Combination of factor VII and anti-factor IX / X bispecific antibodies

Abstract

The present invention relates to a combination of Factor VII and an anti-Factor IX / X bispecific antibody. The present invention relates to a combination comprising a transgenic factor VII and a multispecific antibody against factors IX and X for simultaneous or separate administration.

Description

[0001] This application is a divisional application of PCT application PCT / FR2019 / 051456, titled "Combination of Factor VII and Anti-Factor IX / X Bispecific Antibody", filed on June 14, 2019. The date when the PCT application entered the Chinese national phase is February 7, 2021, and the application number is 201980054066.X.

[0002] The present invention relates to a pharmaceutical composition which can be used for treating coagulation disorders, such as hemophilia A, especially in patients with hemophilia A who have developed factor VIII inhibitory antibodies. Technical Background

[0003] Coagulation involves two pathways, one intrinsic and the other extrinsic, leading to a final common pathway. The combination of the two mechanisms ensures the formation of a solid and flexible anti-blood pressure blood clot. Through the action of thrombin, fibrinogen is chemically modified, resulting in the formation of fibrin. Fibrin is essential for the formation of the clot.

[0004] The intrinsic pathway involves factors present in the bloodstream, and the coagulation process begins within the blood vessels themselves. The extrinsic pathway involves tissue factor that is not normally present in the bloodstream and is released during blood vessel injury.

[0005] Factor VII is a glycoprotein that participates in the extrinsic coagulation pathway. To initiate the coagulation cascade, FVII must be activated to FVIIa. Once activated, FVIIa complexes with the tissue factor (TF) protein associated with two phospholipids, which is released during blood vessel injury. FVIIa alone (not complexed with tissue factor) exhibits low proteolytic activity. Then, the FVIIa-FT complex converts factor X to factor Xa in the presence of calcium ions. This complex also acts on the activation of factor FIX to FIXa, thereby catalyzing the intrinsic pathway. Factor IXa and Xa sequentially activate activated factor VII.

[0006] Factor IX and factor X participate in the intrinsic coagulation pathway. Activated factor IX is capable of activating factor X to factor Xa.

[0007] Factor Xa complexed with activated factor FV and prothrombinase converts prothrombin to thrombin. Then, thrombin acts on fibrinogen to convert it to fibrin and activates FVIII and FV to FVIIIa and FVa, respectively. For its part, prothrombin, in the presence of calcium naturally present in plasma, is capable of activating factor XIII to FXIIIa, which results in the consolidation of the fibrin clot.

[0008] Nevertheless, when a coagulation factor is lacking, the coagulation cascade is interrupted or absent, which we call coagulopathy.

[0009] 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. This is why factor VII (preferably in the activated form) is used to treat certain coagulation disorders presenting as bleeding.

[0010] Thus, factor VII is used to treat patients with hemophilia presenting with a deficiency of factor VIII (hemophilia A) or factor IX (hemophilia B) and patients presenting with a deficiency of other coagulation factors, for example, genetic deficiency of FVII. FVII is also recommended in the treatment of stroke.

[0011] Some hemophilia patients produce antibodies against factor VIII, which is usually administered in concentrated form as a hemophilia treatment. This is the most common complication in current hemophilia treatment.

[0012] Bispecific antibodies targeting FIX or FIXa and FX or FXa, such as emicizumab, are used to treat patients with hemophilia A having anti-factor VIII antibodies. These antibodies functionally replace FVIII by promoting the activation of FX by FIXa by binding these two molecules together. These antibodies have a long-lasting effect.

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

[0014] Therefore, there is a need for a drug combination that allows better management of patients with hemophilia A and more particularly patients with anti-factor VIII. Summary of the Invention

[0016] The present invention proposes a combination of transgenic factor VII with a multispecific antibody against factors IX and X.

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

[0018] Thus, one aspect of the present invention is a pharmaceutical composition comprising:

[0019] a. a transgenic factor VII, and

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

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

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

[0023] Preferably, the transgenic factor VII is human factor VII produced from the mammary epithelial cells of a non-human transgenic mammal, such as a rabbit transgenic for human factor VII.

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

[0025] a. a transgenic factor VII, and

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

[0027] The combination product is for preventing or treating coagulation disorders, such as hemophilia A, and more particularly hemophilia A having a factor VIII inhibitor (FVIII).

[0028] Preferably, the combination product is in the form of a pharmaceutical composition comprising the transgenic factor VII and the antibody.

[0029] Alternatively, the transgenic factor VII and the antibody are in the form of separate compositions, which are suitable for simultaneous or separate (e.g., sequential) administration to a patient.

[0030] In addition, another object of the present invention relates to a kit comprising:

[0031] - a container containing the transgenic factor FVII; and

[0032] - another container containing the antibody against factors IX and X. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 : Evaluation of the synergistic thrombus formation effect of the Sevenfact TM + combination on the plasma of hemophilia A in the first batch. (A) Evaluation of the synergistic thrombus formation effect in terms of PTE, (B) Evaluation of the synergistic thrombus formation effect in terms of the peak of thrombin generation, (C) Evaluation of the synergistic thrombus formation effect in terms of velocity.

[0034] Figure 2 : Evaluation of the synergistic thrombus formation effect of the Sevenfact TM + combination on the plasma of hemophilia A in the second batch. (A) Evaluation of the synergistic thrombus formation effect in terms of PTE, (B) Evaluation of the synergistic thrombus formation effect in terms of the peak of thrombin generation, (C) Evaluation of the synergistic thrombus formation effect in terms of velocity. DETAILED DESCRIPTION OF THE INVENTION

[0036] General Definitions

[0037] The coagulation phenomenon consists of an enzymatic reaction cascade involving coagulation factors, where the coagulation factors exist in the form of zymogens and are converted into their "activated" forms by proteolytic cleavage in the presence of certain cofactors. The activated form of each factor that exists in the inactive precursor form is denoted by the letter a. Thus, FVIIa is generated in vivo by the cleavage of the zymogen into two chains linked by a disulfide bridge by a variety of proteases (FIXa, FXa, FVIIa).

[0038] The term "treatment" generally refers to the improvement, prevention or reversal of a disease or disorder or at least symptoms thereof, such as slowing the progression of the disease or stabilizing symptoms. It also includes delaying the onset of a disease or disorder or at least symptoms thereof.

[0039] The term "prevention" refers to a decrease in the risk of developing or acquiring a specific disease or disorder.

[0040] In the present invention, "patient" or "individual" refers to any mammal, and more particularly to a human of any age, male or female, including children.

[0041] The term "pharmaceutical composition" refers to a preparation that allows the active ingredient to have biological activity and does not contain any other components that are toxic to the individual to whom the composition is administered.

[0042] Transgenic Factor VII

[0043] The term "Factor VII" or "FVII" includes a polypeptide comprising the sequence 1-406 of wild-type human Factor VII (as described in U.S. Patent No. 4,784,950) or FVII from another species (e.g., bovine, porcine, canine, murine). Also included are natural allelic variants of Factor VII, which may exist in any form or degree of glycosylation or other post-translational modification. Thus, the term "Factor VII" also includes FVII variants that have the same or better biological activity relative to the activity of the wild-type, and these variants particularly include polypeptides that differ from wild-type FVII by the insertion, deletion, or substitution of one or more amino acids.

[0044] Unless otherwise indicated, in this specification, the term "Factor VII" refers to uncleaved FVII (zymogen) or activated Factor VII (FVIIa).

[0045] Thus, FVIIa consists of a light chain of 152 amino acids with a molecular weight of approximately 20 kDa and a heavy chain of 254 amino acids with a molecular weight of approximately 30 kDa, which are linked together by a single disulfide bridge (Cys135-Cys262).

[0046] "Recombinant Factor VII" refers to any Factor VII derived from genetic engineering and produced by the expression of the corresponding gene in any microorganism, plant, or transgenic plant. A microorganism refers to any bacterium, fungus, virus, or cell system. Recombinant Factor VII can also be produced by eukaryotic cells in culture, such as plant or mammalian cells, such as animal or human cells.

[0047] "Transgenic Factor VII" refers to any recombinant Factor VII obtained from an animal transgenic for Factor VII.

[0048] "Transgenic animal" refers to any non-human animal having a modified genome in which it is expected to express a protein of interest (here Factor VII). The genomic modification can be caused by the alteration, modification, or insertion of a gene. Such modification can be effected by the use of conventional altering or mutagenic agents or even by site-directed mutagenesis. The genomic modification can also be produced by the insertion or substitution of one or more genes in wild-type or mutant form. Transgenic animals can be selected in a non-limiting manner from rabbits, goats, cows, camels, hamsters, mice, rats, horses, sows, dromedaries, sheep, or llamas. In a particular embodiment, animals that do not express α1,3-galactosyltransferase can be selected.

[0049] The expression "biological activity of factor VIIa" refers to the ability of FVIIa to generate thrombin, for example, on the surface of activated platelets. The activity of factor VII can be evaluated in a variety of ways. For example, the biological activity of FVIIa can be quantified by measuring the ability of an FVII composition to promote blood clotting, using FVII- and thromboplastine-deficient plasma as described, for example, in U.S. Patent 5,997,864. In this test, the biological activity is evaluated relative to a control sample and converted to "FVII units" by comparison with pooled standard human serum containing 1 unit / mL of factor VII activity. Alternatively, the biological activity of factor VII can be quantified by the following steps: (i) measuring the ability of factor VIIa to generate factor Xa in a system containing tissue factor (TF) and factor X surrounded by a lipid membrane (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 by surface plasmon resonance (Persson, FEBS letts, 413:359-363, 1997); (iv) measuring the hydrolysis of a synthetic substrate; or (v) measuring the generation of thrombin in an in vitro system independent of TF.

[0050] In a preferred embodiment, the FVII described herein is a polypeptide, and its peptide sequence can be the peptide sequence of native human FVII, i.e., the sequence present in a person not suffering from an FVII-related disorder. Such techniques are described in the literature EP 0 200421.

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

[0052] "Synergistic" or "synergy" preferably refers to the effect of a combination of two products being greater than twice the sum of the effects of each product used separately. According to the present invention, a synergistic effect is obtained when, with respect to at least one thrombin generation parameter, the use of transgenic FVIIa in combination with a multispecific antibody against factor IX and factor X results in an effect greater than twice the sum of the effect obtained by using transgenic FVIIa alone and the effect obtained by using the multispecific antibody against factor IX and factor X alone. The thrombin generation parameters are selected from peak height, velocity, or potential thrombin generation endogenously (PTE).

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

[0054] In particular embodiments, the multispecific antibodies against Factor IX and Factor X are administered at a concentration below 600 nM, preferably below 550 nM, preferably below 500 nM, preferably below 450 nM, preferably below 400 nM, preferably below 350 nM, preferably below 325 nM.

[0055] In particular embodiments, Factor VII is obtained from the milk of transgenic animals.

[0056] A method for producing a recombinant protein in the milk of a transgenic animal can include the steps of integrating a synthetic DNA molecule (which contains a gene encoding a protein of interest (such as human FVII herein), the gene being under the control of a promoter of a protein that is naturally secreted in milk) into an embryo of a non-human mammal. The embryo is then implanted into a female mammal of the same species. Once the mammal produced from the embryo is fully developed, lactation of the mammal can be induced, and then the milk can be collected. The milk contains the FVII of interest secreted by the transgenic animal.

[0057] An example of protein preparation using the milk of a non-human female mammal is given in patent application EP0527063, and the teachings therein can be reused for preparing the Factor VII of the present invention.

[0058] The mammary gland secretes Factor VII and causes it to be secreted into the milk of the transgenic mammal, participating in the tissue-dependent control of the expression of Factor VII. Such control methods are well known to those skilled in the art. Expression is controlled by sequences that allow expression of the protein in a specific tissue. These are in particular the WAP, β-casein, and β-lactoglobulin promoter sequences and signal peptide sequences; this list is not restrictive.

[0059] In a preferred embodiment, the Factor VII of the present invention is produced in the milk of transgenic rabbits.

[0060] In a particularly advantageous manner, expression is carried out in the mammary gland of rabbits under the control of the β-casein promoter well known to the person skilled in the art. In particular, a plasmid containing the β-casein promoter is constructed by introducing a sequence containing the promoter of the β-casein gene, and this plasmid is produced so as to be able to receive a foreign gene placed under the control of this promoter. The gene encoding human FVII is integrated and placed under the control of the β-casein promoter. The plasmid containing the promoter and the sequence encoding the protein of interest is digested with a restriction enzyme to release a DNA fragment containing the β-casein promoter and the human FVII sequence. After purification, the fragment is introduced by microinjection into the male pronucleus of a wild-type rabbit embryo. The embryos are then cultured and then transferred into the oviducts of hormonally prepared wild-type females. When these female animals give birth, the offspring are evaluated by PCR to identify transgenic animals. The copy number and integrity of the transgene are revealed by Southern techniques from DNA extracted from the resulting young transgenic rabbits. The concentration of human FVII expressed in the milk of female transgenic offspring is evaluated by immunoenzymatic tests.

[0061] In a particular embodiment, factor VII useful in the present invention is obtained by a method comprising the following steps:

[0062] (a) inserting a DNA sequence containing a gene encoding factor VII into a non-human mammalian embryo, said gene being under the transcriptional control of a β-casein promoter,

[0063] (b) transferring the embryo obtained in step (a) into the oviduct of a non-human female mammal so that it develops into an adult non-human mammal,

[0064] (c) inducing lactation in the adult non-human mammal of the female type obtained in step (b) or in the female offspring of said non-human mammal in which the gene and the promoter are present in its genome,

[0065] (d) collecting milk from said non-human mammal, and

[0066] (e) purifying FVII present in the collected milk.

[0067] The FVII used herein has a substantially homogeneous isoelectric point.

[0068] "Isoelectric point" or "pI" refers to the pH at which the net basic charge of a Factor VII or Factor VIIa molecule is zero, i.e., the pH at which the molecule is electrically neutral (zwitterionic form). The isoelectric point of the Factor VII of the present invention can be measured by techniques well known to those skilled in the art such as isoelectric focusing ("IEF"). This electrophoresis technique separates proteins based on their isoelectric points. It consists of the migration of proteins in a pH gradient induced by a uniform current until they reach a pH equal to their specific isoelectric point, at which point they stop migrating due to their zero net charge. IEF gels are used to determine the isoelectric point of a given protein.

[0069] "Substantially homogeneous" means that at least 90%, preferably at least 95% of the Factor VII molecules in the composition have an isoelectric point with a pH unit difference of less than or equal to 1.2. In another embodiment of the present invention, at least 50%, preferably at least 55%, preferably 60% of the transgenic Factor VII molecules in the composition have an isoelectric point with a pH unit difference of less than 1, preferably less than 0.5. In another embodiment of the present invention, at least 50%, preferably at least 55%, preferably 60% of the Factor VII molecules in the composition have an isoelectric point with a pH unit difference of 0.4.

[0070] "N-glycan form" refers to all N-glycan forms present at the two N-glycosylation sites of the Factor VII of the present invention. An N-glycan form is called singly charged if its total charge equals 1. In the present invention, "charge" refers to a phosphate group, a sulfate group or a sialic acid molecule. Thus, an N-glycan form is called singly charged if it contains only one phosphate group or one sulfate group or one sialic acid molecule. In contrast to the term "singly charged", the term "doubly charged" refers to an N-glycan form whose total charge carried equals 2, i.e., it has two charges selected from phosphate groups, sulfate groups and / or sialic acid molecules. In other words, a doubly charged N-glycan form has 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 "triply charged" refers to an N-glycan form whose total charge carried equals 3, i.e., it has three charges selected from phosphate groups, sulfate groups and / or sialic acid molecules. In other words, a triply charged N-glycan form has one sialic acid molecule, 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 molecule 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" refers to an N-glycan form that does not contain any charge.

[0071] The charge of the N-glycan forms of factor VII can be determined by methods well known to those skilled in the art, in particular by ultra-performance liquid chromatography with anion-exchange resin coupled to fluorescence detection (AEX-UPLC / FD). This method is capable of separating different N-glycan forms according to their apparent charge (see in particular Hermentin et al., Glycobiology, Volume 6, Number 2, 1996). In the case of anion-exchange chromatography, a positively charged resin is used as the stationary phase. These positively charged resins are generally composed of crosslinked polymers or gels to which positively charged groups are grafted. In a preferred embodiment of the invention, an aminopropyl type weak anion-exchange column is used.

[0072] In the case of the factor VII compositions of the present invention, it appears that in all N-glycan forms of factor VII 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% carry a single charge. In a preferred embodiment, the factor VII molecules in the composition with N-glycan forms carrying a single charge account for 50% - 95% of the factor VII molecules in the composition, preferably 50% - 90% of the factor VII molecules in the composition, preferably 50% - 80% of the factor VII molecules in the composition, preferably 50% - 75% of the factor VII molecules in the composition, preferably 50% - 70% of the factor VII molecules in the composition, preferably 50 - 65% of the factor VII molecules in the composition, preferably 50% - 60% of the factor VII molecules in the composition.

[0073] The substantially uniform isoelectric point of the factor VII compositions of the present invention is generated by the combination of the glycosylation and γ-carboxylation characteristics of the FVII molecules that make it up.

[0074] The transgenic factor VII used herein is characterized by post-translational modifications. In particular, these are glycosylation modifications, such as having zero or very low amounts of Galα1,3Gal at two N-glycosylation sites in the FVII composition, or still low enough not to be immunogenic. In contrast, the FVII described herein is not plasma FVII, i.e., it is not a purified product from human or animal plasma. More particularly, the transgenic FVII used herein has post-translational modifications, as well as O-glycosylation, γ-carboxylation, and specific disulfide bridges with defined glycan units.

[0075] The FVIIa used herein may include several post-translational modifications: the first 9 or 10 N-terminal glutamic acids are γ-carboxylated, Asp 63 is partially hydroxylated, Ser 52 and Ser60 are O-glycosylated and carry glucose (xylose) and 0-2 fucose units, respectively, Asn 145 and Asn 322 are N-glycosylated mainly through a monosialylated biantennary complex structure.

[0076] Advantageously, at least 80% of the transgenic factor VII molecules used herein are γ-carboxylated on 9 glutamate residues. In another embodiment, at least 85% of said molecules are γ-carboxylated on 9 glutamate residues. In another embodiment, 85%-100%, preferably 90%-100%, preferably 95%-100% of said molecules are γ-carboxylated on 9 glutamate residues. Advantageously, the degree of γ-carboxylation on glutamate residue 35 (Glu 35) of the factor VII molecules of the composition is less than 20%. In another embodiment, the degree of γ-carboxylation of residue Glu35 is less than 15%, preferably less than 10%, preferably less than 5%.

[0077] The Galα1,3Gal unit is a structure consisting of two galactoses linked at α1,3. It is located at the end of the oligosaccharide antenna of the N-linked structure. This unit is known for its immunogenicity. Thus, it is preferred to prepare FVII or FVIIa in which the number of Galα1,3Gal structures is zero or so low that it cannot be distinguished from the background noise obtained by measurements with currently available analytical devices. This expression equivalently represents all transgenic FVIIs in which the amount of Galα1,3Gal is close to that of plasma FVII. Advantageously, the amount of Galα1,3Gal in the FVII compositions described herein is not immunogenic to humans. In addition, the FVII used herein preferably contains two N-glycosylation sites at positions 145 and 322 and two O-glycosylation sites at positions 52 and 60, as does human FVII. At the N-glycosylation site, the oligosaccharide chain is linked to asparagine (N-linked). At the O-glycosylation site, the oligosaccharide chain is linked to serine. For each protein of the composition, the units linked to these amino acids will vary. However, for the entire composition, the amount of each glycan unit, and even each sugar, can be quantified.

[0078] The percentages of the different glycans given in this application do not take into account O-glycosylation.

[0079] Preferably, the FVII composition is characterized in that at least 40% of all glycan units of the FVII in the composition are in the form of monosialylated biantennary glycans. In another embodiment, the monosialylated biantennary form is present at at least 50%. In another embodiment, the monosialylated biantennary form is present at at least 60%, preferably at least 65%, preferably at least 70%.

[0080] Advantageously, the monosialylated biantennary glycan form of FVII is predominant. The FVII composition is characterized in that at least some of the factor VII sialic acid comprises an α2-6 bond.

[0081] Advantageously, at least 65% of the sialic acid of FVII comprises an α2,6 bond. Most advantageously, at least 70%, even 80% and especially at least 90% of the FVII sialic acid comprises an α2,6 bond.

[0082] In a particularly preferred manner, all sialic acids comprise an α2,6 bond, i.e. all sialic acids are bound to galactose via an α2,6 bond. The FVII composition described herein may also comprise sialic acid having an α2-3 bond.

[0083] According to an embodiment of the invention, 65%-100% of the FVII sialic acid comprises an α2,6 bond. More preferably, 70% or 80%-100% of the FVII sialic acid comprises an α2,6 bond.

[0084] Advantageously, in the monosialylated biantennary glycan form of FVII, the predominant glycan form is non-fucosylated.

[0085] Preferably, these non-fucosylated monosialylated biantennary glycan forms are present in the FVII of the composition in an amount of more than 20%. Advantageously, the amount is greater than 25% or greater than 40%. In a particularly advantageous manner, the degree of fucosylation of the FVII composition is 20%-50%. In one embodiment, the degree may be less than 20%.

[0086] 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 factor FVII of the composition are high mannose / hybrids.

[0087] Preferably, the glycosylation characteristics described herein provide improved biological activity and stability of FVII. Factor VII compositions having a substantially uniform isoelectric point facilitate formulation steps at the optimal pH of the pharmaceutical composition, preferably at an optimal pH of 6.0 ± 0.2, by preventing precipitation of FVII. In fact, it is known that at the isoelectric point of a molecule, they will tend to aggregate and precipitate. The factor VII molecules used in the compositions of the present invention have an isoelectric point of 6.6 - 7.0. This results in better stability of the factor VII composition, particularly at pH values below the isoelectric point, and particularly when formulated at pH 6.0. The improved stability of the factor VII composition prevents electrostatic interactions that lead to soluble and insoluble precipitation and aggregation phenomena, and prevents loss of raw materials, thereby preventing a decrease in yield resulting in a decrease in the active ingredient content and a resulting potential decrease in activity.

[0088] In a preferred embodiment, transgenic FVII is produced in the milk of rabbits, enabling the obtaining of a composition in which each factor VII molecule of the composition has two N - glycosylation sites. Preferably, all FVII molecules of the composition have an amount of Galα1,3Gal glycan units of less than 4%, and even no Galα1,3Gal glycan units. Thus, it is advantageous that the transgenic FVII produced by rabbits does not have Galα1,3Gal units.

[0089] FVII can be purified from milk by techniques known to those skilled in the art. For example, as described in U.S. Patent 6,268,487, a method for purifying a protein of interest from milk can include the following steps: a) subjecting the milk to tangential filtration through a membrane of sufficient porosity to form a retentate and a permeate, the permeate containing the exogenous protein, b) passing the permeate through a chromatographic capture device to displace the exogenous protein and obtain an effluent, c) combining the effluent and the retentate, d) repeating steps a) - c) until FVII is separated from lipids and casein micelles and FVII is recovered.

[0090] Advantageously, the FVII of the present invention is in an activated form. In one embodiment, FVII can be activated in vitro by factor Xa, VIIa, IIa, IXa, or XIIa. FVII can also generally be activated during its purification process, particularly by passing through a positively charged chromatographic column.

[0091] Multispecific antibody

[0092] "Multispecific antibody" refers to any antibody having at least two binding sites that are specific for 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 an antigen and substantially not cross - react with other antigens. Advantageously, the antibody has at least 10-6 M, preferably at least 10 -7 M, more preferably at least 10 -8 M, 10 -9 M or 10 -10 The affinity constant kD of M.

[0093] Thus, the antibodies used in the present invention have the ability to specifically bind both activated or non-activated forms of coagulation factor IX and coagulation factor X.

[0094] The antibodies used in the present invention having the ability to specifically bind both coagulation factor IX and coagulation factor X preferably have the ability to act as a substitute for factor VIII (FVIII), indicating that the antibody promotes the activation of FX by FIXa.

[0095] Such multispecific antibodies, preferably bispecific antibodies, can be obtained by a variety of methods known to those skilled in the art, such as by chemical conjugation or by using cell hybridomas (quadromes), which are formed by the fusion between two hybridomas producing two different monoclonal antibodies, and even by genetic recombination.

[0096] Therefore, the polynucleotides encoding such antibodies can be inserted into an expression vector and expressed in host cells or organisms improved by techniques well-known to those skilled in the art.

[0097] The antibodies used herein can have a very simple form, which is constructed from single-chain Fv fragments (scFv) of two or more antibodies linked by a suitable peptide linker.

[0098] "Fv" refers to the smallest antibody fragment that retains the property of recognizing and binding an antigen. The "Fv" fragment is a dimer (V H ) consisting of adjacent variable regions (V L ) carried by the heavy chain (H) and light chain (V L ) (V H + V L dimer).

[0099] Alternatively, it can be a full-length antibody, preferably comprising an Fc region. Several forms are possible. For example, in the first form, the scFv fragment of antibody A is fused to the end (usually the N-terminus) of the heavy chain of antibody B. The resulting antibody has a single type of heavy chain that 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 the second form, the heavy and light chains of antibody A are combined with the heavy and light chains of antibody B. Mutations, such as "knob into holes" (Ridgway et al., Protein Eng, 9, 617-21, 1996; US Patent 7,695,936), can be introduced where appropriate to prevent mismatching.

[0100] In this specification, unless indicated to the contrary, the term "Factor IX" refers to unactivated Factor IX or activated Factor IX (FIXa).

[0101] In this specification, unless indicated to the contrary, the term "Factor X" refers to unactivated Factor X or activated Factor X (FXa).

[0102] In particular, antibodies that recognize (i) FIX and / or FIXa and (ii) FX and / or FXa can be obtained according to the methods described in patent applications WO2005 / 035756, WO2006 / 109592, or WO2012 / 067176.

[0103] In a preferred embodiment, the antibody is emicizumab. For example, the preparation of this antibody is described in patent application WO2018047813 or patent application EP1688488.

[0104] Drug Compositions and Dosages

[0105] Factor VII and the antibody can be formulated in the form of separate drug compositions or combined in the same drug composition.

[0106] In the case of separate administration, FVII and the antibody are formulated in a manner suitable for administration by different routes or the same route.

[0107] Thus, for example, FVII can be administered intravenously, subcutaneously, or intramuscularly.

[0108] For example, the antibody can also be administered intravenously, subcutaneously, or intramuscularly.

[0109] The Factor VII composition can be, for example, as described in patent application WO2010 / 149907.

[0110] Thus, in an example of an embodiment, the composition comprises:

[0111] - Factor VII, preferably in the form of Factor VIIa;

[0112] - Arginine, possibly in the form of hydrochloride;

[0113] - Isoleucine;

[0114] - Lysine;

[0115] - Glycine;

[0116] - Trisodium citrate or calcium chloride;

[0117] - And, if appropriate, polysorbate 80 or polysorbate 20.

[0118] More particularly, the composition may comprise:

[0119] - Factor VII, preferably in the form of Factor VIIa;

[0120] - 10 - 40 g / L of arginine, possibly in the form of hydrochloride;

[0121] - 4.2 - 6.6 g / L of isoleucine;

[0122] - 0.6 - 1.8 g / L of lysine;

[0123] - 0.6 - 1.8 g / L of glycine;

[0124] - 0 - 0.2 g / L of trisodium citrate or 1 - 2 g / L of calcium chloride;

[0125] - And, if appropriate, 0 - 0.5 g / L of polysorbate 80.

[0126] The FVII composition can be stored in liquid or solid form, usually obtained by drying, which optionally further comprises at least one multispecific antibody as described herein. Before drying or after reconstitution into the form of an injectable preparation, the composition disclosed above is determined as a composition in liquid form.

[0127] Drying is a method for advanced water removal. Dehydration aims to eliminate as much water as possible. This phenomenon can be natural or forced. Drying can be carried out by freeze-drying, spray-drying, and spray-freeze-drying.

[0128] The preferred method for obtaining the solid form of the composition for the pharmaceutical use described herein is freeze-drying.

[0129] Lyophilization methods 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].

[0130] Other suitable methods for reducing the humidity or water content of the composition can be envisaged. Preferably, the humidity 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%.

[0131] The solid composition, preferably in lyophilized form, can be dissolved in water for injection (WFI) to obtain a formulation for therapeutic use.

[0132] The injectable formulation can be administered parenterally (intravenously, subcutaneously, intramuscularly), and the amount is evaluated by the practitioner. Administration in liquid form (before drying) or solid form by any route and in any suitable manner is not excluded.

[0133] The dose of FVII useful in the present invention can be determined in a suitable manner according to the type of formulation, the method of administration, the age and weight of the patient, the symptoms of the patient, the severity of the disease, etc.

[0134] The dose of FVII administered according to the present invention can be selected from 270 μg / kg - 2.70 μg / kg. Preferably, the dose of FVII administered is less than 270 μg / kg body weight, preferably less than 225 μg / kg body weight, preferably less than 180 μg / kg body weight, preferably less than 135 μg / kg body weight, preferably less than 90 μg / kg body weight, preferably less than 45 μg / kg body weight, preferably less than 9 μg / kg, preferably less than 5.4 μg / kg, preferably less than 2.7 μg / kg.

[0135] Multispecific antibody compositions, such as emicizumab antibodies, are antibodies as described in, for example, patent applications WO2017 / 188356 and WO2018 / 047813.

[0136] Thus, in an example of an embodiment, the composition is a liquid composition.

[0137] In an example of an embodiment, the composition comprises:

[0138] - an antibody bispecific for factor IX and factor X

[0139] - a surfactant, such as poloxamer 188 or polysorbate 20

[0140] - Histidine - Aspartic acid buffer

[0141] - Arginine.

[0142] More particularly, the composition may comprise:

[0143] - An antibody bispecific for factor IX and factor X at 20 mg / mL - 180 mg / mL,

[0144] - Poloxamer 188 at 0.2 mg / mL - 1 mg / mL,

[0145] - Histidine - Aspartic acid buffer at 10 mM - 40 mM,

[0146] - Arginine at 100 mM - 300 mM,

[0147] At pH 4.5 - 6.5.

[0148] The dose of the multispecific antibody composition for use in the present invention, such as emicizumab antibody, can be appropriately determined according to the type of formulation, administration method, patient age and weight, patient symptoms, severity of the disease, etc. For example, the antibody dose can be 0.3 - 5 mg / kg, preferably at most 3 mg / kg, once a week in the initial stage for 4 weeks, for example, followed by a maintenance dose, preferably lower, such as 1.5 mg / kg, once a week. Preferably, the dose of the antibody administered is less than 5 mg / kg body weight, preferably less than 3 mg / kg body weight, preferably less than 1.5 mg / kg body weight, preferably less than 1 mg / kg body weight, preferably less than 0.5 mg / kg body weight, preferably less than 0.1 mg / kg body weight, preferably less than 0.05 mg / kg body weight.

[0149] The antibody composition for use in the present invention can be administered to a patient by any suitable route, such as intravenously, intramuscularly, intraperitoneally, intraspinally, transdermally, subcutaneously, intraarticularly, sublingually, intrasynovially, orally or by inhalation. Preferably, the intravenous route or the subcutaneous route is advantageous.

[0150] According to a particular embodiment, factor VII and the antibody are administered to the patient simultaneously.

[0151] According to another particular embodiment, factor VII and the antibody are administered to the patient separately, preferably sequentially.

[0152] Therapeutic indications

[0153] The combinations described herein prevent or treat coagulation disorders, particularly hemophilia with factor VIII deficiency (hemophilia A, preferably acquired hemophilia A).

[0154] Preferably, the patient is a hemophilia A patient with anti-factor VIII.

[0155] The combinations described herein prevent or treat coagulation disorders, particularly factor VII deficiency.

[0156] The combinations described herein combine the rapid action of the extrinsic pathway of the FVII-activated coagulation cascade and the prolonged action of the multispecific antibodies of the intrinsic pathway of the activated coagulation cascade described herein. This combination makes it possible to provide better patient management. Examples:

[0157] Example 1: Purification and extraction of transgenic FVII

[0158] The method for the purification and extraction of factor VII implemented in this example is described in application EP12305882. The steps of this method are described below.

[0159] Transgenic rabbit milk is obtained from transgenic rabbit lines. The frozen milk from transgenic rabbits is thawed and concentrated into the form of a transgenic rabbit milk pool.

[0160] The resulting transgenic rabbit milk pool is then subjected to a clarification step using a depth filter with a porosity of 0.2 μm to remove lipids and insoluble compounds. The thus clarified milk is then subjected to a virus inactivation step at 25°C ± 2°C for at least 2 hours using a detergent solvent such as polysorbate 80 or tributyl phosphate. Such treatment effectively inactivates viruses, and particularly non-enveloped viruses. Then, an affinity chromatography step is performed on the clarified and virus-inactivated milk using an affinity ligand specific for factor VII / factor VIIa. The factor VII eluate obtained from this chromatography step is then subjected to ultrafiltration and formulation steps, enabling the production of an intermediate factor VII concentrate with a purity of 95%.

[0161] The intermediate factor VII concentrate is then subjected to a filtration step using a filter with a porosity of 0.1 μm - 0.2 μm, and then a nanofiltration step using filters with porosities of 20 nm and then 15 nm. The product thus obtained and containing factor VII is then subjected to Q Sepharose XL gel chromatography, then a CHT-1 chromatography step, and subsequently a Superdex 200 SEC chromatography. The factor VII concentrate thus obtained is then subjected to a stabilization treatment and then filtered through a filter with a porosity of 0.2 μm.

[0162] The method described thus enables the production of a factor VII concentrate with a purity of approximately 99.9995%.

[0163] Example 2: and Comparison of thrombotic potential Technicians can measure through the following scheme and (also known as emicizumab) for thrombogenic potential.

[0164] Reagents:

[0165] ■ Thrombin calibrator (Stago)

[0166] ■ 5pM PPP reagent (Stago)

[0167] ■ PPP reagent LOW (Stago)

[0168] ■ CK-Prest (Stago)

[0169] ■ Fluo-buffer (Stago)

[0170] ■ Fluo-substrate (Stago)

[0171] ■ FVIII-deficient plasma (Siemens)

[0172] ■ / Transgenic factor VII produced in rabbits 1mg / mL (LFB)

[0173] ■ PNP (Cryopep)

[0174] ■ (NovoNordisk)

[0175] ■ / Emicizumab (Roche / Genentech / )

[0176] Method:

[0177] The thrombin generation test consists of: activating blood coagulation ex vivo with a mixture of tissue factor and phospholipids (TF / PL) or by using cephalin, and then measuring the concentration of thrombin generated over time.

[0178] ● After inducing blood coagulation with TF / P, measure for thrombogenic potential:

[0179] The thrombin generation test is performed in 80 μL of a pooled FVIII-deficient plasma (which mimics plasma from patients with hemophilia A) in the presence of 20 μL of PPP reagent (Stago) containing 0.5 pM tissue factor (TF) and 4 μM phospholipids (PL). By adding 20 μL of the Fluca kit (substrate + CaCl 2) Initiate the reaction, which is the start of measuring thrombin generation.

[0180] The therapeutic dose of FVIIa is 270 μg / kg, equivalent to 6 μg / mL FVIIa in plasma, which takes into account a 100% yield. Then, thrombin generation tests are performed in the presence of 0.5 pM TF / 2 μM PL (thrombin inducer) at doses of 0 μg / mL, 1 μg / mL, 2 μg / mL, 3 μg / mL, 4 μg / mL, 5 μg / mL, and 6 μg / mL. The thrombin generation test is performed in the presence of 0.5 pM TF / 2 μM PL (thrombin inducer) at doses of 0 μg / mL, 1 μg / mL, 2 μg / mL, 3 μg / mL, 4 μg / mL, 5 μg / mL, and 6 μg / mL.

[0181] ● After inducing coagulation with cephalin, measure the thrombotic potential:

[0182] The thrombin generation test is performed in 80 μL of FVIII-deficient plasma pool (which mimics plasma of hemophilia A) in the presence of 20 μL of cephalin (CK-Prest reconstituted with 5 mL of distilled H 2 O).

[0183] The reaction is initiated by adding 20 μL of the Fluca kit (substrate + CaCl 2 ), which is the start of measuring thrombin generation.

[0184] Thrombin generation tests are performed in the presence of 20 μL of cephalin (thrombin inducer) at doses of 0 μg / mL, 1 μg / mL, 2 μg / mL, 3 μg / mL, 4 μg / mL, 5 μg / mL, and 6 μg / mL. The thrombin generation test is performed in the presence of 20 μL of cephalin (thrombin inducer) at doses of 0 μg / mL, 1 μg / mL, 2 μg / mL, 3 μg / mL, 4 μg / mL, 5 μg / mL, and 6 μg / mL.

[0185] ● After inducing coagulation with TF / PL, measure the thrombotic potential:

[0186] The thrombin generation test is performed in 80 μL of FVIII-deficient plasma pool (which mimics plasma of hemophilia A) in the presence of 20 μL of PPP reagent (Stago) containing 0.5 pM tissue factor (TF) and 4 μM phospholipid (PL).

[0187] The reaction is initiated by adding 20 μL of the Fluca kit (substrate + CaCl 2 ), which is the start of measuring thrombin generation.

[0188] Thrombin generation tests are performed in the presence of 0.5 pM TF / 2 μM PL (thrombin inducer) at doses of 0 μg / mL, 1 μg / mL, 2 μg / mL, 3 μg / mL, 4 μg / mL, 5 μg / mL, and 6 μg / mL. The thrombin generation test is performed in the presence of 0.5 pM TF / 2 μM PL (thrombin inducer) at doses of 0 μg / mL, 1 μg / mL, 2 μg / mL, 3 μg / mL, 4 μg / mL, 5 μg / mL, and 6 μg / mL.

[0189] ● After inducing coagulation with cephalin, measure the thrombus formation potential:

[0190] The thrombin generation test is performed in 80 μL of FVIII-deficient plasma pool (which mimics hemophilia A plasma) in the presence of 20 μL of cephalin (CK-Prest reconstituted with 5 mL of distilled H 2 2O).

[0191] The reaction is initiated by adding 20 μL of the Fluca kit (substrate + CaCl 2 2), which is the start of measuring thrombin generation.

[0192] The thrombin generation test is performed in the presence of 20 μL of cephalin at doses of 0 μg / mL, 1 μg / mL, 2 μg / mL, 3 μg / mL, 4 μg / mL, 5 μg / mL, and 6 μg / mL of .

[0193] ● After inducing coagulation with TF / PL, measure the thrombus formation potential:

[0194] The thrombin generation test is performed in 80 μL of FVIII-deficient plasma pool (which mimics hemophilia A plasma) in the presence of 20 μL of PPP reagent (Stago) containing 0.5 pM tissue factor (TF) and 4 μM phospholipid (PL).

[0195] The reaction is initiated by adding 20 μL of the Fluca kit (substrate + CaCl 2 2), which is the start of measuring thrombin generation.

[0196] The bispecific antibody that mimics FVIII function (Roche / Genentech / USA) is used at a maximum concentration of 50 μg / mL, which is the concentration detected during patient treatment (Oldenburg et al., NEJM, 2017). The thrombin generation test is performed in the presence of 0.5 pM TF / 4 μM PL (coagulation inducer) at doses of 0 μg / mL, 10 μg / mL, 20 μg / mL, 30 μg / mL, 40 μg / mL, and 50 μg / mL of

[0197] ● After inducing coagulation with cephalin, measure the thrombus formation potential:

[0198] The thrombin generation test is performed in 80 μL of FVIII-deficient plasma pool (which mimics hemophilia A plasma) in the presence of 20 μL of cephalin (CK-Prest reconstituted with 5 mL of distilled H2 Performed in the presence of O - reconstructed CK - Prest).

[0199] The reaction was initiated by adding 20 μL of the Fluca kit (substrate + CaCl 2 ), which was the start of the thrombin generation assay.

[0200] Then, thrombin generation tests were performed in the presence of 20 μL of cephalin at doses of 0 μg / mL, 10 μg / mL, 20 μg / mL, 30 μg / mL, 40 μg / mL, and 50 μg / mL. For all these tests, the fluorescence that appeared was measured on a Fluoroskan Ascent fluorometer (ThermoLabsystems) at an excitation wavelength of 390 nm and an emission wavelength of 460 nm. Then, thrombinograms (curves showing the change in fluorescence intensity over time) were analyzed using Thrombinoscope

[0201] software, which converted the fluorescence values to nM of thrombin by comparative calculation. TM Thrombin was generated, and the key variables for evaluating the efficacy of different drug products were recorded and compared: endogenous thrombin potential (PTE), peak height, latency, and velocity.

[0202]

[0203] Example 3: and or and Synergistic blood Evaluation of thrombotic potential

[0204] Technicians can determine the thrombotic potential of the combination of and by performing the following protocol.

[0205] Reagents:

[0206] The reagents, devices, and test protocols in FVIII - deficient plasma were the same as those described in Example 2.

[0207] Method:

[0208] ● After inducing coagulation with TF / PL, determine the thrombotic potential of the combination of :

[0209] ​The thrombin generation test was performed in 80 μL of a pooled FVIII-deficient plasma (which mimics hemophilia A plasma) in the presence of 20 μL of PPP reagent (Stago) containing 0.5 pM tissue factor (TF) and 4 μM phospholipids (PL).

[0210] The reaction was initiated by adding 20 μL of the Fluca kit (substrate + CaCl 2 ), which is the start of the thrombin generation assay.

[0211] The thrombin generation test was performed in several combinations in the presence of 0.5 pM TF / 4 μM PL (thrombin inducer). The composition containing the maximum amount of the product consisted of 6 μg / mL at its maximum value and 50 μg / mL and was constituted.

[0212] The thrombotic potential obtained in the presence of the product combination was compared with that of the single product. To account for the synergistic effect of the product combination, lower doses were evaluated to ensure that the thrombin detection was not saturated.

[0213] The tested compositions included:

[0214]

[0215] ● After inducing coagulation with cephalin, the thrombotic potential of the combination was determined:

[0216] The thrombin generation test was performed in 80 μL of a pooled FVIII-deficient plasma (which mimics hemophilia A plasma) in the presence of 20 μL of cephalin (CK-Prest reconstituted with 5 mL of distilled H 2 2O).

[0217] The reaction was initiated by adding 20 μL of the Fluca kit (substrate + CaCl 2 ), which is the start of the thrombin generation assay.

[0218] The thrombin generation test was performed in several combinations in the presence of 20 μL of cephalin. The composition containing the maximum amount of the product consisted of 6 μg / mL at its maximum value and 50 μg / mL and was constituted.

[0219] The thrombotic potential obtained in the presence of the product combination was compared with that of the single product. To account for the synergistic effect of the product combination, lower doses were evaluated to ensure that the thrombin detection was not saturated.

[0220] The tested compositions included:

[0221]

[0222]

[0223] ● After inducing coagulation with TF / PL, measure The combined thrombotic potential:

[0224] The thrombin generation test was performed in 80 μL of pooled FVIII-deficient plasma (which mimics hemophilia A plasma) in the presence of 20 μL of PPP reagent (Stago) containing 0.5 pM tissue factor (TF) and 4 μM phospholipid (PL).

[0225] The reaction was initiated by adding 20 μL of the Fluca kit (substrate + CaCl 2 ), which is the start of measuring thrombin generation.

[0226] The thrombin generation test was performed in several combinations in the presence of 0.5 pM TF / 4 μM PL (coagulation inducer). The composition containing the maximum amount of the product consisted of 6 μg / mL at its maximum and 50 μg / mL constituted.

[0227] The thrombotic potential obtained in the presence of the product combination was compared with that of the single product. To account for the synergistic effect of the product combination, lower doses were evaluated to ensure that the thrombin assay would not be saturated.

[0228] The tested compositions included:

[0229]

[0230] ● After inducing coagulation with cephalin, measure The combined thrombotic potential:

[0231] The thrombin generation test was performed in 80 μL of pooled FVIII-deficient plasma (which mimics hemophilia A plasma) in the presence of 20 μL of cephalin (CK-Prest reconstituted with 5 mL of distilled H 2 O).

[0232] The reaction was initiated by adding 20 μL of the Fluca kit (substrate + CaCl 2 ), which is the start of measuring thrombin generation.

[0233] The thrombin generation test was performed in several combinations in the presence of 20 μL of cephalin. The composition containing the maximum amount of the product consisted of 6 μg / mL at its maximum and 50 μg / mL consists of

[0234] The thrombogenic potential obtained in the presence of the product combination is compared with that of the single product. To account for the synergy of the product combination, lower doses are evaluated to ensure that the thrombin assay is not saturated.

[0235] The compositions tested included:

[0236]

[0237] For all these tests, the fluorescence that appeared was measured on a Fluoroskan Ascent fluorometer (ThermoLabsystems) at an excitation wavelength of 390 nm and an emission wavelength of 460 nm. The thrombinogram (a curve showing the change in fluorescence intensity over time) was then analyzed using Thrombinoscope TM software, which converts the fluorescence values to nM of thrombin by comparison calculations.

[0238] For example, synergy is considered when at least one of the parameters calculated from the thrombin generation test of a given combination is greater than the sum of each such parameter obtained from the individual components (after subtracting the background noise of the assay).

[0239] Example 4: Sevenfact TM , and comparison of the potential of a combination of these two in plasma from patients with hemophilia A:

[0240] Reagents:

[0241] ■ Thrombin calibrator (Stago)

[0242] ■ 1 pM TF PRP reagent (Stago)

[0243] ■ 4 μM PL MP reagent (Stago)

[0244] ■ Fluo-buffer (Stago)

[0245] ■ Fluo-substrate (Stago)

[0246] ■ Sevenfact TM : Recombinant factor VII produced in rabbits, 1 mg / mL (LFB)

[0247] ■ emicizumab (Roche / Genentech / )

[0248] ■ Plasma from patients with hemophilia A (Cryopep)

[0249] ■Owren Koller (Stago)

[0250] Method:

[0251] The thrombin generation test consists of: in vitro activated coagulation, for example using a mixture of tissue factor and phospholipids (TF / PL), and then measuring the concentration of thrombin generated over time. The thrombin generation test is performed using 80 μL of hemophilia A plasma (Cryopep) in the presence of a 20 μL mixture of PRP and MP reagents (Stago) containing 0.5 pM tissue factor and 4 μM phospholipids.

[0252] The reaction is initiated by adding 20 μL of the Fluca kit (substrate + CaCl 2 ), which is the start of the determination of thrombin generation.

[0253] Using a Fluoroskan Ascent device (ThermoLabsystems), fluorescence is measured by fluorometry at an excitation wavelength of 390 nm and an emission wavelength of 460 nm. The thrombinogram is analyzed using Thrombinoscope TM software, which converts the fluorescence intensity to the molar concentration of thrombin (nM) using comparative calculations.

[0254] To measure the thrombotic potential of two molecules, several hemophilia A plasmas were studied. The highest therapeutic dose of FVIIa was 270 μg / kg, corresponding to 6 μg / mL FVIIa (or 120 nM) in plasma. The application of this dose can be considered the maximum potential for thrombin generation. Based on the product concentration in the bloodstream obtained in patients, Sevenfact TM concentrations ranging from 20 - 100 nM were also studied. A bispecific antibody (Roche / Genentech / USA) was used at a maximum concentration of 120 μg / mL. The concentration actually detected during patient treatment was 50 μg / mL (or 300 nM) (Oldenburg et al., NEJM, 2017). Therefore, in this article, it was used at approximately 300 nM (50 μg / mL) for the determination of and the variables studied for the thrombotic potential of Sevenfact TM were:

[0255] - Peak thrombin potential (PTE): the area under the curve representing the total amount of thrombin generated,

[0256] - Peak height: the maximum concentration of thrombin measured, and

[0257] - Thrombin generation rate: The rate of thrombin formation.

[0258] 2 - Results

[0259] 2.1 - Sevenfact TM or Effect on plasma of hemophilia A

[0260] 2.1.1 - Evaluation in the first batch of plasma of hemophilia A

[0261] In this model, very low thrombin generation signals from two compounds were obtained regardless of the concentration used. In fact, at concentrations of 20 and 40 nM, for and Sevenfact TM virtually zero thrombin generation was observed. Using 100 nM Sevenfact TM , a very low thrombin generation peak was observed (Table 1).

[0262]

[0263] Table 1: Thrombin generation parameters from the first batch of plasma of hemophilia A treated with Sevenfact TM or

[0264] Therefore, each molecule used alone induced only very low thrombin generation.

[0265] 2.1.2 - Evaluation in the second batch of plasma of hemophilia A

[0266] The second batch of plasma of hemophilia A was tested. Using and Sevenfact TM again very low thrombin generation was observed, with the maximum thrombin generation peak at a concentration of 100 nM Sevenfact TM (Table 2).

[0267]

[0268] Table 2: Thrombin generation parameters from the second batch of plasma of hemophilia A treated with Sevenfact TM or

[0269] In this model, Sevenfact TM and used alone have low thrombotic potential.

[0270] Example 5: Sevenfact TM + Evaluation of synergistic combination

[0271] 1 - Protocol

[0272] The reagents, devices, and test protocols in plasma of hemophilia A are the same as those described in Example 2.

[0273] 2 - Results

[0274] As observed in Example 2, Sevenfact used alone TM and induces low thrombin generation in plasma of hemophilia A. The synergistic effect of the combination of Sevenfact TM and was investigated. Three concentrations (20 nM, 40 nM, and 100 nM) of Sevenfact were investigated in the presence of a 300 nM TM concentration. For at least one of the thrombin generation test parameters (PTE, thrombin generation peak, and velocity), if the effect of the Sevenfact TM + combination is at least 2 - fold greater than the sum of the effects of Sevenfact and used alone, a synergistic effect is considered.

[0275] 2.1 - Effect of Sevenfact TM and on plasma of hemophilia A after inducing coagulation with TF / PL

[0276] 2.1.1 - Evaluation in the first batch of plasma of hemophilia A

[0277] The results are shown in Table 3 and Figure 1 . At a very low Sevenfact TM concentration of 20 nM, the ratios of PTE ( TM + A), thrombin peak ( Figure 1 B), and velocity ( Figure 1 C) of the Sevenfact TM combination were 2.14, 2.95, and 4.19, respectively. Therefore, a synergistic thrombotic effect was observed even at the lowest test concentration.

[0278] At a concentration of 40 nM, for all test parameters, the ratio was greater than 2. The ratio obtained for PTE was 2.75 ( Figure 1 A), the ratio obtained for the thrombin peak was 3.96 ( Figure 1 B), and the ratio obtained for the velocity reached 6.21 ( Figure 1C). In other words, when Sevenfact is used in combination with TM and , the rate of thrombin formation is 6 times higher.

[0279] At a concentration of 100 nM of Sevenfact TM , the synergistic effect is greatest. At a concentration of 100 nM, for all tested parameters, the ratio is greater than 2. The ratio obtained for PTE is 4.00 ( Figure 1 A), and for the thrombin peak ratio is 4.81 ( Figure 1 B), which indicates that when and Sevenfact TM are used in combination, the maximum concentration of thrombin generated is almost 5 times higher. The corresponding velocity ratio is 9.58 ( Figure 1 C), which indicates that when Sevenfact TM and are used in combination, thrombin generation is almost 10 times faster.

[0280]

[0281] In summary, for all tested concentrations of Sevenfact TM , the combination application of Sevenfact TM and has a synergistic effect on thrombin generation.

[0282] 2.1.2 - Evaluation in the second batch of plasma from patients with hemophilia A

[0283] The results are shown in Table 4 and Figure 2 . At a very low concentration of 20 nM of Sevenfact TM , for the Sevenfact TM + combination, the ratio obtained for the PTE parameter is 2.21 ( Figure 2 A), the ratio obtained for the thrombin peak is 2.34 ( Figure 2 B), and the ratio obtained for the velocity parameter is 2.9 ( Figure 2 C). Therefore, even at the lowest concentration of Sevenfact TM tested, a synergistic thrombotic effect was observed.

[0284] At a concentration of 40 nM, the ratio corresponding to PTE is 2.29 ( Figure 2 A), the ratio corresponding to the thrombin peak is 2.79 ( Figure 2 B), and the ratio corresponding to the velocity is 3.68 ( Figure 2 C), which indicates that SevenfactTM In combination with results in thrombin formation that is approximately 4 times faster.

[0285] At a concentration of 100 nM of Sevenfact TM the synergistic effect is greatest. At a concentration of 100 nM, the ratio corresponding to the thrombin generation peak is 3.41( Figure 2 B), and the ratio corresponding to the rate is 5.63( Figure 2 C), which means that when Sevenfact TM is combined with thrombin generation is almost 6 times faster and the thrombin concentration obtained is almost 4 times higher.

[0286]

[0287] In summary, for all tested concentrations of Sevenfact TM the combination of Sevenfact TM and has a synergistic effect on thrombin generation.

Claims

1. A pharmaceutical composition comprising: a. a transgenic Factor VII, and b. a multispecific antibody against Factor IX and Factor X.

2. The pharmaceutical composition according to claim 1, wherein the transgenic Factor VII is human Factor VII produced by epithelial cells derived from the mammary gland of a transgenic non-human mammal.

3. The pharmaceutical composition according to claim 2, wherein the transgenic mammal is a rabbit.

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

5. A combination product comprising: a. a transgenic Factor VII, and b. a multispecific antibody against Factor IX and Factor X, which is used for preventing or treating a coagulation disorder in a patient.

6. The combination product according to claim 5, which is used for treating hemophilia A.

7. The combination product according to either claim 5 or 6, which is used for treating hemophilia A with Factor VIII inhibitors.

8. The combination product according to claims 5-7, wherein the combination product is in the form of a pharmaceutical composition, such as the pharmaceutical composition defined in any one of claims 1-4.

9. The combination product according to claims 5-8, wherein the Factor VIIa and the antibody are in a form suitable for simultaneous administration to a patient.

10. The combination product according to claims 5-8, wherein the Factor VIIa and the antibody are in a form suitable for separate administration to a patient.

11. A kit comprising: - a container containing transgenic Factor FVII; and - another container containing an antibody against Factor IX and Factor X.

12. A method for treating a coagulation disorder in a patient, the method comprising administering to the patient a transgenic Factor VII and a multispecific antibody against Factor IX and Factor X simultaneously or sequentially.

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

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