The overall goal of this project is to develop a standardized system for """"""""loading"""""""" technetium-99m (99mTc) onto proteins that recognizes cell-surface specific markers associated with onset and progression of human diseases. Using multiple markers for imaging might lead to better characterization of each patient and therefore, to development of personalized treatment regiments and rational selection of patients for experimental therapeutics. However, labeling of proteins for imaging frequently requires highly customized procedures that often result in the loss of protein activity. An attractive alternative would be assembly of targeting imaging complexes using a standardized radiolabeled adapter protein that can be bound to any targeting protein expressed with a corresponding """"""""docking tag"""""""". We have recently developed an adapter/docking tag system for assembly of complexes for targeted radionuclide imaging. We used an 18-127-amino acid (aa) fragment of human RNase I (adapter protein) radiolabeled with technetium-99m (99mTc) as our standardized radiolabeled module. This module was then noncovalently bound to a fusion protein comprised of a 1-15-aa fragment of RNAase I (docking tag) and the 121- aa isoform of vascular endothelial factor (VEGF121), forming a 99mTc/VEGF imaging complex. With this strategy we have achieved selective and specific radionuclide imaging of mouse tumor neovasculature in lesions as small as several millimeters. Our experiments suggest that targeting complexes assembled via adapter/docking tag system can be used for targeted delivery of imaging agents, and specifically for delivery of 99mTc. However, there are several milestones that have to be achieved before standardized assembled complexes are suitable for clinical development. First, we will develop an adapter/docking tag system that is stable in circulation. Second, to improve signal/noise ratio and to minimize exposure to biologically active targeting proteins, we will develop an imaging payload module that can be radiolabeled to 10-30 fold higher specific activity than current 99mTc /protein preparations. Third, using different targeting proteins we will establish to what extent targeting complexes can penetrate into tumors. If these milestones are achieved, assembled targeting imaging complexes might become a powerful tool in diagnostics of patients, development of individualized treatment, and monitoring responses to drug treatment. Furthermore, this approach would allow rapid and uniform conversion of any newly discovered or constructed cell-targeting protein into a powerful imaging tool.
| Backer, Marina V; Levashova, Zoia; Levenson, Richard et al. (2008) Cysteine-containing fusion tag for site-specific conjugation of therapeutic and imaging agents to targeting proteins. Methods Mol Biol 494:275-94 |
| Backer, Marina V; Levashova, Zoya; Patel, Vimalkumar et al. (2007) Molecular imaging of VEGF receptors in angiogenic vasculature with single-chain VEGF-based probes. Nat Med 13:504-9 |
| Blankenberg, Francis G; Backer, Marina V; Levashova, Zoia et al. (2006) In vivo tumor angiogenesis imaging with site-specific labeled (99m)Tc-HYNIC-VEGF. Eur J Nucl Med Mol Imaging 33:841-8 |
