A combination of Factor VII and bispecific anti-Factor IX and Factor X antibodies.

Abstract

To provide a pharmaceutical combination that permits better management of hemophilia A patients, more particularly, patients with anti-factor VIII.SOLUTION: The present invention relates to a combination containing transgenic factor VII and a multispecific antibody against factors IX and X for simultaneous or separate administration.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates in particular to a pharmaceutical composition useful for treating coagulation disorders such as hemophilia A in patients who have type A hemophilia and express factor VIII inhibitor antibodies. [Background technology]

[0002] Blood coagulation involves two pathways, one endogenous and the other exogenous, which ultimately lead to a common pathway. The combination of both mechanisms ensures the formation of a solid, flexible blood clot that resists blood pressure. Through the action of thrombin, fibrinogen undergoes chemical modification, resulting in fibrin formation. Fibrin is essential for blood clot formation.

[0003] The endogenous pathway involves factors present in the bloodstream, and the coagulation process begins within the blood vessel itself. The extrinsic pathway involves tissue factor that is not normally present in the bloodstream and is released during vascular injury.

[0004] Factor VII is a glycoprotein involved in the exogenous coagulation pathway. To initiate the coagulation cascade, FVII must be activated to FVIIa. After activation, FVIIa complexes with tissue factor (TF) protein, which is associated with two phospholipids and released during vascular injury. FVIIa alone (not complexed with tissue factor) exhibits low proteolytic activity. The FVIIa-TF complex then converts factor X to factor Xa in the presence of calcium ions. This complex also acts to activate factor IX to factor IXa, thereby catalyzing the endogenous pathway. Factors IXa and Xa further activate activated factor VII.

[0005] Factor IX and Factor X are involved in the endogenous coagulation pathway. Activated factor IX allows factor X to be activated into factor Xa.

[0006] Factor Xa complexes with activated factor V, and prothrombinase converts prothrombin to thrombin. Thrombin then acts on fibrinogen, converting it to fibrin, and also allows FVIII and FV to be activated to FVIIIa and FVa, respectively. Prothrombin, in part, in the presence of naturally occurring calcium in plasma, enables the activation of factor XIII to FXIIIa, which is responsible for the hardening of fibrin thrombi.

[0007] Nevertheless, if coagulation factors are lacking, the coagulation cascade is interrupted or absent, and next we will discuss abnormal coagulation.

[0008] Activated factor VII acts locally in the presence of tissue factor, which is released after bleeding-causing tissue injury, even in the absence of factor VIII or factor IX. This is why factor VII, preferably in its activated form, is useful in treating certain blood clotting disorders that manifest as bleeding.

[0009] Therefore, factor VII is used to treat hemophilia patients with deficiencies in factor VIII (type A hemophilia) or factor IX (type B hemophilia), as well as patients with other coagulation factor deficiencies, such as the genetic deficiency of FVII. FVII is also recommended for stroke treatment.

[0010] Some hemophilia patients develop antibodies that inhibit factor VIII, which is administered as a treatment for hemophilia, typically in a concentrated form. This is currently the most common complication of hemophilia treatment.

[0011] Bispecific antibodies such as emicizumab, targeting FIX or FIXa and FX or FXa, are used to treat hemophilia A patients with anti-factor VIII antibodies. These antibodies functionally replace FVIII by promoting FIXa-mediated activation of FX, thereby linking these two molecules together. These antibodies have a sustained effect.

[0012] Combinations of recombinant FVIIa derived from cell culture (such as Novoseven® produced in BHK cells) and emicizumab (such as ACE910 or Hemlibra®) have 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), pp. 11-27)). This combination only showed an additive effect on the treatment of coagulation disorders. [Prior art documents] [Patent Documents]

[0013] [Patent Document 1] U.S. Patent No. 4,784,950 [Patent Document 2] U.S. Patent No. 5,997,864 [Patent Document 3] European Patent No. 0200421 [Patent Document 4] European Patent Application No. 0527063 [Patent Document 5] U.S. Patent No. 6,268,487 [Patent Document 6] U.S. Patent No. 7,695,936 [Patent Document 7] International Patent Application No. 2005 / 035756 [Patent Document 8] International Patent Application No. 2006 / 109592 [Patent Document 9] International Patent Application No. 2012 / 067176 [Patent Document 10] International Patent Application No. 2018047813 [Patent Document 11] European Patent Application No. 1688488 [Patent Document 12] International Patent Application No. 2010 / 149907 [License 13] International Patent Application No. 2017 / 188356 [License 14] International Patent Application No. 2018 / 047813 [License 15] European Patent Application No. 12305882 [Non-licensed literature]

[0014] [Non-licensed Document 1] R. HARTMANNら、OR36|Synergistic Effects of a Procoagulant Bispecific Antibody and FEIBA or Factor VIIA on Thrombin Generation (Haemophilia (2017), 23 (Suppl. 2), pages 11~27) [Non-licensed Document 2] Persson, J. Biol. Chem. 272: 19919~19924, 1997 [Non-licensed Document 3] Persson, FEBS letts, 413: 359-363, 1997 [Non-licensed Document 4] Hermentin, Glycobiology, Volume 6, No. 2, 1996 [Non-licensed Document 5] Qu, Blood, 11, pp. 2211-2219, 2008 [Non-licensed Document 6] Ridgway, Protein Eng, 9, pp. 617-21, 1996 [Non-licensed Document 7] Wang, Lyophilization and development of solid protein pharmaceuticals, International Journal of Pharmaceutics, Volume 203, Pages 1~60, 2000 [Non-licensed Document 8] Oldenburg, NEJM, 2017 [Overview of the Initiative] [Problems that the invention aims to solve]

[0015] Therefore, there is a need for drug combinations that enable better management of hemophilia A patients, and more specifically, patients with anti-factor VIII. [Means for solving the problem]

[0016] This invention proposes combining transgenic factor VII with multispecific antibodies against factor IX and factor X.

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

[0018] Therefore, one aspect of the present invention is: a. Transgenic factor VII, and b. A polyspecific antibody, preferably a bispecific antibody against factor IX and factor X, such as emicizumab. It is a pharmaceutical composition containing [a specific ingredient].

[0019] Preferably, factor VII is in the form of activated factor VII (FVIIa).

[0020] In certain embodiments, factor IX is in the form of activated factor IX (FIXa) and / or factor X is in the form of activated factor X (FXa).

[0021] Preferably, the transgenic factor VII is human factor VII derived from non-human transgenic mammals, such as human factor VII produced by mammary epithelial cells of rabbits that are transgenic for human factor VII.

[0022] The present invention a. Transgenic factor VII, and b. Multispecific antibodies against factor IX and factor X for use in the prevention or treatment of coagulation disorders such as hemophilia A, and more specifically, hemophilia A with factor VIII inhibitor (FVIII). We also offer combinations that include this.

[0023] Preferably, the combination is in the form of a pharmaceutical composition containing both transgenic factor VII and the antibody.

[0024] Alternatively, the transgenic factor VII and the antibody may be in the form of separate compositions suitable for simultaneous or separate (e.g., sequential) administration to the patient.

[0025] Another objective of the present invention is, - A container containing transgenic factor VII; and - Another container containing antibodies against Factor IX and Factor X This also applies to kits that include this item. [Brief explanation of the drawing]

[0026] [Figure 1] Evaluation of the synergistic thrombogenic effect of the Sevenfact® + Hemlibra® combination on batch 1 of hemophilia A plasma induced using TF / PL. (A) Evaluation of the synergistic thrombogenic effect on ETP, (B) Evaluation of the synergistic thrombogenic effect on the thrombin production peak, (C) Evaluation of the synergistic thrombogenic effect on velocity. [Figure 2] Evaluation of the synergistic thrombogenic effect of the Sevenfact® + Hemlibra® combination on batch 2 of hemophilia A plasma induced using TF / PL. (A) Evaluation of the synergistic thrombogenic effect on ETP, (B) Evaluation of the synergistic thrombogenic effect on the thrombin production peak, (C) Evaluation of the synergistic thrombogenic effect on velocity. [Modes for carrying out the invention]

[0027] General definition The coagulation phenomenon consists of a cascade of enzymatic reactions involving coagulation factors that exist in the form of pre-enzymes, which are converted to their "activated" form by protein cleavage in the presence of a specific cofactor. The activated form of each factor, which exists in the form of an inactive precursor, is named with the letter 'a'. Therefore, in vivo, FVIIa arises from the cleavage of its enzyme precursor by various proteases (FIXa, FXa, FVIIa) into two chains linked by disulfide crosslinks.

[0028] The term "treatment" or "to treat" generally refers to the improvement, prevention, or recovery of a disease or disorder, or at least its symptoms, such as slowing the progression of the disease or stabilizing its symptoms. This also includes delaying the onset of a disease or disorder, or at least its symptoms.

[0029] The term "prevention" or "preventing" refers to reducing the risk of developing or acquiring a particular disease or disorder.

[0030] In this invention, "patient" or "subject" means any mammal, and more specifically, a human being of any age, male or female, including children.

[0031] The term "pharmaceutical composition" refers to a preparation that enables the biological activity of an active ingredient and does not contain any additional ingredients that are toxic to the target of administration.

[0032] Transgenic Factor VII The term “Factor VII” or “FVII” includes polypeptides containing sequences 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., cattle, pigs, dogs, mice). The term also includes natural allele variants of Factor VII, which may be present in any form or degree of glycosylation or other posttranslational modifications. Thus, the term “Factor VII” also includes FVII variants having the same or better biological activity than wild-type FVII, and these variants include polypeptides in which one or more amino acid insertions, deletions or substitutions differ from wild-type FVII.

[0033] Unless otherwise specified in this document, the term "factor VII" refers to uncleaved FVII (enzyme precursor) or activated factor VII (FVIIa).

[0034] Therefore, 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, linked by a single disulfide bridge (Cys135~Cys262).

[0035] "Recombinant factor VII" refers to any factor VII derived from genetic manipulation and resulting from the expression of the corresponding gene in any microorganism, plant, or transgenic plant. Microorganisms refer to any bacterial, fungal, viral, or cellular system. Recombinant factor VII can also be produced from plant or mammalian cells, such as cultured eukaryotic cells of animals or humans.

[0036] "Transgenic Factor VII" refers to any recombinant Factor VII obtained from a transgenic animal for Factor VII.

[0037] A “transgenic animal” means any non-human animal whose genome has been modified in a way that is intended to enable the expression of a target protein (here, factor VII). Genomic modifications may result from alterations, modifications, or insertions of genes. These alterations may be caused by alterations or mutagenic agents used in conventional methods, or by directional mutagenesis. Genomic modifications may result from the insertion or substitution of wild-type or mutant genes or multiple genes. Transgenic animals may be selected from, without limitation, rabbits, goats, cattle, camels, hamsters, mice, rats, horses, sows, dromedaries, sheep, or llamas. In certain embodiments, animals that do not express α1,3-galactosyltransferase may be selected.

[0038] The term "biological activity of factor VIIa" refers to FVIIa's ability to, for example, produce thrombin on the surface of activated platelets. Factor VII activity can be evaluated in various ways. The biological activity of FVIIa can be quantified, for example, by measuring the ability of an FVII composition to promote blood coagulation by using plasma deficient in FVII and thromboplastin, as described in U.S. Patent No. 5,997,864. In this test, the biological activity is evaluated against a control sample and converted to "FVII units" compared to pooled standard human serum containing 1 unit / mL of factor VII 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 containing tissue factor (TF) and factor X surrounded by a lipid membrane (Persson et al., J. Biol. Chem. 272: pp. 19919-19924, 1997); (ii) measuring the hydrolysis of factor X in an aqueous system; (iii) measuring the physical binding of FVIIa to TF using surface plasmon resonance (Persson, FEBS letts, 413: pp. 359-363, 1997); (iv) measuring the hydrolysis of the synthetic substrate; or (v) measuring thrombin production in an in vitro system independent of TF.

[0039] In preferred embodiments, the FVII described herein is a polypeptide whose peptide sequence may be the sequence of a natural human FVII, i.e., a sequence present in a human without the FVII-related disorder. Such a technique is described in the document European Patent No. 0200421.

[0040] Advantageously, the FVII sequence used in this invention is sequence number 1.

[0041] "Synergistic effect" preferably means the combined effect of two products that is greater than twice the sum of the effects of the individual products produced separately. According to the present invention, a synergistic effect is obtained when, with respect to at least one thrombin production parameter, using transgenic FVIIa in combination with a polyspecific antibody against factor IX and factor X yields an effect greater than twice the sum of the effect obtained by transgenic FVIIa alone and the effect obtained by the polyspecific antibody against factor IX and factor X alone. This thrombin production parameter is selected from peak height, rate, or endogenous thrombin potential (ETP).

[0042] In certain embodiments, FVIIa is administered at a concentration of less than or equal to 105 nM, preferably less than 100 nM.

[0043] In certain embodiments, polyspecific antibodies against factor IX and factor X are administered at concentrations 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, and preferably less than 325 nM.

[0044] In certain embodiments, factor VII is obtained from the milk of a transgenic animal.

[0045] A method for producing recombinant protein in the milk of a transgenic animal may include the following steps: A synthetic DNA molecule containing the gene encoding the protein of interest (here, for example, human FVII) and under the control of a promoter of the protein naturally secreted in milk is incorporated into a non-human mammalian embryo. The embryo is then transplanted into a female mammal of the same species. After the mammal that develops from the embryo has grown sufficiently, milk secretion is induced in the mammal, and then the milk is collected. The milk contains the FVII of interest secreted by the transgenic animal.

[0046] An example of a protein preparation from the milk of a non-human female mammal is given in European Patent Application No. 0527063, and its teachings can be reproduced for the production of factor VII of the present invention.

[0047] The secretion of factor VII by the mammary glands allows factor VII to be secreted into the milk of transgenic animals, but involves tissue-dependent regulation of factor VII expression. Such regulation methods are well known to those skilled in the art. Expression is regulated via sequences that enable the expression of the protein toward specific tissues. These include, in particular, WAP, beta-casein, and beta-lactoglobulin promoter sequences, as well as signal peptide sequences, and the list is not limited.

[0048] In a preferred embodiment, factor VII according to the present invention is produced in the milk of transgenic rabbits.

[0049] In a particular advantageous manner, 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. In particular, plasmids containing the beta-casein promoter are created by introducing a sequence containing the beta-casein gene promoter, and these plasmids are designed to receive exogenous genes under the control of this promoter. The gene encoding human FVII is incorporated and placed under the control of the beta-casein promoter. The plasmid containing this promoter and the sequence encoding the target protein is digested with restriction enzymes to release a DNA fragment containing the beta-casein promoter and the human FVII sequence. After purification, the fragment is introduced into the male pronucleus of wild-type rabbit embryos by microinjection. The embryos are then cultured before being transferred into the hormonally-prepared oviducts of wild-type females. When these females give birth, the offspring are evaluated by PCR to confirm that they are transgenic animals. The copy number and completeness of the trans gene are determined by Southern technique from DNA extracted from the resulting young transgenic rabbits. The concentration of human FVII expressed in the milk of female transgenic offspring is assessed via immunoenzymatic testing.

[0050] In a particular embodiment, the VII factor useful in the present invention is the following step: (a) A step of inserting a DNA sequence containing a gene encoding factor VII into a non-human mammalian embryo, wherein the gene is under the transcriptional control of a beta-casein promoter. (b) A step of transferring the embryo obtained in step a) into the oviduct of a female non-human mammal so that it may develop into an adult non-human mammal. (c) A step of inducing milk secretion in an adult non-human mammal obtained in step b) of the female type, or in a female offspring of the non-human mammal in which the gene and the promoter are present in its genome. (d) the process of collecting milk from the non-human mammal, (e) Process of purifying FVII present in the collected milk It is obtained by a method that includes the following.

[0051] The FVII, which is useful here, has a substantially uniform isoelectric point.

[0052] "Isoelectric point" or "pl" means the pH at which the net elementary charge of the factor VII or factor VIIa molecule is zero, i.e., the pH at which the molecule is electrically neutral (in the form of an amphoteric ion). The isoelectric point of factor VII according to the present invention can be measured by performing techniques well known to those skilled in the art, such as isoelectric focusing electrophoresis ("IEF"). This electrophoretic method separates proteins based on their isoelectric points. Electrophoresis consists of the movement of proteins induced by a uniform current along a pH gradient until the protein reaches a pH equivalent to its specific isoelectric point, at which point the protein stops moving because its net charge becomes zero. An IEF gel is used to determine the isoelectric point of a given protein.

[0053] "Substantially homogeneous" means that at least 90%, preferably at least 95%, of the factor VII molecules in the composition have isoelectric points that fall within 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 isoelectric points that fall within 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 isoelectric points that fall within a pH unit difference of 0.4.

[0054] The term "N-glycan type" refers to all N-glycan types present at the two N-glycosylation sites of factor VII of the present invention. An N-glycan type is called monocharged if its total charge is equal to 1. In this invention, "charge" refers to a phosphate group, a sulfate group, or a sialic acid molecule. Therefore, an N-glycan type is called monocharged if it contains only one phosphate group, one sulfate group, or one sialic acid molecule. In contrast to the term "monocharged," the term "bicharged" means that the total charge carried by the N-glycan type is equal to 2, i.e., the N-glycan type has two charges selected from a phosphate group, a sulfate group, and / or a sialic acid molecule. In other words, a bicharged N-glycan type has one sialic acid molecule and one phosphate group, or one sialic acid molecule and one sulfate group, or two sialic acid groups, or two phosphate groups, or two sulfate groups, or a phosphate group and a sulfate group. The term "tricharged" means that the total charge carried by the N-glycan form is equal to 3, i.e., the N-glycan form has three charges selected from phosphate groups, sulfate groups, and / or sialic acid molecules. In other words, a tricharged 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 groups and one sulfate group, or one sialic acid molecule and two phosphate groups, or one sialic acid molecule 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" means that the N-glycan form contains no charge whatsoever.

[0055] The charge of the N-glycan type of factor VII according to the present invention can be measured by performing methods well known to those skilled in the art, particularly by ultra-high performance liquid chromatography (AEX-UPLC / FD) coupled with fluorescence detection of an anion exchange resin. This method makes it possible to separate different N-glycan types according to their apparent charge (see Hermentin et al., Glycobiology, Vol. 6, No. 2, 1996, in particular). In the context of anion exchange chromatography, a positively charged resin is used as the stationary phase. These positively charged resins are generally composed of cross-linked polymers or gels on which positively charged groups are grafted. In advantageous embodiments of the present invention, an aminopropyl type weak anion exchange column is used.

[0056] In the case of the factor VII composition according to the present invention, of all N-glycan types of factor VII in the composition, at least 50% of the N-glycan types, at least 60%, preferably at least 65%, preferably at least 70%, preferably at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, and preferably at least 95% are thought to be monocharged. In a preferred embodiment, factor VII molecules having monocharged N-glycan types correspond to 50% to 95%, preferably 50% to 90%, preferably 50% to 80%, preferably 50% to 75%, preferably 50% to 70%, preferably 50% to 65%, and preferably 50% to 60% of factor VII in the composition.

[0057] The substantially uniform isoelectric point of the factor VII composition combination according to the present invention arises from the combination of glycosylation and γ-carboxylation properties of the FVII molecules constituting the combination.

[0058] The transgenic factor VII useful here possesses post-translational modification characteristics. In particular, these characteristics include glycosylation modifications such as two N-glycosylation sites with zero or extremely low amounts of Galα1,3Gal in the FVII composition, or amounts low enough not to be immunogenic. In contrast, the FVII described herein is not plasma FVII; that is, this FVII is not a purified product derived from human or animal plasma. More specifically, the transgenic FVII useful here has post-translational modifications, as well as two O-glycosylations, γ-carboxylations, and specific disulfide crosslinks with limited glycan units.

[0059] The useful FVIIa here can contain several posttranslational modifications: the first nine or ten N-terminal glutamates are γ-carboxylated, and Asp 63 It is partially hydroxylated, Ser 52 and Ser 60 These are O-glycosylated, and each is glucose (xylose) 0~2 And it is responsible for the fucose unit, Asn 145 and Asn 322 It is primarily N-glycosylated by monosialylated biantennary complex structures.

[0060] Advantageously, at least 80% of the transgenic factor VII molecules useful here have γ-carboxylation at nine glutamate residues. In another embodiment, at least 85% of the molecules have γ-carboxylation at nine glutamate residues. In yet another embodiment, 85% to 100%, preferably 90% to 100%, and preferably 95% to 100% of the molecules have γ-carboxylation at nine glutamate residues. Advantageously, the degree of γ-carboxylation at glutamate residue 35 (Glu35) of the factor VII molecules of the composition is less than 20%. In yet another embodiment, the degree of γ-carboxylation at residue Glu35 is less than 15%, preferably less than 10%, and preferably less than 5%.

[0061] The Galα1,3Gal unit is a structure composed of two galactoses linked at α1,3. This unit is located at the end of the oligosaccharide antennas of the N-linked structure. This unit is known for its immunogenicity. Therefore, it is preferable to produce FVII or FVIIa with zero or so few Galα1,3Gal structures that they are indistinguishable from background noise obtained by measurements performed by currently available analytical instruments. This phrase equally refers to all transgenic FVIIs in which the amount of Galα1,3Gal is close to the amount of Galα1,3Gal in plasma FVII. Advantageously, the amount of Galα1,3Gal in the FVII compositions described herein is not immunogenic to humans. Furthermore, the FVIIs useful herein preferably contain two N-glycosylation sites at positions 145 and 322, and two O-glycosylation sites at positions 52 and 60, similar to human FVII. In N-glycosylation sites, the oligosaccharide chain is linked to asparagine (N-linking). In O-glycosylation sites, the oligosaccharide chain is linked to serine. The units linked to these amino acids differ for each protein in the composition. However, in the overall composition, the amount of each glycan unit or even the amount of each sugar can be quantified.

[0062] The percentages of different glycans given in this application do not take O-glycosylation into account.

[0063] Preferably, the FVII composition is characterized in that at least 40% of all glycan units of the FVII composition are monosialic acid-added branched glycans. In another embodiment, the monosialic acid-added branched glycans are present in at least 50%. In yet another embodiment, the monosialic acid-added branched glycans are present in at least 60%, preferably at least 65%, and preferably at least 70%.

[0064] Advantageously, the monosialic acid-added branched glycan type of FVII is dominant. The FVII composition is characterized in that at least a portion of the factor VII sialic acid contains an α2-6 linkage.

[0065] Advantageously, at least 65% of FVII sialic acid contains α2,6 bonds. More advantageously, at least 70%, 80%, and especially at least 90% of FVII sialic acid contains α2,6 bonds.

[0066] In a particularly preferred manner, all sialic acids contain an α2,6 linkage, i.e., all sialic acids are bonded to galactose by an α2,6 linkage. The FVII compositions described herein may also include sialic acids having an α2-3 linkage.

[0067] According to embodiments of the present invention, 65% to 100% of FVII sialic acid contains α2,6 bonds. More preferably, 70% or 80% to 100% of FVII sialic acid contains α2,6 bonds.

[0068] Advantageously, among the monosialic acid-added branched glycan types of FVII, the dominant glycan type is not fucosylated.

[0069] Preferably, these nonfucosylated monosialic acid-added branched glycans are present in greater than 20% of the FVII of the composition. Advantageously, this amount is greater than 25% or greater than 40%. In a particularly advantageous form, the degree of fucosylation in the FVII composition is between 20% and 50%. In one embodiment, this degree can be less than 20%.

[0070] In certain embodiments, at least 10%, preferably at least 15%, preferably at least 20%, preferably at least 25% of the N-glycan type of factor VII of the composition is high-mannose / hybrid.

[0071] Preferably, the glycosylation profiles described herein result in improved bioactivity and stability for FVII. Factor VII compositions having substantially uniform isoelectric points facilitate the formulation process at the optimal pH of the pharmaceutical composition, which is 6.0 ± 0.2, by preventing precipitation of FVII at the optimal pH. In fact, it is known that at the isoelectric points of molecules, these factors tend to aggregate and precipitate. The factor VII molecules used in the compositions of the present invention have isoelectric points in the range of 6.6 to 7.0. This results in better stability of the factor VII composition, especially when the factor VII composition is formulated at a pH lower than its isoelectric point, particularly at pH 6.0. Improved stability of the factor VII composition prevents electrostatic interactions that cause soluble and insoluble precipitation and aggregation, and prevents the loss of raw materials, and therefore the potential for reduced yield, which in turn leads to a loss of the amount of active ingredient and thus a loss of activity.

[0072] In a preferred embodiment, the transgenic FVII is produced by rabbits in their milk, and the composition can be obtained if each factor VII molecule of the composition has two N-glycosylation sites. Preferably, the amount of Galα1,3Gal glycan units in all FVII molecules of the composition is less than 4%, or even zero. Thus, advantageously, the transgenic FVII produced by rabbits does not have Galα1,3Gal units.

[0073] FVII can be purified from milk by techniques known to those skilled in the art. For example, the method for purifying the target protein from milk described in U.S. Patent No. 6,268,487 includes the steps of: a) subjecting milk to tangential filtration through a membrane with sufficient porosity to form residues and permeates, where the permeates contain exogenous proteins; b) subjecting the permeates to a chromatographic capture device to obtain eluates in place of the exogenous proteins; c) combining the eluates with the residues; d) repeating steps a) to c) until FVII is separated from lipids and casein micelles, and recovering FVII.

[0074] Advantageously, the FVII of the present invention is in an activated form. In one embodiment, FVII can be activated in vitro by factor Xa, factor VIIa, factor IIa, factor IXa or factor XIIa. FVII can also typically be activated during its purification process, particularly by passing through a positively charged chromatographic column.

[0075] Bispecific antibody The term "bispecific antibody" means any antibody having at least two different binding sites 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 without substantially cross-reacting with another antigen. Advantageously, the antibody has an affinity constant kD of at least 10 -6 M, preferably at least 10 -7 M, more preferably at least 10 -8 M, 10 -9 M, or 10 -10 M.

[0076] Therefore, the antibodies useful in the present invention have the ability to specifically bind to both factor IX and factor X of coagulation, either in an activated or non-activated form.

[0077] The antibody used in this invention has the ability to specifically bind to both coagulation factor IX and coagulation factor X, but preferentially has the ability to act as a substitute for factor VIII (FVIII), which means that this antibody promotes the activation of FX by FIXa.

[0078] Such polyspecific, preferably bispecific, antibodies can be obtained by various methods known to those skilled in the art, for example, by chemical conjugation, or by using quadromas resulting from the fusion of two hybridomas that produce two different monoclonal antibodies, or by genetic recombination.

[0079] Therefore, polynucleotides encoding such antibodies can be inserted into an expression vector and expressed in host cells or organisms adapted by techniques well known to those skilled in the art.

[0080] The antibodies useful here can be constructed from single-stranded Fv fragments (scFv) derived from two or more antibodies, and can be associated with a suitable peptide linker, allowing for a very simple format.

[0081] "Fv" refers to the smallest antibody fragment that retains the property of recognizing and binding to an antigen. The "Fv" fragment is a variable region (V) carried by the heavy chain (H). H ) and the adjacent variable region (V) of the light chain (L) L ) consists of a dimer (V H +V L It is a dimer.

[0082] Alternatively, Fv allows for a full-length antibody, which preferably contains an Fc region. Several formats are feasible. For example, in the first format, the svFv fragment of antibody A is fused to the end (generally the N-terminus) of the heavy chain of antibody B. The resulting antibody has a single type of heavy chain containing 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 containing the VL and CL domains of antibody B (Qu et al., Blood, 11, pp. 2211-2219, 2008). In the second format, the heavy and light chains of antibody A are associated with the heavy and light chains of antibody B. If necessary, mutations, such as "knobs into holes" (Ridgway et al., Protein Eng, 9, pp. 617-21, 1996; U.S. Patent No. 7,695,936), can be introduced to prevent mismatches.

[0083] Unless otherwise specified, the term "factor IX" in this document refers to either inactivated factor IX or activated factor IX (FIXa).

[0084] Unless otherwise specified, the term "Factor X" in this document refers to inactivated factor X or activated factor X (FXa).

[0085] (i) Antibodies that recognize FIX and / or FIXa, and (ii) Antibodies that recognize FX and / or FXa, in particular, can be obtained by the methods described in International Patent Application No. 2005 / 035756, International Patent Application No. 2006 / 109592, and International Patent Application No. 2012 / 067176.

[0086] In a preferred embodiment, the antibody is emicizumab. The production of this antibody is described, for example, in International Patent Application No. 2018047813 or European Patent Application No. 1688488.

[0087] Pharmaceutical composition and dosage Factor VII and the antibody can be formulated in the form of separate pharmaceutical compositions or combined within the same pharmaceutical composition.

[0088] For individual administration, FVII and the antibody can be prescribed in a form suitable for administration via different or the same route.

[0089] Therefore, for example, FVII can be administered intravenously, subcutaneously, or intramuscularly.

[0090] Antibodies can also be administered, for example, intravenously, subcutaneously, or intramuscularly.

[0091] The factor VII composition can be, for example, the composition described in International Patent Application No. 2010 / 149907.

[0092] Therefore, in one example of the embodiment, the composition, - Factor VII, preferably in the form of factor VIIa; - Arginine, preferably in the form of hydrochloride; - Isoleucine; - Lysine; - Glycine; - Trisodium citrate or calcium chloride; - And, if necessary, polysorbate 80 or polysorbate 20 Includes.

[0093] For further details, the composition is - Factor VII, preferably in the form of factor VIIa; - 10-40 g / L of arginine, preferably in the form of hydrochloride; - Isoleucine at 4.2-6.6 g / L; - 0.6~1.8 g / L of lysine; - 0.6~1.8 g / L of glycine; - 0-0.2 g / L of trisodium citrate or 1-2 g / L of calcium chloride; - And, if necessary, 0-0.5 g / L of polysorbate 80 It can include...

[0094] The FVII composition may optionally also include at least one polyspecific antibody described herein, and can be stored in liquid form or in solid form, typically obtained by drying. The compositions disclosed above are determined in relation to the liquid composition before drying or after reconstitution into an injectable formulation.

[0095] Drying is a method for high-stage water removal. This is dehydration aimed at removing as much water as possible. This process can be natural or forced. This drying can be carried out using freeze-drying, spray drying, and spray freeze-drying.

[0096] A preferred method for obtaining the solid form of the pharmaceutical compositions described herein is freeze-drying.

[0097] The freeze-drying method is 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, pp. 1-60, 2000].

[0098] Other suitable methods can be considered for reducing the humidity or moisture content of the composition. Preferably, the humidity is less than or equal to 3% by mass, preferably less than or equal to 2.5%, preferably less than or equal to 2%, and preferably less than or equal to 1.5%.

[0099] Preferably, the solid composition in lyophilized form can be dissolved in water for injection (WFI) to obtain a therapeutic formulation.

[0100] Injectable formulations may be administered parenterally (intravenously, subcutaneously, or intramuscularly) in amounts assessed by the practitioner. Administration in liquid (before drying) or solid form via any appropriate route and by any means is not excluded.

[0101] The effective FVII dosage in this invention can be determined appropriately depending on the type of formulation, administration method, patient age and weight, patient symptoms, disease severity, etc.

[0102] The FVII dose administered according to the present invention can be selected between 270 μg / kg and 2.70 g / 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 body weight, preferably less than 5.4 μg / kg, and preferably less than 2.7 μg / kg.

[0103] The polyspecific antibody composition, such as the emicizumab antibody, is similar to the compositions described in, for example, International Patent Application No. 2017 / 188356 and International Patent Application No. 2018 / 047813.

[0104] Therefore, in one embodiment, the composition is a liquid composition.

[0105] In one example of the embodiment, the composition, - Antibodies that are bispecific to factor IX and factor X, - Surfactants such as poloxamer 188 or polysorbate 20, - Histidine-aspartate buffer, - Arginine Includes.

[0106] For further details, the composition is - Antibodies with bispecificity for factor IX and factor X, ranging from 20 mg / mL to 180 mg / mL. - Poloxamer 188 in concentrations of 0.2 mg / mL to 1 mg / mL - 10 mM to 40 mM histidine-aspartate buffer, - 100mM to 300mM arginine It can be contained within a pH range of 4.5 to 6.5.

[0107] The dosage of the polyspecific antibody composition, such as emicizumab antibody, which is useful in the present invention, can be appropriately determined according to the type of formulation, method of administration, the patient's age and weight, the patient's symptoms, the severity of the disease, etc. The antibody dose is, for example, 0.3 to 5 mg / kg, preferably up to 3 mg / kg once a week during the initial period, which can last for 4 weeks, and then, for example, the maintenance dose is preferably lower, for example, 1.5 mg / kg once a week. Preferably, the dose of 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, and preferably less than 0.05 mg / kg body weight.

[0108] The antibody compositions useful in the present invention can be administered to a patient via any suitable route, for example, intravenously, intramuscularly, intraperitoneally, intracerebrospinally, percutaneously, subcutaneously, intraarticularly, sublingually, intrasynovally, orally, or by inhalation. Intravenous or subcutaneous routes are preferred.

[0109] According to a specific embodiment, Factor VII and the antibody are administered to the patient simultaneously.

[0110] According to another specific embodiment, Factor VII and the antibody are administered to the patient separately, preferably sequentially.

[0111] Signs of treatment The combinations described herein are for preventing or treating coagulation disorders, particularly hemophilia with factor VIII deficiency (type A hemophilia, preferably acquired type A hemophilia).

[0112] Preferably, the patient is a patient with type A hemophilia who has anti-factor VIII.

[0113] The combinations described herein are for the prevention or treatment of coagulation disorders, particularly factor VII deficiency.

[0114] The combination described herein combines the rapid effect of FVII, which activates the exogenous pathway of the coagulation cascade, with the long-term effect of the multispecific antibody described herein, which activates the endogenous pathway of the coagulation cascade. This combination makes it possible to provide better patient management. [Examples]

[0115] (Example 1) Purification and extraction of transgenic FVII The method for purifying and extracting Factor VII performed in this embodiment is the method described in European Patent Application No. 12305882. The steps of this method are described below.

[0116] Transgenic rabbit milk is obtained from transgenic rabbit strains. Frozen milk derived from transgenic rabbits is thawed and concentrated in the form of a pool of transgenic rabbit milk.

[0117] Next, the pool of transgenic rabbit milk obtained in this manner is subjected to a clearing step using a depth filter with a porosity of 0.2 μm to remove lipids and insoluble compounds. The milk thus cleared is then subjected to a virus inactivation step with a detergent solvent, such as polysorbate 80 or tri-n-butyl phosphate, at 25°C ± 2°C for at least 2 hours. Such treatment effectively inactivates viruses, especially non-enveloped viruses. Next, the cleared and virus-inactivated milk is subjected to an affinity chromatography step using an affinity ligand specific to factor VII / factor VIIa. The factor VII eluate obtained from this chromatography step is then subjected to an ultrafiltration and formulation step, thus making it possible to obtain an intermediate factor VII concentrate with a purity of 95%.

[0118] Next, the intermediate factor VII concentrate is subjected to a filtration step using a filter with a porosity of 0.1 μm to 0.2 μm, followed by a nanofiltration step through filters with porosity of 20 nm, and then 15 nm. The resulting product containing factor VII is then subjected to Q Sepharose XL gel chromatography, then CHT-I chromatography, and then Superdex 200 SEC chromatography. The resulting factor VII concentrate is then subjected to a stabilization step, and then filtration through a filter with a porosity of 0.2 μm.

[0119] Therefore, the method described makes it possible to obtain a factor VII concentrate with a purity of approximately 99.9995%.

[0120] (Example 2) Comparison of thrombus formation potential of Novoseven®, Sevenfact®, and Hemlibra® Those skilled in the art can measure the thrombus-forming potential of Novoseven®, Sevenfact®, and Hemlibra® (also known as emicizumab) by following the protocol below.

[0121] reagent: ● Thrombin Calibrator (Stago) ● 5 pM PPP reagent (Stago) ● PPP reagent LOW (Stago Corporation) ● CK-Prest (Stago Corporation) ● Fluo Buffer (Stago Corporation) ● Fluo substrate (Stago) ● FVIII-deficient plasma (Siemens) ● Sevenfact (registered trademark) / Transgenic Factor VII produced in rabbits 1 mg / ml (LFB Corporation) ● PNP (Cryopep Corporation) ● Novoseven (registered trademark) (NovoNordisk Inc.) ● Hemlibra (registered trademark) / Emicizumab (Roche / Genentech / Chugai)

[0122] method: The thrombin production test consists of ex vivo activation of coagulation using a tissue factor-phospholipid mixture (TF / PL) or cephalin, followed by measurement of the concentration of thrombin produced over time.

[0123] • Measuring the thrombus formation potential of Novoseven® after coagulation induction using TF / PL: The thrombin production test is performed in 80 μL of FVIII-deficient plasma, mimicking 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).

[0124] The reaction is initiated by adding 20 μL of the Fluca kit (substrate + CaCl2), and this addition marks the start of the measurement of thrombin production.

[0125] The therapeutic dose of FVIIa is 270 μg / kg, which corresponds to 6 μg / mL of FVIIa in plasma, considering a 100% recovery rate. Next, the thrombin production test is performed in the presence of 0.5 pM TF / 2 μM PL (coagulation-inducing factor) with Novoseven® doses of 0 μg / mL, 1 μg / mL, 2 μg / mL, 3 μg / mL, 4 μg / mL, 5 μg / mL, and 6 μg / mL.

[0126] • Measuring the thrombus formation potential of Novoseven® after coagulation induction with cephalin: The thrombin production 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 H2O).

[0127] The reaction is initiated by adding 20 μL of the Fluca kit (substrate + CaCl2), and this addition marks the start of the measurement of thrombin production.

[0128] The thrombin production test is performed in the presence of 20 μL of cephalin (coagulation-inducing factor) at Novoseven® doses of 0 μg / mL, 1 μg / mL, 2 μg / mL, 3 μg / mL, 4 μg / mL, 5 μg / mL, and 6 μg / mL.

[0129] • Measuring the thrombus formation potential of Sevenfact® after coagulation induction using TF / PL: The thrombin production test is performed in 80 μL of FVIII-deficient plasma, mimicking 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).

[0130] The reaction is initiated by adding 20 μL of the Fluca kit (substrate + CaCl2), and this addition marks the start of the measurement of thrombin production.

[0131] Next, the thrombin production test is performed in the presence of 0.5 pM TF / 2 μM PL (coagulation-inducing factor) with Sevenfact® doses of 0 μg / mL, 1 μg / mL, 2 μg / mL, 3 μg / mL, 4 μg / mL, 5 μg / mL, and 6 μg / mL.

[0132] • Measuring the thrombus formation potential of Sevenfact® after coagulation induction with cephalin: The thrombin production 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 H2O).

[0133] The reaction is initiated by adding 20 μL of the Fluca kit (substrate + CaCl2), and this addition marks the start of the measurement of thrombin production.

[0134] The thrombin production test is performed in the presence of 20 μL of cephalin at Sevenfact® doses of 0 μg / mL, 1 μg / mL, 2 μg / mL, 3 μg / mL, 4 μg / mL, 5 μg / mL, and 6 μg / mL.

[0135] • Measuring the thrombus formation potential of Hemlibra® after coagulation induction using TF / PL: The thrombin production test is performed in 80 μL of FVIII-deficient plasma, mimicking 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).

[0136] The reaction is initiated by adding 20 μL of the Fluca kit (substrate + CaCl2), and this addition marks the start of the measurement of thrombin production.

[0137] Hemlibra® (Roche / Genentech / Chugai, USA), a bispecific antibody that mimics the function of FVIII, is used at a maximum concentration of 50 μg / mL, which is the concentration detected in patients undergoing treatment (Oldenburg et al., NEJM, 2017). Next, the thrombin production test is performed with Hemlibra® doses of 0 μg / mL, 10 μg / mL, 20 μg / mL, 30 μg / mL, 40 μg / mL, and 50 μg / mL in the presence of 0.5 pM TF / 4 μM PL (coagulation-inducing factor).

[0138] • Measuring the thrombus formation potential of Hemlibra® after coagulation induction with cephalin: The thrombin production 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 H2O).

[0139] The reaction is initiated by adding 20 μL of the Fluca kit (substrate + CaCl2), and this addition marks the start of the measurement of thrombin production.

[0140] Next, the thrombin production test is performed in the presence of 20 μL of cephalin with Hemlibra® doses of 0 μg / mL, 10 μg / mL, 20 μg / mL, 30 μg / mL, 40 μg / mL, and 50 μg / mL.

[0141] In all of these tests, fluorescence is measured on a Fluoroskan Ascent fluorophotometer (ThermoLabsystems) at excitation wavelengths of 390 nm and emission wavelengths of 460 nm. Thrombinograms (curves showing fluorescence intensity over time) are then analyzed using Thrombinoscope® software, which converts fluorescence values ​​to nM thrombin through comparative calculations.

[0142] Thrombin is produced, and key variables for evaluating the efficacy of different drugs—endogenous thrombin activity (ETP), peak height, latency, and velocity—are recorded and compared.

[0143] (Example 3): Evaluation of the synergistic thrombus formation potential of Novoseven® and Hemlibra® or SevenFact® and Hemlibra®. Those skilled in the art can measure the thrombus formation potential of the Novoseven® / Hemlibra® and Sevenfact® / Hemlibra® combinations by following the protocol below.

[0144] reagent: The reagents, equipment, and experimental protocol for FVIII-deficient plasma are the same as those described in Example 2.

[0145] method: • Measuring the thrombus formation potential of the Novoseven® + Hemlibra® combination after coagulation induction using TF / PL: The thrombin production test is performed in 80 μL of FVIII-deficient plasma, mimicking 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).

[0146] The reaction is initiated by adding 20 μL of the Fluca kit (substrate + CaCl2), and this addition marks the start of the measurement of thrombin production.

[0147] Thrombin production tests are performed in the presence of 0.5 pM TF / 4 μM PL (coagulation-inducing factor) with several Novoseven® / Hemlibra® combinations. The composition containing the maximum amount of product consists of a maximum of 6 μg / mL Novoseven® and 50 μg / mL Hemlibra®.

[0148] The thrombus formation potential obtained in the presence of a product combination is compared to the potential of a single product. To examine the synergistic effect of the product combination, lower doses are evaluated so that thrombin detection does not saturate.

[0149] The composition to be tested,

[0150] [Table 1]

[0151] It contains.

[0152] • Measuring the thrombus formation potential of the Novoseven® + Hemlibra® combination after coagulation induction using cephalin: The thrombin production 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 H2O).

[0153] The reaction is initiated by adding 20 μL of the Fluca kit (substrate + CaCl2), and this addition marks the start of the measurement of thrombin production.

[0154] Thrombin production tests are performed in the presence of 20 μL of cephalin with several Novoseven® / Hemlibra® combinations. The composition containing the maximum amount of product consists of a maximum of 6 μg / mL of Novoseven® and 50 μg / mL of Hemlibra®.

[0155] The thrombus formation potential obtained in the presence of a product combination is compared to the potential of a single product. To examine the synergistic effect of the product combination, lower doses are evaluated so that thrombin detection does not saturate.

[0156] Compositions to be tested:

[0157] [Table 2]

[0158] It contains.

[0159] • Measuring the thrombus formation potential of the Sevenfact® + Hemlibra® combination after coagulation induction using TF / PL: The thrombin production test is performed in 80 μL of FVIII-deficient plasma, mimicking 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).

[0160] The reaction is initiated by adding 20 μL of the Fluca kit (substrate + CaCl2), and this addition marks the start of the measurement of thrombin production.

[0161] Thrombin production tests are performed in the presence of 0.5 pM TF / 4 μM PL (coagulation-inducing factor) with several Sevenfact® / Hemlibra® combinations. The composition containing the maximum amount of product consists of a maximum of 6 μg / mL of Sevenfact® and 50 μg / mL of Hemlibra®.

[0162] The thrombus formation potential obtained in the presence of a product combination is compared to the potential of a single product. To examine the synergistic effect of the product combination, lower doses are evaluated so that thrombin detection does not saturate.

[0163] Compositions to be tested:

[0164] [Table 3]

[0165] It contains.

[0166] • Measuring the thrombus formation potential of the Sevenfact® + Hemlibra® combination after coagulation induction using cephalin: The thrombin production 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 H2O).

[0167] The reaction is initiated by adding 20 μL of the Fluca kit (substrate + CaCl2), and this addition marks the start of the measurement of thrombin production.

[0168] Thrombin production tests are performed in the presence of 20 μL of cephalin with several Sevenfact® / Hemlibra® combinations. The composition containing the maximum amount of product consists of a maximum of 6 μg / mL of Sevenfact® and 50 μg / mL of Hemlibra®.

[0169] The thrombus formation potential obtained in the presence of a product combination is compared to the potential of a single product. To examine the synergistic effect of the product combination, lower doses are evaluated so that thrombin detection does not saturate.

[0170] Compositions to be tested:

[0171] [Table 4]

[0172] It contains.

[0173] In all of these tests, the appearance of fluorescence is measured on a Fluoroskan Ascent fluorophotometer (ThermoLabsystems) at an excitation wavelength of 390 nm and an emission wavelength of 460 nm. Next, the thrombinogram (a curve showing fluorescence intensity over time) is analyzed using Thrombinoscope® software, which converts the fluorescence values ​​to nM thrombin through comparative calculation.

[0174] For example, a synergistic effect is considered if at least one of the parameters calculated from a thrombin production test for a given combination is greater than the sum of each of these parameters obtained using the individual components and estimated from the experimental background noise.

[0175] (Example 4) Comparison of the potential of Sevenfact®, Hemlibra®, and combinations thereof in hemophilia A plasma. reagent: ● Thrombin Calibrator (Stago) ● 1 pM TF PRP reagent (Stago) ● 4 μM PL MP reagent (Stago) ● Fluo Buffer (Stago Corporation) ● Fluo substrate (Stago) ● Sevenfact (trademark): Transgenic Factor VII produced in rabbits, 1 mg / ml (LFB Corporation) ● Hemlibra (registered trademark): Emicizumab (Roche / Genentech / Chugai) ● Hemophilia A plasma (Cryopep) ● Owren Koller (Stago Company)

[0176] method: The thrombin production test consists of ex vivo activation of coagulation using, for example, a tissue factor and phospholipid mixture (TF / PL), followed by measurement of the concentration of thrombin produced over time. The thrombin production test is performed using 80 μL of hemophilia A plasma (Cryopep) in the presence of 20 μL of a PRP and MP reagent mixture (Stago) containing 0.5 pM tissue factor and 4 μM phospholipid.

[0177] The reaction is initiated by adding 20 μL of the Fluca kit (Fluo substrate + CaCl2), and this addition marks the start of the measurement of thrombin production (TG).

[0178] Fluorescence is measured by fluorescence quantification using a Fluoroskan Ascent instrument (ThermoLabsystems) at an excitation wavelength of 390 nm and an emission wavelength of 460 nm. The thrombinogram is analyzed using Thrombinoscope® software, which employs comparative calculations to convert fluorescence intensity to thrombin molar concentration (nM).

[0179] Several hemophilia A plasma samples are examined to measure the thrombus-forming potential of these two molecules. The maximum therapeutic dose of FVIIa is 270 μg / kg, which corresponds to 6 μg / mL of FVIIa (or 120 nM) in plasma. The use of this dose can be considered the maximum potential for thrombin production. Based on the product concentrations in the bloodstream obtained in patients, Sevenfact® concentrations, including 20 and 100 nM, are also examined. Hemlibra® (Roche / Genentech / Chugai), a bispecific antibody that mimics the function of FVIII, is used at a maximum concentration of 120 μg / mL. The concentration actually detected in patients during treatment is 50 μg / mL (or 300 nM) (Oldenburg et al., NEJM, 2017). Therefore, Hemlibra® is used here at approximately 300 nM (50 μg / mL). The variables examined to measure thrombus formation potential in Hemlibra® and Sevenfact® are: - Endogenous thrombin activity (ETP): The area under the curve representing the total amount of thrombin produced. - Peak height: The maximum concentration of thrombin measured, and - Thrombin production rate: Thrombin formation rate That is the case.

[0180] 2- Results 2.1- Effects of Sevenfact® or Hemlibra® on Hemophilia A plasma 2.1.1- Evaluation of hemophilia A plasma in batch 1 In this matrix, extremely low thrombin production signals are obtained from these two compounds, regardless of the concentration used. In fact, the observed thrombin production is nearly zero at concentrations of 20 and 40 nM for Hemlibra® and Sevenfact®. Using 100 nM Sevenfact®, an extremely low thrombin production peak is observed (Table 1 (Table 5)).

[0181] [Table 5]

[0182] Thus, each molecule used individually only induces extremely low levels of thrombin production.

[0183] 2.1.2- Evaluation of hemophilia A plasma in batch 2 A second batch of hemophilia A plasma was tested. Again, extremely low thrombin production was observed using Hemlibra® and Sevenfact®, with the maximum thrombin production peak occurring at a Sevenfact® concentration of 100 nM (Table 2 (Table 6)).

[0184] [Table 6]

[0185] In this matrix, Sevenfact® and Hemlibra®, used separately, showed low thrombus formation potential.

[0186] (Example 5) Evaluation of the synergistic combination of Sevenfact (trademark) + Hemlibra (registered trademark) 1- Protocol The reagents, equipment, and experimental protocol using hemophilia A plasma are the same as those described in Example 2.

[0187] 2- Results As seen in Example 2, Sevenfact® and Hemlibra® used individually induce low thrombin production in hemophilia A plasma. Here, we investigate the synergistic effect of the Sevenfact® and Hemlibra® combination. We examine Sevenfact® at three concentrations (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 combined effect of Sevenfact® and Hemlibra® taken separately for at least one of the parameters of the thrombin production test (ETP, peak thrombin production, and rate).

[0188] 2.1- Effects of Sevenfact® and Hemlibra® on hemophilia A plasma after coagulation induction with TF / PL 2.1.1- Evaluation of hemophilia A plasma in batch 1 The results are shown in Table 3 (Table 7) and Figure 1. At an extremely low Sevenfact® concentration of 20 nM, the ratios for ETP (Figure 1A), thrombin peak (Figure 1B), and velocity (Figure 1C) for the Sevenfact® + Hemlibra® combination were 2.14, 2.95, and 4.19, respectively. Thus, a synergistic thrombus-forming effect was observed even at the lowest concentrations tested.

[0189] At a concentration of 40 nM, the ratios for all parameters tested were greater than 2. The ratio obtained for ETP was 2.75 (Figure 1A), the ratio obtained for thrombin peak was 3.96 (Figure 1B), and the ratio obtained for velocity reached a value of 6.21 (Figure 1C). In other words, the thrombin formation rate is 6 times higher when Sevenfact® and Hemlibra® are used in combination.

[0190] The synergistic effect is maximized at a Sevenfact® concentration of 100 nM. At a concentration of 100 nM, the ratio is greater than 2 for all parameters tested. The ratio obtained for ETP was 4.00 (Figure 1A), and the ratio obtained for the thrombin peak was 4.81 (Figure 1B), meaning that the maximum concentration of thrombin produced is approximately 5 times higher when Hemlibra® and Sevenfact® are used in combination. The corresponding rate ratio is 9.58 (Figure 1C), meaning that thrombin is produced approximately 10 times higher when Sevenfact® and Hemlibra® are used in combination.

[0191] [Table 7A]

[0192] [Table 7B]

[0193] In conclusion, at all Sevenfact® concentrations tested, Sevenfact® and Hemlibra® used in combination exhibit a synergistic effect on thrombin production.

[0194] 2.1.2- Evaluation of hemophilia A plasma in batch 2 The results are shown in Table 4 (Table 8) and Figure 2. At an extremely low Sevenfact® concentration of 20 nM, the ETP parameter ratio for the Sevenfact® + Hemlibra® combination was 2.21 (Figure 2A), the thrombin peak ratio was 2.34 (Figure 2B), and the velocity parameter ratio was 2.9 (Figure 2C). Thus, a synergistic thrombus-forming effect was observed even at the lowest Sevenfact® concentration tested.

[0195] At a concentration of 40 nM, the ratio corresponding to ETP is 2.29 (Figure 2A), the ratio corresponding to the thrombin peak is 2.79 (Figure 2B), and the ratio corresponding to the velocity is 3.68 (Figure 2C). This means that when Sevenfact® is used in combination with Hemlibra®, thrombin can be formed approximately four times faster.

[0196] The synergistic effect is maximized at a Sevenfact® concentration of 100 nM. At a concentration of 100 nM, the ratio corresponding to the thrombin production peak is 3.41 (Figure 2B), and the ratio corresponding to the rate is 5.63 (Figure 2C). This means that thrombin is produced nearly six times faster when Sevenfact® is used in combination with Hemlibra®, and the thrombin concentration achieved is nearly four times higher.

[0197] [Table 8A]

[0198] [Table 8B]

[0199] In conclusion, at all Sevenfact® concentrations tested, Sevenfact® and Hemlibra® used in combination exhibit a synergistic effect on thrombin production.

Claims

1. a. Transgenic factor VII derived from non-human mammals, and b. Bispecific antibodies against Factor IX and Factor X A pharmaceutical composition containing, A pharmaceutical composition wherein the transgenic factor VII is human activated factor VII derived from the production of epithelial cells of the mammary gland of transgenic rabbits.

2. The pharmaceutical composition according to claim 1, wherein the antibody is emicizumab.

3. For use in the prevention or treatment of coagulation disorders in patients, a. Transgenic factor VII derived from non-human mammals, and b. Bispecific antibodies against Factor IX and Factor X A combination that includes, The transgenic factor VII is a combination of human activated factor VII derived from the production of mammary gland epithelial cells of transgenic rabbits.

4. The combination according to claim 3 for the treatment of hemophilia A.

5. A combination according to claim 3 or 4 for the treatment of hemophilia A with a factor VIII inhibitor.

6. The combination according to any one of claims 3 to 5, wherein the combination is in the form of the pharmaceutical composition according to claim 1 or 2.

7. The combination according to any one of claims 3 to 5, wherein the factor VIIa and the antibody are in a form suitable for simultaneous administration to a patient.

8. The combination according to any one of claims 3 to 5, wherein the factor VIIa and the antibody are in a form suitable for individual administration to a patient.

9. - A container containing transgenic factor VII derived from a non-human mammal, and - Another container containing bispecific antibodies against Factor IX and Factor X A kit that includes, The kit wherein the transgenic factor VII is human activated factor VII derived from the production of epithelial cells of the mammary gland of transgenic rabbits.

10. A combination for the treatment of coagulation disorders in a patient, comprising a transgenic factor VII derived from a non-human mammal, and bispecific antibodies against factors IX and X, wherein the transgenic factor VII and the bispecific antibodies are administered to the patient simultaneously or sequentially. The transgenic factor VII is a combination of human activated factor VII derived from the production of mammary gland epithelial cells of transgenic rabbits.

11. A pharmaceutical composition for treating coagulation disorders in patients, comprising transgenic factor VII derived from a non-human mammal, The aforementioned patient was also administered bispecific antibodies against factor IX and factor X. A pharmaceutical composition wherein the transgenic factor VII is human activated factor VII derived from the production of epithelial cells of the mammary gland of transgenic rabbits.

12. The pharmaceutical composition according to claim 11, wherein the antibody is emicizumab.

13. The pharmaceutical composition according to claim 11 or 12, wherein the patient is administered the transgenic factor VII and the bispecific antibodies against factors IX and X in separate manners.

14. The pharmaceutical composition according to claim 13, wherein the transgenic factor VII and the bispecific antibodies against factors IX and X are administered via different routes of administration.

15. The pharmaceutical composition according to claim 13, wherein the transgenic factor VII and the bispecific antibodies against factors IX and X are administered via the same route of administration.

16. The pharmaceutical composition according to any one of claims 11 to 14, wherein the coagulation disorder is a factor VIII deficiency.

17. The pharmaceutical composition according to claim 16, wherein the coagulation disorder is type A hemophilia.

18. The pharmaceutical composition according to claim 17, wherein the coagulation disorder is acquired type A hemophilia.

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