Xyntha

Six to ten-week-old F8^−/−VWF^+/+ or F8^−/− VWF^−/− mice were immunized with recombinant human B-domain deleted FVIII (rhF8, Xyntha, Pfizer Inc, New York, NY, USA) at a dose of 50 IU kg⁻¹ via the retro-orbital vein injection weekly for 4 weeks total. One week after the last immunization, plasmas were collected and inhibitor titers were determined by Bethesda assay as described in our previous report.[2 (link)] For 2bF8^tg+/− mice, six to ten-week-old animals were immunized with rhF8 at a dose of 600 U kg⁻¹ in the presence of adjuvant (Sigma, St. Louis, MO, USA) twice with a 3-week interval. One week after the second immunization, blood samples were collected and inhibitor titers were determined.
For inhibitor model studies, both chronic and acute models were used. In the chronic inhibitor model, rhF8-immunized F8^−/− mice received bone marrow transplantation (BMT) from 2bF8^tg+/− mice or rhF8-immunized 2bF8^tg+/− mice were used. In the acute model, inhibitory plasmas from rhF8-immunized F8^−/−VWF^−/− mice with high-titer of inhibitors were infused into unimmunized 2bF8 mice with varying VWF conditions to a level of inhibitor titers of 2.5, 25, or 250 BU mL⁻¹ followed by phenotypic correction assessment.

Tail bleeding assays were performed as described (19 (link)). Mice were placed on temperature controlled heating pads (37°C) and anesthetized by isoflurane inhalation. The distal portion of the tail was cut at 1.5 mm diameter after which the tail was immersed in a predefined volume of 37°C saline (0.9% NaCl) for 20 minutes. Blood loss was determined after red cell lysis with 2% acetic acid by measuring the absorbance of hemoglobin at 490 nm and using a hemoglobin standard curve derived from defined blood volumes. Blood loss was expressed in µL/g body weight assuming a hematocrit of 46%. FVIII-deficient mice were injected retro-orbitally 5 minutes prior to tail cut with vehicle, ^superFVa, rhFVIIa, mFVIIa, or rhFVIII (200 units/kg; Xyntha®, Pfizer, NY, NY, USA) in an equal volume (200 µL) of sterile saline for injection (0.9%, Hospira Inc.).
For FVIII inhibitor mice, BalbC mice were injected with the GMA-8015 anti-FVIII antibody (100 µL, 0.25 mg/kg in sterile saline) retro-orbitally 2 hours before tail clip. Prohemostatic agents were injected retro-orbitally in the other eye 5 minutes before tail clip in an equal volume (100 µL) of sterile saline.

One million pltLys-iTreg or purTGFβ-iTreg cells were adoptively transferred by IV injection into F8^null/B6 mice followed by rhF8 (Xyntha; Pfizer Inc., New York, NY) immunization weekly at 50 U/kg per week IV for a total of 4 weeks. One week after the last immunization, blood samples were collected. The titers of anti-FVIII inhibitory antibodies (inhibitors) and total anti-FVIII antibodies were determined by Bethesda assay and enzyme-linked immunosorbent assay (ELISA), respectively, as described in our previous report.^{16 (link),19 (link)} Six days after the iTreg cell transfer, CD4⁺ T cells from peripheral blood were analyzed by flow cytometry for enhanced green fluorescent protein (EGFP) expression to assess the recovery of the infused iTregs. CD45.1 and CD45.2 were used as congenic markers.