WO2015021711A1 - Protéine de fusion améliorée de facteur de coagulation sanguine humain fvii-fc, procédé de préparation s'y rapportant et son utilisation - Google Patents

Protéine de fusion améliorée de facteur de coagulation sanguine humain fvii-fc, procédé de préparation s'y rapportant et son utilisation Download PDF

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WO2015021711A1
WO2015021711A1 PCT/CN2013/089255 CN2013089255W WO2015021711A1 WO 2015021711 A1 WO2015021711 A1 WO 2015021711A1 CN 2013089255 W CN2013089255 W CN 2013089255W WO 2015021711 A1 WO2015021711 A1 WO 2015021711A1
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fusion protein
fvii
hfvii
vfc
human
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Chinese (zh)
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李强
刘长付
冯维
武翠
孙见宇
周若芸
孙乃超
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安源生物科技(上海)有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
    • A61L31/14—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L31/16—Biologically active materials, e.g. therapeutic substances
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/54—Biologically active materials, e.g. therapeutic substances
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/04—Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424—Serine endopeptidases (3.4.21)
    • C12N9/6437—Coagulation factor VIIa (3.4.21.21)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21021—Coagulation factor VIIa (3.4.21.21)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/20—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials
    • A61L2300/252—Polypeptides, proteins, e.g. glycoproteins, lipoproteins, cytokines
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Definitions

  • the present invention relates to an improved human clotting factor FVII Fc fusion protein, a preparation method thereof and use thereof, in particular, a plurality of therapeutic and suspected blood-related diseases. Background technique
  • Coagulation is a process that gradually leads to the formation of fibrin clots, consisting of complex interactions between various blood components (or factors), usually involved in a blood component called the clotting cascade, which is the original enzyme or enzyme.
  • the original protein which is not enzymatically active, is converted to an active enzyme by the action of an activator.
  • the clotting factor FVII is one of these clotting factors.
  • FVII is a vitamin K-dependent plasma glycoprotein that is synthesized in the liver and secreted into the blood as a single-chain protease with a molecular weight of approximately 53 KDa (Broze et al, J o/ C3 ⁇ 4em, 1980, 255: 1242- 1247).
  • the FVII zymogen is hydrolyzed by a protease at a single site, Argl52-Ilel53, to produce a double-strand linked by a disulfide bond, thereby converting to its active form, FVIIa.
  • the single-stranded factor FVII can be hydrolyzed in vitro to the double-stranded factor FVIIa by coagulation factor FXa, factor FXIIa, factor FIXa, factor FVIIa or thrombin.
  • Activated FVIIa comprising NH 2 - terminal derived light chain ( ⁇ 20 KDa) and a heavy chain derived COOH- terminal connected via a single disulfide bond (Cysl35 to Cys262) ( ⁇ 30 KDa).
  • the light chain contains a cell membrane-binding Gla domain and two EGF binding domains, while the heavy chain contains a catalytic domain.
  • FVIIa acts as a serine protease and participates in the extrinsic pathway of the coagulation cascade.
  • tissue Tissue
  • TF membrane glycoprotein tissue factor
  • this combination promotes the complete conversion of FVII to FVIIa, followed by the synergistic action of calcium ions and phospholipids, converting FIX to FIXa, FX It is converted to FXa, which in turn converts prothrombin to thrombin.
  • Thrombin plays a key role in blood coagulation and wound healing.
  • hFVII human FVII
  • chromosome 13 The gene encoding human FVII (hFVII) is located at q34-qter9 on chromosome 13 and consists of 9 exons with a length of 12.8 Kb (O'Hara et al, Proc Natl Acad Sci USA, 1987, 84: 5158-5162).
  • hFVII contains four domains: the amino-terminal Y-carboxyglutamic acid (Gla) domain, two "epithelial growth factor (EGF)" domains and a carboxy-terminal serine protease domain (catalytic domain; Exon la and lb encode signal peptide sequences; exon 2 encodes the Gla domain; exon 3 encodes a short hydrophobic region; exons 4 and 5 encode a class of epidermal growth factor domain; exon 6 to 8 encodes a serine protease catalytic domain (Yoshitake et al, Biochemistry, 1985, 24: 3736-3750).
  • Ga amino-terminal Y-carboxyglutamic acid
  • EGF epidermal growth factor
  • the mature FVII protein undergoes a variety of post-translational modifications, including vitamin K-dependent carboxylation, resulting in the production of 10 gamma-carboxylated glutamic acid residues at the NH2-terminus of the molecule.
  • Other post-translational modifications include hydroxylation (Asp63), N-type glycosylation (Asnl45 and Asn322), and 0-type glycosylation (Ser52 and Ser60).
  • Hemophilia A and B are hereditary diseases caused by defects in factor FVIII and factor FIX, respectively.
  • Protein replacement therapy is a traditional treatment for hemophilia A and B, including intravenous administration of recombinant FVIII or FIX prepared from human plasma or genetically engineered.
  • the most serious medical problem is the production of an alloantibody against an alternative clotting factor, which results in a decrease in therapeutic efficacy or ineffective treatment.
  • All hemophilia A patients produce up to 30% of antibodies against clotting factor FVIII, while those that produce clotting factor FIX antibodies against hemophilia B are less likely to have more serious consequences because they are more immune to induction therapy. Not sensitive.
  • FVIIa in the treatment of hemophilia is based on the low affinity binding of FVIIa to thrombin-activated platelet surface, and by the administration of a pharmacological dose of exogenous FVIIa, thrombin production on the surface of the platelets at the site of injury is enhanced, This is independent of the presence of FVIII/FIX, which bypasses the need for coagulation factor Villa and factor IXa to achieve hemostasis. Therefore, FVIIa can be used for the treatment of hemorrhagic conditions in hemophilia patients with inhibitors. FVIIa is also used to treat patients with congenital FVII deficiency. In addition, FVIIa is increasingly being used for off-lable use, such as treatment of congenital or acquired bleeding disorders in patients with non-hemophilia, trauma or surgery-related bleeding.
  • This drug has been approved worldwide for the treatment of patients with hemophilia A or B due to the production of FVIII or FIX antibodies (inhibitors), patients with congenital FVII deficiency and termination of trauma associated with trauma and/or surgery. Event or prevent bleeding.
  • Therapeutic coagulation proteins including FVIIa
  • FVIIa are rapidly degraded by proteolytic enzymes and are easily neutralized by antibodies, which reduces their half-life and circulating time in the body, thereby limiting their efficacy.
  • the "Region Basis for Approval NovoSeven®" (FDA Reference No. 96-0597) reports that the circulating half-life of recombinant FVIIa in humans is 2.3 hours, while relatively high doses and frequent dosing achieve and maintain the desired therapeutic and prophylactic effects. It is necessary, which results in extremely high cost of treatment and inconvenience in patient treatment. To date, there has been no commercialized recombinant FVIIa with a prolonged plasma half-life.
  • glycosylation sites such as the hyperglycosylated FVIIa, T106N and V253N point mutations being developed by Bayer (see patents US20060252128, EP1549677B and US20100260741) or FVIIa being developed by CSL Behring. - Albumin fusion protein (see patent WO2007090584).
  • a long-acting factor FVII which is being developed by Prolor Biotech in the United States, has a half-life of FVII by attaching a carboxy-terminal (CTP) of hCG to the carboxyl terminus of factor FVII (see US20100317585).
  • the IgG class of immunoglobulins is the most abundant protein in human blood. Their half-life can be as high as 21 days, and the Fc fragment is the main reason for IgG to maintain a longer half-life in vivo, while also stabilizing the protein. Numerous studies have confirmed that the Fc fragment of IgG is linked to the active protein to form a fusion protein, which can increase the in vivo half-life of the active protein. This method has been applied to some clinically important cytokines (eg EPO-Fc, GCSF-Fc, IL2).
  • Fc fusion proteins have varying degrees of in vivo half-life (U.S. Patent No. 5,349,053 and 6224867).
  • the native prototypic or engineered double-headed homodimeric Fc fusion protein is linked by a cysteine residue in the IgG Fc hinge region to form an IgG molecule but no CH1 region and light chain. Due to structural homology, Fc fusion proteins exhibit in vitro pharmacokinetic properties comparable to similar subtypes of human IgG, and the currently available Fc fusion proteins are of this type.
  • the single-headed dimer Fc fusion protein developed by Biogen pou in recent years is a Fc dimer containing a fusion target protein at one end and a blank containing no target protein at the other end.
  • This method is mainly for the following two cases. : First, the recombinant expression of the target protein to be expressed is very difficult, and the correct folding and secretion efficiency of the double-headed homodimeric Fc fusion is low; Second, the target molecule is large, and the double-headed homodimeric Fc fusion may be The steric hindrance of the three-dimensional structure is generated to affect the function of the target protein.
  • the single-head dimer Fc fusion method has been applied to the blood coagulation factors FVIII, FIX and FVII, in order to achieve efficient recombinant expression in CHO cells, and has an extended in vivo half-life, wherein the single-head dimer FVIII-Fc and FIX-
  • the Fc fusion proteins have all been in the clinical research stage and are disclosed in WO2011069164A2 and the patent EP1624891B1, respectively, while the European Patent EP 1624891 B1 also discloses a fusion form of the single-headed dimer FVII and Fc.
  • the present invention relates to a hFVII-L-vFc fusion protein having a greatly extended half-life in vivo, a preparation method and use thereof.
  • the hFVII-L-vFc fusion protein of the present invention comprises, in order from the N-terminus to the C-terminus, a human FVIK flexible peptide linker comprising a plurality of amino acids and a human IgG2 Fc mutant comprising a Pro331 Ser mutation.
  • the flexible peptide linker is preferably pliable and non-immunogenic, and creates a sufficient distance between FVII and Fc to minimize potential interference of the two fusion proteins.
  • a flexible peptide linker comprising from about 6 to 21 amino acids in length comprising the following two or more amino acids is used: glycine, serine, alanine and threonine.
  • a preferred sequence as disclosed in one embodiment of the invention is GlySerGlyGlyGlySerGlyGlyGlyGlyGlySerGlyGlyGlyGlyGlySer 0
  • the amino acid sequence of the fusion protein of the present invention is shown in SEQ ID NO: 2, and the mature protein thereof is an amino acid sequence represented by SEQ ID NO: 2 after excision of the hFVII leader peptide (amino acid residues 1 to 38). It is characterized in that the human IgG2 Fc mutant contains a hinge region, a CH2 and a CH3 region. Its CH2 region contains a basal acid mutation at position 331 (position determined by the EU numbering system), thereby eliminating the effector function of Fc.
  • the present invention discloses a pharmaceutical composition
  • a pharmaceutical composition comprising a pharmaceutically acceptable carrier or adjuvant or diluent, and an effective amount of the hFVII-L-vFc fusion protein of the present invention.
  • the fusion protein of the present invention is generally applied to the prevention and treatment of hemorrhagic diseases in patients with FVII congenital or acquired deficiency and the prevention and treatment of spontaneous or surgical bleeding in patients with hemophilia A or B or other related Hemorrhagic disease.
  • a method of making or producing such a recombinant fusion protein from a cell line derived from a mammalian cell line, such as CHO is disclosed, resulting in production of the recombinant fusion protein every 24 hours in its growth medium.
  • the fusion protein with high expression yield and IgG2 Fc can be efficiently and conveniently purified by Protein A affinity chromatography.
  • the expression level of the fusion protein is high and the purification step is efficient and convenient, which can reduce the production cost.
  • 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 Fc-mediated effector function of IgG functions through two mechanisms: (1) binding to cell surface Fc receptors (FcyRs), digestion of pathogens by phagocytosis or cleavage or killer cells via antibody-dependent cellular cytotoxicity (ADCC) pathway , or (2) binds to Clq of the first complement component C1, elicits a complement-dependent cytotoxicity (CDC) pathway, thereby lysing the pathogen.
  • FcyRs cell surface Fc receptors
  • ADCC antibody-dependent cellular cytotoxicity
  • CDC complement-dependent cytotoxicity
  • IgG1 and IgG3 efficiently bind to FcyRs, and the binding affinity of IgG4 to FcyRs is low, and the binding of IgG2 to FcyRs is too low to be determined, so human IgG2 has almost no ADCC effect.
  • human IgG1 and IgG3 can also efficiently bind to Clq to activate the complement cascade.
  • Human IgG2 binds relatively weakly to Clq, whereas IgG4 does not bind to Clq (Jefferis R et al, Immunol Rev, 1998, 163: 59-76), so human IgG2 CDC effect It should also be weak.
  • the length of the linker peptide is very important for the activity of the fusion protein. It has been reported that erythropoietin (EPO) derivatives (such as dimers) exhibit attenuated fusion proteins containing two intact EPO regions (3 to 7 amino acid peptide linkers) compared to EPO monomers.
  • EPO erythropoietin
  • the length of the peptide which could be interpreted as a fusion protein between the two parts of the connecting peptide is increased, so that the two parts of the molecule to exercise its function, respectively, favors the formation of higher molar ratio of conformational activity.
  • the present inventors first designed five different lengths of glycine-containing and serine-containing flexible linker linkers to obtain a C-terminal fusion protein of hFVII, and transient expression experiments showed that the two amino acid short peptide linker GlySer-linked fusion protein also has Activity, indicating that important functional regions that maintain FVII biological activity are less affected by C-terminal sequences in three-dimensional structure.
  • the linker peptide linker was increased to 16 amino acids GtySerGlyGlyGlySerGlyGlyGlyGlyGlySerGtyGlyGlyGlySer, the FVII biological activity was fully demonstrated.
  • the inventors have also found that the peptide linker added between the hFVII and human IgG2 Fc variants enhances the in vitro biological activity of hFVII-L-Fc in two ways: (1) keeping the Fc region away from the domain on hFVII, and 2) Keep one hFVII away from the domain of another hFVII, thereby reducing the steric hindrance effect.
  • the human IgG2 Fc variant CH2 region contains a point mutation at position 331, thereby reducing the effector function of Fc. Fusion protein and preparation method thereof
  • the fusion protein gene of the present invention is codon-optimized and prepared by a synthetic method.
  • 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 artificial synthesis or traditional subcloning, etc. See J. Sambrook, Molecular Cloning Experiment Guide for specific methods.
  • the coding 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.
  • 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 the promoter controls the transcription of a sequence, then it is operably linked to the coding sequence.
  • Mammalian cell expression vectors can be used, for example, but not limited to, pcDNA3, pIRES, pDR, pBK, pSPORT, etc., which can be used for expression in eukaryotic cell systems.
  • pcDNA3, pIRES, pDR, pBK, pSPORT, etc. 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.
  • the person skilled in the art can insert 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, and obtain the present invention.
  • Recombinant expression vector According to the restriction enzyme map of the known empty-load expression vector, the person skilled in the art can insert 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, and obtain the present invention. Recombinant expression vector.
  • the invention also provides a host cell expressing a fusion protein of the invention, which comprises the coding sequence of the 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 or the like.
  • the cell is a CHO cell, which preferably expresses the fusion protein of the present invention, and obtains a fusion protein having good activity and good stability.
  • the invention also provides a method for producing a fusion protein of the invention by recombinant DNA technology, the steps comprising: 1) providing a nucleic acid sequence encoding a 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, phosphotium phosphate precipitation, lipofection, electroporation, microinjection, viral infection, alkali metal ionization.
  • the fusion protein obtained as described above can be purified to a substantially uniform property, for example, 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.
  • anion exchange chromatography or cation exchange chromatography can be a medium commonly used for protein purification such as agarose, dextran, and polyamide.
  • the Q- or SP- group is a preferred ion exchange group.
  • the purified product may be further purified by hydroxyapatite adsorption chromatography, metal chelate chromatography, hydrophobic interaction chromatography and reversed-phase high performance liquid chromatography.
  • the expressed fusion protein can also be purified using an affinity chromatography column containing a specific antibody, receptor or ligand of the fusion protein.
  • affinity column containing a specific antibody, receptor or ligand of the fusion protein.
  • the fusion polypeptide bound to the affinity column can be eluted by conventional methods such as high salt buffer, pH change, and the like.
  • the present invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising an effective amount (e.g., 0.000001-90 wt%; preferably 0.1-50 wt%; more preferably, 5-40%) of the fusion protein of the present invention, And a pharmaceutically acceptable carrier.
  • 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.
  • “Pharmaceutically acceptable” ingredients are suitable for Humans and/or mammals without excessive adverse side effects (eg, toxicity, irritation, and allergies), ie, substances with a reasonable benefit/risk ratio.
  • pharmaceutically acceptable carrier refers to administration of a therapeutic agent.
  • the carrier includes various co-agents and diluents.
  • Pharmaceutically acceptable carriers include, but are not limited to, saline, buffer, dextrose, water, glycerol, ethanol, and combinations thereof.
  • 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 as a slow-drying 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, pharmacokinetic parameters of the fusion protein such as bioavailability, metabolism, half-life, etc.; severity of the disease to be treated by the patient, patient weight, patient immune status, administration route, etc. . DRAWINGS
  • Figure 1 shows the nucleotide sequence and deduced amino acid sequence of hFVII-L-vFc of the Spel-EcoRI fragment in the PCDNA3 expression vector according to an embodiment of the present invention.
  • Human FVII consists of a signal peptide (1-38) and a mature FVII protein (39-444).
  • the mature fusion protein contains hFVII (39-444), a flexible peptide linker (445-460), and an Fc variant (461-683).
  • Figure 2 shows the gene map of the eukaryotic expression plasmid of the constructed hFVII-L-vFc fusion protein gene.
  • Expression plasmid The full-length 9647 bp contains 10 major gene fragments, including 1. hCMV promoter; 2. target gene hFVII-L-vFc; 3. EMCV IRES; 4. mDHF screening gene; 5. bGH stop sequence; 6. SV40 promoter 7. Caramycin resistance gene; 8. SV40 stop sequence; 9. ColEl replicon; 10. Ampicillin resistance gene.
  • Figure 3 shows the growth trend of the cell line in the spin flask and the concentration trend curve of the hFVII-L-vFc fusion protein.
  • Figure 4 shows the plasma half-life of hFVII-L-vFc purified protein in SD rats.
  • Example 1 Construction of an expression plasmid encoding hFVII-L-vFc fusion protein
  • the target gene sequence encoding the hFVII leader peptide and mature protein is a manually optimized CHO cell-preferred codon obtained by artificial synthesis.
  • a restriction endonuclease site at the 5' and 3' ends of the synthesized fragment, respectively Spel and BamHI.
  • a full-length 1351 b DNA fragment was inserted into the EcoRV restriction site of a transfer vector such as pUC57 to obtain an intermediate plasmid containing the hFVII gene sequence verified by DNA sequencing.
  • the fusion gene of the preferred two-amino acid flexible peptide linker GlySer and the human IgG2 vFc variant (Pro331 Ser mutation) of the present invention is also a manually optimized CHO cell-preferred codon, and the synthesized fragments 5 and 3 have their own A restriction endonuclease site, BamHI and EcoRI, respectively, and an EcoRV site was placed in the middle of the vFc.
  • a full-length 682 b DNA fragment was inserted into the above pUC57 transfer vector containing the hFVII gene using BamHI and EcoRI cleavage sites to obtain a second intermediate plasmid, and the L-vFc sequence was verified by DNA sequencing.
  • the EMCV IRES fragment obtained by artificial synthesis and the mouse dihydrofolate reductase (DHFR) gene were inserted into the EcoRI (5') and Xhol (3') sites of the mammalian cell expression vector PCDNA3 (Invitrogen).
  • the vector PCDNA3-DHFR was obtained, and the new target fragment contained therein was verified by DNA sequencing.
  • the 2069 bp DNA fragment containing the full-length hFVII-L-vFc gene obtained above was transferred from the intermediate vector to the Spel ( 5 ' ) and EcoRI ( 3 ' ) sites of PCDNA-DHFR to obtain PCDNA3- hFVII-L-
  • the vFc expression plasmid also known as PFVII-A.
  • This plasmid contains a cytomegalovirus promoter CMV capable of ensuring stable and highly efficient expression of hFVII-L-vFc.
  • the plasmid also contains a selectable marker which may have ampicillin resistance in bacteria and G418 resistance in mammalian cells.
  • the above-described engineered PCDNA3 expression vector contains the mouse DHFR gene, thereby co-amplifying the hFVII-L-vFc fusion gene and the DHFR gene in the presence of methotrexate (MTX) (USA) Patent No.
  • PFVII-A a longer flexible peptide linker sequence was generated by gene synthesis to examine the effect of peptide linker length on FVII activity. Specifically, pre-set BamHI (5') and EcoRV (3') A single locus was used to insert a flexible peptide linker of different lengths and a partial vFc gene (453 bp) to obtain the other four expression plasmids, also known as PFVII-B, PFVII-C, PFVII-D, PFVII-E, respectively.
  • the number of amino acids in the flexible peptide linker is 6 (sequence is GlySerGlyGlyGlySer ), 11 (sequence is GlySerGlyGlyGlySerGlyGlyGlyGlySly ) , 16 (sequence is GlySerGlyGlyGlySerGlyGlyGlyGlyGlyGlyGlyGlyGlyGlyGlyGlySer ) and 21 (sequence is GlySerGlyGlyGlySlyGlyGlyGlyGlyGlyGlyGlyGlyGlyGlyGlyGlyGlySly ) 0
  • the five expression plasmids obtained in Example 1 were transfected into 3X10 7 CHO-K1 cells using a DNAFect LT reagent (ATGCell) in a 30 ml shake flask, and the transfected cells were grown in serum-free cells containing 100 ng/ml of vitamin K1.
  • the medium was grown for 5 days, and the concentration of the fusion protein in the supernatant was measured by the method detailed in Example 8, and its activity was measured by the method described in Example.
  • the ELISA results showed that the FVII transient expression levels of the five plasmids under these conditions were similar, but their coagulation activities showed a large difference.
  • the FVII supernatant expressed by the PFVII-A plasmid containing the two amino acid linker had the lowest activity, and the FVII supernatant activity expressed by the PFVII-B, PFVII-C, PFVII-D and PFVII-E plasmids was 113% of the PFVII-A, respectively. , 134%, 147% and 155%, indicating that the length of the peptide linker affects FVII activity.
  • the amino acid sequence of the fusion protein obtained from the PFVII-E expression plasmid is shown in SEQ ID NO: 3.
  • Example 3 Selection of stable transfected cell lines expressing fusion proteins
  • PFVII-D (containing the sequence GlySerGlyGlyGlySerGlyGlyGlyGlyGlyGlySerGlyGlyGlyGlyGlySlyGlyGlyGlySly peptide linker) expression plasmid was transfected into a mammalian host cell line to express the hFVII-L-vFc fusion protein.
  • 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 may be used, including phosphoric acid 4 co-collapse, lipofection, and microinjection.
  • Electroporation method Using Gene Pulser Electroporator (Bio-ad Laboratories) set to 250 V and 1050
  • 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 sub-cloning by the limiting dilution method to generate high-level expression fusion.
  • the pores of the protein are 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 sub-cloning by the limiting dilution method to generate high-level expression fusion.
  • the pores of the protein are used to screen the transfectants that are resistant to the selected drugs.
  • MTX drugs to inhibit DHFR gene to achieve co-amplification of DHFR and fusion protein genes.
  • the transfected fusion protein gene was co-amplified in increasing concentrations of MTX growth medium.
  • the subclones obtained by the limiting dilution method can survive and grow slowly in up to 6 g/mL MTX medium, and finally obtain transfectants.
  • the transfectant secretion rate was measured and its cell growth characteristics were further observed.
  • a cell line with a secretion rate of more than 1 (preferably 3-4) ⁇ ⁇ /10 6 (i.e., millions) of cells/24 hours is used for suspension culture and acclimation of the next serum-free growth 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, then the fed-batch culture was performed in a 300 ml shake flask or the perfusion culture was simulated by changing the medium daily.
  • the above CHO-derived cell forest was fed and cultured for 14 days in a 100 ml volume shake flask, and the cumulative yield of recombinant fusion protein expressed was 392 mg/L (see Figure 3), and the viable cell density was up to 11 10 6 . /mL.
  • hFVII-L-vFc recombinant protein In order to obtain more hFVII-L-vFc recombinant protein, it can also be cultured in a 1000 ml shake flask.
  • 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 40-60 mg/L per day, and the viable cell density in the shake flask. Up to 25 10 6 /mL.
  • the biological activities of the assays for recombinant fusion proteins produced by the above two methods are comparable.
  • Example 5 Purification and Qualitative Fusion Proteins
  • the conditioned medium containing the fusion protein obtained in Example 4 was titrated to pH 7.0 with IN NaOH and 5 mM was added.
  • EDTA 0.1% Triton X-100
  • PBS phosphate buffer saline
  • MabSelect TM Protein A column GE Healthcare
  • the effluent fraction was discarded and the column was washed with PBS until the OD value at 280 nm was less than 0.01.
  • the fractions containing the purified protein were pooled and dialyzed against PBS. It was then filtered through a 0.22 micron nitrocellulose filter and stored immediately at -70.
  • the purified hFVII-L-vFc protein has a molecular weight ranging from 80 to 85 kDa as determined by SDS-PAGE under reducing conditions. Under non-reducing conditions, the purified protein migrates to approximately 160-170 kDa.
  • the fusion protein was quantified by BCA protein analysis using BSA as a standard.
  • Example 6 Indirect determination of biological activity of fusion protein by chromogenic substrate method
  • the present invention measures the in vitro enzymatic activity of FVII using a BIOPHEN FVII chromogenic kit (Ref: A221304) manufactured by HYPHEN BioMed.
  • the kit is based on the chromogenic substrate method.
  • FVII is a serine protease that acts on the exogenous coagulation pathway. When FVII binds to tissue factor, it activates clotting factor FX in the presence of phospholipids and Ca 2+ . , transform it into the active form FXa.
  • the FVII 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 the factor FX in the reaction system to cause it to be transferred.
  • the active form FXa, FXa acts on the specific chromogenic substrate SXa-11 in the reaction system, cleaves the substrate and produces ⁇ , and the amount of ⁇ produced directly correlates with the activity of FXa.
  • the concentration of the released pNA was measured by a colorimeter at 405 nm, and the correspondence between the FVII and FXa activities in the test sample was known, thereby calculating the activity of FVII, using normal human plasma as a standard.
  • the hFVII-L-vFc purified sample activity of the above Example 5 indirectly measured by the chromogenic method was equivalent to the direct measurement activity of the coagulation method used in Example 7, and was estimated to be about 1100 to 1300 IU/mg.
  • Example 7 Direct Determination of Biological Activity of Fusion Protein by Coagulation
  • the biological activity of FVII was obtained by correcting the ability of FVII factor-deficient plasma to cause prolonged clotting time.
  • the present invention employs a kit manufactured by STAGO, France STA® - Deficient FVII (Cat. No. 00743).
  • the detection method is to measure the prothrombin time (PT) by diluting the normal human lyophilized plasma with known FVII activity (Unicalibrator, Cat. No. 00625) as a standard and the sample to be tested.
  • the instrument is STAGO START® series blood coagulation.
  • the biological activity of recombinant hFVII produced by HEK cells reported by CSL Behring is 2874 IU/mg, which is equivalent to 144 IU/nM
  • the biological activity of recombinant hFVII fusion albumin (hFVII-FP) produced by HEK or CHO cells is 620-770 IU/mg, equivalent to 69-75 IU/nM (Weimer T et al, Thromb Haemost, 2008, 99: 659-667). Therefore, the biological activity of the hFVII-L-vFc fusion protein of the present invention is better than that of hFVII and hFVII-FP at the same molar ratio.
  • Example 8 Determination of the pharmacokinetics of fusion proteins
  • the purified plasma half-life of the hFVII-L-vFc sample in Example 5 was 18.8 ⁇ 1.5 hours, 17.2 ⁇ 0.8 hours and 16.0 ⁇ 0.5 hours, respectively.
  • the reported recombinant FVII plasma dose half-life is approximately 40-45 minutes, while the recombinant hFVII-FP plasma half-life is 4.5 hours (Weimer T et al, Thromb Haemost, 2008, 99: 659-667).
  • the recombinant hFVII-L-vFc fusion protein of the present invention has a plasma half-life of 16-18 hours, 20 times that of recombinant FVII and 4 times that of hFVII-FP, so the hFVII-L-vFc fusion protein has a greatly extended half-life in vivo.
  • Example 9 Determination of two indexes of coagulation of fusion protein

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Abstract

L'invention porte sur une protéine de fusion recombinée de facteur de coagulation sanguine humain FVII-Fc, sur un procédé de préparation s'y rapportant et sur son utilisation. La protéine de fusion comprend un FVII humain, un lieur peptidique flexible et un variant de Fc d'IgG2 séquentiellement en allant de l'extrémité N à l'extrémité C. Le variant de Fc a une activité lytique et présente des effets secondaires indésirables à médiation par Fc minimaux. La protéine de fusion a une bioactivité supérieure ou similaire à celle du FVII humain et une demi-vie plasmatique fortement prolongée, ce qui améliore de cette manière la pharmacocinétique et l'effet pharmaceutique.
PCT/CN2013/089255 2013-08-16 2013-12-12 Protéine de fusion améliorée de facteur de coagulation sanguine humain fvii-fc, procédé de préparation s'y rapportant et son utilisation WO2015021711A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105567733A (zh) * 2016-01-11 2016-05-11 佛山安普泽生物医药股份有限公司 CHO细胞表达凝血酶-Fc融合蛋白的方法
EP3595707A4 (fr) * 2017-03-14 2021-01-13 Ohio State Innovation Foundation Méthodes et compositions se rapportant à des immunoconjugués igg3 ciblant le facteur tissulaire

Families Citing this family (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103397009B (zh) * 2013-08-16 2015-06-03 安源生物科技(上海)有限公司 改良型人凝血因子FVII-Fc融合蛋白及其制备方法与用途
CN106279437B (zh) 2016-08-19 2017-10-31 安源医药科技（上海）有限公司 高糖基化人凝血因子viii融合蛋白及其制备方法与用途
CN107759694B (zh) * 2016-08-19 2023-01-13 安源医药科技（上海）有限公司 双特异性抗体及其制备方法与用途
CN106279436B (zh) * 2016-08-19 2017-10-31 安源医药科技（上海）有限公司 活化的人凝血因子vii融合蛋白及其制备方法与用途
WO2018032638A1 (fr) 2016-08-19 2018-02-22 安源医药科技（上海）有限公司 Peptide de liaison pour la construction d'une protéine de fusion
US11981718B2 (en) 2020-05-27 2024-05-14 Ampsource Biopharma Shanghai Inc. Dual-function protein for lipid and blood glucose regulation
CN116036244B (zh) * 2023-02-24 2023-09-19 北京基科晟斯医药科技有限公司 培重组人凝血因子VIII-Fc融合蛋白用于治疗含抑制物的血友病A的用途

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101743309A (zh) * 2007-04-13 2010-06-16 催化剂生物科学公司 修饰的因子ⅶ多肽及其应用
CN102083971A (zh) * 2008-04-11 2011-06-01 催化剂生物科学公司 修饰的因子ⅶ多肽及其应用
CN103025358A (zh) * 2010-06-30 2013-04-03 韩美科学株式会社 使用免疫球蛋白片段的因子viia复合物
CN103397009A (zh) * 2013-08-16 2013-11-20 安源生物科技(上海)有限公司 改良型人凝血因子FVII-Fc融合蛋白及其制备方法与用途

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1523504A2 (fr) * 2002-07-12 2005-04-20 Novo Nordisk A/S Compose de liaison au facteur tissulaire
TWI353991B (en) * 2003-05-06 2011-12-11 Syntonix Pharmaceuticals Inc Immunoglobulin chimeric monomer-dimer hybrids
CN101535339B (zh) * 2006-09-01 2014-11-12 诺沃-诺迪斯克保健股份有限公司 改性糖蛋白
EP2591099B1 (fr) * 2010-07-09 2020-11-18 Bioverativ Therapeutics Inc. Facteurs de coagulation chimériques
JP6108630B2 (ja) * 2011-10-06 2017-04-05 ハンミ サイエンス カンパニー リミテッドＨａｎｍｉ Ｓｃｉｅｎｃｅ Ｃｏ．，Ｌｔｄ． 血液凝固因子ＶＩＩ及びＶＩＩａ誘導体、それらを含む結合体及び複合体、並びにそれらの使用
CN102690354B (zh) * 2012-05-14 2015-03-25 安源生物科技(上海)有限公司 重组二聚化抗凝血酶III-Fc融合蛋白及其哺乳动物细胞高效表达系统

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101743309A (zh) * 2007-04-13 2010-06-16 催化剂生物科学公司 修饰的因子ⅶ多肽及其应用
CN102083971A (zh) * 2008-04-11 2011-06-01 催化剂生物科学公司 修饰的因子ⅶ多肽及其应用
CN103025358A (zh) * 2010-06-30 2013-04-03 韩美科学株式会社 使用免疫球蛋白片段的因子viia复合物
CN103397009A (zh) * 2013-08-16 2013-11-20 安源生物科技(上海)有限公司 改良型人凝血因子FVII-Fc融合蛋白及其制备方法与用途

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105567733A (zh) * 2016-01-11 2016-05-11 佛山安普泽生物医药股份有限公司 CHO细胞表达凝血酶-Fc融合蛋白的方法
EP3595707A4 (fr) * 2017-03-14 2021-01-13 Ohio State Innovation Foundation Méthodes et compositions se rapportant à des immunoconjugués igg3 ciblant le facteur tissulaire
US11872194B2 (en) 2017-03-14 2024-01-16 Ohio State Innovation Foundation Methods and compositions related to a tissue factor-targeting IGG3 immunoconjugates

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