Mim8 improves the In Vivo hemostasis of select hemophilia B causing factor IX variants Free

Introduction Emicizumab, a first-generation factor VIIIa (FVIIIa)-mimetic, has revolutionized prophylaxis for people with hemophilia A (HA) by reducing the burden of prophylaxis, including allowing for initiation of prophylaxis during infancy without central catheters. We recently reported that therapeutic amounts of emicizumab (300 nM or 50 µg/mL) can also rescue the procoagulant activity of select hemophilia B (HB)-causing factor IX (FIX) variants that disrupt intrinsic Xase assembly (Lee et al. Blood 2024).These variants were present in 7.1% of people with HB in the My Life Our Future genotyping program. Mim8 (denecimig) is a next-generation FVIIIa-mimetic with distinct binding epitopes and increased potency compared to emicizumab; it is in late-stage clinical development for HA. In the present study, we evaluate the in vitro and in vivo hemostatic ability of Mim8 to rescue the procoagulant activity of HB-causing FIX variants.

Methods 96 HB-causing FIX variants with missense substitutions were transiently transfected in mammalian cell culture and conditioned media was screened for procoagulant rescue with Mim8 in an aPTT-based clotting assay. Recombinant (r) protein of identified FIX variants with substitutions in different protein domains—L6S (Gla), D47E (EGF-like), R333Q (protease), and I397T (protease)—were purified to homogeneity for further characterization. In vivo hemostasis was assessed in HB mice after tail vein transection (TVT) or laser-injury of the cremaster muscle (LICM). Because Mim8 does not interact with mouse FX, human (h) FX as well as hFIX variant protein were intravenously administered with or without Mim8 prior to injury.

Results We identified 43 HB-causing FIX variants with >2-fold enhanced clotting activity with a therapeutic amount (34 nM or 5 µg/mL) of Mim8. Comparisons of Mim8 and emicizumab at equimolar concentrations show Mim8 generally conferred a greater procoagulant effect, though FIX variants with substitutions located near the binding epitope of Mim8 favored emicizumab, likely due to decreased Mim8 binding.

Consistent with the screen, Mim8 and emicizumab increased the aPTT-based specific activity of purified FIX variants 6-60-fold. Therapeutic doses of Mim8 and emicizumab also improved thrombin generation of purified FIX variants from 1-20% wildtype (WT) FIX to 40-80% FIX-WT, depending on the variant and triggering reagent. We also compared the ability of Mim8 and emicizumab to improve thrombin generation to commercial FIX concentrate. In this experiment, the FIX therapeutic equivalence of the FVIIIa-mimetics was 10-60% depending on the FIX variant and triggering reagent.

We next evaluated the in vivo hemostatic ability of Mim8 in HB mice in 2 distinct injury models. In the TVT injury (3-7 mice/cohort), administration of 1.5 mg/kg hFX and hFIX-R333Q or hFIX-I397T modestly improved hemostasis (mean ± SEM) with decreased blood loss (20 ± 4 or 16 ± 3 µL/g) and bleeding time (19 ± 3 or 22 ± 1 minutes) compared to HB mice administer buffer (33 ± 3 µL/g and 31 ± 1 minutes). However, the addition of 1 mg/kg Mim8 substantially improved hemostasis with blood loss (7 ± 2 or 7 ± 1 µL/g) and bleeding time (7 ± 2 or 6 ± 1 minutes) approaching bleeding in wild-type mice (2 ± 1 µL/g and 6 ± 1 minutes). The addition of Mim8 significantly (p < 0.01) improved hemostasis compared to only hFX and hFIX treatment.

In the LICM model (6-7 injuries with 2 mice per cohort), arteriole clot formation was quantified with fluorescently labelled platelets and fibrin. HB mice administered 0.15 mg/kg hFX and FIX-R333Q displayed reduced and delayed platelet and fibrin accumulation at the site of vascular injury. Quantitative analysis as a function of time revealed a mean ± SEM platelet and fibrin area of 100 ± 30 µm² and 50 ± 10 µm² respectively; this clot size is similar to our previous studies of untreated HB mice. However, a significant greater accumulation of platelet and fibrin was observed with the addition 0.1 mg/kg Mim8: platelets and fibrin increased to 350 ± 50 µm² and 200 µm² ± 60 respectively. This clot size approaches the clot size in our previous studies of wild-type mice (platelets 400 ± 40 µm² and fibrin 280 ± 40 µm²).

Conclusion Mim8 improves the in vitro and in vivo hemostatic activity of select HB-causing FIX variants. The magnitude of this improvement for some variants—such as R333Q and I397T, which account for 5.7% of people with HB—is likely therapeutically relevant.