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Affibody Molecules for Epidermal Growth Factor Receptor Targeting In Vivo: Aspects of Dimerization and Labeling Chemistry
Unit of Biomedical Radiation Sciences, Rudbeck Laboratory, Uppsala University.
KTH, School of Biotechnology (BIO), Molecular Biotechnology.
Section of Hospital Physics, Department of Oncology, Uppsala University Hospital.
Affibody AB, Bromma.
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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.

Place, publisher, year, edition, pages
2009. Vol. 50, no 2, 274-283 p.
Keyword [en]
Affibody molecules; EGFR; I-125; In-111; gamma-camera imaging
National Category
Industrial Biotechnology
URN: urn:nbn:se:kth:diva-8280DOI: 10.2967/jnumed.108.055525ISI: 000263487800033ScopusID: 2-s2.0-59249088447OAI: diva2:13559
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: 2010-07-23Bibliographically approved
In thesis
1. Affibody molecules targeting the epidermal growth factor receptor for tumor imaging applications
Open this publication in new window or tab >>Affibody molecules targeting the epidermal growth factor receptor for tumor imaging applications
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.
Trita-BIO-Report, ISSN 1654-2312 ; 2008:2
Affibody, affinity maturation, phage display selection, EGFR, molecular imaging, protein engineering, tumor targeting
National Category
Industrial Biotechnology
urn:nbn:se:kth:diva-4710 (URN)978-91-7178-890-0 (ISBN)
Public defence
2008-05-16, FR4, AlbaNova Universitetscentrum, Roslagstullsbacken 21, Stockholm, 10:00
QC 20100723Available from: 2008-04-25 Created: 2008-04-25 Last updated: 2010-07-23Bibliographically approved

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