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In vivo and in vitro uptake of 111In, delivered with the affibody molecule (ZEGFR:955)2, in EGFR expressing tumour cells
Uppsala Univ, Dept Oncol Radiol & Clin Immunol, Rudbeck Lab.
Uppsala Univ, Dept Oncol Radiol & Clin Immunol, Rudbeck Lab.
KTH, School of Biotechnology (BIO), Molecular Biotechnology.
Uppsala Univ, Dept Oncol Radiol & Clin Immunol, Rudbeck Lab.
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2008 (English)In: Oncology Reports, ISSN 1021-335X, 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.

Place, publisher, year, edition, pages
2008. Vol. 19, no 4, 853-857 p.
Keyword [en]
a431; affibody molecule; EGFR; mouse; radionuclide
National Category
Industrial Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-8278ISI: 000254496100003Scopus ID: 2-s2.0-43849098417OAI: oai:DiVA.org:kth-8278DiVA: diva2:13557
Note
QC 20100723Available 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.
Series
Trita-BIO-Report, ISSN 1654-2312 ; 2008:2
Keyword
Affibody, affinity maturation, phage display selection, EGFR, molecular imaging, protein engineering, tumor targeting
National Category
Industrial Biotechnology
Identifiers
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
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Supervisors
Note
QC 20100723Available from: 2008-04-25 Created: 2008-04-25 Last updated: 2010-07-23Bibliographically approved

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