WO2018032639A1

WO2018032639A1 - Activated human blood-clotting factor vii fusion protein, and manufacturing method and application of same

Info

Publication number: WO2018032639A1
Application number: PCT/CN2016/106012
Authority: WO
Inventors: 李强; 朱文臣; 高永娟; 朱成功; 李媛丽; 王晓山; 孙乃超; 刘宾; 王文文; 李智; 刘婷婷; 朱鹿燕; 李亦清; 任子甲; 朱松林; 肖春峰; 苏鸿声
Original assignee: 安源医药科技（上海）有限公司; 辅仁药业集团有限公司; 旭华（上海）生物研发中心有限公司; 开封制药(集团)有限公司
Priority date: 2016-08-19
Filing date: 2016-11-16
Publication date: 2018-02-22
Also published as: CN106279436A; CN106279436B

Abstract

The present invention discloses a highly glycosylated and activated human blood-clotting factor VII (FVIIa) fusion protein, and a manufacturing method and application of same. The fusion protein comprises, from the N-terminus to the C-terminus, a human (FVIIa), a flexible peptide connector, at least one rigid unit of a human chorionic gonadotropin β-subunit carboxyl terminal peptide, and a half-life extending portion (preferentially selected from a human IgG Fc variant). The fusion protein has a similar level of biological activity as a natural human (FVIIa) and extended in vivo half-life, thereby improving pharmacokinetics and drug efficacy.

Description

Activated human coagulation factor VII fusion protein, preparation method and use thereof

Technical field

The present invention relates to a fusion protein, and more particularly to an activated human Factor VII (FVIIa) fusion protein, a preparation method and use thereof, and particularly to the use for the preparation of a medicament for treating various blood coagulation related diseases.

Background technique

FVII is a vitamin K-dependent plasma glycoprotein that is synthesized in the liver and secreted into the blood as a single-chain proteoprotein with a molecular weight of approximately 53 kDa (Broze et al, J Biol Chem, 1980, 255: 1242-1247) . The FVII zymogen is hydrolyzed by a protease at a single site, Arg152-Ile153, to produce a double-strand linked by a disulfide bond, thereby converting to its active form, FVIIa. Activated FVIIa comprising NH ₂ - terminal derived light chain (about 20 KDa) and COOH- terminal-derived heavy chain (about 30KDa) connected via a single disulfide bond (Cys135 to Cys262). The light chain contains a cell membrane that binds to the Gla domain and two "epidermal growth factor (EGF) domains", while the heavy chain contains a serine protease catalytic domain. In the activated form, FVIIa acts as a serine protease and participates in the extrinsic pathway of the coagulation cascade. The use of FVIIa in the treatment of hemophilia is based on the low affinity binding of FVIIa to thrombin-activated platelet surface. By administering a pharmacological dose of exogenous FVIIa, platelet surface thrombin production at the lesion is enhanced, which is associated with FVIII. /FIX is irrelevant, that is, to bypass the demand for FVIIIa and FIXa to stop bleeding. Therefore, FVIIa can be used for the prevention and treatment of hemorrhagic conditions in hemophilia patients who produce inhibitors. Commercially available recombinant factor VIIa is currently only available

(Novo Nordisk, Denmark). This drug has been approved worldwide for the treatment of hemophilia A or B patients who develop FVIII or FIX antibodies (inhibitors), patients with congenital FVII deficiency, and termination of bleeding events associated with trauma and/or surgery or Prevent bleeding.

However, therapeutic coagulation protein drugs, including FVIIa, are rapidly degraded by proteolytic enzymes and are easily neutralized by antibodies, which results in a significant decrease in their circulating half-life in vivo, thereby limiting their efficacy. Recombinant FVIIa

The circulating half-life in the human body is about 2.3 hours, and it needs to be injected every 2-3 hours.

In order to increase the in vivo half-life of FVIIa, rFVIIa-FP (albumin fusion with albumin and FVIIa) developed by CSL Behring has completed a clinical phase I study, which is serum half in normal subjects. The decay is 3-4 times higher than that of commercially available FVIIa (Golor G et al, J Thromb Haemost, 2013, 11(11): 1977-85), with a biological activity of 620-770 IU/mg, equivalent to 69-75 IU. /nM (Weimer T et al, Thromb Haemost, 2008, 99: 659-667).

The PEGylated liposome formulation of FVIIa, PEPLip-FVIIa (developed by Omri), is also in the early clinical stage and has a serum half-life of only twice that of native FVIIa. In addition, the N-glycosyl-directed PEGylation modification of FVIIa (N7-GP) developed by Novo Nordisk Company was approximately 5-fold longer than that of rFVIIa, but due to the lack of quantitative-effect linearity in clinical phase II data and the occurrence of subjects The allergic reaction, the project has terminated research (Ljung R et al, J Thromb Haemost, 2013, 11 (7): 1260-8).

The rFVIIaFc monomer-dimer hybrid (momer/dimer hybrid) fusion protein developed by Biogen is still in the early stages of research, and the study showed that the terminal half-life of Monomeric rFVIIaFc in hemophilia A mice was 6.6 hours. For the control group rFVIIa

5.5 times. At 5 minutes of intravenous injection of rFVIIaFc, the blood coagulation activity of the mouse returned to 40% of the normal value. However, its activity was abruptly attenuated at the initial stage, and decreased to about 20% at 30 minutes after administration, and 1 hour after administration. Has dropped to about 10%, which is related to

The downward trend is similar in 0 to 1 hour. Although the decrease in the activity of the rFVIIaFc group was slower than that of rFVIIa after 1 hour of administration, the activity had decreased to about 1% after 8 hours of intravenous injection of rFVIIaFc, which was lower than the baseline level of coagulation activity and could not effectively stop bleeding. This suggests that rFVIIaFc can effectively control acute bleeding symptoms in a short period of time and does not maintain the normal coagulation function of the body for a long time, smoothly and effectively. The in vitro coagulation activity test showed that the rFVIIaFc molar specific activity was 1115 IU/nM,

(2511 IU/nM) decreased by about 60%, the researchers believe that this decrease in activity is due to the steric hindrance effect of the fusion ligand Fc, which causes its binding constant (Kd) to decrease with soluble tissue factor (sTF). (J. Salsa et al., Thrombosis Research, 2015, 135: 970-976). In addition, a homodimeric rFVIIa-Fc previously constructed by the present inventors, although its half-life is greatly prolonged, has a problem of reduced activity, and the in vitro activity is only 208-240 IU/nM (Chinese invention patent number: CN104774269B) ).

The carboxy-terminal peptide of the human chorionic gonadotropin (hCG) β-chain (hereinafter referred to as CTP) also has the effect of prolonging the half-life of certain proteins in vivo, and thus some of the extended half-life fractions contained in the fusion proteins disclosed in some patent documents can be selected. Fc, HSA, CTP or other fusion ligands that extend half-life. For example, the rFVIIa-CTP fusion protein developed by OPKO/Prolor, rFVIIa, in series with 1 to 5 CTP molecules, respectively, significantly increases the half-life of FVII in a manner proportional to CTP, when adding 3, 4 or 5 CTP to make FVII half-life, respectively. It is extended by 0.67, 2 and 3 times; however, the addition of CTP reduces the specific activity of rFVIIa, such as rFVIIa-CTP ₃ relative to

The specific activity (U/mg FVIIa) was reduced by about 4 times (Chinese Invention Patent Application No.: CN201380019660.8). In addition, CTP can also be used as a linker sequence to link different subunits of the same protein. For example, among the fusion proteins disclosed in Chinese Patent Nos. CN103539860A, CN103539861A, CN103539868A and CN103539869A, CTP acts as a linker between the beta subunit and the alpha subunit of follicle stimulating hormone; among the fusion proteins disclosed in WO2005058953A2, CTP is used as a linker for Linking the beta and alpha subunits of the glycoprotein hormone.

The present inventors have found that the long-acting rFVIIa disclosed in the prior art has a problem of limited half-life extension, significant decrease in biological activity or strong immunogenicity, mainly because the C-terminus of FVIIa contains a serine protease domain (153-406 residues). Therefore, the fusion of other molecules at the C-terminus affects the catalytic activity and function of FVIIa. In addition, the inventors have found that between FVIIa and an extended half-life portion (eg, an immunoglobulin Fc fragment), only a long stretch of conventional flexible peptide linkers (eg, (GGGGS)n) ligation, but instead the fusion protein pair Proteases are more sensitive, and even worse, such long peptide linkers also allow most of the fusion proteins to be expressed in polymer form with little biological activity. After long-term research, the present inventors have surprisingly discovered that a linker molecule composed of a flexible peptide and CTP is linked to FVIIa and an extended half-life portion (eg, human IgG Fc), thereby maximally retaining the biological activity of FVIIa, and More significantly extend its in vivo half-life of activity.

Summary of the invention

It is an object of the present invention to provide a hyperglycosylated activated human Factor VII fusion protein having a significantly extended half-life and maintaining biological activity similar to recombinant human Factor Vila (FVIIa); The invention also provides a preparation method and use of the fusion protein.

In one aspect, the invention provides an activated human Factor VII fusion protein (hereinafter referred to as a fusion protein) comprising, in order from the N-terminus to the C-terminus, an activated human Factor VII (FVIIa), a flexible peptide linker ( The carboxy terminal peptide rigid unit (hereinafter referred to as CTP rigid unit, denoted as (CTP) _n ), which is L), at least one human chorionic gonadotropin β subunit, preferably n, 1, 2, 3, 4, Or 5) and an extended half-life portion (eg, immunoglobulin Fc fragment, albumin, transferrin or PEG, preferably a human IgG Fc variant (denoted as vFc)). In some preferred embodiments of the invention, the fusion protein is represented by FVIIa-L-CTP _n -vFc.

Wherein the amino acid sequence of human factor VII is at least 80% homologous to native human factor VII; more preferably, the amino acid sequence of human factor VII is at least 90% homologous to native human factor VII; most preferred The human Factor VII comprises the amino acid sequence set forth in SEQ ID NO: 1.

Among them, FVIIa also includes human coagulation factor VII (abbreviated as FVII) which is activated in the applicator or which is activated as FVIIa before application.

Wherein the flexible peptide linker is preferably non-immunogenic and produces a sufficient distance between FVII and Fc to minimize steric effects between each other. Preferably, a flexible peptide linker comprising two or more amino acid residues is used and is selected from the group consisting of Gly (G), Ser (S), Ala (A) and Thr (T).

More preferably, the flexible peptide linker comprises G and S residues. The length of the linker peptide is very important for the activity of the fusion protein. For the purposes of the present invention, the peptide linker may preferably comprise an amino acid sequence formula formed by combining (GS) _a (GGS) _b (GGGS) _c (GGGGS) _d cycle units, wherein a, b, c and d are greater than Or an integer equal to 0, and a+b+c+d≥1.

Specifically, in an embodiment of the invention, the peptide linker may preferably comprise the following sequence:

(i) L1: GSGGGSGGGGSGGGGS (as shown in SEQ ID NO: 2);

(ii) L2: GSGGGGSGGGGSGGGGSGGGGSGGGGS (as shown in SEQ ID NO: 3);

(iii) L3: GGGGSGGGGSGGGGSGGGGS (as shown in SEQ ID NO: 4);

(iv) L4: GSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS (as shown in SEQ ID NO: 5);

(v) L5: GGGSGGGSGGGSGGGSGGGS (as shown in SEQ ID NO: 6);

Wherein the CTP rigid unit is selected from the full length sequence consisting of amino acids 113 to 145 of the carboxy terminus of human chorionic gonadotropin β subunit or a fragment thereof, in particular, the rigid unit comprises SEQ ID NO: 7 The amino acid sequence shown or its truncated sequence.

Preferably, the CTP rigid unit comprises at least 2 glycosylation sites; for example, in a preferred embodiment of the invention, the CTP rigid unit comprises 2 glycosylation sites, exemplarily, the CTP The rigid unit comprises 10 amino acids of the N-terminus of SEQ ID NO: 7, ie SSSS*KAPPPS*, or the CTP rigid unit comprises 14 amino acids of the SEQ ID NO: 7C terminus, ie S*RLPGPS*DTPILPQ; as another example The CTP rigid unit comprises three glycosylation sites, exemplarily, said The CTP rigid unit comprises 16 amino acids of the N-terminus of SEQ ID NO: 7, ie SSSS*KAPPPS*LPSPS*R; as in other embodiments, the CTP rigid unit comprises 4 glycosylation sites, exemplarily, The CTP rigid unit comprises 28, 29, 30, 31, 32 or 33 amino acids and begins at position 113, 114, 115, 116, 117 or 118 of the human chorionic gonadotropin beta subunit, terminating at position 145 Bit. Specifically, the CTP rigid unit comprises 28 amino acids of the N-terminus of SEQ ID NO: 7, namely SSSS*KAPPPS*LPSPS*RLPGPS*DTPILPQ. In this context, * represents a glycosylation site. Each possibility represents a separate embodiment of the invention.

In other embodiments, the CTP rigid units provided herein are at least 70% homologous to the native CTP amino acid sequence; in other embodiments, the CTP rigid units provided herein are at least 80% homologous to the native CTP amino acid sequence; In other embodiments, the CTP rigid units provided herein are at least 90% homologous to the native CTP amino acid sequence; in other embodiments, the CTP rigid units provided herein are at least 95% homologous to the native CTP amino acid sequence.

In a particular embodiment of the invention, the CTP rigid unit may preferably comprise the following sequence:

(i) CTP ¹ : PRFQDSSSSKAPPPSLPSPSRLPGPSDTPILPQ (as shown in SEQ ID NO: 7);

(ii) CTP ² : SSSSKAPPPSLPSPSRLPGPSDTPILPQ (as shown in SEQ ID NO: 8);

(iii) CTP ³ : SSSSKAPPPS (shown as SEQ ID NO: 9);

(iv) CTP ⁴ : SRLPGPSDTPILPQ (shown as SEQ ID NO: 10).

In some embodiments of the invention, the fusion protein comprises one of the above CTP rigid units.

In other embodiments of the invention, the fusion protein comprises more than one of the above CTP rigid units, preferably comprising 2, 3, 4 or 5 of the above CTP rigid units, as in an embodiment of the invention, The fusion protein comprises two CTP ³ rigid units: SSSSKAPPPSSSSSKAPPPS (CTP ³ -CTP ³ , or expressed as (CTP ³ ) ₂ ).

Wherein the extended half-life portion is preferably a self-immunoglobulin IgG, IgM, IgA Fc fragment; more preferably an Fc fragment from human IgG1, IgG2, IgG3 or IgG4 and variants thereof; further, the human IgG Fc variant comprises in the wild At least one amino acid modification in a human IgG Fc, and the variant has reduced effector function (ADCC and/or CDC effect) and/or enhanced binding affinity to the neonatal receptor FcRn. Further, the human IgG Fc variant may be selected from the group consisting of:

(i) vFcγ ₁ : human IgG1 hinge region, CH2 and CH3 region containing the Leu234Val, Leu235Ala and Pro331Ser mutations (such as the amino acid sequence shown in SEQ ID NO: 11);

(ii) vFcγ _2-1 : human IgG2 hinge region, CH2 and CH3 region containing the Pro331Ser mutation (such as the amino acid sequence shown in SEQ ID NO: 12);

(iii) vFcγ _2-2 : human IgG2 hinge region, CH2 and CH3 region containing the Thr250Gln and Met428Leu mutations (such as the amino acid sequence shown in SEQ ID NO: 13);

(iv) vFcγ _2-3 : human IgG2 hinge region, CH2 and CH3 regions (such as the amino acid sequence shown in SEQ ID NO: 14) containing the Pro331Ser, Thr250Gln and Met428Leu mutations.

(v) vFcγ ₄ : human IgG4 hinge region, CH2 and CH3 regions containing the Ser228Pro and Leu235Ala mutations (such as the amino acid sequence shown in SEQ ID NO: 15).

The IgG Fc variants provided by the present invention include, but are not limited to, the five variants described in (i) to (v), and may also be a combination or superposition of two types of functional variant mutation sites between IgG isotypes. The variant as described in (iv) above is a novel variant of the novel IgG2Fc obtained by superimposing the mutation sites in (ii) and (iii).

An Fc variant (vFc) in a fusion protein of the invention, which comprises a hinge region, a CH2 and a CH3 region of human IgG such as human IgG1, IgG2 and IgG4. This CH2 region contains amino acid mutations at positions 228, 234, 235 and 331 (as determined by the EU counting system). It is believed that these amino acid mutations reduce the effector function of Fc. Human IgG2 does not bind to FcyR but shows very weak complement activity. An Fc[gamma]2 variant with a Pro331Ser mutation should have a lower complement activity than native Fc[gamma]2 and is still an Fc[gamma]R non-binding element. IgG4Fc is defective in the activation of the complement cascade and its binding affinity to FcγR is about an order of magnitude lower than that of IgG1. An Fcγ4 variant with a Leu235Ala mutation should exhibit minimal effector function compared to native Fcγ4. Fcγ1 with Leu234Va1, Leu235Ala and Pro331Ser mutations also showed reduced effector function compared to native Fcγ1. These Fc variants are all more suitable for the preparation of FVIIa fusion proteins than native human IgG Fc. The 250 and 428 positions (positions determined by the EU numbering system) contain amino acid mutations that increase the binding affinity of the Fc region to the neonatal receptor FcRn, thereby further extending the half-life (Paul R et al, J Biol Chem, 2004, 279: 6213). -6216); the above two types of functional variants are combined or superimposed to obtain a new combined variant, which reduces the effector function and prolongs its circulating half-life in vivo. The Fc variants of the invention comprise, but are not limited to, mutations at several of the above sites, and substitutions at other sites may be introduced such that the Fc has reduced effector function and/or enhanced binding to the FcRn receptor, while still It does not cause a decrease in Fc variant function/activity or cause a poor conformational change. For common mutation sites, see Shields RL et al, J Biol Chem, 2001, 276(9):6591-604.

In a preferred embodiment of the present invention, the amino acid sequence of the fusion protein is as shown in SEQ ID NO:

According to another aspect of the present invention, a DNA encoding the above fusion protein is provided.

In a preferred embodiment of the invention, the DNA sequence of the fusion protein is set forth in SEQ ID NO: 17.

According to still another aspect of the present invention, a vector comprising the above DNA is provided.

According to still another aspect of the present invention, a host cell comprising the above vector or transfected with the above vector is provided.

In an embodiment of the invention, the host cell is a derivative cell line DXB-11 of CHO.

According to still another aspect of the present invention, a pharmaceutical composition is provided. The pharmaceutical composition comprises a pharmaceutically acceptable carrier, excipient or diluent, and an effective amount of the above fusion protein.

According to another aspect of the invention there is provided a method of making or producing the fusion protein from a cell line derived from a mammalian cell line, such as CHO, comprising the steps of:

(a) introducing a DNA encoding a fusion protein into a CHO cell to generate a CHO-derived cell line;

(b) a high-yield cell line expressing more than 3 μg/10 ⁶ cells per 24 hours during the screening step (a);

(c) culturing step (b) the selected cell strain to express the fusion protein;

(d) harvesting the fermentation broth obtained in step (c), purifying the fusion protein;

(e) activating the purified fusion protein in step (d).

Further, the CHO-derived cell line in the step (a) is DXB-11.

Further, in the step (d), the fusion protein is purified by four-step chromatography, which is ProteinA affinity chromatography, multi-dimensional mode chromatography, anion exchange chromatography and molecular sieve chromatography.

Step Protein A affinity chromatography step, preferably using a commercially available, for example, but not limited to GE's Mabselect ^{^TM,} Mabselect SuRe ^TM, or TOSOH the Toyopearl AF-rProteinA-650F ^TM, or the rProtein A Bestarose ^TM Boge Long , or the world of human and rProtein A Bead ^TM , or Safran Technology's MabPurix ^TM chromatography medium. It utilizes the specific binding of the Fc fragment of the fusion protein (human IgG) to the monoclonal antibody for capture because the proteinA ligand on the affinity medium has a high affinity for the Fc fragment and can specifically bind reversibly with it. X-ray crystallographic analysis showed that the protein A and Fc fragment binding sites are centered on a highly conserved six-membered histidine residue. This histidine residue is uncharged at neutral and alkaline pH because of hydrophobic interaction. Tightly combined. When the pH is lowered or the hydrophobicity is weakened, the histidine residues are mutually repelled upon charging or the solubility of the Fc fusion protein in the solvent is enhanced, and the protein bound to the affinity medium can be eluted. Therefore, in the first step of the present invention, affinity chromatography can rapidly and efficiently capture and purify the fusion protein from the fermentation broth.

The multi-dimensional mode chromatography step of the second step, preferably using commercially available CHT ^(TM) (ceramic hydroxyapatite Form I or Type II) chromatography media (40 [mu]m or 80 [mu]m), such as but not limited to Bio-Rad Corporation. There are 10 Glu residues that can be carboxylated at the N-terminus of the fusion protein, which have a pair of negative charges after carboxylation. The CHT medium is arranged in a repeating geometric pattern of five calcium doublets and a pair of hydroxyl-containing phosphorus triplets, wherein the PO4 ^3- ions are ionically bonded to the positively charged protein and have ion exchange characteristics. The paired negative charge of the Ca ²⁺ ion and the free carboxyl group of the fusion protein is bound by metal chelation, and the binding mode is insensitive to NaCl and can be competitively eluted by a sodium phosphate concentration gradient. According to the literature, the degree of carboxylation of FVIIa is correlated with its biological activity (Hagen FS et al, Proc Natl Acad Sci USA, 1986, 83(8): 2412-2416; US Pat. No. 8,032,224; US Patent No. US 2009/0042784). Therefore, the fusion protein captured by affinity chromatography in the present invention is subjected to a second purification step using CHT to separate a target component having a higher biological activity.

The third step of anion exchange chromatography step, preferably using a commercially available, for example, but not limited to GE's ^{Q Sepharose HP TM, Q Sepharose FF} TM chromatography medium, or the Boge Long ^{Q Bestarose HP TM, Q Bestarose FF} TM layer Analysis of the medium, and the earth or the ^{Q Beads 6HP TM, Q Beads 6FF} TM chromatographic medium is isolated and purified, for removing contaminants. The separation of molecules by ion exchange is based on the difference in their net surface charge. The charge properties of molecules vary widely, and the difference in total charge, charge density, and surface charge distribution of the molecules causes them to differ in their ability to bind to the charge-exchanged ion-exchange chromatography packing. The fusion protein can be separated from the contaminants by progressively increasing the concentration of NaCl in the buffer for competitive elution.

The molecular sieve chromatography step of the fourth step is preferably carried out using a commercially available Superdex 200 ^(TM) such as, but not limited to, GE, or a Chromdex 200 prep grade ^(TM) chromatography medium from Boglon. Molecular sieve chromatography is mainly a chromatographic method for separating according to the size of a protein molecule. Molecular sieve media are porous spherical particles. When different sizes of proteins (like a monomer and polymer of a protein) pass through a packed molecular sieve column, different sizes of proteins travel in different paths in the column, and macromolecules cannot enter. In the pores of the medium, the retention time in the column is relatively short and elutes first; while the small molecules can enter the pores of the medium, and the path passing through the column is long, and then elutes. The fusion protein obtained by the above three-step chromatography in the present invention still has a certain polymer component, which may affect the activation process and the biological activity after activation, and therefore the molecular sieve layer is used to remove the polymer component therein.

Further, the activating step in the step (e) may activate the fusion protein by coagulation factor XII (FXII) activation, on-column self-activation or solution incubation self-activation. The conversion of FVII to active FVIIa needs to be done under the action of FXII (Hedner et al, J Clin Invest, 1983, 71: 1836-1841), or other proteases with trypsin-like activity can also activate FVII (Kisiel et al, Behring Inst Mitt) , 1983, 73: 29-42); FVII may also be activated by FVII itself, but by its serine protease region, by itself, without the use of other proteases. FVII can also be self-activated by binding to a positively charged surface or filler, such as an anion exchange packing (Pedersen AH et al, Biochemistry, 1989, 28: 9331-9336). Increasing the ionic strength, lowering the pH, or increasing the concentration of Ca ²⁺ in the solution can dissociate the activated FVIIa from the positively charged surface or onto the filler (Bioern et al, Research Disclosures, 1986, 269: 564-56). Self-activation under solution conditions is described in US 2007/0129298.

In one embodiment of the invention, the fusion protein is activated by a solution incubation self-activation method, and the activity of the activated fusion protein is >15000 IU/mg.

According to still another aspect of the present invention, the fusion protein is provided for use in the preparation of a medicament for treating or preventing a bleeding disorder. Preferably, the fusion protein is used for the prevention or treatment of hemorrhagic diseases in patients with FVII congenital or acquired deficiency, prevention or treatment of spontaneous or surgical bleeding in patients with hemophilia A or B, or other related Application in bleeding drugs.

It is to be understood that within the scope of the present invention, the various technical features described above and the various technical features specifically described hereinafter (as in the embodiments) may be combined with each other to form a new or preferred technical solution.

The inventors have found that the advantages of the fusion proteins and methods of preparation disclosed and/or described herein can be summarized as follows:

1. The FVIIa fusion protein constructed by the present invention, wherein the Fc fragment is non-lytic, that is, by mutating the complement and receptor binding domains of the Fc fragment, regulating the binding affinity of the Fc to the corresponding receptor, and reducing or eliminating the ADCC and CDC effects. While retaining only the Fc segment to prolong the half-life of the active protein in vivo, it does not produce cytotoxicity. The FVIIa fusion protein developed by Biogen, whose Fc segment is of natural origin, predicts that Fc-mediated adverse effector functions will necessarily increase the patient's therapeutic risk.

2. The half-life of FVIIa fusion protein was about 3 times longer in vivo. The plasma coagulation activity was about 40% after 3 hours of single injection of FP-A, while the activity of the same active group was reduced to 3% after 3 hours; FP- The plasma clotting activity remained above 7% after 12 hours of A administration, and the monomer-dimer heterozygous (Monomeric) rFVIIaFc developed by Biogen (see J. Salsa et al., Thrombosis Research, 2015, 135: 970-976). Compared to, it has a longer half-life of in vivo activity. The fluctuation of drug concentration in serum is reduced, and the safety is improved, which can reduce the frequency of injection and improve the quality of life of patients;

3. The fusion protein constructed by the invention is more efficient and convenient than the Monomeric FVII fusion protein constructed by Biogen, and the production cost can be greatly reduced. Biogen Corporation constructed a dual expression vector for rFVIIIFc and Fc, wherein the Fc molecule was labeled with Flag (European Patent, Publication No. EP1624891B1). The fusion protein fermentation broth expressed by it is expected to contain three forms of products, namely FVII-Fc: FVII-Fc homodimeric (Dimeric) fusion protein, FVII-Fc: FLAG-Fc monomer-dimerization Monomeric fusion protein and FLAG-Fc: FLAG-Fc dimer three products. In one aspect, in the process of expression of the fusion protein, the host cell needs to simultaneously express two single-stranded molecules of FVII-Fc and Fc, and then separately polymerize to form the above three products, thereby greatly reducing the expression efficiency of the final target product; In addition, the other two forms of impurities must be removed during the purification process, which makes the purification process more complicated, the production efficiency is low, and the production cost is also greatly increased. Therefore, the preparation method of the invention has certain technical advantages and price advantages over the Monomeric rFVIIFc fusion protein developed by Biogen, and the expression and purification processes are simpler, more efficient, and the production cost is lower;

4. The specific activity of each batch of purified fusion protein can reach at least 15000 IU/mg, about 2949 IU/nM (2 FVIIa per fusion protein, equivalent to 1474.4 IU/nM FVIIa), and some batches of fusion protein. The activity is even higher than 22470 IU/mg, which is converted to a molar activity of about 4325 IU/nM (2 FVIIa per fusion protein, equivalent to 2162 IU/nM FVIIa), and the listed recombinant FVIIa-Noci (2511 IU/nM) The activity of FVIIa) is comparable, indicating that the fusion protein provided by the present invention has a C-terminally-fused Fc having minimal effect on the activity of FVIIa;

5. A CTP rigid unit constituting the fusion protein, which contains a plurality of O-glycosyl side chains, which can form a relatively stable stereo conformation and can effectively isolate FVIIa from Fc. On the other hand, the CTP rigid unit contains a glycosyl group, and the negatively charged, highly sialylated CTP rigid unit is able to resist the clearance of the kidney and further prolong the half-life of the fusion protein; on the other hand, the protective effect of the CTP rigid unit glycosyl side chain can be In order to reduce the sensitivity of the linker peptide to protease, the fusion protein is not easily degraded in the junction region;

6. The method for preparing the fusion protein provided by the invention has the advantages of high yield. In a preferred embodiment of the present invention, the culture is continuously cultured for 14 days in a 300 ml shake flask, and the cumulative yield reaches 310 mg/L, which can be subjected to process enlargement to realize large-scale industrial production.

Detailed description of the invention:

Flexible linker

The length of the linker peptide is very important for the activity of the fusion protein. A FVII/FVIIa-albumin fusion polypeptide disclosed in WO2007090584A1, in which a different length of a linker peptide is inserted between FVII/FVIIa and albumin, and the biological activity of the FVII/FVIIa fusion protein without the linker is found to be significantly decreased. The FVII/FVIIa-FP fusion protein containing a linker peptide shows that the increase in its biological activity depends on the length of the linker peptide, which may be explained by the increased linker peptide between the two parts of the fusion protein, allowing the two parts of the molecule to exercise their respective The function is conducive to the formation of a higher specific activity conformation.

The present inventors have previously designed three different lengths of flexible linker peptides composed of glycine and serine, and FVII constitutes a fusion protein (without CTP rigid unit) through the Fc junction of the linker peptide and its C-terminal fusion, and transient expression experiments show that The two amino acid short peptide linker GlySer-linked fusion protein showed little activity, indicating that the important functional regions that maintain the biological activity of FVIIa are greatly affected by the C-terminal fusion ligand in three-dimensional structure. When the linker peptide was increased to 16 amino acids, the biological activity of the FVIIa fusion protein was significantly increased, but still much lower than recombinant FVIIa. When the linker peptide is further extended to 37 amino acids, the fusion protein portion secreted by the CHO cell is polymerized and has low activity. This indicates that the problem of the effect of the Fc fragment on FVIIa activity cannot be completely solved by simply extending the length of the linker peptide.

hCG-β carboxy terminal peptide (CTP)

CTP is a short peptide derived from the carboxy terminus of the β-subunit of human chorionic gonadotropin (hCG). Four reproductive-related peptide hormones, follicle stimulating hormone (FSH), luteinizing hormone (LH), thyrotropin (TSH), and chorionic gonadotropin (hCG) contain the same alpha-subunit and their respective specific beta - Yaki. Compared with the other three hormones, the half-life of hCG is significantly prolonged, mainly due to the unique carboxy terminal peptide (CTP) on its β-subunit (Fares FA et al, ProcNatl Acad Sci USA, 1992, 89: 4304-4308) . The natural CTP contains 37 amino acid residues with four O-glycosylation sites and the terminal is sialic acid. Residues. Negatively charged, highly sialylated CTP is resistant to the clearance of the kidneys, thereby prolonging the half-life of the protein in the body. However, the inventors creatively linked at least one CTP polypeptide to a flexible linker of appropriate length, collectively as a linker peptide, for ligation of FVII with an extended half-life moiety (eg, an immunoglobulin Fc fragment).

The present inventors have found that the C-terminal catalytic domain of FVIIa is critical for its function, and the FVIIa spatial conformation is complex and fragile, and the steric hindrance effect of the fusion ligand is highly susceptible to interference with its correct folding. By adding a CTP rigid unit between FVIIa and the Fc variant, an equivalent of a rigid linker peptide is added. This aspect ensures that the N-terminally fused FVIIa does not affect the binding site of the Fc variant to FcRn, thereby affecting the half-life; in addition, the Fc-ProteinA binding site is important for the purification step in the preparation process, and the CTP rigid unit is connected to ensure N The end-fused FVIIa also does not "cover" its binding site to protein A. On the other hand, the addition of a CTP rigid unit also allows the Fc fragment of about 25 kD size to not interfere with the correct folding of the N-terminally fused FVIIa, resulting in a decrease or loss of its biological activity/function. It was also confirmed that simply extending the length of the flexible linker peptide did not improve the effect of the Fc fragment on FVIIa activity. Too long linker peptides also caused the formation of multimers and increased sensitivity to proteases, which were easily degraded, whereas CTP rigid units The addition causes a significant increase in the biological activity of the fusion protein. This may be interpreted as a CTP rigid polypeptide with multiple glycosyl side chains, which can form a stable stereoconfiguration relative to the random coiling of a flexible linker peptide such as (GGGGS)n, which promotes FVIIa and Fc. The segments are independently folded to form the correct three-dimensional conformation without affecting each other's biological activity. On the other hand, the protective effect of the CTP glycosyl side chain can reduce the sensitivity of the linker peptide to proteases, making the fusion protein less susceptible to degradation in the junction region.

IgG Fc variant

Non-lytic Fc variant

The Fc element is derived from the constant region Fc fragment of immunoglobulin IgG, which plays an important role in eradicating the immune defense of pathogens. The effector function of Fc-mediated IgG is exerted through two mechanisms: (1) binding to cell surface Fc receptors (FcγRs), digestion of pathogens by phagocytosis or cleavage or killer cells via antibody-dependent cellular cytotoxicity (ADCC) pathway , or (2) binding to C1q of the first complement component C1, eliciting a complement-dependent cytotoxicity (CDC) pathway, thereby lysing the pathogen. Among the four human IgG subtypes, IgG1 and IgG3 efficiently bind to FcγRs, and the binding affinity of IgG4 to FcγRs is low, and the binding of IgG2 to FcγRs is too low to be determined, so human IgG2 has almost no ADCC effect. In addition, human IgG1 And IgG3 can also effectively bind to C1q to activate the complement cascade. Human IgG2 binds relatively weakly to C1q, whereas IgG4 does not bind to C1q (Jefferis R et al, Immunol Rev, 1998, 163: 59-76), so the human IgG2 CDC effect is also weak. Clearly, none of the native IgG subtypes are well suited for the construction of FVIIa/Fc fusion proteins. In order to obtain non-lytic Fc without effector function, the most effective method is to mutate the complement and receptor binding domain of the Fc fragment, modulate the binding affinity of Fc to related receptors, reduce or eliminate ADCC and CDC effects, and retain only Fc. Long cycle half-life characteristics without cytotoxicity. For more non-lytic Fc variants, the mutation sites can be found in Shields RL et al, J Biol Chem, 2001, 276(9): 6591-604 or Chinese invention patent CN 201280031137.2.

Fc variant with enhanced binding affinity to neonatal receptor (FcRn)

The plasma half-life of IgG depends on its binding to FcRn, which typically binds at pH 6.0 and dissociates at pH 7.4 (plasma pH). By studying the binding sites of the two, the site of binding to FcRn on IgG was engineered to increase the binding ability at pH 6.0. Mutations in some residues of the human Fcγ domain important for binding to FcRn have been shown to increase serum half-life. Mutations in T250, M252, S254, T256, V308, E380, M428 and N434 have been reported to increase or decrease FcRn binding affinity (Roopenian et al, Nat. Rview Immunology 7: 715-725, 2007). Korean Patent No. KR 10-1027427 discloses variants of trastuzumab (Herceptin, Genentech) having increased FcRn binding affinity, and these variants are selected from the group consisting of 257C, 257M, 257L, 257N, 257Y, 279Q, One or more amino acid modifications of 279Y, 308F and 308Y. Korean Patent Publication No. KR 2010-0099179 provides variants of bevacizumab (Avastin, Genentech) and these variants show increased in vivo by amino acid modifications contained in N434S, M252Y/M428L, M252Y/N434S and M428L/N434S half life. In addition, Hinton et al. also found that two mutants, T250Q and M428L, increased the binding to FcRn by 3 and 7 fold, respectively. At the same time, two sites were mutated, and the binding was increased by 28 times. In rhesus monkeys, the M428L or T250QM/428L mutant showed a 2-fold increase in plasma half-life (Paul R. Hinton et al, J Immunol, 2006, 176: 346-356). More mutation sites including Fc variants with enhanced binding affinity to neonatal receptor (FcRn) can be found in Chinese invention patent CN201280066663.2. In addition, studies on T250Q/M428L mutations in the Fc segment of five humanized antibodies not only improved the interaction of Fc with FcRn, but were also administered by subcutaneous injection in subsequent in vivo pharmacokinetic assays, Fc. Mutant antibodies have improved pharmacokinetic parameters compared to wild-type antibodies, such as increased in vivo exposure, reduced clearance, subcutaneous Increased availability (Datta-Mannan A et al. MAbs. Taylor & Francis, 2012, 4(2): 267-273.).

Fusion protein and preparation method thereof

The fusion protein gene of the present invention is codon-optimized and prepared by a synthetic method. According to the nucleotide sequence of the present invention, those skilled in the art can conveniently prepare the nucleic acid of the present invention by various known methods. These methods are not limited to synthetic or traditional subcloning, and the specific method can be found in J. Sambrook, Molecular Cloning Experiment Guide. As an embodiment of the present invention, the nucleic acid sequence of the present invention is constructed by subcloning a nucleotide sequence and then subcloning.

The invention also provides an expression vector for a mammalian cell comprising a fusion protein sequence encoding the invention and an expression control sequence operably linked thereto. By "operably linked" or "operably linked" is meant a condition in which portions of a linear DNA sequence are capable of modulating or controlling the activity of other portions of the same linear DNA sequence. For example, if a promoter controls the transcription of a sequence, then it is operably linked to the coding sequence.

The mammalian cell expression vector can be commercially available, for example, but not limited to, pcDNA3, pIRES, pDR, pBK, pSPORT, etc., which can be used for expression in eukaryotic cell systems. One skilled in the art can also select a suitable expression vector based on the host cell.

According to the restriction enzyme map of the known empty-load expression vector, the skilled person can prepare the present invention by inserting the coding sequence of the fusion protein of the present invention into a suitable restriction site by restriction enzyme cleavage and splicing according to a conventional method. Recombinant expression vector.

The invention also provides a host cell expressing a fusion protein of the invention comprising a coding sequence for a fusion protein of the invention. The host cell is preferably a eukaryotic cell such as, but not limited to, a CHO cell, a COS cell, a 293 cell, an RSF cell, and the like. In a preferred embodiment of the present invention, the cell is a CHO cell which can preferably express the fusion protein of the present invention, and a fusion protein having good activity and good stability can be obtained.

The invention also provides a method for preparing a fusion protein of the invention by recombinant DNA technology, the steps of which comprise:

1) providing a nucleic acid sequence encoding a fusion protein;

2) inserting the nucleic acid sequence of 1) into a suitable expression vector to obtain a recombinant expression vector;

3) introducing the recombinant expression vector of 2) into a suitable host cell;

4) cultivating the transfected host cell under conditions suitable for expression;

5) collecting the supernatant and purifying the fusion protein product;

6) Activate the fusion protein.

Introduction of the coding sequence into a host cell can employ a variety of known techniques in the art such as, but not limited to, calcium phosphate precipitation, lipofection, electroporation, microinjection, viral infection, alkali metal ion methods.

For the culture and expression of host cells, see Olander RM et al, Dev Biol Stand 1996, 86:338. The cells and debris in the suspension can be removed by centrifugation and the supernatant collected.

Regarding the activation method of the fusion protein, the fusion protein can be activated by coagulation factor XII (FXII) activation, on-column autoactivation or solution incubation self-activation.

The fusion protein obtained as described above can be purified to a substantially uniform property, such as a single band on SDS-PAGE electrophoresis. The supernatant is first concentrated, and the concentrate can be further purified by gel chromatography or by ion exchange chromatography. For example, anion exchange chromatography or cation exchange chromatography. The gel matrix may be a medium commonly used for protein purification such as agarose, dextran, polyamide, and the like. The Q- or SP- group is a preferred ion exchange group. Finally, the purified product may be further purified by hydroxyapatite adsorption chromatography, metal chelate chromatography, hydrophobic interaction chromatography and reversed-phase high performance liquid chromatography. All of the above purification steps can utilize different combinations to ultimately achieve a substantially uniform protein purity. The expressed fusion protein can also be purified using an affinity chromatography column containing a specific antibody, receptor or ligand of the fusion protein. Depending on the nature of the affinity column used, the fusion polypeptide bound to the affinity column can be eluted using conventional methods such as high salt buffer, pH change, and the like.

Pharmaceutical composition

The invention also provides a pharmaceutical composition comprising an effective amount (160-360 [mu]g/kg) of a fusion protein of the invention, and a pharmaceutically acceptable carrier. Generally, an effective amount of a fusion protein of the invention can be formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium wherein the pH is usually from about 5 to about 8, preferably, the pH is from about 6 to about 8. The term "effective amount" or "effective amount" refers to an amount that is functional or active to a human and/or animal and that is acceptable to humans and/or animals. A "pharmaceutically acceptable" ingredient is one which is suitable for use in humans and/or mammals without excessive adverse side effects (such as toxicity, irritation, and allergies), i.e., materials having a reasonable benefit/risk ratio. The term "pharmaceutically acceptable carrier" refers to a carrier for the administration of a therapeutic agent, which comprises various excipients and diluents.

Pharmaceutically acceptable carriers include, but are not limited to, saline, buffer, dextrose, water, glycerol, ethanol, and combinations thereof. Usually, the pharmaceutical preparation should be matched to the mode of administration, and the pharmaceutical composition of the present invention can be prepared into an injection form, for example, by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants. The pharmaceutical composition is preferably manufactured under sterile conditions. The amount of active ingredient administered is a therapeutically effective amount. The pharmaceutical preparation of the present invention can also be formulated into a sustained release preparation.

The effective amount of the fusion protein of the present invention may vary depending on the mode of administration and the severity of the disease to be treated and the like. The selection of a preferred effective amount can be determined by one of ordinary skill in the art based on various factors (e.g., by clinical trials). The factors include, but are not limited to, the pharmacokinetic parameters of the fusion protein such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated by the patient, the patient's weight, the patient's immune status, the route of administration, etc. .

DRAWINGS

Figure 1. Nucleotide sequence and deduced amino acid sequence of FP-A fragment of SpeI-EcoRI (labeled with underlined__) in the PCDNA3 expression vector according to an embodiment of the present invention. Human FVII consists of a signal peptide (1-38, underlined __) and a mature FVII protein (39-444). Mature fusion proteins contain hFVII (39-444), flexible peptide linkers (445-471, underlined __), CTP rigid units (472-499, underlined __) and Fc variants (500-722) .

Figure 2. FP-A results by MabSelect chromatography SEC.

Figure 3. FP-A separation of the target component P3SEC by CHT chromatography.

Figure 4. SDS-PAGE electropherograms of FP-A separated components by MabSelect and fractionated P1, P2 and P3 by CHT chromatography.

Figure 5. SEC detection results of FP-A eluted fraction P3 by Q-HP chromatography.

Figure 6. FP-A was analyzed by Superdex 200 chromatography elution component P3-3SEC.

Figure 7. The SEC detection results of the P3-3 component activated for 40 h.

Figure 8. The results of SDS-PAGE electrophoresis were confirmed after activation of P3-3 components for 16-40 hours.

Figure 9. Statistical graph of mouse bleeding time results. Note: ^* p < 0.01, ^*** p < 0.001.

Figure 10. Statistical graph of mouse bleeding volume results. Note: ^** p < 0.01, ^*** p < 0.001.

Figure 11. Comparison of the duration of bleeding after administration of FP-A and Nobel for 1 h and 2 h in HemA mice. Note: compared with HA-N-1h ^{^{group, * P <0.05, *** P}} <0.01; compared with C57-NS ^{^{group, # P <0.05, ### P}} <0.01.

Figure 12. Activity half-life of FP-A and Novo in warfarin rats.

detailed description

Example 1. Construction of an expression plasmid encoding a FVII fusion protein

The gene sequence encoding the FVII leader peptide, mature protein, flexible peptide linker, CTP rigid unit and human IgG vFc variant is a manually optimized CHO cell preferred codon obtained by artificial synthesis. The full-length DNA fragment of the synthetic fusion protein has a restriction endonuclease site at the 5' and 3' ends, respectively, SpeI and EcoRI, and the full-length DNA fragment is inserted into the corresponding cleavage site of the pUC57 transfer vector, and DNA sequencing verification sequence. Then, the full-length gene fragment of the fusion protein obtained above was transferred from the intermediate vector to the SpeI (5') and EcoRI (3') sites of the expression plasmid PTY1A1 using PCDNA3.1 as a template to obtain a high fusion protein. Expression plasmid. The PTY1A1 plasmid includes, but is not limited to, the following important expression components: 1) human cytomegalovirus early promoter and mammalian cells are required for exogenous high expression; 2) dual screening markers with kanamycin resistance in bacteria Sexuality, G418 resistance in mammalian cells; 3) Murine dihydrofolate reductase (DHFR) gene expression cassette, when the host cell is DHFR gene-deficient, methotrexate (MTX) can amplify the fusion gene And the DHFR gene (see U.S. Patent 4,399,216). The fusion protein expression plasmid is then transfected into a mammalian host cell line, and in order to obtain stable high levels of expression, the preferred host cell line is a DHFR enzyme deficient CHO-cell (see U.S. Patent 4,818,679). Two days after transfection, the medium was changed to a screening medium containing 0.6 mg/mL G418, and the cells were planted in a 96-well culture plate at a concentration (5000-10000 viable cells/well) for 10-14 days until large. Discrete cell clones appear. Transfectants resistant to the selected drug were screened by ELISA assay. Subclones were generated to produce high levels of fusion protein wells by limiting dilution of 96-well plates.

As shown in the table below, the present invention constructs a series of rhFVII fusion proteins comprising peptide linkers of different lengths (Linker), CTP rigid units of different compositions, and IgG Fc variant (vFc) elements of several different subtypes. In order to verify that CTP rigid units containing at least one and different lengths can significantly increase the activity of the fusion protein, we constructed fusion proteins FP-A, FP-B, FP-C, FP-D and FP-E; The amino acid and coding nucleotides of FP-A are shown in Figure 1. In order to verify the importance of the CTP rigid unit for the activity of the fusion protein, we also constructed two Fc fusion proteins FP-G and FP-H without CTP rigid unit, and the expression plasmid construction method was the same as above. In addition, we also constructed the FP-F with CTP at the Fc C end. To verify the importance of the location of the CTP rigid unit. See Table 1 for details. The amino acid sequence of each constituent element is shown in the sequence listing.

Table 1. Composition of various FVII fusion proteins

Example 2. Transient expression of each fusion protein and activity assay

8 kinds of expression plasmids obtained in Example 1 was used DNAFect LT reagent ^TM (ATGCell Company) in 30ml shake bottle transfection 3 × 10 ⁷ CHO-K1 cells, the transfected cells were cultured at 1000ng / ml of vitamin K1 The growth was carried out for 5 days in serum-free growth medium, and the concentration of the fusion protein in the supernatant was measured, and its activity was measured by the method described in Example 7 or 8. The ELISA results showed that the transient expression levels (quantity of the substances) of the eight plasmids under these conditions were similar, but their coagulation activities showed a large difference. Among them, we defined the molar activity of FP-A as 100%. The fusion protein supernatants expressed by FP-F, FP-G and FP-H plasmids were less active, only 29.4%, 26.3% and 41.2% of FP-A. The purified proteins were analyzed by SDS electrophoresis, showing these three fusions. The protein fractions showed different degrees of polymerization; while the activities of FP-B, FP-C, FP-D and FP-E were 98.0%, 89.2%, 86.1% and 92.9%, respectively, of FP-A. It was shown that elongation of the peptide linker alone did not effectively improve the activity of the fusion protein, i.e., the steric hindrance effect of each other was not significantly attenuated with the extension of the flexible peptide linker. Even an excessively long peptide linker not only does not increase the activity of the fusion protein, but instead causes the protein to fold in error and is secreted as an inactive multimer (the product in the FP-H fermentation broth is mostly a polymer). We speculate that the reason for the long flexible peptide linker gives FVIIa a higher degree of flexibility, allowing it to rotate freely relative to Fc, which may bring the steric structure of FVIIa closer to the Fc region, and add a CTP rigid unit between the two. On the one hand, a rigid peptide linker is added to make it away from each other. More importantly, the CTP rigid unit contains multiple glycosyl side chains. The CTP rigid unit can form a fixed spatial conformation with respect to the random coil form of the flexible peptide linker. It can effectively separate the different functional regions of the fusion protein, which is more conducive to the two parts independently folded into the correct three-dimensional conformation, maintaining a high activity. We verified the validity of this speculation by comparing the activity of FP-A and FP-F, that is, the activity of FP-F in which CTP rigid unit is placed at Fc C end is only C9.4 rigid unit placed at Fc N end of FP-A 29.4 %, FP-F is similar to FP-G without CTP rigid units. The above results confirmed that the CTP rigid unit is critical for the activity of the fusion protein, and that the CTP rigid unit is placed at the N-terminus of the Fc to effectively increase the activity of the fusion protein.

Example 3. Screening for stable transfected cell lines with high expression of fusion protein

The expression plasmids of FP-A, FP-B, FP-C, FP-D and FP-E described above were transfected into a mammalian host cell line to express the FVII fusion protein. In order to maintain a stable high level of expression, a preferred host cell is a DHFR deficient CHO cell (U.S. Patent No. 4,818,679). A preferred method of transfection is electroporation, and other methods can be used, including calcium phosphate co-precipitation, lipofection, and microinjection. Electroporation method Using a Gene Pulser Electroporator (Bio-Rad Laboratories) set to 300 V voltage and 1050 μFd capacitance, 50 μg of PvuI linearized expression plasmid was added to 2 to 3 × 10 ⁷ cells in a cuvette, and electroporation was performed. The resulting cells were transferred to shake flasks containing 30 ml of growth medium. Two days after transfection, the medium was changed to a growth medium containing 0.6 mg/mL G418, and the cells were seeded in a 96-well culture plate at a concentration for 10-12 days until large discrete cell clones appeared. The anti-human IgG Fc ELISA method is used to screen the transfectants that are resistant to the selected drugs, and the anti-FVII ELISA method can also be used for the quantitative determination of the fusion protein expression, and then the high-level expression fusion is produced by sub-cloning by limiting dilution method. The pores of the protein.

In order to achieve a higher level of expression of the fusion protein, it is preferred to co-amplify with the DHFR gene which is inhibited by the MTX drug. The transfected fusion protein gene was co-amplified with the DHFR gene in growth medium containing increasing concentrations of MTX. Subclones with positive dilution DHFR expression were gradually pressurized, and transfectants capable of growing in up to 6 μM MTX medium were screened, the secretion rate was determined, and a cell line highly expressing the foreign protein was selected. A cell line having a secretion rate of more than about 3 (preferably about 5) IU/10 ⁶ (i.e., millions) of cells per 24 hours is subjected to adaptive suspension culture using serum-free medium, and then the fusion protein is purified using conditioned medium. .

Example 4. Production of fusion protein

The high-yield cell line preferably obtained in Example 3 was first subjected to serum-free domestication culture in a culture dish, and then transferred to a shake flask for suspension and domestication culture. After the cells were adapted to these culture conditions, supplemental flow culture was then carried out in a 300 ml shake flask or perfusion culture was simulated by changing the medium daily. The CHO-derived cell line producing the fusion protein FP-A obtained by the screening of Example 3 was fed and cultured for 14 days in a 300 ml volume shake flask, and the cumulative yield of the expressed recombinant fusion protein was 310 mg/L, and the viable cell density was obtained. Up to 15 × 10 ⁶ / mL. In order to obtain more fusion proteins, it can also be cultured in a 1000 ml shake flask. In another culture method, the above CHO-derived cell strain is changed daily in a 100 ml volume shake flask, and the recombinant fusion protein expressed has a cumulative yield of about 50 mg/L per day, and the viable cell density can reach up to 25 in a shake flask. ×10 ⁶ / mL. The biological activities of the recombinant fusion proteins produced by the above two methods are comparable.

Example 5. Purification and Qualitative Fusion Proteins

In this embodiment, FP-A is taken as an example to describe the purification steps and methods of several fusion proteins obtained in the above embodiments, and the FP-B, FP-C, FP-D and FP-E methods are the same, and the examples are no longer Narration.

The present invention mainly uses four-step chromatography to purify the fusion protein. Affinity chromatography, multidimensional mode chromatography, anion exchange chromatography and molecular sieve chromatography, respectively.

In the first step, affinity chromatography uses the chromatographic medium described in the Summary of the Invention for sample capture. First use balanced buffer: 20-50mM Tris-HCl, 150-500 mM NaCl, pH 6.8-7.2, equilibrate the column 3-5 column volumes (CV); after clarified fermentation broth loading, the load is not high At 30 g/L; equilibrate the column with 3-5 column volumes (CV) using a balanced buffer: 20-50 mM Tris-HCl, 150-500 mM NaCl, pH 6.8-7.2, and rinse the unbound components; Stain buffer: 20 mM Tris, 1.5-2 M NaCl, 0.5-2 M urea, 5-10 mM (EDTA) ₂ Na, pH 6.5-7.0 Wash the column 3-5 column volumes to remove some contaminants; use balanced buffer: 20-50 mM Tris-HCl, 150-500 mM NaCl, pH 6.8-7.2, equilibrated column after 3-5 column volumes (CV); using elution buffer: 100 mM Gly-HCl, 150-300 mM NaCl, pH 3. The target product was eluted 0-3.5, directly added to 1 M Tris, pH 8.0, and neutralized to pH neutral.

The SEC analysis results of the fusion protein purified by the first Mabselect affinity chromatography are shown in Fig. 2.

In the second step, multi-dimensional mode chromatography was used for sample capture using the chromatographic medium of the CHT ceramic hydroxyapatite skeleton of Bio-Rad Company described in the Summary of the Invention. First, use balanced buffer: 20-50mM PB, pH 6.8-7.2, equilibrate the column 3-5 column volumes (CV); sample captured by affinity chromatography, the loading is not higher than 10g / L; Use balanced buffer: 20-50mM PB, pH 6.8-7.2, equilibration column 3-5 Column volume (CV), rinse unbound components, named P1; rinse the column 3-5 column volumes using elution buffer 1:120 mM PB, pH 6.8-7.2, collect the eluted fractions, name P2; rinse the column 3-5 column volumes using elution buffer 2: 200 mM PB, pH 6.8-7.2, and collect the eluted fraction, designated P3. After the isolated P1, P2 and P3 components were activated, the specific activities were determined to be 103.2, 3026.7 and 5705.7 IU/mg, respectively, indicating that there were differences between the different eluting components, indicating the difference in the degree of carboxylation.

The SEC detection result of the fusion protein FP-A by separating the target component P3 by the second step CHT chromatography is shown in FIG. The SDS-PAGE electrophoresis results of the MabSelect separation component and the CHT chromatographic separation components P1, P2 and P3 are shown in Fig. 4.

In the third step, anion exchange chromatography is further separated and purified using the chromatographic medium described in the Summary of the Invention. Balanced column with 3-5 column volumes using balanced Buffer: 20-50 mM Tris-HCl, pH 7.5-8.0; loading of P3 eluting components of CHT chromatography with a loading of no more than 30 g/L; Buffer: 20-50 mM Tris-HCl, pH 7.5-8.0 equilibrium chromatography column 3-5 column volumes; elute the target component using linear gradient elution, elution buffer: 20 mM Tris-HCl, 500 mM NaCl, pH 7 .5-8.0, elution flow rate of 60 cm / h, gradient of 25-30 column volumes. The results showed that the chromatographic peak was a single peak, and the fraction we collected was more than 1/3 of the highest UV absorption. The SEC results of this part are shown in Figure 5.

In the fourth step, molecular sieve chromatography is carried out by using the chromatographic medium described in the section of the invention. First, the column is equilibrated with a balance buffer: 100 mM Hepes, 100 mM NaCl, pH 8.0, and the column volume is 1.5-2 column volumes; the sample loading is not higher than the column. 2% by volume; using a balanced buffer: 100 mM Hepes, 100 mM NaCl, pH 8.0, eluting at a flow rate of 30 cm/h, abandoning the polymer peak in the previous stage, collecting the rising, middle and descending phases of the monomer peak (by chromatography The peak of the highest UV absorption is limited to 1/2, named P3-2, P3-3, and P3-4. The SEC test results of the P3-3 component are shown in Fig. 6.

Example 6. In vitro activation of fusion proteins

The present invention activates the fusion protein by a solution incubation self-activation method. The single component P3-3 of the fusion protein obtained by molecular sieve chromatography was loaded into a 30 kDa ultrafiltration concentrating tube (Corning, Cat. No. 431489), and then centrifuged in a benchtop high-speed cryogenic centrifuge (Eppendorf, model 5810R) to change the buffer to 20 mM PB. , 0.3 M NaCl, 5 mM CaCl ₂ , pH 7.0-7.2, and concentrated to a protein concentration of 3-5 mg/ml (quantified by extinction coefficient method), activated at 4 ° C, activation time 16 to 40 hours. At the end of the activation, the G-25 desalting column was used, and it was replaced with a buffer: 20 mM PB, pH 7.0, and stored at -80 ° C for use.

The biological activity assay described in Example 7 or 8 of the present invention and the reducing SDS-PAGE were used to detect the degree of activation of the chromatographic fractions in vitro for different times. The biological activity results of different activation times are shown in Table 2; the P3-3 components were sampled during the activation process, and the results of the reduced SDS-PAGE of the activated products from 16 to 40 hours are shown in Figure 8, and the SEC detection results after 40 hours of activation are shown. Figure 7. It can be seen that P3-3 has the highest degree of activation after 40 hours of activation. SDS-PAGE gel electrophoresis showed that the P3-3 fraction obtained by the four-step chromatography in FP-A was mainly activated by two main bands, which were about 74.3KDa HC-L-CTP-Fc. (Flexible peptide linker, CTP rigid unit and IgG Fc segment portion linked to the FVIIa heavy chain) and LC of about 24.0 KDa (light chain portion of FVIIa).

Table 2, P3-3 component activation 16-40h biological activity test results

Example 7. Indirect determination of biological activity of fusion protein by chromogenic substrate method

In the present invention, the in vitro enzymatic activity of each fusion protein activated by the method shown in Example 6 was measured using a BIOPHEN FVII chromogenic kit (Ref: A221304) manufactured by HYPHEN BioMed. The kit is based on the chromogenic substrate method. FVIIa is a serine protease that acts on the exogenous coagulation pathway. When FVIIa binds to tissue factor, it activates clotting factor FX in the presence of phospholipids and Ca ²⁺ . To convert it to the active form FXa. The fusion protein to be determined first forms an enzyme complex with a tissue factor derived from rabbit thromboplastin, and then activates a certain concentration (excess) of factor FX in the reaction system to convert it into an active form of FXa, which acts on FXa. The specific chromogenic substrate SXa-11 in the reaction system cleaves the substrate and produces pNA, and the amount of pNA produced directly correlates with the activity of FXa. The concentration of FVIIa and FXa activity in the test sample was determined by measuring the concentration of pNA released at 405 nm with a colorimeter, thereby calculating the activity of FVIIa, using normal human plasma as a standard.

Example 8. Direct determination of the biological activity of a fusion protein by coagulation

Determination of the biological activity of FVIIa by coagulation is obtained by correcting the ability of FVIIa-factor-deficient plasma to cause prolonged clotting time. Using the kit produced by STAGO, France

- Deficient FVII (Cat. No. 00743). The detection method firstly mixes diluted human lyophilized plasma of known FVII activity (Unicalibrator, Cat. No. 00625) with VII matrix plasma, determines prothrombin time (PT), establishes a standard curve, and then tests The plasma was moderately diluted and mixed with spent FVII matrix plasma for PT assay. The logarithmic equation of the activity percentage C (%) and the PT time t(s) fitted by the standard curve can be used to measure the activity of the sample FVIIa, and the result is expressed as a percentage of normal plasma (%). The correspondence between the percentage activity (%) of the standard provided in the cartridge and the international enzyme activity unit IU is 100%=1 IU, and accordingly, the specific activity of the enzyme of the sample FVII to be tested can be calculated, and the unit is IU/mg.

The bioactivity assay results of the separation components in each chromatographic step for 24 h in vitro are shown in Table 3. Among them, the activity data of the target component P3-3 finally obtained by four-step chromatography activated for 16-40 hours is shown in Table 2. The purified FP-A effective single component P3-3 has a biological activity of 22470 IU/mg for 40 h, and a molar activity of about 4417 IU/nM (each fusion protein contains 2 FVIIa, equivalent to 2208.7 IU/ nM FVIIa), comparable to the activity of the marketed recombinant FVIIa, Noci (2511 IU/nM FVIIa), indicating that the fusion protein provided by the present invention has a minimal effect on the activity of FVIIa by the C-terminally fused F. Its activity is also significantly higher than that of other reported recombinant FVIIa. For example, the recombinant hFVII biological activity produced by HEK cells reported by CSL Behring Company is 2874 IU/mg, which is equivalent to 144 IU/nM. The biological activity of the fusion protein hFVII-FP produced by HEK or CHO cells is 620-770 IU/mg, which is equivalent to 69-75 IU/nM (Weimer T et al., Thromb Haemost, 2008, 99: 659-667).

Table 3. Results of biological activity determination of isolated components in vitro for 24 h in each chromatographic step

Example 9. Hemostatic effect of FP-A on acute hemorrhage in hemophilia A mice

We evaluated the FVIII factor gene knockout homozygous hemophilia A mouse (hemophilia A, HA, purchased from Shanghai Southern Model Biological Research Center) tail vein bleeding (TMT) bleeding model to evaluate the example 5 The hemostatic activity of the prepared activated fusion protein FP-A (single component P3-3) in HA mice. Male HA mice of 10-12 weeks old (purchased from Shanghai Southern Model Biological Research Center) were selected. After one week of adaptive feeding, the mice were randomly divided into 5 groups, 8 in each group. The mice were anesthetized with 0.8% pentobarbital sodium (Sigma) at a dose of 0.1 ml/10 g, and then injected intravenously with 7,000 (HA-F-0.7W group) and 21,000 (HA-F-2.1W group). , 70,000 (HA-F-7W group) and 210,000 IU/kg (HA-F-21W group) of FP-A and 100,000 IU/kg of Noki (

Novo Nordisk) (HA-N group). After 5 minutes of administration, the mouse was cut at the distal end of the mouse. The cut-off area was 3 mm in diameter, and the tail end was quickly immersed in 14 mL, 37 ° C, containing 0.1% BSA (Amresco) physiological saline (0.9% NaCl, NS). Centrifuge the tube and start timing. If bleeding stops within 30 minutes, record the bleeding time. If the bleeding time exceeds 30 minutes, the rat tail is ligated and burned with a hot metal rod to stop bleeding. The bleeding time is recorded as 1800 seconds. At the same time, calculate the amount of blood loss. Blood loss (ml) = (centrifuge tube weight after blood collection (g) - centrifuge tube weight (g) before blood collection) / 1.05. Another 10-12 weeks old, wild-type male C57BL/6J (purchased from Shanghai Southern Model Biological Research Center) mice (n=8) were given intravenous saline (purchased from Hunan Kelun Pharmaceutical Co., Ltd.) as a normal control group. (C57-NS group), the operation method is the same as the administration group. All data in mean ± standard error

T-test analysis was used for comparison between the experimental groups. The analysis software used Graphpad Prism 5.0, p<0.05 was considered statistically significant.

From the statistical analysis of the bleeding time and the amount of bleeding in each group of animals in Fig. 9 and Fig. 10, after 5 minutes of administration of 70,000 IU/kg FP-A in HA mice, the bleeding time and the amount of bleeding were close to those of the C57-NS group. The coagulation effect is obvious, indicating that FP-A can be used as an effective clotting agent for acute bleeding in clotting factors such as hemophilia; after administration of FP-A 210,000 IU/kg, mice also have C57 in bleeding time and bleeding volume. - The NS group is close. Compared with the FP-A 7,000 IU/kg group, the bleeding time of the FP-A 70,000 IU/kg group or the FP-A 210,000 IU/kg group was significantly shortened (p<0.001; p<0.001), and the amount of bleeding was also significantly reduced. (p<0.001; p<0.01) with a dose-effect relationship (see Table 4 and Table 5 for detailed results).

Table 4, bleeding time data statistics table (unit: second)

Note: Group 1 (C57-NS): C57BL/6J mice were given saline group; Group 2 (HA-N): HA mice were given 10,000 IU/kg Novo group; Group 3 (HA-F-0.7W) ): HA mice were given to the FP-A 7,000 IU/kg group; Group 4 (HA-F-2.1W): HA mice were given to the FP-A 21,000 IU/kg group; Group 5 (HA-F-7W) : HA mice were given to the FP-A 70,000 IU/kg group; Group 6 (HA-F-21W): HA mice were given to the FP-A 210,000 IU/kg group.

Table 5, blood loss data statistics table (unit: mL)

Note: Grouped as in Table 4.

Example 10. Long-term study of clotting of FP-A in hemophilia A mice

This experiment was designed to investigate the long-term effects of FP-A (single-component P3-3 activated sample) on coagulation in HA mice. One week after 16-20 weeks old male HA mice (purchased from Shanghai Southern Model Biological Research Center), they were randomly divided into 3 groups, 6 in each group. Two groups were given 300,000 IU/kg of FP-A, and the other group was given 300,000 IU/kg of Noci (

Novo Nordisk)). At the same time, wild-type male C57BL/6J mice (purchased from Shanghai Southern Model Biological Research Center) were given normal saline at 16-20 weeks old as a normal control group (n=6). The two groups of HA mice given FP-A were subjected to a tail-breaking test at 1 h and 2 h after administration; the HA mice given Novo were tail-tested 1 h after administration; C57BL/6J normal control group (C57-NS) Group) Mice were subjected to a tail-break test 2 h after injection. Refer to Example 9 for the specific operation method. All data in mean ± standard error

As shown in Figure 11, after 1 hour of administration, the bleeding time of the mice was 30 minutes, indicating that it had no procoagulant hemostasis effect (HA-N-1h group), and FP-A 1h (HA-F- After 1h group and 2h (HA-F-2h group), hemostasis was still effective, and the bleeding time was significantly shorter than that of Novo group (P<0.01; P<0.05). This indicates that FP-A has a significantly prolonged active half-life relative to Novo. See Table 6 for specific values.

Table 6. Statistics of bleeding time data (unit: s)

Example 11. In vivo activity half-life of FP-A

This experiment was to investigate the active half-life of FP-A (single component P3-3) on warfarin-induced rat coagulopathy model. Experiments were performed according to methods reported in the literature (Joe Salas et al, Thrombosis Research, 2015, 135(5): 970-976 or Gerhard Dickneite et al, Thrombosis Research, 2007, 119: 643-651), selecting 8-12 weeks old 200- Sixteen 220 g SD rats (purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd., license number: SCXK (Beijing) 2012-0001) were randomly divided into two groups of 8 rats each. Oral administration of 2.5 mg/kg warfarin (Orion Corporation, Finland, batch number: 1569755), and intravenous administration of 10,000 IU/kg of FP-A or Noci after 24 hours.

Novo Nordisk)). The FP-A group was collected at 0.05, 0.5, 1, 2, 3, 5, 8, and 12 h after administration; the Noqi group was collected at 0.05, 0.5, 1, 2, 3, and 5 h after administration. The blood sample was taken as an anticoagulant with a final concentration of 0.013 M sodium citrate, and the supernatant was taken by centrifugation at 3000 rpm for 10 min. The activity of the sample was measured according to Example 8 and the activity half-life was calculated.

As shown in Figure 12, the active half-life of FP-A was measured to be 3.03 ± 0.35 h; the active half-life of Novo was 1.01 ± 0.16 h. Compared with the isoactive Noci, the activity half-life of FP-A in rats was extended by about 3 times, and the plasma coagulation activity was about 40% after a single injection of FP-A for 3 hours, and the activity of the same active Noki group was 3 hours later. It has been reduced to 3%; plasma clotting activity remains above 7% after 12 hours of FP-A administration.

All documents mentioned in the present application are hereby incorporated by reference in their entirety in their entireties in the the the the the the the the In addition, it should be understood that various modifications and changes may be made by those skilled in the art in the form of the appended claims.

Claims

An activated human factor VII fusion protein comprising, in order from the N-terminus to the C-terminus, an activated human coagulation factor VII, a flexible peptide linker, and at least one human chorionic gonadotropin beta subunit carboxy terminal peptide rigid The unit and the extended half-life portion; wherein the extended half-life portion is selected from the group consisting of a human immunoglobulin Fc fragment and variants thereof, albumin, transferrin or PEG.
The fusion protein of claim 1 wherein the fusion protein is glycosylated.
The fusion protein according to claim 2, wherein the fusion protein is glycosylated by expression in mammalian cells.
The fusion protein according to claim 3, wherein the fusion protein is glycosylated by expression in Chinese hamster ovary cells.
The fusion protein according to claim 1, wherein said human coagulation factor VII comprises the amino acid sequence set forth in SEQ ID NO: 1, or the amino acid sequence of said human coagulation factor VII and SEQ ID NO: 1 The amino acid sequences shown have at least 90% identity.
The fusion protein according to claim 1, wherein the flexible peptide linker comprises two or more amino acids selected from the group consisting of G, S, A and T residues.
The fusion protein according to claim 6, wherein said flexible peptide linker has an amino acid sequence formula formed by combining (GS) a (GGS) b (GGGS) c (GGGGS) d cyclic units, wherein a, b , c and d are integers greater than or equal to 0, and a+b+c+d≥1.
The fusion protein of claim 7 wherein said flexible peptide linker is preferably from the group consisting of:

(i) GSGGGSGGGGSGGGGS;

(ii) GSGGGGSGGGGSGGGGSGGGGSGGGGS;

(iii) GGGGSGGGGSGGGGSGGGGS;

(iv) GSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS;

(v) GGGSGGGSGGGSGGGSGGGS.
The fusion protein according to claim 1, wherein the carboxy terminal peptide rigid unit of the human chorionic gonadotropin beta subunit comprises the amino acid sequence set forth in SEQ ID NO: 7 or a truncated sequence thereof, wherein The truncated sequence comprises at least 2 glycosylation sites.
The fusion protein according to claim 9, wherein the carboxy terminal peptide rigid unit of the human chorionic gonadotropin beta subunit comprises the following amino acid sequence:

(i) SSSSKAPPPSLPSPSRLPGPSDTPILPQ;

(ii) PRFQDSSSSKAPPPSLPSPSRLPGPSDTPILPQ;

(iii) SSSSKAPPPS;

(iv) SRLPGPSDTPILPQ.
The fusion protein according to claim 1, wherein the carboxy terminal peptide rigid unit of the human chorionic gonadotropin beta subunit has at least 70% of the rigid unit in the fusion protein of claim 9 or 10. 80%, 90% or 95% identity.
The fusion protein according to claim 1, wherein the fusion protein comprises a carboxy terminal peptide rigid unit of 1, 2, 3, 4 or 5 human chorionic gonadotropin beta subunits.
The fusion protein of claim 1, wherein the human immunoglobulin Fc variant has a reduced ADCC effect and/or a CDC effect, and/or an enhanced binding affinity to an FcRn receptor.
The fusion protein of claim 13 wherein said Fc variant is selected from the group consisting of:

(i) human IgG1 hinge region, CH2 and CH3 regions containing the Leu234Val, Leu235Ala and Pro331Ser mutations;

(ii) human IgG2 hinge region, CH2 and CH3 regions containing the Pro331Ser mutation;

(iii) human IgG2 hinge region, CH2 and CH3 regions containing the Thr250Gln and Met428Leu mutations;

(iv) human IgG2 hinge region, CH2 and CH3 regions containing the Pro331Ser, Thr250Gln and Met428Leu mutations;

(v) Human IgG4 hinge region, CH2 and CH3 regions containing the Ser228Pro and Leu235Ala mutations.
The fusion protein according to claim 1, wherein the amino acid sequence of the fusion protein is shown in SEQ ID NO: 16.
The fusion protein according to claim 1, wherein the fusion protein has an activity of > 15000 IU/mg, and/or the fusion protein has at least a 1-fold increase in circulating half-life in the animal.
A DNA molecule encoding the fusion protein of any of claims 1-16.
The DNA molecule according to claim 17, wherein the sequence of the DNA molecule is as shown in SEQ ID NO: 17.
A vector comprising the DNA molecule of claim 17 or 18.
A host cell comprising the vector of claim 19 or transfected with the vector of claim 19.
A pharmaceutical composition comprising a pharmaceutically acceptable carrier, excipient or diluent, and an effective amount of the fusion protein of any of claims 1-16.
A method of preparing a fusion protein according to any one of claims 1 to 16, the method comprising:

(a) introducing a DNA encoding a fusion protein according to claim 17 or 18 into a CHO cell to generate a CHO-derived cell line;

(b) a high-yielding cell line expressing more than 3 μg/10 6 (million) cells per 24 hours in its growth medium in the screening step (a);

(c) culturing step (b) the selected cell strain to express the fusion protein;

(d) harvesting the fermentation broth obtained in step (c), purifying the fusion protein;

(e) activating the purified fusion protein in step (d).
The method according to claim 22, wherein the fusion protein purification process in the step (d) comprises Protein A affinity chromatography, multidimensional mode chromatography, anion exchange chromatography and molecular sieve chromatography.
The method according to claim 23, wherein said Protein A affinity chromatography in said step (d) uses a Mabselect chromatography medium; and said multidimensional mode chromatography uses CHT ceramic hydroxyapatite type II chromatography a medium; and the anion exchange chromatography uses a Q Sepharose High Performance chromatography medium; and the molecular sieve chromatography uses a Superdex 200 chromatography medium.
The method according to claim 22, wherein the activating step in step (e) of the method employs factor XII activation, on-column self-activation or solution self-activation.
A fusion protein according to any one of claims 1 to 16 for use in the manufacture of a medicament for preventing or treating a bleeding disorder.
The use according to claim 26, comprising the treatment or prevention of spontaneous bleeding, intraoperative or postoperative bleeding of a patient with hemophilia A who produces a FVIII antibody or a hemophilia A patient who produces a FIX antibody, The use of a drug for the prevention or treatment of a bleeding disorder in a patient with congenital or acquired FVII deficiency, and/or treatment of a patient with platelet insufficiency.