EP3452586A1

EP3452586A1 - Factor x variants

Info

Publication number: EP3452586A1
Application number: EP17727638.3A
Authority: EP
Inventors: Abdessatar Sami Chtourou; Jean-Luc Plantier; Toufik ABACHE
Original assignee: LFB SA
Current assignee: LFB SA
Priority date: 2016-05-06
Filing date: 2017-05-05
Publication date: 2019-03-13
Also published as: CA3023337A1; CN109415713A; WO2017191424A1; KR20190003742A; FR3050992A1; US20190338269A1

Abstract

The invention relates to a protein that is a factor X variant comprising a mutated sequence of SEQ ID NO:1; at its N-terminus, said protein comprises the signal peptide of sequence SEQ ID NO:7 and a propeptide that differs from the natural factor X propeptide.

Description

Factor X mutants

The present invention relates to factor X mutants, and their use for the treatment of blood coagulation disorders.

Factor X is a protein found in the blood. This protein plays an important role in the coagulation cascade. Blood clotting is a complex process that prevents the flow of blood through damaged vessels. As soon as a vessel is broken, the elements responsible for coagulation interact with each other to form a plug, the platelet nail, where the vessel is broken. Coagulation factors are required to hold the platelet nail in place and stabilize the clot.

The formation of a normal clot takes place in four stages:

Step 1 The blood vessel is damaged.

Step 2 The blood vessel contracts to restrict blood supply to the injured area.

Step 3 Platelets adhere to the subendothelial space exposed during vessel injury and to the walls of the stimulated blood vessels. Platelets spread out, this is called platelet adhesion. These spread platelets release substances that activate other nearby platelets to agglomerate at the lesion site to form the platelet nail. This is called platelet aggregation.

Step 4 The surface of the activated platelets thus constitutes a surface on which blood coagulation can take place. Coagulation proteins circulating in the blood (including factor X) are activated on the surface of platelets and form a fibrin clot.

These coagulation proteins (i.e., factors I, II, V, VIII, IX, X, XI, XII and XIII, as well as von Willebrand factor) function in a chain reaction. . the cascade of coagulation. Factor X in activated form (Xa) intervenes more particularly in the activation of prothrombin (factor II) in thrombin (factor Ha), in particular when it is complexed with activated c-factor V to form the prothrombinase complex. This factor is an essential element in the coagulation cascade.

When this factor is lacking, bleeding occurs, such as epistaxis (nose bleeds), hemarthrosis (effusion of blood in a joint cavity) or gastrointestinal bleeding. Factor X deficiency is extremely rare. Its transmission is autosomal recessive, and its prevalence is 1: 1,000,000.

Activation of the FX intervenes:

or very early in the initiation step of the coagulation cascade by the factor VIIa / f tissue factor complex, in a weakly effective reaction which leads to the formation of thrombin traces;

or during the amplification step of the coagulation cascade resulting from a positive feedback control performed by the traces of thrombin leading to the activation of factors VIII and IX.

These last two factors are missing in hemophiliacs A and B, causing a haemorrhagic disorder that can be fatal without treatment. The absence of these factors does not allow generating sufficient amounts of activated factor X to control the bleeding.

The first 42 amino acids of the factor X light chain (residues 1-42 of SEQ ID NO: 5) represent the "Gla" domain, phospholipid binding domain. The "Gla" domain contains 11 residues of glutamic acid (Glu) all or partially post-translational (gamma-carboxylated) to give γ-carboxyglutamic acid (Gla). Factor X is thus a coagulation protein whose biological activity depends on the degree of gamma-carboxylation of its "Gla" domain.

All "Gla" proteins or Gla domain proteins are vitamin K-dependent. Vitamin K is a fat-soluble vitamin implicated in gamma carboxylation of protein residues of glutamates to form gamma-carboxyglutamate residues. Gamma-carboxyglutamate residues are essential for the biological activity of all proteins possessing Gla domains, in particular via a high affinity binding to calcium ions.

The presence of gamma-carboxylated Glu residues (also referred to as Gla residues) in vitamin K-dependent proteins has been shown to be essential for their functional activity. Thus, the presence of Glu residues on factor X and their level of gamma-carboxylation is essential to the functional activity of activated factor X. Thus, there is a need for a modified thrombin-activated factor X having a gamma-carboxylation level which would allow effective coagulation in the absence of factor VIII and / or factor IX by direct use. traces of thrombin generated during the initiation of coagulation. The inventors have identified factor X-specific mutants (also called X variant factors), which are efficiently activated by thrombin, thus making it possible to restore coagulation in the absence of factor VIII, factor IX. Preferably, factor X-specific mutants identified by the inventors can also restore coagulation in the absence of factor X. Indeed, as demonstrated by example, these factor X mutants can be activated by thrombin, and allow efficient coagulation. even in the absence of factor VIII and / or factor IX and / or endogenous factor X.

These factor X mutants advantageously have a high level of gamma-carboxylation. By high degree of gamma-carboxylation is meant a gamma-carboxylation level of at least 20%, preferably at least 25%, 30%, 35%, 40%, 45%, 50%, 55%. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% of gamma-carboxylation of plasma factor X, considered 100%.

The present invention relates to a protein which is a variant of factor X comprising a mutated sequence of SEQ ID NO: 1, said protein comprising at its N-terminus the natural signal peptide of factor X represented by the sequence SEQ ID NO: 7 , and a propeptide different from the natural propeptide of factor X. The present invention relates to a protein comprising a mutated sequence of SEQ ID NO: 1, said mutated sequence of SEQ ID NO: 1 comprising an A, A ', B, C or C mutation, wherein:

mutation A consists in the substitution of amino acids 43 to 52 of the sequence SEQ ID NO: 1 by a sequence chosen from DFLAEGLTPR, KATN * ATLSPR and KATXATLSPR,

mutation A 'consists of the substitution of amino acids 47 to 52 of the sequence SEQ ID NO: 1 by a sequence chosen from TSKLTR, FNDFTR, LSSMTR, PPSLTR and LSCGQR,

mutation B consists of the insertion of a sequence chosen from DFLAEGLTPR, KATN * ATLSPR, KATXATLSPR, TSKLTR, FNDFTR, LSSMTR, PPSLTR and LSCGQR, between amino acids 52 and 53 of the sequence SEQ ID NO: 1,

mutation C consists in the insertion of a sequence chosen from DFLAEGLTPR, KATN * ATLSPR and KATXATLSPR, between amino acids 52 and 53 of the sequence SEQ ID NO: 1, and in the deletion of amino acids 4 to 13 of the sequence SEQ ID NO: 1, mutation C consists in the insertion of a sequence selected from TSKLTR, FNDFTR, LSSMTR, PPSLTR and LSCGQR, between amino acids 52 and 53 of the sequence SEQ ID NO: 1, and in the deletion of amino acids 4 to 9 of the sequence SEQ ID NO: 1,

where N * is an optionally glycosylated asparagine, and

said protein comprising at its N-terminal end the signal peptide of sequence SEQ ID NO: 7 fused to a propeptide different from the natural propeptide of factor X. Preferably, the present invention relates to a protein comprising a mutated sequence of SEQ ID NO: 1 , said mutated sequence of SEQ ID NO: 1 comprising a mutation consisting of the insertion of a sequence selected from DFLAEGLTPR, KATN * ATLSPR, KATXATLSPR, TSKLTR, FNDFTR, LSSMTR, PPSLTR and LSCGQR, between amino acids 52 and 53 of the sequence SEQ ID NO: 1 (ie mutation B above),

where N * is an optionally glycosylated asparagine, and said protein comprising at its N-terminal end the signal peptide of sequence SEQ ID NO: 7 fused to a propeptide of a coagulation factor different from factor X. Such a protein according to the invention is a mutated factor X, which is effective in the treatment of coagulation disorders.

In particular, a sequence coding for a mutated factor X according to the invention and comprising a combination of sequences from the N-terminus to the C-terminus, namely:

a propeptide different from the natural propeptide of factor X

the signal peptide of sequence SEQ ID NO: 7

a mutated sequence of SEQ ID NO: 1 according to the invention makes it possible to directly impact the gamma-carboxylation of the expressed factor X, increased with respect to a factor X comprising a mutated sequence of SEQ ID NO: 1 according to the invention, but not comprising at the N-terminal end the signal peptide of sequence SEQ ID NO: 7 fused to a propeptide of a coagulation factor different from factor X. Preferably, such factor X mutants have a gamma carboxylation of at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% of gamma-carboxylation of plasma factor X, considered 100%.

Another subject of the invention is a polynucleotide encoding said protein.

Another subject of the invention is an expression vector comprising said polynucleotide.

Another subject of the invention is a host cell comprising said expression vector or said polynucleotide.

Another object of the invention is the use of said protein as a medicament. In particular, said protein may be used for the treatment of blood coagulation disorders, in particular hemorrhagic disorders, such as haemophilia A, B and C (factor XI deficiency), or even deficits in factor X, or even the need for emergency coagulation to replace Factor VIIa. When a powerful and rapid procoagulant response is required, said protein can be used in combination with other hemostatic molecules, such as factor VIIa and / or fibrinogen, or even in combination with procoagulant compounds (platelet transfusion, procoagulant mixture such as FEIBA, Kaskadil, Kanokad etc.), which may enhance the effectiveness of treatment .

The term "protein" refers to an amino acid sequence having more than 100 amino acids. Preferably, the protein consists of an amino acid sequence having between 100 and 1000 amino acids, preferably between 120 and 600 amino acids. A factor X variant according to the invention advantageously has a high level of gamma carboxylation. By high gamma-carboxylation level is meant a gamma-carboxylation level of at least 20%, preferably at least 25%, 30%, 35%, 40%, 45%, 50%, 55%. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% of gamma-carboxylation of plasma factor X, considered 100%.

The calculation of said gamma-carboxylation level can be carried out by any conventional technique for detecting and then quantifying Gla residues, such as the ELISA technique using an anti-Gla antibody for the capture of gamma-carboxylated X factors. For example, the optical density measurement obtained for a factor X varying according to the invention can be related to the measurement of optical density obtained for the same amount of plasma factor X obtained according to the same production method, considered as the reference measurement. gamma-carboxylation rate at 100%.

Preferably, a factor X variant according to the invention comprises at least 2 gamma-carboxylated Glu residues (Le, Gla residues) among 1 1 Glu, at least 3 gamma-carboxylated residues among 11 Glu, at least 4 gamma-carboxylated residues. of 11 Glu, at least 5 gamma-carboxylated residues among 11 Glu, at least 6 gamma-carboxylated residues among 1 1 Glu, at least 7 gamma-carboxylated residues among 1 1 Glu, at least 8 gamma-carboxylated residues among 11 Glu, at least 9 gamma-carboxylated residues among 11 Glu, at least 10 gamma-carboxylated residues among 11 Glu, or 11 gamma-carboxylated residues. Preferably, a variant of factor X according to the invention comprises at least 10 of the 11 gamma-carboxylic acid residues mentioned above, present on the Gla domain of the light chain factor X, gamma-carboxylated. Thus, preferably, a factor X variant according to the invention comprises 10 gamma-carboxylated Glu residues (10 Gla residues). More preferentially, a factor X variant according to the invention comprises 11 gamma-carboxylated Glu residues (11 residues). Gla)

According to a particular aspect, the invention relates to a composition of factor X variants according to the invention, advantageously having a high level of gamma-carboxylation within the composition. High level of gamma-carboxylation within the composition means a gamma-carboxylation level of at least 20%, preferably at least 25%, 30%, 35%, 40%, 45%, 50%. %, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% of gamma carboxylation in a plasma factor X composition, considered 100%.

In this case, a factor X variant composition according to the invention may comprise a homogeneous gamma-carboxylated factor X population for the number of Gla residues. Preferably, a factor X variant composition according to the invention comprises a population of gamma-carboxylated X-factors each comprising 10 Gla residues. Preferably, a factor X variant composition according to the invention comprises a population of gamma-carboxylated X-factors each comprising 11 Gla residues. Alternatively, a factor X variant composition according to the invention may comprise a heterogeneous gamma-carboxylated factor X population, comprising at least two gamma-carboxylated factor X populations not comprising the same number of gamma-carboxylated Gla residues. For example, a factor X variant composition according to the invention may comprise 50% variants comprising 10 Gla residues and 50% variants comprising 11 Gla residues.

Factor X, also called Stuart-Power factor, is encoded by the F10 gene and refers to serine protease EC3.4.21.6. Factor X is composed of a heavy chain, 306 amino acids, and a light chain, 139 amino acids.

Factor X is a 488 amino acid protein consisting of a signal peptide, a propeptide, and light and heavy chains. Human X factor can be found in UniProtKB under accession number P00742. Its primary structure is illustrated in Figure 1.

The protein is translated as a prepropeptide. After cleavage of the signal peptide, the propeptide is finally cleaved, resulting in a light chain and a heavy chain (142 and 306 amino acids respectively) (zymogen). Following the onset of coagulation, the heavy chain is finally activated by cleavage of the activation peptide, to contain only 254 amino amino acids (the first 52 amino acids are cleaved during treatment): it is the heavy chain of the factor Xa (SEQ ID NO: 6).

The human factor X prepropeptide corresponds to SEQ ID NO: 4. The unactivated human factor X heavy chain corresponds to SEQ ID NO: 1, and the light chain corresponds to SEQ ID NO: 5. heavy chain corresponds to SEQ ID NO: 3, and comprises 52 amino acids. The signal peptide corresponds to SEQ ID NO: 7, and comprises 31 amino acids. The natural propeptide of factor X corresponds to SEQ ID NO: 8, and comprises 9 amino acids.

SEQ ID NO: 2 is identical to amino acids 1 to 182 of SEQ ID NO: 4.

SEQ ID NO: 1 is identical to amino acids 183 to 488 of SEQ ID NO: 4.

The factor Xa heavy chain (SEQ ID NO: 6) corresponds to SEQ ID NO: 1, in which the activation peptide represented by SEQ ID NO: 3 was cleaved.

In the context of the invention, the term "natural propeptide of factor X" preferably refers to a variant of the natural propeptide of human factor X represented by the sequence SEQ ID NO: 9 which comprises 9 amino acids.

The protein according to the invention is a mutant (or variant) factor X.

The preferred mutation according to the invention consists in the insertion of a sequence chosen from DFLAEGLTPR, KATN * ATLSPR, KATXATLSPR, TSKLTR, FNDFTR, LSSMTR, PPSLTR and LSCGQR, between amino acids 52 and 53 of the sequence SEQ ID NO: 1 ("mutation B"), where N * is an optionally glycosylated asparagine. Preferably, the mutation according to the invention consists of the insertion of the sequence DFLAEGLTPR between amino acids 52 and 53 of the sequence SEQ ID NO: 1.

The sequence SEQ ID NO: 1 comprising a mutation according to the invention is also called "mutated sequence of SEQ ID NO: 1". In other words, preferably, the sequence SEQ ID NO: 1 comprising a mutation according to the invention consists of the sequence SEQ ID NO: 3, fused at its C-terminal end to a sequence chosen from DFLAEGLTPR, KATN * ATLSPR , KATXATLSPR, TSKLTR, FNDFTR, LSSMTR, PPSLTR and LSCGQR ("mutation B"), where N * is an optionally glycosylated asparagine, itself fused at its C-terminus to the sequence SEQ ID NO: 6.

When the mutation consists of the insertion of the DFLAEGLTPR sequence, the mutated sequence of SEQ ID NO: 1 corresponds to SEQ ID NO: 11. The propeptide used in the protein according to the invention is different from the natural propeptide of factor X. Preferably, the propeptide used in the protein according to the invention is that of a vitamin K dependent protein. Preferably, the propeptide used in the protein according to the invention is chosen from the propeptide of the protein S, the propeptide of the protein Z, the propeptide of FIX, the propeptide of one of the proteins GAS6, BGP, MGP, and PRGP1, PRGP2, TMG3 and TMG4, the propeptide of thrombin, the propeptide of factor VII, the propeptide of protein C, including their natural isoforms or modified versions thereof. By "modified version" is meant that the propeptide used is truncated, and optionally comprises the insertion of one or more amino acids, for example at its N-terminus. Preferably, the propeptide used in the protein according to the invention is that of a coagulation factor different from factor X. Thus, the propeptide used in the protein according to the invention is preferably the natural propeptide of a different coagulation factor. Preferably, the propeptide is selected from the propeptide of thrombin, the propeptide of factor VII, the propeptide of protein C, their natural isoforms or modified versions thereof. Preferably, the propeptide of factor VII according to the invention corresponds to isoform A of the propeptide of factor VII (or "FVIIvl") and has for sequence SEQ ID NO: 14. Preferably, the propeptide of factor VII according to the invention corresponds to to the isoform B of the factor VII propeptide (or "FVIIv2") and has the sequence SEQ ID NO: 15. More preferentially, the propeptide is chosen from the sequences SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16. More preferably, the propeptide has the sequence SEQ ID NO: 14. Preferably , the signal peptide of sequence SEQ ID NO: 7 fused to a propeptide different from the natural propeptide of factor X is chosen from the sequences SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21 . More preferably, the signal peptide of sequence SEQ ID NO: 7 fused to a propeptide different from the natural propeptide of factor X is represented by the sequence SEQ ID NO: 19.

The particular combination of the sequence SEQ ID NO: 19 to a mutated sequence of SEQ ID NO: 1 according to the invention makes it possible to directly impact the gamma-carboxylation of the mutated factor X comprising such a combination, with respect to a mutated factor X comprising a mutated sequence of SEQ ID NO: 1 according to the invention, but not comprising at its N-terminal end the sequence SEQ ID NO: 19.

The protein according to the invention preferably comprises an intermediate sequence, between the signal peptide of sequence SEQ ID NO: 7 fused to a propeptide different from the natural propeptide of factor X and the mutated sequence of SEQ ID NO: 1. intermediate sequence is fused at its N-terminus, to the signal peptide of sequence SEQ ID NO: 7 fused to a propeptide different from the natural propeptide of factor X, and at its C-terminus, to the mutated sequence of SEQ ID NO: 1. Preferably, said intermediate sequence is the sequence of the light chain of factor X, preferably the sequence SEQ ID NO: 5.

The protein according to the invention thus preferably comprises, from the N-terminus to the C-terminus:

the signal peptide of sequence SEQ ID NO: 7 fused to a propeptide different from the natural propeptide of factor X, and then

the sequence SEQ ID NO: 5, then

said mutated sequence of SEQ ID NO: 1. More particularly, the protein according to the invention comprises, in the N- to C-terminal direction, the signal peptide of sequence SEQ ID NO: 7 fused to a propeptide different from the natural propeptide of factor X, then the sequence SEQ ID NO: 5, then the mutated sequence of SEQ ID NO: 1.

The protein according to the invention preferably comprises, from the N-terminal to the C-terminal end:

the sequence SEQ ID NO: 5, then

the sequence SEQ ID NO: 11.

Thus, the protein according to the invention preferably comprises, from the N-terminus to the C-terminal end: the signal peptide of sequence SEQ ID NO: 7 fused to a propeptide different from the natural propeptide of factor X, fused to the sequence SEQ ID NO: 17.

Preferably, the protein according to the invention comprises, preferably consists of, a sequence chosen from SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24 and SEQ ID NO: 25. More preferably, the protein according to the invention comprises, preferably consists of, the sequence SEQ ID NO: 23.

The mutated sequence of SEQ ID NO: 1 comprising an A, A ', B, C or C mutation may further comprise at least one mutation of at least one amino acid that does not alter the functional activity of the protein according to invention. Preferably, the mutated sequence of SEQ ID NO: 1 comprising an A, A ', B, C or C mutation and further comprising at least one additional mutation of at least one amino acid that does not impair the functional activity, has at least 80% identity, at least 81% at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity with the mutated sequence of SEQ ID NO: 1 comprising only an A, A ', B, C or C mutation. The protein according to the invention may also be fused at the C-terminal to at least one wild-type immunoglobulin fragment, optionally mutated. By wild-type immunoglobulin fragment is meant a fragment selected from wild-type Fc fragments and wild-type scFc fragments, optionally mutated.

By "Fc fragment" is meant the constant region of a full length immunoglobulin excluding the first immunoglobulin constant region domain (i.e. CH1-CL). Thus the Fc fragment refers to a homodimer, each monomer comprising the last two constant domains of IgA, IgD, IgG (ie CH2 and CH3), or the last three constant domains of IgE and IgM (ie CH2, CH3 and CH4), and the flexible N-terminal hinge region of these domains. The Fc fragment, when it is derived from IgA or IgM, may comprise the J chain. Preferably, the Fc region of an IgG1 is composed of the flexible N-terminal hinge and the CH2-CH3 domains. that is, the portion from amino acid C226 to the C-terminus, the numbering being indicated according to the EU number or equivalent in Kabat.

By "scFc fragment"("single chain Fc") is meant a single chain Fc fragment, obtained by genetic fusion of two Fc monomers linked by a polypeptide linker. The linker may be - (GGGGS) n-, where n is an integer of 1 to 3. The scFc folds naturally into a functional dimeric Fc region. The scFc fragment preferably has the sequence SEQ ID NO: 42 (which corresponds to SEQ ID NO: 36 fused to C-terminal at -GGGGS- fused at C-terminal to SEQ ID NO: 37) optionally followed by a lysine . The fusion of the protein according to the invention with at least one fragment of wild-type immunoglobulin (in particular a Fc or scFc fragment) at the C-terminal, makes it possible to improve the stability and the retention of the protein in the body, and thus its bioavailability; it also improves its half-life in the body. In addition, it may make it possible to simplify the purification of the molecule obtained by targeting or by using the Fc fragment during one of the purification steps. Preferably, the wild-type Fc fragment is chosen from the sequence SEQ ID NO: 36 and the sequence SEQ ID NO: 37 optionally followed by a C-terminal lysine (226 or 227 amino acids respectively for SEQ ID NO: 36, 231 or 232 amino acids respectively for SEQ ID NO: 37). The Fc fragment corresponding to the sequence SEQ ID NO: 36 comprises the constant domains CH2 and CH3 of a wild type IgG and the partial hinge region in N-terminal (DKTHTCPPCP, SEQ ID NO: 38). The fragment corresponding to the sequence SEQ ID NO: 37 comprises the constant domains CH2 and CH3 of a wild type IgG and the entire hinge region at the N-terminal (sequence EPKSSDKTHTCPPCP, SEQ ID NO: 39, variant of the present natural sequence on a wild-type IgG, sequence EPKSCDKTHTCPPCP, SEQ ID NO: 81). Preferably, the protein according to the invention fused to a C-terminal wild type Fc fragment has the sequence SEQ ID NO: 40 optionally followed by a C-terminal lysine. Its corresponding nucleic acid has the sequence SEQ ID NO: 41 optionally followed by a codon coding for a C-terminal lysine. Alternatively and preferably, its nucleic acid was obtained by gene synthesis with codon optimization for Homo sapiens and has the sequence SEQ ID NO: 82. Preferably, the protein according to the invention fused to a C-terminal wild type scFc fragment has the sequence SEQ ID NO: 43 optionally followed by a C-terminal lysine. Its corresponding nucleic acid has the sequence SEQ ID NO: 44 optionally followed by a codon coding for a C-terminal lysine.

The wild-type Fc fragment or the wild-type scFc fragment used according to the invention can be mutated according to the "knobs-into-holes" technology. This technology is described in the application WO96 / 27011 of Genentech: it consists in obtaining heterodimers, which comprise and preferably pair at a constant CH3 domain of antibodies. These heterodimers, preferably 2 Fc fragments or one scFc, comprise various point mutations, which induce a "protuberance-in-cavity" (knobs-into-holes) interface. A first mutation on the first monomer induces a protuberance, and a second mutation on the second monomer induces a cavity, so that the heterodimer pairs preferentially.

Preferably, the first monomer (ie an Fc fragment or Fc monomer of the scFc fragment) comprises the T366Y mutation, and the second monomer (ie an Fc fragment or Fc monomer of the scFc fragment) comprises the Y407T mutation. The sequences described in this application can be summarized as follows: SEQ ID NO: Protein

1 Human Factor X heavy chain (306 amino acids), including the activation peptide

Signal Peptide, Propeptide, and Human Factor X Light Chain (182 amino acids)

3 Peptide for activation of the heavy chain (52 amino acids)

4 Human Factor X Prepropeptide (488 amino acids)

5 Light chain of human factor X (142 amino acids)

6 Human Factor X Heavy Chain Activated (FXa) (254 amino acids)

7 Signal peptide of human factor X (31 amino acids)

8 Human Factor X Propeptide (9 amino acids)

9 Propeptide Variant of Human Factor X (9 amino acids)

FX-WT (488 amino acids)

Mutation sequence of SEQ ID NO: 1 comprising the insertion of DFLAEGLTPR between amino acids 52 and 53

(mutation according to the invention)

12 Anti-Gla Aptamer

13 Propeptide thrombin (19 amino acids)

14 Propeptide Factor VII Version 1 ("FVIIvl") (40 amino acids)

Propeptide of factor VII version 2 ("FVIIv2") (18 amino acids)

16 Propeptide of protein C (24 amino acids)

17 FX-IIa (458 amino acids)

Signal peptide of human factor X fused to the propeptide of thrombin

Signal peptide of human factor X fused to FVIIvl

Signal peptide of human factor X fused to FVIIv2 Signal peptide of human factor X fused to the propeptide of protein C

Protein according to the invention (SEQ ID NO: 18 fused to

SEQ ID NO: 17)

Protein according to the invention (SEQ ID NO: 19 fused to

SEQ ID NO: 17)

Protein according to the invention (SEQ ID NO: 20 fused to

SEQ ID NO: 17)

Protein according to the invention (SEQ ID NO: 21 fused to

SEQ ID NO: 17)

Nucleic Sequence Encoding for SEQ ID NO: 7

Sequence encoding SEQ ID NO: 13

Nucleic Sequence Encoding SEQ ID NO: 14

Nucleic Sequence Encoding for SEQ ID NO: 15

Nucleic Sequence Encoding SEQ ID NO: 16

31 Nucleic Sequence Encoding for SEQ ID NO: 17

Nucleic Sequence Encoding for SEQ ID NO: 22

33 Sequence encoding SEQ ID NO: 23

Nucleic Sequence Encoding for SEQ ID NO: 24

Nucleic Sequence Encoding for SEQ ID NO: 25

36 Wild Fc Fragment, possibly followed by Lysine

37 SEQ ID NO: 36 including the entire hinge region in

N-terminal, possibly followed by lysine

38 Partial hinge region in N-terminal

39 Whole hinge region in N-terminal

Protein SEQ ID NO: 23 fused to the wild-type Fc fragment SEQ ID NO: 36, optionally followed by lysine

Nucleic acid encoding the protein SEQ ID NO: 40, optionally followed by a codon encoding a lysine 42 Wild scFc fragment, possibly followed by lysine

Protein SEQ ID NO: 23 fused to the wild scFc fragment SEQ ID NO: 42, optionally followed by lysine

Nucleic acid encoding the protein SEQ ID NO: 43 optionally followed by a codon encoding a lysine

45 wild human VKORC1

46 Nucleic acid encoding the SEQ ID subunit

NO: 45

47 Signal peptide of prothrombin

48 Factor VII signal peptide

49 Signal peptide of protein C

50 to 80 Sample primers

81 Whole hinge region native to N-terminal

Optimized nucleic acid sequence encoding the protein SEQ ID NO: 40, optionally followed by a codon encoding a lysine

Another subject of the invention is a nucleic acid (polynucleotide) coding for said protein. Preferably, the nucleic acid is chosen from the sequences SEQ ID NO: 32 to 35.

Another subject of the invention is an expression vector comprising said polynucleotide encoding said protein, or an expression cassette comprising said polynucleotide. According to the invention, the expression vectors suitable for use according to the invention may comprise at least one expression control element operably linked to the nucleic acid sequence. The expression control elements are inserted into the vector and make it possible to regulate the expression of the nucleic acid sequence. Examples of expression control elements include lac systems, lambda phage promoter, yeast promoters or viral promoters. Other operational elements may be incorporated, as a leader sequence, termination codons, polyadenylation signals and sequences necessary for transcription and subsequent translation of the nucleic acid sequence into the host system. It will be understood by those skilled in the art that the correct combination of expression control elements depends on the chosen host system. It will also be understood that the expression vector should contain the additional elements necessary for the subsequent transfer and replication of the expression vector containing the nucleic acid sequence in the host system.

Such vectors are easily constructed using conventional or commercially available methods.

Preferably, the expression vector used is a polycistronic vector comprising a polynucleotide encoding a protein according to the invention, a polynucleotide encoding the enzyme VKOR, preferably for subunit 1 of the vitamin K epoxide reductase complex ( VKORC1) wild-type human, and optionally a polynucleotide encoding furin, and / or a polynucleotide encoding an Fc fragment in the context of the production of variants according to the invention fused to an Fc fragment. The co-expression of furin optimizes the natural cleavage within the cell at the natural cleavage sites present on factor X (RRKR). Preferably, the expression vector used is a bicistronic comprising a polynucleotide encoding a protein according to the invention, and a polynucleotide encoding the enzyme VKOR, preferably for subunit 1 of the vitamin K epoxide reductase complex ( VKORC1) wild human.

Subunit 1 of the wild-type human vitamin K epoxide reductase (VKORC1) complex is the catalytic subunit of the complex; it is a protein of 163 amino acids. The sequence of this wild-type human subunit can be found in UniProt under accession number Q9BQB6 (SEQ ID NO: 45). The nucleic acid encoding this protein has the sequence SEQ ID NO: 46. This protein is present in the endoplasmic reticulum of the cells. The polynucleotide encoding the VKOR enzyme may also be a polynucleotide encoding a mutated VKOR.

Preferably, alternatively, the expression vectors used are as many vectors as polynucleotides to be expressed, one comprising a polynucleotide encoding a protein according to the invention, another comprising a polynucleotide encoding the VKOR enzyme mentioned above, optionally another comprising a polynucleotide encoding furin, and / or another comprising a polynucleotide encoding an Fc or scFc fragment; in the context of the production of variants according to the invention fused to an Fc or scFc fragment.

Another subject of the invention is a recombinant cell comprising an expression vector as described above, or a polynucleotide as described above. According to the invention, examples of host cells that can be used are eukaryotic cells, such as animal, plant, insect and yeast cells; and prokaryotic cells, such as E. coli. The means by which the vector carrying the gene can be introduced into the cells include microinjection, electroporation, transduction or transfection using DEAE-dextran, lipofection, calcium phosphate or other procedures. known to those skilled in the art. In a preferred embodiment, expression vectors for expression in eukaryotic cells are used. Examples of such vectors include viral vectors such as retroviruses, adenovirus, herpes virus, vaccinia virus, variola virus, poliovirus, lentivirus, bacterial expression vectors or plasmids such as pcDNA5. Preferred eukaryotic cell lines include COS cells, CHO cells, HEK cells including HEK293 (ATCC # CRL1573), YB2 / 0 cells, BHK cells, PerC6 cells, HeLa cells, NIH / 3T3 cells, T2 cells, dendritic cells or monocytes. Preferably, the cells used are HEK cells. More preferably, the cells used are YB2 / 0 cells. More preferably, the cells used are CHO cells.

Another subject of the invention is a method for producing a protein according to the invention, said protein comprising a light chain (preferably SEQ ID NO: 5), comprising:

a) the expression of a polycistronic vector, preferably bicistronic, in a host cell, preferably an HEK cell, more preferably YB2 / 0, even more preferably a CHO cell, said vector comprising a polynucleotide encoding a protein according to the invention, and a polynucleotide encoding the enzyme VKOR, preferably for subunit 1 of the wild-type human vitamin K epoxide reductase (VKORC1) complex, preferably in the presence of vitamin K;

b) culturing said host cell;

c) recovering the cellular supernatant;

d) optionally at least one of the steps selected from:

the clarification of the supernatant, optionally followed by a filtration step, the concentration of the supernatant,

the neutralization of activated proteases by the addition of protease inhibitors; e) purification of the protein according to the invention by passage of the production supernatant obtained in c) or d) on an aptamer column capable of binding to the Gla domain of factor X. All culture conditions well known to the those skilled in the art can be used for culturing the host cell in step b). For example, any mode of production can be chosen, the culture can thus be performed in production mode batch, fedbatch, continuous infusion or XD process, without being limiting. The concentration of the supernatant optionally carried out in step d) can be carried out by any well-known technique, such as by passing on concentration cassettes, by tangential filtration, or by using chromatography columns to concentrate the product. Another subject of the invention is a method for producing a protein according to the invention, said protein comprising a light chain (preferably SEQ ID NO: 5), comprising:

a) the expression of two expression vectors, one comprising a polynucleotide encoding a protein according to the invention, and the other comprising a polynucleotide encoding the VKOR enzyme mentioned above, in a host cell preferably a CHO cell, preferably in the presence of vitamin K; b) culturing said host cell; c) recovering the cellular supernatant;

d) optionally at least one of the steps selected from:

the neutralization of activated proteases by the addition of protease inhibitors; e) purification of the protein according to the invention by passage of the production supernatant obtained in c) or d) on an aptamer column capable of binding to the Gla domain of factor X. The two processes mentioned above advantageously allow obtaining factor X mutants according to the invention having a gamma-carboxylation level identical to that of plasma factor X, close to 100%.

The aptamers used in the methods described above are in particular those described in the patent application WO2011 / 012831. In particular, the aptamer used has the following sequence:

5 'CCACGACCTCGCACATGACTTGAAGTAAAACGCGAATTAC 3' (SEQ ID NO: 12).

Advantageously, this aptamer specifically binds to the biologically active forms of factor X. Thus, the methods for producing a protein according to the invention, comprising a purification step using an aptamer column described above, make it possible to obtaining biologically active forms of factor X.

The protein according to the invention can be produced in the milk of transgenic animals.

In this case, according to a first aspect, the expression of the polynucleotide encoding the protein according to the invention is controlled by a mammalian casein promoter or a mammalian whey promoter, said promoter not naturally controlling the transcription of said gene, and the polynucleotide further containing a secretion sequence of the protein. The secretion sequence comprises a secretion signal interposed between the gene and the promoter.

The transgenic animal used is capable not only of producing the desired protein, but also of transmitting this ability to its offspring. The secretion of the protein in the milk facilitates purification and avoids the use of blood products. The animal can thus be chosen from the mouse, the goat, the rabbit, the sheep or the cow.

The protein according to the invention can be used as a medicament. Therefore, the protein according to the invention can be introduced into a pharmaceutical composition. In particular, the protein according to the invention can be used for the treatment of coagulation disorders, in particular hemorrhagic disorders.

The pharmaceutical composition of the invention may be combined with pharmaceutically acceptable excipients, and optionally extended release matrices, such as biodegradable polymers, to form a therapeutic composition. The pharmaceutical composition of the present invention may be administered orally, sublingually, subcutaneously, intramuscularly, intravenously, intraarterially, intrathecally, intraocularly, intracerebrally, transdermally, locally or rectally. The active ingredient, alone or in combination with another active ingredient, can then be administered in unit dosage form, in admixture with conventional pharmaceutical carriers. Unit dosage forms include oral forms such as tablets, capsules, powders, granules and oral solutions or suspensions, sublingual and oral forms of administration, aerosols, subcutaneous implants, transdermal, topical, intraperitoneal, intramuscular, intravenous, subcutaneous, intrathecal, intranasal administration forms and rectal administration forms.

Preferably, the pharmaceutical composition contains a pharmaceutically acceptable carrier for a formulation that can be injected. It may be in particular isotonic, sterile, saline solutions (with monosodium or disodium phosphate, sodium chloride, potassium chloride, calcium or magnesium chloride and the like, or mixtures of such salts), or freeze-dried compositions which, when adding sterilized water or physiological saline as appropriate, allow the constitution of injectable solutions.

Suitable dosage forms for injectable use include sterile aqueous solutions or dispersions, oily formulations, including sesame oil, peanut oil, and sterile powders for the preparation extemporaneous sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that it must be injected by syringe. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.

The dispersions according to the invention can be prepared in glycerol, liquid polyethylene glycols or mixtures thereof, or in oils. Under normal conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

The pharmaceutically acceptable carrier may be a solvent or dispersion medium containing, for example, water, ethanol, a polyol (eg, glycerine, propylene glycol, polyethylene glycol, and the like), suitable mixtures of these, and / or vegetable oils. The proper fluidity can be maintained, for example, by the use of a surfactant, such as lecithin. Prevention of the action of microorganisms can be caused by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid or thimerosal. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions may be caused by the use in the compositions of agents delaying absorption, for example, aluminum monostearate or gelatin. Sterile injectable solutions are prepared by incorporating the active ingredients in the required amount in the appropriate solvent with several of the other ingredients listed above, if appropriate, followed by sterilization by filtration. In general, the dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle that contains the basic dispersion medium and the other required ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and lyophilization. During formulation, the solutions will be administered in a manner compatible with the dosage formulation and in a therapeutically effective amount. The formulations are easily administered in a variety of dosage forms, such as the injectable solutions described above, but drug release capsules and the like can also be used. For parenteral administration in an aqueous solution for example, the solution should be suitably buffered and the liquid diluent rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this regard, sterile aqueous media that can be used are known to those skilled in the art. For example, a dose may be dissolved in 1 ml of isotonic NaCl solution and then added to 1000 ml of appropriate liquid, or injected at the proposed site of the infusion. Certain dosage variations will necessarily occur depending on the condition of the subject being treated.

The pharmaceutical composition of the invention can be formulated in a therapeutic mixture comprising about 0.0001 to 1.0 milligrams, about 0.001 to 0.1 milligrams, about 0.1 to 1.0 milligrams, or about 10 milligrams per dose or more. Multiple doses may also be administered. The level of therapeutically effective dose specific for a particular patient will depend on a variety of factors, including the disorder being treated and the severity of the disease, the activity of the specific compound employed, the specific composition used, the age, the body weight, general health, sex and diet of the patient, the time of administration, the route of administration, the rate of excretion of the specific compound used, the duration of treatment, or the drugs used in parallel.

The protein according to the invention can also be used as a product of a gene or cell therapy.

For this purpose, the present invention also relates to an expression vector comprising a polynucleotide encoding a protein according to the invention, said polynucleotide being as described above. This expression vector can be used as a drug, preferably as a gene therapy drug.

This expression vector can also be used as a cell therapy drug: in this case, it is intended to be injected ex vivo to a sample of patient cells, before their reinjection. The following examples are given to illustrate various embodiments of the invention.

Legend of figures

Figure 1: Primary structure of human factor X

Figure 2: OptiHEK-VKOR-FX-IIa bicistronic vector Figure 3: Final vector FVIIvl-psFX-IIa-F2

Figure 4: Evaluation of the gamma-carboxylation level of the different FX variants Figure 5: Evaluation of the FX after purification

2 μg of product / track were deposited.

A, SDS-PAGE of plasma FX (lane 2), FX-IIa-F2 of immunopurified CHO (lane 4), and FX-IIa-F2 of aptamopurified CHO (lane 3).

B, SDS-PAGE of the plasma FX (lane 2) and FX-IIa-F2 of aptamopurified HEK (lane 3). In lane 1: molecular weight markers or MW (values in kDa are indicated on the left of the figure). NR: unreduced products; DTT-R: products reduced to DTT.

Figure 6: Activation of FX variants by the RVV-X fraction

Plasma (?), Plasma FX (?), FVIIv1-psFX-IIa-F2 derived from aptamopurified HEK (o), FVIIv1-psFX-IIa-F2-VKOR from aptamopurified HEK (□), FVIIvl- psFX-IIa-F2 from aptamopurified HEK (Δ).

FIG. 7: Activation of FVIIa Complex FX / Tissue Factor (FT) FXa Plasma Factor (·), Plasma FX (B), FVIIv1-psFX-IIa-F2-VKOR Aptamopurified FX from HEK (□), FVIIv1-psFX- IIa-F2 aptamopurified from HEK (Δ). Figure 8: Generation of thrombin by FVIII deficient plasma modified FX

Thrombin generation was monitored over time in normal or factor VIII deficient plasma stimulated by 0.5 μM tissue factor in the presence of phospholipids. Signal obtained with a normal plasma (·); signal obtained in plasma deficient in FVIII (o); in the presence of 0, 1 U / ml of recombinant FVIII (□); in the presence of 1 U / ml of recombinant FVIII (■); in the presence of 10 μs / l (Δ) or 20 μg / ml (A) of aptamopurified FVIIv1-psFX-IIa-F2 from HEK. Figure 9: Evaluation of FX-Fc after purification

400 ng of product / track were filed.

SDS-PAGE of Plasma FX (lanes 2/6), FVIIvl-psFX-IIa-F2-Fc of aptamopurified CHO (lane 3/4/7/8) and FVIIvl-psFX-IIa-F2-Fc of HEK (lane 5) / 9).

In lanes 1 and 10: molecular weight markers or MW (values in kDa are indicated on the left of the figure). NR: unreduced products; DTT-R: products reduced to DTT.

Figure 10: Evaluation of the binding of FX-Fc to phospholipids

Plasma FX (x), FVIIv1-psFX-IIa-F2-Fc YB2 / 0 supernatant (o) or aptamopurified (·), FVIIv1-psFX-IIa-F2-Fc from HEK supernatant (□) or aptamopurified (■), CHO-F supernatant (Δ) or aptamopurified (A) FVIIvl-psFX-IIa-F2-Fc.

Figure 11: Activation of variant FX-Fc by thrombin

Plasma FX (x), aptamopurified YB2 / 0 FVIIv1-psFX-IIa-F2-Fc (aptamopurified HEK-FVIIv1-psFX-IIa-F2-Fc), CHO-FVIIv1-psFX-IIa-F2-Fc F aptamopurified (A).

Figure 12: Generation of Thrombin by FX-Fc Modified to FVIII-deficient Plasma

Thrombin generation was monitored over time in normal or factor VIII deficient plasma stimulated by 0.5 μM tissue factor in the presence of phospholipids. Signal obtained with a normal plasma (·); in the presence of 0.1 U / ml of Recombinant FVIII (□); in the presence of 1 U / ml of recombinant FVIII (■); in the presence of 4 μg / ml of aptamopurified FVIIv1-psFX-IIa-F2-Fc of YB2 / 0 (Δ); in the presence of 4 μg / ml of aptamopurified FVIIv1-psFX-IIa-F2-Fc of HEK (A); in the presence of 4 μg / ml of aptamopurified FVIIv1-psFX-IIa-F2-Fc of CHO (♦).

Example 1: Generation of expression vectors containing modified propeptides

Construction of a bicistronic expression vector expressing a modified FX and human VKOR

A non-commercial expression vector (OptiCHO) was used to insert, by In fusion ligation at the NheI-SwaI restriction sites, a polynucleotide encoding the modified FX. Briefly, the OptiCHO expression vector was digested with the NheI-SwaI restriction enzymes and then gel-purified using the Nucleospin extract II kit (Macherey Nagel).

The modified FX polynucleotide was obtained by assembly PCR using as template a vector containing a polynucleotide encoding the wild-type FX. The primers used are:

-5'FXWT: ACCAGCTGCTAGCAAGCTTGCCG (SEQ ID NO: 50) and

-3'FX-2b:

GTCAGGCCCTCGGCCAGGAAGTCCCTAGTCAGATTGTTATCGCCTCTTTCAG GC (SEQ ID NO: 51) for the first PCR, and

-5'FX-lb AGGGCCTGACCCCTAGGATCGTGGGAGGACAGGAGTGCAAGGA (SEQ ID NO: 52) and

-3'FX-SwaI GAAACTATTTAAATGGATCCTCACTTGCCGTCAATCAGC (SEQ ID NO: 53) for the second PCR.

The fragment of interest obtained by PCR was then cloned by ligation In Fusion into the OptiCHO vector digested beforehand at the NheI and Swal restriction sites. The polynucleotide sequence encoding human VKOR obtained by gene synthesis with codon optimization for Homo sapiens. It was extracted from a parental vector (OptiHEK-v3-VKOR) with the whole of the promoter unit (CMV enhancer, RSV promoter, polynucleotide, BGH poly A termination signal by Ascl-SpeI digestion. It was introduced into the vector previously constructed by ligation on the same Ascl-SpeI restriction sites. Human VKOR contained in the C-terminal position a hexahistidine tag.

The final vector obtained containing 2 transcription units (bicistronic vector coding for FX-modified on the one hand and human VKOR on the other hand) was named OptiHEK-VKOR-FXIIa (FIG. 2).

Construction of bicistronic expression vectors expressing human VKOR and a modified FX containing a signal peptide and / or a propeptide different from that of FX wt

Preparation of the final expression vector for ligation

The optiHEK-VKOR-FXIIa bicistronic vector which contains the transcription unit (UT) coding for FX-modified on the one hand and for human VKOR on the other hand was digested with Spel-SwaI restriction enzymes allowing obtain 2 fragments of 6904 and 3510 bp respectively. The 6904bp fragment (digested vector) was gel purified using the Nucleospin extract II kit (Macherey Nagel).

Preparation of a promoter UT allowing expression of the modified FX

From a vector containing a polynucleotide encoding the modified FX, the FX promoter unit was amplified by PCR using the primers:

3UTFX: GGTGGCGGCAAGCTTGCTAGC (SEQ ID NO: 54) and

5UTFX: CCTTGGGCAATAAATACTAGTGGCGTTAC (SEQ ID NO: 55).

The amplicon obtained with the Kappa Hifi polymerase was then digested with the restriction enzymes NheI and Spel to obtain a final fragment of 1983bp. The fragment was purified on agarose gel and extracted using the Nucleospin extract II kit (Macherey

Nagel).

Preparation of signal peptide and propeptide inserts for the expression of FX variants The signal peptide (PS) and the propeptide of FX-WT were replaced by those of prothrombin or FVII Isoform vl (A) or FVII Isoform v2 (B ) or protein C. For this the same strategy was applied each time (in the case of protein C, 3 primers were used to carry out assembly PCR):

the signal peptide and the propeptide of interest were obtained by PCR from a vector containing the corresponding nucleotide sequences using respectively the following primers:

^■ prothrombin:

5PSth primers: AAGCTTGCCGCCACCATGGCTCACGTCCGAGGGCTG (SEQ ID NO: 56) and

3PSth:

CTTCATTTCCTCCAGGAAAGAGTTGGCTCTCCGCACCCGCTGCAGC (SEQ ID NO: 57)

^■ FVII isoform vl (A):

primers 5PSFVII: AAGCTTGCCGCCACCATGGTGTCTCAGGCTCTGCGGC (SEQ ID NO: 58) and

3PSFVII:

CAGGAAAGAGTTGGCCCTTCTCCTTCTATGCAGCACTCCATG (SEQ ID NO: 59) ■ FVII isoform v2 (B):

primers 5PSFVII: AAGCTTGCCGCCACCATGGTGTCTCAGGCTCTGCGGC (SEQ ID NO: 60) and

3FVIIv2: GTCACGAACACAGCAGCCAGACATCCCTGCAGTC (SEQ ID NO: 61)

^■ Protein C: primers

Protein 1: AAGCTTGCCGCCACCATGTGGCAGCTGACCAGCCTGCTGCTGTTC GTGGCCACATG (SEQ ID NO: 62),

Protein2: GAGCTGCTGAACACGCTATCCAGAGGGGCGGGTGTGCCAGAGAT GCCCCATGTGGCCACG (SEQ ID NO: 63),

Protein3: TTCAGCAGCTCTGAGCGGGCCCACCAGGTGCTGCGGATCAGAAAG AGAGCCAACTCTTTC (SEQ ID NO: 64) The sequence of the modified FX without the signal peptide and without the FX-WT propeptide was obtained by PCR with primers 5FX: GCCAACTCTTTCCTGGAGGAAATGAAG (SEQ ID NO: 65) and 3FXIIa: AGCTCTAGACAATTGATTTAAATGGATCCTCAC (SEQ ID NO: 66) (1142bp amplicon) .

PCR assembly was performed between these 2 PCR products

A recombination ligation (ligation In Fusion) was performed between this assembly PCR product, the promoter UT and the digested final vector, previously prepared. Cloning efficiency was verified by colony PCR with primers 5'EFla: GTGGAGACTGAAGTTAGGCCAG (SEQ ID NO: 67) and 2BGHpA and sequencing with 5'EFla primer:

GTGGAGACTGAAGTTAGGCCAG (SEQ ID NO: 68) and 5FXseq: GGAGGCACTATCCTGAGCGAG (SEQ ID NO: 69).

The following bicistronic vectors have thus been obtained:

• Proth-FX-IIa-F2: PS + Prothrombin Propeptide-FX Modified + Human VKOR WT

• FVIIv1-FX-IIa-F2: PS + propeptide FVII isoform vl-FX modified + human VKOR WT

• FVIIv2-FX-IIa-F2: PS + propeptide FVII isoform v2-FX modified + human VKOR WT

• protc-FX-IIa-F2: PS + propeptide protein C-FX modified + human VKOR WT

Replacement of the PS by the FX-WT PS:

From the 4 final vectors obtained, the following strategy was implemented in order to replace only the signal peptide with that of the FX-WT:

1) From a vector containing the nucleotide sequence of the modified FX, we obtained by PCR the sequence corresponding to the PS of the FX-WT with the primers PSlfxWT and PS2fxWT. 2) On each of the 4 final vectors we obtained by PCR the sequence corresponding to that of the modified FX without signal peptide using the following primers:

• proth-FX-IIa-F2: primers 3FXIIA:

AGCTCTAGACAATTGATTTAAATGGATCCTCAC (SEQ ID NO: 70)

AND PSWT-P TH:

GACGGGAGCAGGCCCAGCATGTCTTCCTGGCACCACAG (SEQ ID NO: 71)

• FVIIvl-FX-IIa-F2: primers 3FXIIA:

AGCTCTAGACAATTGATTTAAATGGATCCTCAC (SEQ ID NO: 72)

AND PSWT-FVII vl:

CGGGAGCAGGCCGCTGGCGGCGTCGCTAAGGC (SEQ ID NO: 73)

• FVIIv2-FX-IIa-F2: primers 3FXIIA:

AGCTCTAGACAATTGATTTAAATGGATCCTCAC (SEQ ID NO: 74) AND PSWT-FVII v2:

CGGGAGCAGGCCGCTGTGTTCGTGACCCAGGAAGAG (SEQ ID NO: 75)

• protc-FX-IIa-F2: primers 3FXIIA:

AGCTCTAGACAATTGATTTAAATGGATCCTCAC (SEQ ID NO: 76) AND PSWT-PROT:

CGGGAGCAGGCCACACCCGCCCCTCTGGATAGCG (SEQ ID NO: 77)

An assembly PCR was then carried out between amplicon obtained in step 1 (FX WT PS) and each of those obtained in step 2 (modified FX without signal peptide).

A recombination ligation (ligation In Fusion) was performed between these assembly PCR products, the promoter UT and the digested final vector, previously prepared. The cloning efficiency was verified by PCR on colonies with the primers:

3) 5'EFla: GTGGAGACTGAAGTTAGGCCAG (SEQ ID NO: 78) and

4) 2BGHpA and sequencing with primers

5'EF1A: GTGGAGACTGAAGTTAGGCCAG (SEQ ID NO: 79) and 5FXSEQ: GGAGGCACTATCCTGAGCGAG (SEQ ID NO: 80). We thus obtained the following final bicistronic vectors:

• proth-psFX-IIa-F2: PS FXwt + prothrombin propeptide-modified FX + human VKOR WT

• FVIIv1-psFX-IIa-F2: PS FXwt + propeptide FVII modified A-FX isoform + human VKOR WT (FIG. 3)

• FVIIv2-psFX-IIa-F2: PS FXwt + propeptide FVII isoform B-FX modified + human VKOR WT

• protc-psFX-IIa-F2: PS FXwt + propeptide modified protein C-FX + human VKOR WT.

The different sequences used in the examples are represented in the following Table 1:

Proth- psFX- QHVFLAPQQARS

LLQRVRR ANSFLEEMKKGHLERECMEETCSYEEARE

He has*

(SEQ ID (SEQ ID NO: 13) VFEDSDKTNEFWNKYKDGDQCETSPCQN

QGKCKDGLGEYTCTCLEGFEGKNCELFTR

NO: 21)

KLCSLDNGDCDQFCHEEQNSVVCSCARGY

FVIIvl- AGGVAKASGGET

TLADNGKACIPTGPYPCGKQTLERRKRSV

psFX- RDMPWKPGPHR

AQATSSSGEAPDSITWKPYDAADLDPTENP

IIa * (SEQ MGRPLHL VFVTQEEAHGVL

FDLLDFNQTQPERGDNNLTRDFLAEGLTPR

ID VLLSASLA HRRRR IVGGQECKDGECPWQALLINEENEGFCGG

NO: 22) GLLLLGES (SEQ ID NO: 14)

TILSEFYILTAAHCLYQAKRFKVRVGDRNT

FVIIv2- LFIRREQA

EQEEGGEAVHEVEVVIKHNRFTKETYDFDI

psFX- (SEQ ID AVFVTQEEAHGV

AVLRLKTPITFRMNVAPACLPERDWAEST

Ila * NO: 7) LHRRRR

LMTQKTGIVSGFGRTHEKGRQSTRLKMLE

(SEQ ID (SEQ ID NO: 15)

VPYVDRNSCKLSSSFIITQNMFCAGYDTKQ

NO: 23)

EDACQGDSGGPHVTRFKDTYFVTGIVSWG

ProtC- EGCARKGKYGIYTKVTAFLKWIDRSMKTR psFX- TPAPLDSVFSSSE GLPKAKSHAPEVITSSPLK

Ila * RAHQVLRIRKR (SEQ ID NO: 17)

(SEQ ID (SEQ ID NO: 16)

NO: 24)

mutant according to the invention

Example 2 Production of FX Containing Modified Propeptides in the Production Line HEK 293 Freestyle

1. Reagents

F17 Freestyle ™ Culture Medium

L-glutamine

Transfection medium of HEK cells: Opti-MEM

Vitamin KI 2. Protocol

Wild type factor X and modified FX were produced in HEK-293-Feestyle eukaryotic cells (HEK 293F) in transient expression.

HEK 293F were cultured in F17 medium, supplemented with 8 mM L-glutamine, under stirring conditions at 135 rpm in a controlled atmosphere (8% CO ₂ ) at 37 ° C. On the day before the day of transfection, the cells were seeded at a density of 7 × 10 ⁵ cells / ml. On the day of the transfection, the DNA (30 μg) and 60 μl of transfection agent (AT) were preincubated separately in Opti-MEM medium for 5 minutes and then mixed and incubated for 20 minutes to allow the formation of the DNA / AT complex. The whole was added to a cell preparation of 1.10 ⁶ cells / ml in a volume of 30 ml.

In the case of co-transfections, the 2 vectors were added at different ratios to obtain a total amount of DNA of 20-30 μg. Immediately after transfection, vitamin K1 (5 μg / ml) was added to the medium. Transfection rates were assessed the day after transfection with a control plasmid expressing GFP (Green Fluorescent Protein). The productions were made in "batch" mode for 7 days. At the end of production, the cells and the supernatant were separated by centrifugation. The cells were removed and the supernatant was harvested, supplemented with 2mM PMSF and 10mM benzamidine, filtered in 0.22μιη, concentrated 10X and frozen.

Example 3 Production of FX Containing Modified Propeptides in the CHO-S Production Line 1. Reagents

Culture medium proCH04

L-glutamine

CHO-S cell transfection medium: Opti-Pro S FM

Vitamin Kl

2. Protocol

Wild-type factor X and modified FX were produced in CHO-S (Invitrogen) eukaryotic cells in transient expression.

The CHO-S were cultured in proCH04 medium, supplemented with 4 mM L-glutamine, under stirring conditions at 135 rpm in a controlled atmosphere (8% CO ₂ ) at 37 ° C. On the day before transfection, the cells were seeded at a density of 6.10 ⁵ cells / ml.

On the day of the transfection, the DNA (37.5 μg) and 37.5 μΐ of transfection agent (AT) were pre-incubated separately in Opti-Pro SFM for 5 minutes and then mixed and incubated for 20 minutes to allow the formation of the DNA / AT complex. The whole was added to a cell preparation of 1.10 ⁶ cells / ml in a volume of 30 ml.

In the case of co-transfections, the 2 vectors were added at different ratios to obtain a total amount of DNA of 20-45 μg. Immediately after transfection, vitamin K1 (5 μg / ml) was added to the medium. Transfection rates were assessed the day after transfection with a control plasmid expressing GFP. The productions were made in "batch" mode for 7 days. At the end of production, the cells and the supernatant were separated by centrifugation. The cells were removed and the supernatant was harvested, supplemented with 2mM PMSF and 10mM benzamidine, filtered in 0.22μιη, concentrated 10X and frozen.

EXAMPLE 4 Quantification of Gamma-carboxylation of X Factors Produced 1- Experimental Protocol: Measurement of Factor X Concentration

Factor X concentration was measured using the commercial ELISA Zymutest Factor X (HYPHEN BioMed ref RK033A) following the recommendations from the manufacturer. The concentrations were measured in duplicate using antigen values located in the linear detection zone of the assay. To ensure that the introduced mutations do not interfere with the measurement of the concentration, the FX were deposited in identical quantities and revealed by immunoblotting with a polyclonal antibody different from that used in ELISA (Polyclonal anti-human antibody FX (CRYOPEP cat n ° PAHFX-S) or staining after SDS-PAGE (data not shown).

FX variant concentrations present in supernatants of transfected HEK cells were measured to deposit the same amount of FX on the anti-Gla ELISA.

2- Experimental protocol: Gamma-carboxylation rate measurement

The gamma-carboxylation rate was measured using a laboratory-based ELISA using the ELISA assay antibody Zymutest Factor X (Hyphen) and the anti-Gla antibody (Sekisui) as the capture antibody.

The anti-Gla antibody (200 μl at 5 μg / ml) was incubated overnight at room temperature (RT). After incubation, the plate was saturated with PBS + 1% BSA (250 μl / well) for 2 h at RT. After washing, 200 μl of sample at 0.2 μg / ml or standards (consisting of a mixture of different ratios of plasma FX and factor X produced in CHO (non-gamma-carboxylated) were deposited for 2 hours at RT. the anti-FX antibody coupled to the peroxidase (200 μl of the ZYMUTEST kit) was diluted in the buffer provided and incubated for 1 h at RT After washing, the revelation was made by adding 200 μl of TMB for 8 minutes. revelation was stopped by 50 μΐ _^ of 0.45 M sulfuric acid and the optical density was read at 450 nm.

3- Results

HEK cells naturally produce ectopic FX in a non-gamma carboxylated form (not shown). To advantageously increase the level of gamma-carboxylation, the FX was co-transfected in the presence of VKOR. This co-transfection can be done either by treating the cells with two vectors (Opti-HEK-FX-IIa-F2) or by using a bicistronic vector carrying the two cDNAs (Opti-HEK-VKOR-IIa). In both cases, the gamma-carboxylation rate was 11.55% and 10.20% plasma FX respectively (Table 2). Integral substitution of the signal peptide of FX and propeptide with those of FVII (v1 and v2), prothrombin or protein C did not significantly increase the level of gamma-carboxylation (6.7 to 24.5%). ). However, the combination with FVIIv1 (FVIIv1-FX-IIa-F2) is the most effective of the 4 to 24.5%.

Chimeric constructs were then constructed retaining the FX signal peptide and inserting the previously used propeptides. The new constructs thus generated are surprisingly advantageous in terms of gamma-carboxylation, especially that with the propeptide FVIIvl (FVIIvl-psFX-IIa-F2) for which a rate of 52% was observed.

Thus, this latter construction makes it possible to increase the gamma-carboxylation rate by 4.7 times. The set of individual measurements has been presented in FIG. 4 which shows the superiority of the use of the combination FX and the propeptide FVIIvl.

Table 2: Evaluation of gamma-carboxylation of the different mutants

Example 5 Purification of FX Containing Altered Propeptides on an Aptamer Column Capable of Setting the Factor X Gla Domain 1. Protocol

The concentrated culture supernatant from HEK or CHO was thawed at 37 ° C. It was then diluted to ½ in equilibration buffer (50 mM Tris HCl, 10 mM CaCl ₂ , pH 7.5) and then purified on an anti-Gla aptamer column which had been previously equilibrated in the same buffer. The column was washed with 12 column volumes in equilibration buffer. FX was then eluted with 50 mM Tris-HCl buffer, 10 mM EDTA, pH 7.5. The column was returned to equilibration buffer (25 column volumes) before storage at 4 ° C. FX was treated with 2mM PMSF, concentrated, and stored at -80 ° C. 2. Results

FX-FIIa-F2 produced from CHO or HEK were purified on an aptamer recognizing the gamma-carboxyl domain. The CHO product was purified according to a standard immunopurification protocol or by aptamopurification. The purified products were controlled by SDS-PAGE 4-10% (Figure 5A). The two recombinant products showed a similar profile following acrylamide separation with or without DTT reduction (Figure 5 A, lane 4 and 3). Not reduced, the products appeared as a single band at around 60-65 kDa. Their migration was a little slower than that of plasma FX (Figure 5 A, lane 2) because the products have 10 additional amino acids. The product reduction completely separates the heavy chain (48 kDa) from the light chain (17 kDa). The recombinant FX showed a similar profile irrespective of their mode of purification. The light chain of the three purified X factors migrated in the same way, as expected.

The aptamopurified HEK product was compared to plasma FX (Figure 5B). The product was pure with homogeneity and appeared as a single migrating band at a molecular weight slightly higher than that of plasma FX as previously seen (Figure 5B, lane 3). The reduction of the product shows that the migration difference is carried by the heavy chain. These data show that aptamopurification, for example of FX-IIa-F2, makes it possible to obtain a pure product with homogeneity after a single purification step.

EXAMPLE 6 Measurement of the Activation of X Variant Factors Produced in HEK by the RVV-X Venom Fraction

1. Experimental protocol

Activation of FX variants produced by HEK cells was measured following the incubation of aptamopurified X factors in the presence of the anti-factor X fraction of venom of the Russell's viper (RVV-X). Activated control factor X, venom X moiety (RVV-X) and pNAPEP 1065 substrate were commercially available (e.g. Haematologic Technologies).

Activation was studied at 37 ° C in the following buffer: 25 mM HEPES, pH 7.4, 0.175 M NaCl, 5 mM CaCl ₂ , 5 mg / ml BSA. For concentrations of 0 to 100 nM FX, a concentration of 200 mU / ml of RVV-X was used. After 5 min of incubation, the reaction was stopped in 50 mM Tris buffer, pH 8.8, 0.475 M NaCl, 9 mM EDTA. The amount of FXa generated was monitored by measuring the rate of hydrolysis of the pNAPEP 1065 (250 μΜ) substrate at 405 nm. 2. Results

Purified factor X variants were incubated with RVV-X. The generation of FXa was measured following this treatment from different concentrations of FX. The presence of FXa was quantified by the rate of appearance of the pNAPEP 1065 product in solution (in mUDO / min). This generation is a reflection of the recognition and cleavage of FX by the RVV-X as well as the FXa's ability to recognize the FX substrate. The average onset rates were made for the different initial FX concentrations and this value was reduced as a percentage of the FX-WT value.

The controls of the FX already activated and the FX processed by the RVV-X both gave a positive signal of the same order of magnitude (Figure 6). The FXa control was considered the 100%. Both variant X-factor constructs were activated at about 60% FXa (54% for Opti-HEK-VKOR-IIa and 63% for FVIIv1-psFX-IIa-F2). This The result was not surprising because activation with RVV-X is not sensitive to gamma-carboxylation. This result has however advantageously shown that the modification of the propeptide does not lead to a loss of the chromogenic activity of FXa.

EXAMPLE 7 Measurement of the Activation of the X Variants Produced in HEK by the Factor VIIa / Tissue Factor (FT) Complex

1. Experimental protocol

Activation of variant FX produced by HEK cells was measured following incubation of the aptamopurified product in the presence of 50 μM FVIIa and tissue factor. In a flat-bottomed plate, the FVIIa complex (100 μΐ to 100 μM) - FT was added to the various dilutions of FX (100 μΐ). After 10 min, the mixture (20 μl) was removed and deposited in 180 μl of STOP buffer (50 mM Tris, 9 mM EDTA, 475 mM NaCl, pH 8.8). The PNAPEP substrate diluted ½ in PPI water (50 μl) was added and an immediate reading in kinetic mode every 25 seconds was made for 10 min at 405 nm.

2. Results

The controls represented by the already activated plasma FX and the plasma FX treated with FVIIa / TF both gave a positive signal of the same order of magnitude (Figure 7). Plasma FXa control was considered 100%. The two variant X factor constructs were activated at 27% FXa for Opti-HEK-VKOR-IIa and 36% for FVIIv1-psFX-IIa-F2. This activity is sensitive to the rate of gamma-carboxylation. Modification of the propeptide allowed FVIIv1-psFX-IIa-F2 to have an activity of 142% of that of the molecule containing the wild-type propeptide. These results indicate that the increase in the level of gamma-carboxylation makes it possible to increase the procoagulant activity of the FXa variant relative to the control molecule. EXAMPLE 8 Measurement in Thrombin Generation Time (TGT) of the Procoagulant Factor of Variant X Factors: Activation of the Extrinsic Coagulation Pathway (FT 1 μM / PL 4 μM) in FVIII-deficient Plasma 1. Experimental Protocol

1.1. Reagents

Thrombin calibrator, PPP reagent low, CK-Perst, Fluca Kit (Fluo-buffer + Fluo- substrate) and PNP were commercially available, for example at Stago. FVIII-deficient plasma (e.g. Siemens Healthcare) and recombinant human factor VIII control comes from Baxter (Advate).

1.2. Protocol

The thrombin generation test consists in activating ex vivo coagulation using a mixture of tissue factor and phospholipids (activation of the extrinsic coagulation pathway) and then measuring the thrombin concentration generated over time. .

The thrombin generation tests were performed on 80 μl of a pool of plasma containing purified product or controls, in the presence of 20 μl of PPP reagent containing in the end 1 μM of Tissue Factor (TF) and 4 μl of phospholipids. (PL). Different plasmas can be used: normal plasma, deficient factor X, deficient factor VIII, deficient plasma factor IX or deficient plasma FXI The reaction was started by the addition of 20 μΐ _^ Fluca-kit (substrate + CaCl ₂ ) which constitutes the beginning of the measurement of the appearance of thrombin. The appearance of fluorescence was measured on a Fluoroskan Ascent fluorimeter (ThermoLabsystems) at an excitation wavelength of 390 nm and at an emission wavelength of 460 nm. Thrombinograms (curves representing the fluorescence intensity versus time) were then analyzed using the Thrombinoscope ™ software which converts the fluorescence value into nM thrombin by comparative calculation.

2. Results Plasmas Unicalibrator, as well as plasmas deficient in FVIII reconstituted with 0, 0.1 or 1 U / ml of recombinant FVIII were used as controls. The aptamopurified FVIIv1-psFX-IIa-F2 was used at 10 and 20 μg / ml.

As expected following the activation of coagulation by tissue factor, the FVIII deficient plasma gave the weakest signal, corresponding to the background noise of the experiment (Figure 8). Plasma Unicalibrator provided a weaker signal than FVIII deficient plasma reconstituted by FVIII concentrations (0.1 or 1 U / ml). Variant FX with a modified propeptide has the ability to correct a FVIII deficient plasma as effectively as FVIII. A dose response was observed with a lag time that is shortened as the dose increases and an amplitude increases. The amplitude of the signal, however, did not completely reach reconstitution with 1 U / ml of FVIII but was much higher than that of normal plasma. Consequently, the increase in gamma-carboxylation obtained for a factor X which varies according to the invention, advantageously coupled with an aptamopurification, has made it possible to obtain a perfectly active variant factor X which effectively replaces FVIII.

Example 9 Production of FX-Fc Containing a Modified Propeptide in the YB2 / 0 Production Line

1. Reagents

Culture medium: EMabprol

L-Glutamine 200mM

Geneticin 50 mg / ml

LSI 00

Vitamin Kl

2. Protocol

The modified FX FVIIv1-psFX-IIa-F2-Fc was cloned into a bicistronic vector optimized for expression in the YB2 / 0 line in which a nucleic sequence coding for human furine at the level of the second was introduced. transcription unit. The amount of vector required for transfection was then prepared and linearized at the EcoRV restriction site. After centrifugation, the YB2 / 0 cells were taken up in a volume making it possible to obtain a cell density of 1.10 cells / ml. The transfection was carried out by electroporation using a specific kit (ref: EB110, Ozyme) at 5 × 10 ⁶ cell / ml in the presence of 61 μg of bicistronic vector containing the sequence FVIIv1-psFX-IIa-F2-Fc. and the sequence of human furin. After transfection, the cells were resuspended in 75 cm ² flasks. Selection pressure was added three days after transfection by adding G418 at 0.6 g / l. The selection pressure was maintained for 14 days and then the cells were frozen. Productions of the FVIIv1-psFX-IIa-F2-Fc molecule were initiated by seeding the selected YB2 / 0 cells at a density of 3.10 ⁵ cells / ml in Emabprol medium containing 4 mM glutamine. For production, a fed-batch mode was applied for 12 days by adding glucose and glutamine based on pre-determined cell density.

At the end of production, the cells and the supernatant were separated by centrifugation. The cells were removed and the supernatant was harvested, supplemented with 2mM PMSF and 10mM benzamidine, 5X concentrate, filtered in 0.22μιη, and then frozen.

Example 10 Purification of FX-Fc Containing Modified Propeptides on an Aptamer Column Capable of Fixing the Gla Domain of Factor X

1. Protocol

FVIIv1-psFX-IIa-F2-Fc was produced in HEK293F, CHO-S and YB2 / 0 as described in Examples 2, 3 and 9.

The concentrated culture supernatant from HEK, YB2 / 0 or CHO-S was thawed at 37 ° C. and then filtered on Nalgene 0.2 μιη unit (aPES). For a volume of supernatant after filtration, a volume of 50 mM Tris-HCl buffer pH 7.5 was added. QAE Sephadex A50 gel (0.25% w / v; GE Healthcare) was added and the whole was stirred for one hour at + 4 ° C. The gel was loaded into a column body, washed with the equilibration buffer and the molecules of interest were eluted with 50 mM Tris-HCl buffer pH7.5 500 mM NaCl. The eluate was then frozen at -80 ° C before aptamopurification. The thawed eluate was diluted ½ in equilibration buffer (50 mM Tris HCl, 10 mM CaCl ₂ , pH 7.5) and then purified on an anti-Gla aptamer column which had been previously equilibrated in the same buffer. . The column was washed with 12 column volumes in equilibration buffer. FVIIv1-psFX-IIa-F2-Fc was then eluted with 50 mM Tris-HCl buffer, 10 mM EDTA, pH 7.5. The column was returned to equilibration buffer (25 column volumes + 0.01% sodium azide) before storage at 4 ° C. FVIIv1-psFX-IIa-F2-Fc was treated with 0.01 mM GGACK inhibitor (Cryopep), dialyzed against 0.9% NaCl, concentrated, and stored at -80 ° C. 2. Results

FVIIv1-psFX-IIa-F2-Fc produced from CHO-S, YB2 / 0 or HEK were purified on an aptamer recognizing the gamma-carboxyl domain. The purified products from CHO-S or HEK were controlled by SDS-PAGE 4-10% (Figure 9). They showed a similar profile following acrylamide separation with or without DTT reduction (Figure 9, lanes 3-5, 7-9). Not reduced, the products appeared in the form of a majority band at about 250 kDa migrating very differently from that of the plasma FX (67 kDa, Figure 9, lane 1) because the products are grafted to an Fc fragment. A minority band at 135 kDa was also detected only in CHO products. The reduction of the products completely separates the Fc-grafted heavy chain (81 kDa) from the light chain (17 kDa). FX-Fc variants showed a similar profile regardless of their mode of production. The light chain of the three purified X factors migrated in the same way, as expected, with however greater heterogeneity in the product derived from CHO. Note that a profile similar to that of HEK was obtained with the product from YB2 / 0 (not shown).

These data show that aptamopurification, for example FVIIv1-psFX-IIa-F2-Fc, makes it possible to obtain a pure product with homogeneity after a single purification step, even if this material is produced from different cell lines. The presence of a modified propeptide does not affect the ability of the product to be purified by this method.

Example 11: Binding of FX-Fcs Produced in Different Phospholipid Cell Lines 1. Protocol

The phopholipids were diluted to 12.5 μΜ in absolute ethanol and then loaded into 96-wells. They were incubated overnight at room temperature without a lid. The wells were then saturated for 2 h with 50 mM Tris buffer, 150 mM NaCl, CaCl _2.

10 mM, 1% BSA, pH 7.5. After saturation, the wells were washed 5 times with the washing / dilution buffer (50 mM Tris, 150 mM NaCl, 10 mM CaCl ₂ , 0.1% BSA, pH 7.5) and the samples were deposited at different levels. dilutions and incubated for 2 hours at room temperature. They were then washed 5 times before incubation with the anti-FX antibody coupled with peroxidase. The wells were washed 5 times before revelation: 3 minutes at room temperature with TMB (Zymutest FX kit). The reaction was stopped by the addition of 50 μl of 0.45 M sulfuric acid. The OD at 450 nm were read after shaking the plate slightly. 2. Results

Plasma factor X control (x) binds as expected to phospholipids depending on the starting concentration. The signal tends to saturation. Unpurified FVIIvl-psFX-IIa-F2-Fc present in the supernatants of CHO-S, HEK293 and YB2 / 0 and the same aptamopurified products were evaluated. All supernatant, non-purified forms are less well-bound to phospholipids than aptamopurified forms. This signal difference may be due either to the presence of inhibitory molecules or binding competitors in the supernatants or, most likely, that the supernatants contain a fraction of the weakly gamma-carboxylated product and that they bind therefore less well with phospholipids. Passage on the aptamopurification column has increased the ability of the three products to bind phospholipids: the product from CHO is the one for which the signal has been the most improved, then comes the product YB2 / 0 and then the HEK293. After aptamopurification, all the products bound to the phospholipids giving a signal at least as strong as that of the plasma FX or even higher for the products HEK293 and

YB2 / 0. These data show that FX-IIa-F2-Fc produced in the presence of a modified propeptide (eg FVIIv1-psFX-IIa-F2-Fc) have an excellent ability to bind to phospholipids, a binding known to be mediated by different gamma-carboxylation sites.

Example 12 Activation of FX-Fc Produced in Various Cell Lines by Thrombin

1. Protocol

The experiments and dilutions were conducted in the following reaction buffer: 25 mM Hepes, 175 mM NaCl, 5 mg / ml BSA, 5 mM CaCl ₂ pH 7.4. The standard range was prepared as follows: in a flat-bottomed plate, 100 μΐ r-Hirudine at 50 nM + 100 μΐ of each dilution of FXa + 50 μΐ of PNAPEP substrate diluted ½ in PPL water Immediate reading in kinetic mode all 25 seconds for 10 min at 405 nm was performed. The tests were carried out by adding to 100 μl of sample at 200 nM, 100 μl thrombin at 20 nM final concentrations 100 nM FX / 10 nM Ha.

The mixture was then incubated at 37 ° C. and then at different times 0, 0.5, 1, 2, 3.5, 6 and 8 h, a 20 μΐ aliquot was taken and placed in a well of a flat-bottom microplate containing 180 μl of r-Hirudine at 50 nM. The pNAPEP 1065 substrate (50 μl) diluted ½ in PPI water was added and an immediate kinetic reading every 25 seconds was carried out for 10 min at 405 nm.

2. Results

The incubation of plasma factor X in the presence of thrombin did not lead to the appearance of FXa activity confirming that this molecule is not activatable by thrombin.

In contrast, the FVIIv1-psFX-IIa-F2-Fc molecule produced in three HEK293, CHO-S and YB2 / 0 cell lines was sensitive to this activation and showed FXa linearly over time. The amount of FXa generated was slightly higher with the product from YB2 / 0.

These data indicate that the presence of a different antigen than factor X does not alter the ability of the molecule to be activated by thrombin. EXAMPLE 13 Measurement in Thrombin Generation Time (TGT) of the procoagulant capacity of FX-IIa-F2-Fc: Activation of the extrinsic coagulation pathway (FT 1 μM / PL 4 μM) in FVIII deficient plasma

1. Protocol

A protocol identical to that described in Example 8 was applied.

2. Results

Controls, factor VIII-deficient plasma reconstituted with 0.1 U / ml of factor VIII (□) or 1 U / ml of factor VIII (■) allow thrombin generation which increases with the level of FVIII. Normal plasma gives a median signal between these two conditions (·). The presence of the FVIIv1-psFX-IIa-F2-Fc molecule (4 μg / ml) makes it possible to correct the deficiency of FVIII. The factor produced in HEK293 gives a stronger signal than that of normal plasma with identical lagtime. It is, however, a little higher than that of deficient plasma + 1 U / ml factor VIII.

The product from YB2 / 0 also gives a powerful signal but with an increased lagtime. The product from CHO gives a more moderate signal with a lagtime still increased but able to correct the deficiency in FVIII.

These data show that FVIIv1-psFX-IIa-F2-Fc possessing the propeptide as described in SEQ ID NO: 14 and produced in different cell lines has the ability to restore factor VIII deficiency.

Claims

A protein which is a factor X variant comprising a mutated sequence of SEQ ID NO: 1, characterized in that said mutated sequence of SEQ ID NO: 1 comprises an A, A ', B, C or C mutation, wherein :

where N * is an optionally glycosylated asparagine, and

said protein comprising at its N-terminus the signal peptide of sequence SEQ ID NO: 7 fused to a propeptide different from the natural propeptide of factor X.

2. Protein according to claim 1, characterized in that the mutated sequence of SEQ ID NO: 1 comprises mutation B.

3. Protein according to one of claims 1 to 2, characterized in that the propeptide different from the natural propeptide factor X is selected from the propeptide of thrombin, the propeptide factor VII, the propeptide of protein C, and their modified versions.

4. Protein according to one of claims 1 to 3, characterized in that the propeptide is selected from the sequences SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16.

5. Protein according to one of claims 1 to 4, characterized in that the signal peptide of sequence SEQ ID NO: 7 fused to a propeptide different from the natural propeptide factor X is selected from the sequences SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 2l.

6. Protein according to one of claims 1 to 5, characterized in that it comprises, between the signal peptide sequence SEQ ID NO: 7 fused to a propeptide different from the natural propeptide factor X, and the mutated sequence of SEQ ID NO: 1, an intermediate sequence.

7. Protein according to claim 6, characterized in that the intermediate sequence is the sequence of the light chain of factor X, preferably the sequence SEQ ID NO: 5.

8. Protein according to one of claims 1 to 7, characterized in that it comprises, from the N-terminus to C-terminus:

the sequence SEQ ID NO: 5, then

said mutated sequence of SEQ ID NO: 1.

9. Protein according to one of claims 1 to 8, characterized in that it comprises, from the N-terminus to C-terminus:

the sequence SEQ ID NO: 5, then

the sequence SEQ ID NO: 11.

10. Protein according to one of claims 1 to 9, characterized in that it comprises, preferably consists of, a sequence selected from SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24 and SEQ ID NO: 25.

11. Protein according to one of claims 1 to 10, characterized in that it is fused at the C-terminal end to at least one wild-type Fc fragment or at least one wild-type scFc fragment, optionally mutated.

12. Protein according to claim 11, characterized in that the wild-type Fc fragment has the sequence SEQ ID NO: 36 or SEQ ID NO: 37, optionally followed by a C-terminal lysine.

13. Protein according to claim 12, characterized in that the wild-type scFc fragment has the sequence SEQ ID NO: 42.

14. Protein according to one of claims 11 to 13, characterized in that it has the sequence SEQ ID NO: 40 or SEQ ID NO: 43.

15. Protein according to one of claims 11 to 14, characterized in that the wild-type Fc fragment or the wild-type scFc fragment is mutated to include the T366Y or Y407T mutation.

16. A nucleic acid characterized in that it encodes the protein according to one of claims 1 to 15.

17. Nucleic acid according to claim 16, characterized in that it is chosen from the sequences SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34 and SEQ ID NO: 35.

An expression cassette comprising the nucleic acid of claim 16 or 17.

Expression vector, characterized in that it comprises the expression cassette according to claim 18.

An expression vector according to claim 19 for use as a medicament, preferably as a gene therapy medicament.

21. A recombinant cell comprising the nucleic acid according to claim 16 or 17, said nucleic acid being preferentially included in the vector according to claim 19.

22. Protein according to one of claims 1 to 15 for its use as a medicament.

23. Protein according to one of claims 1 to 15 for use in treating bleeding disorders.

24. Process for producing a protein according to one of claims 7 to 15, comprising:

a) the expression of a polycistronic vector, preferably bicistronic, in a host cell, preferably a CHO cell, said vector comprising a polynucleotide encoding a protein according to the invention, and a polynucleotide encoding the VKOR enzyme, preferably for subunit 1 of the wild-type human vitamin K epoxide reductase (VKORC1) complex, preferably in the presence of vitamin K;

b) culturing said host cell;

c) recovering the cellular supernatant;

d) optionally at least one of the steps selected from:

the neutralization of activated proteases by the addition of protease inhibitors; e) purification of the protein according to the invention by passing the production supernatant obtained in c) or d) on an aptamer column capable of binding to the Gla domain of factor X.

25. Process for producing a protein according to one of claims 7 to 15, comprising:

a) the expression of two expression vectors, one comprising a polynucleotide encoding a protein according to the invention, and the other comprising a polynucleotide encoding the VKOR enzyme mentioned above, in a host cell preferably a CHO cell, preferably in the presence of vitamin K; b) culturing said host cell;

c) recovering the cellular supernatant;

d) optionally at least one of the steps selected from: