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Engineering and characterization of a bispecific HER2 × EGFR-binding affibody molecule
KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
KTH, Skolan för bioteknologi (BIO), Nanobioteknologi.
KTH, Skolan för bioteknologi (BIO), Nanobioteknologi.
KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Cellens fysik.ORCID-id: 0000-0003-0578-4003
Visa övriga samt affilieringar
2009 (Engelska)Ingår i: Biotechnology and applied biochemistry, ISSN 0885-4513, E-ISSN 1470-8744, Vol. 54, s. 121-131Artikel i tidskrift (Refereegranskat) Published
Abstract [en]

HER2 (human epidermal-growth-factor receptor-2; ErbB2) and EGFR (epidermal-growth-factor receptor) are overexpressed in various forms of cancer, and the co-expression of both HER2 and EGFR has been reported in a number of studies. The simultaneous targeting of HER2 and EGFR has been discussed as a strategy with which to potentially increase efficiency and selectivity in molecular imaging and therapy of certain cancers. In an effort to generate a molecule capable of bispecifically targeting HER2 and EGFR, a gene fragment encoding a bivalent HER2-binding affibody molecule was genetically fused in-frame with a bivalent EGFR-binding affibody molecule via a (G(4)S)(3) [(Gly(4)-Ser)(3)]-encoding gene fragment. The encoded 30 kDa affibody construct (Z(HER2))(2)-(G(4)S)(3)-(Z(EGFR))(2), with potential for bs (bispecific) binding to HER2 and EGFR, was expressed in Escherichia coli and characterized in terms of its binding capabilities. The retained ability to bind HER2 and EGFR separately was demonstrated using both biosensor technology and flow-cytometric analysis, the latter using HER2- and EGFR-overexpressing cells. Furthermore, simultaneous binding to HER2 and EGFR was demonstrated in: (i) a sandwich format employing real-time biospecific interaction analysis where the bs affibody molecule bound immobilized EGFR and soluble HER2; (ii) immunofluorescence microscopy, where the bs affibody molecule bound EGFR-overexpressing cells and soluble HER2; and (iii) a cell-cell interaction analysis where the bs affibody molecule bound HER2-overexpressing SKBR-3 cells and EGFR-overexpressing A-431 cells. This is, to our knowledge, the first reported bs affinity protein with potential ability for the simultaneous targeting of HER2 and EGFR. The potential future use of this and similar constructs, capable of bs targeting of receptors to increase the efficacy and selectivity in imaging and therapy, is discussed.

Ort, förlag, år, upplaga, sidor
2009. Vol. 54, s. 121-131
Nyckelord [en]
bispecifc (bs) affibody molecule; human epidermal-growth-factor receptor-3 (ErbB); microwell array; protein engineering; tumour targeting
Nationell ämneskategori
Industriell bioteknik
Identifikatorer
URN: urn:nbn:se:kth:diva-11662DOI: 10.1042/BA20090096ISI: 000270769000006Scopus ID: 2-s2.0-70350561680OAI: oai:DiVA.org:kth-11662DiVA, id: diva2:279096
Anmärkning
QC 20100728Tillgänglig från: 2009-12-01 Skapad: 2009-12-01 Senast uppdaterad: 2017-12-12Bibliografiskt granskad
Ingår i avhandling
1. Microwell devices for single-cell analyses
Öppna denna publikation i ny flik eller fönster >>Microwell devices for single-cell analyses
2009 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
Abstract [en]

Powerful tools for detailed cellular studies are emerging, increasing the knowledge ofthe ultimate target of all drugs: the living cell. Today, cells are commonly analyzed inensembles, i.e. thousands of cells per sample, yielding results on the average responseof the cells. However, cellular heterogeneity implies the importance of studying howindividual cells respond, one by one, in order to learn more about drug targeting andcellular behavior. In vitro assays offering low volume sampling and rapid analysis in ahigh-throughput manner are of great interest in a wide range of single-cellapplications.

This work presents a microwell device in silicon and glass, developed using standardmicrofabrication techniques. The chip was designed to allow flow-cytometric cellsorting, a controlled way of analyzing and sorting individual cells for dynamic cultureand clone formation, previously shown in larger multiwell plates only. Dependent onthe application, minor modifications to the original device were made resulting in agroup of microwell devices suitable for various applications. Leukemic cancer cellswere analyzed with regard to their clonogenic properties and a method forinvestigation of drug response of critical importance to predict long-term clinicaloutcome, is presented. Stem cells from human and mouse were maintainedpluripotent in a screening assay, also shown useful in studies on neural differentiation.For integrated liquid handling, a fluidic system was integrated onto the chip fordirected and controlled addition of reagents in various cell-based assays. The chip wasproduced in a slide format and used as an imaging tool for low-volume sampling withthe ability to run many samples in parallel, demonstrated in a protein-binding assay fora novel bispecific affinity protein. Moving from cells and proteins into geneticanalysis, a method for screening genes from clones in a rapid manner was shown bygene amplification and mutation analysis in individual wells. In summary, a microwelldevice with associated methods were developed and applied in a range of biologicalinvestigations, particularly interesting from a cell-heterogeneity perspective.

Ort, förlag, år, upplaga, sidor
Stockholm: KTH, 2009. s. xii, 80
Serie
Trita-BIO-Report, ISSN 1654-2312 ; 2009:23
Nyckelord
microwell, miniaturization, microfluidics, cell culture, single-cell, clone, imaging, stem cell, cancer, low volume, high-throughput
Nationell ämneskategori
Biokemi och molekylärbiologi
Identifikatorer
urn:nbn:se:kth:diva-11665 (URN)978-91-7415-477-1 (ISBN)
Disputation
2009-12-11, FR4 Oscar Klein, AlbaNova, Roslagstullsbacken, Stockholm, 10:00 (Engelska)
Opponent
Handledare
Anmärkning
QC 20100728Tillgänglig från: 2009-12-01 Skapad: 2009-12-01 Senast uppdaterad: 2011-11-23Bibliografiskt granskad

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Förlagets fulltextScopushttp://www.babonline.org/bab/054/bab0540121.htm

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Brismar, HjalmarStåhl, Stefan

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Friedman, MikaelaLindström, SaraAndersson-Svahn, HeleneBrismar, HjalmarStåhl, Stefan
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