 kDa) resulted in significantly lower tumor accumulation, similar to values observed for scFvs, whereas valency had no influence on accumulation. PEGylation of the DARPins increased the circulation half-life, leading to higher tumor accumulation (13.4% ID/g after 24 hours) but lower tumor-to-blood ratios. Affinity was less important for tumor uptake of the PEGylated constructs. We conclude that two regimes exist for delivering high levels of drug to a tumor: small proteins with very high affinity, such as unmodified DARPins, and large proteins with extended half-life, such as PEGylated DARPins, in which the importance of affinity is less pronounced.