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Step-wise down regulationof the epidermal growth factor receptor by affinity-based intracellular redirection
KTH, School of Biotechnology (BIO), Molecular Biotechnology.
KTH, School of Biotechnology (BIO), Molecular Biotechnology.ORCID iD: 0000-0002-5391-600X
(English)Manuscript (preprint) (Other academic)
National Category
Other Basic Medicine
Identifiers
URN: urn:nbn:se:kth:diva-116890OAI: oai:DiVA.org:kth-116890DiVA: diva2:601449
Note

QS 2013

Available from: 2013-01-29 Created: 2013-01-29 Last updated: 2013-01-29Bibliographically approved
In thesis
1. Engineering of Affibody molecules for Radionuclide Molecular Imaging and Intracellular Targeting
Open this publication in new window or tab >>Engineering of Affibody molecules for Radionuclide Molecular Imaging and Intracellular Targeting
2013 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Affibody molecules are small (7 kDa) affinity proteins of non-immunoglobulin origin that have been generated to specifically interact with a large number of clinically important molecular targets.

In this thesis, Affibody molecules have been employed as tracers for radionuclide molecular imaging of HER2- and IGF-1R-expressing tumors, paper I-IV, and for surface knock-down of EGFR, paper V. In paper I, a tag with the amino acid sequence HEHEHE was fused to the N-terminus of a HER2-specific Affibody molecule, (ZHER2), and was shown to enable facile IMAC purification and efficient tri-carbonyl 99mTc-labeling. In vivo evaluation of radioactivity uptake in different organs showed an improved biodistribution, including a 10-fold lower radioactivity uptake in liver, compared to the same construct with a H6-tag. In paper II, it was further shown that an N-terminally placed HEHEHE-tag on ZHER2 provided lower unspecific uptake of radioactivity in liver compared to its H6-tagged counterpart even when radiolabeling was at the C-terminus using alternative chemistries to attach 99mTc, 111In or 125I. In paper III, the H6-tag’s composition and position was varied with regards to charge, hydrophobicity and its C- or N-terminal placement on ZHER2. Among the ten variants investigated, it was found that an N-terminal HEHEHE-tag provided the most favorable overall biodistribution profile and that introduction of hydrophobic and positively charged amino acids provoked liver uptake of radioactivity. In paper IV, the HEHEHE-tag was shown to enable IMAC purification and tri-carbonyl 99mTc-labeling of an IGF-1R-specific Affibody molecule and improved its overall biodistribution when compared to the same construct with a H6-tag. In paper V, the aim was to develop an intracellular receptor-entrapment system to reduce the surface levels of EGFR. An EGFR-specific Affibody molecule was expressed as a fusion to different mutants of an intracellular transport protein in SKOV-3 cells, resulting in a collection of cell lines with 50%, 60%, 80% and 96% reduced surface level of EGFR. Analysis of the proliferation rate of these cell lines showed that a modest reduction (15%) in proliferation occurs between 60% and 80% reduction of the surface level of EGFR.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2013. xi, 65 p.
Series
Trita-BIO-Report, ISSN 1654-2312 ; 2013:2
Keyword
Affibody molecules, affinity proteins, radionuclide molecular imaging, intracellular targeting, EGFR, HER2, IGF-1R
National Category
Biochemistry and Molecular Biology Medical Biotechnology
Research subject
SRA - Molecular Bioscience
Identifiers
urn:nbn:se:kth:diva-116884 (URN)978-91-7501-613-9 (ISBN)
Public defence
2013-02-20, FR4, AlbaNova University Center Roslagstullsbacken 21, Stockholm, 10:30 (English)
Opponent
Supervisors
Funder
Swedish Research CouncilKnut and Alice Wallenberg Foundation
Note

QC 20130129

Available from: 2013-01-29 Created: 2013-01-29 Last updated: 2013-01-29Bibliographically approved

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Gräslund, Torbjörn

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