Characterization of HMD4-2 and MHD4-46 and effect of HMD4-2 on tumor growth. (A) Flow cytometric analysis showed the specific bindings of HMD4-2 to mouse DLL4 and MHD4-46 to human DLL4. Bold histogram shows the staining with biotinylated HMD4-2 or MHD4-46 followed by PE-labeled streptavidin. Thin histogram shows the staining with biotinylated control hamster IgG or control mouse IgG1 followed by PE-labeled streptavidin. (B) The antibodies blocked Notch1-Fc and Notch4-Fc binding to DLL4. Binding of mouse Notch1-Fc (top left) and mouse Notch4-Fc (top right) to mouse Dll4/CHO cells was blocked by the indicated doses (μg) of HMD4-2. Binding of human Notch1-Fc (bottom left) and mouse Notch4-Fc (bottom right) to human Dll4/CHO cells was blocked by the indicated doses (μg) of MHD4-46. The shaded histograms indicate the background staining without Notch-Fc. (C) HMD4-2 inhibited subcutaneous tumor growth in mouse tumor models with KLN205 (left) and LLC (right). The vertical axis showed the tumor volume and horizontal axis showed the time after implantation. The values represent the mean plus or minus SE (n = 7-10 per group). *P < .05 versus control, §P < .05 versus HMD4-2 low dose, †P < .05 versus HMD4-2 high dose, by Student unpaired t test or 1-way ANOVA with Fisher least-significant-difference test at each time point.