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Design of a modular protein-based MRI contrast agent for targeted application.

Grum D, Franke S, Kraff O, Heider D, Schramm A, Hoffmann D, Bayer P - PLoS ONE (2013)

Bottom Line: Low molecular weight MRI contrast agents currently in clinical use suffer either from a lack of specificity for tumour tissue or from low relaxivity and thus low contrast amplification.In this study, we present the newly designed two domain fusion protein Zarvin, which is able to bind to therapeutic IgG antibodies suitable for targeting, while facilitating contrast enhancement through high affinity binding sites for Gd(3+).We show that the Zarvin fold is stable under serum conditions, specifically targets a cancer cell-line when bound to the Cetuximab IgG, and allows for imaging with high relaxivity, a property that would be advantageous for the detection of small tumours and metastases at 1.5 or 3 T.

View Article: PubMed Central - PubMed

Affiliation: Research Group Structural and Medicinal Biochemistry, Centre for Medical Biotechnology, University of Duisburg-Essen, Essen, Germany.

ABSTRACT
Magnetic resonance imaging (MRI) offers a non-radioactive alternative for the non-invasive detection of tumours. Low molecular weight MRI contrast agents currently in clinical use suffer either from a lack of specificity for tumour tissue or from low relaxivity and thus low contrast amplification. In this study, we present the newly designed two domain fusion protein Zarvin, which is able to bind to therapeutic IgG antibodies suitable for targeting, while facilitating contrast enhancement through high affinity binding sites for Gd(3+). We show that the Zarvin fold is stable under serum conditions, specifically targets a cancer cell-line when bound to the Cetuximab IgG, and allows for imaging with high relaxivity, a property that would be advantageous for the detection of small tumours and metastases at 1.5 or 3 T.

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Binding properties and relaxometric properties of Zarvin. (A) Cartoon representation of Zarvin bound to the Fc part of an IgG antibody. Two calcium ions (spheres) are bound to Parvalbumin (green), which is connected with the Z domain (violet) via a decaglycine linker (grey). (B) Fluorescence anisotropy titration experiment. Increasing amounts of the monoclonal IgG antibody Cetuximab were added to a 100 nM concentration of Zarvin-Atto-465. (C) Confocal microscopic analysis of the complex Cetuximab:Zarvin-D72C-Atto 594 binding to the EGF receptor located in the cell membrane of A431 cells. Left, cell assembly; right, single cell; control experiments (Figure S4) (D) Relaxometric properties of Zarvin:(Gd3+)2 at three different field strengths employing an inversion recovery TSE experiment. A diluted solution of rising concentrations of Zarvin:(Gd3+)2 was investigated to find the limiting concentration which still produces a visible contrast towards the buffer control (0 µM). The picture is displayed with an inversion time TI which zeroes the signal of the buffer control (appears black).
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pone-0065346-g001: Binding properties and relaxometric properties of Zarvin. (A) Cartoon representation of Zarvin bound to the Fc part of an IgG antibody. Two calcium ions (spheres) are bound to Parvalbumin (green), which is connected with the Z domain (violet) via a decaglycine linker (grey). (B) Fluorescence anisotropy titration experiment. Increasing amounts of the monoclonal IgG antibody Cetuximab were added to a 100 nM concentration of Zarvin-Atto-465. (C) Confocal microscopic analysis of the complex Cetuximab:Zarvin-D72C-Atto 594 binding to the EGF receptor located in the cell membrane of A431 cells. Left, cell assembly; right, single cell; control experiments (Figure S4) (D) Relaxometric properties of Zarvin:(Gd3+)2 at three different field strengths employing an inversion recovery TSE experiment. A diluted solution of rising concentrations of Zarvin:(Gd3+)2 was investigated to find the limiting concentration which still produces a visible contrast towards the buffer control (0 µM). The picture is displayed with an inversion time TI which zeroes the signal of the buffer control (appears black).

Mentions: First, Zarvin was modelled and studied using in-silico methods. For this purpose three-dimensional models of Zarvin were generated with the MODELLER 9.1 software, using the Z domain (PDB entry 1q2n) and the calcium bound structure of S55D/E59D alpha-Parvalbumin (PDB entry 1s3p) as templates. The C-terminus of the Z domain was linked to the N-terminus of Parvalbumin using a decaglycine peptide (Figure 1A).


Design of a modular protein-based MRI contrast agent for targeted application.

Grum D, Franke S, Kraff O, Heider D, Schramm A, Hoffmann D, Bayer P - PLoS ONE (2013)

Binding properties and relaxometric properties of Zarvin. (A) Cartoon representation of Zarvin bound to the Fc part of an IgG antibody. Two calcium ions (spheres) are bound to Parvalbumin (green), which is connected with the Z domain (violet) via a decaglycine linker (grey). (B) Fluorescence anisotropy titration experiment. Increasing amounts of the monoclonal IgG antibody Cetuximab were added to a 100 nM concentration of Zarvin-Atto-465. (C) Confocal microscopic analysis of the complex Cetuximab:Zarvin-D72C-Atto 594 binding to the EGF receptor located in the cell membrane of A431 cells. Left, cell assembly; right, single cell; control experiments (Figure S4) (D) Relaxometric properties of Zarvin:(Gd3+)2 at three different field strengths employing an inversion recovery TSE experiment. A diluted solution of rising concentrations of Zarvin:(Gd3+)2 was investigated to find the limiting concentration which still produces a visible contrast towards the buffer control (0 µM). The picture is displayed with an inversion time TI which zeroes the signal of the buffer control (appears black).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3675113&req=5

pone-0065346-g001: Binding properties and relaxometric properties of Zarvin. (A) Cartoon representation of Zarvin bound to the Fc part of an IgG antibody. Two calcium ions (spheres) are bound to Parvalbumin (green), which is connected with the Z domain (violet) via a decaglycine linker (grey). (B) Fluorescence anisotropy titration experiment. Increasing amounts of the monoclonal IgG antibody Cetuximab were added to a 100 nM concentration of Zarvin-Atto-465. (C) Confocal microscopic analysis of the complex Cetuximab:Zarvin-D72C-Atto 594 binding to the EGF receptor located in the cell membrane of A431 cells. Left, cell assembly; right, single cell; control experiments (Figure S4) (D) Relaxometric properties of Zarvin:(Gd3+)2 at three different field strengths employing an inversion recovery TSE experiment. A diluted solution of rising concentrations of Zarvin:(Gd3+)2 was investigated to find the limiting concentration which still produces a visible contrast towards the buffer control (0 µM). The picture is displayed with an inversion time TI which zeroes the signal of the buffer control (appears black).
Mentions: First, Zarvin was modelled and studied using in-silico methods. For this purpose three-dimensional models of Zarvin were generated with the MODELLER 9.1 software, using the Z domain (PDB entry 1q2n) and the calcium bound structure of S55D/E59D alpha-Parvalbumin (PDB entry 1s3p) as templates. The C-terminus of the Z domain was linked to the N-terminus of Parvalbumin using a decaglycine peptide (Figure 1A).

Bottom Line: Low molecular weight MRI contrast agents currently in clinical use suffer either from a lack of specificity for tumour tissue or from low relaxivity and thus low contrast amplification.In this study, we present the newly designed two domain fusion protein Zarvin, which is able to bind to therapeutic IgG antibodies suitable for targeting, while facilitating contrast enhancement through high affinity binding sites for Gd(3+).We show that the Zarvin fold is stable under serum conditions, specifically targets a cancer cell-line when bound to the Cetuximab IgG, and allows for imaging with high relaxivity, a property that would be advantageous for the detection of small tumours and metastases at 1.5 or 3 T.

View Article: PubMed Central - PubMed

Affiliation: Research Group Structural and Medicinal Biochemistry, Centre for Medical Biotechnology, University of Duisburg-Essen, Essen, Germany.

ABSTRACT
Magnetic resonance imaging (MRI) offers a non-radioactive alternative for the non-invasive detection of tumours. Low molecular weight MRI contrast agents currently in clinical use suffer either from a lack of specificity for tumour tissue or from low relaxivity and thus low contrast amplification. In this study, we present the newly designed two domain fusion protein Zarvin, which is able to bind to therapeutic IgG antibodies suitable for targeting, while facilitating contrast enhancement through high affinity binding sites for Gd(3+). We show that the Zarvin fold is stable under serum conditions, specifically targets a cancer cell-line when bound to the Cetuximab IgG, and allows for imaging with high relaxivity, a property that would be advantageous for the detection of small tumours and metastases at 1.5 or 3 T.

Show MeSH
Related in: MedlinePlus