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Direct comparison of In-111-labelled two-helix and three-helix Affibody molecules for in vivo molecular imaging
KTH, School of Biotechnology (BIO), Molecular Biotechnology.ORCID iD: 0000-0003-4409-9236
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2012 (English)In: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 39, no 4, 693-702 p.Article in journal (Refereed) Published
Abstract [en]

Radiolabelled Affibody molecules have demonstrated a potential for visualization of tumour-associated molecular targets. Affibody molecules (7 kDa) are composed of three alpha-helices. Recently, a smaller two-helix variant of Affibody molecules (5.1 kDa) was developed. The aim of this study was to compare two- and three-helix HER2-targeting Affibody molecules directly in vivo. The three-helix Affibody molecule ABY-002 and the two-helix Affibody molecule PEP09239 were labelled with In-111 at the N-termini via DOTA chelator. Tumour-targeting properties were directly compared at 1 and 4 h after injection in mice bearing SKOV-3 xenografts with high HER2 expression and LS174T xenografts with low HER2 expression. The dissociation constants (K (D)) for HER2 binding were 78 pM for the three-helix Affibody molecule and 2.1 nM for the two-helix Affibody molecule. In-111-PEP09239 cleared more rapidly from the blood. In xenografts with high HER2 expression, the uptake of In-111-ABY-002 was significantly higher than that of In-111-PEP09239. The tumour-to-blood ratio was higher for In-111-PEP09239 at 4 h after injection, while there was no significant difference in other tumour-to-organ ratios. The tumour uptake of In-111-ABY-002 was eightfold higher than that of In-111-PEP09239 in xenografts with low expression. Tumour-to-blood ratios were equal in this case, but other tumour-to-organ ratios were appreciably higher for the three-helix variant. For tumours with high HER2 expression, two-helix HER2-targeting Affibody molecules can provide higher tumour-to-blood ratio at the cost of lower tumour uptake. In the case of low expression, both tumour uptake and tumour-to-organ ratios are appreciably higher for three-helix than for two-helix HER2-targeting Affibody molecules.

Place, publisher, year, edition, pages
2012. Vol. 39, no 4, 693-702 p.
Keyword [en]
111In-ABY-002, 111In-PEP09239, Affibody, HER2 targeting
National Category
Medical Laboratory and Measurements Technologies
URN: urn:nbn:se:kth:diva-59008DOI: 10.1007/s00259-011-2016-5ISI: 000302287500017PubMedID: 22170322ScopusID: 2-s2.0-84862514780OAI: diva2:474606
Swedish Research Council
QC 20120507Available from: 2012-01-09 Created: 2012-01-09 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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