Abstract
P 082
Sustained expression of an anti-VEGF F(ab) antibody fragment in different cell lines in vitro
Sebastian Schmidt, Nina Wagner, Tobias Wimmer, Markus Preising, Birgit Lorenz
Universitätsklinikum Gießen und Marburg GmbH, Standort Gießen, Klinik und Poliklinik für Augenheilkunde, Labor für molekulare Ophthalmologie, Gießen
Objective
Ocular neovascularisation due to uncontrolled growth of new vessels into the retina following overexpression of vascular endothelial growth factor (VEGF) is the main cause of visual impairment in retinal neovascular diseases such as age-related macular degeneration (AMD) or diabetic retinopathy (DR). Intraocular injections of molecules that block the activity of VEGF, like Bevacizumab (Avastin®) or Ranibizumab (Lucentis®), represent the current therapy of choice. However, the short half life of these molecules necessitates repeated injections of the drug into the vitreous, which implies the possibility for negative side effects of the surgical procedure. The purpose of this study was the development of a vector system that allows for the expression of anti-VEGF F(ab) antibody fragments in different eukaryotic cell lines in vitro.
Methods
Both antibody chains, the light and part of the heavy chain that constitute the anti-VEGF F(ab) antibody fragment, were designed in frame with a secretory leader sequence, de novo synthesized and cloned into an expression cassette containing an internal ribosomal entry site (IRES). Expression of the transgenes was placed under the control of a strong viral CMV promoter. The vector plasmid was expressed in several cell lines in vitro, including HEK 293, HeLa, and retinal cell lines arpe19 and Y79. Expression of both chains was assessed by Western Blot under reducing and non-reducing conditions, and the biological function of the heterodimeric molecule was studied by a cell proliferation assay.
Results
Medium of transfected cell lines displayed in Western blot analysis bands at a size of approximately 25 and 50 kDa, which represents monomeric and dimeric molecules of the expressed F(ab) chains. Usind this medium in a cell proliferation assay resulted in the reduction of VEGF activity in vitro.
Conclusions
Both chains of the anti-VEGF F(ab) antibody fragment can be produced in eukaryotic cell lines in vitro and detected by Western blot analysis. The peptides assemble into heterodimeric molecules containing one heavy and one light chain and are biologically active.
The results of this study may allow for the development of an alternative treatment strategy for patients with AMD or DR, in which the anti-VEGF F(ab) antibody fragment is expressed directly in retinal cells following vector mediated gene transfer.
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