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Chris Hughes, Neil O’Flynn, Maureen Gatherer, Srini Goverdhan, Martin Glennie, Andrew Lotery; Novel Antiangiogenic Antibodies For Treating Wet Age-Related Macular Degeneration. Invest. Ophthalmol. Vis. Sci. 2014;55(13):1798.
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Current anti-angiogenic treatments for wet age-related macular degeneration (AMD) require multiple injections, are expensive and prone to complications. Gene therapy could be an elegant solution for providing a long-term source of anti-angiogenic proteins after a single administration. Recent research has shown that combination therapies such as vascular endothelial growth factor (VEGF) and platelet derived growth factor (PDGFbb) blocking appear to have an enhanced anti-angiogenic effect compared to monotherapy. We therefore aimed to produce a panel of anti-VEGF and anti-PDGFRβ blocking antibodies, and to systematically test these recombinant proteins both in vitro and in vivo to determine suitability for incorporating into gene therapy vectors.
Recombinant anti-VEGF and anti-PDGFRβ antibodies in the mIgG1, mIgG2a, and scFv formats were produced by transfecting 293F cells. Proteins secreted into the supernatant were then purified by affinity chromatography. Antibodies were verified in vitro by SDS-PAGE, Western Blot, ELISA, and in a bioassay. Their activity in vivo was tested in laser-induced choroidal neovascularisation (CNV) C57BL/6 mouse models by intravitreal injections (both 3 days before and 3 days after laser induction of CNV, n=5). Subsequent fundus fluorescein angiography (FFA; using micron-3 imaging) and immunohistolologic analysis (using anti-collagen IV) was used to quantify any anti-angiogenic effects on CNV.
All antibodies were produced to a high level of purity as demonstrated by a single band on SDS-PAGE, and the anticipated molecular weight was observed. The identity of the proteins was further confirmed by Western Blot. The antibodies were found to bind VEGF or PDGFRβ in ELISAs, and anti-VEGF antibodies were also shown to neutralise VEGF active in a bioassay. Moreover, when the anti-VEGF (mIgG1 format) was administered CNV mice, there was a significant decrease in area of measured CNV both on FFA (p=0.03) and on immunohistology (p=0.01) compared to PBS controls.
We have successfully produced a panel of novel anti-angiogenic antibodies, whose function has been verified in vitro. Our preliminary in vivo data suggest a therapeutic effect for these anti-angiogenic antibodies. Further studies are being done to confirm the efficacy of the scFv formats of anti-VEGF and anti-PDGFRβ, both individually and in combination.
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