Table 1.
Characteristics of anti-fVIII MAbs.
Figure 1.
Epitope map of non-overlapping MAbs.
The relative epitopes of anti-fVIII MAbs are shown. MAbs 4A4, 2–54, 1D4 and 2–93 target non-overlapping epitopes in the A2 domain. MAbs G38 and 2–113 target non-overlapping epitopes in the A3 domain at residues 1690–1817 and 1818–1916, respectively. MAbs I109 and 2–77 target non-overlapping epitopes in the C2 domain. [13].
Figure 2.
Amount of fVIII needed to restore thrombin generation of rfVIIa.
Varying concentrations of fVIII were mixed 1∶1 with rfVIIa at a final concentration of 2.25 µg/ml to determine the level of fVIII activity necessary for restoration of thrombin generation of rfVIIa to fVIII at 1 U/ml. The resulting data was transformed using manufacturer’s software to yield thrombin generation curves (A), peak thrombin concentration (B) and index velocity (C). Error bars represent sample standard deviation.
Figure 3.
Inhibition of thrombin generation by group 1 anti-fVIII MAbs.
Thrombin generation curves are shown for fVIII deficient plasma (negative control), fVIII deficient plasma with 1 U/ml of fVIII (positive control) and 1 U/ml of fVIII in the presence of 5 µg/ml of specified group 1 MAb. Data represent mean ± sample standard deviation for 3 experiments where duplicates of each measurement were taken and averaged.
Table 2.
TGA parameters for anti-fVIII MAbs.
Figure 4.
Time dependent inhibition of thrombin generation by group 2 anti-fVIII MAbs.
Thrombin generation curves are shown for fVIII deficient plasma (negative control), fVIII deficient plasma with 1 U/ml of fVIII (positive control) and 1 U/ml of fVIII in the presence of 5 µg/ml of specified group 2 MAb at both the immediate and 1 hr time points (A) Thrombin generation curves comparing fVIII alone, rfVIIa alone, and fVIII and rfVIIa in the presence of MAb at the 1 hr time points (B). Graphs shown are representative of 3 independent experiments.
Figure 5.
Thrombin generation in the presence of group 2 anti-fVIII MAbs.
Thrombin generation curves are shown comparing fVIII alone, rfVIIa alone, and fVIII and rfVIIa in the presence of MAb at the 1 hr time points. Graphs shown are representative of 3 independent experiments.
Figure 6.
Influence of inhibitory titer on residual fVIII activity and peak thrombin generation.
The inhibitor titer for each MAb was compared with the peak thrombin generation of fVIII alone or fVIII + rfVIIa at the immediate (solid circles, r2 = 0.36) or 1 hr (open circles, r2 = 0.09) time points (A.) and with residual fVIII activity at the immediate (solid circles, r2 = 0.004) or 1 hr (r2 = 0.07) time points (B.).