24 Inhibition of VEGF-Mediated Processes Based on its ability to bind and inhibit VEGF, KSI-301 is expected to behave pharmacologically similar to Lucentis, EYLEA and Avastin to decrease the leakage of blood proteins and fluid into the retina. In fact, in vitro testing of KSI-301 against Lucentis, EYLEA and Avastin in their respective ability to inhibit VEGF-mediated endothelial cell proliferation (a key component of neovascularization) in primary human retina microvascular endothelial cells, or HRMVECs, showed that KSI-301 inhibited proliferation to approximately the same degree as EYLEA and with greater potency than Lucentis or Avastin. In addition, KSI-301 displayed a superior maximal inhibition of VEGF-mediated proliferation relative to EYLEA and Avastin. Figure: Effects of KSI-301, Lucentis, EYLEA and Avastin on HRMVEC proliferation. Molecule IC50 (nM) Maximal Inhibition (%) KSI-301 ........................................................................................................................ 0.96±0.18 64.74±2.36 OG1950 ........................................................................................................................ 0.85±0.07 58.92±5.30 Ranibizumab (Lucentis) ............................................................................................... 1.25±0.14 60.96±2.53 Aflibercept (EYLEA)................................................................................................... 0.74±0.10 53.93±4.91 Bevacizumab (Avastin)................................................................................................ 1.25±0.36 38.98±6.18 Table: IC50 Values and maximal inhibition of anti-VEGF agents on VEGF-mediated proliferation of HRMVECs. IC50 values were calculated from concentration of anti-VEGF agents. All values shown as average with standard deviation. To mimic in vivo conditions where endothelial cells and pericytes coexist in blood vessels, a three-dimensional co- culture of HRMVECs and human mesenchymal pericytes, or HMPs, grown on beads was established. This model was then used to test the ability of KSI-301 to inhibit VEGF-mediated vascular sprouting compared to Lucentis and EYLEA. The average number of sprouts per bead and the length per sprout were analyzed under each treatment condition.