March 2012
Volume 53, Issue 14
ARVO Annual Meeting Abstract  |   March 2012
Biological Activity of Different Transgenic Ranibizumab Compositions
Author Affiliations & Notes
  • Knut Stieger
    Department of Ophthalmology, Justus-Liebig-University Giessen, Giessen, Germany
  • Tobias Wimmer
    Department of Ophthalmology, Justus-Liebig-University Giessen, Giessen, Germany
  • Birgit Lorenz
    Department of Ophthalmology, Justus-Liebig-University Giessen, Giessen, Germany
  • Footnotes
    Commercial Relationships  Knut Stieger, None; Tobias Wimmer, None; Birgit Lorenz, None
  • Footnotes
    Support  DFG STI 597/2-1
Investigative Ophthalmology & Visual Science March 2012, Vol.53, 444. doi:
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      Knut Stieger, Tobias Wimmer, Birgit Lorenz; Biological Activity of Different Transgenic Ranibizumab Compositions. Invest. Ophthalmol. Vis. Sci. 2012;53(14):444.

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

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Purpose: : 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). The intraocular expression of anti-VEGF molecules potentially represents a therapeutic strategy to block neovascularization in these pathologies. The aim of this study was to characterize the optimal composition of the expression cassette for the production of these molecules in standard cell lines in vitro.

Methods: : Both antibody chains of Ranibizumab, the light and part of the heavy chain were designed containing secretory leader sequences or restriction sites for subsequent subcloning. The fragments were either expressed separately using an IRES containing expression cassette (Ra01), or were cloned together into one reading frame containing either a glycine or glycine-proline anchor in between (Ra02-Ra06). Plasmids were transfected into HEK293 and Hela cell lines and the expression of the molecules verified by Western blot analysis. A Ranibizumab specific ELISA was developed in order to measure the concentration of the anti-VEGF molecules. The biological activity was tested using HUVEC (human umbilical vein endothelial cell) tube formation assays and HUVEC migration assays.

Results: : All Ra compositions were detected in the supernatant of transfected Hela and HEK293 cells. Generation of long cellular tubes in the HUVEC tube formation assay, which is predominantly due to VEGF activity, was reduced to 50% with Ra01 and similar levels were achieved with Ra02-Ra06. VEGF activity in the HUVEC migration assay was reduced by about 50% for all Ra compositions. Similar VEGF inhibition results were obtained using commercially available recombinant Ranibizumab (Lucentis®) at equal concentrations.

Conclusions: : Transgenic Ranibizumab, either expressed separately or as one molecule have similar inhibitory effects on VEGF activity as commercially available Ranibizumab in vitro. These results lay the foundation for the development of an alternative treatment strategy for patients with AMD or DR, in which Ranibizumab is produced at low doses directly in retinal cells.

Keywords: vascular endothelial growth factor • gene transfer/gene therapy • neovascularization 

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