Combination of factor vii and a Anti-factor ix and factor x bispecific antibody

Abstract

The invention relates to a combination containing transgenic factor VII and a multispecific antibody against factors IX and X for simultaneous or separate administration.

Description

[0001] Combination of Factor VII and a bispecific antibody against Factors IX and X

[0002] The invention relates to pharmaceutical compositions useful for the treatment of a coagulation disorder, such as hemophilia A, especially in a patient with type A hemophilia with development of factor VIII inhibitory antibodies.

[0003] Technological background

[0004] Blood clotting involves two pathways, one intrinsic and the other extrinsic, which converge into a common final pathway. The combination of these two mechanisms ensures the formation of a solid and flexible blood clot that resists blood pressure. Under the influence of thrombin, fibrinogen undergoes chemical modifications that result in the formation of fibrin. Fibrin is essential for clot formation.

[0005] The intrinsic pathway involves factors present in the bloodstream, and the coagulation process begins within the blood vessel itself. The extrinsic pathway, on the other hand, involves tissue factors not normally present in the bloodstream, which are released during vascular injury.

[0006] Factor VII is a glycoprotein involved in the extrinsic pathway of blood coagulation. To initiate the coagulation cascade, factor VII must be activated to factor VIIa. Once activated, factor VIIa binds to tissue factor (TF), a phospholipid-associated protein released upon vascular injury. Factor VIIa alone (not bound to tissue factor) exhibits weak proteolytic activity. The FVIIa-TF complex then converts factor X to factor Xa in the presence of calcium ions. This complex also activates factor IX to factor Xa, thus catalyzing the intrinsic pathway. Factors IXa and Xa, in turn, activate factor VII.

[0007] Factor IX and factor X are involved in the intrinsic pathway of coagulation. Activated factor IX allows the activation of factor X to factor Xa.

[0008] Factor Xa, complexed with activated factor V and prothrombinase, converts prothrombin to thrombin. Thrombin then acts on fibrinogen, converting it to fibrin and also activating factor VIII and factor V into factor VIIa and factor Va, respectively. Thrombin, in turn, activates factor XIII into factor IIIa, which is responsible for the consolidation of the fibrin clot in the presence of calcium, naturally present in the plasma.

[0009] However, when a coagulation factor is missing, the coagulation cascade is interrupted or deficient, resulting in abnormal coagulation. Activated factor VII acts locally in the presence of tissue factor released after tissue injury that causes bleeding, even in the absence of factor VIII or IX. Therefore, factor VII, preferably in its activated form, is used to treat certain blood clotting disorders that manifest as bleeding.

[0010] Factor VII is used to treat patients with hemophilia who have a deficiency in factor VIII (hemophilia A) or factor IX (hemophilia B), as well as patients with other coagulation factor deficiencies, such as an inherited deficiency in factor VII. Factor VII is also recommended for the treatment of strokes.

[0011] Some hemophilia patients develop inhibitory antibodies against Factor VIII, which is administered, usually in concentrated form, as a treatment for hemophilia. This is currently the most frequent complication of hemophilia treatment.

[0012] Bispecific antibodies targeting FIX or FIXα and FX or FXα, such as emicizumab, are used to treat patients with hemophilia A who have factor VIII inhibitors. These antibodies functionally replace FVIII by promoting FX activation through FIXα by bringing these two molecules into close proximity. These antibodies have a long-lasting effect.

[0013] The combination of recombinant FVIIa from cell culture (such as Novoseven®, produced in BHK cells) with emicizumab (such as ACE910 or Hemlibra®) has been tested (R. HARTMANN, et al. OR36 / Synergistic Effects of a Procoagulant Bispecific Antibody and FEIBA or Factor VII A on Thrombin Generation (Haemophilia (2017), 23 (Suppl. 2), 11-27)). This combination shows only an additive effect on the treatment of coagulation disorders.

[0014] There is therefore a need for a pharmaceutical combination that allows for better management of hemophilia A patients, and more particularly of patients with anti-factor VIII.

[0015] Summary of the invention

[0016] The invention proposes to combine transgenic factor Vil with a multispecific antibody directed against factors IX and X.

[0017] According to the invention, the combination of factor VII obtained by transgenesis and antibodies directed against factor IX and factor X induces a synergistic effect in the treatment of coagulation disorders, and particularly for the treatment of hemophilia A patients with anti-FVI 11 inhibitors and FVII deficient patients.

[0018] One aspect of the invention is therefore a pharmaceutical composition comprising:

[0019] a. of the transgenic factor Vil, and

[0020] b. a multispecific antibody, preferably bispecific, directed against factor IX and factor X, such as emicizumab.

[0021] Preferably, factor Vil is in the form of activated factor Vil (FVIIa).

[0022] In one particular embodiment, factor IX is in the form of activated factor IX (FIXa), and / or factor X is in the form of activated factor X (FXa).

[0023] Preferably, said transgenic factor Vil is a human factor Vil produced by the epithelial cells of the mammary glands of a transgenic non-human mammal, for example a transgenic rabbit for human factor Vil.

[0024] The invention also provides a combination product comprising:

[0025] a. of the transgenic factor Vil, and

[0026] b. a multispecific antibody directed against factor IX and factor X,

[0027] for its use in the prevention or treatment of a coagulation disorder, such as hemophilia A, more specifically hemophilia A with factor VIII (FVIII) inhibitors. Preferably, the combination product is in the form of a pharmaceutical composition that comprises both the transgenic factor VII and said antibody.

[0028] Alternatively, the transgenic factor Vil and antibody are in the form of separate compositions, suitable for simultaneous or separate (e.g. sequential) administration to the patient.

[0029] Another object of the invention relates to a kit comprising:

[0030] A container containing a transgenic FVII factor; and

[0031] Another container containing an antibody directed against factor IX and factor X.

[0032] List of Fiaures:

[0033] Figure 1: Evaluation of the synergistic thrombogenic effect of the Sevenfact™ + Hemlibra® combination on batch 1 of Hemophilia A plasma after TF / PL induction. (A) Evaluation of the synergistic thrombogenic effect on PTE, (B) Evaluation of the synergistic thrombogenic effect on peak thrombin generation, (C) Evaluation of the synergistic thrombogenic effect on velocity.

[0034] Figure 2: Evaluation of the synergistic thrombogenic effect of the Sevenfact™ + Hemlibra® combination on batch 2 of Hemophilia A plasma after TF / PL induction. (A) Evaluation of the synergistic thrombogenic effect on PTE, (B) Evaluation of the synergistic thrombogenic effect on peak thrombin generation, (C) Evaluation of the synergistic thrombogenic effect on velocity.

[0035] Detailed description of the invention

[0036] General definitions

[0037] The coagulation process involves a cascade of enzymatic reactions involving coagulation factors present as proenzymes which, in the presence of certain cofactors, are converted by proteolytic cleavage into their "activated" form. The activated form of each factor present as an inactive precursor is designated by the letter a. Thus, FVIIa results, in vivo, from the cleavage of zymogen by different proteases (FIXa, FXa, FVIIa) into two chains joined by a disulfide bridge.

[0038] The term "treatment" or "to treat" generally refers to the improvement, prevention, or reversal of a disease or disorder, or at least of a symptom, for example, slowing the progression of a disease or stabilizing a symptom. It also includes delaying the onset of a disease or disorder, or at least of a symptom.

[0039] The term "prevention" or "prevent" refers to a reduction of the risk of developing or acquiring a specified disease or disorder.

[0040] For the purposes of this invention, "patient" or "subject" means any mammal, and more particularly human beings, male or female, of any age, including children.

[0041] The term "pharmaceutical composition" refers to preparations that allow the biological activity of the active ingredients and do not contain any additional components that are toxic to the subjects to whom the composition is administered.

[0042] Transgenic Factor VII

[0043] The term “Factor VII” or “FVII” includes polypeptides comprising the 1-406 sequence of wild-type human Factor VII (as described in US patent 4,784,950), or of FVII derived from another species (e.g., bovine, porcine, canine, murine). It further includes any naturally occurring allelic variations of Factor VII, and any form or degree of glycosylation or other post-translational modification. The term “Factor VII” also includes variants of FVII that exhibit the same or greater biological activity than the wild-type form; these variants include, in particular, polypeptides differing from wild-type FVII by the insertion, deletion, or substitution of one or more amino acids.

[0044] Unless otherwise indicated, in this description the term “Factor Vil” refers indifferently to uncleaved FVII (zymogen) or activated Factor Vil (FVIIa).

[0045] FVIIa is therefore composed of a light chain of 152 amino acids with a molecular weight of about 20 kDa and a heavy chain of 254 amino acids with a molecular weight of about 30 kDa linked together by a single disulfide bridge (Cys135-Cys262).

[0046] The term "recombinant factor VII" refers to any genetically engineered factor VII resulting from the expression of the corresponding gene in any microorganism, plant, or transgenic plant. Microorganism is defined as any bacterial, fungal, viral, or cellular system. Recombinant factor VII can also be produced from cultured eukaryotic cells, such as plant or mammalian cells, for example, animal or human cells.

[0047] By "transgenic factor VII" is meant any recombinant factor Vil obtained from an animal transgenic for factor Vil.

[0048] The term "transgenic animal" refers to any non-human animal with a modified genome designed to allow the expression of a protein of interest (in this case, factor Vil). The genome modification may result from the alteration, modification, or insertion of a gene. This modification may be due to the action of conventional altering or mutagenic agents or carried out by site-directed mutagenesis. The genome modification may also result from the insertion or replacement of genes in their wild-type or mutated form. The transgenic animal may be chosen, without limitation, from among rabbits, goats, cows, camels, hamsters, mice, rats, horses, sows, dromedaries, sheep, and llamas. In one particular embodiment, an animal not expressing α1,3-galactosyltransferase may be chosen.

[0049] The term "factor VIIa biological activity" refers to the ability of FVIIa to generate thrombin, for example, on the surface of activated platelets. Factor VIIa activity can be assessed in various ways. For example, FVIIa biological activity can be quantified by measuring the ability of an FVII composition to promote blood clotting using FVII- and thromboplastin-deficient plasma, as described, for instance, in US Patent No. 5,997,864. In this assay, biological activity is evaluated relative to a control sample and converted into "FVII units" by comparison with pooled standard human serum containing 1 unit / ml of Factor VIIa activity.Alternatively, the biological activity of Factor VII can be quantified by (i) measuring the ability of Factor VIIa to produce Factor Xa in a system comprising tissue factor (TF) enclosed in a lipid membrane and Factor X (Persson et al. J. Biol. Chem. 272: 19919-19924, 1997); (ii) measuring the hydrolysis of Factor X in an aqueous system; (iii) measuring the physical binding of FVIIa to TF via surface plasmon resonance (Persson, FEBS Letts, 413:359-363, 1997); (iv) measuring the hydrolysis of a synthetic substrate; or (v) measuring thrombin generation in a TF-independent in vitro system.

[0050] In a preferred embodiment, the FVII described here is a polypeptide whose peptide sequence can be that of natural human FVII, i.e., the sequence present in humans without FVII-related disorders. Such a sequence is described in document EP 0 200 421.

[0051] Advantageously, the FVII sequence used in the invention is the SEQ ID NO: 1 sequence.

[0052] By "synergy" or "synergistic effect," it is preferably understood that the effect of combining two products is greater than twice the sum of the effects of each product taken individually. According to the present invention, a synergistic effect is obtained when the use of a transgenic FVIIa in combination with a multispecific antibody directed against factor IX and factor X results in an effect greater than twice the sum of the effect obtained with a transgenic FVIIa alone and the effect obtained with a multispecific antibody directed against factor IX and factor X alone, on at least one thrombin generation parameter. The thrombin generation parameter is chosen from peak height, velocity, or endogenous thrombin potential (ETP).

[0053] In one particular embodiment, FVIIa is administered at a concentration less than or equal to 105 nM, preferably less than 100 nM.

[0054] In one particular embodiment, the multispecific antibody directed against factor IX and factor X is administered at a concentration of less than 600 nM, preferably less than 550 nM, preferably less than 500 nM, preferably less than 450 nM, preferably less than 400 nM, preferably less than 350 nM, preferably less than 325 nM. In another particular embodiment, factor V1 is obtained from the milk of a transgenic animal.

[0055] A method for producing a recombinant protein in the milk of a transgenic animal may involve the following steps: a synthetic DNA molecule containing a gene encoding a protein of interest (here, for example, human FVII), this gene being under the control of a promoter of a protein naturally secreted in milk, is inserted into a non-human mammalian embryo. The embryo is then placed in a female mammal of the same species. Once the mammal derived from the embryo has developed sufficiently, lactation is induced, and the milk is collected. The milk then contains the FVII of interest secreted by the transgenic animal.

[0056] An example of protein preparation in the milk of a female mammal other than a human is given in patent application EP0527063, the teachings of which can be taken up for the production of Factor Vil of the invention.

[0057] The secretion of factor Vil by the mammary glands, enabling its release into the milk of the transgenic mammal, necessitates tissue-dependent control of factor Vil expression. Such control methods are well known to those skilled in the art. Expression control is achieved through sequences that direct the protein to a specific tissue. These include, but are not limited to, WAP, beta-casein, and beta-lactoglobulin promoter sequences, as well as signal peptide sequences.

[0058] In a preferred embodiment, the Vil factor according to the invention is produced in the milk of transgenic rabbits.

[0059] Advantageously, expression in rabbit mammary glands is carried out under the control of the beta-casein promoter, which is well known to those skilled in the art. Specifically, a plasmid containing the beta-casein promoter is created by introducing a sequence containing the beta-casein gene promoter. This plasmid is designed to accommodate a foreign gene that is dependent on this promoter. The gene encoding human FVII is integrated and placed under the control of the beta-casein promoter. The plasmid containing the promoter and the sequence encoding the protein of interest is digested with restriction enzymes to release the DNA fragment containing the beta-casein promoter and the human FVII sequence. After purification, the fragments are introduced by microinjection into the male pronucleus of wild-type rabbit embryos.The embryos are then cultured before being transferred into the oviducts of hormonally prepared wild-type females. Upon birth, the offspring are evaluated by PCR to identify transgenic animals. The transgene copy number and integrity are determined using Southern blotting on DNA extracted from the resulting transgenic rabbits. The concentration of human FVII expressed in the milk of the transgenic female offspring is assessed using enzyme-linked immunosorbent assays.

[0060] In a particular embodiment, the Vil factor useful in the invention is obtained by a process comprising the following steps:

[0061] (a) the insertion of a DNA sequence comprising a gene encoding factor Vil into a non-human mammalian embryo, said gene being under the transcriptional control of the beta-casein promoter,

[0062] (b) the transfer of embryos obtained in step a) into the oviduct of female non-human mammals so that they develop into an adult non-human mammal,

[0063] (c) induction of lactation in the adult non-human mammal obtained in step b) of female type or in a female offspring of that non-human mammal in which the gene and promoter are present in its genome,

[0064] (d) the collection of milk from said non-human mammal, and

[0065] (e) the purification of FVII present in the collected milk.

[0066] The transgenic FVII useful here exhibits a substantially homogeneous isoelectric point.

[0067] The term "isoelectric point" or "pi" refers to the pH at which the net elementary charge of the factor VII or factor VI molecule is zero, i.e., the pH at which the molecule is electrically neutral (zwitterionic form). The isoelectric point of factor VII according to the invention can be measured using a technique well known to those skilled in the art, such as isoelectric focusing ("IEF"). This electrophoretic technique allows the separation of proteins based on their isoelectric point. It consists of the migration of proteins, induced by a uniform electric current, down a pH gradient until they reach a pH equivalent to their specific isoelectric point, at which point they cease to migrate since their net charge is zero. IEF gels are used to determine the isoelectric point of a given protein.

[0068] By "substantially homogeneous," it is understood that at least 90%, preferably at least 95%, of the Factor VII molecules in the composition have an isoelectric point within a pH unit deviation of 1.2 or less. In another embodiment of the invention, at least 50%, preferably at least 55%, preferably 60% of the transgenic Factor VII molecules in the composition have an isoelectric point within a pH unit deviation of less than 1, preferably less than 0.5. In another preferred embodiment, at least 50%, preferably at least 55%, preferably 60% of the Factor VII molecules in the composition have an isoelectric point within a pH unit deviation of 0.4.

[0069] The term "N-glycan forms" refers to all N-glycan forms present at the two N-glycosylation sites of factor VII of the invention. N-glycan forms are said to be single-charged if their total charge is equal to 1. For the purposes of this invention, "charge" means a phosphate group, a sulfate group, or a sialic acid molecule. Thus, N-glycan forms are said to be single-charged if they contain only a phosphate group, a sulfate group, or a sialic acid molecule. In contrast to the term "single-charged," the term "double-charged" means that the total charge carried by the N-glycan forms is equal to 2; that is, they have two charges chosen from among a phosphate group, a sulfate group, and / or a sialic acid molecule.In other words, doubly charged N-glycan forms possess one sialic acid molecule and one phosphate group, or one sialic acid molecule and one sulfate group, or two sialic acid molecules, or two phosphate groups, or two sulfate groups, or one phosphate group and one sulfate group. The term "tricharged" means that the total charge carried by the N-glycan forms is equal to 3; that is, they have three charges chosen from among a phosphate group, a sulfate group, and / or a sialic acid molecule.In other words, tricharged N-glycan forms possess one sialic acid molecule and one phosphate group and one sulfate group, or two sialic acid molecules and one phosphate group, or two sialic acid molecules and one sulfate group, or one sialic acid group and two phosphate groups, or one sialic acid group and two sulfate groups, or one phosphate group and two sulfate groups, or one sulfate group and two phosphate groups, or three sialic acid molecules, or three phosphate groups, or three sulfate groups. The term "neutral," on the other hand, means that the N-glycan forms contain no charge.

[0070] The charge of the N-glycan forms of factor Vil according to the invention can be measured using a technique well known to those skilled in the art, in particular by ultra-high-performance liquid chromatography with an anion-exchange resin coupled to fluorescence detection (AEX-UPLC / FD). This method allows the separation of the different N-glycan forms according to their apparent charge (see, in particular, Hermentin et al., Glycobiology, vol. 6, no. 2, 1996). In anion-exchange chromatography, a positively charged resin is used as the stationary phase. These positively charged resins generally consist of a cross-linked polymer or gel onto which positively charged groups are grafted. In an advantageous embodiment of the invention, a weak anion-exchange column of the aminopropyl type is used.

[0071] In the case of the Factor Vil composition according to the invention, it appears that among all the N-glycan forms of Factor Vil in the composition, at least 50% of the N-glycan forms, at least 60% of the N-glycan forms, preferably at least 65%, preferably at least 70%, preferably at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, are monocharged.In a preferred embodiment, factor Vil molecules with single-charged N-glycan forms represent between 50% and 95% of the factor Vil molecules in the composition, preferably between 50% and 90% of the factor Vil molecules in the composition, preferably between 50% and 80% of the factor Vil molecules in the composition, preferably between 50% and 75% of the factor Vil molecules in the composition, preferably between 50% and 70% of the factor Vil molecules in the composition, preferably between 50% and 65% of the factor Vil molecules in the composition, preferably between 50% and 60% of the factor Vil molecules in the composition.

[0072] The substantially homogeneous isoelectric point of the factor Vil composition of the combination according to the invention results from the combination of the glycosylation and γ-carboxylation properties of the FVII molecules that compose it.

[0073] The transgenic factor VII used here exhibits post-translational modifications. These include glycosylation modifications, such as two N-glycosylation sites with zero or very low levels of Gala1,3Gal in the FVII composition, or levels low enough to be non-immunogenic. In contrast, the FVII described here is not plasma-derived FVII; that is, it is not a product purified from human or animal plasma. More specifically, the transgenic FVII used here exhibits post-translational modifications, as well as two O-glycosylation sites with defined glycan motifs, a γ-carboxylation site, and specific disulfide bridges.

[0074] The FVIIa used here may have several post-translational modifications: the first nine or ten N-terminal glutamic acids are γ-carboxylated, Asp63 is partially hydroxylated, Sefe and Ser6o are O-glycosylated and carry the Glucose(Xylose)o-2 and Fucose motifs, respectively, and Asni45 and Asn322 are N-glycosylated, primarily by monosialylated bientenenous complex structures. Advantageously, at least 80% of the transgenic factor VII molecules used here have γ-carboxylation on nine glutamic acid residues. In another embodiment, at least 85% of these molecules have γ-carboxylation on nine glutamic acid residues. In another embodiment, between 85% and 100%, preferably between 90% and 100%, preferably between 95% and 100% of said molecules exhibit g-carboxylation on nine glutamic acid residues.Advantageously, the degree of γ-carboxylation on the glutamic acid residue 35 (Glu 35) of the factor VII molecules in the composition is less than 20%. In another embodiment, the degree of γ-carboxylation of the Glu35 residue is less than 15%, preferably less than 10%, preferably less than 5%.

[0075] The Gala1,3Gal motif is a structure composed of two galactoses linked at α1,3. It is located at the tips of the oligosaccharide antennae of N-linked structures. This motif is known for its immunogenicity. Therefore, it is preferable to produce FVII or FVIIa with a Gala1,3Gal structure content that is zero or so low that it cannot be distinguished from background noise obtained by measurements performed with currently available analytical equipment. This term is equivalent to any transgenic FVII with a Gala1,3Gal content close to that of plasma FVII. Advantageously, the Gala1,3Gal content of the FVII composition described here is not immunogenic to humans. In addition, the FVII useful here preferably has, like human FVII, two N-glycosylation sites, at positions 145 and 322, and two O-glycosylation sites, at positions 52 and 60. In an N-glycosylation site, the oligosaccharide chains are linked to an asparagine (N-linked).In an O-glycosylation site, oligosaccharide chains are linked to a serine residue. The specific motifs linked to these amino acids will differ for each protein in the composition. However, it is possible to quantify the proportion of each glycan motif, or even each sugar, for the entire composition.

[0076] The percentages of the different glycans given in this application do not take into account O-glycosylation.

[0077] Preferably, the composition of FVII is characterized in that, among all the glycan motifs of FVII in the composition, at least 40% are monosialylated biantennate glycan forms. In another embodiment, the monosialylated biantennate forms are present at a minimum of 50%. In yet another embodiment, the monosialylated biantennate forms are present at a minimum of 60%, preferably at least 65%, and preferably at least 70%.

[0078] Advantageously, the biantennate, monosialylated glycan forms of FVII are predominant. The composition of FVII is characterized in that at least some of the sialic acids of factor VII involve α2-6 linkages. Advantageously, at least 65% of the sialic acids of FVII involve α2,6 linkages. Very advantageously, at least 70%, or even 80%, and in particular, at least 90% of the sialic acids of FVII involve α2,6 linkages.

[0079] Preferably, all sialic acids involve α2,6 bonds, meaning that all sialic acids are linked to galactose by an α2,6 bond. The FVII composition described here may further include sialic acids with α2-3 bonds.

[0080] According to embodiments of the invention, 65% to 100% of the sialic acids of FVII involve α2,6 bonds. More preferably, 70% or 80% to 100% of the sialic acids of FVII involve α2,6 bonds.

[0081] Advantageously, among the monosialylated bientennate glycan forms of FVII, the majority of glycan forms are non-fucosylated.

[0082] Preferably, these non-fucosylated monosialylated bientennate glycan forms are present in the FVII composition at a level exceeding 20%. Advantageously, this level is greater than 25%, or even greater than 40%. Particularly advantageously, the fucosylation level of the FVII composition is between 20% and 50%. In one embodiment, this level may be less than 20%.

[0083] In a particular embodiment, at least 10%, preferably at least 15%, preferably at least 20%, preferably at least 25% of the N-glycan forms of the factors Vil in the composition are high mannose / hybrid.

[0084] Preferably, the glycosylation profile described herein provides FVII with enhanced biological activity and stability. Factor Vil compositions exhibiting a substantially homogeneous isoelectric point facilitate the formulation step at an optimal pH, preferably 6.0 ± 0.2, of pharmaceutical compositions by preventing FVII precipitation. Indeed, it is known that at the isoelectric point of a molecule, molecules tend to aggregate and precipitate. The factor Vil molecules used in the composition of the invention have an isoelectric point between 6.6 and 7.0. This results in improved stability of the factor Vil composition, particularly when formulated at a pH below the isoelectric point, and especially at pH 6.0.Improving the stability of the factor VII composition prevents electrostatic interactions responsible for precipitation and aggregation of both soluble and insoluble components, thus avoiding raw material loss and a decrease in yield, which leads to a loss of the active ingredient and potentially a loss of activity. In a preferred embodiment, the transgenic FVII is produced by the rabbit in its milk, resulting in a composition where each factor VII molecule has two N-glycosylation sites. Preferably, all FVII molecules in the composition have a Gala1,3Gal glycan motif content of less than 4%, or even zero. Advantageously, the transgenic FVII produced by the rabbit lacks the Gala1,3Gal glycan motif.

[0085] FVII can be purified from milk by techniques known to those skilled in the art. For example, a method for purifying a protein of interest from milk, as described in US patent 6,268,487, may include the following steps: a) subjecting the milk to tangential flow filtration through a membrane of sufficient porosity to form a retentate and a permeate, the permeate containing the exogenous protein; b) subjecting the permeate to a chromatographic capture apparatus so as to displace the exogenous protein and obtain an effluent; c) combining the effluent and the retentate; d) repeating steps a) through c) until FVII is separated from lipids and casein micelles, and FVII is recovered.

[0086] Advantageously, the FVII of the invention is in an activated form. In one embodiment, FVII can be activated in vitro by factors Xa, VIla, lia, IXa, or XIla. FVII can also typically be activated during its purification process, in particular by passing through positively charged chromatography columns.

[0087] Multispecific antibody

[0088] A "multispecific antibody" is defined as any antibody possessing at least two binding sites specific to at least two different antigens, or different epitopes of the same antigen. The term "specific" means that the antibody has the ability to recognize and bind one antigen, substantially without cross-reactivity with another antigen. Advantageously, the antibody exhibits, with respect to each antigen, an affinity constant Kd of at least 10 6 M, preferably at least 10 7 M, preferably at least 10 more8 M, 10- 9 M, or 10- 10 Mr.

[0089] Thus, the antibodies useful in the invention have the ability to bind specifically to both coagulation factor IX and coagulation factor X in activated or non-activated form.

[0090] The antibody used in the invention, which has the ability to bind specifically to both coagulation factor IX and coagulation factor X, preferentially has the ability to act as a substitute for factor VIII (FVI II), meaning that the antibody promotes the activation of FX by FIXa.

[0091] Such multispecific antibodies, preferably bispecific, can be obtained by various methods known to those skilled in the art, for example by chemical conjugation, or by using quadromes, which result from the fusion between two hybridomas producing two different monoclonal antibodies; or by genetic recombination.

[0092] Polynucleotides encoding such antibodies can thus be inserted into expression vectors and expressed in suitable host cells or organisms by techniques well known to those skilled in the art.

[0093] The antibodies useful here can be very simple in format, constructed from single-chain Fv fragments (scFv) of two or more antibodies, linked by a suitable peptide linker. "Fv" refers to the smallest antibody fragment that retains antigen recognition and binding properties. An "Fv" fragment is a dimer (VH + VL dimer) consisting of a variable region (VH) carried by a heavy chain (H) and an adjacent variable region (VL) carried by a light chain (L).

[0094] Alternatively, they can be full-length antibodies, preferably containing an Fc region. Several formats are possible. For example, in one format, scFv fragments of an antibody A are fused to the ends (usually N-terminal) of the heavy chains of an antibody B. The resulting antibody has a single type of heavy chain, which contains the VH, CH1, CH2, and CH3 domains of antibody B and the VH and VL domains of antibody A, and a single type of light chain that contains the VL and CL domains of antibody B (Qu et al. Blood, 111, 2211-2219, 2008). In a second format, the heavy chain and light chain of an antibody A are associated with the heavy chain and light chain of an antibody B. Where appropriate, mutations, for example of the "knob into holes" type (Ridgway et al, Protein Eng, 9, 617-21, 1996; US patent 7,695,936) can be introduced to avoid mismatches.

[0095] Unless otherwise stated, in this description the term “Factor IX” refers to both unactivated Factor IX and activated Factor IX (FIXa).

[0096] Unless otherwise specified, in this description, the term "Factor X" refers to either non-activated Factor X or activated Factor X (FXa). An antibody recognizing (i) FIX and / or FIXa, and (ii) FX and / or FXa can be obtained, in particular, by methods described in patent applications W02005 / 035756, W02006 / 109592, or W02012 / 067176.

[0097] In a preferred embodiment, said antibody is emicizumab. The production of this antibody is described, for example, in patent application WO2018047813 or patent application EP1688488.

[0098] Pharmaceutical compositions and dosages

[0099] Factor VII and antibodies can be formulated as separate pharmaceutical compositions or combined within a single pharmaceutical composition. In the case of separate administration, FVII and antibodies can be formulated to suit administration via different or the same routes.

[0100] Thus, FVII can be administered, for example, intravenously, subcutaneously, or intramuscularly.

[0101] The antibody can also be administered, for example, intravenously, subcutaneously, or intramuscularly.

[0102] A composition of Factor VII may, for example, be such as that described in patent application W02010 / 149907.

[0103] Thus, in one example, the composition includes:

[0104] factor Vil, preferably in the form of factor Vlla;

[0105] arginine, possibly in hydrochloride form;

[0106] isoleucine;

[0107] lysine;

[0108] - glycine;

[0109] trisodium citrate or calcium chloride;

[0110] and, where applicable, polysorbate 80 or polysorbate 20.

[0111] More specifically, the composition may include:

[0112] factor Vil, preferably in the form of factor Vlla;

[0113] 10 to 40 g / l of arginine, possibly in the form of hydrochloride;

[0114] from 4.2 to 6.6 g / l of isoleucine;

[0115] 0.6 to 1.8 g / l of lysine;

[0116] 0.6 to 1.8 g / l of glycine;

[0117] from 0 to 0.2 g / l of trisodium citrate or from 1 to 2 g / L of calcium chloride;

[0118] and, where applicable, 0 to 0.5 g / L of polysorbate 80. The FVII composition, which may also include at least one multispecific antibody as described herein, can be stored in liquid or solid form, typically obtained by desiccation. The concentrations disclosed above are determined with respect to the compositions in liquid form, before desiccation, or after reconstitution as an injectable preparation.

[0119] Desiccation is a process of water removal to an advanced stage. It involves dehydration aimed at eliminating as much water as possible. This phenomenon can be natural or induced. This desiccation can be achieved using freeze-drying, spray drying, and cryo-spray drying techniques.

[0120] The preferred method of obtaining the solid form of the pharmaceutical composition described herein is lyophilization.

[0121] The methods of lyophilization are well known to those skilled in the art, see for example [Wang et al, Lyophilization and development of solid protein pharmaceuticals, International Journal of Pharmaceutics, Vol 203, p 1-60, 2000].

[0122] Other suitable processes for reducing the degree of moisture or the water content of the composition are conceivable. Preferably the degree of moisture is less than or equal to 3% by weight, preferably less than or equal to 2.5%, preferably less than or equal to 2%, preferably less than or equal to 1.5%.

[0123] The solid composition, preferably in lyophilized form, can be dissolved in water for injection (or "water for injection or WFI") to obtain a formulation for therapeutic use.

[0124] The injectable formulation can be administered parenterally (intravenously, subcutaneously, intramuscularly), in a quantity determined by the practitioner. Administration of the liquid form (before drying) or the solid form, by any appropriate route and means, is not excluded.

[0125] The dosage of FVII useful in the invention can be appropriately determined according to the type of formulation, the method of administration, the age and weight of the patient, the patient's symptoms, the severity of the disease, etc.

[0126] The dose of FVII to be administered according to the invention can advantageously be chosen between 270 pg / kg and 2.70 pg / kg. Preferably the dose of FVII to be administered is less than 270 pg / kg of body weight, preferably it is less than 225 pg / kg of body weight, preferably it is less than 180 pg / kg of body weight, preferably it is less than 135 pg / kg of body weight, preferably it is less than 90 pg / kg of body weight, preferably it is less than 45 pg / kg of body weight, preferably it is less than 9 pg / kg, preferably it is less than 5.4 pg / kg, preferably it is less than 2.7 pg / kg. A multispecific antibody composition, such as the emicizumab antibody, is for example such as those described in patent applications WO2017 / 188356 and WO2018 / 047813.

[0127] Thus, in one example of implementation, the composition is a liquid composition.

[0128] In an example of a finished product, the composition includes:

[0129] bispecific antibody against factor IX and factor X,

[0130] a surfactant such as poloxamer 188 or polysorbate 20

[0131] histidine buffer - aspartic acid

[0132] arginine

[0133] More specifically, the composition may include:

[0134] from 20 mg / mL to 180 mg / mL of bispecific antibody for factor IX and factor X, from 0.2 mg / mL to 1 mg / mL of poloxamer 188,

[0135] 10 mM to 40 mM histidine-aspartic acid buffer

[0136] from 100 mM to 300 mM of arginine,

[0137] at a pH between 4.5 and 6.5

[0138] The dosage of the multispecific antibody composition, such as the emicizumab antibody used in the invention, can be appropriately determined according to the type of formulation, the method of administration, the patient's age and weight, the patient's symptoms, the severity of the disease, etc. The antibody dose can be, for example, from 0.3 to 5 mg / kg, preferably a maximum of 3 mg / kg once a week during an initiation period, which can last, for example, 4 weeks, followed by a maintenance dose, which is preferably lower, for example, 1.5 mg / kg once a week. Preferably, the administered antibody dose is less than 5 mg / kg of body weight, preferably less than 3 mg / kg of body weight, preferably less than 1.5 mg / kg of body weight, preferably less than 1 mg / kg of body weight, preferably less than 0 mg / kg of body weight.5 mg / kg body weight, preferably less than 0.1 mg / kg body weight, preferably less than 0.05 mg / kg body weight.

[0139] The antibody composition useful in the invention can be administered to a patient via any suitable route, for example, intravenously, intramuscularly, intraperitoneally, intracerebrospinally, transdermally, subcutaneously, intra-articularly, sublingually, intrasynovially, orally, or by inhalation. Preferably, the intravenous or subcutaneous route is preferred.

[0140] According to a particular embodiment, factor Vil and the antibody are administered simultaneously to the patient.

[0141] According to another particular embodiment, factor Vil and the antibody are administered separately to a patient, preferably sequentially.

[0142] Therapeutic indications

[0143] The combination described here makes it possible to prevent or treat coagulation disorders, including hemophilias with a deficiency in factor VIII (hemophilia type A, preferably acquired hemophilia A).

[0144] Preferably, the patients are patients who have type A hemophilia, with anti-Factor VIII.

[0145] The combination described here makes it possible to prevent or treat coagulation disorders, including deficiencies in factor VII.

[0146] The combination described here combines the rapid effect of factor VII, which activates the extrinsic pathway of the coagulation cascade, with the prolonged effect of the multispecific antibodies described here, which activate the intrinsic pathway of the coagulation cascade. This combination allows for improved patient management.

[0147] Examples:

[0148] Example 1: Purification and extraction of transgenic FVII

[0149] The purification and extraction process for factor Vil implemented in this example is that described in application EP12305882. The steps of this process are described below. Transgenic rabbit milk is obtained from the transgenic rabbit line. Frozen transgenic rabbit milk is thawed and concentrated into a transgenic rabbit milk pool.

[0150] The resulting pool of transgenic rabbit milk is then clarified using a depth filter with a porosity of 0.2 µm to remove lipids and insoluble compounds. The clarified milk then undergoes a viral inactivation step by treatment with a detergent-type solvent, such as Polysorbate 80 or Tri-n-Butyl Phosphate, at 25°C ± 2°C for at least two hours. This treatment effectively inactivates viruses, particularly non-enveloped viruses. The clarified and virally inactivated milk is then subjected to affinity chromatography using a factor VI l / factor VI la specific affinity ligand. The factor VI eluate obtained from this chromatography step is then ultrafiltered and formulated to produce an intermediate factor VI concentrate with a purity of 95%.

[0151] The intermediate factor Vil concentrate is then subjected to a filtration step using a filter with a porosity of 0.1 µm to 0.2 µm, followed by a nanofiltration step using filters with a porosity of 20 nm and then 15 nm. The resulting product containing factor Vil is then subjected to a Q-Sepharose XL gel chromatography step, followed by a CHT-I chromatography step and then a SEC Superdex 200 chromatography step. The resulting factor Vil concentrate is then subjected to a stabilization step and further filtration through a filter with a porosity of 0.2 µm.

[0152] The process described thus makes it possible to obtain a concentrate of factor Vil having a purity of approximately 99.9995%.

[0153] Example 2: Comparison of the thromboembolic potential of Novoseven®, Sevenfact® and

[0154] Hemlibra®

[0155] A person skilled in the art can measure the thrombogenic potential of Novoseven®, Sevenfact® and Hemlibra® (also called Emicizumab) by carrying out the following protocol.

[0156] Reagents:

[0157] ■ thrombin calibrator (Stago)

[0158] ■ PPP 5 pM reagent (Stago)

[0159] ■ PPP low reactive (Stago)

[0160] ■ CK-Prest (Stago)

[0161] ■ Fluo-buffer (Stago)

[0162] ■ Fluorescent substrate (Stago)

[0163] ■ FVIII-deficient plasma (Siemens)

[0164] ■ Sevenfact® / Transgenic Factor Vil produced in rabbits 1 mg / ml (LFB)

[0165] ■ PNP (Cryopep)

[0166] ■ Novoseven® (NovoNordisk)

[0167] ■ Hemlibra® / Emicizumab (Roche / Genentech / Chugai) Method:

[0168] The thrombin generation test involves activating coagulation ex vivo either with a mixture of tissue factor and phospholipids (TF / PL), or by using cephalin and then measuring the concentration of thrombin generated over time.

[0169] • Measurement of the thrombogenic potential of Novoseven® after coagulation induction with TF / PL:

[0170] The thrombin generation test is performed on 80 pL of a pool of FVIII-deficient plasma that mimics hemophilia A plasma in the presence of 20 pL of PPP reagent (Stago) containing 0.5 pM Tissue Factor (TF) and 4 pM phospholipids (PL). The reaction is initiated by the addition of 20 pL of Fluca-kit (substrate + CaCh), which marks the beginning of the thrombin generation measurement.

[0171] The therapeutic dose of FVIIa is 270 pg / kg, which corresponds to 6 pg / mL of FVIIa in the plasma, assuming 100% recovery. The thrombin generation test is performed at Novoseven® doses of 0 pg / mL, 1 pg / mL, 2 pg / mL, 3 pg / mL, 4 pg / mL, 5 pg / mL, and 6 pg / mL, in the presence of 0.5 pM TF / 2 pM PL (coagulation inducer).

[0172] • Measurement of the thrombogenic potential of Novoseven® after induction of coagulation with cephalin:

[0173] The thrombin generation test is performed on 80 pL of an FVIII deficient plasma pool that mimics hemophilia A plasma in the presence of 20 pL of cephalin (CK-Prest reconstituted with 5 mL of distilled H2O).

[0174] The reaction is initiated by the addition of 20 pL of Fluca-kit (substrate + CaC₂) which is the start of the measurement of thrombin generation.

[0175] The thrombin generation test is performed at Novoseven® doses of 0 pg / mL, 1 pg / mL, 2 pg / mL, 3 pg / mL, 4 pg / mL, 5 pg / mL, and 6 pg / mL, in the presence of 20 pL of cephalin (coagulation inducer).

[0176] • Measurement of the thrombogenic potential of Sevenfact® after coagulation induction with TF / PL:

[0177] The thrombin generation test is performed on 80 pL of a pool of FVIII-deficient plasma that mimics hemophilia A plasma in the presence of 20 pL of PPP reagent (Stago) containing 0.5 pM Tissue Factor (TF) and 4 pM phospholipids (PL). The reaction is initiated by the addition of 20 pL of Fluca-kit (substrate + CaCh), which marks the start of the thrombin generation measurement. The thrombin generation test is performed at Sevenfact® doses of 0 pg / mL, 1 pg / mL, 2 pg / mL, 3 pg / mL, 4 pg / mL, 5 pg / mL, and 6 pg / mL, in the presence of 0.5 rM TF / 2 mM PL (coagulation inducer).

[0178] • Measurement of the thrombogenic potential of Sevenfact® after induction of coagulation with cephalin:

[0179] The thrombin generation test is performed on 80 pL of an FVIII deficient plasma pool that mimics hemophilia A plasma in the presence of 20 pL of cephalin (CK-Prest reconstituted with 5 mL of distilled HO).

[0180] The reaction is initiated by the addition of 20 pL of Fluca-kit (substrate + CaCh) which is the start of the measurement of thrombin generation.

[0181] The thrombin generation test is performed at Sevenfact® doses of 0 pg / mL, 1 pg / mL, 2 pg / mL, 3 pg / mL, 4 pg / mL, 5 pg / mL, and 6 pg / mL, in the presence of 20 pL of cephalin.

[0182] • Measurement of the thrombogenic potential of Hemlibra® after coagulation induction with TF / PL:

[0183] The thrombin generation test is performed on 80 pL of an FVIII deficient plasma pool that mimics hemophilia A plasma in the presence of 20 pL of PPP reagent (Stago) containing 0.5 pM Tissue Factor (TF) and 4 pM phospholipids (PL).

[0184] The reaction is initiated by the addition of 20 pL of Fluca-kit (substrate + CaC₂) which is the start of the measurement of thrombin generation.

[0185] Hemlibra® (Roche / Genentech / Chugai, USA), a bispecific antibody mimicking the function of FVIII, is used at the maximum concentration of 50 pg / mL, which is the concentration detected in the patient under treatment (Oldenburg et al. NEJM, 2017). The thrombin generation test is performed at Hemlibra® doses of 0 pg / mL, 10 pg / mL, 20 pg / mL, 30 pg / mL, 40 pg / mL, and 50 pg / mL, in the presence of 0.5 pM TF / 4 pM PL (coagulation inducer).

[0186] • Measurement of the thrombogenic potential of Hemlibra® after induction of coagulation with cephalin

[0187] The thrombin generation test is performed on 80 pL of an FVIII deficient plasma pool that mimics hemophilia A plasma in the presence of 20 pL of cephalin (CK-Prest reconstituted with 5 mL of distilled HO).

[0188] The reaction is initiated by the addition of 20 pL of Fluca-kit (substrate + CaCh), which marks the beginning of the thrombin generation measurement. The thrombin generation test is performed at Hemlibra® doses of 0 pg / mL, 10 pg / mL, 20 pg / mL, 30 pg / mL, 40 pg / mL, and 50 pg / mL, in the presence of 20 pL of cephalin.

[0189] For all these tests, the appearance of fluorescence is measured on a Fluoroskan Ascent fluorometer (ThermoLabsystems) at an excitation wavelength of 390 nm and an emission wavelength of 460 nm. The thrombinograms (curves representing the fluorescence intensity over time) are then analyzed using Thrombinoscope™ software, which converts the fluorescence value into nM of thrombin by comparative calculation.

[0190] Thrombin generation is performed and key variables for evaluating the potency of different drugs are recorded and compared: endogenous thrombin potential (ETP), peak height, latency time and velocity.

[0191] Example 3: Evaluation of the thromboembolic potentials of Novoseven® and

[0192] Hemlibra® or SevenFact® and Hemlibra®

[0193] A person skilled in the art can measure the thrombogenic potential of Novoseven® / Hemlibra® and Sevenfact® / Hemlibra® combinations by carrying out the following protocol.

[0194] Reagents:

[0195] The reagents, the automated system, and the experimental protocol in FVIII-deficient plasma are identical to those described in Example 2.

[0196] Method :

[0197] • Measurement of the thrombogenic potential of the Novoseven® + Hemlibra® combination after coagulation induction with TF / PL:

[0198] The thrombin generation test is performed on 80 pL of an FVIII deficient plasma pool that mimics hemophilia A plasma in the presence of 20 pL of PPP reagent (Stago) containing 0.5 pM Tissue Factor (TF) and 4 mM phospholipids (PL).

[0199] The reaction is initiated by the addition of 20 pL of Fluca-kit (substrate + CaCh) which is the start of the measurement of thrombin generation.

[0200] The thrombin generation test is performed in the presence of 0.5 pM TF / 4 pM PL (coagulation inducer) on several Novoseven® / Hemlibra® combinations. The composition containing the highest amount of product consists of 6 pg / mL of Novoseven® and 50 pg / mL of Hemlibra®, at their maximum concentrations.

[0201] The thrombogenic potential obtained with the combination of products is compared to the potential of the individual products. To consider a synergistic effect of the product combination, lower doses are evaluated to ensure that thrombin detection is not saturated.

[0202] The tested compositions contain:

[0203] • Measurement of the thrombogenic potential of the Novoseven® + Hemlibra® combination after induction of coagulation with cephalin

[0204] The thrombin generation test is performed on 80 pL of an FVIII deficient plasma pool that mimics hemophilia A plasma in the presence of 20 pL of cephalin (CK-Prest reconstituted with 5 mL of distilled H2O).

[0205] The reaction is initiated by the addition of 20 pL of Fluca-kit (substrate + CaCh) which is the start of the measurement of thrombin generation.

[0206] The thrombin generation test is performed in the presence of 20 pL of cephalin on several Novoseven® / Hemlibra® combinations. The composition containing the highest quantity of product consists of 6 pg / mL of Novoseven® and 50 pg / mL of Hemlibra®, at their maximum concentration.

[0207] The thrombogenic potential obtained with the combination of products is compared to the potential of the individual products. To consider a synergistic effect of the product combination, lower doses are evaluated to ensure that thrombin detection is not saturated.

[0208] The tested compositions contain:

[0209] • Measurement of the thrombogenic potential of the Sevenfact® + Hemlibra® combination after coagulation induction with TF / PL

[0210] The thrombin generation test is performed on 80 pL of an FVIII deficient plasma pool that mimics hemophilia A plasma in the presence of 20 pL of PPP reagent (Stago) containing 0.5 pM Tissue Factor (TF) and 4 pM phospholipids (PL).

[0211] The reaction is initiated by the addition of 20 pL of Fluca-kit (substrate + CaC₂) which is the start of the measurement of thrombin generation.

[0212] The thrombin generation test is performed in the presence of 0.5 pM TF / 4 pM PL (coagulation inducer) on several Sevenfact® / Hemlibra® combinations. The composition containing the highest amount of product consists of 6 pg / mL of Sevenfact® and 50 pg / mL of Hemlibra®, at their maximum concentrations.

[0213] The thrombogenic potential obtained with the combination of products is compared to the potential of the individual products. To consider a synergistic effect of the product combination, lower doses are evaluated to ensure that thrombin detection is not saturated.

[0214] The tested compositions contain:

[0215]

[0216] • Measurement of the thrombogenic potential of the Sevenfact® + Hemlibra® combination after induction of coagulation with cephalin

[0217] The thrombin generation test is performed on 80 pL of an FVIII deficient plasma pool that mimics hemophilia A plasma in the presence of 20 pL of cephalin (CK-Prest reconstituted with 5 mL of distilled H2O).

[0218] The reaction is initiated by the addition of 20 pL of Fluca-kit (substrate + CaCh) which is the start of the measurement of thrombin generation.

[0219] The thrombin generation test is performed in the presence of 20 pL of cephalin using several Sevenfact® / Hemlibra® combinations. The composition with the highest concentration consists of 6 pg / mL of Sevenfact® and 50 pg / mL of Hemlibra® at their maximum concentrations. The thrombogenic potential obtained with the combination of products is compared to the potential of the individual products. To consider a synergistic effect of the combination, lower doses are evaluated to ensure that thrombin detection is not saturated.

[0220] The tested compositions contain:

[0221]

[0222] For all these tests, the appearance of fluorescence is measured on a Fluoroskan Ascent fluorometer (ThermoLabsystems) at an excitation wavelength of 390 nm and an emission wavelength of 460 nm. The thrombinograms (curves representing the fluorescence intensity over time) are then analyzed using Thrombinoscope™ software, which converts the fluorescence value into nM of thrombin by comparative calculation.

[0223] A synergistic effect is considered, for example, when at least one of the parameters calculated from the thrombin generation test for a given combination is greater than the sum of each of these parameters obtained with the components alone, deduced from the background noise of the experiment.

[0224] Example 4: Comparison of the potential of Sevenfact™, Hemlibra® and the combination of the two in hemophilic plasma A:

[0225] Reagents:

[0226] ■thrombin calibrator (Stago)

[0227] ■ PRP reagent 1 pM TF (Stago)

[0228] ■ MP 4mM PL reagent (Stago)

[0229] ■ Fluo-buffer (Stago)

[0230] ■ Fluorescent substrate (Stago)

[0231] ■ Sevenfact™: Transgenic Factor Vil produced in rabbits 1 mg / mL (LFB)

[0232] ■ Hemlibra®: Emicizumab (Roche / Genentech / Chugaï)

[0233] ■ Hemophilia A Plasma (Cryopep)

[0234] ■ Owren Koller (Stago) Method:

[0235] The thrombin generation assay involves activating coagulation ex vivo, for example with a mixture of tissue factor and phospholipids (TF / PL), and then measuring the concentration of thrombin generated over time. Thrombin generation assays are performed with 80 pL of hemophilic A plasma (Cryopep) in the presence of 20 pL of a mixture of PRP and MP reagents (Stago) containing 0.5 pM of tissue factor and 4 mM of phospholipids.

[0236] The reaction is initiated by adding 20 pL of Fluca-kit (Fluo substrate + CaCh), which corresponds to the starting point for measuring thrombin (TG) generation.

[0237] Fluorescence is measured by fluorimetry using the Fluoroskan Ascent instrument (ThermoLabsystems) at an excitation wavelength of 390 nm and an emission wavelength of 460 nm. Thrombinograms are analyzed using Thrombinoscope™ software, which converts fluorescence intensity into a molar concentration of thrombin (nM) by comparative calculation.

[0238] To measure the thrombogenic potential of the two molecules, several hemophilia A plasma samples were studied. The highest therapeutic dose of FVIIa was 270 pg / kg, corresponding to 6 pg / mL of FVIIa (or 120 nM) in the plasma. The use of this dose can be considered a maximum potential for thrombin generation. Based on circulating concentrations of the product obtained in patients, Sevenfact™ concentrations between 20 and 100 nM were also studied. Hemlibra® (Roche / Genentech / Chugai, USA), a bispecific antibody mimicking the function of FVIII, was used at the maximum concentration of 120 pg / mL. The concentration currently detected in patients undergoing treatment was 50 pg / mL (or 300 nM) (Oldenburg et al. NEJM, 2017). Thus, Hemlibra® is used here at approximately 300 nM (50 pg / mL). The variables studied to measure the thrombogenic potential of Hemlibra®, from Sevenfact™, are:

[0239] Endogenous thrombin potential (ETP): area under the curve representing the total amount of thrombin generated,

[0240] peak height: maximum measured thrombin concentration, and

[0241] Thrombin generation velocity: rate of thrombin formation.

[0242] 2 - Results

[0243] 2.1 - Effect of Sevenfact™ or Hemlibra® on Hemophilia A plasma

[0244] 2.1.1 - Evaluation in batch 1 of Hemophilia A plasma. In this matrix, very weak thrombin generation signals from both compounds are obtained, regardless of the concentrations used. Indeed, the observed thrombin generation is virtually zero for Hemlibra® and Sevenfact™ at concentrations of 20 and 40 nM. With 100 nM of Sevenfact™, a very weak thrombin generation peak is observed (Table 1).

[0245]

[0246] Table 1: Thrombin generation parameters from batch 1 of Hemophilia A plasma treated with Sevenfact™ or Hemlibra®

[0247] Thus, each molecule used individually induces only a very small generation of thrombin.

[0248] 2.1.2 - Evaluation in batch 2 of Hemophilia A plasma

[0249] A second batch of Hemophilia A plasma was tested. Again, very low thrombin generation was observed with the use of Hemlibra® and Sevenfact™, with a maximum thrombin generation peak at a Sevenfact™ concentration of 100 nM (Table 2).

[0250]

[0251] Table 2: Thrombin generation parameters from batch 2 of Hemophilia A plasma treated with Sevenfact™ or Hemlibra®. In this matrix, Sevenfact™ and Hemlibra® used separately have a low thrombogenic potential. Example 5: Evaluation of the synergistic thrombogenic potential of the Sevenfact™ + Hemlibra® combination

[0252] 1 - Protocol

[0253] The reagents, the automated system, and the experimental protocol in hemophilia A plasma are identical to those described in Example 2.

[0254] 2 - Results

[0255] As seen in Example 2, Sevenfact™ and Hemlibra® used individually induce low thrombin generation in Hemophilia A plasma. The synergistic effect of the Sevenfact™ and Hemlibra® combination is investigated here. Three concentrations of Sevenfact™ are studied (20 nM, 40 nM, and 100 nM) in the presence of a 300 nM Hemlibra® concentration. A synergistic effect is considered if the effect of the Sevenfact™ + Hemlibra® combination is at least twice the sum of the effects of Sevenfact™ and Hemlibra® taken separately for at least one of the parameters of the thrombin generation assay (PTE, peak thrombin generation, and velocity).

[0256] 2.1 - Effect of Sevenfact™ and Hemlibra® on Hemophilia A plasmas after coagulation induction with TF / PL

[0257] 2.1.1 - Evaluation in batch 1 of hemophilia A plasma

[0258] The results are presented in Table 3 and Figure 1. At a very low concentration of Sevenfact™ of 20 nM, the ratios for PTE (Figure 1A), for thrombin peak (Figure 1B) and for velocity (Figure 1C) of the Sevenfact™ + Hemlibra® combination are respectively 2.14, 2.95 and 4.19. Thus, even at the lowest concentration tested, a synergistic thrombogenic effect is observed.

[0259] At a concentration of 40 nM, the ratio for all tested parameters is greater than 2. The ratio obtained for PTE is 2.75 (Figure 1A), the ratio obtained for the thrombin peak is 3.96 (Figure 1B), and the ratio obtained for velocity reaches a value of 6.21 (Figure 1C). In other words, the rate of thrombin formation is multiplied by 6 when Sevenfact™ and Hemlibra® are used in combination.

[0260] The synergistic effect is greatest at a concentration of 100 nM of Sevenfact™. At this concentration, the ratio for all tested parameters is greater than 2. The ratio obtained for PTE is 4.00 (Figure 1A), and the ratio for peak thrombin is 4.81 (Figure 1B), meaning that the maximum concentration of thrombin generated is almost 5 times greater when Hemlibra® and Sevenfact™ are used in combination. The ratio corresponding to velocity is 9.58 (Figure 1C), meaning that thrombin is generated almost 10 times faster when Sevenfact™ and Hemlibra® are used in combination.

[0261] o *

[0262] O

[0263] O

[0264]

[0265]

[0266]

[0267] Table 3: Thrombin generation parameters from batch 1 of Hemophilia A plasma treated with the Sevenfact™ + n H Hemlibra® S combination

[0268] In conclusion, for all concentrations of Sevenfact™ tested, Sevenfact™ and Hemlibra® used in combination have a synergistic effect on thrombin generation.

[0269] 2.1.2 - Evaluation in batch 2 of Hemophilia A plasma

[0270] The results are presented in Table 4 and Figure 2. At a very low concentration of Sevenfact™ of 20 nM, a ratio of 2.21 was obtained for the PTE parameter (Figure 2A), a ratio of 2.34 was obtained for the thrombin peak (Figure 2B), and a ratio of 2.9 was obtained for the velocity parameter (Figure 2C) of the Sevenfact™ + Hemlibra® combination. Thus, even at the lowest tested concentration of Sevenfact™, a synergistic thrombogenic effect was observed.

[0271] At a concentration of 40 nM, the ratio corresponding to PTE is 2.29 (Figure 2A), that corresponding to the thrombin peak is 2.79 (Figure 2B) and the ratio corresponding to velocity is 3.68 (Figure 2C), which means that the use of Sevenfact™ in combination with Hemlibra® allows thrombin formation approximately 4 times faster.

[0272] The synergistic effect is greatest at a concentration of 100 nM of Sevenfact™. At a concentration of 100 nM, the ratio corresponding to the peak thrombin generation is 3.41 (Figure 2B) and that corresponding to the velocity is 5.63 (Figure 2C), which means that thrombin is generated almost 6 times faster and the thrombin concentration achieved is multiplied by almost 4 when Sevenfact™ is used in combination with Hemlibra®.

[0273] o

[0274] O

[0275]

[0276] Table 4: Thrombin generation parameters from lot 2 of hemophilic plasma treated with the Sevenfact™ + Hemlibra® n H combination

[0277] So

[0278] O o

[0279] In conclusion, for all concentrations of Sevenfact™ tested, Sevenfact™ and Hemlibra® used in combination have a synergistic effect on thrombin generation.

Claims

Demands 1. Pharmaceutical composition comprising: a. of the transgenic Factor Vil, and b. a multispecific antibody directed against factor IX and factor X.

2. Pharmaceutical composition according to claim 1, wherein said transgenic factor Vil is a human factor Vil produced by the epithelial cells of the mammary glands of a transgenic non-human mammal.

3. Pharmaceutical composition according to claim 2, wherein said transgenic mammal is the rabbit.

4. Pharmaceutical composition according to any one of claims 1 to 3, wherein the antibody is emicizumab.

5. Combination product comprising: a. of the transgenic Factor Vil, and b. a multispecific antibody directed against factor IX and factor X, for its use in the prevention or treatment of a coagulation disorder in a patient.

6. Combination product according to claim 5, in the treatment of hemophilia A.

7. Combination product according to one of claims 5 or 6, in the treatment of hemophilia A with factor VIII inhibitors.

8. Combination product for its use according to claims 5 to 7, said combination product being in the form of a pharmaceutical composition as defined in any one of claims 1 to 4.

9. Combination product for its use according to claims 5 to 8, said factor VI la and said antibody being in a form suitable for simultaneous administration to the patient.

10. Combination product for use according to claims 5 to 8, said factor VI and said antibody being in forms suitable for separate administration to said patient.

1. Kit comprising A container containing a transgenic FVII factor; and Another container containing an antibody directed against factor IX and factor X.

12. Method for treating a coagulation disorder in a patient, which method comprises the simultaneous or sequential administration to said patient of transgenic Factor VII and a multispecific antibody directed against factor IX and factor X.

13. Use of a combination of transgenic Factor VII and a multispecific antibody directed against factor IX and factor X for the treatment of a coagulation disorder in a patient, preferably hemophilia A with factor VIII inhibitors.