June 2013
Volume 54, Issue 15
Free
ARVO Annual Meeting Abstract  |   June 2013
Functional characterization of recombinant anti-VEGF variants in vitro
Author Affiliations & Notes
  • Tobias Wimmer
    Department of Ophthalmology, Justus-Liebig University Giessen, Giessen, Germany
  • Birgit Lorenz
    Department of Ophthalmology, Justus-Liebig University Giessen, Giessen, Germany
  • Knut Stieger
    Department of Ophthalmology, Justus-Liebig University Giessen, Giessen, Germany
  • Footnotes
    Commercial Relationships Tobias Wimmer, None; Birgit Lorenz, Optos (F); Knut Stieger, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2013, Vol.54, 3284. doi:
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      Tobias Wimmer, Birgit Lorenz, Knut Stieger; Functional characterization of recombinant anti-VEGF variants in vitro. Invest. Ophthalmol. Vis. Sci. 2013;54(15):3284.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: Most retinal neovascular disorders are caused by an up-regulation of the vascular endothelial growth factor (VEGF). Especially disorders like age-related macular degeneration (AMD), diabetic retinopathy (DR) and retinal vein occlusion (RVO) are treated with repeated injections of anti-VEGF molecules like Bevacizumab (Avastin®), Ranibizumab (Lucentis®), or Aflibercept (Eyelea®). Repeated injections of anti-VEGF molecules can be connected to severe side effects like endophthalmitis and can represent a financial burden to the patients. The aim of this project is to develop an alternative strategy i.e. controlled expression of anti-VEGF molecules by the retina itself.

Methods: The DNA sequences of the light and the heavy chain of the Ranibizumab F(ab)-fragment including secretory leader sequences were cloned into pIREShrGFP1a separated by an internal ribosomal entry site (IRES) (Ra01). This construct was PCR mutated to generate a Ranibizumab variant (Ra02), which is expressed as just one molecule containing a 6x glycine linker. HEK293 were transfected with both constructs, presence in the culture medium verified by western blot analysis and the concentrations measured with a custom made Ranibizumab/Lucentis® ELISA. VEGF binding affinities were determined in a cell free binding assay and the biological activity evaluated in a HUVEC (human umbilical endothelial cell) migration assay.

Results: Ra01 and Ra02 were detected in cell culture medium. The concentrations were 374,1 ng/ml and 403,4 ng/ml for Ra01 and Ra02, respectively. VEGF binding affinity was significantly lower for Ra01 and Ra02 compared to injectable Lucentis® for each tested concentration. The inhibition of VEGF induced endothelial cell migration by Ra01 and Ra02 was comparable with Lucentis® over all tested concentrations, but the maximum inhibitory effect of Ra01 and Ra02 was reached at lower doses compared to Lucentis®.

Conclusions: Ra01 and Ra02 can be produced in eukaryotic cells in vitro and display comparable biological activities as commercially available Lucentis®. In vivo studies in human VEGF overexpressing mice are ongoing. These results are the basis for a gene-based therapy of human neovascular disorders.

Keywords: 412 age-related macular degeneration • 609 neovascularization • 748 vascular endothelial growth factor  
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