Increasing the Net Negative Charge by Replacement of DOTA Chelator with DOTAGA Improves the Biodistribution of Radiolabeled Second-Generation Synthetic Affibody Molecules

A promising strategy to enable patient stratification for targeted therapies is to monitor the target expression in a tumor by radionuclide molecular imaging. Affibody molecules (7 kDa) are nonimmunoglobulin scaffold proteins with a 25-fold smaller size than intact antibodies. They have shown an apparent potential as molecular imaging probes both in preclinical and clinical studies. Earlier, we found that hepatic uptake can be reduced by the incorporation of negatively charged purification tags at the N-terminus of Affibody molecules. We hypothesized that liver uptake might similarly be reduced by positioning the chelator at the N-terminus, where the chelator-radionuclide complex will provide negative charges. To test this hypothesis, a second generation synthetic anti-HER2 Z(HER2:2891) Affibody molecule was synthesized and labeled with In-111 and Ga-68 using DOTAGA and DOTA chelators. The chelators were manually coupled to the N-terminus of Z(HER2:2891) forming an amide bond. Labeling DOTAGA-Z(HER2:2891) and DOTA-Z(HER2:2891) with Ga-68 and In-111 resulted in stable radioconjugates. The tumor-targeting and biodistribution properties of the In-111- and Ga-68-labeled conjugates were compared in SKOV-3 tumor-bearing nude mice at 2 h postinjection. The HER2-specific binding of the radioconjugates was verified both in vitro and in vivo. Using the DOTAGA chelator gave significantly lower radioactivity in liver and blood for both radionuclides. The In-111-labeled conjugates showed more rapid blood clearance than the Ga-68-labeled conjugates. The most pronounced influence of the chelators was found when they were labeled with Ga-68. The DOTAGA chelator gave significantly higher tumor-to-blood (61 +/- 6 vs 23 +/- 5, p < 0.05) and tumor-to-liver (10.4 +/- 0.6 vs 4.5 +/- 0.5, p < 0.05) ratios than the DOTA chelator. This study demonstrated that chelators may be used to alter the uptake of Affibody molecules, and most likely other scaffold-based imaging probes, for improvement of imaging contrast.