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Evaluation of backbone-cyclized HER2-binding 2-helix Affibody molecule for In Vivo molecular imaging
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences. (Tolmachev)
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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2013 (English)In: Nuclear Medicine and Biology, ISSN 0969-8051, E-ISSN 1872-9614, Vol. 40, no 3, 378-386 p.Article in journal (Refereed) Published
Abstract [en]

Introduction

Affibody molecules, small scaffold proteins, have demonstrated an appreciable potential as imaging probes. Affibody molecules are composed of three alpha-helices. Helices 1 and 2 are involved in molecular recognition, while helix 3 provides stability. The size of Affibody molecules can be reduced by omitting the third alpha-helix and cross-linking the two remaining, providing a smaller molecule with better extravasation and quicker clearance of unbound tracer. The goal of this study was to develop a novel 2-helix Affibody molecule based on backbone cyclization by native chemical ligation (NCL).

Methods

The HER2-targeting NCL-cyclized Affibody molecule ZHER2:342min has been designed, synthesized and site-specifically conjugated with a DOTA chelator. DOTA-ZHER2:342min was labeled with 111In and 68 Ga. The binding affinity of DOTA-ZHER2:342min was evaluated in vitro. The targeting properties of 111In- and 68 Ga-DOTA-ZHER2:342min were evaluated in mice bearing SKOV-3 xenografts and compared with the properties of 111In- and 68 Ga-labeled PEP09239, a DOTA-conjugated 2-helix Affibody analogue cyclized by a homocysteine disulfide bridge.

Results

The dissociation constant (KD) for DOTA-ZHER2:342min binding to HER2 was 18 nM according to SPR measurements. DOTA-ZHER2:342min was labeled with 111In and 68 Ga. Both conjugates demonstrated bi-phasic binding kinetics to HER2-expressing cells, with KD1 in low nanomolar range. Both variants demonstrated specific uptake in HER2-expressing xenografts. Tumor-to-blood ratios at 2 h p.i. were 6.1 ± 1.3 for 111In- DOTA-ZHER2:342min and 4.6 ± 0.7 for 68 Ga-DOTA-ZHER2:342min. However, the uptake of DOTA-ZHER2:342min in lung, liver and spleen was appreciably higher than the uptake of PEP09239-based counterparts.

Conclusions

Native chemical ligation enables production of a backbone-cyclized HER2-binding 2-helix Affibody molecule (ZHER2:342min) with low nanomolar target affinity and specific tumor uptake.

Place, publisher, year, edition, pages
2013. Vol. 40, no 3, 378-386 p.
Keyword [en]
Affibody, HER2, 2-helix protein, SPPS, Native chemical ligation, biodistribution
National Category
Radiology, Nuclear Medicine and Medical Imaging
Identifiers
URN: urn:nbn:se:uu:diva-198614DOI: 10.1016/j.nucmedbio.2012.12.009ISI: 000316507400011OAI: oai:DiVA.org:uu-198614DiVA: diva2:617275
Note

NOTICE: this is the author's version of a work that was accepted for publication in Nuclear Medicine and Biology. Changes resulting from the publishing process, such as editing, corrections, structural formatting, and other quality control mechanisms may not be reflected in this document. Changes may have been made to this work since it was submitted for publication. A definitive version was subsequently published in:Honarvar H, Jokilaasko N, Andersson K, Malmberg J, Rosik D, Orlova A, Eriksson Karlström A, Tolmachev V, Järver P. Evaluation of Backbone-Cyclized HER2-Binding Two-Helix Affibody Molecule for In Vivo Molecular Imaging. Nucl Med Biol, 40,3, 2013 Apr; :378-86 

DOI 10.1016/j.nucmedbio.2012.12.009.

http://www.sciencedirect.com/science/article/pii/S0969805112003204

Available from: 2013-04-22 Created: 2013-04-22 Last updated: 2017-12-06Bibliographically 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.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1237
Keyword
Affibody molecules, HER2, Molecular imaging, Radionuclide targeted therapy, Radionuclide molecular imaging, Labeling chemistry
National Category
Medical and Health Sciences
Research subject
Biomedical Radiation Science
Identifiers
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)
Opponent
Supervisors
Available from: 2016-08-31 Created: 2016-07-06 Last updated: 2016-09-05

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