ALBERT

Description: Factor VIII functions as a cofactor for factor IXa in the anionic phospholipid surface-dependent conversion of factor X to Xa. It is well-known that the A2 and A3 domains of factor VIII interact with the catalytic domain and EGF2 domain of factor IXa, respectively. Recently, Furie et al. have reported that the Gla domain of factor IXa (factor IXa-GD) interacts with the light chain of factor VIII. However, the factor IXa-GD-interactive site on the light chain remained to be investigated. In the current study, the recombinant C2 (rC2) domain of factor VIII was prepared using a yeast secretion system. ELISA-based assay in the absence of phospholipid showed the Glu-Gly-Arg-active site modified factor IXa (EGR-factor IXa) bound to the immobilized rC2 domain dose-dependently, and the binding ability was maximum under the condition of 150 mM NaCl/1 mM CaCl2. This binding was competitively inhibited by the addition of excess of factor VIII or rC2 domain, supporting the specificity of this interaction. Furthermore, the presence of high ionic strength and the metal-ion chelator EDTA blocked this binding by ∼95 and ∼75%, respectively. Surface plasmon resonance-based assay showed that the binding affinity (Kd) of rC2 domain for EGR-factor IXa was 108 ± 15.5 nM. GD less-factor IXa, deleting the GD completely, failed to bind to rC2 domain. A monoclonal antibody against factor IXa-GD specific for calcium-dependent conformation (mAbIXa-GD) also inhibited (∼ 95%) the rC2 domain binding to EGR-factor IXa in a dose-dependent manner (IC50; 758 nM), suggesting the authentic of the C2 domain and factor IXa-GD interaction. The addition of rC2 domain or mAbIXa-GD inhibited the factor IXa-catalyzed factor X activation with factor VIIIa in the absence of phospholipid (IC50; 15.7 μM or 43.2 nM, respectively), whilst both any little affected in the absence of factor VIIIa. In addition, the ∼8-kDa C2 fragment obtained by V8 protease digestion (residues 2182–2259) bound directly to EGR-factor IXa. Taken together, these results indicate that factor VIII C2 domain directly interacts with factor IXa-GD via both the electrostatic- and calcium-dependent interactions. Furthermore, our results provide the first evidence for an essential role of the C2 domain in the association between factor VIII and factor IXa in the factor Xase complex.

Description: Factor VIIa (FVIIa), complexed with tissue factor (TF), is a trigger of blood coagulation through activation of factor X in the initiation phase. FVIIa can catalyze intrinsic clotting factors such as not only factor IX, but also factor VIII (FVIII). However the role and the mechanisms of the FVIIa-catalyzed FVIII are poorly understood. We first examined FVIIa-catalyzed FVIII activation in the presence of phospholipid (PL) using a one-stage clotting assay. The levels of FVIII activity elevated rapidly by ~4-fold within 30 sec after the addition of FVIIa (1 nM)-TF (1 nM)complex, and subsequently decreased to the initial level within 20 min. This time-dependent reaction was enhanced by the presence of TF and PL in dose-dependent manners, but was moderately inhibited (~50%) in the presence of von Willebrand factor at physiological concentration of 10 μg/mL. FVIII cleavage was evaluated using western blotting immediately after the addition of FVIIa-TF complex. The heavy chain of FVIII was proteolyzed more rapidly (at 15 sec) by cleavages at Arg740 (A2-B junction) and Arg372 (A1-A2 junction) by FVIIa-TF complex, whilst the cleavage at Arg336 in the A1 domain was appeared at ~2.5 min. However little cleavage of the light chain of FVIII was observed, supporting that cleavages at Arg740/Arg372 and Arg336 by FVIIa-TF complex contribute to the up- and down-regulation of FVIII(a) activity, respectively. Of interest, no proteolysis of isolated intact heavy chain was observed, indicating that the proteolysis of the heavy chain was governed by the presence of the light chain. Compared to FVIII activation by thrombin (0.1–1 nM), the activation by FVIIa (0.1–1 nM)-TF (1 nM) complex was observed more rapidly. The activation rate observed by the addition of FVIIa-TF complex was ~50-fold greater than that by thrombin. Kinetics by the chromogenic Xa generation assay showed the catalytic efficiency (kcat/Km; 8.9 min−1/32.8 nM) on FVIIa-TF complex-catalyzed FVIII activation showed ~4-fold greater than that on thrombin-catalyzed activation (kcat/Km; 7.5 min−1/86.4 nM). Furthermore, the catalytic efficiencies on cleavages at Arg740 and Arg372 of FVIII by FVIIa-TF complex were ~3- and ~20-fold greater compared to those by thrombin, respectively. These findings suggested that FVIIa-TF complex was a greater FVIII activator than thrombin in very early phase. In order to localize the binding region for FVIIa, we evaluated the interactions between FVIII subunit and Glu-Gly-Arg-active site modified FVIIa, lacking enzymatic activity, in a surface plasmon resonance-based assay. The heavy chain of FVIII bound to EGR-FVIIa with higher affinity than the light chain (Kd; 2.1 and 45 nM, respectively). Binding was particularly evident with the A2, A3, and C2 domains (Kd; 34, 37, and 44 nM, respectively), whilst the A1 domain failed to bind. In conclusion, we demonstrated that FVIIa-TF complex rapidly activated FVIII by proteolysis of the heavy chain and the activation was governed by the presence of the light chain. Furthermore, present results suggested the role of TF-dependent FVIII activation by FVIIa which is responsible for the initiation phase of blood coagulation.

Description: Background Emicizumab (HEMLIBRA®) is a factor (F) VIII function-mimetic therapeutic bispecific antibody (BsAb) to FIXa and FX able to prevent bleeding in persons with hemophilia A (PwHA) when injected subcutaneously once every 1, 2 or 4 weeks. To develop a next generation version, we sought an agent able to keep hemostatic potential in non-hemophilic range with more convenient dosing regimen (dosing frequency/volume). We successfully created the emicizumab-based engineered four-chain BsAbs, NXT series. Among these, we selected NXT007 as a clinical candidate. Objectives The aim of this study is to clarify the in vitro and in vivo properties of NXT007 and predict its therapeutic potency non-clinically. Methods We evaluated the pharmacological activities of NXT007 in vitro using a thrombin generation assay (TGA) with FVIII-deficient patient plasma, and in vivo by inducing bleeding in FVIII-neutralizing antibody-treated acquired hemophilia A cynomolgus monkey (cyno) model. To clarify the FVIII-cofactor activity of NXT007, we performed an enzymatic kinetics analysis of FIXa-catalyzed FX activation with and without NXT007, as well as surface plasmon resonance analysis to determine the dissociation constant (KD) of NXT007 to FIX, FIXa, FX and FXa. We obtained its pharmacokinetic (PK) profile in non-human primates in a single dose SC/IV study. Results In vitro addition of NXT007 at 30 μg/mL increased the peak height of TGA in FVIII-deficient plasma to the same levels achieved by recombinant human FVIII at 40-100 IU/dL (FXIa-triggering) or 100-150 IU/dL (tissue factor-triggering). A single bolus intravenous injection of NXT007 (0.075 mg/kg) ameliorated bleeding symptoms in the cyno model to similar as a twice daily intravenous injection of recombinant porcine FVIII (20 U/kg). The in vitro and in vivo results were roughly concordant. NXT007 increased the turnover rate (kcat) of FIXa-catalyzed FX activation by approximately 4,000-folds compared to the condition without cofactor. The impact of NXT007 on the kcat was similar to that of emicizumab. As for binding affinities, the KD values of NXT007 to FIX, FIXa, FX and FXa were 1.08, 0.728, 0.0538 and 0.0231 μM, respectively in buffer solution. Compared to emicizumab, NXT007 bound more strongly to FX/FXa and with similar affinity to FIX/FIXa. This means that NXT007 would have an ability to form more FIX-BsAb-FX ternary complex than emicizumab. Calculated using the above KD values, at 30 μg/mL of BsAb the estimated concentration of FIX-NXT007-FX ternary complex in plasma is approximately 10-fold higher than that of the FIX-emicizumab-FX ternary complex which is roughly concordant with the difference in their FVIII equivalent thrombin generation activity. Prothrombin time (PT) was not clearly prolonged suggesting minimal impact on FX function by in vitro addition of NXT007 at up to 30 μg/mL, which was enough to induce sufficient thrombin burst in FVIII-deficient plasma as described above. A half-life of NXT007 was 19.6 to 24.4 days (0.02-2 mg/kg, SC) and SC bioavailability was 84.4% (2 mg/kg) in the in vivo cyno PK study, in which no obvious change in plasma FIX or FX levels was observed after 0.02-2 mg/kg single SC administration. Conclusions Based on the nonclinical results, NXT007, delivered in every-4-week SC injections, will keep a non-hemophilic range of equivalent FVIII thrombin generation in PwHA, Compared with emicizumab, NXT007's improved cofactor activity may be attributed to its more efficient ternary complex formation while keeping turnover rate with minimal impact on FX function suggested by PT value and antigen accumulation. A phase 1/2 clinical study of NXT007 is now on-going (NXTAGE; JapicCTI-194919). Disclosures Yamaguchi: Chugai Pharmaceutical Co., Ltd: Current Employment. Soeda:Chugai Pharmaceutical Co., Ltd.: Current Employment. Sato:Chugai Pharmaceutical Co., Ltd.: Current Employment. Shibahara:Chugai Pharmaceutical Co., Ltd.: Current Employment. Koga:Chugai Pharmaceutical Co., Ltd.: Current Employment. Ichiki:Chugai Pharmaceutical Co., Ltd.: Current Employment. Joyashiki:Chugai Pharmaceutical Co., Ltd.: Current Employment. Teranishi:Chugai Pharmaceutical Co., Ltd.: Current Employment. Nishimura:Chugai Pharmaceutical Co., Ltd.: Current Employment. Shiraiwa:Chugai Pharmaceutical Co., Ltd.: Current Employment. Kitamura:Chugai Pharmaceutical Co., Ltd.: Current Employment. Igawa:Chugai Pharmaceutical Co., Ltd.: Current Employment. Konishi:Chugai Pharmaceutical Co., Ltd.: Current Employment. Kitazawa:Chugai Pharmaceutical Co., Ltd.: Current Employment.