Page 158 - 2020_09-Haematologica-web

T. Abache et al.
the potency of the traces of thrombin generated during the initiation of coagulation. These are a short interfering (si)RNA inhibiting the expression of antithrombin and several monoclonal antibodies targeting the tissue factor pathway inhibitor.9,10 Downmodulation of the anticoagu- lant system (activated protein C or protein S) also enters this category.11,12 Another NRF aims to substitute the func- tion of FVIII. A bispecific antibody called emicizumab, which facilitates the interaction of endogenous FIX/FIXa with factor X (FX), demonstrated its potency in this set- ting.13-15 These NRF are pharmacological agents with a mechanism of action that is independent of the fate of FVIII or VWF, thus offering drugs with a longer half-life, for the patients’ comfort, while restoring a partial but clin- ically sufficient coagulation.
A third, proposed NRF strategy is to redirect the acti- vation of FX.16 FX is at the crossroads of the intrinsic and extrinsic coagulation pathways and is responsible for the activation of prothrombin to thrombin. Rendering FX activatable to thrombin allows the thrombin that appears during the initiation of coagulation to generate larger amounts of FXa. These supplemental amounts will be enough to bypass the need for FVIIIa, the natural amplifier of coagulation. Such a modified FX was demonstrated in vitro to correct FVIII-deficient plasma.16 In this study, a second generation of recombinant throm- bin-activatable FX (actiten) was created, in which, notably, the activation peptide was preserved in order to maintain FX pharmacokinetics in vivo.17 The ability of this molecule to correct several coagulation factor deficien- cies was assessed in vitro and in a rabbit antibody- induced model of hemophilia A.
Methods
Material
The materials used in this study are listed in the Online Supplement.
Recombinant protein preparation
The generation and production of actiten and recombinant FX constructs are explained in detail in the Online Supplement.
Purification using an anti-Gla aptamer
Re-calcified supernatant was loaded on an anti-Gla aptamer col- umn containing the proprietary Mapt1.2CSO aptamer (Eurogentec, Liège, Belgium). The equilibration buffer was 50 mM Tris-HCl, 10 mM CaCl2, pH 7.5. After a wash with the equilibra- tion buffer + 300 mM NaCl, the bound compounds were eluted with 50 mM Tris-HCl, 10 mM EDTA, pH 7.5. Fractions correspon- ding to the protein peak were pooled. Eventual traces of FXa were inhibited by 10 μM GGACK and the eluate was concentrated with a 50 kDa tangential flow filtration system then dialyzed in NaCl 0.9% and stored at -80°C. The absence of FXa was con- trolled by incubating the actiten preparation (100 nM) with FXa substrate (pNAPEP 1065) for 30 min at 37°C.
Antigen dosage, electrophoresis and immunoblotting
An enzyme-linked immunosorbent assay (ELISA) for FX (Zymutest Factor X, Hyphen, France) was performed according to the manufacturer’s instructions.
For sodium dodecylsulfate polyacrylamide gel electrophoresis, proteins were deposited on Gel NuPAGE Novex Bis-Tris 4-12% and migrated at 200 V. The gel was stained with PageBlue Protein
Staining Solution or transferred onto a polyvinylidene fluoride membrane and revealed by anti-Gla or anti-FX antibody.
Binding to phospholipids
FX binding was evaluated by ELISA on phospholipid dry-coated plates. Details of the protocol are provided in the Online Supplement.
Activation of actiten
All activation reactions of actiten were conducted in Hepes buffer and were stopped in EDTA-containing buffers before the amount of FXa generated was measured following the degrada- tion of pNAPEP 1065. A standard curve of human FXa was used as the reference. The conditions for each activation are described in detail in the Online Supplement.
In vitro evaluation of the thrombotic potential of actiten
FX-deficient plasma was re-calcified to a final concentration of 7.5 mM calcium. Plasma-derived FX (pdFX), pdFX + plasma- derived activated FX (pdFXa) or actiten was incubated at 37°C until clotting of the mixture. A similar experiment was conducted in FVIII-deficient plasma spiked with FVIII, pdFXa or actiten.
Thrombin generation assay
The performance of the thrombin generation assay was based on the Calibrated Automated Thrombogram method developed by Hemker et al.18 The values of the assay are the means of dupli- cates of two to five independent experiments. Normal plasma val- ues were from 13 independent assays. Values for actiten (20 μg/mL) are from at least three independent experiments.
In vivo evaluation of actiten
In vivo studies were conducted in accordance with procedures
approved by the Institutional Animal Care and Use Committee (CEEA26: A16_004). The rabbit model was rendered hemophiliac following infusion of anti-FVIII and the bleeding assay is described in the Online Supplement.
Statistical analysis
In vitro data are presented as means ± standard deviation. In vivo data were analyzed using Prism 5 software (GraphPad Software Inc.) and are represented as means ± standard error of mean. In addition, all statistical comparisons were carried out using the nonparametric two-tailed Mann-Whitney test. Statistically signif- icant differences are indicated in the figures: *P<0.05 and **P<0.01.
Results
Expression of actiten, a thrombin-sensitive factor X
A FX sensitive to thrombin was generated by inserting a 10-amino acid polypeptide between the carboxyl termi- nus of the activation peptide and the amino terminus of the heavy chain. The polypeptide was composed of six amino acids of fibrinopeptide A and four amino acids forming a thrombin cleavage site. The resulting molecule was called actiten (Figure 1). It was expressed in the HEK293F cell line and purified using a proprietary aptamer column recognizing the g-carboxylated (Gla) domain of the coagulation factor. The molecule was immunodetect- ed by anti-Gla monoclonal and polyclonal anti-human FX antibodies (Figure 2). In native conditions, purified actiten appeared as a unique band at 64-66 kDa. Under reducing conditions, the anti-Gla antibody revealed the FX light
2336
haematologica | 2020; 105(9)