Abstract
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.
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References
Ahlgren S, Tolmachev V (2010) Radionuclide molecular imaging using Affibody molecules. Curr Pharm Biotechnol 11:581–589
Ahlgren S, Orlova A, Rosik D, Sandström M, Sjöberg A, Baastrup B, Widmark O, Fant G, Feldwisch J, Tolmachev V (2008) Evaluation of maleimide derivative of DOTA for site-specific labeling of recombinant Affibody molecules. Bioconjug Chem 19:235–243
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 50:781–789
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 51:1131–1138
Ahlgren S, Andersson K, Tolmachev V (2010b) Kit formulation for 99mTc-labeling of recombinant anti-HER2 Affibody molecules with a C-terminally engineered cysteine. Nucl Med Biol 37:539–546
Baum RP, Prasad V, Müller D, Schuchardt C, Orlova A, Wennborg A, Tolmachev V, Feldwisch J (2010) Molecular imaging of HER2-expressing malignant tumors in breast cancer patients using synthetic 111In- or 68Ga-labeled Affibody. J Nucl Med 51:892–897
Britz-Cunningham SH, Adelstein SJ (2003) Molecular targeting with radionuclides: state of the science. J Nucl Med 44:1945–1961
Cameron DA, Stein S (2008) Drug insight: intracellular inhibitors of HER2—clinical development of lapatinib in breast cancer. Nat Clin Pract Oncol 5:512–520
Chang HR (2010) Trastuzumab-based neoadjuvant therapy in patients with HER2-positive breast cancer. Cancer 116:2856–2867
Citri A, Yarden Y (2006) EGF-ERBB signalling: towards the systems level. Nat Rev Mol Cell Biol 7:505–516
Ekblad T, Tran T, Orlova A et al (2008) Development and preclinical characterisation of 99mTc-labelled Affibody molecules with reduced renal uptake. Eur J Nucl Med Mol Imaging 35:2245–2255
Ekblad T, Orlova A, Feldwisch J, Wennborg A, Eriksson Karlström A, Tolmachev V (2009) Positioning of 99mTc-chelators influences radiolabeling, stability and biodistribution of Affibody molecules. Bioorg Med Chem Lett 19:3912–3914
Engfeldt T, Orlova A, Tran T et al (2007a) Imaging of HER2-expressing tumours using a synthetic Affibody molecule containing the 99mTc-chelating mercaptoacetyl-glycyl-glycyl-glycyl (MAG3) sequence. Eur J Nucl Med Mol Imaging 34:722–733
Engfeldt T, Tran T, Orlova A, Widström Ch, Eriksson Karlström A, Tolmachev V (2007b) 99mTc-chelator engineering to improve tumour targeting properties of a HER2-specific Affibody molecule. Eur J Nucl Med Mol Imaging 34:1843–1853
Hynes NE, MacDonald G (2009) ErbB receptors and signaling pathways in cancer. Curr Opin Cell Biol 21:177–184
Löfblom J, Feldwisch J, Tolmachev V, Carlsson J, Ståhl S, Frejd FY (2010) Affibody molecules: engineered proteins for therapeutic, diagnostic and biotechnological applications. FEBS Lett 584:2670–2680
Mankoff DA (2009) Molecular imaging to select cancer therapy and evaluate treatment response. Q J Nucl Med Mol Imaging 53:181–192
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
Nygren PA (2008) Alternative binding proteins: affibody binding proteins developed from a small three-helix bundle scaffold. FEBS J 275:2668–2676
Orlova A, Tolmachev V, Pehrson R, Lindborg M, Tran T, Sandström M, Nilsson FY, Wennborg A, Abrahmsén L, Feldwisch J (2007) Synthetic affibody molecules: a novel class of affinity ligands for molecular imaging of HER2-expressing malignant tumors. Cancer Res 67:2178–2186
Orlova A, Magnusson M, Eriksson T, Nilsson M, Larsson B, Höiden-Guthenberg I, Widström C, Carlsson J, Tolmachev V, Ståhl S, Nilsson F (2006) Tumor imaging using a picomolar affinity HER2 binding Affibody molecule. Cancer Res 66:4339–4348
Tolmachev V (2008) Imaging of HER-2 overexpression in tumors for guiding therapy. Curr Pharm Des 14:2999–3011
Tolmachev V, Nilsson FY, Widström C, Andersson K, Rosik D, Gedda L, Wennborg A, Orlova A (2006) 111In-benzyl-DTPA-ZHER2:342, an affibody-based conjugate for in vivo imaging of HER2 expression in malignant tumors. J Nucl Med 47:846–853
Tolmachev V, Stone-Elander S, Orlova A (2010a) Current approaches to the use of radiolabeled tyrosine kinase-targeting drugs for patient stratification and treatment response monitoring: prospects and pitfalls. Lancet Oncol 11:992–1000
Tolmachev V, Hofström C, Malmberg J et al (2010b) HEHEHE-tagged affibody molecule may be purified by IMAC, is conveniently labeled with [99(m)Tc(CO)3](+), and shows improved biodistribution with reduced hepatic radioactivity accumulation. Bioconjug Chem 21:2013–2022
Tolmachev V, Velikyan I, Sandström M, Orlova A (2010c) A HER2-binding Affibody molecule labelled with 68Ga for PET imaging: direct in vivo comparison with the 111In-labelled analogue. Eur J Nucl Med Mol Imaging 37:1356–1367
Tran T, Engfeldt T, Orlova A et al (2007a) In vivo evaluation of cysteine-based chelators for attachment of 99mTc to tumor-targeting Affibody molecules. Biocojug Chem 18:549–558
Tran T, Engfeldt T, Orlova A et al (2007b) 99mTc-maEEE-ZHER2:342, an Affibody molecule-based tracer for detection of HER2-expression in malignant tumors. Bioconjug Chem 18:1956–1964
Tran T, Ekblad T, Orlova A et al (2008) Effects of Lysine-containing mercaptoacetyl-based Chelators on the Biodistribution of 99mTc-labeled anti-HER2. Bioconjug Chem 19:2568–2576
Tran TA, Rosik D, Abrahmsén L et al (2009) Design, synthesis and biological evaluation of a HER2-specific affibody molecule for molecular imaging. Eur J Nucl Med Mol Imaging 36:1864–1873
Wållberg H, Orlova A (2008) Slow internalization of anti-HER2 synthetic affibody monomer 111In-DOTA-ZHER2:342-pep2: implications for development of labeled tracers. Cancer Biother Radiopharm 23:435–442
Wållberg H, Ahlgren S, Widström C, Orlova A (2010) Evaluation of the radiocobalt-labeled [MMA-DOTA-Cys61]-Z HER2:2395(-Cys) affibody molecule for targeting of HER2-expressing tumors. Mol Imaging Biol 12:54–62
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 52:461–469
Wållberg H, Löfdahl PÅ, Tschapalda K, Uhlén M, Tolmachev V, Nygren PÅ, Ståhl S (2011b) Affinity recovery of eight HER2-binding affibody variants using an anti-idiotypic affibody ligand. Protein Exp Purif 76:127–135
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This research was financially supported by grants from the Swedish Cancer Society (Cancerfonden) and the Swedish Research Council (Vetenskapsrådet).
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Altai, M., Wållberg, H., Orlova, A. et al. Order of amino acids in C-terminal cysteine-containing peptide-based chelators influences cellular processing and biodistribution of 99mTc-labeled recombinant Affibody molecules. Amino Acids 42, 1975–1985 (2012). https://doi.org/10.1007/s00726-011-0927-x
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DOI: https://doi.org/10.1007/s00726-011-0927-x