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On the Design of Affibody Molecules for Radiolabeling and In Vivo Molecular Imaging
KTH, School of Biotechnology (BIO), Molecular Biotechnology. (Prof. Amelie Eriksson Karlström)ORCID iD: 0000-0003-4409-9236
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.
Series
Trita-BIO-Report, ISSN 1654-2312
Keyword [en]
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
Identifiers
URN: urn:nbn:se:kth:diva-117862ISBN: 978-91-7501-635-1 (print)OAI: oai:DiVA.org:kth-117862DiVA: diva2:603189
Public defence
2013-03-08, F2, Lindstedtsvägen 28, KTH, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

QC 20130203

Available from: 2013-02-06 Created: 2013-02-05 Last updated: 2013-02-06Bibliographically approved
List of papers
1. Design, synthesis and biological evaluation of a multifunctional HER2-specific Affibody molecule for molecular imaging
Open this publication in new window or tab >>Design, synthesis and biological evaluation of a multifunctional HER2-specific Affibody molecule for molecular imaging
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2009 (English)In: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 36, no 11, 1864-1873 p.Article in journal (Refereed) Published
Abstract [en]

The purpose of this study was to design and evaluate a novel platform for labelling of Affibody molecules, enabling both recombinant and synthetic production and site-specific labelling with Tc-99m or trivalent radiometals. The HER2-specific Affibody molecule PEP05352 was made by peptide synthesis. The chelator sequence SECG (serine-glutamic acid-cysteine-glycine) was anchored on the C-terminal to allow Tc-99m labelling. The cysteine can alternatively serve as a conjugation site of the chelator DOTA for indium labelling. The resulting Tc-99m- and In-111-labelled Affibody molecules were evaluated both in vitro and in vivo. Both conjugates retained their capacity to bind to HER2 receptors in vitro and in vivo. The tumour to blood ratio in LS174T xenografts was 30 at 4 h post-injection for both conjugates. Biodistribution data showed that the Tc-99m-labelled Affibody molecule had a fourfold lower kidney accumulation compared with the In-111-labelled Affibody molecule while the accumulation in other organs was similar. Gamma camera imaging of the conjugates could clearly visualise the tumours 4 h after injection. Incorporation of the C-terminal SECG sequence in Affibody molecules provides a general multifunctional platform for site-specific labelling with different nuclides (technetium, indium, gallium, cobalt or yttrium) and for a flexible production (chemical synthesis or recombinant).

Keyword
HER2, Affibody molecules, Peptide synthesis, Imaging
National Category
Industrial Biotechnology Medical and Health Sciences
Identifiers
urn:nbn:se:kth:diva-32789 (URN)10.1007/s00259-009-1176-z (DOI)000270980400017 ()19504093 (PubMedID)2-s2.0-72149106173 (Scopus ID)
Note

QC 20110426

Available from: 2011-04-26 Created: 2011-04-20 Last updated: 2017-12-11Bibliographically approved
2. Direct comparison of In-111-labelled two-helix and three-helix Affibody molecules for in vivo molecular imaging
Open this publication in new window or tab >>Direct comparison of In-111-labelled two-helix and three-helix Affibody molecules for in vivo molecular imaging
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2012 (English)In: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 39, no 4, 693-702 p.Article in journal (Refereed) Published
Abstract [en]

Radiolabelled Affibody molecules have demonstrated a potential for visualization of tumour-associated molecular targets. Affibody molecules (7 kDa) are composed of three alpha-helices. Recently, a smaller two-helix variant of Affibody molecules (5.1 kDa) was developed. The aim of this study was to compare two- and three-helix HER2-targeting Affibody molecules directly in vivo. The three-helix Affibody molecule ABY-002 and the two-helix Affibody molecule PEP09239 were labelled with In-111 at the N-termini via DOTA chelator. Tumour-targeting properties were directly compared at 1 and 4 h after injection in mice bearing SKOV-3 xenografts with high HER2 expression and LS174T xenografts with low HER2 expression. The dissociation constants (K (D)) for HER2 binding were 78 pM for the three-helix Affibody molecule and 2.1 nM for the two-helix Affibody molecule. In-111-PEP09239 cleared more rapidly from the blood. In xenografts with high HER2 expression, the uptake of In-111-ABY-002 was significantly higher than that of In-111-PEP09239. The tumour-to-blood ratio was higher for In-111-PEP09239 at 4 h after injection, while there was no significant difference in other tumour-to-organ ratios. The tumour uptake of In-111-ABY-002 was eightfold higher than that of In-111-PEP09239 in xenografts with low expression. Tumour-to-blood ratios were equal in this case, but other tumour-to-organ ratios were appreciably higher for the three-helix variant. For tumours with high HER2 expression, two-helix HER2-targeting Affibody molecules can provide higher tumour-to-blood ratio at the cost of lower tumour uptake. In the case of low expression, both tumour uptake and tumour-to-organ ratios are appreciably higher for three-helix than for two-helix HER2-targeting Affibody molecules.

Keyword
111In-ABY-002, 111In-PEP09239, Affibody, HER2 targeting
National Category
Medical Laboratory and Measurements Technologies
Identifiers
urn:nbn:se:kth:diva-59008 (URN)10.1007/s00259-011-2016-5 (DOI)000302287500017 ()22170322 (PubMedID)2-s2.0-84862514780 (Scopus ID)
Funder
Swedish Research Council
Note
QC 20120507Available from: 2012-01-09 Created: 2012-01-09 Last updated: 2017-12-08Bibliographically approved
3. 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
Open this publication in new window or tab >>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
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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.

Keyword
HER2, Affibody molecule, PET, (18)F, ovarian cancer
National Category
Radiology, Nuclear Medicine and Medical Imaging
Identifiers
urn:nbn:se:kth:diva-75531 (URN)10.2967/jnumed.111.093047 (DOI)000298660900003 ()22173842 (PubMedID)2-s2.0-84855390257 (Scopus ID)
Funder
Swedish Research Council
Note
QC 20120206Available from: 2012-02-06 Created: 2012-02-06 Last updated: 2017-12-08Bibliographically approved
4. Incorporation of a Triglutamyl Spacer Improves the Biodistribution of Synthetic Affibody Molecules Radiofluorinated at the N-Terminus via Oxime Formation with F-18-4-Fluorobenzaldehyde
Open this publication in new window or tab >>Incorporation of a Triglutamyl Spacer Improves the Biodistribution of Synthetic Affibody Molecules Radiofluorinated at the N-Terminus via Oxime Formation with F-18-4-Fluorobenzaldehyde
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2014 (English)In: Bioconjugate chemistry, ISSN 1043-1802, E-ISSN 1520-4812, Vol. 25, no 1, 82-92 p.Article in journal (Refereed) Published
Abstract [en]

Affibody molecules are a class of affinity agents for molecular imaging based on a non-immunoglobulin protein scaffold. Previous studies have demonstrated high contrast for in vivo imaging of cancer-associated molecular abnormalities using Affibody molecules. Using the radionuclide F-18 for labeling and PET as the imaging modality, the sensitivity of molecular imaging using Affibody molecules can be further increased. The use of oxime formation between an aminooxy-functionalized peptide and F-18-fluorobenzaldehyde (F-18-FBA) is a promising way of radiolabeling of targeting peptides. However, previous studies demonstrated that application of this method to Affibody molecules is associated with high liver uptake. We hypothesized that incorporation of a triglutamyl spacer between the aminooxy moiety and the N-terminus of a synthetic Affibody molecule would decrease the hepatic uptake of the F-18-N-(4-fluorobenzylidine)oxime) (F-18-FBO)-labeled tracer. To verify this, we have produced two variants of the HER2-targeting Z(HER2:342) Affibody molecule by peptide synthesis: OA-PEP4313, where aminooxyacetic acid was conjugated directly to the N-terminal alanine, and OA-E-3-PEP4313, where a triglutamyl spacer was introduced between the aminooxy moiety and the N-terminus. We have found that the use of the spacer is associated with a minor decrease of affinity, from K-D = 49 pM to K-D = 180 pM. Radiolabeled F-18-FBO-E-3-PEP4313 demonstrated specific binding to HER2-expressing ovarian carcinoma SKOV-3 cells and slow internalization. Biodistribution studies in mice demonstrated that the use of a triglutamyl linker decreased uptake of radioactivity in liver 2.7-fold at 2 h after injection. Interestingly, radioactivity uptake in kidneys was also reduced (2.4-fold). Experiments in BALB/C nu/nu mice bearing SKOV-3 xenografts demonstrated HER2-specific uptake of F-18-FBO-E-3-PEP4313 in tumors. At 2 h pi, the tumor uptake (20 +/- 2% ID/g) exceeded uptake in liver 5-fold and uptake in kidneys 3.6-fold. The tumor-to-blood ratio was 21 +/- 3. The microPET/CT imaging experiment confirmed the biodistribution data. In conclusion, the use of a triglutamyl spacer is a convenient way to improve the biodistribution profile of Affibody molecules labeled at the N-terminus using F-18-FBA. It provides a tracer capable of producing high-contrast images of HER2-expressing tumors.

Keyword
Positron-Emission-Tomography, In-Vivo, Anti-Her2 Affibody, Labeling Methods, Her2 Expression, Immuno-Pet, Peptides, Affinity, Proteins, Receptor
National Category
Medical Laboratory and Measurements Technologies
Identifiers
urn:nbn:se:kth:diva-117890 (URN)10.1021/bc400343r (DOI)000330018400011 ()2-s2.0-84892751639 (Scopus ID)
Funder
Swedish Cancer SocietySwedish Research CouncilVinnova
Note

QC 20140221. Updated from manuscript to article in journal.

Available from: 2013-02-06 Created: 2013-02-06 Last updated: 2017-12-06Bibliographically approved

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