Emicizumab, A Bispecific Antibody to Factors IX/IXa and X/Xa, Does Not Interfere with Antithrombin or TFPI Activity In Vitro

Abstract

Emicizumab is a humanized bispecific antibody that binds simultaneously to factor (F) IXa and FX replacing the cofactor function of FVIIIa. Because emicizumab recognizes FIX/FIXa and FX/FXa, a question may arise whether emicizumab competes with antithrombin (AT) and/or tissue factor pathway inhibitor (TFPI), thereby enhancing overall hemostatic potential by blocking their antihemostatic effects. To address this question, we performed enzymatic assays using purified coagulation factors to confirm whether emicizumab interferes with the action of AT on FIXa or FXa, or with the action of TFPI on FXa. In those assays, we found no interference of emicizumab on the actions of AT and TFPI. We next assessed emicizumab's influences on the anticoagulation actions of AT or TFPI in thrombin generation assays triggered with FXIa or tissue factor (TF) in AT-depleted or TFPI-depleted plasma supplemented with AT or TFPI in vitro. In those assays, we employed anti-FIXa and anti-FX monospecific one-armed antibodies derived from emicizumab instead of emicizumab itself so as to prevent emicizumab's FVIIIa cofactor activity from boosting thrombin generation. Consequently, we found that neither anti-FIXa, anti-FX monospecific antibody, nor the mixture of the two interfered with the anticoagulation actions of AT or TFPI in plasma. Although emicizumab can bind to FIXa and FXa, our results showed no interference of emicizumab with the action of AT or TFPI on FIXa or FXa. This indicates that the presence of emicizumab is irrelevant to the action of AT and TFPI, and thus should not alter the coagulant/anticoagulant balance related to AT and TFPI.

Keywords

emicizumab - antithrombin - TFPI - coagulation factors - hemophilia A

Authors' Contributions

H.K., T.K., M.N-S., T.S., A.U., and A.M. designed and performed the in vitro studies. M.H., H.K., T.K., K.F-O., Y.K., K.N., and M.S. contributed to the direction of this research and interpretation of the data. M.N-S. wrote this manuscript.

• References

• 1 Kitazawa T, Igawa T, Sampei Z. , et al. A bispecific antibody to factors IXa and X restores factor VIII hemostatic activity in a hemophilia A model. Nat Med 2012; 18 (10) 1570-1574
• 2 Sampei Z, Igawa T, Soeda T. , et al. Identification and multidimensional optimization of an asymmetric bispecific IgG antibody mimicking the function of factor VIII cofactor activity. PLoS One 2013; 8 (02) e57479
• 3 Muto A, Yoshihashi K, Takeda M. , et al. Anti-factor IXa/X bispecific antibody (ACE910): hemostatic potency against ongoing bleeds in a hemophilia A model and the possibility of routine supplementation. J Thromb Haemost 2014; 12 (02) 206-213
• 4 Muto A, Yoshihashi K, Takeda M. , et al. Anti-factor IXa/X bispecific antibody ACE910 prevents joint bleeds in a long-term primate model of acquired hemophilia A. Blood 2014; 124 (20) 3165-3171
• 5 Shima M, Hanabusa H, Taki M. , et al. Factor VIII-mimetic function of humanized bispecific antibody in hemophilia A. N Engl J Med 2016; 374 (21) 2044-2053
• 6 Oldenburg J, Mahlangu JN, Kim B. , et al. Emicizumab prophylaxis in hemophilia A with inhibitors. N Engl J Med 2017; 377 (22) 2194-2195
• 7 Pasi KJ, Rangarajan S, Georgiev P. , et al. Targeting of antithrombin in hemophilia A or B with RNAi therapy. N Engl J Med 2017; 377 (09) 819-828
• 8 Chowdary P, Lethagen S, Friedrich U. , et al. Safety and pharmacokinetics of anti-TFPI antibody (concizumab) in healthy volunteers and patients with hemophilia: a randomized first human dose trial. J Thromb Haemost 2015; 13 (05) 743-754
• 9 Hilden I, Lauritzen B, Sørensen BB. , et al. Hemostatic effect of a monoclonal antibody mAb 2021 blocking the interaction between FXa and TFPI in a rabbit hemophilia model. Blood 2012; 119 (24) 5871-5878
• 10 Monahan PE. Emerging genetic and pharmacologic therapies for controlling hemostasis: beyond recombinant clotting factors. Hematology (Am Soc Hematol Educ Program) 2015; 2015: 33-40
• 11 Patnaik MM, Moll S. Inherited antithrombin deficiency: a review. Haemophilia 2008; 14 (06) 1229-1239
• 12 Girard TJ, Warren LA, Novotny WF. , et al. Functional significance of the Kunitz-type inhibitory domains of lipoprotein-associated coagulation inhibitor. Nature 1989; 338 (6215): 518-520
• 13 Kitazawa T, Esaki K, Tachibana T. , et al. Factor VIIIa-mimetic cofactor activity of a bispecific antibody to factors IX/IXa and X/Xa, emicizumab, depends on its ability to bridge the antigens. Thromb Haemost 2017; 117 (07) 1348-1357
• 14 Uchida N, Sambe T, Yoneyama K. , et al. A first-in-human phase 1 study of ACE910, a novel factor VIII-mimetic bispecific antibody, in healthy subjects. Blood 2016; 127 (13) 1633-1641
• 15 Okuda M, Yamamoto Y. Usefulness of synthetic phospholipid in measurement of activated partial thromboplastin time: a new preparation procedure to reduce batch difference. Clin Lab Haematol 2004; 26 (03) 215-223
• 16 Coagulation Sequence and Structure Database (CoagBase). Available at: http://www.isth.org/?page=CaogBase . Accessed June 16, 2017
• 17 Girard TJ, Broze Jr GJ. Tissue factor pathway inhibitor. Methods Enzymol 1993; 222: 195-209
• 18 Yada K, Nogami K, Matsumoto T. , et al. Activated protein C–catalyzed factor Va inactivation predominantly contributes to the downregulation of coagulation rather than factor VIIIa inactivation. Blood 2014; 124 (21) 4222