Figure 1 from Inhibition of Notch4 Using Novel Neutralizing Antibodies Reduces Tumor Growth in Murine Cancer Models by Targeting the Tumor Endothelium

Development and validation of 6-3-A6 and E7011 as Notch4 inhibitors. A, Schematic of the immunizing epitope used to generate clone 6-3-A6. EGF, EGF repeats; Igk, immunoglobulin κ chain; SEAP, secreted embryonic alkaline phosphatase. B, Binding assay of the fully humanized E7011 and 6-3-A6 fused to human Fc for immobilization. Affinity to recombinant human Notch4 was calculated with surface plasmon resonance. C, Affinity of 6-3-A6 and E7011 to Raji cell lines generated to overexpress full-length human Notch1, Notch2, Notch3, and Notch4 (hNotch1 to 4, respectively). Binding was determined by MFI with flow cytometry analysis (n = 3). D, Luciferase reporter assay using a Notch4-ECD/Notch1-ICD-Gal4 fusion protein expressed in bEnd.3-immortalized ECs. Luminescence was measured with increasing concentrations of added E7011 or 6-3-A6. Statistical analysis performed with one-way ANOVA with the Dunnett multiple comparison test, plotted as average ± SEM; **, P < 0.01; ****, P < 0.0001.