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Position for Site-Specific Attachment of a DOTA Chelator to Synthetic Affibody Molecules Has a Different Influence on the Targeting Properties of 68Ga- Compared to 111In-Labeled Conjugates.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Preclinical PET Platform.ORCID iD: 0000-0001-6120-2683
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2014 (English)In: Molecular Imaging, ISSN 1535-3508, E-ISSN 1536-0121, Vol. 13, 1-12 p.Article in journal (Refereed) Published
Abstract [en]

AbstractAffibody molecules, small (7 kDa) scaffold proteins, are a promising class of probes for radionuclide molecular imaging. Radiolabeling of Affibody molecules with the positron-emitting nuclide 68Ga would permit the use of positron emission tomography (PET), providing better resolution, sensitivity, and quantification accuracy than single-photon emission computed tomography (SPECT). The synthetic anti-HER2 ZHER2:S1 Affibody molecule was conjugated with DOTA at the N-terminus, in the middle of helix 3, or at the C-terminus. The biodistribution of 68Ga- and 111In-labeled Affibody molecules was directly compared in NMRI nu/nu mice bearing SKOV3 xenografts. The position of the chelator strongly influenced the biodistribution of the tracers, and the influence was more pronounced for 68Ga-labeled Affibody molecules than for the 111In-labeled counterparts. The best 68Ga-labeled variant was 68Ga-[DOTA-A1]-ZHER2:S1, which provided a tumor uptake of 13 ± 1 %ID/g and a tumor to blood ratio of 39 ± 12 at 2 hours after injection. 111In-[DOTA-A1]-ZHER2:S1 and 111In-[DOTA-K58]-ZHER2:S1 were equally good at this time point, providing a tumor uptake of 15 to 16 %ID/g and a tumor to blood ratio in the range of 60 to 80. In conclusion, the selection of the best position for a chelator in Affibody molecules can be used for optimization of their imaging properties. This may be important for the development of Affibody-based and other protein-based imaging probes.

Place, publisher, year, edition, pages
2014. Vol. 13, 1-12 p.
National Category
Biochemistry and Molecular Biology Radiology, Nuclear Medicine and Medical Imaging
Identifiers
URN: urn:nbn:se:uu:diva-232934DOI: 10.2310/7290.2014.00034ISI: 000349631400003PubMedID: 25249017OAI: oai:DiVA.org:uu-232934DiVA: diva2:750256
Funder
Swedish Research CouncilSwedish Cancer Society
Available from: 2014-09-27 Created: 2014-09-27 Last updated: 2017-12-05Bibliographically approved
In thesis
1. Affibody Molecules for PET Imaging
Open this publication in new window or tab >>Affibody Molecules for PET Imaging
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Optimization of Affibody molecules would allow for high contrast imaging of cancer associated surface receptors using molecular imaging. The primary aim of the thesis was to develop Affibody-based PET imaging agents to provide the highest possible sensitivity of RTK detection in vivo. The thesis evaluates the effect of radiolabelling chemistry on biodistribution and targeting properties of Affibody molecules directed against HER2 and PDGFRβ. The thesis is based on five published papers (I-V).

Paper I. The targeting properties of maleimido derivatives of DOTA and NODAGA for site-specific labelling of a recombinant HER2-binding Affibody molecule radiolabelled with 68Ga were compared in vivo. Favourable in vivo properties were seen for the Affibody molecule with the combination of 68Ga with NODAGA.

Paper II. The aim was to compare the biodistribution of 68Ga- and 111In-labelled HER2-targeting Affibody molecules containing DOTA, NOTA and NODAGA at the N-terminus. This paper also demonstrated favourable in vivo properties for Affibody molecules in combination with 68Ga and NODAGA placed on the N-terminus.

Paper III.  The influence of chelator positioning on the synthetic anti-HER2 affibody molecule labelled with 68Ga was investigated. The chelator DOTA was conjugated either at the N-terminus, the middle of helix-3 or at the C-terminus of the Affibody molecules. The N-terminus placement provided the highest tumour uptake and tumour-to-organ ratios.

Paper IV. The aim of this study was to evaluate if the 68Ga labelled PDGFRβ-targeting Affibody would provide an imaging agent suitable for PDGFRβ visualization using PET. The 68Ga labelled conjugate provided high-contrast imaging of PDGFRβ-expressing tumours in vivo using microPET as early as 2h after injection.

Paper V. This paper investigated if the replacement of IHPEM with IPEM as a linker molecule for radioiodination of Affibody molecules would reduce renal retention of radioactivity. Results showed that the use of the more lipophilic linker IPEM reduced the renal radioactivity retention for radioiodinated Affibody molecules.

In conclusion, this thesis clearly demonstrates that the labelling strategy is of great importance with a substantial influence on the targeting properties of Affibody molecules and should be taken under serious considerations when developing new imaging agents.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2015. 70 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1125
Keyword
Affibody molecules, Molecular imaging, PET, Radiolabelling, HER2, PDGFRβ
National Category
Medical and Health Sciences
Research subject
Biomedical Radiation Science
Identifiers
urn:nbn:se:uu:diva-259410 (URN)978-91-554-9299-1 (ISBN)
Public defence
2015-10-03, Fåhraeussalen, Rudbecklaboratoriet, Dag Hammarskjölds väg 20, 751 85, Uppsala, 09:00 (English)
Opponent
Supervisors
Available from: 2015-09-03 Created: 2015-08-03 Last updated: 2015-10-01
2. 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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Honarvar, HadisStrand, JoannaOrlova, AnnaSelvaraju, Ram KumarTolmachev, Vladimir

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