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Imaging of Human Epidermal Growth Factor Receptor Type 2 Expression with (18)F-Labeled Affibody Molecule Z(HER2:2395) in a Mouse Model for Ovarian Cancer
KTH, School of Biotechnology (BIO), Molecular Biotechnology.ORCID iD: 0000-0003-4409-9236
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2012 (English)In: Journal of Nuclear Medicine, ISSN 0161-5505, E-ISSN 1535-5667, Vol. 53, no 1, 146-153 p.Article in journal (Refereed) Published
Abstract [en]

Affibody molecules are small (7 kDa) proteins with subnanomolar targeting affinity. Previous SPECT studies in xenografts have shown that the Affibody molecule (111)In-DOTA-Z(HER2:2395) can discriminate between high and low human epidermal growth factor receptor type 2 (HER2)-expressing tumors, indicating that radiolabeled Affibody molecules have potential for patient selection for HER2-targeted therapy. Compared with SPECT, PET with positron-emitting radionuclides, such as (18)F, may improve imaging of HER2 expression because of higher sensitivity and improved quantification of PET. The aim of the present study was to determine whether the (18)F-labeled NOTA-conjugated Affibody molecule Z(HER2:2395) is a suitable agent for imaging of HER2 expression. The tumor-targeting properties of (18)F-labeled Z(HER2:2395) were compared with (111)In- and (68)Ga-labeled Z(HER2:2395) in mice with HER2-expressing SK-OV-3 xenografts. Methods: Z(HER2:2395) was conjugated with NOTA and radiolabeled with (18)F, (68)Ga, and (111)In. Radiolabeling with (18)F was based on the complexation of Al(18)F by NOTA. The 50% inhibitory concentration values for NOTA-Z(HER2:2395) labeled with (19)F, (69)Ga, and (115)In were determined in a competitive cell-binding assay using SK-OV-3 cells. Mice bearing subcutaneous SK-OV-3 xenografts were injected intravenously with radiolabeled NOTA-Z(HER2:2395). One and 4 h after injection, PET/CT or SPECT/CT images were acquired, and the biodistribution was determined by ex vivo measurement. Results: The 50% inhibitory concentration values for (19)F-, (69)Ga-, and (115)In-NOTA-Z(HER2:2395) were 5.0, 6.3, and 5.3 nM, respectively. One hour after injection, tumor uptake was 4.4 +/- 0.8 percentage injected dose per gram (% ID/g), 5.6 +/- 1.6 % ID/g, and 7.1 +/- 1.4 % ID/g for (18)F-, (68)Ga-, and (111)In-NOTA-Z(HER2:2395), respectively, and the respective tumor-to-blood ratios were 7.4 +/- 1.8, 8.0 +/- 1.3, and 4.8 +/- 1.3. Tumor uptake was specific, because uptake could be blocked efficiently by coinjection of an excess of unlabeled Z(HER2:2395). PET/CT and SPECT/CT images clearly visualized HER2-expressing SK-OV-3 xenografts. Conclusion: This study showed that (18)F-NOTA-Z(HER2:2395) is a promising new imaging agent for HER2 expression in tumors. Affibody molecules were successfully labeled with (18)F within 30 min, based on the complexation of Al(18)F by NOTA. Further research is needed to determine whether this technique can be used for patient selection for HER2-targeted therapy.

Place, publisher, year, edition, pages
2012. Vol. 53, no 1, 146-153 p.
Keyword [en]
HER2, Affibody molecule, PET, (18)F, ovarian cancer
National Category
Radiology, Nuclear Medicine and Medical Imaging
URN: urn:nbn:se:kth:diva-75531DOI: 10.2967/jnumed.111.093047ISI: 000298660900003PubMedID: 22173842ScopusID: 2-s2.0-84855390257OAI: diva2:490858
Swedish Research Council
QC 20120206Available from: 2012-02-06 Created: 2012-02-06 Last updated: 2013-02-06Bibliographically approved
In thesis
1. On the Design of Affibody Molecules for Radiolabeling and In Vivo Molecular Imaging
Open this publication in new window or tab >>On the Design of Affibody Molecules for Radiolabeling and In Vivo Molecular Imaging
2013 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Affibody molecules have lately shown great potential as tools for in vivo molecular imaging. These small, 3-helical bundles, with their highly stable protein scaffold, are well suited for the often harsh conditions of radiolabeling. Their small size allows for rapid clearance from the blood circulation which permits the collection of images already within hours after injection. This thesis includes four papers aimed at engineering different variants of a HER2-binding Affibody molecule to enable effective  and  flexible  radiolabeling  and  enhancing  the  molecular  imaging  in  terms  of  imaging contrast and resolution.

In paper I an Affibody molecule was engineered to function as a multifunctional platform for site-specific labeling with different nuclides for radionuclide imaging. This was done using only natural amino  acids,  thereby  allowing  for  both  synthetic  and  recombinant  production.  By  grafting  the amino acid sequence -GSECG to the C-terminal of our model-protein, a HER2-binding Affibody molecule, we enabled site specific labeling with both trivalent radiometals and with  99m Tc. Maleim-ide-DOTA was conjugated to the cysteine residue for labeling with  111 In, while the peptide sequence was able to chelate  99m Tc directly. This approach can also be used for site-specific labeling with other probes available for thiol-chemistry, and is applicable also to other protein scaffolds.

In paper II we investigated the impact of size and affinity of radiolabeled Affibody molecules on tumor targeting and image contrast. Two HER2-targeting Affibody molecules, a two-helix (~5 kDa) and a three-helix (~7 kDa) counterpart, were synthetically produced, labeled with  111 In via chelation by  DOTA  and  directly  compared  in  terms  of  biodistribution  and  targeting  properties.  Results showed  that  the  smaller  variant  can  provide  higher  contrast  images,  at  the  cost  of  lower  tumor uptake,  in  high-expressing  HER2-tumors.  However,  neither  the  tumor  uptake  nor  the  contrast of the two-helix variant is sufficient to compete with the three-helix molecule in tumors with low expression of HER2.

In paper III and IV we were aiming to find methods to improve the labeling of Affibody molecules with  18 F for PET imaging. Current methods are either complex, time-consuming or generate heavily lipophilic conjugates. This results in low yields of radiolabeled tracer, low specific activity left for imaging, undesirable biodistribution or a combination thereof. In paper III we demonstrate a swift and efficient 2-step, 1-pot method for labeling HER2-binding Affibody molecules by the formation of aluminum  18 F-fluoride (Al 18 F) and its chelation by NOTA, all in 30 min. The results show that the  18 F-NOTA-approach is a very promising method of labeling Affibody molecules with  18 F and further investigation of this scheme is highly motivated. In the last paper we pursued the possibility of decreasing the high kidney retention that is common among small radiotracers with residual-izing radiometabolites. In this work  18 F-4-fluorobenzaldehyde (FBA) was conjugated to a synthetic HER2-targeting Affibody molecule via oxime ligation. However, to avoid elevated liver retention, as seen in previous studies with this kind of label, a hydrophilic triglutamyl spacer between the aminooxy moiety and the N-terminal was introduced. A comparison of the two constructs (with and without the triglutamyl spacer) showed a clear reduction of retention in both kidney and liver in NMRI mice at 2 h p.i. when the spacer was included. In the light of these promising results, further studies including tumor-bearing mice, are in preparation.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2013. xi, 55 p.
Trita-BIO-Report, ISSN 1654-2312
Affibody molecule, AC/DC, radionuclide molecular imaging, HER2, SPECT, PET, biodistribution, peptide synthesis, radiolabeling
National Category
Biochemistry and Molecular Biology
Research subject
SRA - Molecular Bioscience
urn:nbn:se:kth:diva-117862 (URN)978-91-7501-635-1 (ISBN)
Public defence
2013-03-08, F2, Lindstedtsvägen 28, KTH, Stockholm, 10:00 (English)

QC 20130203

Available from: 2013-02-06 Created: 2013-02-05 Last updated: 2013-02-06Bibliographically approved

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