WO2020063562A1 - 突变型单链人凝血因子viii融合蛋白及其制备方法与用途 - Google Patents

突变型单链人凝血因子viii融合蛋白及其制备方法与用途 Download PDF

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WO2020063562A1
WO2020063562A1 PCT/CN2019/107432 CN2019107432W WO2020063562A1 WO 2020063562 A1 WO2020063562 A1 WO 2020063562A1 CN 2019107432 W CN2019107432 W CN 2019107432W WO 2020063562 A1 WO2020063562 A1 WO 2020063562A1
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fusion protein
human
fviii
amino acid
acid sequence
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PCT/CN2019/107432
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English (en)
French (fr)
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高永娟
贾世香
郑云程
金莹莹
王著
董炤
陈思
孙乃超
李强
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安源医药科技（上海）有限公司
旭华（上海）生物研发中心有限公司
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Priority to US17/280,343 priority Critical patent/US20220033476A1/en
Priority to EP19865552.4A priority patent/EP3858865A4/en
Priority to CN201980059489.0A priority patent/CN112673026A/zh
Priority to BR112021005831-1A priority patent/BR112021005831A2/pt
Publication of WO2020063562A1 publication Critical patent/WO2020063562A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/745—Blood coagulation or fibrinolysis factors
    • C07K14/755—Factors VIII, e.g. factor VIII C (AHF), factor VIII Ag (VWF)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/36—Blood coagulation or fibrinolysis factors
    • A61K38/37—Factors VIII
    • 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
    • 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
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/59—Follicle-stimulating hormone [FSH]; Chorionic gonadotropins, e.g.hCG [human chorionic gonadotropin]; Luteinising hormone [LH]; Thyroid-stimulating hormone [TSH]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/31—Fusion polypeptide fusions, other than Fc, for prolonged plasma life, e.g. albumin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/90—Fusion polypeptide containing a motif for post-translational modification
    • C07K2319/91—Fusion polypeptide containing a motif for post-translational modification containing a motif for glycosylation

Definitions

  • the present invention relates to the field of fusion proteins, and more particularly, to a fusion protein of mutant human coagulation factor VIII (FVIII), and a preparation method and application thereof, in particular to the application of treating various coagulation-related diseases.
  • FVIII mutant human coagulation factor VIII
  • Coagulation factor VIII also known as anti-hemophilia factor
  • FVIII Coagulation factor VIII
  • a large number of studies on the molecular genetics of FVIII have shown that the absence of FVIII in the X-linked genes of the sex chromosome can lead to hemophilia type A. According to statistics, the prevalence of hemophilia A in male population is 1/5000, accounting for more than 80% of the total number of hemophilia.
  • the currently commonly used treatment for hemophilia A is replacement therapy, which supplements FVIII that is lacking in patients with hemophilia.
  • the mature FVIII consists of light and heavy chains with a molecular weight of approximately 280 kDa and has a domain of A1-A2-B-A3-C1-C2. Intracellular proteolysis of Arg-1648 residues in the B domain produces a heterogeneous heavy chain (A1-A2-B) of 90-200 kDa and a light chain of 80 kDa (domain A3-C1-C2) .
  • the heavy and light chains are combined into heterodimers through divalent metal ion-dependent links.
  • the dimers of the heavy and light chains bind von Willebrand (vWF) with high affinity to protect them from premature degradation.
  • vWF von Willebrand
  • FVIII is hydrolyzed and cleaved by activated coagulation factors FX (FXa) and thrombin FII (FIIa) at the Arg372 and Arg740 positions in the heavy chain and Arg1689 position in the light chain, resulting in the release of vWF factor and the production of activated FVIII II Polymer (FVIIIa), in the presence of Ca 2+ , it forms a tight complex with the activated coagulation factors FIX (FIXa) and FX on the surface of phospholipids. FX is then activated by FIXa, and the activated FX is dissociated from the complex.
  • FXa activated coagulation factors
  • FIIa thrombin FII
  • thrombinogen is converted to thrombin, which converts fibrinogen directly to fibrin.
  • FVIII can enhance the catalytic efficiency of FIXa to activate FX by several orders of magnitude.
  • FVIII Due to the complex structure and large molecular weight of FVIII protein, FVIII is very unstable and easily inactivated during the process of raw material plasma collection and recombinant purification. FVIII is relatively short-lived. Especially in aqueous solutions, factors such as storage temperature, inorganic salt ions, trace amounts of proteases, and other proteins involved in coagulation (especially vWF and albumin) will affect the stability of FVIII molecules. FVIII freeze-dried product must be safe and sterile during injection after reconstitution, and the infusion must be completed within a specified time, generally no more than 4 hours. On the other hand, violent shaking during lyophilization and reconstitution can also cause protein structure damage and inactivate FVIII. Therefore, the development of FVIII molecules with better stability can improve the flexibility of FVIII products in clinical applications, which is very important for greatly improving the safety of patients and the quality of life.
  • the B domain of FVIII contains 18 N glycosylation sites, has no known function in coagulation and has no homology with other proteins, and FVIII molecules with deleted B-domain still have good procoagulant activity. Eaton et al. Disclosed that a FVIII molecule with 766 amino acids (797 to 1562) deleted from the central B domain region maintained its biological activity. In addition, the expression of FVIII in mammalian cells was higher than Full-length molecules, and exhibit faster and higher activation rates than full-length molecules (Eaton et al., Biochemistry, 1986, 25: 8343-8347).
  • Afstyla is a new recombinant single-chain human FVIII product. It is currently the only single-chain coagulation factor product approved for the fusion of light and heavy chains of hemophilia A. Because of its strong affinity for vascular pseudohemophilic factor (VWF), it has higher molecular stability and longer duration of efficacy. In clinical studies, Afstyla has excellent hemostatic and bleeding prevention effects in both prophylaxis and on-demand treatment. In both regimens, the dosage of Afstyla is lower. It also has very good security. Compared with the standard care drug Octocog alfa, the protein configuration of Afstyla is more stable and the effect is relatively durable, but it still needs to be injected 2-3 times a week.
  • WO2013106789 discloses a chimeric polypeptide (FVIIIFc) comprising a FVIII portion and an Fc portion.
  • the terminal half-life of the chimeric FVIII polypeptide is twice as long as that of rFVIII, and the frequency of administration of preventive treatment is twice a week. To maintain the level of FVIII activity at 1-3%.
  • CTP is a short peptide derived from the carboxy terminus of the ⁇ -subunit of human chorionic gonadotropin (hCG) and contains multiple O-glycosylation sites. This negatively charged, highly sialylated peptide is covalently linked to the C-terminus of other proteins, which can resist the clearance of the kidney, thereby extending the half-life of the target protein linked to it in the body.
  • hCG human chorionic gonadotropin
  • the present invention creatively uses a CTP polypeptide having multiple O-glycosyl sites as part of a linking peptide for linking single-chain FVIII and Fc fragments, rather than placing it at the C-terminus as a fusion ligand, because it has
  • the natural glycosylation site can not only further extend the half-life of the fusion protein and improve bioavailability, but also cooperate with the conventional flexible GS flexible linker peptide to form a stable three-dimensional conformation, which promotes the independent folding of single-chain FVIII and Fc segments.
  • the correct three-dimensional conformation greatly reduces the steric hindrance effect of the fusion ligand Fc on the single-chain FVIII, and maintains its high biological activity.
  • the protection of the CTP glycosyl side chain can reduce the sensitivity of the linker peptide to proteases.
  • the invention provides a recombinant mutant single-chain coagulation factor VIII Fc fusion protein, which has a prolonged in vivo active half-life and a biological activity similar to that of recombinant FVIII.
  • the present invention provides a method for efficiently and stably expressing the fusion protein.
  • the fusion protein expressed by the method has high yield, good stability during preparation and storage, and its biological activity and marketed recombinant FVIII factors. Similar advantages.
  • the inventors of the present application surprisingly found that the stability of the constructed recombinant mutant single-chain FVIII fusion protein was substantially enhanced, the fusion protein could prevent protease cleavage in the cell, and the obtained fusion protein showed better after purification Stability, showing good bioavailability when applied subcutaneously.
  • the present invention relates to a recombinant mutant single-chain coagulation factor VIII fusion protein.
  • the fusion protein contains a single-chain human FVIII (scFVIII) having a deleted B-domain in sequence from the N-terminus to the C-terminus.
  • CTP rigid unit carboxy terminal peptide rigid unit of at least one human chorionic gonadotropin beta subunit (hereinafter referred to as (CTP) n , preferably, n is 1,2, 3, 4, or 5) and an extended half-life portion (eg, an immunoglobulin Fc segment, albumin, transferrin, or PEG, preferably a human IgG Fc variant (denoted as vFc)).
  • CTP rigid unit expressed as (CTP) n , preferably, n is 1,2, 3, 4, or 5
  • an extended half-life portion eg, an immunoglobulin Fc segment, albumin, transferrin, or PEG, preferably a human IgG Fc variant (denoted as vFc)
  • the fusion protein is represented as scFVIII-L-CTP-vFc.
  • the scFVIII has amino acids 765 to 1651 deleted compared to the full-length human wild-type FVIII amino acid sequence shown in SEQ ID NO: 1; specifically, the scFVIII has the amino acid sequence described in SEQ ID NO: 2 .
  • the flexible peptide linker is preferably non-immunogenic, and generates a sufficient spatial distance between scFVIII and Fc to minimize the steric effect between each other.
  • a flexible peptide linker consisting of two or more amino acid residues is used and is selected from the following amino acids: Gly (G), Ser (S), Ala (A), and Thr (T).
  • 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.
  • the peptide linker may preferably comprise a general formula for an amino acid sequence formed by a combination of (GS) a (GGS) b (GGGS) c (GGGGS) d cycle units, where a, b, c and d are greater than Or an integer equal to 0, and a + b + c + d ⁇ 1.
  • the peptide linker may preferably include the following sequence:
  • the CTP rigid unit is selected from a full-length sequence or a fragment thereof composed of amino acids 113 to 145 of the carboxy terminus of the human chorionic gonadotropin beta subunit, and specifically, the CTP rigid unit comprises, for example, SEQ ID NO: The amino acid sequence shown in 3 or its truncated sequence.
  • the random curl of the CTP rigid linker peptide with multiple glycosylation sites relative to the flexible linker peptide can form a stable three-dimensional conformation, which promotes the independent folding of scFVIII and Fc segments to form the correct three-dimensional conformation without affecting the respective organisms. active.
  • the protection of the CTP glycosyl side chain can reduce the sensitivity of the linker peptide to proteases.
  • the CTP rigid unit includes at least 2 glycosylation sites; for example, in a preferred embodiment of the present invention, the CTP rigid unit includes 2 glycosylation sites.
  • the CTP The rigid unit contains 10 amino acids at the end of SEQ ID NO: 3N, namely SSSS * KAPPPS *; or the rigid unit of CTP contains 14 amino acids at the end of SEQ ID NO: 3C, namely S * RLPGPS * DTPILPQ; for another example, another embodiment
  • the CTP rigid unit includes three glycosylation sites.
  • the CTP rigid unit includes 16 amino acids at the SEQ ID NO: 3N terminus, that is, SSSS * KAPPPS * LPSPS * R; for another example, others
  • the CTP rigid unit comprises 4 glycosylation sites.
  • the CTP rigid unit comprises 28, 29, 30, 31, 32 or 33 amino acids and starts from human chorionic gonadotropin
  • the beta subunit is at position 113, 114, 115, 116, 117, or 118 and ends at position 145.
  • the CTP rigid unit contains 28 amino acids at SEQ ID NO: 3N terminus, that is, SSSS * KAPPPS * LPSPS * RLPGPS * DTPILPQ.
  • * represents a glycosylation site.
  • Each possibility represents a separate embodiment of the invention.
  • the CTP rigid unit provided by the present invention is at least 70% homologous to the natural CTP amino acid sequence; in other embodiments, the CTP rigid unit provided by the present invention is at least 80% homologous to the natural CTP amino acid sequence; In other embodiments, the CTP rigid units provided by the present invention are at least 90% homologous to the natural CTP amino acid sequence; in other embodiments, the CTP rigid units provided by the present invention are at least 95% homologous to the natural CTP amino acid sequence.
  • the fusion protein includes one of the aforementioned CTP rigid units. In other embodiments of the present invention, the fusion protein includes two or more of the above-mentioned CTP rigid units, and preferably includes two, three, four, or five of the above-mentioned CTP rigid units.
  • the extended half-life portion is preferably an 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 At least one amino acid modification in human IgG type Fc, and the variant has reduced effector function (ADCC and / or CDC effect) and / or enhanced binding affinity to neonatal receptor FcRn.
  • human IgG and Fc variants can be selected from the group:
  • vFc ⁇ 1 human IgG1 hinge region, CH2, and CH3 regions (including the amino acid sequence shown in SEQ ID NO: 4) containing Leu234Val, Leu235Ala, and Pro331Ser mutations;
  • vFc ⁇ 2-1 human IgG2 hinge region, CH2 and CH3 regions containing Pro331Ser mutation (as shown in SEQ ID NO: 5);
  • vFc ⁇ 2-2 human IgG2 hinge region, CH2 and CH3 regions containing Thr250Gln and Met428Leu mutations (such as the amino acid sequence shown in SEQ ID NO: 6);
  • vFc ⁇ 2-3 human IgG2 hinge region, CH2 and CH3 regions (including the amino acid sequence shown in SEQ ID NO: 7) containing Pro331Ser, Thr250Gln and Met428Leu mutations;
  • (v) vFc ⁇ 4 human IgG4 hinge region, CH2 and CH3 regions containing Ser228Pro and Leu235Ala mutations (as shown in SEQ ID NO: 8).
  • 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 the combination or superposition of mutation sites of two types of functional variants between IgG isotypes,
  • the variant as described in (iv) above is a new combination variant of IgG2Fc obtained by superimposing the mutation sites in (ii) and (iii).
  • the Fc variant (vFc) in the fusion protein of the present invention contains the hinge region, CH2 and CH3 regions of human IgG such as human IgG1, IgG2 and IgG4.
  • This CH2 region contains amino acid mutations at positions 228, 234, 235, and 331 (determined by the EU counting system). These amino acid mutations are believed to reduce the effector function of Fc.
  • Human IgG2 does not bind Fc ⁇ R, but shows extremely weak complement activity.
  • the Fc ⁇ 2 variant with the Pro331Ser mutation should have lower complement activity than the native Fc ⁇ 2 and still be an Fc ⁇ R non-binding element.
  • IgG4 Fc is defective in activating the complement cascade, and its binding affinity to Fc ⁇ R is about one order of magnitude lower than IgG1.
  • Fc ⁇ 4 variants with a Leu235Ala mutation should exhibit minimal effector function.
  • Fc ⁇ 1 with Leu234Val, Leu235Ala, and Pro331Ser mutations also showed reduced effector functions compared to native Fc ⁇ 1.
  • These Fc variants are more suitable for preparing FVIII fusion proteins than natural human IgG and Fc.
  • the 250 and 428 positions 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 with each other to obtain a new combined variant, which reduces the effector function and prolongs its half-life.
  • the Fc variants of the present invention include but are not limited to mutations at the above-mentioned several sites.
  • amino acid sequence of the fusion protein is shown in SEQ ID NO: 9;
  • a DNA encoding the above-mentioned fusion protein is provided.
  • the DNA sequence of the fusion protein is shown in SEQ ID NO: 10.
  • a vector comprising the above-mentioned DNA.
  • a host cell comprising the above-mentioned vector or transfected with the above-mentioned vector.
  • the host cell is a CHO-derived cell line DXB-11.
  • a pharmaceutical composition includes a pharmaceutically acceptable carrier, excipient or diluent, and an effective amount of the above-mentioned fusion protein.
  • a method for preparing or producing the fusion protein from a mammalian cell line comprising the following steps:
  • step (d) Harvesting the fermentation broth obtained in step (c), and separating and purifying the fusion protein.
  • the CHO-derived cell line in step (a) is DXB-11.
  • the cell culture may be performed by a batch, perfusion or fed-batch culture method.
  • step (d) the fusion protein is purified by four-step chromatography, which are affinity chromatography, hydrophobic chromatography, anion exchange chromatography, and molecular sieve chromatography, respectively.
  • the present invention further gives preferred conditions in combination with Example 5.
  • the activity of the fusion protein prepared by the above method is> 6000 IU / mg.
  • fusion protein in the manufacture of a medicament for the prevention or treatment of a bleeding disease or event caused by FVIII deficiency or functional deficiency.
  • the disease includes type A (or type A) hemophilia.
  • type A or type A hemophilia.
  • the fusion protein of the present invention plays a role in controlling or preventing bleeding.
  • the mutant single-chain FVIII fusion protein constructed by the present invention has an Fc segment that is non-cleavable, that is, by mutating the complement and receptor binding domains of the Fc fragment, regulating the binding affinity of Fc to the corresponding receptor, reducing or eliminating ADCC and CDC effects, while only retaining the Fc segment to extend the half-life of the active protein in vivo, but does not produce cytotoxicity.
  • the mutant single-chain FVIII fusion protein provided by the present invention comprises a rigid CTP polypeptide having multiple glycosyl side chains, and it can form a stable three-dimensional conformation with respect to the random coil of a flexible linker peptide such as (GGGGS) n.
  • This "blocking" effect promotes the independent folding of FVIII and Fc segments to form the correct three-dimensional conformation without affecting each other's biological activity.
  • CTP contains multiple O-modified oligosaccharide groups. Negatively charged, highly sialylated CTP can resist the clearance of the kidney and further extend the half-life of the fusion protein.
  • the protective effect of the CTP glycosyl side chain can reduce the connection The sensitivity of the peptide to proteases makes it difficult for the fusion protein to be degraded in the junction region.
  • the fusion protein of the present invention has good in vitro stability regardless of fermentation, purification and storage.
  • the activity of the mutant single-chain FVIII fusion protein was significantly higher than that between Arg1648 and Glu1649 (encoded according to the full-length sequence of human wild-type FVIII SEQ ID ID: 1) at 25 ° C for 7 days.
  • Chain FVIII Fc fusion protein drug, and loss of activity is less than 20%.
  • the method for preparing the fusion protein provided by the present invention has a high yield. After 14 days of cultivation in a 300 ml shake flask, the cumulative yield can reach at least 200 mg / L, which can be scaled up to achieve large-scale industrial production.
  • CTP is a short peptide derived from the carboxy terminus of the ⁇ -subunit of human chorionic gonadotropin (hCG).
  • FSH follicle stimulating hormone
  • LH luteinizing hormone
  • TSH thyroid stimulating hormone
  • hCG chorionic gonadotropin
  • Natural CTP contains 37 amino acid residues, has 4 O-glycosylation sites, and terminates in sialic acid residues. Negatively charged, highly sialylated CTP can resist the clearance effect of the kidney, thereby prolonging the half-life of the circulation in the body (Fares, et al., Proc. Natl, Acad, Sci USA, 1992, 89 (10): 4304-4308).
  • the present invention creatively links at least one CTP polypeptide to a flexible linker peptide of appropriate length as a linker peptide for linking FVIII to an extended half-life portion (eg, an immunoglobulin Fc fragment).
  • the present invention has found that by adding a CTP peptide between FVIII and an Fc variant, it is equivalent to adding a segment of rigid linker peptide.
  • This aspect ensures that the N-terminally fused FVIII does not affect the binding site of the Fc variant and FcRn, thereby affecting the half-life; in addition, the Protein A binding site of Fc is important for the purification step in the preparation process, and CTP is connected to ensure N- The end-fused FVIII will not "cover" its binding site with protein A, so it is possible to choose a cheaper and more suitable filler to purify the fusion protein and reduce the purification cost.
  • CTP also makes the Fc fragment of about 25kDa size not interfere with the correct folding of the N-terminally fused FVIII, resulting in a decrease or loss of its biological activity / function.
  • Rigid CTP polypeptides with multiple glycosyl side chains compared to the random coils of flexible linker peptides such as (GGGGS) n, can form stable stereo conformations. This "blocking" effect promotes the independent folding of FVIII and Fc segments. Correct three-dimensional conformation without affecting each other's biological activity.
  • the protection of the CTP glycosyl side chain can reduce the sensitivity of the linker peptide to the protease, making the fusion protein difficult to be degraded in the linker region.
  • the Fc element is derived from the constant region Fc fragment of the immunoglobulin IgG, which plays an important role in eliminating the immune defense of the pathogen.
  • Fc-mediated effector function of IgG is exerted through two mechanisms: (1) binding to cell surface Fc receptors (Fc ⁇ Rs), digestion of pathogens by phagocytosis or lysis or killer cells through antibody-dependent cytotoxicity (ADCC) pathway , Or (2) Binding to C1q of the first complement component C1, triggering the complement-dependent cytotoxicity (CDC) pathway, thereby lysing the pathogen.
  • IgG1 and IgG3 can effectively bind Fc ⁇ Rs, IgG4 and Fc ⁇ Rs have low binding affinity, and IgG2 and Fc ⁇ Rs have low binding assays, so human IgG2 has almost no ADCC effect.
  • human IgG1 and IgG3 can effectively bind C1q to activate the complement cascade.
  • Human IgG2 binds to C1q relatively weakly, while IgG4 does not bind to C1q (Jefferis et al., Immunol Rev. 1998, 163: 59-76), so human IgG2 CDC effect is also weak.
  • Fc variant with enhanced affinity for neonatal receptor (FcRn) FcRn
  • the plasma half-life of IgG depends on its binding to FcRn, which generally binds at pH 6.0 and dissociates at pH 7.4 (plasma pH). Through the study of the binding sites of the two, the binding site of IgG to FcRn was modified to increase its binding capacity at pH 6.0. Mutations in some residues of the human Fcy domain that have been shown to be important for binding to FcRn can 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 trastuzumab (Herceptin, Genentech) variants with increased FcRn binding affinity, and these variants comprise a member 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.
  • Hinton et al. also found that T250Q and M428L 2 mutants increased binding to FcRn by 3 and 7 times, respectively. Mutation at two sites simultaneously increased binding 28-fold. In rhesus monkeys, M428L or T250QM / 428L mutants show a two-fold increase in plasma half-life (Paul R. Hinton et al., J Immunol, 2006, 176: 346-356). For more mutation sites contained in Fc variants with enhanced binding affinity to the neonatal receptor (FcRn), see Chinese invention patent CN201280066663.2.
  • the fusion protein gene of the present invention is prepared by an artificial synthesis method with optimized codons.
  • those skilled in the art can conveniently prepare the encoding nucleic acid of the present invention by various known methods. These methods are not limited to artificial synthesis or traditional subcloning. For specific methods, see J. Sambrook, "Molecular Cloning Experiment Guide”.
  • a nucleic acid coding sequence of the present invention is constructed by a method of synthesizing a nucleotide sequence in sections and performing subcloning.
  • the present invention also provides an expression vector for mammalian cells, comprising the sequence encoding the fusion protein of the present invention and an expression control sequence operatively linked thereto.
  • the term "operably linked” or “operably linked” refers to a condition in which certain parts of a linear DNA sequence can regulate or control the activity of other parts of the same linear DNA sequence. For example, if a promoter controls the transcription of a sequence, it is operably linked to a coding sequence.
  • telomeres For mammalian cell expression vectors, commercially available vectors such as, but not limited to, pcDNA3, pIRES, pDR, pBK, pSPORT, and the like can be used for eukaryotic cell system expression. Those skilled in the art can also select a suitable expression vector according to the host cell.
  • restriction map of a known empty-loaded expression vector those 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 splicing and splicing according to conventional methods to prepare the present invention.
  • Recombinant expression vector Recombinant expression vector.
  • the invention also provides a host cell expressing the fusion protein of the invention, which contains 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, and the like.
  • the cell is a CHO cell, which can better express the fusion protein of the present invention, and a fusion protein with good activity and stability can be obtained.
  • the present invention also provides a method for preparing the fusion protein of the present invention by using recombinant DNA technology, the steps include:
  • the coding sequence can be introduced into the host cell using a variety of known techniques in the art, such as, but not limited to, calcium phosphate precipitation, liposome transfection, electroporation, microinjection, virus infection method, and alkali metal ion method.
  • the fusion protein obtained by the above preparation can be purified to a substantially uniform nature, such as a single or specific band on SDS-PAGE electrophoresis.
  • the expression supernatant is first concentrated.
  • the concentrated solution can be further purified by gel chromatography, or purified by ion exchange chromatography. For example, anion exchange chromatography or cation exchange chromatography.
  • the gel matrix can be agarose, dextran, polyamide and other commonly used media for protein purification.
  • the Q- or SP- group is a more ideal ion-exchange group.
  • hydroxyapatite adsorption chromatography, metal chelation chromatography, hydrophobic interaction chromatography and reversed-phase high-performance liquid chromatography can be used to further refine and purify the purified product. All the above purification steps can be used in 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 characteristics of the affinity column used, conventional methods, such as high-salt buffer, pH change, and other methods can be used to elute the fusion polypeptide bound to the affinity column.
  • the invention also provides a pharmaceutical composition containing an effective amount of the fusion protein of the invention, and a pharmaceutically acceptable carrier.
  • an effective amount of the fusion protein of the present invention can be formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium, where the pH is usually about 5-8, preferably, the pH is about 6-8.
  • the term "effective amount” or “effective dose” refers to an amount that is functional or active in humans and / or animals and acceptable to humans and / or animals.
  • “Pharmaceutically acceptable” ingredients are suitable for use in humans and / or mammals without excessive adverse side effects (such as toxicity, irritation and allergies), i.e. substances with a reasonable benefit / risk ratio.
  • pharmaceutically acceptable carrier refers to a carrier for the administration of a therapeutic agent and includes a variety of adjuvants and diluents.
  • Pharmaceutically acceptable carriers include, but are not limited to: saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof.
  • the pharmaceutical preparation should match the mode of administration, and the pharmaceutical composition of the present invention can be prepared in the form of injections, for example, by using normal saline or an aqueous solution containing glucose and other adjuvants by conventional methods.
  • the pharmaceutical composition is preferably manufactured under sterile conditions.
  • the active ingredient is administered in a therapeutically effective amount.
  • the pharmaceutical preparation of the present invention can also be made into a sustained-release preparation.
  • the effective amount of the fusion protein according to the present invention may vary depending on the mode of administration and the severity of the disease to be treated.
  • the selection of a preferred effective amount can be determined by one of ordinary skill in the art based on various factors (e.g., through a clinical trial).
  • 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 weight of the patient, the immune status of the patient, the route of administration, etc. .
  • Figure 3 Effect of fusion protein SS-F8 on saphenous artery bleeding time in SD rats.
  • Figure 4 Effect of fusion protein SS-F8 on plasma APTT in SD rats.
  • Example 1 Construction of an expression plasmid encoding a mutant single-chain FVIII fusion protein
  • the gene sequences encoding FVIII leader peptide, FVIII protein with partially deleted B-domain, flexible peptide linker, CTP rigid unit and human IgG vFc variants are all artificially optimized CHO cell preference codons and obtained by artificial synthesis.
  • the 5 'and 3' ends of the full-length DNA fragment of the synthesized fusion protein each have a restriction endonuclease site, SpeI and EcoRI, respectively.
  • the full-length DNA fragment is inserted between the corresponding restriction sites of the pUC57 transfer vector. DNA sequencing verified its sequence.
  • the full-length gene fragment of the fusion protein obtained above was transferred from the intermediate vector to the corresponding restriction site of the self-made expression plasmid PXY1A1M to obtain a fusion protein high expression plasmid.
  • the PXY1A1M plasmid contains but is not limited to the following important expression components: 1) early human cytomegalovirus promoter and enhancer required for exogenous high expression in mammalian cells; 2) dual screening markers, which have kanamycin resistance in bacteria Resistance, G418 resistance in mammalian cells; 3) mouse dihydrofolate reductase (DHFR) gene expression box, when the host cell is DHFR gene-deficient, methotrexate (MTX) can co-amplify the fusion gene And the DHFR gene (see US Patent 4,399,216).
  • DHFR mouse dihydrofolate reductase
  • the fusion protein expression plasmid is then transfected into a mammalian host cell line.
  • the preferred host cell line is a DHFR enzyme-deficient CHO-cell (see US Patent No. 4,818,679).
  • the medium was changed to a screening medium containing 0.6mg / mL G418.
  • Cells were planted in a 96-well culture plate at a certain concentration (5000-10000 live cells / well) and cultured for 10-14 days until large. Discrete cell clones appeared.
  • ELISA analysis was used to screen transfectants resistant to the selected drug. By limiting dilution of a 96-well culture plate, subcloning produces wells with high levels of fusion protein.
  • the present invention constructs a series of mutant single-chain FVIII fusion proteins, which contain peptide linkers (Linker) of different lengths, CTP rigid units of different compositions, and IgG Fc variant (vFc) elements of several different subtypes to verify Effect of linker peptide and Fc variant on the activity of mutant single-chain FVIII fusion protein. See Table 1 for details. The amino acid sequence of each component is shown in the sequence listing.
  • the expression plasmid of the above-mentioned fusion protein was transfected into a mammalian host cell line to express a mutant single-chain FVIII fusion protein.
  • the preferred host cell is a DHFR-deficient CHO cell (see US Pat. No. 4,818,679).
  • One preferred transfection method is electroporation, but other methods can also be used, including calcium phosphate co-sedimentation, liposome transfection, and microinjection.
  • the electroporation method uses a Gene Pulser Electroporator (Bio-Rad Laboratories) set to a voltage of 300 V and a capacitance of 1050 ⁇ Fd.
  • PvuI linearized expression plasmid 50 ⁇ g of PvuI linearized expression plasmid is added to 2 to 3 ⁇ 10 7 cells placed in a cuvette.
  • the perforated 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 certain concentration, and cultured for 10-12 days until large discrete cell clones appeared.
  • the anti-human IgG Fc analysis method was used to screen transfectants resistant to the selected drug, and then subcloned by limiting dilution to produce wells expressing the fusion protein at high levels.
  • amplification of the DHFR gene inhibited by MTX drugs is appropriate.
  • the transfected fusion protein gene was co-amplified with the DHFR gene.
  • Subclones that were positive for DHFR expression were diluted, and the transfectants that could grow in up to 6 ⁇ M MTX medium were screened and the secretion rate was determined.
  • Cell lines with high expression of foreign proteins were selected.
  • Cell lines with a secretion rate in excess of about 1 (preferably about 3) IU / 10 6 (ie millions) cells / 24 hours are subjected to adaptive suspension culture using serum-free medium, and then fused with conditioned medium protein.
  • the high-yield cell strain preferably obtained in Example 2 was first subjected to serum-free acclimatization culture in a petri dish, and then transferred to a shake flask for suspension acclimatization culture. After the cells are adapted to these culture conditions, then fed-feed culture is performed in a 300ml shake flask or perfusion culture is simulated by changing the medium every day.
  • the CHO-derived cell strain producing the fusion protein SS-F8 obtained from the screening in Example 2 was fed and fed in a 300 ml volume shake flask for 14 days. The cumulative yield of the expressed recombinant fusion protein reached 200 mg / L, and the viable cell density It can reach up to 15 ⁇ 10 6 cells / mL.
  • 1000ml shake flask culture can also be used.
  • the above-mentioned CHO-derived cell strain is changed daily in a 100 ml volume shake flask, and the cumulative daily expression of the recombinant fusion protein expressed is about 20 mg / L.
  • the maximum density of live cells in the shake flask can reach 30. ⁇ 10 6 cells / mL.
  • the biological activity of the recombinant fusion proteins produced by the above two methods is comparable.
  • the invention mainly uses a four-step chromatography method to purify the fusion protein SS-F8.
  • Affinity chromatography, anion exchange chromatography, hydrophobic chromatography, and molecular sieve chromatography The protein purification instrument used in this example is AKTA Pure 25M from GE, USA.
  • the reagents used in this example were purchased from Sinopharm Chemical Reagent Co., Ltd., purity is analytical grade).
  • the first step is affinity chromatography: GE's VIII Select affinity chromatography medium is used for sample capture, concentration, and removal of some contaminants.
  • the clarified fermentation broth was loaded at a linear flow rate of 50-100 cm / h, and the load was not higher than 50,000 IU / ml.
  • the equilibrium buffer was used: 10 mM HEPES, 150 mM NaCl, 5 mM CaCl 2 , 0.05% Tween- 80, pH 6.8-7.2, equilibrate the chromatography column with a linear flow rate of 50-100 cm / h for 3-5 column volumes (CV), rinse unbound components; use decontamination buffer 1: 10mM HEPES, 1M NaCl, 25mM CaCl 2 , 0.05% Tween-80, pH 6.8-7.2, rinse the column with 3-5 column volumes at a linear flow rate of 50-100cm / h to remove some of the contaminants; use an equilibrium buffer: 10mM HEPES, 150mM NaCl, 25mM CaCl 2 , 0.05% Tween-80, pH 6.8-7.2, equilibrate the chromatography column with a linear flow rate of 50-100 cm / h for 3-5 column volumes (CV); then use an elution buffer: 20 mM His-HC
  • anion exchange chromatography use Q-HP from Borglon or other commercially available anion exchange chromatography media (such as Q HP from GE, Toyopearl GigaCap Q-650 from TOSOH, DEAE Beads 6FF from Tianheren) , Generik MC-Q from Saifen Technology, Fractogel EMD TMAE from Merck, and Q Ceramic HyperD from Pall) were used for intermediate purification to isolate structural variants and further remove HCP, DNA and other pollutants.
  • Q HP from GE
  • Toyopearl GigaCap Q-650 from TOSOH
  • DEAE Beads 6FF from Tianheren
  • Generik MC-Q from Saifen Technology
  • Fractogel EMD TMAE from Merck
  • Q Ceramic HyperD from Pall
  • equilibration buffer 20 mM His-HCl, 100 mM NaCl, 10 mM CaCl 2 , 0.02% Tween 80, pH 7.0-7.5, and rinse the chromatography column with a linear flow rate of 50-100 cm / h for 3-5 column volumes (CV);
  • the target protein separated by affinity chromatography in the first step is diluted 5-10 times, and the sample is loaded after reducing the concentration of organic matter, and the load is controlled at 5000-10000IU / ml.
  • Step 3 Hydrophobic Chromatography: Use Butyl HP from Borglon or other commercially available hydrophobic chromatography media (such as GE's Butyl HP, TOSOH's Toyopearl Butyl-650, Tiandiren and Butyl Beads 4FF, Saifen Technology) Generik MC30-HIC Butyl and Merck's Fractogel EMD Propyl) were used for intermediate purification to reduce the polymer content.
  • hydrophobic Chromatography media such as GE's Butyl HP, TOSOH's Toyopearl Butyl-650, Tiandiren and Butyl Beads 4FF, Saifen Technology
  • the second step of the anion exchange chromatography eluent still contains a certain proportion of polymers because of the various reasons for the formation of the polymers Including the polymerization of unchanged structure and the polymerization of changed structure, their biological activities differ greatly, so it will cause greater interference to the analysis of biological activity.
  • the target protein is polymerized, there are differences in properties between the polymer and the monomer, including charge characteristics and hydrophobicity. We use the difference in hydrophobicity to separate the two. Since the final purification step is molecular sieve chromatography, purification is performed using Butyl HP with the goal of partially removing the polymer to less than 10%.
  • Debuffer elute 3-5 column volumes (CV) at a linear flow rate not higher than 60 cm / h, collect the eluted fractions in sections, and submit them to SEC-HPLC for inspection.
  • the target components with a monomer percentage greater than 90% were combined for further chromatography.
  • the fourth step is molecular sieve chromatography: using GE's superdex 200 or other commercially available molecular sieve media (such as Chromdex 200 prep grade from Boglong) for separation, the goal is to reduce the polymer content to ⁇ 5%, and further reduce the key Contaminant content.
  • the loading amount is not higher than 3% of the column volume, and the sample is rinsed at a linear flow rate of 20 cm / h.
  • the eluted components are collected in turn, and the SEC test is combined.
  • the SEC-HPLC purity results and SDS-PAGE electrophoresis results of the samples are shown in Figures 1 and 2.
  • the SEC-HPLC results showed that the purity of the main peak of the purified fusion protein was more than 98%; the SDS-PAGE electrophoresis band pattern was in line with expectations.
  • Example 5 In vitro determination of fusion protein in vitro activity by chromogenic substrate method
  • the activity of the mutant single-chain FVIII fusion protein can be determined using a chromogenic substrate method.
  • This example was determined using the Biophen FVIII: C kit (HYPHEN BioMed, Ref. 221402).
  • the detection principle is as follows: When activated by thrombin, FVIII: C is combined with FIXa to form an enzyme complex in the presence of phospholipids and calcium ions. , Which in turn can activate factor X into its active form Xa.
  • the factor Xa formed by activation can in turn cleave its specific chromogenic substrate (SXa-11), releasing the chromophore pNA.
  • Example 6 Study on the stability of purified fusion protein
  • the mutant single-chain FVIII fusion protein was stored at 25 ° C for several days, and the effect of this condition on the activity of the fusion protein was examined.
  • the drug stability test box (purchased from Shanghai-Heng Scientific Instrument) was set at a test temperature of 25 ° C and a humidity of 75%.
  • SS-F8 and double-stranded eight-factor control drug DS-F8 (the fusion protein retains the protease cleavage site between human wild-type FVIII Arg1648 and Glu1649, and its amino acid sequence is as shown in SEQ ID NO: 11 (Indicated)
  • Each is packed in 8 pieces, each 200 ⁇ l, and stored in a drug stability test box. Take one SS-F8 and DS-F8 aliquots each, and measure the activity of the fusion protein according to the method described in Example 5 above, and record it as the activity value on the first day (d1).
  • SS-F8 only decreased protein activity by about 10% after 5 days at 25 ° C, and the protein activity of DS-F8 decreased by more than 25%.
  • the rate of DS-F8 activity decreased after 7 days at room temperature. It is more significant than SS-F8, and SS-F8 still retains more than 80% of protein activity after 7 days of standing, which shows that the stability of SS-F8 fusion protein is significantly better than DS-F8.
  • Blood coagulation is actually an enzymatic reaction of a series of coagulation factors.
  • the whole coagulation process is divided into three stages: the first stage is blood thrombin formation; the second stage is thrombin formation; the third stage is fibrin formation, of which FVIII, FIX It is an endogenous coagulation factor, and FVII is an exogenous coagulation factor.
  • Bleeding time is the time required to bleed naturally to stop bleeding after the skin capillary is punctured.
  • the procoagulant effect of the fusion protein is detected by observing the effect of the mutant single-chain FVIII fusion protein on the bleeding time of the saphenous artery in SD rats.
  • mice Seven-week-old SD rats (purchased from Shanghai Slark Experimental Animal Co., Ltd.) were randomly divided into two groups.
  • SS-F8 was administered intravenously in a single dose of 200 IU / head
  • the control group was administered the same volume of physiological saline.
  • the rats were induced with anesthesia. After sterilizing the surgical site, cut the skin from the knee to the arteries at the base of the leg about 1 cm from the inner ankle, cut off the subcutaneous tissue and the protective film on the blood vessel, and sequentially expose the venous blood vessels, arterial blood vessels and nerves.
  • Example 8 Direct determination of biological activity of fusion protein by coagulation method
  • Seven-week-old SD rats (purchased from Shanghai Slark Experimental Animal Co., Ltd.) were randomly divided into 12 groups. After inducing anesthesia in rats, under continuous anesthesia, cut with a scalpel along the midline of the abdomen, and take 10-12 ml of blood from the abdominal aorta. Take the above blood sample, centrifuge at 1500 rpm for 30 min at 20 ° C, separate the supernatant plasma, and add each into labeled 1.5 ml centrifuge tubes.
  • the whole plasma and the test drugs SS-F8 and DS-F8 were respectively prepared into test samples with a concentration of 25-1000 IU / ml at a volume ratio of 6: 1, and the control group was added with a dilution solution at a volume ratio of 6: 1.
  • the experimental data is expressed as mean ⁇ standard deviation (Means ⁇ SD).
  • SPSS is used 18.0 software, one-way analysis of variance or student's-test; if it is not normally distributed, use the nonparametric test Kruskal-wallis test or Mann-whitney test. P ⁇ 0.05 has a significant difference, and P ⁇ 0.01 has a very significant difference.
  • test drugs SS-F8 and DS-F8 have anticoagulant effects on the plasma of normal rats.
  • the APTT of the test drugs DS-F8 and SS-F8 Compared with the solvent control group, the time decreased by 33.65% and 31.86% respectively; the EC 50 values of DS-F8 and SS-F8 were 139.4IU / ml and 115.6IU / ml, respectively, and the EC 50 values of the test drug SS-F8 were low.
  • DS-F8 lower doses can be foreseen.
  • the time of APTT is significantly shortened, and there is a certain dose-effect relationship.
  • mice The acute hemostatic effect of SS-F8 in HA mice was evaluated using the Tail Clipping Bleeding Model of the VIII gene knockout homozygous hemophilia A (HA) mice.
  • HA mice 6-7 week old male HA mice (from Shanghai N forcing Model Biotechnology Co., Ltd.) were selected and randomly divided into 2 groups after one week of adaptive breeding, which were the HA mouse blank vehicle control group and SS-F8 G.
  • Male C57BL / 6 mice purchased from Shanghai Slark Experimental Animal Co., Ltd.
  • Anesthetized mice were intraperitoneally injected with 1% sodium pentobarbital (Merck) at a dose of 7.5 ml / kg, and administered by tail vein injection.
  • 1% sodium pentobarbital Merck
  • mice normal control group and HA mice blank vehicle control group were intravenously administered 10 ml / kg of vehicle, and the administration group was given 112 IU / kg of SS-F8 intravenously.
  • the tail was cut off at a distance of 15 mm from the end of the tail with a surgical blade, and the wound was quickly immersed in 13 mL of physiological saline preheated at 37 ° C. The timing and blood collection were started, and the total bleeding time and bleeding within 30 min after the tail was recorded. the amount.
  • Blood loss (ml) (weight of centrifuge tube after blood collection (g)-weight of centrifuge tube before blood collection (g)) / 1.0.
  • the comparison between the experimental groups was analyzed by T-test test, and the analysis software was Graphpad Prism 8.0, p ⁇ 0.05 was considered statistically significant. See Table 2 for detailed results.
  • the median bleeding volume was 438.9 ⁇ l and 790.00 ⁇ l (p ⁇ 0.0001), and the median bleeding time was The numbers are 739s and 1800s (p ⁇ 0.0001), and the procoagulant effect is obvious, indicating that SS-F8 can be used as an effective coagulant for hemorrhage caused by hemophilia A factor VIII deficiency.

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Abstract

本发明公开了一种突变型重组单链人凝血因子VIII(FVIII)融合蛋白、其制备方法及用途。该融合蛋白从N端到C端依次包含缺失部分B-结构域的突变型单链人FVIII、柔性肽接头、至少1个人绒毛膜促性腺激素β亚基羧基末端肽刚性单元和延长半衰期部分(优选人IgG Fc变体)。该融合蛋白具有与重组FVIII类似的生物学活性及延长的体内活性半衰期，以及更好的体外和体内稳定性，从而改善融合蛋白的药代动力学和药效。

Description

突变型单链人凝血因子VIII融合蛋白及其制备方法与用途 技术领域
本发明涉及融合蛋白领域，更具体地，涉及一种突变型人凝血因子VIII(FVIII)的融合蛋白及其制备方法和用途，特别是治疗多种凝血相关疾病的用途。
背景技术
凝血因子VIII(FVIII)，又称抗血友病因子，在内源性凝血体系中具有十分重要的作用。FVIII分子遗传学的大量研究结果表明性染色体X-连锁基因中缺乏FVIII会导致A型血友病。据统计，血友病A在男性人群中患病率为1/5000，占血友病总数的80％以上。血友病A目前常用的治疗方法为替代治疗，即补充血友病患者所缺乏的FVIII。
成熟的FVIII由大约分子量为280kDa的轻链和重链组成，具有A1-A2-B-A3-C1-C2的结构域。在细胞内，对B结构域中Arg-1648残基进行的蛋白酶解产生90-200kDa的大小不均一的重链(A1-A2-B)和80kDa的轻链(结构域A3-C1-C2)。重链和轻链通过二价金属离子依赖性的链接结合为异源二聚体。在血浆中，重链和轻链的二聚体再以高亲和力结合冯.维勒布兰德因子(von Willebrand，vWF)，保护其免于成熟前降解。血浆中非活化FVIII结合vWF的半衰期约为12小时。FVIII在重链内的Arg372和Arg740位置，和在轻链内的Arg1689位置被活化的凝血因子FX(FXa)及凝血酶FII(FIIa)水解切割而活化，导致vWF因子释放并产生活化的FVIII二聚体(FVIIIa)，在Ca 2+存在下，它与磷脂表面上活化的凝血因子FIX(FIXa)和FX形成紧密复合物，FX接着被FIXa激活，活化的FX从复合物中解离出来，转而将凝血酶原转化为凝血酶，后者能将纤维蛋白原直接转化成纤维蛋白。作为一种凝血系统的辅助因子，FVIII能使FIXa对FX的活化催化效率增强几个数量级。
由于FVIII蛋白质结构复杂，分子量大，致使FVIII很不稳定，在原料血浆收集及重组纯化制备等过程中容易失活。FVIII的有效期相对短暂，特别在水溶液中，保存温度、无机盐离子、微量蛋白酶以及参与凝血的其他蛋白质(特别是vWF和白蛋白)等因素都会对FVIII分子的稳定性产生影响。FVIII冻干品复溶后注射给药的过程中，必须确保安全、无菌，且要在规定时间内输液完毕，一般不超过4h。另一方面，冻干复溶过程中的剧烈振荡也会导致蛋白结构受损从而使FVIII失活。因此，研发具有更好稳定性的FVIII分子，能够提高FVIII制品在临床应用中的灵活性，对大幅提高患者的用药安全和生活质量而言至关重要。
FVIII的B结构域含有18个N糖基化位点，在凝血中没有已知功能且与其他蛋白质没有同源性，B-结构域缺失的FVIII分子仍具有良好的促凝血活性。Eaton等公开了一种从中心B结构域区域缺失766个氨基酸(797到1562)的FVIII分子保持了其生物活性，此外，缺失B结构域区域的FVIII在哺乳动物细胞中的表达量是高于全长分子的，并且表现出比全长分子更快和更高的激活率(Eaton等，Biochemistry,1986,25:8343-8347)。其它研究也表明，大部分B结构域缺失(Ser743到Gln1638)对FVIII的功能活性没有影响，且显著降低了分子量(减少38％)，增加了真核细胞中的表达水平(Peters R T等,J Thromb Haemost,2013,11(1):132-141；Sandberg H等,Semin Hematol,2001,38:4-12)。
Afstyla是一种新型重组单链人FVIII产品，是目前唯一获批治疗A型血友病的轻重链融合在一起的单链凝血因子产品。由于其对血管假性血友病因子(VWF)具有极强的亲和性，因而具有更高的分子稳定性和更长的疗效持续时间。在临床研究中，Afstyla在预防性治疗(prophylaxis)和按需治疗(on-demand treatment)均表现出优秀的止血和预防出血功效，在2种给药方案中，Afstyla的用药剂量均较低，同时也具有非常好的安全性。相较于标准护理药物Octocog alfa，Afstyla的蛋白构型更为稳定，疗效也相对持久，但仍需每周注射2-3次。
为了延长FVIII的体内功能半衰期，现有技术将FVIII与PEG、人血清白蛋白(HSA)、转铁蛋白或IgG Fc等延长半衰期部分连接。目前Novo Nordisk(N8-GP)，Bayer(BAY94-9027)和Baxter(BAX 855)公司均开发了PEG化的长效FVIII产品，并已进入临床研究，然而，在该蛋白制备工艺中增加了PEG与FVIII化学缀合的额外步骤，降低了最终产率、加大了制备成本。另一方面，药代动力学研究数据显示，PEG化的FVIII并未获得显著延长的半衰期(Tiede A等,J Thromb Haemost,2013,11:670-678；Turecek PL等,Hamostaseologie,2012,32Suppl1:S29-38)。WO2013106789公开了一种包含FVIII部分和Fc部分的嵌合多肽(FVIIIFc)，所述嵌合FVIII多肽的终末半衰期相较于rFVIII延长了两倍，预防性治疗的给药频次为每周两次，以维持FVIII活性的水平在1-3％。
CTP是一段来自人绒毛膜促性腺激素(hCG)的β-亚基羧基末端的短肽，含有多个O-糖基化位点。这种带负电、高度唾液酸化的的肽段与其它蛋白的C末端共价连接，能够抵抗肾脏对其的清除作用，从而延长与之连接的目标蛋白在体内的半衰期。一些专利文献公开CTP可以作为融合蛋白中包含的延长半衰期部分。本发明创造性地将具有多个O-糖基位点的CTP多肽作为连接肽的一部分，用于连接单链FVIII和Fc片段，而不是放在C末端作为融合配体发挥作用，因它具有的天然糖基化位点，不仅能使融合蛋白的半衰期进一步延长，生物利用度 提高，同时与常规的柔性GS柔性连接肽相互配合，形成稳定的立体构象，促使单链FVIII和Fc段独立折叠形成正确三维构象，大大降低了融合配体Fc对单链FVIII的位阻效应，使其保持了较高的生物学活性。另外，CTP糖基侧链的保护作用可以降低连接肽对蛋白酶的敏感性。
综上，虽然目前有许多研究提供了有效的重组FVIII蛋白制备方案，然而相比全长FVIII而言，FVIII衍生物的异源数目、结构和凝血酶活化的不稳定仍然是一个严峻的问题。此外，由于许多B-结构域缺失的FVIII作为融合蛋白表达，非天然的氨基酸序列给药后可能出现新的免疫原性。FVIII分子的稳定性对于储存和临床应用而言也至关重要。在这种情况下，希望开发活化、稳定、安全的FVIII衍生物，能够具有同样的凝血酶活性、提高的产量和更长的半衰期，可以通过降低注射频率来维持预防性治疗，有助于改善治疗效果。
发明内容
本发明提供一种重组突变型单链凝血因子VIII的Fc融合蛋白，具有延长的体内活性半衰期且与重组FVIII相似的生物学活性。此外，本发明提供了一种高效、稳定表达所述融合蛋白的方法，该方法表达的融合蛋白具有产量高、在制备和存储过程中稳定性好，并且其生物活性和已上市的重组FVIII因子相似的优点。本申请的发明人令人惊讶的发现构建的重组突变型单链FVIII融合蛋白的稳定性实质地增强，融合蛋白在细胞内可以阻止蛋白酶剪切，得到的融合蛋白在纯化后显示出更好的稳定性，皮下施用时显示出良好的生物利用率。
本发明的第一方面，本发明涉及一种重组突变型单链凝血因子VIII融合蛋白，所述融合蛋白从N端至C端依次含有缺失部分B-结构域的单链人FVIII(scFVIII)、柔性肽接头(Linker，L)、至少一个人绒毛膜促性腺激素β亚基的羧基末端肽刚性单元(以下简称CTP刚性单元，表示为(CTP) n，较优地，n为1,2,3,4,或5)和延长半衰期部分(如，免疫球蛋白Fc段、白蛋白、转铁蛋白或PEG，优选人IgG Fc变体(表示为vFc))。本发明的一些优选实施例中，所述融合蛋白表示为scFVIII-L-CTP-vFc。
其中，所述scFVIII其和SEQ ID NO：1所示全长人野生型FVIII氨基酸序列相比缺失了765至1651位氨基酸；具体地，所述scFVIII具有如SEQ ID NO：2所述的氨基酸序列。
其中，所述柔性肽接头优选非免疫原性的，并且在scFVIII和Fc之间产生足够的空间距离，使相互之间的位阻效应降至最低。较佳地，使用含有2个或更多个氨基酸残基组成的柔性肽接头，且选自下列几种氨基酸：Gly(G)、Ser(S)、Ala(A)和Thr(T)。
更优选地，所述柔性肽接头包含G和S残基。连接肽的长度对融合蛋白的活性非常重要。 对本发明而言，所述肽接头可优选地包含以(GS) a(GGS) b(GGGS) c(GGGGS) d循环单元组合形成的氨基酸序列通式，其中a，b，c和d是大于或等于0的整数，且a+b+c+d≥1。
具体地，本发明的实施例中，所述肽接头可优选地包含如下序列：
(i)L1：GSGGGSGGGGSGGGGS；
(ii)L2：GSGGGGSGGGGSGGGGSGGGGSGGGGS；
(iii)L3：GGGGSGGGGSGGGGSGGGGS；
(iv)L4：GSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS；
(v)L5：GGGSGGGSGGGSGGGSGGGS。
其中，所述CTP刚性单元选自人绒毛膜促性腺激素β亚基羧基末端第113至145位氨基酸所组成的全长序列或其片段，具体地，所述CTP刚性单元包含如SEQ ID NO：3所示氨基酸序列或其截短的序列。首先，这种人体内天然存在的含有多个糖基化位点的CTP多肽是非免疫原性的。其次，含有多个糖基化位点的CTP刚性连接肽相对于柔性连接肽的无规则卷曲，它可以形成稳定的立体构象，促使scFVIII和Fc段独立折叠形成正确三维构象而互不影响各自生物活性。另外，CTP糖基侧链的保护作用可以降低连接肽对蛋白酶的敏感性。
优选地，所述CTP刚性单元包含至少2个糖基化位点；例如，本发明的一优选实施例中，所述CTP刚性单元包含2个糖基化位点，示例性地，所述CTP刚性单元包含SEQ ID NO：3N端的10个氨基酸，即SSSS*KAPPPS*；或所述CTP刚性单元包含SEQ ID NO：3C端的14个氨基酸，即S*RLPGPS*DTPILPQ；又如，另一实施例中，所述CTP刚性单元包含3个糖基化位点，示例性地，所述CTP刚性单元包含SEQ ID NO：3N端的16个氨基酸，即SSSS*KAPPPS*LPSPS*R；再如，另一些实施例中，所述CTP刚性单元包含4个糖基化位点，示例性地，所述CTP刚性单元包含28、29、30、31、32或33个氨基酸并开始于人绒毛膜促性腺激素β亚基的第113、114、115、116、117或118位，终止于第145位。具体地，所述CTP刚性单元包含SEQ ID NO：3N端的28个氨基酸，即SSSS*KAPPPS*LPSPS*RLPGPS*DTPILPQ。在本文中，*代表糖基化位点。每种可能性都代表本发明的独立实施方式。
在另一些实施例中，本发明提供的CTP刚性单元与天然CTP氨基酸序列至少70％同源；在另一些实施例中，本发明提供的CTP刚性单元与天然CTP氨基酸序列至少80％同源；在另一些实施例中，本发明提供的CTP刚性单元与天然CTP氨基酸序列至少90％同源；在另一些实施例中，本发明提供的CTP刚性单元与天然CTP氨基酸序列至少95％同源。
本发明具体实施例中所述CTP刚性单元可优选地包含如下序列：
(i)CTP 1：PRFQDSSSSKAPPPSLPSPSRLPGPSDTPILPQ；
(ii)CTP 2：SSSSKAPPPSLPSPSRLPGPSDTPILPQ；
(iii)CTP 3：SSSSKAPPPS；
(iv)CTP 4：SRLPGPSDTPILPQ。
本发明一些实施例中，所述融合蛋白包含1个上述CTP刚性单元。本发明另一些实施例中，所述融合蛋白包含2个或2个以上的上述CTP刚性单元，优选地，包含2，3，4或5个上述CTP刚性单元。
其中，延长半衰期部分优选自免疫球蛋白IgG、IgM、IgA Fc片段；更优选自人IgG1、IgG2、IgG3或IgG4及其变体的Fc片段；进一步地，所述人IgG Fc变体包含位于野生型人IgG Fc中的至少一种氨基酸修饰，且变体具有降低的效应子功能(ADCC和/或CDC效应)和/或与新生儿受体FcRn的结合亲和力增强。进一步地，人IgG Fc变体可选自下组：
(i)vFcγ 1：含有Leu234Val、Leu235Ala和Pro331Ser突变的人IgG1绞链区、CH2和CH3区域(如SEQ ID NO：4所示氨基酸序列)；
(ii)vFcγ 2-1：含有Pro331Ser突变的人IgG2绞链区、CH2和CH3区域(如SEQ ID NO：5所示氨基酸序列)；
(iii)vFcγ 2-2：含有Thr250Gln和Met428Leu突变的人IgG2绞链区、CH2和CH3区域(如SEQ ID NO：6所示氨基酸序列)；
(iv)vFcγ 2-3：含有Pro331Ser、Thr250Gln和Met428Leu突变的人IgG2绞链区、CH2和CH3区域(如SEQ ID NO：7所示氨基酸序列)；
(v)vFcγ 4：含有Ser228Pro和Leu235Ala突变的人IgG4绞链区、CH2和CH3区域(如SEQ ID NO：8所示氨基酸序列)。
本发明所提供的IgG Fc变体包含但不限于(i)～(v)中所述5种变体，还可以是IgG同种亚型间两类功能变体突变位点的组合或叠加，如上述(iv)中所述变体即是由(ii)和(iii)中的突变位点相叠加所获得的新的IgG2Fc的组合变体。
本发明所述融合蛋白中的Fc变体(vFc)，它含有人IgG如人IgG1、IgG2和IgG4的绞链区、CH2和CH3区域。这种CH2区域在228、234、235和331位(由EU计数系统确定)含有氨基酸突变。据信这些氨基酸突变能降低Fc的效应子功能。人IgG2不结合FcγR，但显示出极弱的补体活性。具有Pro331Ser突变的Fcγ2变体应比天然Fcγ2的补体活性更低，而且依 旧是FcγR非结合子。IgG4 Fc在激活补体级联中有缺陷，且它与FcγR的结合亲和力比IgG1低约一个数量级。与天然Fcγ4相比，具有Leu235Ala突变的Fcγ4变体应表现出最小的效应子功能。具有Leu234Val、Leu235Ala和Pro331Ser突变的Fcγ1也表现出比天然Fcγ1降低的效应子功能。这些Fc变体都比天然人IgG Fc更适于制备FVIII融合蛋白。而250和428位(由EU编号体系确定的位置)含有氨基酸突变，使得Fc区与新生儿受体FcRn的结合亲和力增加，从而进一步延长半衰期(Paul R等,J Biol Chem,2004,279:6213–6216)；上述两类功能变体的相互组合或叠加，获得新的组合变体，使其效应子功能降低的同时且延长了其半衰期。本发明所述Fc变体包含却不局限于上述几个位点的突变，也可引入其它位点的替换使得Fc具有降低的效应子功能和/或与FcRn受体的结合力增强，同时还不会致使Fc变体功能/活性降低或引起不良的构象变化，常见的突变位点可以参见Shields RL等,J Biol Chem,2001,276(9):6591-604。
本发明的一优选实施例中，所述融合蛋白的氨基酸序列如SEQ ID NO：9所示；
根据本发明的另一个方面，提供一种编码上述融合蛋白的DNA。
本发明的一优选实施例中，所述融合蛋白的DNA序列如SEQ ID NO：10所示。
根据本发明的再一个方面，提供一种载体，该载体包含上述DNA。
根据本发明的再一个方面，提供一种宿主细胞，该宿主细胞包含上述载体，或者转染了上述的载体。
在本发明的具体实施方式中，宿主细胞是CHO的衍生细胞株DXB-11。
根据本发明的第五方面，提供一种药物组合物。该药物组合物包括药学上可接受的载体、赋形剂或稀释剂，以及有效量的上述融合蛋白。
根据本发明的另一方面提供了一种从哺乳动物细胞系如CHO衍生的细胞系制备或生产所述融合蛋白的方法，包括以下步骤：
(a)将编码所述融合蛋白的DNA引入CHO细胞，生成CHO衍生的细胞系；
(b)筛选步骤(a)中在其生长培养基中每24小时期间内，表达超过1IU/10 6个细胞的高产量细胞株；
(c)培养步骤(b)筛选到的细胞株，表达融合蛋白；
(d)收获步骤(c)得到的发酵液，并分离纯化融合蛋白。
进一步地，所述步骤(a)中CHO衍生细胞系为DXB-11。
进一步地，所述步骤(c)中，细胞培养可选用分批、灌流或流加培养方法。
进一步地，所述步骤(d)中采用四步层析法对融合蛋白进行纯化，分别为亲和层析、疏 水层析、阴离子交换层析和分子筛层析。本发明结合实施例5进一步给出其优选条件。
本发明优选实施例中，采用上述方法制备得到的融合蛋白的活性>6000IU/mg。
根据本发明的第六方面，提供所述融合蛋白在制备用于预防或治疗因FVIII缺乏或功能缺陷导致的出血性疾病或事件的药物中的应用。
进一步地，所述疾病包括甲型(或称A型)血友病。在甲型血友病患者的自发出血事件、手术预防、围手术期处理或手术治疗中，本发明所述融合蛋白起到控制或预防出血发生的作用。
本发明所公开和/或所记载的融合蛋白及其制备方法的优点可以概括如下：
1、本发明构建的突变型单链FVIII融合蛋白，其Fc段是非裂解性的，即通过对Fc片段的补体、受体结合域进行突变，调节Fc与相应受体的结合亲和力，降低或消除ADCC和CDC效应，而只保留Fc段延长活性蛋白体内半衰期的作用，却不产生细胞毒性。
2、本发明提供的突变型单链FVIII融合蛋白包含具有多个糖基侧链的刚性CTP多肽，相对于(GGGGS)n这类柔性连接肽的无规则卷曲，它可以形成稳定的立体构象，这种“阻隔”作用促使FVIII和Fc段独立折叠形成正确的三维构象而互不影响各自的生物活性。CTP含有多个O型修饰的寡糖基，带负电、高度唾液酸化的CTP能够抵抗肾脏对其清除作用，进一步延长融合蛋白的半衰期；再一方面，CTP糖基侧链的保护作用可以降低连接肽对蛋白酶的敏感性，使融合蛋白不易在连接区被降解。
3、本发明所述融合蛋白无论在发酵、纯化过程以及储存过程中均具有良好的体外稳定性。25度室温储存7日，突变型单链FVIII融合蛋白的活性显著高于Arg1648和Glu1649间(根据人野生型FVIII全长序列SEQ ID NO：1编码)蛋白酶切位点未被失活的重组双链FVIII Fc融合蛋白药物，且活性丧失小于20％。
4、本发明提供的所述融合蛋白的制备方法，产量较高，在300ml摇瓶中培养14天，累积产量至少可达到200mg/L，可进行工艺放大，实现大规模工业化生产。
发明详述：
hCG-β羧基末端肽(CTP)
CTP是一段来自人绒毛膜促性腺激素(hCG)的β-亚基羧基末端的短肽。四种与生殖相关的多肽类激素促卵泡激素(FSH)、黄体生成素(LH)、促甲状腺素(TSH)和绒毛膜促性腺激素(hCG)含有相同的α-亚基和各自特异的β-亚基。与其它三种激素相比，hCG体内半衰期明显延长，这主要来源于其β-亚基上特有的羧基末端肽(CTP)(Fares FA等,Proc Natl Acad Sci  USA,1992,89(10):4304-4308)。天然CTP含有37个氨基酸残基，具有4个O-糖基化位点，终端是唾液酸残基。带负电、高度唾液酸化的CTP能够抵抗肾脏对其的清除作用，从而延长体内循环半衰期(Fares F A等,Proc Natl Acad Sci USA,1992,89(10):4304-4308)。然而，本发明创造性地将至少一个CTP多肽与适当长度的柔性连接肽连接，共同作为连接肽，用于连接FVIII与延长半衰期部分(如，免疫球蛋白Fc片段)。
本发明发现，通过在FVIII与Fc变体间增加CTP肽，相当于增加了一段刚性连接肽。这一方面保证了N-端融合的FVIII不会影响Fc变体与FcRn的结合位点，从而影响半衰期；另外Fc的Protein A结合位点对于制备工艺中纯化步骤很重要，连接CTP保证N-端融合的FVIII也不会“罩住”它与protein A的结合位点，因而可选择更便宜和更适用的填料纯化融合蛋白，降低纯化成本。另一方面，CTP的添加也使得约25kDa大小的Fc片段不会干扰N-端融合的FVIII的正确折叠，造成其生物学活性/功能的下降或丧失。具有多个糖基侧链的刚性CTP多肽，相对于(GGGGS)n这类柔性连接肽的无规则卷曲，它可以形成稳定的立体构象，这种“阻隔”作用促使FVIII和Fc段独立折叠形成正确的三维构象而互不影响各自的生物活性。再一方面，CTP糖基侧链的保护作用可以降低连接肽对蛋白酶的敏感性，使融合蛋白不易在连接区被降解。
IgG Fc变体
非裂解性Fc变体
Fc元件来源于免疫球蛋白IgG的恒定区Fc片段，它在消灭病原体的免疫防御中起重要作用。Fc介导的IgG的效应子功能发挥通过两种机制：(1)与细胞表面Fc受体(FcγRs)结合，由吞噬作用或裂解作用或杀伤细胞通过抗体依赖性细胞毒性(ADCC)途径消化病原体，或(2)与第一补体成分C1的C1q结合，引发补体依赖性细胞毒性(CDC)途径，从而裂解病原体。在四种人IgG亚型中，IgG1和IgG3能有效结合FcγRs，IgG4与FcγRs的结合亲和力较低，而IgG2与FcγRs的结合低得难以测定，所以人IgG2几乎没有ADCC效应。此外，人IgG1和IgG3还能有效结合C1q而激活补体级联反应。人IgG2与C1q结合相对弱，而IgG4不与C1q结合(Jefferis R等,Immunol Rev,1998,163:59-76)，所以人IgG2CDC效应也较弱。显然，没有一种天然IgG亚型是非常适合构建FVIII-Fc融合蛋白的。为了得到不具效应子功能的非裂解性Fc，最有效方法是对Fc片段上补体、受体结合域突变改造，调节Fc与相关受体的结合亲和力，降低或消除ADCC和CDC效应，只保留功能蛋白的生物学活性和Fc段长效体内半衰 期，而不产生细胞毒性。更多的非裂解性Fc变体所包含突变位点可以参见Shields RL等,J Biol Chem,2001,276(9):6591-604或中国发明专利CN 201280031137.2。
与新生儿受体(FcRn)结合亲和力增强的Fc变体
IgG的血浆半衰期取决于它与FcRn的结合，一般在pH 6.0时结合，在pH 7.4(血浆pH)时解离。通过对两者结合位点的研究，改造IgG上与FcRn结合的位点，使之在pH 6.0时结合能力增加。已经证明对于结合FcRn重要的人Fcγ结构域的一些残基的突变可增加血清半衰期。已报道T250、M252、S254、T256、V308、E380、M428和N434中的突变可增加或降低FcRn结合亲和力(Roopenian等,Nat.Rview Immunology7:715-725,2007)。韩国专利号KR 10-1027427公开了具有增加的FcRn结合亲和力的曲妥珠单抗(赫赛汀，Genentech)变体，并且这些变体包含选自257C、257M、257L、257N、257Y、279Q、279Y、308F和308Y的一个或更多个氨基酸修饰。韩国专利公开号KR 2010-0099179提供了贝伐单抗(阿瓦斯汀，Genentech)变体并且这些变体通过包含在N434S、M252Y/M428L、M252Y/N434S和M428L/N434S的氨基酸修饰显示增加的体内半衰期。此外，Hinton等也发现T250Q和M428L 2个突变体分别使与FcRn的结合增加3和7倍。同时突变2个位点，则结合增加28倍。在恒河猴体内，M428L或T250QM/428L突变体显示血浆半衰期增加2倍(Paul R.Hinton等,J Immunol,2006,176:346-356)。更多的与新生儿受体(FcRn)结合亲和力增强的Fc变体所包含突变位点可以参见中国发明专利CN201280066663.2。此外，有研究对五种人源化抗体的Fc段进行T250Q/M428L突变不仅改善了Fc与FcRn的相互作用，且在随后的体内药代动力学试验中，发现以皮下注射给药，Fc突变抗体与野生型抗体相比药代动力学参数有所改善，如体内暴露量增加、清除率降低、皮下生物利用度提高(Datta-Mannan A等.MAbs.Taylor&Francis,2012,4(2):267-273.)。
融合蛋白及其制备方法
本发明融合蛋白基因是密码子优化过的由人工合成方法制备。根据本发明所述的核苷酸序列，本领域技术人员可方便的用各种已知方法制得本发明的编码核酸。这些方法不限于人工合成或传统亚克隆等，具体方法可参见J.萨姆布鲁克，《分子克隆实验指南》。作为本发明的一种实施方式，通过分段合成核苷酸序列再进行亚克隆的方法来构建本发明的编码核酸序列。
本发明还提供了一种哺乳动物细胞的表达载体，包含编码本发明的融合蛋白序列以及与之 操作性相连的表达调控序列。所述的“操作性相连”或“可操作地连于”指这样一种状况，即线性DNA序列的某些部分能够调节或控制同一线性DNA序列其它部分的活性。例如，如果启动子控制序列的转录，那么它就是可操作地连于编码序列。
哺乳动物细胞表达载体可采用市售的例如但不限于：pcDNA3、pIRES、pDR、pBK、pSPORT等可用于真核细胞系统表达的载体。本领域技术人员还可以根据宿主细胞来选择合适的表达载体。
根据已知空载表达载体的酶切图谱，本领域技术人员可按照常规方法通过限制性酶剪切与拼接，将本发明的融合蛋白的编码序列插入合适的限制性位点，制得本发明的重组表达载体。
本发明还提供了表达本发明融合蛋白的宿主细胞，其中含有本发明的融合蛋白的编码序列。所述的宿主细胞优选的是真核细胞，例如但不限于CHO细胞，COS细胞，293细胞，RSF细胞等。作为本发明的优选方式，所述的细胞是CHO细胞，其可较佳地表达本发明的融合蛋白，可获得活性和稳定性良好的融合蛋白。
本发明还提供一种用重组DNA技术制备本发明融合蛋白的方法，其步骤包括：
1)提供编码融合蛋白的核酸序列；
2)将1)的核酸序列插入到合适的表达载体，获得重组表达载体；
3)将2)的重组表达载体导入合适的宿主细胞；
4)在适合表达的条件下培养转染宿主细胞；
5)收集上清液，并纯化融合蛋白产物。
将所述编码序列导入宿主细胞可采用本领域的多种已知技术，例如但不限于：磷酸钙沉淀，脂质体转染，电穿孔，微注射，病毒感染法，碱金属离子法。
有关宿主细胞的培养和表达可参见Olander RM等，Dev Biol Stand 1996，86:338。可通过离心去除悬浮液中的细胞和残渣，收集上清液。
可将上述制备获得的融合蛋白纯化为基本均一的性质，例如在SDS-PAGE电泳上呈单一或特定条带。首先将表达上清浓缩,浓缩液可采用凝胶层析的方法进一步加以纯化，或采用离子交换层析的方法纯化。例如阴离子交换层析或阳离子交换层析。凝胶基质可为琼脂糖、葡聚糖、聚酰胺等常用于蛋白纯化的介质。Q-或SP-基团是较为理想的离子交换基团。最后，还可用羟基磷灰石吸附层析，金属螯合层析，疏水相互作用层析和反相高效液相色谱等方法对上述纯化产物进一步精制纯化。上述所有纯化步骤可利用不同的组合，最终使蛋白纯度达到基本均一。还可利用含有所述融合蛋白的特异性抗体、受体或配体的亲和层析柱对表达的融合蛋白进行纯 化。根据所使用的亲和柱的特性，可利用常规的方法，如高盐缓冲液、改变pH等方法洗脱结合在亲和柱上的融合性多肽。
药物组合物
本发明还提供了一种药物组合物，它含有有效剂量的本发明的融合蛋白，以及药学上可接受的载体。通常，可将有效量的本发明融合蛋白配制于无毒的、惰性的和药学上可接受的水性载体介质中，其中pH通常约为5-8，较佳地，pH约为6-8。术语“有效量”或“有效剂量”是指可对人和/或动物产生功能或活性的且可被人和/或动物所接受的量。“药学上可接受的”的成分是适用于人和/或哺乳动物而无过度不良副反应(如毒性、刺激和变态反应)的，即具有合理的效益/风险比的物质。术语“药学上可接受的载体”指用于治疗剂给药的载体，包括各种辅形剂和稀释剂。
药学上可接受的载体包括(但并不限于)：盐水、缓冲液、葡萄糖、水、甘油、乙醇、及其组合。通常药物制剂应与给药方式相匹配，本发明的药物组合物可以被制成针剂形式，例如用生理盐水或含有葡萄糖和其他辅剂的水溶液通过常规方法进行制备。所述的药物组合物宜在无菌条件下制造。活性成分的给药量是治疗有效量。本发明的药物制剂还可制成缓释制剂。
本发明所述的融合蛋白的有效量可随给药的模式和待治疗的疾病的严重程度等而变化。优选的有效量的选择可以由本领域普通技术人员根据各种因素来确定(例如通过临床试验)。所述的因素包括但不限于：所述的融合蛋白的药代动力学参数例如生物利用率、代谢、半衰期等；患者所要治疗的疾病的严重程度、患者体重、患者免疫状况、给药途径等。
附图说明
图1、纯化后融合蛋白SS-F8的SEC-HPLC峰谱图。
图2、纯化后融合蛋白SS-F8的SDS-PAGE电泳图。
图3、融合蛋白SS-F8对SD大鼠隐动脉出血时间的影响。
图4、融合蛋白SS-F8对SD大鼠血浆APTT的影响。
图5、小鼠出血量结果统计图，*p<0.05，****p<0.0001。
图6、小鼠出血时间结果统计图，ns p＞0.05，****p<0.0001。
具体实施方式
实施例1、构建编码突变型单链FVIII融合蛋白的表达质粒
编码FVIII前导肽、B-结构域部分缺失的FVIII蛋白、柔性肽接头、CTP刚性单元和人IgG vFc变体的基因序列都是人工优化过的CHO细胞偏爱密码子，经人工合成获得。所合成融合蛋白全长DNA片段的5’和3’端各有一个限制性酶内切位点，分别为SpeI和EcoRI，全长DNA片段插入至pUC57转移载体相应酶切位点间，并由DNA测序验证其序列。
将上述获得的融合蛋白全长基因片段从中间载体转移到自制表达质粒PXY1A1M的相应酶切位点间，得到融合蛋白高表达质粒。PXY1A1M质粒包含但不限于以下重要表达元器件：1)人巨细胞病毒早期启动子和哺乳动物细胞外源高表达所需增强子；2)双重筛选标记物，在细菌中具有卡那霉素抗性，在哺乳动物细胞中具有G418抗性；3)鼠二氢叶酸还原酶(DHFR)基因表达框，当宿主细胞为DHFR基因缺陷型时，氨甲蝶呤(MTX)能共扩增融合基因和DHFR基因(参见美国专利US 4,399,216)。再将融合蛋白表达质粒转染入哺乳动物宿主细胞系，为了获得稳定高水平的表达，优选的宿主细胞系是DHFR酶缺陷型CHO-细胞(参见美国专利US 4,818,679)。转染两天后，将培养基换成含0.6mg/mL G418的筛选培养基，细胞以一定浓度(5000-10000个活细胞/孔)种植在96孔培养板里，培养10-14天直至大的离散细胞克隆出现。用ELISA分析方法，筛选对选择用药具有抗性的转染子。通过极限稀释96孔培养板，亚克隆产生高水平融合蛋白的孔。
本发明构建了一系列突变型单链FVIII融合蛋白，它含有不同长度的肽接头(Linker)、不同组成的CTP刚性单元以及几种不同亚型的IgG Fc变体(vFc)元件组成，以验证连接肽、Fc变体对突变型单链FVIII融合蛋白活性的影响。详见表1。各组成元件的氨基酸序列见序列表。
表1、各种单链FVIII融合蛋白组成
Figure PCTCN2019107432-appb-000001
实施例2、筛选高表达融合蛋白的稳定转染细胞系
将上述融合蛋白的表达质粒转染入哺乳动物宿主细胞系，以表达突变型单链FVIII融合蛋白。为了维持稳定的高水平表达，优选的宿主细胞是DHFR缺陷型的CHO细胞(参见美国专利US 4,818,679)。一种优选转染方法是电穿孔，也可以使用其它方法，包括磷酸钙共沉降、脂质体转染和微注射等。电穿孔方法应用设置为300V电压和1050μFd电容的Gene Pulser Electroporator(Bio-Rad Laboratories公司)，往放置在比色杯内的2～3×10 7个细胞中加入50μg PvuI线性化的表达质粒，电穿孔后的细胞转移至含30ml生长培养基的摇瓶中。转染两天后，将培养基换成含0.6mg/mL G418的生长培养基，细胞以一定浓度种植在96孔培养板里，培养10-12天直至大的离散细胞克隆出现。用抗人IgG Fc的ELISA分析方法，筛选对选择用药具有抗性的转染子，然后通过极限稀释法亚克隆产生高水平表达融合蛋白的孔。
为了实现融合蛋白较高水平的表达，宜用受MTX药物抑制的DHFR基因进行共扩增。在含有递增浓度MTX的生长培养基中，用DHFR基因共扩增转染的融合蛋白基因。极限稀释DHFR表达阳性的亚克隆，逐步加压并筛选出能在高达6μM MTX培养基中生长的转染子，测定其分泌率，筛选出高表达外源蛋白的细胞系。将分泌率超过约1(较佳地约3)IU/10 6(即百万)个细胞/24小时的细胞系使用无血清培养基的进行适应性悬浮培养，然后再用条件培养基纯化融合蛋白。
实施例3、生产融合蛋白
将实施例2优选得到的高产量细胞株首先在培养皿中进行无血清驯化培养，然后转移到摇瓶中进行悬浮驯化培养。待细胞适应这些培养条件后，然后在300ml摇瓶中进行补料流加培养或通过每天更换培养基的办法模拟灌流培养。由实施例2筛选得到的生产融合蛋白SS-F8的CHO衍生的细胞株在300ml体积的摇瓶中补料流加培养14天，其表达的重组融合蛋白累积产量达到200mg/L，活细胞密度最高可达到15×10 6个/mL。为了得到更多融合蛋白，也可以选用1000ml摇瓶培养。另一种培养方法，上述CHO衍生的细胞株在100ml体积的摇瓶中每天更换培养基，其表达的重组融合蛋白每天累积产量约为20mg/L，在摇瓶中活细胞密度最高可达到30×10 6个/mL。以上两种方法生产的重组融合蛋白的测定的生物学活性相当。
实施例4、纯化与定性融合蛋白
本发明主要采用四步层析法对融合蛋白SS-F8进行纯化。分别为亲和层析、阴离子交换层 析、疏水层析和分子筛层析(本实施例采用的蛋白纯化仪为美国GE公司的AKTA pure 25M。本实施例中采用的试剂均购自国药集团化学试剂有限公司，纯度均为分析级)。
第一步，亲和层析：采用GE公司的VIII Select亲和层析介质进行样品捕获、浓缩以及部分污染物的去除。首先使用平衡buffer：10mM HEPES，150mM NaCl，25mM CaCl 2，0.05％Tween-80，pH 6.8-7.2，以50-100cm/h的线性流速平衡层析柱3-5个柱体积(CV)；将经过澄清后的发酵液以50-100cm/h的线性流速上样，载量不高于50000IU/ml；上样完毕后，使用平衡buffer：10mM HEPES，150mM NaCl，5mM CaCl 2，0.05％Tween-80，pH 6.8-7.2，以50-100cm/h的线性流速平衡层析柱3-5个柱体积(CV)，冲洗未结合的组份；使用去污buffer 1：10mM HEPES，1M NaCl，25mM CaCl 2，0.05％Tween-80，pH 6.8-7.2，以50-100cm/h的线性流速冲洗层析柱3-5个柱体积，去除部分污染物；使用平衡buffer：10mM HEPES，150mM NaCl，25mM CaCl 2，0.05％Tween-80，pH 6.8-7.2，以50-100cm/h的线性流速平衡层析柱3-5个柱体积(CV)；之后使用洗脱buffer：20mM His-HCl，25mM CaCl 2，0.02％Tween 80，45％丙二醇，pH 6.8-7.2，以不高于50cm/h的线性流速洗脱目标产物，收集目标峰。
第二步，阴离子交换层析：使用博格隆公司的Q-HP或其它市售的阴离子交换层析介质(例如GE的Q HP、TOSOH的Toyopearl GigaCap Q-650、天地人和的DEAE Beads 6FF，赛分科技的Generik MC-Q、Merck的Fractogel EMD TMAE、Pall的Q Ceramic HyperD F)进行中间纯化，分离结构变异体、进一步去除HCP、DNA等污染物。首先使用平衡buffer：20mM His-HCl，100mM NaCl，10mM CaCl 2，0.02％Tween 80，pH 7.0-7.5，以50-100cm/h的线性流速冲洗层析柱3-5个柱体积(CV)；经第一步亲和层析分离得到的目标蛋白稀释5-10倍，降低有机物浓度后上样，载量控制在5000-10000IU/ml；上样完毕，使用平衡buffer：20mM His-HCl，100mM NaCl，10mM CaCl 2，0.02％Tween 80，pH 7.0-7.5，以50-100cm/h的线性流速冲洗层析柱3-5个柱体积(CV)；之后使用wash buffer：20mM His-HCl，500mM NaCl，10mM CaCl 2，0.02％Tween 80，pH 7.0-7.5，以50-100cm/h的线性流速冲洗层析柱3-5个柱体积(CV)，去除部分杂蛋白，之后采用层级梯度的盐浓度进行洗脱，洗脱buffer：20mM His-HCl，1M NaCl，10mM CaCl 2，0.02％Tween 80，pH 7.0-7.5，条件为洗脱buffer从50％和60％，洗脱3-5个柱体积(CV)，线性流速控制在不高于50cm/h，对洗脱组分进行分段收集，分别送样进行蛋白含量、SEC-HPLC、活性和HCP含量检测。经蛋白浓度测定，及蛋白活性测定。
第三步，疏水层析：使用博格隆公司的Butyl HP或其它市售的疏水层析介质(例如GE的Butyl HP、TOSOH的Toyopearl Butyl-650、天地人和的Butyl Beads 4FF，赛分科技的Generik  MC30-HIC Butyl、Merck的Fractogel EMD Propyl)进行中间纯化，用于降低聚合体含量，第二步阴离子交换层析洗脱液中仍含有一定比例的聚合体，因为聚合体的形成原因多样，包括结构未改变的聚合和结构发生变化的聚合，它们的生物学活性差别较大，因此对于生物学活性的分析带来较大的干扰。目标蛋白聚合以后，聚合体和单体之间存在性质上的差异，包括电荷特性以及疏水性，我们使用疏水性的差异对二者进行分离。因为纯化最后步骤是分子筛层析，所以使用Butyl HP进行纯化，目标是部分去除聚合体，使其含量低于10％。首先，使用平衡buffer：20mM His-HCl，1.5M NaCl，5mM CaCl 2，0.02％Tween 80，pH 6.8-7.2，以50-100cm/h的线性流速平衡层析柱3-5个柱体积(CV)；第二步阴离子交换层析分离得到的目标蛋白用buffer：2M(NH 4) 2SO 4调电导，然后上样，载量控制在<20000IU/ml；上样完毕后，使用平衡buffer：20mM His-HCl，1.5M NaCl，5mM CaCl 2，0.02％Tween 80，pH 6.8-7.2，以50-100cm/h的线性流速冲洗层析柱3-5个柱体积(CV)；之后使用wash buffer：20mM His-HCl，1.5M NaCl，5mM CaCl 2，0.02％Tween 80，pH 6.8-7.2，以50-100cm/h的线性流速冲洗层析柱3-5个柱体积(CV)，去除部分聚合体；最后进行目标蛋白洗脱，使用洗脱buffer：20mM His-HCl，5mM CaCl 2，0.02％Tween 80，50％乙二醇，pH 6.8-7.2，分别以20％、40％、100％洗脱buffer，以不高于60cm/h的线性流速洗脱3-5个柱体积(CV)，对洗脱组分进行分段收集，分别送检SEC-HPLC。将单体百分比大于90％的目标组分合并进行下一步层析。
第四步，分子筛层析：使用GE的superdex 200或其它市售的分子筛介质(例如博格隆公司的Chromdex 200 prep grade)进行分离，目标是降低聚合体含量至<5％，并进一步降低关键污染物的含量。使用平衡buffer：10mM His-HCl，150mM NaCl，2mM CaCl 2，10mM蔗糖，0.02％Tween 80，pH 6.8-7.2，以20-40cm/h的线性流速冲洗层析柱2个柱体积(CV)；上样量不高于柱体积的3％，以20cm/h的线性流速冲洗，依次收集洗脱组份，SEC检测合并。
样品的SEC-HPLC纯度结果及SDS-PAGE电泳结果分见图1和图2，其中SEC-HPLC结果显示，纯化后融合蛋白的主峰纯度达98％以上；SDS-PAGE电泳带型符合预期，非还原电泳包含未加工融合蛋白条带(约390kDa)，在另一常见酶切位点E720处被切割而脱落两条重链片段后所形成的(LC-L-CTP-Fc) 2二聚片段(约210kDa)，以及LC-L-CTP-Fc单链片段(约100kDa)和HC片段(约90kDa)；还原后可得清晰的HC-LC-L-CTP-Fc(约190kDa)、LC-L-CTP-Fc(约105kDa)和HC(约90kDa)单链条带。得到的蛋白质组份的比活在6000-8000IU/mg。
实施例5.发色底物法间接测定融合蛋白体外活性
突变型单链FVIII融合蛋白的活性可采用发色底物法测定。本实施例采用Biophen FVIII:C试剂盒(HYPHEN BioMed，Ref.221402)测定，其检测原理如下：当被凝血酶激活后，FVIII:C在磷脂和钙离子存在下，与FIXa结合形成酶复合物，继而可激活因子X转变成其活性形式Xa。激活形成的因子Xa继而可使其特异性发色底物(SXa-11)发生裂解，释放发色基团pNA。在405nm下测定所产生pNA的量，即可知与其量直接成正比关系的FXa的活性大小，其中在体系中因子IXa和因子X的含量是一定且过量的，FXa的活性仅与FVIIIa的含量多少直接相关。以本法测定突变型单链FVIII融合蛋白的比活性约为6000-8000IU/mg。
实施例6、纯化的融合蛋白的稳定性研究
为进一步验证突变型单链FVIII融合蛋白在25度室温条件下稳定性，将突变型单链FVIII融合蛋白在25度室温条件下存放数天，考察该条件对融合蛋白活性的影响。
药品稳定性试验箱(购自上海—恒科学仪器)设置试验温度为25℃，湿度为75％。将稀释至相同浓度的SS-F8和双链八因子对照药物DS-F8(该融合蛋白保留了人野生型FVIII Arg1648和Glu1649间的蛋白酶剪切位点，其氨基酸序列如SEQ ID NO：11所示)各分装8支，每支200μl，放入药品稳定性试验箱保存。各取一支SS-F8和DS-F8分装蛋白，根据上述实施例5所述的方法，检测融合蛋白的活性，记为第一天(d1)的活性值。之后分别在室温条件(温度：25℃，湿度75％)下放置d3、d5、d7和d14后，分别检测突变型单链FVIII融合蛋白的活性。结果显示，25度室温条件下放置5天后，SS-F8仅下降约10％的蛋白活性，而DS-F8的蛋白活性下降了25％以上；室温放置7天中，DS-F8的活性降低速率较SS-F8而言更显著，且放置7天后SS-F8仍保有80％以上的蛋白活性，由此可见SS-F8融合蛋白的稳定性明显优于DS-F8。
实施例7、融合蛋白的药效学研究
血液凝固实际是一系列凝血因子的酶促反应，整个凝血过程分为三个阶段：第一阶段为血液凝血活酶形成；第二阶段凝血酶形成；第三阶段纤维蛋白形成，其中FVIII、FIX属于内源性凝血因子，而FVII是外源性凝血因子。出血时间是表示皮肤毛细血管被刺破后自然出血到自然止血所需的时间，通过观察突变型单链FVIII融合蛋白对SD大鼠隐动脉出血时间的影响来检测融合蛋白的促凝血作用。
选取7周龄的SD大鼠(购自上海斯莱克实验动物有限公司)，随机分为2组。给药组以 200IU/只单次静脉给予SS-F8，对照组给以相同体积的生理盐水。给药3h后，对大鼠进行诱导麻醉。对手术位置消毒处理后，由内脚踝往上约1cm位置处，经膝关节至腿根部动脉处剪开皮肤，分离皮下组织和血管上保护膜，依次暴露静脉血管、动脉血管和神经。在膝关节位置找到隐动脉及其分支，用显微直镊锐性分离静脉血管旁5mm*1mm处肌肉位置，从内侧挑起静脉、动脉及神经；用显微剪剪断隐静脉和隐动脉，不得触碰和损伤神经，看到血液涌出即为起始出血时间，记为t1。每隔30s观察一次出血部位，直到看到不出血位置，计为止血时间t2。计算t2-t1，记录出血时间。对数据进行统计分析，实验数据以均数±标差(Means±SD)表示，若数据符合正态分布，则采用SPSS 18.0软件，单因素方差分析或者student’s-test；若非正态分布，采用非参数检验Kruskal-wallis检验或者Mann-whitney检验，P≤0.05具有显著性差异，P≤0.01有非常显著性差异。
实验结果如图3所示，在预防给药后，SD大鼠手术导致隐动脉出血后，SS-F8与生理盐水组的出血时间有明显的统计学差异。在200IU/只剂量下，预防性给予受试药SS-F8对该出血模型有明显地止血作用。
实施例8、凝血法直接测定融合蛋白的生物学活性
通过测定活化部分凝血活酶时间(activated partial thromboplastin time，APTT)，观察SS-F8对SD大鼠血浆的体外抗凝血作用。
选取7周龄的SD大鼠(购自上海斯莱克实验动物有限公司)，随机分为12组。将大鼠诱导麻醉后，在持续麻醉状态下，以手术刀沿腹部正中线划开，自腹部主动脉取血10-12ml。取上述血样，20℃下以1500rpm离心30min，分离上清血浆，分别加入到标记好的1.5ml离心管中。将全血浆与受试药物SS-F8和DS-F8按体积比6:1，分别配制成25-1000IU/ml浓度的待测样品，对照组按体积比6：1加入稀释液。以全自动血凝仪(CS-2000i，Sysmex公司)检测上述样品的APTT值。根据公式计算APTT变化率＝(给药组APTT值-对照组APTT值)/对照组APTT值，实验数据以均数±标差(Means±SD)表示，若数据符合正态分布，则采用SPSS 18.0软件，单因素方差分析或者student’s-test；若非正态分布，采用非参数检验Kruskal-wallis检验或者Mann-whitney检验，P≤0.05具有显著性差异，P≤0.01有非常显著性差异。
从图4中可以看出，受试药SS-F8和DS-F8对正常大鼠的血浆有抗凝血作用，在高浓度1000IU/ml下，受试药DS-F8和SS-F8的APTT时间与溶剂对照组比较，分别下降了33.65％ 和31.86％；DS-F8和SS-F8的EC 50值分别是139.4IU/ml和115.6IU/ml，受试药SS-F8的EC 50值低于DS-F8，可以预见更低的给药剂量。随着各受试药浓度的增加，APTT的时间明显缩短，且有一定的量效关系。
实施例9、融合蛋白对血友病A小鼠急性出血的止血作用
以VIII因子基因剔除纯合子甲型血友病(hemophilia A，HA)小鼠断尾出血模型(the Tail Clip Bleeding Model)评估SS-F8在HA小鼠体内的急性止血作用。选取6-7周龄雄性HA小鼠(来源于上海南方模式生物科技股份有限公司)，适应性饲养一周后将小鼠随机分为2组，分别为HA小鼠空白溶媒对照组和SS-F8给药组。选取雄性C57BL/6小鼠(购自上海斯莱克实验动物有限公司)作为正常对照组。以1％戊巴比妥钠(Merck公司)按照7.5ml/kg剂量腹腔注射麻醉小鼠，尾静脉注射给药，C57BL/6小鼠正常对照组和HA小鼠空白溶媒对照组静脉给予10ml/kg溶媒，给药组静脉给予112IU/kg SS-F8。动物给药15分钟后，采用手术刀片在距离尾末端15mm处断尾，迅速将伤口浸入37℃预热的13mL生理盐水中，开始计时并收集血液，记录断尾后30min内总出血时间和出血量。失血量(ml)＝(采血后离心管重量(g)-采血前离心管重量(g))/1.0。各实验组间比较采用T-test检验分析，分析软件采用Graphpad Prism8.0，p<0.05认为有统计学意义。详细结果见表2。
从图5和图6中各组动物的出血量和出血时间和的统计结果分析，HA小鼠空白溶媒对照组与C57BL/6正常对照组相比，出血量中位数为790.00μl和141.15μl，差异极显著(P＜0.0001)，出血时间中位数为1800s和497s，差异极显著(P＜0.0001)，表明HA小鼠在断尾模型上与正常小鼠相比具有明显的出血特征。HA小鼠给予SS-F8 112IU/kg后，其出血量和出血时间与HA小鼠空白溶媒对照组相比，出血量中位数为438.9μl和790.00μl(p＜0.0001)，出血时间中位数为739s和1800s(p＜0.0001)，促凝血效果明显，说明SS-F8可以作为甲型血友病凝血因子VIII缺乏症发生急性出血情况的有效凝血剂。
表2、各组出血量、出血时间数据统计
Figure PCTCN2019107432-appb-000002
虽然说明并描述了本发明的优选例，应理解本领域的技术人员可根据本文的教导做出各种改变，这些改变不违背本发明的范围。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可对本发明做各种修改或改动，这些等价形式同样落后于本申请所附权利要求书所限定的范围。

Claims (17)

  1. 一种人凝血因子VIII的融合蛋白，所述融合蛋白从N端至C端依次包含B-结构域部分缺失的突变型单链人凝血因子VIII、柔性肽接头、至少1个人绒毛膜促性腺激素β亚基羧基末端肽刚性单元和延长半衰期部分；其中，所述单链人凝血因子VIII氨基酸序列如SEQ ID NO：2所示；其中，延长半衰期部分选自免疫球蛋白Fc段、白蛋白、转铁蛋白或PEG，优选人IgG Fc变体)。
  2. 如权利要求1所述的融合蛋白，其特征在于，所述融合蛋白是糖基化的，优选是通过在哺乳动物细胞(如，中国仓鼠卵巢细胞)中表达而糖基化的。
  3. 如权利要求1所述的融合蛋白，其特征在于，所述B-结构域部分缺失的突变型单链人凝血因子VIII包含如SEQ ID NO：2所示的氨基酸序列，或者所述单链人凝血因子VIII的氨基酸序列与如SEQ ID NO：2所示的氨基酸序列有至少90％的同一性。
  4. 如权利要求1所述的融合蛋白，其特征在于，所述柔性肽接头含有2个或多个选自G、S、A和T残基的氨基酸，
    优选地，所述柔性肽接头具有以(GS)a(GGS)b(GGGS)c(GGGGS)d循环单元组合形成的氨基酸序列通式，其中a，b，c和d是大于或等于0的整数，且a+b+c+d≥1，
    更优选地，所述柔性肽接头优选自下组：
    (i)GSGGGSGGGGSGGGGS；
    (ii)GSGGGGSGGGGSGGGGSGGGGSGGGGS；
    (iii)GGGGSGGGGSGGGGSGGGGS；
    (iv)GSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS；
    (v)GGGSGGGSGGGSGGGSGGGS。
  5. 如权利要求1所述的融合蛋白，其特征在于，所述人绒毛膜促性腺激素β亚基的羧基末端肽刚性单元包含如SEQ ID NO：3所示氨基酸序列或其截短的序列，其中，所述截短的序列包含至少2个糖基化位点，优选地，所述人绒毛膜促性腺激素β亚基的羧基末端肽刚性单元包含以下氨基酸序列：
    (i)PRFQDSSSSKAPPPSLPSPSRLPGPSDTPILPQ；
    (ii)SSSSKAPPPSLPSPSRLPGPSDTPILPQ；
    (iii)SSSSKAPPPS；
    (iv)SRLPGPSDTPILPQ。
  6. 如权利要求1所述的融合蛋白，其特征在于，所述人绒毛膜促性腺激素β亚基的羧基末端 肽刚性单元与权利要求5所述融合蛋白中的羧基末端肽刚性单元氨基酸序列至少具有70％，80％，90％或95％的同一性。
  7. 如权利要求1所述的融合蛋白，其特征在于，所述融合蛋白包含1、2、3、4或5个人绒毛膜促性腺激素β亚基的羧基末端肽刚性单元。
  8. 如权利要求1所述的融合蛋白，其特征在于，所述人IgG Fc变体具有降低的ADCC效应和/或CDC效应和/或与FcRn受体的结合亲和力增强，优选地，所述Fc变体选自：
    (i)含有Leu234Val、Leu235Ala和Pro331Ser突变的人IgG1绞链区、CH2和CH3区域；
    (ii)含有Pro331Ser突变的人IgG2绞链区、CH2和CH3区域；
    (iii)含有Thr250Gln和Met428Leu突变的人IgG2绞链区、CH2和CH3区域；
    (iv)含有Pro331Ser、Thr250Gln和Met428Leu突变的人IgG2绞链区、CH2和CH3区域；
    (v)含有Ser228Pro和Leu235Ala突变的人IgG4绞链区、CH2和CH3区域。
  9. 如权利要求1所述的融合蛋白，其特征在于，所述融合蛋白的氨基酸序列如SEQ ID NO：9所示。
  10. 如权利要求1-9中任一项所述的融合蛋白，其特征在于，所述融合蛋白的活性>6000IU/mg。
  11. 编码如权利要求1-10中任一项所述的融合蛋白的DNA，优选所述DNA的序列如SEQ ID NO：10所示。
  12. 一种载体，其特征在于，包含如权利要求11所述的DNA。
  13. 一种宿主细胞，其特征在于，包含如权利要求12所述的载体，或者转染了权利要求12所述的载体。
  14. 一种药物组合物，其特征在于，包含药学上可接受的载体、赋形剂或稀释剂，以及有效剂量的如权利要求1-10中任一项所述的融合蛋白。
  15. 一种如权利要求1-10中任一项所述的融合蛋白的制备方法，所述方法包括：
    (a)将权利要求11所述编码融合蛋白的DNA序列引入CHO细胞，生成CHO衍生的细胞系；
    (b)筛选步骤(a)中在其生长培养基中每24小时期间内，表达超过1IU/10 6(百万)个细胞的高产细胞株；
    (c)培养步骤(b)筛选到的细胞株，表达融合蛋白；
    (d)收获步骤(c)得到的发酵液，并分离纯化融合蛋白。
  16. 如权利要求15所述的方法，其特征在于，所述步骤(d)中融合蛋白纯化过程包含亲和层析、疏水层析、阴离子交换层析和分子筛层析。
  17. 一种如权利要求1-10中任一项所述的融合蛋白在制备用于预防或治疗出血性疾病(例如，用于FVIII先天性或获得性缺乏症患者的出血性疾病的预防或治疗、血友病A患者的自发或手术性出血的预防或治疗)的药物中应用。
PCT/CN2019/107432 2018-09-26 2019-09-24 突变型单链人凝血因子viii融合蛋白及其制备方法与用途 WO2020063562A1 (zh)

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CN201980059489.0A CN112673026A (zh) 2018-09-26 2019-09-24 突变型单链人凝血因子viii融合蛋白及其制备方法与用途
BR112021005831-1A BR112021005831A2 (pt) 2018-09-26 2019-09-24 proteína de fusão de fator viii de coagulação humana de cadeia simples mutado, método de preparação para isso e uso do mesmo

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