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Development of Affibody molecules for radionuclide molecular imaging and therapy of cancer
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science.
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.
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1237
Keyword [en]
Affibody molecules, HER2, Molecular imaging, Radionuclide targeted therapy, Radionuclide molecular imaging, Labeling chemistry
National Category
Medical and Health Sciences
Research subject
Biomedical Radiation Science
URN: urn:nbn:se:uu:diva-298740ISBN: 978-91-554-9624-1OAI: oai:DiVA.org:uu-298740DiVA: diva2:947052
Public defence
2016-09-24, Fåhraeus Hall, Dag Hammarskjölds väg 20, Uppsala, 09:30 (English)
Available from: 2016-08-31 Created: 2016-07-06 Last updated: 2016-09-05
List of papers
1. Influence of DOTA Chelator Position on Biodistribution and Targeting Properties of In-111-Labeled Synthetic Anti-HER2 Affibody Molecules
Open this publication in new window or tab >>Influence of DOTA Chelator Position on Biodistribution and Targeting Properties of In-111-Labeled Synthetic Anti-HER2 Affibody Molecules
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2012 (English)In: Bioconjugate chemistry, ISSN 1043-1802, E-ISSN 1520-4812, Vol. 23, no 8, 1661-1670 p.Article in journal (Refereed) Published
Abstract [en]

Affibody molecules are a class of affinity proteins. Their small size (7 kDa) in combination with the high (subnanomolar) affinity for a number of cancer-associated molecular targets makes them suitable for molecular imaging. Earlier studies demonstrated that the selection of radionuclide and chelator may substantially influence the tumor-targeting properties of affibody molecules. Moreover, the placement of chelators for labeling of affibody molecules with Tc-99m at different positions in affibody molecules influenced both blood clearance rate and uptake in healthy tissues. This introduces an opportunity to improve the contrast of affibody-mediated imaging. In this comparative study, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) was conjugated to the synthetic affibody molecule Z(HER2:S1) at three different positions: DOTA-A1-Z(HER2:S1) (N-terminus), DOTA-K58-Z(HER2:S1) (C-terminus), and DOTA-K50-Z(HER2:S1) (middle of helix 3). The affinity for HER2 differed slightly among the variants and the K-D values were determined to be 133 pM, 107 pM and 94 pM for DOTA-A1-Z(HER2:S1), DOTA-K50-Z(HER2:S1), and DOTA-K58-Z(HER2:S1), respectively. Z(HER2:S1) K50-DOTA showed a slightly lower melting point (57 degrees C) compared to DOTA-A1-Z(HER2:S1) (64 degrees C) and DOTA-K5S-Z(HER2:S1) (62 degrees C), but all variants showed good refolding properties after heat treatment All conjugates were successfully labeled with In-III resulting in a radiochemical yield of 99% with preserved binding capacity. In vitro specificity studies using SKOV-3 and LS174T cell lines showed that the binding of the radiolabeled compounds was HER2 receptor mediated, which also was verified in vivo using BALB/C nu/nu mice with LS174T and Ramos lymphoma xenografts. The three conjugates all showed specific uptake in L5174T xenografts in nude mice, where DOTA-A1-Z(HER2:S1) and DOTA-K58-Z(HER2:S1) showed the highest uptake. Overall, DOTA-K58-Z(HER2:S1) provided the highest tumor-to-blood ratio, which is important for a high contrast imaging. In conclusion, the positioning of the DOTA chelator influences the cellular processing and the biodistribution pattern of radiolabeled affibody molecules, creating preconditions for imaging optimization.

National Category
Chemical Sciences
urn:nbn:se:uu:diva-181413 (URN)10.1021/bc3002369 (DOI)000307487300016 ()

Anna Perols and Hadis Honarvar contributed equally to this study

Available from: 2012-09-28 Created: 2012-09-24 Last updated: 2016-08-26Bibliographically approved
2. 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
urn:nbn:se:uu:diva-232934 (URN)10.2310/7290.2014.00034 (DOI)25249017 (PubMedID)
Available from: 2014-09-27 Created: 2014-09-27 Last updated: 2016-08-26Bibliographically approved
3. Evaluation of backbone-cyclized HER2-binding 2-helix Affibody molecule for In Vivo molecular imaging
Open this publication in new window or tab >>Evaluation of backbone-cyclized HER2-binding 2-helix Affibody molecule for In Vivo molecular imaging
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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]


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).


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.


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.


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.

Affibody, HER2, 2-helix protein, SPPS, Native chemical ligation, biodistribution
National Category
Radiology, Nuclear Medicine and Medical Imaging
urn:nbn:se:uu:diva-198614 (URN)10.1016/j.nucmedbio.2012.12.009 (DOI)000316507400011 ()

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.


Available from: 2013-04-22 Created: 2013-04-22 Last updated: 2016-08-26Bibliographically approved
4. Selection of an optimal cysteine-containing peptide-based chelator for labeling of affibody molecules with 188Re
Open this publication in new window or tab >>Selection of an optimal cysteine-containing peptide-based chelator for labeling of affibody molecules with 188Re
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2014 (English)In: European Journal of Medicinal Chemistry, ISSN 0223-5234, E-ISSN 1768-3254, Vol. 87, 519-528 p.Article in journal (Refereed) Published
Abstract [en]

Affibody molecules constitute a class of small (7 kDa) scaffold proteins that can be engineered to have excellent tumor targeting properties. High reabsorption in kidneys complicates development of affibody molecules for radionuclide therapy. In this study, we evaluated the influence of the composition of cysteine-containing C-terminal peptide-based chelators on the biodistribution and renal retention of 188Re-labeled anti-HER2 affibody molecules. Biodistribution of affibody molecules containing GGXC or GXGC peptide chelators (where X is G, S, E or K) was compared with biodistribution of a parental affibody molecule ZHER2:2395 having a KVDC peptide chelator. All constructs retained low picomolar affinity to HER2-expressing cells after labeling. The biodistribution of all 188Re-labeled affibody molecules was in general comparable, with the main observed difference found in the uptake and retention of radioactivity in excretory organs. The 188Re-ZHER2:V2 affibody molecule with a GGGC chelator provided the lowest uptake in all organs and tissues. The renal retention of 188Re-ZHER2:V2 (3.1 ± 0.5 %ID/g at 4 h after injection) was 55-fold lower than retention of the parental 188Re-ZHER2:2395 (172 ± 32 %ID/g). We show that engineering of cysteine-containing peptide-based chelators can be used for significant improvement of biodistribution of 188Re-labeled scaffold proteins, particularly reduction of their uptake in excretory organs.

Rhenium-188; Affibody; HER2; Peptide-based chelator; Biodistribution; Radionuclide therapy
National Category
Medical Biotechnology
urn:nbn:se:uu:diva-235383 (URN)10.1016/j.ejmech.2014.09.082 (DOI)000363431300049 ()25282673 (PubMedID)
Available from: 2014-11-01 Created: 2014-11-01 Last updated: 2016-08-26Bibliographically approved
5. Feasibility of Affibody Molecule-Based PNA-Mediated Radionuclide Pretargeting of Malignant Tumors
Open this publication in new window or tab >>Feasibility of Affibody Molecule-Based PNA-Mediated Radionuclide Pretargeting of Malignant Tumors
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2016 (English)In: Theranostics, ISSN 1838-7640, E-ISSN 1838-7640, Vol. 6, no 1, 93-103 p.Article in journal (Refereed) Published
Abstract [en]

Affibody molecules are small (7 kDa), non-immunoglobulin scaffold proteins with a potential as targeting agents for radionuclide imaging of cancer. However, high renal re-absorption of Affibody molecules prevents their use for radionuclide therapy with residualizing radiometals. We hypothesized that the use of Affibody-based peptide nucleic acid (PNA)-mediated pretargeting would enable higher accumulation of radiometals in tumors than in kidneys. To test this hypothesis, we designed an Affibody-PNA chimera ZHER2:342-SR-HP1 containing a 15-mer HP1 PNA recognition tag and a complementary HP2 hybridization probe permitting labeling with both (125)I and (111)In. (111)In-ZHER2:342-SR-HP1 bound specifically to HER2-expressing BT474 and SKOV-3 cancer cells in vitro, with a KD of 6±2 pM for binding to SKOV-3 cells. Specific high affinity binding of the radiolabeled complementary PNA probe (111)In-/(125)I-HP2 to ZHER2:342-SR-HP1 pre-treated cells was demonstrated. (111)In-ZHER2:342-SR-HP1 demonstrated specific accumulation in SKOV-3 xenografts in BALB/C nu/nu mice and rapid clearance from blood. Pre-saturation of SKOV-3 with non-labeled anti-HER2 Affibody or the use of HER2-negative Ramos xenografts resulted in significantly lower tumor uptake of (111)In-ZHER2:342-SR-HP1. The complementary PNA probe (111)In/(125)I-HP2 accumulated in SKOV-3 xenografts when ZHER2:342-SR-HP1 was injected 4 h earlier. The tumor accumulation of (111)In/(125)I-HP2 was negligible without ZHER2:342-SR-HP1 pre-injection. The uptake of (111)In-HP2 in SKOV-3 xenografts was 19±2 %ID/g at 1 h after injection. The uptake in blood and kidneys was approximately 50- and 2-fold lower, respectively. In conclusion, we have shown that the use of Affibody-based PNA-mediated pretargeting enables specific delivery of radiometals to tumors and provides higher radiometal concentration in tumors than in kidneys.

Affibody; peptide nucleic acid; radionuclide pretargeting; scaffold protein; HER2
National Category
Clinical Medicine
urn:nbn:se:uu:diva-273542 (URN)10.7150/thno.12766 (DOI)000371806600001 ()26722376 (PubMedID)
Swedish Cancer Society, 2012/354Swedish Research Council, 521-2012-2228Swedish Research Council, 621-2013-5135

De två första författarna delar förstaförfattarskapet och de två sista författarna delar sistaförfattarskapet.

Available from: 2016-01-15 Created: 2016-01-15 Last updated: 2016-08-26Bibliographically approved

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