Targeting key angiogenic pathways with a bispecific CrossMAb optimized for neovascular eye diseases (2016)
Figure 2

Figure 2. Inhibition of vessel leakiness and lesion number by combined inhibition of VEGF-A and ANG-2 in the model of sCNV in JR5558 mice using fluorescence angiography.

A,B) Real-time qPCR analysis of ANG-2 levels in retina and RPE/choroid complexes of JR5558 and C57Bl/6J (C57) mice. A) Relative expression levels of ANG-2 were significantly increased in the RPE/choroid complexes of JR5558 mice in comparison to C57, at 50 and 62 days old (P = 0.022 at D50 and P = 0.042 and at D62). B) By contrast, retina levels of ANG-2 were not significantly different between C57 and JR5558 mice, indicating neovascularization is driven locally by ANG-2. SEM is shown as error bar with n = 4 animals per group. *Denotes statistical significance of JR5558 mice compared to wild type C57bl6 using unpaired T-test for each time point analyzed separately.

C) Schematic presentation of experimental design. Mice received IP injections of CrossMAb anti-VEGF-A/ANG-2 (species crossreactive, B20.4.1 and LC10), anti ANG-2 (LC10), an anti-VEGF-A IgG1 (B20.4.1) or IgG control at postnatal D14 and D19 followed by fluorescence angiography at D26 (early intervention), results demonstrated in D–K. Alternatively, mice received antibody at postnatal D47 and D55 followed by fluorescence angiography at D60 (late intervention), results demonstrated in L and M.

D–I) Representative examples of fluorescence angiograms of IgG control (D), anti-VEGF-A (E), anti-ANG-2 (F) (all the 5 mg/kg) and three doses of anti-VEGF-A/ANG-2 (G,H,I), (at 3, 5, and 10 mg/kg).

J,K) Bar graph of numbers of spontaneously occurring lesion number (J) and area by fluorescence angiography (K) after two weekly doses of antibody (antibodies at 5 mg/kg IP and anti-VEGF-A/ANG-2 at 10 [high], 5 [mid], and 3 [low] mg/kg IP) followed by analysis a week later in the early intervention model.

L,M) Bar graph of numbers of spontaneously occurring lesion numbers (L) and area by fluorescence angiography (M) after two weekly doses of antibody (3 mg/kg IP) followed by analysis a week later in the late intervention model.

SEM is shown as error bars with n = 8 animals per group and significance indicated by * using ANOVA (J, K: ****, P < 0.0001; L: **, P < 0.0018; M: *, P < 0.031) followed by Tukey’s multiple t-test in J-M). In J) IgG control is significant against anti-VEGF-A (****, P < 0.0001), anti-ANG-2 (**, P = 0.0069), anti-VEGF-A/ANG-2 low (*, P = 0.0194), -mid and –high (****, P < 0.0001). Furthermore, anti-VEGF-A/ANG-2 high is significant against anti-VEGF-A (*, P = 0.0428), anti-ANG-2 (****, P < 0.0001) and anti-VEGF-A/ANG-2 low (****, P < 0.0001). Finally anti-ANG-2 is significantly against anti-VEGF-A/ANG-2 mid (**,P < 0.0041). In K) IgG control is significant against anti-VEGF-A (***, P = 0.0003), anti-VEGF-A/ANG-2 mid and –high (both ****, P < 0.0001). Furthermore, anti-VEGF-A/ANG-2 high is significant against anti-VEGF-A (**, P = 0.0037), anti-ANG-2 (****, P < 0.0001) and anti-VEGF-A/ANG-2 low (**, P = 0.0001). Finally anti-ANG-2 is significantly against anti-VEGF-A/ANG-2 mid (**, P < 0.0022) and anti-VEGF-A/ANG-2 low against -mid (*, P = 0.022). In L) IgG control is significantly different from anti-ANG-2 (*, P = 0.023) and anti-VEGF-A/ANG-2 (*, P = 0.014). Vehicle is different from anti-ANG-2 (*, P = 0.024) and anti-VEGF-A/ANG-2 (*, P = 0.016). In M) IgG control is significantly different from anti-ANG-2 (*, P = 0.044) and anti-VEGF-A/ANG-2 (*, P = 0.049). D, day; IP, intraperitoneal; sCNV, spontaneous choroidal neovascularization