In vivo analysis of FVIII-KB013bv. (A) FVIII-deficient mice were given BDD-FVIII (open circles) or FVIII-KB013bv (closed circles) at a dose of 250 U/kg via tail vein injection; then, blood samples were taken at various time points, and plasma was analyzed for residual FVIII activity. Plotted are FVIII activities relative to the activity at t = 3 minutes, which was arbitrarily set at 100%. Each data point represents mean ± SD of 3 to 5 mice, and each mouse was bled 1 to 2 times. (B) FVIII-deficient mice received BDD-FVIII or FVIII-KB013bv (500 U/kg) via tail vein injection, and 24 hours after injection a tail clip–bleeding assay was performed. Clipped tails were immersed in saline at 37°C, and blood was collected for 30 minutes. Blood loss for each individual mouse is indicated. (C) Mice were given BDD-FVIII or FVIII-KB013bv (50 U/kg) at days 0, 7, 14, 21, and 40 via tail vein injection. At day 43, blood samples were taken, and plasma was analyzed for the presence of murine anti-FVIII antibodies. Briefly, wells coated with BDD-FVIII were incubated with murine plasma, and bound murine antibodies were detected via peroxidase-labeled polyclonal goat anti-mouse antibodies. As standard, a monoclonal anti-FVIII antibody was used. The limit of detection (LOD) in this assay was 0.1 μg/mL. The immune response for each individual mouse is presented. Statistical analyses were performed using a Mann-Whitney U test. (D) According to Sorvillo et al,⁹  the VWF-FVIII complex separates at the cellular surface of antigen-presenting cells, with FVIII being endocytosed and most of the VWF molecules remaining outside the cell. (E) Based on the model described by Sorvillo et al, we anticipate that the complex between FVIII-KB013bv and VWF will not dissociate, and consequently, there will be reduced uptake of FVIII-KB013bv by antigen-presenting cells. Fewer FVIII-derived peptides will then be presented to T cells, and in turn, there will be reduced development of anti-FVIII antibodies.