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Labelling of a polypeptide conjugate binder for the C-reactive protein with 68Ga for PET imaging
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry. (Lars Baltzer)
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry. (Bengt Långström)
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
(English)Manuscript (preprint) (Other academic)
Abstract [en]

A polypeptide conjugate that binds the C-reactive protein (CRP) with high affinity and specificity in human serum has been labelled with the short-lived radionuclide 68Ga (t1/2 = 68 min) for PET imaging. The binder molecule was formed by conjugating phosphocholine to the side chain of a lysine residue of a 42-residue designed polypeptide via an aliphatic spacer. For radiolabelling, a DOTA group was covalently attached to the polypeptide via a PEG spacer linked to the side chain of a Cys residue. The affinity of the binder for CRP is due to the interactions between CRP and the phosphocholine group as well as between CRP and the polypeptide scaffold itself. The affinity for CRP of the polypeptide conjugate was not affected by the incorporation of the DOTA and the dissociation constant was shown by fluorescence titration to be in the low nM range. Life times of polypeptide conjugates in human serum have been shown to depend on the level of incorporation of residues that are not recognized by proteases and thus to be tunable by design. The apparent half life of the polypeptide scaffold without conjugated groups was 10–20 min at 37 °C in human serum whereas the half-life of the labelled CRP binder was more than 24 h under the same conditions. The molecular versatility of peptides and peptide conjugates has therefore been shown to make them excellent carriers of multiple functions for in vivo applications, as illustrated here by the combination of molecular recognition with short-lived radioisotopes for PET imaging.

National Category
Chemical Sciences
Identifiers
URN: urn:nbn:se:uu:diva-108591OAI: oai:DiVA.org:uu-108591DiVA: diva2:271583
Available from: 2009-10-12 Created: 2009-09-23 Last updated: 2010-01-14
In thesis
1. Development of 18F- and 68Ga-Labelled Tracers: Design Perspectives and the Search for Faster Synthesis
Open this publication in new window or tab >>Development of 18F- and 68Ga-Labelled Tracers: Design Perspectives and the Search for Faster Synthesis
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

This thesis deals with the design of 18F- and 68Ga-labelled positron emission tomography (PET) tracers and the development of technologies that enable faster and simpler preparation with high specific radioactivity. Techniques like microwave heating and reducing the concentrations of the precursor were investigated with this perspective. A few applications were explored using molecular design perspectives.

A nucleophilic 18F-labelling strategy using perfluoro-containing leaving groups was explored. We observed that [18F]fluoride was interacting with the perfluoro alkyl chains of the substrate, preventing the nucleophilic substitution from taking place. When a perfluoroaryl group was instead used in the leaving group, the substitution took place and purification by fluorous solid-phase extraction was possible.

18F-Labelled analogues of the monoamine oxidase-A inhibitor harmine were prepared by one-step nucleophilic fluorinations and evaluated by in vitro autoradiography, showing high specific binding.

Biotin analogues labelled with 18F and 68Ga were prepared and their binding to avidin evaluated. All analogues retained their binding ability and will be further evaluated in transplantation models with avidin-coated islets of Langerhans.

Peptide design perspectives were used in some examples where the Arg-Gly-Asp (RGD) sequence and a single-chain version of vascular endothelial growth factor (VEGF) protein functionalized with 2,2',2'',2'''-(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrayl)tetraacetic acid (DOTA) or 2,2',2''-(1,4,7-triazonane-1,4,7-triyl)triacetic acid (NOTA) as chelators were labelled with 68Ga. The RGD motif and VEGF have high affinity for, respectively, αvβ3 integrin and VEGFR-2 receptor that are overexpressed in angiogenesis process. The 68Ga-labelled scVEGF maintained its functional activity in vitro.

A polypeptide conjugate containing phosphocholine, which has affinity for the C-reactive protein released during the inflammatory process, was labelled with 68Ga for the development of an imaging agent for inflammation in vivo.

Finally [18F]/19F exchange in fluorine-containing compounds was studied in order to investigate whether the exchange reaction can be of practical use for labelling.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2009. 48 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 678
Keyword
PET, nucleophilic 18F-fluorination, perfluoro, F-SPE, molecular design, 68Ga, chelators, DOTA, NOTA, microwave, halogen exchange, harmine, biotin, RGD, VEGF, CRP
National Category
Chemical Sciences
Research subject
Organic Chemistry
Identifiers
urn:nbn:se:uu:diva-108629 (URN)978-91-554-7626-7 (ISBN)
Public defence
2009-11-26, C8:305, BMC, Husargatan 3, 751 23 Uppsala, Uppsala, 13:15 (English)
Opponent
Supervisors
Available from: 2009-11-05 Created: 2009-09-24 Last updated: 2011-03-03Bibliographically approved

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Blom, Elisabeth

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