Introduction: Coagulation Factor VIII (FVIII) mimetics is a new class of molecules restoring the hemostatic capacity in blood lacking FVIII irrespective of the presence of inhibitory anti-FVIII antibodies. FVIII mimetics are bispecific antibodies bridging FIXa and FX on platelet surface, enhancing FX activation and thereby coagulation. Despite a significant reported prophylactic effect of the FVIII-mimetic emicizumab (Hemlibra®) in persons with Hemophilia A with and without inhibitors [Oldenburg et al., NEJM, 2017; Mahlangu et al., NEJM, 2018], bleeding may still occur during treatment, requiring intervention with FVIII or bypassing agents, i.e. recombinant FVIIa (rFVIIa) or activated prothrombin complex concentrate (aPCC). The combined treatment with emicizumab and aPCC has under certain conditions led to thrombotic events and microangiopathies in the treated patients, whereas the combination with rFVIIa has been safe [Levy et al., J Thromb Haemost, 2019]. We have developed a novel, next-generation FVIII mimetic antibody, Mim8, that potently enhances coagulation in pre-clinical models [manuscript in preparation]. We evaluated the combination of Mim8 with recombinant FVIII (rFVIII), rFVIIa, and aPCC by thrombin generation assay (TGA) and by thromboelastography (TEG), in haemophilia A condition.
Material and methods: Mim8 was tested in a range including the expected therapeutic concentration (10 - 50 nM, based on pre-clinical data) and studied in parallel with a sequence identical analogue (SIA) of emicizumab (concentrations including the therapeutic range of ⁓350 nM [Oldenburg et al., NEJM, 2017]). The compounds were tested in combinations with rFVIII, rFVIIa or aPCC. For TGA, coagulation was initiated with addition of FXIa or tissue factor (TF), and peak thrombin is reported. For TEG, haemophilia A was induced in whole blood from healthy volunteers by adding an anti-FVIII polyclonal antibody, and clot formation initiated by TF. The clot time (R-time) and clot development (alpha-angle) are reported. Under the chosen experimental conditions, TEG is particularly useful for studying the interaction between Mim8 and rFVIIa as clinically relevant doses of rFVIIa (i.e. 25 and 75 nM) can normalize haemophilic blood in TEG, while a very poor response is observed in TGA.
Results: In TGA, Mim8 alone induced a concentration-dependent increase in thrombin generation with both triggers (TF or FXIa). In combination with rFVIII, Mim8 generated thrombin levels within normal range and did not inhibit the effect of 1 IU/mL rFVIII. The combined addition of aPCC 1 IU/mL and Mim8 generated TF-triggered thrombin peaks up to thirteen-fold higher than in normal plasma, suggesting synergistic effect between Mim8 and aPCC. Similar data were obtained when aPCC and emicizumab SIA were combined. When triggered by FXIa, the thrombin peaks generated by the combination of aPCC 1 IU/mL and Mim8 were also above normal plasma values. In contrast, the combination of rFVIIa (up to 75 nM) and Mim8 (up to 50 nM) showed an additive effect in TF-triggered assays i.e. the thrombin peak did not increase more than the individual contribution from each molecule. In TEG, Mim8 alone normalized clot time of haemophilia A blood. When rFVIIa (up to 75 nM) was combined with Mim8, clot time stayed within normal range. In addition, clot time induced by up to 50 nM of Mim8 in combination with 75 nM rFVIIa was still significantly longer than emicizumab SIA 350 nM + 1 IU/mL aPCC (P < 0.01). Also, the combination of rFVIIa and Mim8 at any concentrations tested only increased clot development, or alpha-angle, up to normal blood levels. In contrast, the addition of only 0.25 IU/mL aPCC combined with 5 nM of Mim8, or only 0.1 IU/mL aPCC combined with 50 nM Mim8, decreased the clot time below normal value.
Conclusions: Using TGA and TEG, Mim8, at the anticipated clinical concentrations, did not interfere with the effect of rFVIII, i.e. only an additive effect of Mim8 and rFVIII was observed. Similarly, rFVIIa had an additive effect with Mim8. However, as expected, aPCC demonstrated a synergistic effect when combined with Mim8. Clot formation parameters and thrombin peak exceeded normal levels, even at a reduced concentration of aPCC. The data obtained with Mim8 and emicizumab SIA were comparable, suggesting that the synergistic effect with aPCC is a class effect.
Burnier:Novo Nordisk A/S: Employment. Hermit:Novo Nordisk A/S: Employment, Equity Ownership, Patents & Royalties: Patents. Hilden:Novo Nordisk A/S: Employment, Equity Ownership, Patents & Royalties. Ezban:Novo Nordisk A/S: Employment. Kjellev:Novo Nordisk A/S: Employment, Equity Ownership.
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