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Affibody Molecules for PET Imaging
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology. (Vladimir Tolmachev)
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 [en]
Affibody molecules, Molecular imaging, PET, Radiolabelling, HER2, PDGFRβ
National Category
Medical and Health Sciences
Research subject
Biomedical Radiation Science
Identifiers
URN: urn:nbn:se:uu:diva-259410ISBN: 978-91-554-9299-1 (print)OAI: oai:DiVA.org:uu-259410DiVA: diva2:844633
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
List of papers
1. Influence of Nuclides and Chelators on Imaging Using Affibody Molecules: Comparative Evaluation of Recombinant Affibody Molecules Site-Specifically Labeled with 68Ga and 111In via Maleimido Derivatives of DOTA and NODAGA
Open this publication in new window or tab >>Influence of Nuclides and Chelators on Imaging Using Affibody Molecules: Comparative Evaluation of Recombinant Affibody Molecules Site-Specifically Labeled with 68Ga and 111In via Maleimido Derivatives of DOTA and NODAGA
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2013 (English)In: Bioconjugate chemistry, ISSN 1043-1802, E-ISSN 1520-4812, Vol. 24, no 6, 1102-1109 p.Article in journal (Refereed) Published
Abstract [en]

Accurate detection of cancer-associated molecular abnormalities in tumors could make cancer treatment more personalized. Affibody molecules enable high contrast imaging of tumor-associated protein expression shortly after injection. The use of the generator-produced positron-emitting radionuclide 68Ga should increase sensitivity of HER2 imaging. The chemical nature of radionuclides and chelators influences the biodistribution of Affibody molecules, providing an opportunity to further increase the imaging contrast. The aim of the study was to compare maleimido derivatives of DOTA and NODAGA for site-specific labeling of a recombinant ZHER2:2395 HER2-binding Affibody molecule with 68Ga. DOTA and NODAGA were site-specifically conjugated to the ZHER2:2395 Affibody molecule having a C-terminal cysteine and labeled with 68Ga and 111In. All labeled conjugates retained specificity to HER2 in vitro. Most of the cell-associated activity was membrane-bound with a minor difference in internalization rate. All variants demonstrated specific targeting of xenografts and a high tumor uptake. The xenografts were clearly visualized using all conjugates. The influence of chelator on the biodistribution and targeting properties was much less pronounced for 68Ga than for 111In. The tumor uptake of 68Ga-NODAGA-ZHER2:2395 and 68Ga-DOTA-ZHER2:2395 and tumor-to-blood ratios at 2 h p.i. did not differ significantly. However, the tumor-to-liver ratio was significantly higher for 68Ga-NODAGA- ZHER2:2395 (8 ± 2 vs 5.0 ± 0.3) offering the advantage of better liver metastases visualization. In conclusion, influence of chelators on biodistribution of Affibody molecules depends on the radionuclides and reoptimization of labeling chemistry is required when a radionuclide label is changed.

National Category
Basic Medicine
Identifiers
urn:nbn:se:uu:diva-203054 (URN)10.1021/bc300678y (DOI)000320898900030 ()23705574 (PubMedID)
Note

De två (2) första författarna delar förstaförfattarskapet.

Available from: 2013-07-02 Created: 2013-07-02 Last updated: 2017-12-06Bibliographically approved
2. Influence of Macrocyclic Chelators on the Targeting Properties of 68Ga-Labeled Synthetic Affibody Molecules: Comparison with 111In-Labeled Counterparts
Open this publication in new window or tab >>Influence of Macrocyclic Chelators on the Targeting Properties of 68Ga-Labeled Synthetic Affibody Molecules: Comparison with 111In-Labeled Counterparts
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2013 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 8, no 8, e70028- p.Article in journal (Refereed) Published
Abstract [en]

Affibody molecules are a class of small (7 kDa) non-immunoglobulin scaffold-based affinity proteins, which have demonstrated substantial potential as probes for radionuclide molecular imaging. The use of positron emission tomography (PET) would further increase the resolution and quantification accuracy of Affibody-based imaging. The rapid in vivo kinetics of Affibody molecules permit the use of the generator-produced radionuclide 68Ga (T1/2 = 67.6 min). Earlier studies have demonstrated that the chemical nature of chelators has a substantial influence on the biodistribution properties of Affibody molecules. To determine an optimal labeling approach, the macrocyclic chelators 1,4,7,10-tetraazacylododecane-1,4,7,10-t​etraaceticacid (DOTA), 1,4,7-triazacyclononane-N,N,N-triacetic acid (NOTA) and 1-(1,3-carboxypropyl)-1,4,7- triazacyclononane-4,7-diacetic acid (NODAGA) were conjugated to the N-terminus of the synthetic Affibody molecule ZHER2:S1 targeting HER2. Affibody molecules were labeled with 68Ga, and their binding specificity and cellular processing were evaluated. The biodistribution of 68Ga-DOTA-ZHER2:S1, 68Ga-NOTA-ZHER2:S1 and 68Ga-NODAGA-ZHER2:S1, as well as that of their 111In-labeled counterparts, was evaluated in BALB/C nu/nu mice bearing HER2-expressing SKOV3 xenografts. The tumor uptake for 68Ga-DOTA-ZHER2:S1 (17.9±0.7%IA/g) was significantly higher than for both 68Ga-NODAGA-ZHER2:S1 (16.13±0.67%IA/g) and 68Ga-NOTA-ZHER2:S1 (13±3%IA/g) at 2 h after injection. 68Ga-NODAGA-ZHER2:S1 had the highest tumor-to-blood ratio (60±10) in comparison with both 68Ga-DOTA-ZHER2:S1 (28±4) and 68Ga-NOTA-ZHER2:S1 (42±11). The tumor-to-liver ratio was also higher for 68Ga-NODAGA-ZHER2:S1 (7±2) than the DOTA and NOTA conjugates (5.5±0.6 vs.3.3±0.6). The influence of chelator on the biodistribution and targeting properties was less pronounced for 68Ga than for 111In. The results of this study demonstrate that macrocyclic chelators conjugated to the N-terminus have a substantial influence on the biodistribution of HER2-targeting Affibody molecules labeled with 68Ga.This can be utilized to enhance the imaging contrast of PET imaging using Affibody molecules and improve the sensitivity of molecular imaging. The study demonstrated an appreciable difference of chelator influence for 68Ga and 111In.

National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:uu:diva-205597 (URN)10.1371/journal.pone.0070028 (DOI)000324518400033 ()23936372 (PubMedID)
Available from: 2013-08-20 Created: 2013-08-20 Last updated: 2017-12-06Bibliographically approved
3. 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.
Open this publication in new window or tab >>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.
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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.

National Category
Biochemistry and Molecular Biology Radiology, Nuclear Medicine and Medical Imaging
Identifiers
urn:nbn:se:uu:diva-232934 (URN)10.2310/7290.2014.00034 (DOI)000349631400003 ()25249017 (PubMedID)
Funder
Swedish Research CouncilSwedish Cancer Society
Available from: 2014-09-27 Created: 2014-09-27 Last updated: 2017-12-05Bibliographically approved
4. Gallium-68-Labeled Affibody Molecule for PET Imaging of PDGFRβ Expression in Vivo
Open this publication in new window or tab >>Gallium-68-Labeled Affibody Molecule for PET Imaging of PDGFRβ Expression in Vivo
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2014 (English)In: Molecular Pharmaceutics, ISSN 1543-8384, E-ISSN 1543-8392, Vol. 11, no 11, 3957-3964 p.Article in journal (Refereed) Published
Abstract [en]

Platelet-derived growth factor receptor β (PDGFRβ) is a transmembrane tyrosine kinase receptor involved, for example, in angiogenesis. Overexpression and excessive signaling of PDGFRβ has been observed in multiple malignant tumors and fibrotic diseases, making this receptor a pharmaceutical target for monoclonal antibodies and tyrosine kinase inhibitors. Successful targeted therapy requires identification of responding patients. Radionuclide molecular imaging would enable determination of the PDGFRβ status in all lesions using a single noninvasive repeatable procedure. Recently, we have demonstrated that the affibody molecule Z09591 labeled with 111In can specifically target PDGFRβ-expressing tumors in vivo. The use of positron emission tomography (PET) as an imaging technique would provide superior resolution, sensitivity, and quantitation accuracy. In this study, a DOTA-conjugated Z09591 was labeled with the generator-produced positron emitting radionuclide 68Ga (T1/2 = 67.6 min, Eβ + max = 1899 keV, 89% β+). 68Ga-DOTA-Z09591 retained the capacity to specifically bind to PDGFRβ-expressing U-87 MG glioma cells. The half-maximum inhibition concentration (IC50) of 68Ga-DOTA-Z09591 (6.6 ± 1.4 nM) was somewhat higher than that of 111In-DOTA-Z09591 (1.4 ± 1.2 nM). 68Ga-DOTA-Z09591 demonstrated specific (saturable) targeting of U-87 MG xenografts in immunodeficient mice. The tumor uptake at 2 h after injection was 3.7 ± 1.7% IA/g, which provided a tumor-to-blood ratio of 8.0 ± 3.1. The only organ with higher accumulation of radioactivity was the kidney. MicroPET imaging provided high-contrast imaging of U-87 MG xenografts. In conclusion, the 68Ga-labeled affibody molecule Z09591 is a promising candidate for further development as a probe for imaging PDGFRβ expression in vivo using PET.

National Category
Medical Biotechnology
Identifiers
urn:nbn:se:uu:diva-235390 (URN)10.1021/mp500284t (DOI)000344307700020 ()24972112 (PubMedID)
Funder
Swedish Cancer SocietySwedish Research Council
Available from: 2014-11-01 Created: 2014-11-01 Last updated: 2017-12-05Bibliographically approved
5. Site-Specific Radioiodination of HER2-Targeting Affibody Molecules using 4-Iodophenethylmaleimide Decreases Renal Uptake of Radioactivity
Open this publication in new window or tab >>Site-Specific Radioiodination of HER2-Targeting Affibody Molecules using 4-Iodophenethylmaleimide Decreases Renal Uptake of Radioactivity
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2015 (English)In: ChemistryOpen, ISSN 2191-1363, Vol. 4, no 2, 174-182 p.Article in journal (Refereed) Published
Abstract [en]

Affibody molecules are small scaffold-based affinity proteins with promising properties as probes for radionuclide-based molecular imaging. However, a high reabsorption of radiolabeled Affibody molecules in kidneys is an issue. We have shown that the use of I-125-3-iodo-((4-hydroxyphenyl)ethyl)maleimide (IHPEM) for site-specific labeling of cysteine-containing Affibody molecules provides high tumor uptake but low radioactivity retention in kidneys. We hypothesized that the use of 4-iodophenethylmaleimide (IPEM) would further reduce renal retention of radioactivity because of higher lipophilicity of radiometabolites. An anti-human epidermal growth factor receptor type2 (HER2) Affibody molecule (Z(HER2:2395)) was labeled using I-125-IPEM with an overall yield of 45 +/- 3%. I-125-IPEM-Z(HER2:2395) bound specifically to HER2-expressing human ovarian carcinoma cells (SKOV-3 cell line). In NMRI mice, the renal uptake of I-125-IPEM-Z(HER2:2395) (24 +/- 2 and 5.7 +/- 0.3%IAg(-1)at 1 and 4 h after injection, respectively) was significantly lower than uptake of I-125-IHPEM-Z(HER2:2395) (50 +/- 8 and 12 +/- 2%IAg(-1)at 1 and 4 h after injection, respectively). In conclusion, the use of a more lipophilic linker for the radioiodination of Affibody molecules reduces renal radioactivity.

Keyword
affibody molecules, drug design, iodophenethylmaleimide, radiolabeling, radiopharmaceuticals
National Category
Chemical Sciences
Identifiers
urn:nbn:se:uu:diva-253262 (URN)10.1002/open.201402097 (DOI)000353653800014 ()25969816 (PubMedID)
Available from: 2015-05-26 Created: 2015-05-25 Last updated: 2017-12-04Bibliographically approved

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