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Synthesis and chemoselective intramolecular crosslinking of a HER2-binding Affibody
KTH, Skolan för bioteknologi (BIO).
Uppsala Univ, Rudbeck Lab, Unit Biomed Radiat Sci.
Uppsala Univ, Rudbeck Lab, Unit Biomed Radiat Sci.
Univ Cambridge, Dept Chem.
Vise andre og tillknytning
2009 (engelsk)Inngår i: Biopolymers, ISSN 0006-3525, E-ISSN 1097-0282, Vol. 92, nr 2, s. 116-123Artikkel i tidsskrift (Fagfellevurdert) Published
Abstract [en]

The human epidermal growth factor receptor HER2 has emerged as an important target for molecular imaging of breast cancer. This article presents the design and synthesis of a HER2-targeting affibody molecule with improved stability and tumor targeting capacity and with potential use as an imaging agent. The 58 aa three-helix bundle protein was assembled using solid-phase peptide synthesis, and a chemoselective ligation strategy was used to establish an intramolecular thioether bond between the side chain thiol group of a cysteine residue, positioned in the loop between helices I and II, and a chloroacetyl group on the side chain amino group of the C-terminal lysine residue. The tethered protein offered an increased thermal stability, with a melting temperature of 64 degrees C, compared to 54 degrees C for the linear control. The ligation did not have a major influence on the HER2 binding affinity, which was 320 and 380 pM for the crosslinked and linear molecules, respectively. Biodistribution studies were performed both in normal and tumor-bearing mice to evaluate the impact of the crosslinking on the in vivo behavior and on the tumor targeting performance. The distribution pattern was characterized by a low uptake in all organs except kidney, and rapid clearance from blood and normal tissue. Crosslinking of the protein resulted in a significantly increased tumor accumulation, rendering the tethered HER2-binding affibody molecule a valuable lead in the development of superior HER2 imaging agents.

sted, utgiver, år, opplag, sider
2009. Vol. 92, nr 2, s. 116-123
Emneord [en]
affibody molecules; chemoselective ligation; thioalkylation; HER2; molecular imaging; tumor targeting
HSV kategori
Identifikatorer
URN: urn:nbn:se:kth:diva-9725DOI: 10.1002/bip.21142ISI: 000264691400005Scopus ID: 2-s2.0-67449106599OAI: oai:DiVA.org:kth-9725DiVA, id: diva2:127223
Merknad
QC 20100716. Uppdaterad från submitted till published (20100716)Tilgjengelig fra: 2008-12-03 Laget: 2008-12-03 Sist oppdatert: 2017-12-14bibliografisk kontrollert
Inngår i avhandling
1. Chemical Synthesis of Affibody Molecules for Protein Detection and Molecular Imaging
Åpne denne publikasjonen i ny fane eller vindu >>Chemical Synthesis of Affibody Molecules for Protein Detection and Molecular Imaging
2008 (engelsk)Doktoravhandling, med artikler (Annet vitenskapelig)
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.

sted, utgiver, år, opplag, sider
Stockholm: KTH, 2008. s. viii, 84
Serie
Trita-BIO-Report, ISSN 1654-2312 ; 2008:22
Emneord
Affibody, peptide synthesis, protein detection, molecular imaging
HSV kategori
Identifikatorer
urn:nbn:se:kth:diva-9626 (URN)978-91-7415-152-7 (ISBN)
Disputas
2008-12-12, D2, Lindstedtsv 5, Stockholm, 10:00 (engelsk)
Opponent
Veileder
Merknad
QC 20100719Tilgjengelig fra: 2008-11-21 Laget: 2008-11-21 Sist oppdatert: 2010-07-19bibliografisk kontrollert

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