Journal: Nature Communications

Article Title: Targeted inhibition of activated protein C by a non-active-site inhibitory antibody to treat hemophilia

doi: 10.1038/s41467-020-16720-9

Figure Lengend Snippet: a – d Affinity measurement by SPR for type I and type II mAbs: Biacore sensograms showing the association and dissociation curves for antibody binding to hAPC at concentrations of 1.56, 3.125, 6.25, 12.5, 25, 50, 100, and 200 nM ( a , b ) or to hPC at concentrations of 1.56, 3.125, 6.25, 12.5, 25, 50, 100, 200, 400, and 1000 nM ( c , d ). Black traces represent experimental data, and red traces represent the corresponding fits. Type I showed a slower on-rate ( k a , 1.5 × 10 5 M −1 s −1 ) and off-rate ( k d , 1.9 × 10 −3 s −1 ) than type II ( k a , 6.1 × 10 5 M −1 s −1 ; k d , 6.2 × 10 −3 s −1 ). e , f Antibody-binding specificity by ELISA for type I mAb ( e ) and type II mAb ( f ) bound to hAPC (circle) or hPC (square). ELISA results are shown as mean ± SDs from triplicate wells for each antibody concentration, and experiments were repeated >3 times. g Schematic representation of the binding sites on APC protease domain for type I, type II, and R41C17 mAbs. R41C17, an anti-APC Gla-domain antibody, was used as control. h Type II but not type I bound the complex of PPACK-hAPC. PPACK-hAPC or hAPC was coated onto a MaxiSorp plate at 100 ng per well overnight. IgG or Fab at concentrations starting at 20 nM were tested for binding. SPR surface plasmon resonance, ELISA enzyme-linked immunosorbent assay, hAPC human activated protein C, hPC human protein C, RU relative unit, OD490 absorbance at 490 nm, PPACK Phe–Pro–Arg–chloromethylketone, RFU relative fluorescence unit, IgG immunoglobulin G, Fab antigen-binding fragment.

Article Snippet: Type I anti-APC mAb was identified by panning the n-CoDeR ® phage-display library of human antibody Fab fragments (BioInvent International AB).

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Concentration Assay, SPR Assay, Fluorescence

Journal: Nature Communications

Article Title: Targeted inhibition of activated protein C by a non-active-site inhibitory antibody to treat hemophilia

doi: 10.1038/s41467-020-16720-9

Figure Lengend Snippet: a APC amidolytic activity is defined by the maximum velocity ( V max ) of its hydrolyzing reaction toward the small chromogenic substrate spectrozyme PCa, and percent inhibition of APC amidolytic activity by mAbs is shown in Supplementary Fig. . Data are expressed as mean ± SD. N = 4–6. b Both mAbs protect FVa from APC-mediated proteolysis. In the absence of APC, the activity of FVa in the prothrombinase assay was designated as 100%. In the absence of added antibodies, APC proteolyzed FVa and reduced its activity to 6.4 ± 0.3% (mean ± SD). Assays were repeatedly performed in triplicates; a typical curve is shown. c , d TM–TGA in normal human plasma (FACT): c ETP and d peak thrombin levels. e , f TGA on EA.hy926 endothelial cells: e baseline thrombin levels (nM) shown as a representative experiment and f ETP as %NP (percent of normal pooled plasma). Data represent mean values ± SD from three independent experiments. g – l Plasma-clotting assays where IC 50 values in nM of mAbs are compared in a table below each panel. Protac-aPTT using FACT: g clotting time in seconds (s), h percent inhibition. Protac-aPTT using HemA plasma: i clotting time in seconds (s) and j percent inhibition. Protac-aPTT using FVIII Ab-treated FACT: k clotting time in seconds (s) and l percent inhibition. In c – l , experiments were run with replicate samples in each assay, and data reflect the average of two replicates. Assays were repeated ≥3 times. FACT normal human plasma, Protac a protein C activator from A. contortrix venom.

Article Snippet: Type I anti-APC mAb was identified by panning the n-CoDeR ® phage-display library of human antibody Fab fragments (BioInvent International AB).

Techniques: Activity Assay, Inhibition, Coagulation

Journal: Nature Communications

Article Title: Targeted inhibition of activated protein C by a non-active-site inhibitory antibody to treat hemophilia

doi: 10.1038/s41467-020-16720-9

Figure Lengend Snippet: a Histone-mediated cytotoxicity assay using HUVECs (% live cells after 2 h). Positive (50 µg/mL histone 3, 0% live cells, black bar) and negative (no histone 3, 70% live cells, open bar) controls. Reduction of histone cytotoxicity by hAPC (20 nM, gray bars) in the presence of type I mAb (light gray) or type II mAb (dark gray). The results are shown as mean + SD of three independent experiments. b Effect of mAbs on PAR1 cleavage of SEAP–PAR1 reporter construct on transfected HEK293/wt-EPCR cells. Shown are mean ± SEM of n = 4 performed on two independent cell seedings. c PAR1 cleavage on EA.hy926 endothelial cells. The ATAP antibody reports PAR1 cleavage at Arg46 . Shown are mean ± SD of N = 10. * denotes p < 0.05 tested with ANOVA with Dunnett’s multiple-comparison test in both ( c ) and ( d ). d , e APC (50 nM)-mediated endothelial barrier protection in response to thrombin (2 nM)-induced barrier disruption of an EA.hy926 endothelial cell monolayer was determined using the iCelligence system. d Permeability is expressed as the percentage of maximal barrier disruption induced by thrombin (100%) in the absence of APC. Shown are mean ± SEM of N = 4. e Effect of the mAbs on APC’s barrier protection is expressed as the percentage of the maximal barrier-protective effect of APC in the absence of mAbs. Shown are mean ± SEM of N = 3. f Effect of the type II mAb on the inactivation of APC in human plasma by SERPINs. Data represent mean values ± SD from at least three independent experiments. HUVEC human umbilical vein endothelial cells, SEAP–PAR1 secreted embryonic alkaline phosphatase (SEAP) fused to the N terminus of human PAR1, HEK293/wt-EPCR HEK293 cells with stable expression of wild-type human EPCR, SERPINs serine protease inhibitors.

Article Snippet: Type I anti-APC mAb was identified by panning the n-CoDeR ® phage-display library of human antibody Fab fragments (BioInvent International AB).

Techniques: Cytotoxicity Assay, Construct, Transfection, Permeability, Expressing