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Preclinical evaluation of anti-HER2 Affibody molecules site-specifically labeled with 111In using a maleimido derivative of NODAGA
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences. (Vladimir Tolmachev)
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Section of Medical Physics.
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2012 (English)In: Nuclear Medicine and Biology, ISSN 0969-8051, E-ISSN 1872-9614, Vol. 39, no 4, 518-529 p.Article in journal (Refereed) Published
Abstract [en]


Affibody molecules have demonstrated potential for radionuclide molecular imaging. The aim of this study was to synthesize and evaluate a maleimido derivative of the 1,4,7-triazacyclononane-1-glutaric acid-4,7-diacetic acid (NODAGA) for site-specific labeling of anti-HER2 Affibody molecule.


The maleimidoethylmonoamide NODAGA (MMA-NODAGA) was synthesized and conjugated to ZHER2:2395 Affibody molecule having a C-terminal cysteine. Labeling efficiency, binding specificity to and cell internalization by HER2-expressing cells of [111In-MMA-NODAGA-Cys61]-ZHER2:2395 were studied. Biodistribution of [111In-MMA-NODAGA-Cys61]-ZHER2:2395 and [111In-MMA-DOTA-Cys61]-ZHER2:2395 was compared in mice.


The affinity of [MMA-NODAGA-Cys61]-ZHER2:2395 binding to HER2 was 67 pM. The 111In-labeling yield was 99.6%±0.5% after 30 min at 60°C. [111In-MMA-NODAGA-Cys61]-ZHER2:2395 bound specifically to HER2-expressing cells in vitro and in vivo. Tumor uptake of [111In-MMA-NODAGA-Cys61]-ZHER2:2395 in mice bearing DU-145 xenografts (4.7%±0.8% ID/g) was lower than uptake of [111In-MMA-DOTA-Cys61]-ZHER2:2395 (7.5%±1.6% ID/g). However, tumor-to-organ ratios were higher for [111In-MMA-NODAGA-Cys61]-ZHER2:2395 due to higher clearance rate from normal tissues.


MMA-NODAGA is a promising chelator for site-specific labeling of targeting proteins containing unpaired cysteine. Appreciable influence of chelators on targeting properties of Affibody molecules was demonstrated.

Place, publisher, year, edition, pages
2012. Vol. 39, no 4, 518-529 p.
National Category
Medical and Health Sciences
URN: urn:nbn:se:uu:diva-164424DOI: 10.1016/j.nucmedbio.2011.10.013ISI: 000303790600008PubMedID: 22172396OAI: oai:DiVA.org:uu-164424DiVA: diva2:525013
Available from: 2012-05-04 Created: 2011-12-20 Last updated: 2015-03-24Bibliographically approved
In thesis
1. Tumour Targeting using Radiolabelled Affibody Molecules: Influence of Labelling Chemistry
Open this publication in new window or tab >>Tumour Targeting using Radiolabelled Affibody Molecules: Influence of Labelling Chemistry
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Affibody molecules are promising candidates for targeted radionuclide-based imaging and therapy applications. Optimisation of targeting properties would permit the in vivo visualization of cancer-specific surface receptors with high contrast. In therapy, this may increase the ratio of radioactivity uptake between tumour and normal tissues.  This thesis work is based on 5 original research articles (papers I-V) and focuses on optimisation of targeting properties of anti-HER2 affibody molecules by optimising the labelling chemistry.

Paper I and II report the comparative evaluation of the anti-HER2 ZHER2:2395 affibody molecule site specifically labelled with 111In (suitable for SPECT imaging) and 68Ga (suitable for PET imaging) using the thiol reactive derivatives of DOTA and NODAGA as chelators. The incorporation of different macrocyclic chelators and labelling with different radionuclides modified the biodistribution properties of affibody molecules. This indicates that the labelling strategy may have a profound effect on the targeting properties of radiotracers and must be carefully optimized.

Paper III reports the study of the mechanism of renal reabsorption of anti-HER2 ZHER2:2395 affibody molecule. An unknown receptor (not HER2) is suspected to be responsible for the high reabsorption of ZHER2:2395 molecules in the kidneys.

Paper IV reports the optimization and development of in vivo targeting properties of 188Re-labelled anti-HER2 affibody molecules. By using an array of peptide based chelators, it was found that substitution of one amino acid by another or changing its position can have a dramatic effect on the biodistribution properties of 188Re-labelled affibody molecules. This permitted the selection of –GGGC chelator whichdemonstrated the lowest retention of radioactivity in kidneys compared to other variants and showed excellent tumour targeting properties.

Paper V reports the preclinical evaluation of 188Re-ZHER2:V2 as a potential candidate for targeted radionuclide therapy of HER2-expressing tumours. In vivo experiments in mice along with dosimetry assessment in both murine and human models revealed that future human radiotherapy studies using 188Re-ZHER2:V2 may be feasible.

It would be reasonable to believe that the results of optimisation of anti-HER2 affibody molecules summarized in this thesis can be of importance for the development of other scaffold protein-based targeting agents.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2014. 77 p.
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1013
HER2, Affibody molecule, Radionuclide molecular maging, Targeted radionuclide therapy, Labeling chemistry
National Category
Medical and Health Sciences
urn:nbn:se:uu:diva-229090 (URN)978-91-554-8983-0 (ISBN)
Public defence
2014-09-20, Rudbecksalen, DagHammarskjölds väg 20, Uppsala, 09:00 (English)
Available from: 2014-08-28 Created: 2014-07-29 Last updated: 2014-09-08

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