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Design, synthesis and biological evaluation of a multifunctional HER2-specific Affibody molecule for molecular imaging
KTH, School of Biotechnology (BIO), Molecular Biotechnology.ORCID iD: 0000-0003-4409-9236
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2009 (English)In: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 36, no 11, 1864-1873 p.Article in journal (Refereed) Published
Abstract [en]

The purpose of this study was to design and evaluate a novel platform for labelling of Affibody molecules, enabling both recombinant and synthetic production and site-specific labelling with Tc-99m or trivalent radiometals. The HER2-specific Affibody molecule PEP05352 was made by peptide synthesis. The chelator sequence SECG (serine-glutamic acid-cysteine-glycine) was anchored on the C-terminal to allow Tc-99m labelling. The cysteine can alternatively serve as a conjugation site of the chelator DOTA for indium labelling. The resulting Tc-99m- and In-111-labelled Affibody molecules were evaluated both in vitro and in vivo. Both conjugates retained their capacity to bind to HER2 receptors in vitro and in vivo. The tumour to blood ratio in LS174T xenografts was 30 at 4 h post-injection for both conjugates. Biodistribution data showed that the Tc-99m-labelled Affibody molecule had a fourfold lower kidney accumulation compared with the In-111-labelled Affibody molecule while the accumulation in other organs was similar. Gamma camera imaging of the conjugates could clearly visualise the tumours 4 h after injection. Incorporation of the C-terminal SECG sequence in Affibody molecules provides a general multifunctional platform for site-specific labelling with different nuclides (technetium, indium, gallium, cobalt or yttrium) and for a flexible production (chemical synthesis or recombinant).

Place, publisher, year, edition, pages
2009. Vol. 36, no 11, 1864-1873 p.
Keyword [en]
HER2, Affibody molecules, Peptide synthesis, Imaging
National Category
Industrial Biotechnology Medical and Health Sciences
URN: urn:nbn:se:kth:diva-32789DOI: 10.1007/s00259-009-1176-zISI: 000270980400017PubMedID: 19504093ScopusID: 2-s2.0-72149106173OAI: diva2:412848

QC 20110426

Available from: 2011-04-26 Created: 2011-04-20 Last updated: 2013-02-06Bibliographically approved
In thesis
1. On the Design of Affibody Molecules for Radiolabeling and In Vivo Molecular Imaging
Open this publication in new window or tab >>On the Design of Affibody Molecules for Radiolabeling and In Vivo Molecular Imaging
2013 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Affibody molecules have lately shown great potential as tools for in vivo molecular imaging. These small, 3-helical bundles, with their highly stable protein scaffold, are well suited for the often harsh conditions of radiolabeling. Their small size allows for rapid clearance from the blood circulation which permits the collection of images already within hours after injection. This thesis includes four papers aimed at engineering different variants of a HER2-binding Affibody molecule to enable effective  and  flexible  radiolabeling  and  enhancing  the  molecular  imaging  in  terms  of  imaging contrast and resolution.

In paper I an Affibody molecule was engineered to function as a multifunctional platform for site-specific labeling with different nuclides for radionuclide imaging. This was done using only natural amino  acids,  thereby  allowing  for  both  synthetic  and  recombinant  production.  By  grafting  the amino acid sequence -GSECG to the C-terminal of our model-protein, a HER2-binding Affibody molecule, we enabled site specific labeling with both trivalent radiometals and with  99m Tc. Maleim-ide-DOTA was conjugated to the cysteine residue for labeling with  111 In, while the peptide sequence was able to chelate  99m Tc directly. This approach can also be used for site-specific labeling with other probes available for thiol-chemistry, and is applicable also to other protein scaffolds.

In paper II we investigated the impact of size and affinity of radiolabeled Affibody molecules on tumor targeting and image contrast. Two HER2-targeting Affibody molecules, a two-helix (~5 kDa) and a three-helix (~7 kDa) counterpart, were synthetically produced, labeled with  111 In via chelation by  DOTA  and  directly  compared  in  terms  of  biodistribution  and  targeting  properties.  Results showed  that  the  smaller  variant  can  provide  higher  contrast  images,  at  the  cost  of  lower  tumor uptake,  in  high-expressing  HER2-tumors.  However,  neither  the  tumor  uptake  nor  the  contrast of the two-helix variant is sufficient to compete with the three-helix molecule in tumors with low expression of HER2.

In paper III and IV we were aiming to find methods to improve the labeling of Affibody molecules with  18 F for PET imaging. Current methods are either complex, time-consuming or generate heavily lipophilic conjugates. This results in low yields of radiolabeled tracer, low specific activity left for imaging, undesirable biodistribution or a combination thereof. In paper III we demonstrate a swift and efficient 2-step, 1-pot method for labeling HER2-binding Affibody molecules by the formation of aluminum  18 F-fluoride (Al 18 F) and its chelation by NOTA, all in 30 min. The results show that the  18 F-NOTA-approach is a very promising method of labeling Affibody molecules with  18 F and further investigation of this scheme is highly motivated. In the last paper we pursued the possibility of decreasing the high kidney retention that is common among small radiotracers with residual-izing radiometabolites. In this work  18 F-4-fluorobenzaldehyde (FBA) was conjugated to a synthetic HER2-targeting Affibody molecule via oxime ligation. However, to avoid elevated liver retention, as seen in previous studies with this kind of label, a hydrophilic triglutamyl spacer between the aminooxy moiety and the N-terminal was introduced. A comparison of the two constructs (with and without the triglutamyl spacer) showed a clear reduction of retention in both kidney and liver in NMRI mice at 2 h p.i. when the spacer was included. In the light of these promising results, further studies including tumor-bearing mice, are in preparation.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2013. xi, 55 p.
Trita-BIO-Report, ISSN 1654-2312
Affibody molecule, AC/DC, radionuclide molecular imaging, HER2, SPECT, PET, biodistribution, peptide synthesis, radiolabeling
National Category
Biochemistry and Molecular Biology
Research subject
SRA - Molecular Bioscience
urn:nbn:se:kth:diva-117862 (URN)978-91-7501-635-1 (ISBN)
Public defence
2013-03-08, F2, Lindstedtsvägen 28, KTH, Stockholm, 10:00 (English)

QC 20130203

Available from: 2013-02-06 Created: 2013-02-05 Last updated: 2013-02-06Bibliographically approved

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