Order of amino acids in C-terminal cysteine-containing peptide-based chelators influences cellular processing and biodistribution of 99mTc-labeled recombinant Affibody molecules
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Affibody molecules constitute a novel class of molecular display selected affinity proteins based on non-immunoglobulin scaffold. Preclinical investigations and pilot clinical data have demonstrated that Affibody molecules provide high contrast imaging of tumor-associated molecular targets shortly after injection. The use of cysteine-containing peptide-based chelators at the C-terminus of recombinant Affibody molecules enabled site-specific labeling with the radionuclide 99mTc. Earlier studies have demonstrated that position, composition and the order of amino acids in peptide-based chelators influence labeling stability, cellular processing and biodistribution of Affibody molecules. To investigate the influence of the amino acid order, a series of anti-HER2 Affibody molecules, containing GSGC, GEGC and GKGC chelators have been prepared and characterized. The affinity to HER2, cellular processing of 99mTc-labeled Affibody molecules and their biodistribution were investigated. These properties were compared with that of the previously studied 99mTc-labeled Affibody molecules containing GGSC, GGEC and GGKC chelators. All variants displayed picomolar affinities to HER2. The substitution of a single amino acid in the chelator had an appreciable influence on the cellular processing of 99mTc. The biodistribution of all 99mTc-labeled Affibody molecules was in general comparable, with the main difference in uptake and retention of radioactivity in excretory organs. The hepatic accumulation of radioactivity was higher for the lysine-containing chelators and the renal retention of 99mTc was significantly affected by the amino acid composition of chelators. The order of amino acids influenced renal uptake of some conjugates at 1 h after injection, but the difference decreased at later time points. Such information can be helpful for the development of other scaffold protein-based imaging and therapeutic radiolabeled conjugates.
KeywordsAffibody molecule Technetium-99m Molecular imaging HER2 C-terminal cysteine Peptide-based chelator
This research was financially supported by grants from the Swedish Cancer Society (Cancerfonden) and the Swedish Research Council (Vetenskapsrådet).
- Ahlgren S, Wållberg H, Tran TA, Widström C, Hjertman M, Abrahmsén L, Berndorff D, Dinkelborg LM, Cyr JE, Feldwisch J, Orlova A, Tolmachev V (2009) Targeting of HER2-expressing tumors using a site-specifically 99mTc-labeled recombinant Affibody molecule ZHER2:2395 with C-terminal engineered cysteine. J Nucl Med 50:781–789PubMedCrossRefGoogle Scholar
- Ahlgren S, Orlova A, Wållberg H, Hansson M, Sandström M, Lewsley R, Wennborg A, Abrahmsén L, Tolmachev V, Feldwisch J (2010a) Targeting of HER2-expressing tumors using 111In-ABY-025, a second generation Affibody molecule with a fundamentally re-engineered scaffold. J Nucl Med 51:1131–1138PubMedCrossRefGoogle Scholar
- Miao Z, Levi J, Cheng Z (2010) Protein scaffold-based molecular probes for cancer molecular imaging. Amino Acids. doi: 10.1007/s00726-010-0503-9
- Wållberg H, Orlova A, Altai M, Widström C, Hosseinimehr SJ, Malmberg J, Ståhl S, Tolmachev V (2011a) Molecular design and optimization of 99mTc-labeled recombinant affibody molecules improves their biodistribution and imaging properties. J Nucl Med 52:461–469Google Scholar