Gla-domainless Factor Xa as a new treatment of hemorrhages – MINITEN

Prior to the MINITEN project, a factor X gla domain truncated (GD-FXa) has been generated and demonstrated its ability to efficiently restore coagulation in hemophilia A plasmas with inhibitors.
The aim of the MINITEN 36-months project was to improve its production level, establish a purification process and characterize its potential as an anti-hemophilic agent by inhibiting Tissue Factor Plasma Inhibitor (TFPI) and as an antidote to direct oral anticoagulant (DOA). Several modifications were performed to reinforce the interaction of the molecule with TFPI or with novel oral anticoagulants as well as its stability in vivo. In addition, the docking of GPAD-FXa with the anti-Xa was performed using computational techniques.
To face with a GPAD-FXa molecule that displays a short half-life in plasma, fusing GPAD-FXa with molecules that were demonstrated to increase the circulating protein half-life such as Fc fragment from IgG as well as point mutations are proposed. The immunogenicity of the molecule will also be evaluated by in silico and in vitro available techniques.
Pure and active TFPI is required to test the enzymatic properties of GPAD-FXa and to rationalize the interest of the mutations introduced. The production of this product was assayed in different production systems.A functionnal TFPI was obtained and is now ready to be evaluated in the presence of Miniten.

A lead molecule, GPAD2opt-FXa (GFXA), is produced in HEK Free Style without serum following a stable a transfection. It possesses a sequence optimized for human expression, a synthetic MB7signal peptide, an additional furin cleavage site between the heavy and the light chain. The molecule is produced at a workable amount. The amounts produced allowed to perform in vivo assays.
A two steps purification process (heparin column + benzamidine column) provide a material sufficiently pure to envisage its characterization. The resulting product is sensitive to degradation. The addition of anti-proteases compounds when material is collected is mandatory.
Following purification, the molecule kept up to 60 % of the FXa chromogenic activity. The molecule was able to restore coagulation in a dose dependent-manner on FVIII, FIX but not FX depleted plasmas. GFXA is also functional when inhibitors (anti-FVIII) are present.
GFXA was assayed in vitro on plasma incubated with therapeutic doses of rivaroxaban and fondaparinux and corrected both plasmas in a dose dependent manner.
These data indicate that GFXA demonstrated in vitro its expected potential in the two indications targeted.
A series of DOAC were docked in the active site of the enzyme: rivaroxaban, apixaban and razaxaban. The docking scores were very good and similar. The theoretical binding affinities remained similar for all DOA. Increasing the binding affinity of DOA was thus not possible to envisage, regarding the very good and similar docking scores obtained. To get more reliable insight into the role of each residue in the binding affinities of the DOA, we performed molecular dynamics simulations of the complexes between rivaroxaban and respectively the wild-type and the S195 enzymes. This identifies the interaction hot spots between the enzyme and the inhibitor. Two residues showing non-favorable interaction energies were proposed for mutation to obtain enzymes with enhanced affinities for DOACs.

The lead molecule GFXA has now to be studied in vivo in the two targeted indications. Hemophilia A and B mice colonies are available at LFB and will be used in the anti-hemophilic indication. A model evaluating the blood loss is currently used. To evaluate the DOA antidote potential, a wild-type mouse model under rivaroxaban was preliminary used. However, further tried are required to improve the robustness of the model.
Several targeted mutations were made to improve the interaction with TFPI, with DOA and to improve the resistance of the molecule. Mutated molecules need to be produced and characterized.
The interaction between GFXA and TFPI is to be evaluated. This will serve as a basis for the mutated molecule studied.
Long-lasting molecules were generated and have also to be produced and analyzed.

1-9th International PPB Meeting-Laguna di Chia-Italy. May 11-15th. Oral Communication. J-L Plantier et al. “MINITEN, a recombinant truncated activated factor X with dual function”
2-XXVth ISTH Congress, Toronto, Canada. Poster. June 20-25th. J-L Plantier et al. ”GENERATION OF A RECOMBINANT TRUNCATED ACTIVATED FACTOR X AS ANTI-HEMOPHILIC AGENT”
3- XXVth ISTH Congress, Toronto, Canada. Poster. June 20-25th. M Castellan et al. ”Production of highly active recombinant human tissue factor pathway inhibitor”
4-Patent: Facteur X dépourvu de domaine gla. N°FR2014/051542. PCT déposé le 20 juin 2014.

Hemophilia is a bleeding disorder due to the deficiency in coagulation factors VIII (hemophilia A) or IX (hemophilia B). The therapeutic strategies developed to date rely on the replacement of the missing or dysfunctional FVIII or FIX.
The development of antibodies, called inhibitors, inhibiting the activity of the therapeutic factor is the most serious and costly complication. Inhibitors formation is observed in 30% of severe hemophilia A patients and 3% of hemophilia B patients; anti-factor VIII antibodies may also appear as an autoimmune acquired hemophilia. The first treatment to eradicate inhibitors is the immune tolerance induction. For acute bleeding episodes in patients with acquired hemophilia or with inhibitors that cannot be tolerized, bypassing agents (FEIBA: activated prothrombin complex, or NovoSeven, (rFVIIa)) are used. However, a substantial number of patients do not respond to these agents, with still some cases of fatal issues. Therefore, limitations in the treatment of hemophilia with inhibitors and the substantiality of the market highlight the opportunities to develop alternative therapeutic strategies to address the unmet needs, both in terms of safety and efficacy.

The anticoagulant market has recently evolved with novel oral anticoagulants (NOA) replacing vitamin K antagonists or heparins. These chemical molecules possess a high specificity and affinity, targeting activated factor X (Rivaroxaban) or thrombin (Dabigatran; Argatroban). There is a consensus emerging to preferentially target FXa as it amplifies the hemostatic response. However, these molecules are used without any antidote and dramatic issues have been reported.

Our aim is to provide an innovative approach for the treatment of bleeding episodes of hemophilia particularly those with inhibitors and under NOA treatments. This approach, called MiniTen, is based on the use of a truncated form of FXa acting as molecular bait for Tissue Factor Pathway Inhibitor (TFPI) and for anti-Xa molecules. TFPI is a rational target in hemophilia since it controls directly the initiation complex of coagulation. Several experimental evidences showed that ex vivo and in vivo blocking TFPI restores coagulation in the absence of FVIII or FIX. A competitive advantage of this truncated factor Xa is a lack of procoagulant activity and therefore of thrombotic risks.

This application aims to strengthen a research project already developed in close collaboration between an academic laboratory TIMC-IMAG Team TheREx (UMR5525-Grenoble) and LFB Biotechnologies a company with an historical expertise in coagulation products.
TheREx team has developed, patented and published a gamma carboxyglutamic-domainless-factor Xa (GD-FXa) that is able to be used in vitro as an anti-hemophilic treatment. In addition, TheREx team has identified and protected a series of mutations that could improve its binding to TFPI and its efficiency. Expression vectors coding for recombinant wild-type and mutated GD-FXa were constructed, and the industrial partner has demonstrated that the patented mutants were more effective to restore thrombin generation in hemophilia plasmas. Among the mutated forms, a modified molecule (GPAD-FXa) was homogeneously produced and was able to restore coagulation at doses corresponding to 5% of circulating FX: it was identified as a lead compound.

The project is thus mature for programmed transfer from the academic laboratory to the industrial partner to produce the candidate drug and start animal experiments that are prerequisites to phase I/II in human. During the project, the academic and industrial partners will continue to improve the molecule, both in terms of quantities and quality. Moreover, during the course of the project, breakthrough insights into the real nature of blood coagulation are expected, especially while determining the nanostructure of the fibrin clot in the presence of the new medicament.