Angiopoietin-1 Antibody (MM0019-26B10) - Azide and BSA Free NB110-85464 from Novus Biologicals, a Bio-Techne Brand

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EMBO molecular medicine
November 01 2016

Fig 1: Structural characteristics and functional binding properties of CrossMAb RG7716 Structural presentation of CrossMAb RG7716 with substituted amino acids highlighted and colored. Amino acids corresponding to individual point mutations ensuring correct and efficient heavy chain heterodimerization (“knobs-into-holes” [green] and additional disulfide bridge [yellow]), abolishing Fc receptor functionality (Fc? receptors I, II, and III, [pink] and FcRn [blue]) are highlighted.Summary table of experimental binding affinities of the parental components targeting VEGF-A (ranibizumab) and ANG-2 (LC10) as well as of RG7716 measured by isothermal titration calorimetry. RG7716 binds VEGF-A121 and VEGF-A165 with an affinity comparable to ranibizumab (3.3 vs. 3.1 nM). ANG-2 binding of RG7716 is slightly decreased to 22 nM compared to a-ANG-2 IgG1 (LC10) with 4.1 nM. No binding to ANG-1 was detectable for RG7716 or a-ANG-2 IgG1 (LC10).Inhibition of ANG-1-induced endothelial cell survival was tested in the presence of increasing concentrations of LC10 (anti-ANG-2 IgG, ANG-2 binding part in RG7716) and LC08 (ANG-1 and ANG-2 binding antibody). An average of three independent experiments is shown with SEM.The ability of RG7716 to bind to ANG-2 and VEGF-A simultaneously and independent is demonstrated by a surface plasmon resonance experiment. RG7716 is captured via an anti-Fc antibody on the chip surface. The green curve depicts the experiment with ANG-2 in the first and VEGF-A in the second binding event. The red curve shows the binding events in the other sequence.
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EMBO molecular medicine
November 01 2016

Fig 2: Vitreous concentrations of angiopoietins in patients newly diagnosed with retinal diseases and cell model of barrier breakdown testing the interaction of VEGF-A and angiopoietins A, BBox plots of vitreal ANG-1 (A) and ANG-2 (B) levels from newly diagnosed patients with wAMD, DR, proliferative DR and RVO compared to controls (macular hole). The interquartile range of the data is indicated by the box. A nonparametric Kruskal–Wallis analysis followed by Dunn's method for multiple comparisons was used to show significant differences of the groups to control which are indicated by asterisks. ANG-1 levels did not differ significantly, but ANG-2 levels were significantly different: control vs. AMD (*, P = 0.0451), vs. DR (****, P < 0.0001), pDR (****, P < 0.0001), and RVO (****, P < 0.0001). CSchematic of cellular experiments to measure transendothelial resistance in human endothelial cells using transwell filters and CellZscope technology.D24-h concentration of ANG-2 in supernatants of the basal side of the culture stimulated with 5 ng/ml of VEGF-A, 200 ng/ml of ANG-1, or the combination. Error bars show SEM with one-sided ANOVA (P < 0.0001) and Tukey's multiple t-test for five independent experiments indicating significance for control vs. VEGF-A (**, P = 0.0028); VEGF-A vs. ANG-1 (****, P < 0.0001); ANG-1 vs. VEGF-A and ANG1 (**, P = 0.0061); VEGF-A vs. VEGF-A and ANG-1 (ns, P = 0.0625).EHuman endothelial cells plated on filters were assessed for endothelial barrier function over time after the addition of 5 ng/ml VEGF-A alone or in combination with 200 ng/ml ANG-1 or 10 µg/ml anti-ANG-2 or all three test items. The final time point was used for statistical analysis. Error bars show SEM with one-sided ANOVA (P < 0.0001) and Tukey's multiple t-test for three independent experiments indicating significance of VEGF-A vs. untreated (****, P < 0.0001); vs. VEGF-A and ANG-1 and anti-ANG-2 (***, P = 0.0004); vs. VEGF-A and anti-ANG-2 (*, P = 0.038); vs. VEGF-A and ANG-1 (*, P = 0.040). Furthermore, VEGF-A and anti-ANG-2 and ANG-1 are significantly different vs. VEGF-A and ANG-1 (*, P = 0.0451); vs. VEGF-A and anti-ANG-2 (*, P = 0.0474); vs. untreated (*, P = 0.0492). Finally untreated is significantly different vs. VEGF-A and ANG-1 (***, P = 0.0009) and VEGF-A and anti-ANG-2 (***, P = 0.0008).Data information: ANOVA, analysis of variance; SEM, standard error of the mean; ANG-2, angiopoietin-2; DR, diabetic retinopathy; RVO, retinal vein occlusion; wAMD, wet age-related macular degeneration; pDR, proliferative diabetic retinopathy; vs., versus.