Single-Chain Fragment VEGF Antibody (HR6) effects as an inhibitor on Choroidal Neovascularization

Purpose: Evaluation the effect of single chain variable fragment antibody (scFv) in inhibition of angiogenesis in an animal model of laser induced choroidal neovascularization(CNV)Methods: In this study, a recombinant single-chain antibody (HR6) was derived from Tomlinson(i+j) phage display library, transferred and expressed in non-suppressor HB2151 bacteria. HR6 was purified by NI-NTA affinity chromographyand analyzed with Westernblot technique. In vivo assays were designed in 2 phases. In phase ǀ, to test retinal toxicity, fifteen, 6-8 old weeks male c57bl6 mice, were divided in 3 groups. Each group received intravitreal HR6 injection of 0.5 μg, 1μg and 1.5 μg/1 μl, respectively. Ophthalmic examination and electroretinography (ERG) were performed after 7 and 28 days. In phase ǁ, CNV was induced via laser burns in right eyes of 25 mice. The animals were divided into control and treatment groups. Three of which received the same HR6 doses of ERG assay, one group received bevacizumab as positive control the same dose as clinical use and another group received PBS intravitrealy. Two weeks later, CNV scores were detected in sclerochoroidal flatmounts, stained with antibodies against intercellular adhesion molecule-2. CNV area were measured using flurosence microscopy and image j software.Results: In phase 1, ERG results were unremarkable in terms of retinal toxicity in treatment and control groups. The flat mount analysis have shown significant reduction of formation the new blood vessels compared to control group in three different doses of intravitreal injection of HR6 antibody (p o.oo1). Conclusion: HR6 as a scFv antibody, because of its small size compared to complete antibody as Bevacizumab, improve tissue penetration and lower immunogenicity. In this study we showed that intravitreal injection of up to 1.5 μg/1 of this antibody has no toxicity on retina and is effective to reduce laser induced choroidal neovascularization