Consistent clinical factor VIII equivalency is unlikely for non-factor therapies in hemophilic mice

Abstract

Non-factor therapies are changing the treatment paradigm in hemophilia A, which was previously dominated by replacement- therapy using factor VIII (FVIII) concentrates. However, the FVIII equivalence of these new therapies has remained unclear, since in vitro assays generate variable responses. Here we used four different in vivo bleeding models to compare FVIII to emicizumab and to a sequence-identical analog of the tissue factor pathway inhibitor-targeting antibody marstacimab (SIA-marstacimab). The severity of these models was variable, each requiring different doses of FVIII to reduce blood loss to levels of wild-type mice. For example, a dose of 2.5 IU/kg FVIII was needed for full correction in the tail vein transection (TVT) model, whereas 25 IU/kg was needed in the saphenous vein puncture (SVP) model. Intermediate doses were required in the tail artery transection (TAT) model (5 IU/kg) and tail clip model (7.5 IU/kg). Importantly, FVIII treatment produced stable clots, without spontaneous rebleeds being observed. Both emicizumab and SIA-marstacimab (used at therapeutic doses of 55 μg/mL and 16 μg/mL, respectively) displayed a variable, model-dependent FVIII equivalence. For example, emicizumab proved equivalent to 5 IU/kg FVIII in the tail clip model, and to 10 IU/kg in the SVP model. Strikingly, both emicizumab and SIA-marstacimab treatment resulted in spontaneous rebleeds in the TVT, TAT and tail clip models, further distinguishing them from FVIII treatment. Our data suggest that there is unlikely to be a single FVIII equivalence for emicizumab, SIA-marstacimab, and similar molecules, because their activity is dependent on local conditions and severity of the injury.

Figure 1.

Different bleeding models used in this study. In the tail vein transection model, the lateral vein is transected in a template-dependent manner, allowing for a reproducible incision in terms of depth and location. In the tail artery transection model, the ventral artery is transected, also in a template-dependent manner to ensure reproducible incisions in terms of depth and location. In the tail clip model, the terminal 3 mm of the tail tip is severed. In the saphenous vein puncture model, the saphenous vein is punctured. Subsequently, every time a clot is formed, it is removed.

Figure 2.

Tail vein transection model without clot removal. (A) Factor VIII (FVIII)-deficient mice were given intravenously vehicle, FVIII (2.5 IU/kg), emicizumab (5 mg/kg) or sequence-identical analog of marstacimab (SIA-marstacimab) (1 mg/kg). Estimated plasma concentrations at time of injury were 5 IU/dL FVIII, 55 μg/ mL emicizumab or 16 μg/mL SIA-marstacimab. Wild-type (wt) mice were used as control. Mice were monitored for 45 minutes (min). During the 45-min observation time, periods of bleeding and bleeding arrest were noted. Bleeding patterns of 4 mice representative for each group are presented. (B) Blood loss for each individual mouse included in the study. (C) Statistical analysis of data presented in (B). Statistical analysis was performed via one-way ANOVA with Tukey’s correction for multiple comparisons. Conditions that are above the threshold of statistical significance (P<0.05) are indicated in red.

Figure 3.

Tail vein transection model with clot removal. (A) Factor VIII (FVIII)-deficient mice were given intravenously vehicle, various doses of FVIII (2.5 and 5 IU/kg), emicizumab (5 mg/kg) or sequence-identical analog of marstacimab (SIA-marstacimab) (1 mg/kg). Estimated plasma concentrations at time of injury were 5 IU/dL FVIII, 10 IU/dL FVIII, 55 μg/mL emicizumab or 16 μg/mL SIA-marstacimab. Wild-type (wt) mice were used as control. If mice were not bleeding at 15 minutes (min) and/or 30 min, clots were dislodged. Mice were monitored for 45 min. During the 45-min observation time, periods of bleeding and bleeding arrest were noted. Bleeding patterns of 4 mice representative for each group are presented. For FVIII-treated mice, data for 4 mice receiving the lowest dose are depicted. (B) Blood loss for each individual mouse included in the study. (C) Statistical analysis of data presented in (B). Statistical analysis was performed via one-way ANOVA with Tukey’s correction for multiple comparisons. Conditions that are above the threshold of statistical significance (P<0.05) are indicated in red.

Figure 4.

Tail artery transection model. (A) Factor VIII (FVIII)-deficient mice were given intravenously vehicle, various doses of FVIII (2.5, 5, and 10 IU/kg), emicizumab (5 mg/kg) or sequence-identical analog of marstacimab (SIA-marstacimab) (1 mg/kg). Estimated plasma concentrations at time of injury were 5 IU/dL FVIII, 10 IU/dL FVIII, 20 IU/dL FVIII, 55 μg/mL emicizumab or 16 μg/mL SIA-marstacimab. Wild-type (wt) mice were used as control. If mice were not bleeding at 15 minutes (min), clots were dislodged. Mice were monitored for 30 min. During the 30-min observation time, periods of bleeding and bleeding arrest were noted. Bleeding patterns of 4 mice representative for each group are presented. For FVIII-treated mice, data for mice receiving the two lowest doses are depicted. (B) Blood loss for each individual mouse included in the study. (C) Statistical analysis of data presented in (B). Statistical analysis was performed via one-way ANOVA with Tukey’s correction for multiple comparisons. Conditions that are above the threshold of statistical significance (P<0.05) are indicated in red.

Figure 5.

Tail clip model. (A) Factor VIII (FVIII)-deficient mice were given intravenously vehicle, various doses of FVIII (2.5, 5, 10, and 20 IU/kg), emicizumab (5 mg/kg) or sequence-identical analog of marstacimab (SIA-marstacimab) (1 mg/kg). Estimated plasma concentrations at time of injury were 5 IU/dL FVIII, 10 IU/dL FVIII, 20 IU/dL FVIII, 40 IU/dL, 55 μg/mL emicizumab or 16 μg/ mL SIA-marstacimab. Wild-type (wt) mice were used as control. Mice were monitored for 30 minutes (min). During the 30-min observation time, periods of bleeding and bleeding arrest were noted. Bleeding patterns of 4 mice representative for each group are presented. For FVIII-treated mice, data for mice receiving 5 or 7.5 IU/ kg are depicted. (B) Blood loss for each individual mouse included in the study. (C) Statistical analysis of data presented in (B). Statistical analysis was performed via one-way ANOVA with Tukey’s correction for multiple comparisons. Conditions that are above the threshold of statistical significance (P<0.05) are indicated in red.

Figure 6.

Saphenous vein puncture model. (A) Factor VIII (FVIII)-deficient mice were given intravenously vehicle, various doses of FVIII (2, 10, and 25 IU/kg), emicizumab (5 mg/kg) or sequence-identical analog of marstacimab (SIA-marstacimab) (1 mg/kg). Plasma concentrations at time of injury were 4 IU/dL FVIII, 20 IU/dL FVIII, 50 IU/dL FVIII, 55 μg/mL emicizumab or 16 μg/mL SIA-marstacimab. Wild-type (wt) mice were used as control. Mice were monitored for 30 minutes (min). Presented is the number of clots over 30 min generated for each individual mouse included in the study. (B) Statistical analysis of data presented in (A). Statistical analysis was performed via one-way ANOVA with Tukey’s correction for multiple comparisons. Conditions that are above the threshold of statistical significance (P<0.05) are indicated in red.

Figure 7.

Overview of the protective levels of factor VIII. (A) Schematic depiction on the potential relation between factor VIII (FVIII) levels and the extent of protection they provide under different conditions, based on consensus proposed in several guidance articles. Note that these values may vary between individuals. Red line simulates plasma levels of FVIII following intravenous infusion; pink horizontal bar mimics steady-state levels of emicizumab and marstacimab during subcutaneous prophylactic treatment. Based on the data generated in this study, it appears that emicizumab and sequence-identical analog of marstacimab (SIA-marstacimab) display a model-dependent, variable FVIII equivalence that does not seem to exceed 20 IU/dL (as is observed in the saphenous vein puncture model). (B) Summary of the hemostatic responses upon emicizumab treatment in the various bleeding models. (C) Summary of the hemostatic responses upon SIA-marstacimab treatment in the various bleeding models.