Efficacy of simultaneous VEGF-A/ANG-2 neutralization in suppressing spontaneous choroidal neovascularization

Abstract

This study independently confirms in vivo in the JR5558 mouse model of aberrant retinal angiogenesis, that simultaneous VEGF‐A and ANG‐2 neutralization using a bispecific anti‐VEGF‐A/ANG‐2 antibody reduces vascular leakage, immune reactivity and apoptosis more effectively than either agent alone.

Figure 1. Reduction of vessel leakiness and lesion number by combined neutralization of VEGF‐A and ANG‐2 in the JR5558 mice using FFA

(A) Study schematic. Baseline FFA was carried out at P44‐45. Antibody injections were given IP on P46 and P53. Post‐treatment FFA was at P60 and tissue harvest at P61. (B) Bar/scatter graph of baseline lesion numbers. Lesions were counted in all mice prior to study start. Combined total lesions from left and right eyes were calculated, and then, animals were assigned to treatment groups ensuring no statistically significant differences between groups (P > 0.05). (C, D) Bar/scatter graphs showing numbers of spontaneously occurring lesions (C) and area by fluorescence angiography (D) after two weekly doses of antibody (IgG, anti‐VEGF‐A, anti‐ANG‐2 at 5 mg/kg IP, and anti‐VEGF‐A/ANG‐2 at 10 mg/kg IP) followed by analysis a week after the last treatment. (E) Representative examples of fluorescence fundus angiograms from the untreated (top left), IgG control (top middle; 10 mg/kg), anti‐VEGF‐A (bottom left; 5 mg/kg), anti‐ANG‐2 (bottom middle; 5 mg/kg), and anti‐VEGF‐A/ANG‐2 (bottom right; 10 mg/kg) groups. Data information: SEM is shown as error bars with n = 9–10 animals (B) or n = 19–20 (C, D) eyes per group and significance indicated by asterisks using ANOVA (B: P > 0.05; C: P < 0.0001; D: P < 0.0001, followed by Newman–Keul's multiple comparison test in C, D). In (C), untreated is significantly different versus IgG control (*P < 0.05) and anti‐VEGF‐A/ANG‐2 (***P < 0.001). IgG control is significantly different versus anti‐VEGF‐A (****P < 0.0001), anti‐ANG‐2 (****P < 0.0001), and anti‐VEGF‐A/ANG‐2 (****P < 0.0001). In (D), untreated is significantly different versus IgG control (*P < 0.05), anti‐VEGF‐A (***P < 0.001), anti‐ANG‐2 (***P < 0.001), and anti‐VEGF‐A/ANG‐2 (****P < 0.0001). IgG control is significantly different versus anti‐VEGF‐A (****P < 0.0001), anti‐ANG‐2 (****P < 0.0001), and anti‐VEGF‐A/ANG‐2 (****P < 0.0001). Anti‐VEGF‐A/ANG‐2 is significantly different versus anti‐VEGF‐A (*P < 0.05) and anti‐ANG‐2 (**P < 0.01). FFA, fluorescein fundus angiography; P, post‐natal day; AB, antibody; IP, intraperitoneal; SEM, standard error of the mean; ANOVA, analysis of variance.Source data are available online for this figure.

Figure 2. Combined neutralization of VEGF‐A and ANG‐2 reduced the number of Iba1⁺ macrophages in the JR5558 mice

(A) Bar/scatter graph of the number of Iba1⁺ cells in the subretinal space of mice treated with 10 mg/kg IgG control, 5 mg/kg anti‐VEGF‐A, 5 mg/kg anti‐ANG‐2, 10 mg/kg anti‐VEGF‐A/ANG‐2, or left untreated. Data are shown as mean ± SEM with n = 5 animals per group, and asterisk denotes significant changes after one‐way ANOVA and Tukey's multiple t‐test (Tukey–Kramer HSD). Anti‐VEGF‐A/ANG‐2 is significantly different from IgG control (*P < 0.02). (B) Representative examples of flat‐mounted RPE/choroid Iba1 (red) and IB4 counterstain (green) of untreated (top left), IgG control (top middle; 10 mg/kg), anti‐VEGF‐A (bottom left; 5 mg/kg), anti‐ANG‐2 (bottom middle; 5 mg/kg), and anti‐VEGF‐A/ANG‐2 (bottom right; 10 mg/kg). Adjustment of brightness to 100 using Photoshop CS6 was applied equally to all images, and non‐tissue background was removed using Lasso tool. Inserts show selected areas. Scale bars = 500 μm. SEM, standard error of the mean; ANOVA, analysis of variance.Source data are available online for this figure.