| 1. |
- Feldwisch, Joachim, et al.
(författare)
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Design of an optimized scaffold for affibody molecules.
- 2010
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Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 0022-2836 .- 1089-8638. ; 398:2, s. 232-247
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Tidskriftsartikel (refereegranskat)abstract
- Affibody molecules are non-immunoglobulin-derived affinity proteins based on a three-helical bundle protein domain. Here, we describe the design process of an optimized Affibody molecule scaffold with improved properties and a surface distinctly different from that of the parental scaffold. The improvement was achieved by applying an iterative process of amino acid substitutions in the context of the human epidermal growth factor receptor 2 (HER2)-specific Affibody molecule Z(HER2:342). Replacements in the N-terminal region, loop 1, helix 2 and helix 3 were guided by extensive structural modeling using the available structures of the parent Z domain and Affibody molecules. The effect of several single substitutions was analyzed followed by combination of up to 11 different substitutions. The two amino acid substitutions N23T and S33K accounted for the most dramatic improvements, including increased thermal stability with elevated melting temperatures of up to +12 degrees C. The optimized scaffold contains 11 amino acid substitutions in the nonbinding surface and is characterized by improved thermal and chemical stability, as well as increased hydrophilicity, and enables generation of identical Affibody molecules both by chemical peptide synthesis and by recombinant bacterial expression. A HER2-specific Affibody tracer, [MMA-DOTA-Cys61]-Z(HER2:2891)-Cys (ABY-025), was produced by conjugating MMA-DOTA (maleimide-monoamide-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) to the peptide produced either chemically or in Escherichia coli. ABY-025 showed high affinity and specificity for HER2 (equilibrium dissociation constant, K(D), of 76 pM) and detected HER2 in tissue sections of SKOV-3 xenograft and human breast tumors. The HER2-binding capacity was fully retained after three cycles of heating to 90 degrees C followed by cooling to room temperature. Furthermore, the binding surfaces of five Affibody molecules targeting other proteins (tumor necrosis factor alpha, insulin, Taq polymerase, epidermal growth factor receptor or platelet-derived growth factor receptor beta) were grafted onto the optimized scaffold, resulting in molecules with improved thermal stability and a more hydrophilic nonbinding surface.
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| 2. |
- Tolmachev, Vladimir, et al.
(författare)
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Tumor Targeting Using Affibody Molecules : Interplay of Affinity, Target Expression Level, and Binding Site Composition
- 2012
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Ingår i: Journal of Nuclear Medicine. - : Society of Nuclear Medicine. - 0161-5505 .- 1535-5667 .- 2159-662X. ; 53:6, s. 953-960
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Tidskriftsartikel (refereegranskat)abstract
- Radionuclide imaging of cancer-associated molecular alterations may contribute to patient stratification for targeting therapy. Scaffold high-affinity proteins, such as Affibody molecules, are a new, promising class of probes for in vivo imaging. Methods. The effects of human epidermal growth factor receptor 2 (HER2) affinity and binding site composition of HER2-binding Affibody molecules, and of the HER2 density on the tumor targeting, were studied in vivo. The tumor uptake and tumor-to-organ ratios of Affibody molecules with moderate (dissociation constant [K-D)] 10(-9) M) or high (K-D = 10(-10) M) affinity were compared between tumor xenografts with a high (SKOV-3) and low (LS174T) HER2 expression level in BALB/C nu/nu mice. Two Affibody molecules with similar affinity (K-D = 10(-10) M) but having alternative amino acids in the binding site were compared. Results. In SKOV-3 xenografts, uptake was independent of affinity at 4 h after injection, but high-affinity binders provided 2-fold-higher tumor radioactivity retention at 24 h. In LS174T xenografts, uptake of high-affinity probes was already severalfold higher at 4 h after injection, and the difference was increased at 24 h. The clearance rate and tumor-to-organ ratios were influenced by the amino acid composition of the binding surface of the tracer protein. Conclusion. The optimal affinity of HER2-binding Affibody molecules depends on the expression of a molecular target. At a high expression level (>10(6) receptors per cell), an affinity in the low-nanomolar range is sufficient. At moderate expression, subnanomolar affinity is desirable. The binding site composition can influence the imaging contrast. This information may be useful for development of imaging agents based on scaffold affinity proteins.
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| 3. |
- Tran, Thuy A., 1980-, et al.
(författare)
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Design, synthesis and biological evaluation of a multifunctional HER2-specific Affibody molecule for molecular imaging
- 2009
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Ingår i: European Journal of Nuclear Medicine and Molecular Imaging. - : Springer Science and Business Media LLC. - 1619-7070 .- 1619-7089. ; 36:11, s. 1864-1873
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Tidskriftsartikel (refereegranskat)abstract
- Purpose: The purpose of this study was to design and evaluate a novel platform for labelling of Affibody molecules, enabling for both recombinant and synthetic production and for site-specific labelling with 99mTc or trivalent radiometals. Methods: The HER2-specific Affibody molecule PEP05352 was made by peptide synthesis. The chelator sequence SECG (serine-glutamic acid-cysteine-glycine) was anchored on the C-terminal to allow 99mTc-labelling. The cysteine can alternatively serve as a conjugation site of the chelator DOTA for indium-labelling. The resulting 99mTc- and 111In-labelled Affibody molecules were evaluated both in vitro and in vivo. Results: Both conjugates retained their capacity to bind to HER2 receptors in vitro and in vivo. The tumour-to-blood ratio in LS174T xenografts was 30 at 4 h p.i. for both conjugates. Biodistribution data showed that 99mTc-labelled Affibody molecule had 4-fold lower kidney accumulation compared with 111In-labelled Affibody molecule while the accumulation in other organs was similar. Gamma-camera imaging of the conjugates could clearly visualise the tumours 4 h after injection. Conclusions: Incorporation of C-terminal SECG sequence in Affibody molecules provides a general multifunctional platform for site-specific labelling with different nuclides (technetium, indium, gallium, cobalt, or yttrium) and for a flexible production (chemical synthesis or recombinant).
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