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Influence of DOTA Chelator Position on Biodistribution and Targeting Properties of In-111-Labeled Synthetic Anti-HER2 Affibody Molecules
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Preclinical PET Platform.
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2012 (English)In: Bioconjugate chemistry, ISSN 1043-1802, E-ISSN 1520-4812, Vol. 23, no 8, 1661-1670 p.Article in journal (Refereed) Published
Abstract [en]

Affibody molecules are a class of affinity proteins. Their small size (7 kDa) in combination with the high (subnanomolar) affinity for a number of cancer-associated molecular targets makes them suitable for molecular imaging. Earlier studies demonstrated that the selection of radionuclide and chelator may substantially influence the tumor-targeting properties of affibody molecules. Moreover, the placement of chelators for labeling of affibody molecules with Tc-99m at different positions in affibody molecules influenced both blood clearance rate and uptake in healthy tissues. This introduces an opportunity to improve the contrast of affibody-mediated imaging. In this comparative study, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) was conjugated to the synthetic affibody molecule Z(HER2:S1) at three different positions: DOTA-A1-Z(HER2:S1) (N-terminus), DOTA-K58-Z(HER2:S1) (C-terminus), and DOTA-K50-Z(HER2:S1) (middle of helix 3). The affinity for HER2 differed slightly among the variants and the K-D values were determined to be 133 pM, 107 pM and 94 pM for DOTA-A1-Z(HER2:S1), DOTA-K50-Z(HER2:S1), and DOTA-K58-Z(HER2:S1), respectively. Z(HER2:S1) K50-DOTA showed a slightly lower melting point (57 degrees C) compared to DOTA-A1-Z(HER2:S1) (64 degrees C) and DOTA-K5S-Z(HER2:S1) (62 degrees C), but all variants showed good refolding properties after heat treatment All conjugates were successfully labeled with In-III resulting in a radiochemical yield of 99% with preserved binding capacity. In vitro specificity studies using SKOV-3 and LS174T cell lines showed that the binding of the radiolabeled compounds was HER2 receptor mediated, which also was verified in vivo using BALB/C nu/nu mice with LS174T and Ramos lymphoma xenografts. The three conjugates all showed specific uptake in L5174T xenografts in nude mice, where DOTA-A1-Z(HER2:S1) and DOTA-K58-Z(HER2:S1) showed the highest uptake. Overall, DOTA-K58-Z(HER2:S1) provided the highest tumor-to-blood ratio, which is important for a high contrast imaging. In conclusion, the positioning of the DOTA chelator influences the cellular processing and the biodistribution pattern of radiolabeled affibody molecules, creating preconditions for imaging optimization.

Place, publisher, year, edition, pages
2012. Vol. 23, no 8, 1661-1670 p.
National Category
Chemical Sciences
URN: urn:nbn:se:uu:diva-181413DOI: 10.1021/bc3002369ISI: 000307487300016OAI: oai:DiVA.org:uu-181413DiVA: diva2:557599

Anna Perols and Hadis Honarvar contributed equally to this study

Available from: 2012-09-28 Created: 2012-09-24 Last updated: 2016-08-26Bibliographically approved
In thesis
1. Development of Affibody molecules for radionuclide molecular imaging and therapy of cancer
Open this publication in new window or tab >>Development of Affibody molecules for radionuclide molecular imaging and therapy of cancer
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Affibody molecules are a promising class of scaffold-based targeting proteins for radionuclide-based imaging and therapy of cancer. This thesis work is based on 5 original research articles (papers I-V), which focus on optimization of molecular design of HER2-binding Affibody variants for high contrast imaging of this predictive biomarker as well as development of Affibody molecules suitable for radionuclide-based targeted therapies. 

Papers I and II were dedicated to evaluation of the influence of the macrocyclic chelator DOTA positioning at N-terminus, in the middle of helix-3 and at C terminus of a synthetic Affibody molecule, ZHER2:S1. These synthetic variants were labelled with different radionuclides i.e. 111In and 68Ga to study also the effect of different labels on their biodistribution properties.

In paper III a 2-helix variant, Z342min, was developed using native ligation cyclization to cross-link helices one and two resulting in a stable 2-helix scaffold and characterized in vivo. This study was performed with the aim to obtain structure-properties relationship for development of smaller Affibody molecules.  

Papers IV and V were devoted to development of therapeutic strategies. In paper IV, a series of peptide based chelators was investigated for labelling of Affibody molecules with 188Re to provide low renal retention. In paper V, a pretargeting approach using peptide nucleic acid was investigated. These studies were performed with the aim to overcome the high renal retention of Affibody molecules when labelled with residualizing therapeutic radionuclides. Otherwise, the particle emitting radiometals could damage the kidneys more than the tumours.

The results obtained for anti-HER2 Affibody molecules summarized in this thesis might be of importance for the development of other scaffold protein based targeting agents.


Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2016. 71 p.
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1237
Affibody molecules, HER2, Molecular imaging, Radionuclide targeted therapy, Radionuclide molecular imaging, Labeling chemistry
National Category
Medical and Health Sciences
Research subject
Biomedical Radiation Science
urn:nbn:se:uu:diva-298740 (URN)978-91-554-9624-1 (ISBN)
External cooperation:
Public defence
2016-09-24, Fåhraeus Hall, Dag Hammarskjölds väg 20, Uppsala, 09:30 (English)
Available from: 2016-08-31 Created: 2016-07-06 Last updated: 2016-09-05

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