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Imaging of HER2-expressing tumours using a synthetic Affibody molecule containing the 99mTc-chelating mercaptoacetyl-glycyl-glycyl-glycyl (MAG3) sequence
KTH, School of Biotechnology (BIO), Molecular Biotechnology.
Biomedical Radiation Sciences, Rudbeck Laboratory, Uppsala University.
Biomedical Radiation Sciences, Rudbeck Laboratory, Uppsala University.
Biomedical Radiation Sciences, Rudbeck Laboratory, Uppsala University.
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2007 (English)In: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 34, no 5, 722-733 p.Article in journal (Refereed) Published
Abstract [en]

Purpose  Expression of human epidermal growth factor receptor type 2 (HER2) in malignant tumours possesses well-documented prognostic and predictive value. Non-invasive imaging of expression can provide valuable diagnostic information, thereby influencing patient management. Previously, we reported a phage display selection of a small (about 7 kDa) protein, the Affibody molecule ZHER2:342, which binds HER2 with subnanomolar affinity, and demonstrated the feasibility of targeting of HER2-expressing xenografts using radioiodinated ZHER2:342. The goal of this study was to develop a method for 99mTc labelling of ZHER2:342 using the MAG3 chelator, which was incorporated into ZHER2:342 using peptide synthesis, and evaluate the targeting properties of the labelled conjugate. Methods  MAG3-ZHER2:342 was assembled using Fmoc/tBu solid phase peptide synthesis. Biochemical characterisation of the agent was performed using RP-HPLC, ESI-MS, biosensor studies and circular dichroism. A procedure for 99mTc labelling in the presence of sodium/potassium tartrate was established. Tumour targeting was evaluated by biodistribution study and gamma camera imaging in xenograft-bearing mice. Biodistribution of 99mTc-MAG3-ZHER2:342 and 125I-para-iodobenzoate -ZHER2:342 was compared 6 h p.i. Results  Synthetic MAG3-ZHER2:342 possessed an affinity of 0.2 nM for HER2 receptors. The peptide was labelled with 99mTc with an efficiency of about 75–80%. Labelled 99mTc-MAG3-ZHER2:342 retained capacity to bind specifically HER2-expressing SKOV-3 cells in vitro. 99mTc-MAG3-ZHER2:342 showed specific tumour targeting with a contrast similar to a radioiodinated analogue in mice bearing LS174T xenografts. Gamma camera imaging demonstrated clear and specific visualisation of HER2 expression. Conclusion  Incorporation of a mercaptoacetyl-containing chelating sequence during chemical synthesis enabled site-specific 99mTc labelling of the ZHER2:342 Affibody molecule with preserved targeting capacity.

Place, publisher, year, edition, pages
2007. Vol. 34, no 5, 722-733 p.
Keyword [en]
NEGATIVE BREAST-CANCER; DISSEMINATED PERITONEAL DISEASE; IN-VITRO CHARACTERIZATION; HER-2/NEU ONCOPROTEIN; HER2 EXPRESSION; CORRESPONDING METASTASES; COMBINATORIAL LIBRARIES; C-ERBB-2 EXPRESSION; DIFFERENT CHELATORS; ANTI-HER2 AFFIBODY
National Category
Radiology, Nuclear Medicine and Medical Imaging
Identifiers
URN: urn:nbn:se:kth:diva-9721DOI: 10.1007/s00259-006-0266-4ISI: 000246095900013Scopus ID: 2-s2.0-34047191078OAI: oai:DiVA.org:kth-9721DiVA: diva2:127219
Note
QC 20100715Available from: 2008-12-03 Created: 2008-12-03 Last updated: 2010-07-15Bibliographically approved
In thesis
1. Chemical Synthesis of Affibody Molecules for Protein Detection and Molecular Imaging
Open this publication in new window or tab >>Chemical Synthesis of Affibody Molecules for Protein Detection and Molecular Imaging
2008 (English)Doctoral thesis, comprehensive summary (Other scientific)
Abstract [en]

Proteins are essential components in most processes in living organisms. The detection and quantification of specific proteins can be used e.g. as measures of certain physiological conditions, and are therefore of great importance. This thesis focuses on development of affinity-based bioassays for specific protein detection. The use of Affibody molecules for specific molecular recognition has been central in all studies in this thesis. Affibody molecules are affinity proteins developed by combinatorial protein engineering of the 58-residue protein A-derived Z domain scaffold. In the first paper, solid phase peptide synthesis is investigated as a method to generate functional Affibody molecules. Based on the results from this paper, chemical synthesis has been used throughout the following papers to produce Affibody molecules tailored with functional groups for protein detection applications in vitro and in vivo.

 

In paper I, an orthogonal protection scheme was developed to enable site-specific chemical introduction of three different functional probes into synthetic Affibody molecules. Two of the probes were fluorophores that were used in a FRET-based binding assay to detect unlabeled target proteins. The third probe was biotin, which was used as an affinity handle for immobilization onto a solid support. In paper II, a panel of Affibody molecules carrying different affinity handles were synthesized and evaluated as capture ligands on microarrays. Paper III describes the synthesis of an Affibody molecule that binds to the human epidermal growth factor receptor type 2, (HER2), and the site-specific incorporation of a mercaptoacetyl-glycylglycylglycine (MAG3) chelating site in the peptide sequence to allow for radiolabeling with 99mTc. The derivatized Affibody molecule was found to retain its binding capacity, and the 99mTc-labeling was efficient and resulted in a stable chelate formation. 99mTc-labeled Affibody molecules were evaluated as in vivo HER2-targeting imaging agents in mice. In the following studies, reported in papers IV-VI, the 99mTc-chelating sequence was engineered in order to optimize the pharmacokinetic properties of the radiolabeled Affibody molecules and allow for high-contrast imaging of HER2-expressing tumors and metastatic lesions. The main conclusion from these investigations is that the biodistribution of Affibody molecules can be dramatically modified by amino acid substitutions directed to residues in the MAG3-chelator. Finally, paper VII is a report on the chemical synthesis and chemoselective ligation to generate a cross-linked HER2-binding Affibody molecule with improved thermal stability and tumor targeting capacity.

 

Taken together, the studies presented in this thesis illustrate how peptide synthesis can be used for production and modification of small affinity proteins, such as Affibody molecules for protein detection applications.

Place, publisher, year, edition, pages
Stockholm: KTH, 2008. viii, 84 p.
Series
Trita-BIO-Report, ISSN 1654-2312 ; 2008:22
Keyword
Affibody, peptide synthesis, protein detection, molecular imaging
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-9626 (URN)978-91-7415-152-7 (ISBN)
Public defence
2008-12-12, D2, Lindstedtsv 5, Stockholm, 10:00 (English)
Opponent
Supervisors
Note
QC 20100719Available from: 2008-11-21 Created: 2008-11-21 Last updated: 2010-07-19Bibliographically approved

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Eriksson Karlström, Amelie

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