Activated Protein C-Catalyzed Inactivation Pathway Down-Regulates NXT007-Mediated Hemostatic Function

Activated protein C (APC) together with protein S (PS) inactivates factor (F)Va and FVIIIa and efficient FVIIIa inactivation requires not only PS but also cofactor function of FV. Any impairment in these inactivation pathways is one of the most common risks of venous thrombosis. NXT007, emicizumab (Emi)-based engineered therapeutic bispecific antibody (Ab), is currently undergoing the phase 1 clinical trial. NXT007 increased coagulation potential of FVIII-deficient plasma up to the similar level to healthy controls in tissue factor (TF)-triggered thrombin generation (TG; Yamaguchi, ASH 2020). Additionally, concomitant use of NXT007 and bypassing agents could be useful for improving coagulation potentials in FVIII-deficient samples (Ogiwara, ASH 2021). However, the role of APC-dependent inactivation in NXT007-mediated hemostasis reactions remains to be investigated. Here, we examined the regulatory effects by APC in NXT007-driven hemostasis using a purified FXa generation assay and TF/ellagic acid-triggered thrombin generation assay (TGA).

We firstly investigated coagulation potentials in FVIII-deficient plasma (ΔFVIII) spiked with NXT007 (3-100 μg/mL) or Emi (50 μg/mL). Peak thrombin (PeakTh) in ΔFVIII spiked with NXT007 was increased in a NXT007 dose-dependent manner. ΔFVIII spiked with NXT007 (3 μg/mL) showed higher PeakTh (355±74 nM) than emicizumab (50 μg/mL; 274±48 nM). PeakTh in ΔFVIII after the addition of NXT007 (10, 30, and 100 μg/mL) were 419±12, 454±13, and 484±10 nM, respectively. The coagulant effects of NXT007 (30 and 100 μg/mL) were comparable to pooled normal plasma (PNP). To evaluate the impact of APC-mediated down regulation on the hemostatic enhancement by NXT007 in ΔFVIII, TGA in ΔFVIII reacted with NXT007 (10 μg/mL) in the presence of APC (0-16 nM) was examined. PeakTh was decreased in an APC-dose dependent manner. Comparing with PeakTh without and with APC (16 nM), PeakTh was reduced from 457±11 nM to 331±1.4 nM and the %inhibition was 28%. We next evaluated the effects of APC on TG in PNP with or without an anti-FVIII polyclonal Ab (FVIIIAb) (10 BU/mL). The %inhibition of PeakTh in PNP with APC (16 nM) was 30%, indicating that the effect of APC-mediated inactivation was not disturbed by the presence of NXT007. To clarify the association of NXT007 alone with APC-catalyzed inactivation, we examined a purified FXa generation with NXT007 (10 μg/mL) or with FVIIIa (2 nM) after the addition of APC (0-2 nM) and PS (5 nM). FXa generation with FVIIIa was reduced by ~60% at maximum level in an APC dose-dependent manner, whereas that with NXT007 was not impaired, indicating that NXT007 itself was not affected by APC-catalyzed inactivation.

To elucidate the contribution of APC-catalyzed inactivation of FV(a) in NXT007-catalyzed hemostatic regulation, we evaluated the effect of APC on the TG with NXT007 (10 μg/mL) and FV (7.5-30 nM; corresponded to 25-100% of physiological concentration) in the FV-deficient plasma with FVIIIAb (10 BU/mL). The PeakTh in the presence of NXT007 and FV (7.5-30 nM) without APC was 310-340 nM and the %inhibition of PeakTh by the addition of APC (16 nM) was ~35%, showing that NXT007 showed sufficient coagulation potential even at low concentration of FV and that the inhibition of TG by APC-catalyzed FV(a) inactivation was not disturbed by the presence of NXT007 even at FV (7.5 nM). In addition, the influence on TG with APC in the FV-Leiden (FV-L) plasma without or with FVIIIAb and NXT007 (10 μg/mL) were investigated. The %inhibition of PeakTh by APC (16 nM) in FV-L plasma was ~20%. While, the %inhibition of PeakTh by APC (16 nM) in FV-L plasma with FVIIIAb and NXT007 was similar to FV-L plasma alone (~20%), indicating that APC could regulate NXT007-catalyzed hemostatic reaction by inactivation of FV(a) under the FV-L condition. We conclude that APC-catalyzed inactivation mechanisms could play a significant role on the down-regulation of coagulation in NXT007-mediated hemostasis.

Nakajima:Takeda Pharmaceutical company: Research Funding. Ogiwara:Chugai Pharmaceutical Co., Ltd.: Research Funding. Kitazawa:Chugai Pharmaceutical Co., Ltd.: Current Employment. Shima:F. Hoffmann-La Roche Ltd.: Speakers Bureau; CSL Behring: Honoraria, Research Funding, Speakers Bureau; Chugai Pharmaceutical Co., Ltd.: Consultancy, Honoraria, Research Funding, Speakers Bureau; Takeda: Honoraria, Research Funding; Ono Yakuhin: Speakers Bureau; Nara Medical University: Current Employment; UniQure: Consultancy; Fujimoto Seiyaku: Consultancy, Honoraria, Speakers Bureau; Sanofi: Consultancy, Honoraria, Speakers Bureau; Bayer Yakuhin: Honoraria; Novo Nordisk: Honoraria, Speakers Bureau. Nogami:Chugai: Honoraria, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Speakers Bureau; Chugai, Sanofi, Takeda, Bayer, Novo Nordisk, CSL Behring, KMBio, Fujimoto Seiyaku, Sekisu Medical, Sysmex,: Honoraria; Chugai, Sanofi, Takeda, Bayer, Novo Nordisk, KMBio, CSL Behring, Fujimoto Seiyaku, Sekisui Medical, Sysmex, AsahiKasei: Research Funding.