In Vitro and In Vivo Characterization of Marstacimab, an Anti-TFPI Antibody

Hemophilia A and B are X-linked genetic disorders resulting from functional deficiencies of the intrinsic coagulation plasma proteins Factor VIII (FVIII) or Factor IX (FIX), respectively. An approach to achieving hemostatic pharmacology in hemophilia is to augment the extrinsic cascade is to neutralize Tissue Factor Pathway Inhibitor (TFPI). TFPI is a multi-Kunitz (K) domain inhibitor which binds to and inhibits Factor Xa via the K2 domain and Factor VIIa/Tissue Factor activity (K1 domain). Marstacimab is a fully human monoclonal antibody that binds to and neutralizes TPFI activity and is under development for treatment of hemophilia. To support lead identification during late discovery, five anti-tissue factor pathway inhibitor (TFPI) antibodies (EC₅₀<5nM) were evaluated in an in vivo rabbit pharmacokinetics/pharmacodynamics (PK/PD) study and in vitro using rabbit and human hemostatic assays. Marstacimab displayed potent in vitro activities, and potent pharmacology in shortening the dilute prothrombin time (dPT) assay (1.3 nM) and in the thrombin generation assay (TGA) (2.8 nM). Following an intravenous administration in rabbits, marstacimab and the other anti-TFPI antibodies exhibited target-mediated drug disposition (TMDD) with a half-life less than 2 days, when compared to a control IgG. The pharmacokinetics of anti-TFPI antibody can be described using a Michaelis-Menten mechanistic model in which in vivo Km and Vmax were estimated for the observed nonlinear clearance. The estimated pharmacokinetic parameters for marstacimab was 45 and 38 mL/kg for the central and peripheral volume of distribution, 23 nM for Km, 131 nM/kg/hr for Vmax. In addition, the correlation between in vitro Kd, in vivo Km and in vitro/in vivo potency were assessed. Compared to other anti-TFPI antibodies, marstacimab exhibited reduced clearance and a good in vitro-in vivo relationship. Second, the binding epitopes were further characterized using competitive surface plasmon resonance (SPR) and marstacimab has distinct binding epitopes from the other antibodies. Lastly, the potential human pharmacokinetics and efficacy were evaluated using allometry scaling and in vitro potency, the results predict a minimal target occupancy of 76% following a weekly subcutaneous administration of marstacimab. These studies indicate that the differential influence of the TFPI-binding epitopes on the pharmacokinetics, but not on the pharmacodynamics. The selection strategy could be applicable to the other antibody discovery and development.