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

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 FVIIIequivalence 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 analogue of the tissue factor pathway inhibitor-targeting antibody marstacimab (SIA-marstacimab). The severity of these models was variable, each requiring different doses of FVIII needed to reduce blood loss to levels of wild-type mice. For instance, 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 microgram/ml and 16 microgram/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 a single FVIIIequivalence is unlikely to exist for emicizumab, SIA-marstacimab and similar molecules, because their activity is dependent on local conditions and severity of the injury.