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 99mTc-maEEE-ZHER2:342, an Affibody Molecule-Based Tracer for the Detection of HER2 Expression in Malignant Tumors
Division of Biomedical Radiation Sciences, Rudbeck Laboratory, Uppsala University.
KTH, School of Biotechnology (BIO), Molecular Biotechnology.
Division of Biomedical Radiation Sciences, Rudbeck Laboratory, Uppsala University.
Medical Radiation Physics, Uppsala University Hospital, Uppsala University.
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2007 (English)In: Bioconjugate chemistry, ISSN 1043-1802, E-ISSN 1520-4812, Vol. 18, no 6, 1956-1964 p.Article in journal (Refereed) Published
Abstract [en]

Detection of HER2-overexpression in tumors and metastases is important for the selection of patients who will benefit from trastuzumab treatment. Earlier investigations showed successful imaging of HER2-positive tumors in patients using indium- or gallium-labeled Affibody molecules. The goal of this study was to evaluate the use of 99mTc-labeled Affibody molecules for the detection of HER2 expression. The Affibody molecule ZHER2:342 with the chelator sequences mercaptoacetyl-Gly-Glu-Gly (maGEG) and mercaptoacetyl-Glu-Glu-Glu (maEEE) was synthesized by peptide synthesis and labeled with technetium-99m. Binding specificity, cellular retention, and in vitro stability were investigated. The biodistribution of 99mTc-maGEG-ZHER2:342 and 99mTc-maEEE-ZHER2:342 was compared with 99mTc-maGGG-ZHER2:342 in normal mice, and the tumor targeting properties of 99mTc-maEEE-ZHER2:342 were determined in SKOV-3 xenografted nude mice. The results showed that the Affibody molecules were efficiently labeled with technetium-99m. The labeled conjugates were highly stable in vitro with preserved HER2-binding capacity. The use of glutamic acid in the chelator sequences for 99mTc-labeling of ZHER2:342 reduced the hepatobiliary excretion 3-fold with a single Gly-to-Glu substitution and 10-fold with three Gly-to-Glu substitutions. 99mTc-maEEE-ZHER2:342 showed a receptor-specific tumor uptake of 7.9 ± 1.0 %IA/g and a tumor-to-blood ratio of 38 at 4 h pi. Gamma-camera imaging with 99mTc-maEEE-ZHER2:342 could detect HER2-expressing tumors in xenografts already at 1 h pi. It was concluded that peptide synthesis for the coupling of chelator sequences to Affibody molecules for 99mTc labeling is an efficient way to modify the in vivo kinetics. Increased hydrophilicity, combined with improved stability of the mercaptoacetyl-triglutamyl chelator, resulted in favorable biodistribution, making 99mTc-maEEE-ZHER2:342 a promising tracer for clinical imaging of HER2 overexpression in tumors.

Place, publisher, year, edition, pages
2007. Vol. 18, no 6, 1956-1964 p.
Keyword [en]
National Category
Biochemistry and Molecular Biology
URN: urn:nbn:se:kth:diva-9723DOI: 10.1021/bc7002617ISI: 000251166400035ScopusID: 2-s2.0-36849020126OAI: diva2:127221
QC 20100716Available from: 2008-12-03 Created: 2008-12-03 Last updated: 2010-07-16Bibliographically 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.
Trita-BIO-Report, ISSN 1654-2312 ; 2008:22
Affibody, peptide synthesis, protein detection, molecular imaging
National Category
Industrial Biotechnology
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)
QC 20100719Available from: 2008-11-21 Created: 2008-11-21 Last updated: 2010-07-19Bibliographically approved

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