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Vineeta Tripathi, Jestin George, Sterenn Davis, Sidath E Liyanage, James W B Bainbridge, John Greenwood, Stephen E Moss, ; Development of an anti-angiogenic therapeutic antibody against LRG1. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):2278.
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Neovascularisation and vascular remodelling are key clinical features of several pathologies, including neovascular age-related macular degeneration (AMD), diabetic retinopathy (DR) and solid tumors. Regulation or inhibition of vascular changes in these conditions has been shown to slow or arrest disease, but the number of anti-angiogenic therapeutics available is limited. We have identified a protein named leucine-rich alpha-2-glycoprotein-1 (LRG1), of previously unknown function that we have shown to be a potent regulator of angiogenesis. The purpose of this study is to generate a function-blocking monoclonal antibody (mAb) against LRG1.
Wild-type and Lrg1-/- mice were immunised with recombinant human LRG1 and > 100 mAbs were obtained for characterisation. mAbs were tested for blocking activity in a range of in vitro, ex vivo and in vivo angiogenesis assays, and were also analysed by surface plasmon resonance (SPR) to determine binding kinetics and affinities. mAbs were further evaluated for species cross-reactivity and selectivity.
From an initial pool of 102 mAbs raised against recombinant human LRG1 we isolated a subset that demonstrated efficacy in a range of ex vivo, in vitro and in vivo angiogenesis assays. Using real-time binding and dissociation assays to determine the binding kinetics of mAbs to rhLRG1, we identified 25 mAbs with KD < 1 nM. Next, we tested the function-blocking efficacy of all 102 mAbs in an in vitro human umbilical vein endothelial cell co-culture assay. We identified 73 antibodies showing at least 50 % blocking of tube formation. 25 of these 73 antibodies had also showed KD < 1 nM in our binding experiments. Therefore, we further tested these 25 mAbs in the ex vivo mouse foetal pre-metatarsal assay. 12 mAbs showed a 10-50% reduction in sprouting and these were then tested in in vivo rat and mouse laser-induced Choroidal Neovascularisation (CNV) assays, from which we identified a lead that exhibited ~40 % blocking of CNV formation. We are now in the process of humanisation of this lead monoclonal antibody prior to evaluation in a Phase I/IIa clinical trial.
We have identified LRG1 as a potential therapeutic target for vascular retinopathies. After performing a functional and biophysical screen we have identified a lead mAb for onward humanisation and therapeutic testing.
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