31 IL-6 and VEGF mediated proliferation of HUVECs The ability of the TAF protein of KSI-501 to inhibit IL-6 and VEGF mediated angiogenic functions was tested in a Human Vascular Endothelial Cell, or HUVEC, proliferation assay as shown in the figure below. Importantly, the concentrations of VEGF and IL-6 used to stimulate proliferation were below the saturation point for each individual stimulant and under these conditions VEGF and IL-6 showed some synergy for growth. The presence of TAF protein significantly attenuated proliferation to approximately 50% of maximal growth, while neither EYLEA nor control anti-IL-6 alone had quantifiable effects. These data provide supporting evidence that KSI-501 can synergistically abrogate endothelial cell proliferation that is driven by concurrent inflammatory and VEGF mediated signaling. Figure: VEGF/IL-6 mediated HUVEC proliferation in the presence of inhibitors IL-6 and VEGF mediated tubule formation of HUVECs: TAF protein of KSI-501 was also tested in an endothelial cell tubule formation assay. Treatment of HUVECs seeded on an extracellular basement membrane matrix (Matrigel) with VEGF and IL-6 together stimulate tubule formation to a higher degree than either treatment alone. The TAF protein of KSI-501 demonstrated superior inhibition of this tubule formation when compared to EYLEA or control anti-IL-6 antibody. Furthermore, quantification of the effects of each inhibitor on twenty parameters of HUVEC tubule formation show that the TAF protein significantly inhibited 17 of 20 angiogenic parameters versus control (compared to 4 of 20 for EYLEA and 7 of 20 for control anti-IL-6 antibody). TAF protein was statistically better than EYLEA and anti-IL-6 control in 12 of 20 parameters. Figure: Quantification of IL-6/VEGF mediated HUVEC tubule formation in the presence or absence of inhibitor molecules using the Angiogenesis Analyzer plugin for ImageJ log[Antibody] nM % Untreated 150 100 50 0 -3 -2 -1 0 1 2 KSI-501 protein Eylea Anti-IL6 3 Average % Control Control 125 150 175 Branching interval 75 50 25 0 Mesh index No branches Nb extrem. Nb isol. seg. Nb junctions Nb master junction Nb master segments Nb meshes Nb nodes Nb peaces Nb segments tot branches length tot branching length tot isol. branches length tot length tot master segment length tot meshes area tot segments length KSI-501 Anti-IL6 Eylea