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

Abstract

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

Description

[0001] The present invention relates to pharmaceutical compositions which can be used to treat coagulation disorders, such as hemophilia A, particularly in patients with hemophilia A who produce factor VIII inhibitory antibodies. Technical Background

[0002] Blood coagulation involves two pathways, one intrinsic and the other extrinsic, leading to a final common pathway. The combination of both mechanisms ensures the formation of a solid and flexible blood pressure-resistant blood clot. Through the action of thrombin, fibrinogen undergoes chemical modification, leading to the formation of fibrin. Fibrin is essential for the formation of a clot.

[0003] The intrinsic pathway involves factors that are present in the bloodstream, and the clotting 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 that participates in the extrinsic coagulation pathway. In order to start the coagulation cascade, FVII must be activated to FVIIa. Once activated, FVIIa is compounded with the tissue factor (TF) protein associated with two phospholipids, which is released during vascular injury. Independent FVIIa (not compounded with tissue factor) shows low proteolytic activity. Then, the FVIIa-FT complex converts factor X into factor Xa in the presence of calcium ions. The complex also works on activating factor FIX into FIXa, thereby catalyzing the intrinsic pathway. Factor IXa and Xa activate activated factor VII in turn.

[0005] Factor IX and Factor X participate in the intrinsic coagulation pathway. Activated Factor IX is able to activate Factor X to Factor Xa.

[0006] Factor Xa, compounded with activated factor FV and prothrombinase, converts prothrombin into thrombin. Thrombin then acts on fibrinogen to convert it into fibrin and activates FVIII and FV to FVIIIa and FVa, respectively. For its part, prothrombin, in the presence of calcium naturally present in blood plasma, can activate factor XIII to FXIIIa, which results in the consolidation of fibrin clots.

[0007] Nevertheless, when coagulation factors are deficient, the coagulation cascade is interrupted or absent, which we call coagulation disorder.

[0008] Activated factor VII acts locally in the presence of tissue factor released after tissue damage causing bleeding, even in the absence of factor VIII or IX. This is why factor VII, preferably in activated form, is used to treat certain coagulation disorders that present as bleeding.

[0009] Thus, factor VII is used to treat hemophiliacs who present a deficiency of factor VIII (hemophilia A) or factor IX (hemophilia B) and patients who present other coagulation factor deficiencies, for example, a hereditary deficiency of FVII. FVII is also recommended in the treatment of stroke.

[0010] Some hemophiliacs develop antibodies that inhibit factor VIII, which is usually administered in concentrated form as a hemophilia treatment. This is the most common complication of current hemophilia treatments.

[0011] Bispecific antibodies targeting FIX or FIXa and FX or FXa, such as emicizumab, are used to treat hemophilia A patients with anti-factor VIII antibodies. These antibodies functionally replace FVIII by facilitating the activation of FX by FIXa by bringing the two molecules together. These antibodies have a long-lasting effect.

[0012] Recombinant FVIIa from cell culture (e.g. BHK cells) and emicizumab (e.g. ACE910 or ) (R. HARTMANN et al., OR36 | Synergistic Effects of a Procoagulant Bispecific Antibody and FEIBA or FactorVIIA on Thrombin Generation (Haemophilia (2017), 23 (Supplement 2), 11-27)). This combination only shows an additive effect on the treatment of coagulation disorders.

[0013] Therefore, there is a need for drug combinations that allow for better management of hemophilia A patients and more particularly patients with anti-factor VIII. SUMMARY OF THE INVENTION

[0015] The present invention proposes combining transgenic factor VII with multispecific antibodies against factors IX and X.

[0016] According to the invention, the inventive combination of transgenic factor VII and antibodies against factor IX and factor X 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.

[0017] Therefore, one aspect of the present invention is a pharmaceutical composition comprising:

[0018] a. Transgenic Factor VII, and

[0019] b. Multispecific antibodies, preferably bispecific antibodies against Factor IX and Factor X, such as emicizumab.

[0020] Preferably, the Factor VII is in the form of activated Factor VII (FVIIa).

[0021] In particular 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).

[0022] Preferably, the transgenic Factor VII is human Factor VII derived from mammary epithelial cells produced by a non-human-transgenic mammal, such as a rabbit transgenic for human Factor VII.

[0023] The present invention also provides a combination product comprising:

[0024] a. Transgenic Factor VII, and

[0025] b. Multispecific antibodies against factors IX and X,

[0026] The combination product is used to prevent or treat coagulation disorders, such as hemophilia A, more particularly hemophilia A with factor VIII inhibitors (FVIII).

[0027] Preferably, the combination product is in the form of a pharmaceutical composition comprising transgenic Factor VII and the antibody.

[0028] Alternatively, the transgenic Factor VII and the antibody are in the form of separate compositions suitable for simultaneous or separate (eg sequential) administration to a patient.

[0029] Furthermore, another object of the present invention relates to a kit comprising:

[0030] - a container comprising transgenic factor FVII; and

[0031] - Another container comprising antibodies against Factor IX and Factor X. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 : After induction with TF / PL, Evaluation of the synergistic thrombotic effect of the combination on the first batch of hemophilia A plasma. (A) Evaluation of the synergistic thrombotic effect in terms of PTE, (B) Evaluation of the synergistic thrombotic effect in terms of thrombin generation peak, (C) Evaluation of the synergistic thrombotic effect in terms of speed.

[0033] Figure 2 : After induction with TF / PL, Evaluation of the synergistic thrombotic effect of the combination on the second batch of hemophilia A plasma. (A) Evaluation of the synergistic thrombotic effect in terms of PTE, (B) Evaluation of the synergistic thrombotic effect in terms of thrombin generation peak, (C) Evaluation of the synergistic thrombotic effect in terms of speed. DETAILED DESCRIPTION OF THE INVENTION

[0035] General Definition

[0036] The coagulation phenomenon consists of a cascade of enzymatic reactions involving coagulation factors, which exist as zymogens and, in the presence of certain cofactors, are converted into their "activated" forms by proteolytic cleavage. The activated form of each factor, which exists as an inactive precursor, is represented by the letter a. Thus, FVIIa is produced in vivo by the cleavage of the zymogen into two chains connected by a disulfide bridge by various proteases (FIXa, FXa, FVIIa).

[0037] The term "treat" generally refers to the improvement, prevention or reversal of a disease or disorder or at least symptoms, 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.

[0038] The term "prevent" refers to the reduction of the risk of developing or acquiring a particular disease or disorder.

[0039] In the present invention, "patient" or "individual" refers to any mammal, and more particularly, a human being of any age, male or female, including children.

[0040] The term "pharmaceutical composition" refers to a preparation that allows the active ingredient to possess biological activity and does not contain any other components that are toxic to the subject to which the composition is administered.

[0041] Transgenic Factor VII

[0042] The term "factor VII" or "FVII" includes polypeptides comprising sequence 1-406 of wild-type human factor VII (as described in U.S. Patent No. 4,784,950) or FVII derived from other species (e.g., cattle, pigs, dogs, mice). Also included are natural allele variants of factor VII, which may exist in any form or degree of glycosylation or other post-translational modifications. Therefore, the term "factor VII" also includes FVII variants, which have the same or better biological activity relative to the activity of the wild type, and these variants particularly include polypeptides that are different from wild-type FVII by insertion, deletion or substitution of one or more amino acids.

[0043] Unless otherwise indicated, in the present specification, the term "factor VII" refers to uncleaved FVII (zymogen) or activated factor VII (FVIIa).

[0044] Thus, FVIIa consists of a 152 amino acid light chain of approximately 20 kDa molecular weight and a 254 amino acid heavy chain of approximately 30 kDa molecular weight linked together by a single disulfide bridge (Cys135-Cys262).

[0045] "Recombinant Factor VII" refers to any Factor VII derived from genetic engineering and produced by expression of the corresponding gene in any microorganism, plant or transgenic plant. Microorganism refers to any bacteria, fungi, virus or cell system. Recombinant Factor VII can also be produced by eukaryotic cells in culture, such as plants or mammalian cells, such as animal or human cells.

[0046] "Transgenic Factor VII" refers to any recombinant Factor VII obtained from an animal that is transgenic for Factor VII.

[0047] "Transgenic animal" refers to any non-human animal with a modification in its genome that is expected to enable the expression of a protein of interest (herein Factor VII). The genome modification can be caused by the alteration, modification or insertion of a gene. Such modifications can be attributed to conventionally used alterations or mutagens or even by directed mutagenesis. The genome modification can also be produced by the insertion or replacement of one or more genes in wild-type or mutant form. The transgenic animal can be selected from rabbits, goats, cattle, camels, hamsters, mice, rats, horses, sows, dromedaries, sheep or llamas in a non-limiting manner. In a particular embodiment, an animal that does not express α1,3-galactosyltransferase can be selected.

[0048] 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 quantitatively measured by measuring the ability of the FVII composition to promote blood clots, by using FVII and thromboplastine (thromboplastine) deficiency plasma as described in, for example, U.S. Patent No. 5,997,864. In this test, biological activity is evaluated relative to a control sample, and is converted into "FVII units" by comparing it with a pooled standard human serum comprising 1 unit / mL of factor VII activity. Alternatively, the biological activity of Factor VII is quantified by: (i) measuring the ability of Factor VIIa to generate Factor Xa in a system comprising 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 that is independent of TF.

[0049] In a preferred embodiment, the FVII described herein is a polypeptide, the peptide sequence of which may be the peptide sequence of native human FVII, ie, the sequence present in humans who do not suffer from a disorder associated with FVII. Such technology is described in document EP 0 200421.

[0050] Advantageously, the FVII sequence used in the present invention is SEQ ID NO:1.

[0051] "Synergy" or "synergistic effect" preferably means that the effect of the combination of two products is greater than twice the sum of the effects of each product taken separately. According to the present invention, a synergistic effect is obtained when the combined use of transgenic FVIIa and a multispecific antibody to factor IX and factor X is able to obtain an effect greater than twice the sum of the effect obtained using transgenic FVIIa alone and the effect obtained using the multispecific antibody to factor IX and factor X alone in at least one thrombin generation parameter. The thrombin generation parameter is selected from peak height, velocity or endogenous thrombin generation potential (PTE).

[0052] In a particular embodiment, FVIIa is administered at a concentration lower than or equal to 105 nM, preferably lower than 100 nM.

[0053] In a particular embodiment, the multispecific antibody against Factor IX and Factor X is administered at a concentration of less than 600 nM, preferably less than 550 nM, preferably less than 500 nM, preferably less than 450 nM, preferably less than 400 nM, preferably less than 350 nM, preferably less than 325 nM.

[0054] In a particular embodiment, Factor VII is obtained from the milk of a transgenic animal.

[0055] The method for producing recombinant protein in the milk of transgenic animals may include the following steps: a synthetic DNA molecule (which comprises a gene encoding a protein of interest (such as human FVII herein), which is under the control of a promoter of a protein naturally secreted in the milk) is integrated into the embryo of a non-human mammal. The embryo is then implanted in a female mammal of the same species. Once the mammal produced by the embryo is fully developed, mammalian lactation can be induced, and milk is then collected. The milk comprises the FVII of interest secreted by the transgenic animal.

[0056] An example of protein preparation using milk of non-human female mammals is given in patent application EP0527063, the teachings of which can be re-used for the preparation of Factor VII of the present invention.

[0057] The mammary gland secretes Factor VII, causing it to be secreted into the milk of transgenic mammals, and is involved in controlling the expression of Factor VII in a tissue-dependent manner. Such control methods are well known to the skilled person. Expression is controlled by sequences that allow the protein to be expressed in specific tissues. These are in particular WAP, β-casein and β-lactoglobulin promoter sequences and signal peptide sequences; this list is not limiting.

[0058] In a preferred embodiment, Factor VII of the present invention is produced in the milk of transgenic rabbits.

[0059] In a particularly advantageous manner, expression is carried out in the mammary gland of rabbits under the control of the well-known beta casein promoter of the technician. In particular, a plasmid comprising a beta casein promoter is constructed by importing a sequence comprising a beta casein gene promoter, which is produced so as to be able to receive an exogenous gene placed under the control of the promoter. The gene encoding human FVII is integrated and placed under the control of the beta casein promoter. The plasmid comprising a promoter and a sequence encoding a protein of interest is digested with a restriction enzyme to release a DNA fragment comprising a beta casein promoter and a human FVII sequence. After purification, the fragment is introduced into the male pronucleus of a wild-type rabbit embryo by microinjection. The embryo is then cultured and then transferred to the wild-type female oviduct prepared by hormones. When these female animals give birth, offspring are evaluated by PCR to determine transgenic animals. The number of copies of the transgene and its integrity are revealed by Southern technology from the DNA extracted from the young transgenic rabbit obtained. The concentration of human FVII expressed in the milk of female transgenic offspring is evaluated by immunoenzyme test.

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

[0061] (a) inserting into a non-human mammal embryo a DNA sequence comprising a gene encoding Factor VII, said gene being under the transcriptional control of a beta casein promoter,

[0062] (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,

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

[0064] (d) collecting milk from said non-human mammal, and

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

[0066] As used herein, FVII has a substantially homogeneous isoelectric point.

[0067] "Isoelectric point" or "pi" refers to the pH at which the net elementary 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 Factor VII of the present invention can be measured by implementing techniques well known to the skilled person, such as isoelectric focusing ("IEF"). This electrophoretic technique separates proteins based on their isoelectric points. It consists of uniform current-induced protein migration in a pH gradient until they reach a pH equal to their specific isoelectric point, at which point they stop migrating because their net charge is zero. IEF gels are used to determine the isoelectric point of a given protein.

[0068] "Substantially homogeneous" means that at least 90%, preferably at least 95% of the Factor VII molecules of the composition have an isoelectric point with a pH unit difference of less than or equal to 1.2. In another embodiment of the invention, at least 50%, preferably at least 55%, preferably 60% of the transgenic Factor VII molecules of 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 invention, at least 50%, preferably at least 55%, preferably 60% of the Factor VII molecules of the composition have an isoelectric point with a pH unit difference of 0.4.

[0069] "N-glycan form" refers to all N-glycan forms present in two N-glycosylation sites of Factor VII of the present invention. If their total charge is equal to 1, then the N-glycan form is referred to as singly charged. In the present invention, "charge" refers to a phosphate group, a sulfate group or a sialic acid molecule. Therefore, if they only contain one phosphate group or one sulfate group or one sialic acid molecule, then the N-glycan form is referred to as singly charged. In contrast to the term "single charge", the term "double charge" refers to that the total charge carried by the N-glycan form is equal to 2, i.e., they have two charges selected from phosphate groups, sulfate groups and / or sialic acid molecules. In other words, the double-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" means that the total charge carried by the N-glycan forms is equal to 3, i.e. they have three charges selected from phosphate groups, sulfate groups and / or sialic acid molecules. In other words, the triply charged N-glycan forms have 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" means that the N-glycan forms do not contain any charge.

[0070] Can be by implementing the well-known method of technical personnel, particularly by using the ultra-high performance liquid chromatography (AEX-UPLC / FD) of the anion exchange resin of coupled fluorescence detection to measure the charge of the N- polysaccharide form of factor VII.This method can separate different N- polysaccharide forms according to its apparent charge (especially referring to Hermentin et al., Glycobiology, the 6th volume, the 2nd phase, 1996).In the case of anion exchange chromatography, positively charged resin is used as stationary phase.These positively charged resins are usually composed of cross-linked polymers or gels, on which positively charged groups are grafted.In an advantageous embodiment of the present invention, aminopropyl type weak anion exchange column is used.

[0071] In the case of the factor VII composition of the present invention, it appears that of all the N-glycan forms of the factor VII of the composition, at least 50% of the N-glycan forms, at least 60% of the N-glycan forms, preferably at least 65%, preferably at least 70%, preferably at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95% are singly charged. In a preferred embodiment, the factor VII molecules with singly charged N-glycan forms account for 50%-95% of the factor VII molecules of the composition, preferably 50%-90% of the factor VII molecules of the composition, preferably 50%-80% of the factor VII molecules of the composition, preferably 50%-75% of the factor VII molecules of the composition, preferably 50%-70% of the factor VII molecules of the composition, preferably 50-65% of the factor VII molecules of the composition, preferably 50%-60% of the factor VII molecules of the composition.

[0072] The substantially uniform isoelectric point of the combined Factor VII composition of the invention results from a combination of the glycosylation and gamma-carboxylation properties of the FVII molecules that comprise it.

[0073] Transgenic factor VII used herein has the characteristics of post-translational modification. In particular, these are glycosylation modifications, such as two N-glycosylation sites with zero or very low amounts of Galα1,3Gal in the FVII composition, or still low enough to be non-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 modification, and two O-glycosylation, γ-carboxylation and specific disulfide bridges with determined glycan units.

[0074] As used herein, FVIIa may include several post-translational modifications: the first 9 or 10 N-terminal glutamic acids are γ-carboxylated, Asp 63 Partially hydroxylated, Ser 52 and Ser60 O-glycosylated and with glucose (xylose) 0-2 and fucose units, Asn 145 and Asn 322 They are N-glycosylated primarily through monosialylated biantennary complex structures.

[0075] Advantageously, at least 80% of the transgenic Factor VII molecules used herein have γ-carboxylation on 9 glutamic acid residues. In another embodiment, at least 85% of the molecules have γ-carboxylation on 9 glutamic acid residues. In another embodiment, 85%-100%, preferably 90%-100%, preferably 95%-100% of the molecules have γ-carboxylation on 9 glutamic acid residues. Advantageously, the γ-carboxylation degree on the glutamic acid residue 35 (Glu 35) of the Factor VII molecule of the composition is less than 20%. In another embodiment, the γ-carboxylation degree of residue Glu35 is less than 15%, preferably less than 10%, preferably less than 5%.

[0076] The Galα1,3Gal unit is a structure consisting of two galactoses connected at α1,3. It is located at the end of the oligosaccharide antennae of the N-linked structure. This unit is well known for its immunogenicity. Therefore, it is preferred to prepare FVII or FVIIa, the amount of which is zero or so low that it cannot be distinguished from the background noise obtained by the measurement by the currently available analytical device. This expression equivalently represents all transgenic FVII, the amount of which is close to the amount of plasma FVII. Advantageously, the amount of Galα1,3Gal of the FVII composition 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 be different. However, for the entire composition, the amount of each glycan unit, or even each sugar, can be quantified.

[0077] The percentages of different glycans given in this application do not take into account O-glycosylation.

[0078] Preferably, the FVII composition is characterized in that, of all the glycan units of FVII in the composition, at least 40% are in the form of monosialylated biantennary glycans. In another embodiment, the monosialylated biantennary forms are present in at least 50%. In another embodiment, the monosialylated biantennary forms are present in at least 60%, preferably at least 65%, preferably at least 70%.

[0079] 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 acids comprise α2-6 linkages.

[0080] Advantageously, at least 65% of the sialic acids of FVII comprise α2,6 bonds. Very advantageously, at least 70%, even 80% and in particular at least 90% of the sialic acids of FVII comprise α2,6 bonds.

[0081] In a particularly preferred manner, all sialic acids contain α2,6 bonds, ie, all sialic acids are bound to galactose via α2,6 bonds. The FVII compositions described herein may also contain sialic acids having α2-3 bonds.

[0082] According to an embodiment of the present invention, 65%-100% of the FVII sialic acid comprises α2,6 linkages. More preferably, 70% or 80%-100% of the FVII sialic acid comprises α2,6 linkages.

[0083] Advantageously, among the monosialylated biantennary glycan forms of FVII, the major glycan form is non-fucosylated.

[0084] Preferably, these non-fucosylated monosialylated biantennary glycan forms are present in the FVII of the composition in an amount exceeding 20%. Advantageously, this 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, this degree may be less than 20%.

[0085] 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 / hybrid.

[0086] Preferably, the glycosylation characteristics described herein provide improved biological activity and stability of FVII. Factor VII compositions with substantially uniform isoelectric points can facilitate the formulation step at the optimum pH of the pharmaceutical composition, preferably at an optimum pH of 6.0 ± 0.2, by preventing FVII precipitation. In fact, it is known that at the isoelectric point of the molecule, they will have a tendency to aggregate and precipitate. The factor VII molecules used in the composition of the present invention have an isoelectric point of 6.6-7.0. This leads to better stability of the factor VII composition, particularly at a pH below the isoelectric point, and particularly when formulated at pH 6.0. The improvement in the stability of the factor VII composition prevents electrostatic interactions that cause soluble and insoluble precipitation and aggregation phenomena, and prevents loss of raw materials, thereby preventing a decrease in yield and causing a decrease in the content of active ingredients and a resulting potential decrease in activity.

[0087] In a preferred embodiment, transgenic FVII is produced in rabbit milk, thereby being able to obtain 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 less than 4%, or even no Galα1,3Gal glycan units. Therefore, it is advantageous that the transgenic FVII produced by rabbits does not have Galα1,3Gal units.

[0088] FVII can be purified from milk by techniques known to the skilled artisan. For example, as described in U.S. Pat. No. 6,268,487, a method for purifying a protein of interest from milk can include the following steps: a) tangentially filtering the milk through a membrane of sufficient porosity to form a retentate and a permeate, the permeate comprising 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 recovering FVII.

[0089] Advantageously, 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 be activated in its purification process generally, particularly by passing through a positively charged chromatographic column.

[0090] Multispecific Antibodies

[0091] A "multispecific antibody" refers to any antibody having at least two binding sites specific for at least two different antigens or different epitopes of the same antigen. The term "specificity" refers to the ability of the antibody to recognize and bind to an antigen without substantially cross-reacting with additional antigens. Advantageously, the antibody has at least 10 binding sites specific for each antigen.-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.

[0092] Therefore, the antibodies used in the present invention have the ability to specifically bind to both activated or inactivated forms of coagulation factor IX and coagulation factor X.

[0093] The antibody used in the present invention having the ability to specifically bind to both coagulation factor IX and coagulation factor X preferably has the ability to serve as a substitute for factor VIII (FVIII), which indicates that the antibody promotes the activation of FX by FIXa.

[0094] Such multispecific antibodies, preferably bispecific antibodies, can be obtained by various methods known to the skilled person, for example by chemical conjugation or by using cell hybridomas (quadromes), which are produced by fusion between two hybridomas producing two different monoclonal antibodies, or even by genetic recombination.

[0095] Thus, polynucleotides encoding such antibodies may be inserted into expression vectors and expressed in host cells or organisms modified by techniques well known to the skilled artisan.

[0096] The antibodies used herein may be in a very simple form constructed from single-chain Fv fragments (scFv) from two or more antibodies connected by a suitable peptide linker.

[0097] "Fv" refers to the smallest antibody fragment that retains the ability to recognize and bind to an antigen. The "Fv" fragment is the variable region (V) carried by the heavy chain (H) H ) and light chain (V L ) carries the adjacent variable region (V L ) composed of dimers (V H +V L dimer).

[0098] 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, which comprises 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 (Qu et al., Blood, 111, 2211-2219, 2008) comprising the VL and CL domains of antibody B. In the second form, the heavy chain and light chain of antibody A are combined with the heavy chain and light chain of antibody B. Where appropriate, mutations such as "knob into holes" (Ridgway et al., Protein Eng, 9, 617-21, 1996; U.S. Patent 7,695,936) can be introduced to prevent mispairing.

[0099] In the present specification, unless otherwise indicated, the term "factor IX" refers to inactivated factor IX or activated factor IX (FIXa).

[0100] In the present specification, unless otherwise indicated, the term "factor X" refers to inactivated factor X or activated factor X (FXa).

[0101] In particular, according to the methods described in patent applications WO2005 / 035756, WO2006 / 109592 or WO2012 / 067176, antibodies recognizing (i) FIX and / or FIXa and (ii) FX and / or FXa can be obtained.

[0102] 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.

[0103] Drug composition and dosage

[0104] Factor VII and the antibody may be formulated as separate pharmaceutical compositions, or combined in the same pharmaceutical composition.

[0105] In the case of separate administration, the FVII and the antibody are formulated in a manner suitable for administration by different routes or the same route.

[0106] Thus, for example, FVII can be administered intravenously, subcutaneously, or intramuscularly.

[0107] For example, the antibody can also be administered intravenously, subcutaneously, or intramuscularly.

[0108] The Factor VII composition may be, for example, as described in patent application WO 2010 / 149907.

[0109] Thus, in an example of an embodiment, the composition comprises:

[0110] - Factor VII, preferably in the form of Factor VIIa;

[0111] - Arginine, possibly in the form of its hydrochloride;

[0112] -Isoleucine;

[0113] - lysine;

[0114] -Glycine;

[0115] - trisodium citrate or calcium chloride;

[0116] - and, if appropriate, polysorbate 80 or polysorbate 20.

[0117] More particularly, the composition may comprise:

[0118] - Factor VII, preferably in the form of Factor VIIa;

[0119] -10-40 g / L of arginine, possibly in the form of hydrochloride;

[0120] -4.2-6.6 g / L isoleucine;

[0121] - 0.6-1.8 g / L lysine;

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

[0123] -0-0.2g / L trisodium citrate or 1-2g / L calcium chloride;

[0124] - and, if appropriate, 0-0.5 g / L polysorbate 80.

[0125] The FVII composition can be stored in liquid or solid form, usually obtained by drying, and optionally further comprises at least one multispecific antibody as described herein. Before drying or after reconstitution into an injection formulation, the composition disclosed above is determined as a composition in liquid form.

[0126] Drying is a method used 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.

[0127] A preferred method of obtaining a solid form of the compositions described herein for pharmaceutical use is freeze drying.

[0128] Freeze-drying methods are well known to the skilled person, see for example [Wang et al., Lyophilization and development of solid protein pharmaceuticals, International Journal of Pharmaceutics, Vol. 203, p 1-60, 2000].

[0129] Other suitable methods for reducing the moisture or water content of the composition can be envisaged. Preferably, the moisture content 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%.

[0130] The solid composition, preferably in freeze-dried form, may be dissolved in water for injection (WFI) to give a formulation for therapeutic use.

[0131] The injectable preparation can be administered parenterally (intravenously, subcutaneously, intramuscularly) in an amount estimated by the practitioner. Administration of liquid forms (before drying) or solid forms by any route and in any appropriate manner is not excluded.

[0132] The dose of FVII that can be used in the present invention can be determined in an appropriate manner depending on the type of preparation, administration method, age and weight of the patient, symptoms of the patient, severity of the disease, and the like.

[0133] The FVII dosage used according to the present invention can be selected at 270 μ g / kg-2.70 μ g / kg. Preferably, the FVII dosage used is lower than 270 μ g / kg body weight, preferably lower than 225 μ g / kg body weight, preferably lower than 180 μ g / kg body weight, preferably lower than 135 μ g / kg body weight, preferably lower than 90 μ g / kg body weight, preferably lower than 45 μ g / kg body weight, preferably lower than 9 μ g / kg, preferably lower than 5.4 μ g / kg, preferably lower than 2.7 μ g / kg.

[0134] Multispecific antibody compositions, e.g., emicizumab antibodies are, e.g., antibodies as described in patent applications WO2017 / 188356 and WO2018 / 047813.

[0135] Thus, in an example of an embodiment, the composition is a liquid composition.

[0136] In an example of one embodiment, the composition comprises:

[0137] - Antibodies with bispecificity for Factor IX and Factor X

[0138] - Surfactants such as poloxamer 188 or polysorbate 20

[0139] -Histidine-aspartate buffer

[0140] -Arginine.

[0141] More particularly, the composition may comprise:

[0142] - 20 mg / mL to 180 mg / mL of a bispecific antibody for Factor IX and Factor X,

[0143] -0.2mg / mL-1mg / mL of Poloxamer 188,

[0144] - 10mM-40mM histidine-aspartate buffer,

[0145] -100mM-300mM arginine,

[0146] At pH 4.5-6.5.

[0147] The dosage of the multispecific antibody composition of the present invention, such as emicizumab antibody, can be appropriately determined according to the type of formulation, method of administration, patient age and weight, patient symptoms, severity of the disease, etc. For example, the antibody dose can be 0.3-5 mg / kg, preferably up to 3 mg / kg, once a week in the initial stage, which can last 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.

[0148] The antibody compositions used in the present invention can be administered to patients by any suitable route, such as intravenous, intramuscular, intraperitoneal, intracerebrospinal, transdermal, subcutaneous, intraarticular, sublingual, intrasynovial, oral or by inhalation. Preferably, the intravenous route or the subcutaneous route is advantageous.

[0149] According to a particular embodiment, Factor VII and the antibody are administered to the patient simultaneously.

[0150] According to another particular embodiment, Factor VII and the antibody are administered to the patient separately, preferably sequentially.

[0151] Indications for treatment

[0152] The combinations described herein prevent or treat coagulation disorders, in particular hemophilia presenting with factor VIII deficiency (hemophilia A, preferably acquired hemophilia A).

[0153] Preferably, the patient is a hemophilia A patient with anti-factor VIII.

[0154] The combinations described herein prevent or treat coagulation disorders, in particular Factor VII deficiency.

[0155] The combination described herein combines the rapid action of FVII activating the extrinsic pathway of the coagulation cascade and the prolonged action of the multispecific antibodies described herein activating the intrinsic pathway of the coagulation cascade. This combination makes it possible to provide better patient treatment. Example

[0156] Example 1: Purification and extraction of transgenic FVII

[0157] The method for purification and extraction of Factor VII implemented in this example is described in application EP 12305882. The steps of this method are described below.

[0158] Transgenic rabbit milk is obtained from a transgenic rabbit strain. Frozen milk from transgenic rabbits is thawed and concentrated in the form of a transgenic rabbit milk pool.

[0159] The transgenic rabbit milk pool thus obtained is then subjected to a clarification step using a depth filter having a porosity of 0.2 μm to remove lipids and insoluble compounds. The milk thus clarified is then subjected to a virus inactivation step at least 2 hours at 25°C ± 2°C by a detergent solvent such as polysorbate 80 or tri-n-butyl phosphate. Such treatments effectively inactivate viruses, and particularly non-enveloped viruses. The clarified and virus-inactivated milk is then subjected to an affinity chromatography step using an affinity ligand specific for factor VII / factor VIIa. The factor VII eluate obtained from the chromatographic step is then subjected to ultrafiltration and a formulation step, thereby being able to obtain an intermediate factor VII concentrate having a purity of 95%.

[0160] The intermediate factor VII concentrate is then subjected to a filtration step using a filter having a porosity of 0.1 μm-0.2 μm, followed by a nanofiltration step through a filter having a porosity of 20 nm and then 15 nm. The product thus obtained and containing factor VII is then subjected to Q Sepharose XL gel chromatography, followed by a CHT-1 chromatography step, followed by Superdex 200SEC chromatography. The factor VII concentrate thus obtained is then subjected to a stabilization treatment and then filtered through a filter having a porosity of 0.2 μm.

[0161] The process thus described makes it possible to obtain a Factor VII concentrate having a purity of about 99.9995%.

[0162] Embodiment 2: and The ratio of thrombotic potential Comparison

[0163] A technician can determine by performing the following protocol and (also known as emicizumab) for its thrombotic 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 1 mg / mL (LFB)

[0173] ■PNP (Cryopep)

[0174] ■ (NovoNordisk)

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

[0176] method:

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

[0178] ●After inducing coagulation with TF / P, measure Thrombotic potential:

[0179] The thrombin generation test was performed in 80 μL of FVIII-deficient plasma pool (which simulates hemophilia A plasma) in the presence of 20 μL PPP reagent (Stago) containing 0.5 pM tissue factor (TF) and 4 μM phospholipids (PL). The reaction was started by adding 20 μL Fluca kit (substrate + CaCl2), which was the start of the determination of thrombin generation.

[0180] The therapeutic dose of FVIIa was 270 μg / kg, which was equivalent to 6 μg / mL FVIIa in plasma, considering a 100% yield. Then, at 0 μg / mL, 1 μg / mL, 2 μg / mL, 3 μg / mL, 4 μg / mL, 5 μg / mL and 6 μg / mL, the yield of FVIIa was 100%. At the dose of 50 μg / ml, thrombin generation assay was performed in the presence of 0.5 pM TF / 2 μM PL (coagulation inducer).

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

[0182] The thrombin generation assay was performed in 80 μL of FVIII-deficient plasma pool (which simulates hemophilia A plasma) in the presence of 20 μL of phospholipids (CK-Prest reconstituted with 5 mL of distilled H2O).

[0183] The reaction was initiated by adding 20 μL of Fluca kit (substrate + CaCl2), which is the start of the measurement of thrombin generation.

[0184] At 0μg / mL, 1μg / mL, 2μg / mL, 3μg / mL, 4μg / mL, 5μg / mL and 6μg / mL At the dose, thrombin generation test was performed in the presence of 20 μL of phospholipids (coagulation inducer).

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

[0186] Thrombin generation assay was performed in 80 μL of FVIII-deficient plasma pool (which simulates 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).

[0187] The reaction was initiated by adding 20 μL of Fluca kit (substrate + CaCl2), which is the start of the measurement of thrombin generation.

[0188] At 0μg / mL, 1μg / mL, 2μg / mL, 3μg / mL, 4μg / mL, 5μg / mL and 6μg / mL At the dose of 50 μg / ml, thrombin generation assay was performed in the presence of 0.5 pM TF / 2 μM PL (coagulation inducer).

[0189] ●After inducing coagulation with cephalin, the Thrombotic potential:

[0190] The thrombin generation assay was performed in 80 μL of FVIII-deficient plasma pool (which simulates hemophilia A plasma) in the presence of 20 μL of phospholipids (CK-Prest reconstituted with 5 mL of distilled H2O).

[0191] The reaction was initiated by adding 20 μL of Fluca kit (substrate + CaCl2), which is the start of the measurement of thrombin generation.

[0192] At 0μg / mL, 1μg / mL, 2μg / mL, 3μg / mL, 4μg / mL, 5μg / mL and 6μg / mL At the dose, thrombin generation test was performed in the presence of 20 μL of cephalin.

[0193] ●After inducing coagulation with TF / PL, measure Thrombotic potential:

[0194] Thrombin generation assay was performed in 80 μL of FVIII-deficient plasma pool (which simulates 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).

[0195] The reaction was initiated by adding 20 μL of Fluca kit (substrate + CaCl2), which is the start of the measurement of thrombin generation.

[0196] Bispecific antibodies that mimic FVIII function (Roche / Genentech / USA) was used at a maximum concentration of 50 μg / mL, which was the concentration detected during patient treatment (Oldenburg et al., NEJM, 2017). At the dose of 50 μg / ml, thrombin generation assay was performed in the presence of 0.5 pM TF / 4 μM PL (coagulation inducer).

[0197] ●After inducing coagulation with cephalin, the Thrombotic potential:

[0198] The thrombin generation assay was performed in 80 μL of FVIII-deficient plasma pool (which simulates hemophilia A plasma) in the presence of 20 μL of phospholipids (CK-Prest reconstituted with 5 mL of distilled H2O).

[0199] The reaction was initiated by adding 20 μL of Fluca kit (substrate + CaCl2), which is the start of the measurement of thrombin generation.

[0200] Then at 0 μg / mL, 10 μg / mL, 20 μg / mL, 30 μg / mL, 40 μg / mL and 50 μg / mL At the dose, thrombin generation test was performed in the presence of 20 μL of cephalin.

[0201] For all these tests, the fluorescence that emerged was measured on a Fluoroskan Ascent fluorometer (ThermoLabsystems) at an excitation wavelength of 390 nm and an emission wavelength of 460 nm. TM Thrombinograms (curves showing the change in fluorescence intensity over time) were analyzed by software, which converted the fluorescence values ​​into nM of thrombin by comparative calculation.

[0202] Thrombin is generated and the key variables for evaluating the efficacy of different drug products: intrinsic thrombin potential (PTE), peak height, latency and velocity are recorded and compared.

[0203] Embodiment 3: and or and The Evaluation of thrombotic potential

[0204] A skilled person can determine by performing the following protocol and The thrombotic potential of the combination.

[0205] Reagents:

[0206] The reagents, apparatus and experimental protocol in FVIII-deficient plasma were the same as those described in Example 2.

[0207] method:

[0208] ●After inducing coagulation with TF / PL, measure Combined thrombotic potential:

[0209] Thrombin generation assay was performed in 80 μL of FVIII-deficient plasma pool (which simulates 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 Fluca kit (substrate + CaCl2), which is the start of the measurement of thrombin generation.

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

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

[0213] The compositions tested contained:

[0214]

[0215] ●After inducing coagulation with cephalin, the Combined thrombotic potential:

[0216] The thrombin generation assay was performed in 80 μL of FVIII-deficient plasma pool (which simulates hemophilia A plasma) in the presence of 20 μL of phospholipids (CK-Prest reconstituted with 5 mL of distilled H2O).

[0217] The reaction was initiated by adding 20 μL of Fluca kit (substrate + CaCl2), which is the start of the measurement of thrombin generation.

[0218] Thrombin generation test in several The combination was performed in the presence of 20 μL of phospholipids. The composition containing the maximum amount of product was composed of 6 μg / mL at its maximum value. and 50 μg / mL constitute.

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

[0220] The compositions tested contained:

[0221]

[0222]

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

[0224] Thrombin generation assay was performed in 80 μL of FVIII-deficient plasma pool (which simulates 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).

[0225] The reaction was initiated by adding 20 μL of Fluca kit (substrate + CaCl2), which is the start of the measurement of thrombin generation.

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

[0227] The thrombotic potential obtained in the presence of the product combination was compared to the potential of the individual products. 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 compositions tested contained:

[0229]

[0230] ●After inducing coagulation with cephalin, the Combined thrombotic potential:

[0231] Thrombin generation assays were performed in 80 μL of FVIII-deficient plasma pool (which simulates hemophilia A plasma) in the presence of 20 μL of phospholipids (CK-Prest reconstituted with 5 mL of distilled H2O).

[0232] The reaction was initiated by adding 20 μL of Fluca kit (substrate + CaCl2), which is the start of the measurement of thrombin generation.

[0233] Thrombin generation test in several The combination was performed in the presence of 20 μL of phospholipids. The composition containing the maximum amount of product was composed of 6 μg / mL at its maximum value. and 50 μg / mL constitute.

[0234] The thrombotic potential obtained in the presence of the product combination was compared to the potential of the individual products. To account for the synergistic effect of the product combination, lower doses were evaluated to ensure that the thrombin assay would not be saturated.

[0235] The compositions tested contained:

[0236]

[0237] For all these tests, the fluorescence that emerged was measured on a Fluoroskan Ascent fluorometer (ThermoLabsystems) at an excitation wavelength of 390 nm and an emission wavelength of 460 nm. TM The thrombinogram (a curve showing the change in fluorescence intensity over time) was analyzed by software, which converted the fluorescence values ​​into nM of thrombin by comparative calculation.

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

[0239] Example 4: Sevenfact TM , Comparison of the potency of these two combinations in hemophilia A plasma:

[0240] Reagents:

[0241] ■Thrombin calibrator (Stago)

[0242] ■1pM TF PRP reagent (Stago)

[0243] ■4μM PL MP reagent (Stago)

[0244] ■Fluo-buffer (Stago)

[0245] ■Fluo-Substrate (Stago)

[0246] ■Sevenfact TM :Transgenic Factor VII produced in rabbits 1mg / mL (LFB)

[0247] ■ Emicizumab (Roche / Genentech / )

[0248] ■Hemophilia A plasma (Cryopep)

[0249] ■Owren Koller (Stago)

[0250] method:

[0251] The thrombin generation test consists of activating coagulation ex vivo, 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 20 μL of a mixture of PRP and MP reagent (Stago) containing 0.5 pM tissue factor and 4 μM phospholipids.

[0252] The reaction was initiated by adding 20 μL of Fluca kit (substrate + CaCl2), which is the start of the measurement of thrombin generation.

[0253] Fluorescence was measured by fluorometry at an excitation wavelength of 390 nm and an emission wavelength of 460 nm using a Fluoroskan Ascent apparatus (ThermoLabsystems). TM The thrombinograms were analyzed by software that converted the fluorescence intensity to molar concentration (nM) of thrombin using a comparative calculation.

[0254] To measure the thrombotic potential of the two molecules, several hemophilia A plasmas were studied. The highest therapeutic dose of FVIIa was 270 μg / kg, which is equivalent to 6 μg / mL FVIIa (or 120 nM) in plasma. The use of this dose can be regarded as the maximum potential for thrombin generation. Based on the product concentrations obtained in the bloodstream of patients, Sevenfact was also studied containing 20-100 nM. TM Concentration. Bispecific antibodies that activate FVIII function (Roche / Genentech / USA) is used at a maximum concentration of 120 μg / mL. The actual concentration detected during patient treatment is 50 μg / mL (or 300 nM) (Oldenburg et al., NEJM, 2017). Therefore, about 300 nM (50 μg / mL) is used in this article. For determination and Sevenfact TM The variables studied for thrombotic potential were:

[0255] - Endogenous 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: How fast thrombin is formed.

[0258] 2- Results

[0259] 2.1-Sevenfact TM or Effects on Hemophilia A Plasma

[0260] 2.1.1-Evaluation of Hemophilia A Plasma Batch 1

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

[0262]

[0263] Table 1: From Sevenfact TM or Thrombin generation parameters of batch 1 of processed hemophilia A plasma

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

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

[0266] Testing of Batch 2 of Hemophilia A plasma. Use and Sevenfact TM Again, very low thrombin generation was observed, with 100 nM Sevenfact TM There was a maximum thrombin generation peak at concentration (Table 2).

[0267]

[0268] Table 2: From Sevenfact TM or Thrombin generation parameters of the second batch of processed hemophilia A plasma

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

[0270] Embodiment 5: Evaluation of synergistic combinations

[0271] 1-Scheme

[0272] The reagents, apparatus and experimental protocol in hemophilia A plasma were the same as those described in Example 2.

[0273] 2- Results

[0274] As observed in Example 2, Sevenfact used alone TM and Inducing hypothrombin generation in hemophilia A plasma. This paper studies Sevenfact TM and The synergistic effect of the combination. Sevenfact TM For at least one of the thrombin generation test parameters (PTE, thrombin generation peak and velocity), if The combination is more effective than Sevenfact and If the sum of the effects of the two agents is at least 2 times, a synergistic effect is considered.

[0275] 2.1- Sevenfact after TF / PL induction of coagulation TM and Effects on Hemophilia A Plasma

[0276] 2.1.1-Evaluation of Hemophilia A Plasma Batch 1

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

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

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

[0280]

[0281] In summary, for all tested Sevenfact TM Concentration, combined application of Sevenfact TM and Both have a synergistic effect on thrombin generation.

[0282] 2.1.2-Evaluation of the second batch of hemophilia A plasma

[0283] The results are shown in Table 4 and Figure 2 At a very low concentration of 20 nM Sevenfact TM At concentrations, The ratio for the PTE parameter is 2.21 ( Figure 2 A), the ratio obtained for the thrombin peak was 2.34 ( Figure 2 B), and the ratio obtained for the speed parameter is 2.9 ( Figure 2 C) Therefore, even in the test of the minimum Sevenfact TM Concentrations of 5.5 and 2.5 were also observed to induce thrombosis.

[0284] At a concentration of 40 nM, the ratio corresponding to PTE was 2.29 ( Figure 2 A), corresponding to the ratio of the thrombin peak is 2.79 ( Figure 2 B), and the corresponding speed ratio is 3.68 ( Figure 2 C) This shows that Sevenfact TM and The combined application enabled thrombin formation to be approximately 4 times faster.

[0285] Sevenfact at 100nM TMAt a concentration of 100 nM, the synergistic effect is the greatest. At a concentration of 100 nM, the ratio corresponding to the thrombin generation peak is 3.41 ( Figure 2 B), and the corresponding speed ratio is 5.63 ( Figure 2 C) This means that when Sevenfact TM and When used in combination, thrombin generation was almost 6 times faster and the thrombin concentration achieved was almost 4 times greater.

[0286]

[0287] In summary, for all tested Sevenfact TM Concentration, combined application of Sevenfact TM and Both have a synergistic effect on thrombin generation.

Claims

1. A pharmaceutical composition comprising: a. transgenic factor VII, wherein the transgenic factor VII is human factor VII produced by epithelial cells of the mammary gland of a transgenic rabbit, and b. Emicizumab.

2. A combination product comprising: a. transgenic factor VII, wherein the transgenic factor VII is human factor VII produced by epithelial cells of the mammary gland of a transgenic rabbit, and b. Emicizumab.

3. The combination product according to claim 2, wherein the transgenic Factor VII and the emicizumab are in a form suitable for simultaneous administration to a patient.

4. The combination product according to claim 2, wherein the transgenic Factor VII and the emicizumab are in a form suitable for separate administration to a patient.

5. A medicine kit comprising: - a container comprising transgenic factor VII, wherein the transgenic factor VII is human factor VII produced by epithelial cells derived from the mammary gland of a transgenic rabbit; and - Another container containing emicizumab.

6. Use of a combination of transgenic factor VII and emicizumab in the preparation of a medicament for treating hemophilia A with factor VIII inhibitors, wherein the transgenic factor VII is human factor VII produced by epithelial cells derived from the mammary gland of a transgenic rabbit.

7. The use according to claim 6, wherein transgenic Factor VII and emicizumab are administered simultaneously.

8. The use according to claim 6, wherein the transgenic Factor VII and emicizumab are administered sequentially.

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