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99mTc-chelator engineering to improve tumour targeting properties of a HER2-specific Affibody molecule
KTH, School of Biotechnology (BIO).
Unit of Biomedical Radiation Sciences, Rudbeck Laboratory, Uppsala University.
Unit of Biomedical Radiation Sciences, Rudbeck Laboratory, Uppsala University.
Section of Hospital Physics, Department of Oncology, Uppsala University Hospital.
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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 11, 1843-1853 p.Article in journal (Refereed) Published
Abstract [en]

Purpose  Monitoring HER2 expression is crucial for selection of breast cancer patients amenable to HER2-targeting therapy. The Affibody molecule ZHER2:342 binds to HER2 with picomolar affinity and enables specific imaging of HER2 expression. Previously, ZHER2:342 with the additional N-terminal mercaptoacetyl-glycyl-glycyl-glycyl (maGGG) sequence was labelled with 99mTc and demonstrated specific targeting of HER2-expressing xenografts. However, hepatobiliary excretion caused high radioactivity accumulation in the abdomen. We investigated whether the biodistribution of ZHER2:342 can be improved by substituting glycyl residues in the chelating sequence with more hydrophilic seryl residues.

Methods  The Affibody molecule ZHER2:342, carrying the chelators mercaptoacetyl-glycyl-seryl-glycyl (maGSG), mercaptoacetyl-glycyl-D-seryl-glycyl [maG(D-S)G] and mercaptoacetyl-seryl-seryl-seryl (maSSS), were prepared by peptide synthesis and labelled with 99mTc. The differences in the excretion pathways were evaluated in normal mice. The tumour targeting capacity of 99mTc-maSSS-ZHER2:342 was studied in nude mice bearing SKOV-3 xenografts and compared with the capacity of radioiodinated ZHER2:342.

Results  A shift towards renal excretion was obtained when glycine was substituted with serine in the chelating sequence. The radioactivity in the gastrointestinal tract was reduced threefold for the maSSS conjugate in comparison with the maGGG conjugate 4 h post injection (p.i.). The tumour uptake of 99mTc-maSSS-ZHER2:342 was 11.5 ± 0.5% IA/g 4 h p.i., and the tumour-to-blood ratio was 76. The pharmacokinetics and uptake characteristics of technetium-labelled ZHER2:342 were better than those of radioiodinated ZHER2:342.

Conclusion  The introduction of serine residues in the chelator results in better tumour imaging properties of the Affibody molecule ZHER2:342 compared with glycyl-containing chelators and is favourable for imaging of tumours and metastases in the abdominal area.

Place, publisher, year, edition, pages
2007. Vol. 34, no 11, 1843-1853 p.
Keyword [en]
affibody molecule; HER2; peptide synthesis; technetium-99m; tumour targeting
National Category
Radiology, Nuclear Medicine and Medical Imaging
URN: urn:nbn:se:kth:diva-9726DOI: 10.1007/s00259-007-0474-6ISI: 000250205400017ScopusID: 2-s2.0-35348984582OAI: diva2:127225
QC 20100719Available from: 2008-12-03 Created: 2008-12-03 Last updated: 2010-07-19Bibliographically 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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Engfeldt, TorunEriksson Karlström, Amelie
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