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Affibody molecules targeting the epidermal growth factor receptor for tumor imaging applications
KTH, School of Biotechnology (BIO), Molecular Biotechnology.
2008 (English)Doctoral thesis, comprehensive summary (Other scientific)
Abstract [en]

Tumor targeting and molecular imaging of protein markers specific for or overexpressed in tumors can add useful information in deciding upon treatment and assessing the response to treatment for a cancer patient. The epidermal growth factor receptor (EGFR) is one such tumor-associated receptor, which expression is abnormal or upregulated in various cancers and associated with a poor patient prognosis. It is therefore considered a good target for imaging and therapy. Monoclonal antibodies and recently also antibody fragments have been investigated for in vivo medical applications, like therapy and imaging. In molecular imaging a small sized targeting agent is favorable to give high contrast and therefore, antibody fragments and lately also small affinity proteins based on a scaffold structure constitute promising alternatives to monoclonal antibodies. Affbody molecules are such affinity proteins that are developed by combinatorial protein engineering of the 58 amino acid residue Z-domain scaffold, derived from protein A.

In this thesis, novel Affibody molecules specific for the EGFR have been selected from a combinatorial library using phage display technology. Affibody molecules with moderate high affinity demonstrated specific binding to native EGFR on the EGFR-expressing epithelial carcinoma A431 cell line. Further cellular assays showed that the EGFR-binding Affibody molecules could be labeled with radiohalogens or radiometals with preserved specific binding to EGFR-expressing cells. In vitro, the Affibody molecule demonstrated a high uptake and good retention to EGFR-expressing cells and was found to internalize. Furthermore, successful imaging of tumors in tumor-bearing mice was demonstrated. Low nanomolar or subnanomolar affinities are considered to be desired for successful molecular imaging and a directed evolution to increase the affinity was thus performed. This resulted in an approximately 30-fold improvement in affinity, yielding EGFR-binding Affibody molecules with KD´s in the 5-10 nM range, and successful targeting of A431 tumors in tumor-bearing mice. To find a suitable format and labeling, monomeric and dimeric forms of one affinity matured binder were labeled with 125I and 111In. The radiometal-labeled monomeric construct, 111In-labeled-ZEGFR:1907, was found to provide the best tumor-to-organ ratio due to good tumor localization and tumor retention. The tumor-to-blood ratio, which is often used as a measure of contrast, was 31±8 at 24 h post injection and the tumor was clearly visualized by gamma-camera imaging.

Altogether, the EGFR-binding Affibody molecule is considered a promising candidate for further development of tumor imaging tracers for EGFR-expressing tumors and metastases. This could simplify the stratification of patients for treatment and the assessment of the response of treatment in patients.

Place, publisher, year, edition, pages
Stockholm: KTH , 2008. , xii, 102 p.
Series
Trita-BIO-Report, ISSN 1654-2312 ; 2008:2
Keyword [en]
Affibody, affinity maturation, phage display selection, EGFR, molecular imaging, protein engineering, tumor targeting
National Category
Industrial Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-4710ISBN: 978-91-7178-890-0 (print)OAI: oai:DiVA.org:kth-4710DiVA: diva2:13560
Public defence
2008-05-16, FR4, AlbaNova Universitetscentrum, Roslagstullsbacken 21, Stockholm, 10:00
Opponent
Supervisors
Note
QC 20100723Available from: 2008-04-25 Created: 2008-04-25 Last updated: 2010-07-23Bibliographically approved
List of papers
1. Phage display selection of Affibody molecules with specific binding to the extracellular domain of the epidermal growth factor receptor
Open this publication in new window or tab >>Phage display selection of Affibody molecules with specific binding to the extracellular domain of the epidermal growth factor receptor
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2007 (English)In: Protein Engineering Design & Selection, ISSN 1741-0126, E-ISSN 1741-0134, Vol. 20, no 4, 189-199 p.Article in journal (Refereed) Published
Abstract [en]

Affibody molecules specific for the epidermal growth factor receptor (EGFR) have been selected by Phage display technology from a combinatorial protein library based on the 58-residue, protein A-derived Z domain. EGFR is overexpressed in various malignancies and is frequently associated with poor patient prognosis, and the information provided by targeting this receptor could facilitate both patient diagnostics and treatment. Three selected Affibody variants were shown to selectively bind to the extracellular domain of EGFR (EGFR-ECD). Kinetic biosensor analysis revealed that the three monomeric Affibody molecules bound with similar affinity, ranging from 130 to 185 nM. Head-to-tail dimers of the Affibody molecules were compared for their binding to recombinant EGFR-ECD in biosensor analysis and in human epithelial cancer A431 cells. Although the dimeric Affibody variants were found to bind in a range of 2550 nM affinities in biosensor analysis, they were found to be low nanomolar binders in the cellular assays. Competition assays using radiolabeled Affibody dimers confirmed specific EGFR-binding and demonstrated that the three Affibody molecules competed for the same epitope. Immunofluorescence microscopy demonstrated that the selected Affibody dimers were initially binding to EGFR at the cell surface of A431, and confocal microscopy analysis showed that the Affibody dimers could thereafter be internalized. The potential use of the described Affibody molecules as targeting agents; for radionuclide based imaging applications in various carcinomas ils discussed.

Keyword
affibody; EGFR; phage display; selection; targeting
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-8276 (URN)10.1093/protein/gzm011 (DOI)000246966800005 ()2-s2.0-34347380371 (Scopus ID)
Note
QC 20100723Available from: 2008-04-25 Created: 2008-04-25 Last updated: 2017-12-14Bibliographically approved
2. Cellular studies of binding, internalization and retention of a radiolabeled EGFR-binding affibody molecule
Open this publication in new window or tab >>Cellular studies of binding, internalization and retention of a radiolabeled EGFR-binding affibody molecule
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2007 (English)In: Nuclear Medicine and Biology, ISSN 0969-8051, E-ISSN 1872-9614, Vol. 34, no 6, 609-618 p.Article in journal (Refereed) Published
Abstract [en]

Introduction: The cellular binding and processing of an epidermal growth factor receptor (EGFR) targeting affibody molecule, (Z(EGFR:955))(2), was studied. This new and small molecule is aimed for applications in nuclear medicine. The natural ligand epidermal growth factor (EGF) and the antibody cetuximab were studied for comparison.

Methods: All experiments were made with cultured A431 squamous carcinoma cells. Receptor specificity, binding time patterns, retention and preliminary receptor binding site localization studies were all made after (125) I, labeling. Internalization was studied using Oregon Green 488, Alexa Fluor 488 and CypHer5E markers.

Results: [(125) I](Z(EGFR:955))(2) and [(125) I]cetuximab gave a maximum cellular uptake of I-125 within 4 to 8 h of incubation, while [(125) I]EGF gave a maximum uptake already after 2 h. The retention studies showed that the cell-associated fraction of 125 1 after 48 It of incubation was similar to 20% when delivered as [(125) I](Z(FGFR:955))(2) and similar to 25% when delivered as [I-125] cetuximab. [(125) I]EGF-mediated delivery gave a faster (125) I release, where almost all cell-associated radioactivity had disappeared within 24 It. All three substances were internalized as demonstrated with confocal microscopy. Competitive binding studies showed that both EGF and cetuximab inhibited binding Of (Z(EGFR:955))(2) and indicated that the three substances competed for an overlapping binding site.

Conclusion: The results gave information on cellular processing of radionuclides when delivered with (Z(EGFR:955))(2) in comparison to delivery with EGF and cetuximab. Competition assays suggested that [I-125](Z(EGFR:955))(2) bind to Domain III of EGFR. The affibody molecule (Z(EGFR:955))(2) can be a candidate for EGFR imaging applications in nuclear medicine.

Keyword
A431; affibody molecule; EGFR; internalization; radionuclide; retention
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-8277 (URN)10.1016/j.nucmedbio.2007.05.010 (DOI)000249157300003 ()2-s2.0-34547830391 (Scopus ID)
Note
QC 20100723Available from: 2008-04-25 Created: 2008-04-25 Last updated: 2017-12-14Bibliographically approved
3. In vivo and in vitro uptake of 111In, delivered with the affibody molecule (ZEGFR:955)2, in EGFR expressing tumour cells
Open this publication in new window or tab >>In vivo and in vitro uptake of 111In, delivered with the affibody molecule (ZEGFR:955)2, in EGFR expressing tumour cells
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2008 (English)In: Oncology Reports, ISSN 1021-335X, E-ISSN 1791-2431, Vol. 19, no 4, 853-857 p.Article in journal (Refereed) Published
Abstract [en]

The epidermal growth factor receptor, EGFR, is overexpressed in many carcinomas. Targeting this receptor with radionuclides is important for imaging and therapy applications in nuclear medicine. We investigated the in vitro and in vivo properties of a new high affinity EGFR binding affibody molecule, (Z(EGFR:955))(2), when conjugated with CHXA"-DTPA and labelled with In-111. The binding time patterns and retention studies were performed using cultured squamous carcinoma A431 cells that overexpress EGFR. In the in vivo studies, female BALB/c nu/nu mice carrying tumours from xenografted A431 cells were used. The in vitro studies showed EGFR specific binding, high uptake and good retention of In-111 when delivered as [In-111](Z(EGFR:955))(2). The retention after 72 h of incubation was 38.0 +/- 1.15% of the initial level. The biodistribution study showed a tumour specific In-111 uptake of 3.8 +/- 1.4% of injected dose per gram turnout tissue 4 h post-injection. The tumour to blood ratio was 9.1 and the tumours could easily be visualized with a gamma camera at this time-point. In-111 delivered with [In-111](Z(EGFR:955))(2) gave an EGFR specific uptake and the results indicated that the (Z(EGFR:955))(2) affibody molecule is a candidate for radionuclide-based tumour imaging. Potential therapy applications are discussed.

Keyword
a431; affibody molecule; EGFR; mouse; radionuclide
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-8278 (URN)000254496100003 ()2-s2.0-43849098417 (Scopus ID)
Note
QC 20100723Available from: 2008-04-25 Created: 2008-04-25 Last updated: 2017-12-14Bibliographically approved
4. Directed evolution to low nanomolar affinity of a tumor-targeting epidermal growth factor receptor-binding Affibody molecule
Open this publication in new window or tab >>Directed evolution to low nanomolar affinity of a tumor-targeting epidermal growth factor receptor-binding Affibody molecule
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2008 (English)In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 376, no 5, 1388-1402 p.Article in journal (Refereed) Published
Abstract [en]

The epidermal growth factor receptor 1 (EGFR) is overexpressed in various malignancies and is associated with a poor patient prognosis. A small, receptor-specific, high-affinity imaging agent would be a useful tool in diagnosing malignant tumors and in deciding upon treatment and assessing the response to treatment. We describe here the affinity maturation procedure for the generation of Affibody molecules binding with high affinity and specificity to EGFR. A library for affinity maturation was constructed by rerandomization of selected positions after the alignment of first-generation binding variants. New binders were selected with phage display technology, using a single oligonucleotide in a single-library effort, and the best second-generation binders had an approximately 30-fold improvement in affinity (K-d = 5-10 nM) for the soluble extracellular domain of EGFR in biospecific interaction analysis using Biacore. The dissociation equilibrium constant, Kd, was also determined for the Affibody with highest affinity using EGFR-expressing A431 cells in flow cytometric analysis (K-d = 2.8 nM). A retained high specificity for EGFR was verified by a dot blot assay showing staining only of EGFR proteins among a panel of serum proteins and other EGFR family member proteins (HER2, HER3, and HER4). The EGFR-binding Affibody molecules were radiolabeled with indium-111, showing specific binding to EGFR-expressing A431 cells and successful targeting of the A431 tumor xenografts with 4-6% injected activity per gram accumulated in the tumor 4 h postinjection.

Keyword
Affibody; EGFR; tumor targeting; phage display selection; affinity maturation
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-8279 (URN)10.1016/j.jmb.2007.12.060 (DOI)000254585900014 ()2-s2.0-39149087035 (Scopus ID)
Note
QC 20100723Available from: 2008-04-25 Created: 2008-04-25 Last updated: 2017-12-14Bibliographically approved
5. Affibody Molecules for Epidermal Growth Factor Receptor Targeting In Vivo: Aspects of Dimerization and Labeling Chemistry
Open this publication in new window or tab >>Affibody Molecules for Epidermal Growth Factor Receptor Targeting In Vivo: Aspects of Dimerization and Labeling Chemistry
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2009 (English)In: Journal of Nuclear Medicine, ISSN 0161-5505, E-ISSN 1535-5667, Vol. 50, no 2, 274-283 p.Article in journal (Refereed) Published
Abstract [en]

Noninvasive detection of epidermal growth factor receptor (EGFR) expression in malignant tumors by radionuclide molecular imaging may provide diagnostic information influencing patient management. The aim of this study was to evaluate a novel EGFR-targeting protein, the Z(EGFR:1907) Affibody molecule, for radionuclide imaging of EGFR expression, to determine a suitable tracer format (dimer or monomer) and optimal label. Methods: An EGFR-specific Affibody molecule, ZEGFR:1907, and its dimeric form, (Z(EGFR:1907))(2), were labeled with In-111 using benzyl-diethylenetriaminepentaacetic acid and with I-125 using p-iodobenzoate. Affinity and cellular retention of conjugates were evaluated in vitro. Biodistribution of radiolabeled Affibody molecules was compared in mice bearing EGFR-expressing A431 xenografts. Specificity of EGFR targeting was confirmed by comparison with biodistribution of non-EGFR-specific counterparts. Results: Head-to-tail dimerization of the Affibody molecule improved the dissociation rate. In vitro, dimeric forms demonstrated superior cellular retention of radioactivity. For both molecular set-ups, retention was better for the In-111-labeled tracer than for the radioiodinated counterpart. In vivo, all conjugates accumulated specifically in xenografts and in EGFRexpressing tissues. The retention of radioactivity in tumors was better in vivo for dimeric forms; however, the absolute uptake values were higher for monomeric tracers. The best tracer, In-111-labeled Z(EGFR:1907), provided a tumor-to-blood ratio of 100 (24 h after injection). Conclusion: The radiometal-labeled monomeric Aff ibody molecule Z(EGFR:1907) has a potential for radionuclide molecular imaging of EGFR expression in malignant tumors.

Keyword
Affibody molecules; EGFR; I-125; In-111; gamma-camera imaging
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-8280 (URN)10.2967/jnumed.108.055525 (DOI)000263487800033 ()2-s2.0-59249088447 (Scopus ID)
Note
QC 20100723. Tidigare titel: Affibody molecules for EGFR targeting in vivo: aspects of dimerization and labeling chemistryAvailable from: 2008-04-25 Created: 2008-04-25 Last updated: 2017-12-14Bibliographically approved

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