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Development and preclinical characterisation of 99mTc-labelled Affibody molecules with reduced renal uptake
School of Biotechnology, Royal Institute of Technology, Stockholm, Sweden.
Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för onkologi, radiologi och klinisk immunologi.
Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för onkologi, radiologi och klinisk immunologi.ORCID-id: 0000-0001-6120-2683
Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för onkologi, radiologi och klinisk immunologi, Enheten för onkologi.
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2008 (Engelska)Ingår i: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 35, nr 12, s. 2245-2255Artikel i tidskrift (Refereegranskat) Published
Abstract [en]

Purpose  Affibody molecules are low molecular weight proteins (7 kDa), which can be selected to bind to tumour-associated target proteins with subnanomolar affinity. Because of rapid tumour localisation and clearance from nonspecific compartments, Affibody molecules are promising tracers for molecular imaging. Earlier, 99mTc-labelled Affibody molecules demonstrated specific targeting of tumour xenografts. However, the biodistribution was suboptimal either because of hepatobiliary excretion or high renal uptake of the radioactivity. The goal of this study was to optimise the biodistribution of Affibody molecules by chelator engineering.

Materials and methods  Anti-HER2 ZHER2:342 Affibody molecules, carrying the mercaptoacetyl-glutamyl-seryl-glutamyl (maESE), mercaptoacetyl-glutamyl-glutamyl-seryl (maEES) and mercaptoacetyl-seryl-glutamyl-glutamyl (maSEE) chelators, were prepared by peptide synthesis and labelled with 99mTc. The tumour-targeting capacity of these conjugates was compared with each other and with the best previously available conjugate, 99mTc-maEEE-ZHER2:342, in nude mice bearing SKOV-3 xenografts. The tumour-targeting capacity of the most promising conjugate, 99mTc-maESE-ZHER2:342, was compared with radioiodinated ZHER2:342. Results  All novel conjugates demonstrated successful tumour targeting and a low degree of hepatobiliary excretion. The renal uptakes of serine-containing conjugates, 33 ± 5, 68 ± 21 and 71 ± 10%IA/g, for99mTc-maESE-ZHER2:342, 99mTc-maEES-ZHER2:342 and 99mTc-maSEE-ZHER2:342, respectively, were significantly reduced in comparison with 99mTc-maEEE-ZHER2:342 (102 ± 13%IA/g). For 99mTc-maESE-ZHER2:342, a tumour uptake of 9.6 ± 1.8%IA/g and a tumour-to-blood ratio of 58 ± 6 were reached at 4 h p.i. Conclusions  A combination of serine and glutamic acid residues in the chelator sequence confers increased renal excretion and relatively low renal uptake of 99mTc-labelled Affibody molecules. In combination with preserved targeting capacity, this improved imaging of targets in abdominal area.

Ort, förlag, år, upplaga, sidor
2008. Vol. 35, nr 12, s. 2245-2255
Nyckelord [en]
Affibody molecule, HER2, Renal uptake, Technetium-99m, Tumour targeting
Nationell ämneskategori
Medicin och hälsovetenskap
Identifikatorer
URN: urn:nbn:se:uu:diva-17054DOI: 10.1007/s00259-008-0845-7ISI: 000261654000012PubMedID: 18594815OAI: oai:DiVA.org:uu-17054DiVA, id: diva2:44825
Tillgänglig från: 2008-06-16 Skapad: 2009-03-25 Senast uppdaterad: 2017-12-08Bibliografiskt granskad
Ingår i avhandling
1. Chemical Synthesis of Affibody Molecules for Protein Detection and Molecular Imaging
Öppna denna publikation i ny flik eller fönster >>Chemical Synthesis of Affibody Molecules for Protein Detection and Molecular Imaging
2008 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
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.

Ort, förlag, år, upplaga, sidor
Stockholm: KTH, 2008. s. viii, 84
Serie
Trita-BIO-Report, ISSN 1654-2312 ; 2008:22
Nyckelord
Affibody, peptide synthesis, protein detection, molecular imaging
Nationell ämneskategori
Industriell bioteknik
Identifikatorer
urn:nbn:se:kth:diva-9626 (URN)978-91-7415-152-7 (ISBN)
Disputation
2008-12-12, D2, Lindstedtsv 5, Stockholm, 10:00 (Engelska)
Opponent
Handledare
Anmärkning
QC 20100719Tillgänglig från: 2008-11-21 Skapad: 2008-11-21 Senast uppdaterad: 2010-07-19Bibliografiskt granskad

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