Evaluation of the combined effects of emicizumab and marstacimab in plasma from Hemophilia A patients using thrombin generation assay

Abstract Background: Patients with Hemophilia A (HA) may be treated with prophylactic emicizumab therapy, a bispecific monoclonal antibody that promotes coagulation by bridging factor IX (FIX) and factor X (FX). Marstacimab has been recently approved for the prevention of bleeding events in patients with hemophilia A and B. Marstacimab is a human monoclonal antibody targeting the K2 domain of TFPI to improve thrombin generation and restore hemostasis. As both agents are non-replacement therapies with distinct mechanisms of action and half-life, transitioning between them raises questions about the potential risk of excess thrombin generation (TG) and safety. Objective: To test the potential additive effect of marstacimab on thrombin generation ex vivo using plasma collected from a cohort of eight severe hemophilia A (HA) patients receiving emicizumab as part of their standard prophylactic treatment regimen. Methods: Platelet poor plasma was obtained from adult male patients (age 20-54) with severe HA on stable prophylaxis with emicizumab for at least 6 months and did not receive any additional hemostatic therapy in the previous 4 weeks. Samples were spiked with various concentrations of marstacimab (4, 16, 80 μg/mL). Additionally, to mimic the effect of addition of FVIII products as in the case of the treatment of a breakthrough bleed, we spiked the plasma with FVIII (0.4 IU/mL). Tissue factor (Recombiplastin 2G, HemosIL) was used as trigger, and the assay was allowed to run for 90 minutes, with fluorescence readings occurring at 1-minute intervals throughout. TG was performed using a SpectraMax M5 plate reader with SoftMax Pro v.7 software (Molecular Devices). TG parameters (peak thrombin, lag time, and endogenous thrombin potential [ETP]) were determined analyzing data using the Technothrombin TGA Evaluation Software (Technoclone). Commercial normal pooled plasma (NPP) and FVIII depleted plasma (F8DP), plasma from healthy donors (CTRL), and HA plasma with residual <1% FVIII activity spiked with 0.2 IU/mL and 0.8 IU/mL were used as controls. Results: In plasma from healthy donors, ETP, peak thrombin and lag time values ranged from 2,552 to 2,872 nM*min, 124 to 223 nM, and 11-13 minutes respectively. There was no significant thrombin generation detected in severe HA plasma used as a control. When F8DP was supplemented with 0.8 IU/mL mean (range) values for ETP, peak thrombin and lag time were 3,059 nM*min (2603-3302 nM*min), 157.4 nM (122.6-212.5 nM), and 13.2 minutes (12-14 minutes), respectively. At baseline, plasma from patients receiving emicizumab showed a mean ETP of 3,177.3 (range: 2212-3778 nM*min), mean peak thrombin of 94 nM (range: 49.8-109.4 nM) and mean lag time of 13.3 minutes (range: 12-15.5 minutes). Addition of marstacimab to plasmas of patients on treatment with emicizumab resulted in a significant improvement in all the parameters tested. When spiked with16 μg/mL of marstacimab (expected average steady state marstacimab concentration in patients), mean ETP and peak thrombin were higher than baseline and closer to the range of values obtained in healthy controls and NPP. The lag time was shortened and similar to controls and NPP. At the highest concentration tested (marstacimab 80 μg/mL) mean ETP was 3,503.4 nM*min (range: 3212-3987 nM*min), mean peak thrombin was 189.8 nM (range: 175.2-208.2 nM) and mean lag time was reduced to 9 minutes (range: 8-11 minutes). Additionally, patient plasma samples spiked with both marstacimab 80 μg/mL and FVIII 0.4 IU/mL showed no significant changes in the mean ETP, (3,460 nM*min, range: 2688-3902 nM*min), a modest increase in mean peak thrombin (202.4 nM; range:181.2-241 nM), and similar mean lag time (9.9 minutes; range: 9-12 minutes) compared to the spike with Marstacimab 80 μg/mL only. Conclusion: Our data show that addition of marstacimab to plasma from patients on prophylaxis with emicizumab resulted in a dose-dependent increase in peak thrombin generation and ETP, while also reducing lag time. These changes consistently remained mostly within the range observed in healthy donors. Overall, no evidence of excessive thrombin generation was detected, even at the highest concentrations of marstacimab tested. While the clinical safety of switching between emicizumab and marstacimab remains to be established in future clinical studies, these findings provide supportive evidence for absence of excessive thrombin generation.