WO2018032786A1 - 人凝血因子ix融合蛋白及其制备方法与用途 - Google Patents

人凝血因子ix融合蛋白及其制备方法与用途 Download PDF

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WO2018032786A1
WO2018032786A1 PCT/CN2017/079872 CN2017079872W WO2018032786A1 WO 2018032786 A1 WO2018032786 A1 WO 2018032786A1 CN 2017079872 W CN2017079872 W CN 2017079872W WO 2018032786 A1 WO2018032786 A1 WO 2018032786A1
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fusion protein
human
fix
amino acid
ctp
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PCT/CN2017/079872
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French (fr)
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高永娟
陈思
李子瑞
屠晓平
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 US16/604,081 priority Critical patent/US11472863B2/en
Priority to RU2019135518A priority patent/RU2736339C9/ru
Priority to CN201780000362.2A priority patent/CN108137708B/zh
Priority to CA3059994A priority patent/CA3059994C/en
Publication of WO2018032786A1 publication Critical patent/WO2018032786A1/zh

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Definitions

  • the present invention relates to the field of fusion proteins, and more particularly to a fusion protein of human coagulation factor IX (FIX), a preparation method thereof and use thereof, particularly for the treatment of various coagulation-related diseases.
  • FIX human coagulation factor IX
  • Hemophilia B is a chain recessive genetic disease whose pathogenesis is a mutation in the human coagulation factor IX (FIX) gene located on the X chromosome, resulting in a significant decrease in the content or activity of the coagulation factor in plasma, thereby making endogenous The sexual coagulation pathway is impeded and normal coagulation cannot be performed. It is estimated that the total number of hemophilia B patients in China is about 20,000, accounting for 15%-20% of hemophilia, and the incidence of hemophilia B in men is 1/30000, which is very rare among women. The most common treatment currently used is FIX drug replacement therapy, including plasma enrichment source FIX and recombinant cell expression source FIX.
  • FIX drug replacement therapy including plasma enrichment source FIX and recombinant cell expression source FIX.
  • FIX Human Factor IX
  • FIX Human Factor IX
  • FIX plays an important role in the endogenous coagulation pathway.
  • Activated FIXa can activate FX with FXa together with activated FVIII (FVIIIa), phospholipids and Ca 2+ to initiate a coagulation pathway and exert a hemostatic effect.
  • FVIIIa activated FVIII
  • More than 100 FIX mutations have been documented in the current study; some of them do not cause any clinical symptoms, but others lead to significant bleeding disorders. If left untreated, hemophilia B can cause bleeding in muscles, joints, and body cavities that are beyond control after injury and can result in death. In the past, the treatment of this disease was mainly the administration of FIX prepared from human plasma.
  • FIX hepatitis C virus
  • the half-life of commercially available recombinant FIX is relatively short, only 18 hours, which makes hemophilia patients need to receive frequent intravenous administration in the emergency on-demand treatment after hemorrhage or in the pre-hemorrhagic prophylaxis.
  • Type B hemophilia patients are recommended to receive FIX twice a week for 40 to 100 IU/kg to prevent bleeding events. Therefore, the development of long-acting recombinant FIX preparations with extended plasma half-life can not only reduce the number of medications, but also reduce the physical and mental burden of patients, and greatly improve patient compliance.
  • FIX in vivo functional half-life of FIX
  • extended half-life moieties such as PEG, human serum albumin (HSA), XTEN, CTP or IgG Fc.
  • PEG polyethylene glycol
  • HSA human serum albumin
  • CTP CTP or IgG Fc
  • Novo Nordisk's N9-GP PEGylated
  • CSL Behring's FIX-FP HSA fusion protein
  • CTP fusion protein OPKO/Prolor's long-acting FIX-CTP
  • the results of the N9-GP clinical trial showed that the cumulative half-life of FIX was prolonged by a factor of five (the average half-life was 110 h). However, one severe hypersensitivity reaction was observed in the trial and three non-inhibitory antibodies were produced.
  • the immunogenicity of N9-GP remains to be further studied (Collins PW et al, Blood, 2014, 124(26): 3880-3886).
  • the FIX-FP clinical trial results showed that the FIX-FP half-life was 89-96 hours, and the patient did not have a special immune response.
  • the FIX-CTP model in the hemophilia B mouse model showed a four-fold increase in FIX half-life, a decrease in bleeding frequency and time, but a decrease in FIX activity (Hart G et al., Haemophilia, 2012, 18:32.).
  • FIX-Fc The first FIX and Fc fusion protein (FIX-Fc) was approved by the US FDA in March 2014 under the trade name Alprolix (Biogen Idec) and is currently the only approved recombinant long-acting FIX drug.
  • Alprolix is a fusion protein formed by covalently binding a single FIX molecule at the N-terminus of the double-stranded Fc fragment of human IgG1, and is recombinantly expressed by HEK-293H cells. Clinical studies have shown that Alprolix has a half-life of 57-86 hours and a frequency of up to 7 or 10 days for prophylactic administration. Currently, Alprolix has been approved for listing in several countries around the world.
  • CTP is a short peptide derived from the carboxy terminus of the ⁇ -subunit of human chorionic gonadotropin (hCG), which has been shown to have an extended half-life in vivo.
  • Chinese patents CN103539860A and CN103539861A disclose a fusion protein which uses a CTP as a linker to link the ⁇ subunit and the ⁇ subunit of FSH to prolong the in vivo half-life of the fusion protein.
  • Patent WO2013121416 discloses a long acting coagulation factor IX comprising at least one CTP linked to the carboxy terminus of coagulation factor IX.
  • FIX-(CTP) 3 containing 3 tandem CTPs exhibit improved pharmacokinetic properties relative to rhFIX, FIX-CTP or FIX-CTP-CTP, and FIX-CTP has comparable in vitro activity and half-life to rhFIX
  • the half-life of FIX-CTP-CTP in rats and FIX-deficient mice was 3 times that of rhFIX, and FIX-(CTP) 3 was 2.5-4 times that of rhFIX.
  • FIX-(CTP) 3 showed reduced clotting activity in the in vivo coagulation test.
  • the present inventors did not use CTP as a single linker or as an extended half-life part according to the prior art, but instead linked it to a flexible peptide linker (for example, (GGGGS)n) to form a flexible peptide linker containing GS and a plurality of sugar-based side chains.
  • a flexible peptide linker for example, (GGGGS)n
  • a hybrid linker peptide of a rigid CTP peptide linker disposed between the FIX and the extended half-life portion (eg, an immunoglobulin Fc fragment, but excluding the CTP suggested by the prior art) to form a new FIX fusion protein, It not only further prolongs the half-life, but also reduces the immunogenicity, improves the bioavailability, greatly reduces the steric hindrance effect of the fusion ligand Fc on FIX, and maintains good biological activity and function.
  • the extended half-life portion eg, an immunoglobulin Fc fragment, but excluding the CTP suggested by the prior art
  • the present invention provides an Fc fusion protein of a hyperglycosylated homodimeric coagulation factor IX (FIX) having an extended in vivo activity half-life, lower immunogenicity and similar biological activity as recombinant FIX.
  • FIX hyperglycosylated homodimeric coagulation factor IX
  • the present invention provides a method for efficiently and stably expressing the fusion protein, which expresses a fusion protein with high yield, good stability during preparation and storage, and its biological activity is similar to that of the marketed recombinant FIX.
  • a high glycosylated FIX fusion protein comprising human coagulation factor IX (hFIX), a flexible peptide linker (Linker, L), at least one person from N-terminus to C-terminus is provided
  • the carboxy terminal peptide rigid unit (CTP) and the extended half-life portion of the chorionic gonadotropin beta subunit eg, immunoglobulin Fc segment, albumin, transferrin or PEG, preferably human IgG Fc variant (denoted as vFc)
  • the fusion protein is represented by hFIX-L-CTP-vFc.
  • the hFIX is wild type or a mutant thereof; further, the wild type hFIX has an amino acid sequence as shown in SEQ ID NO: 1; preferably, the mutant hFIX and SEQ ID NO: 1 are The amino acid sequence is at least 85% homologous; more preferably, the mutant hFIX is at least 90% homologous to the amino acid sequence set forth in SEQ ID NO: 1. Most preferably, the mutant hFIX is at least 95% homologous to the amino acid sequence set forth in SEQ ID NO:1.
  • the flexible peptide linker is preferably non-immunogenic and creates a sufficient spatial distance between hFIX and Fc to minimize steric effects between each other.
  • a flexible peptide linker comprising two or more amino acid residues is used and is selected from the group consisting of Gly (G), Ser (S), Ala (A) and Thr (T).
  • 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 an amino acid sequence formula formed by combining (GS) a (GGS) b (GGGS) c (GGGGS) d cycle units, wherein a, b, c and d are greater than Or an integer equal to 0, and a+b+c+d ⁇ 1.
  • the peptide linker may preferably comprise the following sequence:
  • the CTP rigid unit is selected from the full length sequence consisting of amino acids 113 to 145 of the carboxy terminus of human chorionic gonadotropin ⁇ subunit or a fragment thereof, in particular, the CTP rigid unit comprises SEQ ID NO: The amino acid sequence shown in 2 or its truncated sequence.
  • the CTP rigid linker peptide containing multiple glycosylation sites can form a stable stereoconfiguration with respect to the random coiling of the flexible linker peptide, which promotes the FIX and Fc segments to fold independently to form the correct three-dimensional conformation without affecting the respective organisms. active.
  • the protective effect of the CTP glycosyl side chain can reduce the sensitivity of the linker peptide to proteases.
  • the CTP rigid unit comprises at least 2 glycosylation sites; for example, in a preferred embodiment of the invention, the CTP rigid unit comprises 2 glycosylation sites, exemplarily, the CTP The rigid unit comprises 10 amino acids of the N-terminus of SEQ ID NO: 2, ie SSSS*KAPPPS*; or the CTP rigid unit comprises 14 amino acids of the C-terminus of SEQ ID NO: 2, ie S*RLPGPS*DTPILPQ; as another example Wherein the CTP rigid unit comprises 3 glycosylation sites, exemplarily, the CTP rigid unit comprises 16 amino acids of the N-terminus of SEQ ID NO: 2, ie SSSS*KAPPPS*LPSPS*R; In an embodiment, the CTP rigid unit comprises four glycosylation sites, exemplarily, the CTP rigid unit comprises 28, 29, 30, 31, 32 or 33 amino acids and begins with human chorionic gonadotropin The 113th, 114th, 115th, 116th, 117th or 118th position of
  • the CTP rigid unit comprises 28 amino acids of the N-terminus of SEQ ID NO: 2, namely SSSS*KAPPPS*LPSPS*RLPGPS*DTPILPQ. in the text,* Represents a glycosylation site. Each possibility represents a separate embodiment of the invention.
  • the CTP rigid units provided herein are at least 70% homologous to the native CTP amino acid sequence; in other embodiments, the CTP rigid units provided herein are at least 80% homologous to the native CTP amino acid sequence; In other embodiments, the CTP rigid units provided herein are at least 90% homologous to the native CTP amino acid sequence; in other embodiments, the CTP rigid units provided herein are at least 95% homologous to the native CTP amino acid sequence.
  • the fusion protein comprises one of the above CTP rigid units. In other embodiments of the invention, the fusion protein comprises two or more of the above CTP rigid units, preferably comprising 2, 3, 4 or 5 of the above CTP rigid units, for example, an embodiment of the invention
  • the fusion protein comprises two CTP 3 rigid units: SSSSKAPPPSSSSSKAPPPS (CTP 3- CTP 3 , or expressed as (CTP 3 ) 2 ).
  • the extended half-life portion is preferably a self-immunoglobulin IgG, IgM, IgA Fc fragment; more preferably an Fc fragment from human IgG1, IgG2, IgG3 or IgG4 and variants thereof; further, the human IgG Fc variant comprises in the wild At least one amino acid modification in a human IgG Fc, and the variant has reduced effector function (ADCC and/or CDC effect) and/or enhanced binding affinity to the neonatal receptor FcRn. Further, the human IgG Fc variant may be selected from the group consisting of:
  • vFc ⁇ 1 human IgG1 hinge region, CH2 and CH3 region containing the Leu234Val, Leu235Ala and Pro331Ser mutations (such as the amino acid sequence shown in SEQ ID NO: 3);
  • vFc ⁇ 2-1 human IgG2 hinge region, CH2 and CH3 region containing the Pro331Ser mutation (such as the amino acid sequence shown in SEQ ID NO: 4);
  • vFc ⁇ 2-2 human IgG2 hinge region, CH2 and CH3 region containing the Thr250Gln and Met428Leu mutations (such as the amino acid sequence shown in SEQ ID NO: 5);
  • vFc ⁇ 2-3 human IgG2 hinge region, CH2 and CH3 regions containing the Pro331Ser, Thr250Gln and Met428Leu mutations (such as the amino acid sequence shown in SEQ ID NO: 6);
  • (v) vFc ⁇ 4 human IgG4 hinge region, CH2 and CH3 region containing the Ser228Pro and Leu235Ala mutations (such as the amino acid sequence shown in SEQ ID NO: 7).
  • An Fc variant (vFc) in a fusion protein of the invention which comprises a hinge region, a CH2 and a CH3 region of human IgG such as human IgG1, IgG2 and IgG4.
  • This CH2 region contains amino acid mutations at positions 228, 234, 235 and 331 (as determined by the EU counting system). It is believed that these amino acid mutations reduce the effector function of Fc.
  • Human IgG2 does not bind to FcyR but shows very weak complement activity.
  • An Fc[gamma]2 variant with a Pro331Ser mutation should have a lower complement activity than native Fc[gamma]2 and is still an Fc[gamma]R non-binding element.
  • IgG4Fc is defective in the activation of the complement cascade and its binding affinity to Fc ⁇ R is about an order of magnitude lower than that of IgG1.
  • An Fc ⁇ 4 variant with a Leu235Ala mutation should exhibit minimal effector function compared to native Fc ⁇ 4.
  • Fc ⁇ 1 with Leu234Val, Leu235Ala and Pro331Ser mutations also showed reduced effector function compared to native Fc ⁇ 1.
  • These Fc variants are all more suitable for the preparation of FIX fusion proteins than native human IgG 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 to obtain a new combined variant, which reduces the effector function and prolongs its half-life.
  • the Fc variants of the invention comprise, but are not limited to, mutations at several of the above sites, and substitutions at other sites may be introduced such that the Fc has reduced effector function and/or enhanced binding to the FcRn receptor, while still It does not cause a decrease in Fc variant function/activity or cause a poor conformational change.
  • Common mutation sites can be found in Shields RL et al, J Biol Chem, 2001, 276(9):6591-604.
  • amino acid sequence of the fusion protein is set forth in SEQ ID NO: 8.
  • a DNA encoding the above fusion protein is provided.
  • the DNA sequence of the fusion protein is set forth in SEQ ID NO: 9.
  • a vector comprising the above DNA is provided.
  • a host cell comprising the above vector or transfected with the above vector is provided.
  • the host cell is a CHO derived cell line DXB-11.
  • a pharmaceutical composition includes pharmaceutically acceptable Accepted carrier, excipient or diluent, and an effective amount of the above fusion protein.
  • a method of making or producing the fusion protein from a cell line derived from a mammalian cell line, such as CHO comprising the steps of:
  • step (c) culturing step (b) the selected cell strain to express the fusion protein
  • the CHO-derived cell line in the step (a) is DXB-11.
  • the cell culture may be a batch, perfusion or fed culture method.
  • the fusion protein is purified by four-step chromatography, which is affinity chromatography, hydrophobic chromatography, anion exchange chromatography and molecular sieve chromatography.
  • the present invention is further given its preferred conditions in conjunction with Example 5.
  • the fusion protein prepared by the above method has an activity of >200 IU/mg.
  • a use of the fusion protein for the preparation of a medicament for preventing or treating a bleeding disorder or event caused by a deficiency or malfunction of FIX comprising preparation for FIX congenital or acquired deficiency
  • FIX congenital or acquired deficiency For the prevention or treatment of hemorrhagic diseases in patients with septic disease, or for the prevention or treatment of spontaneous or surgical bleeding in patients with hemophilia B.
  • the fusion ligand human IgG Fc variant used in the fusion protein is non-lytic, which reduces the effector function triggered by binding to Fc ⁇ Rs and Clq.
  • the fusion protein of the present invention can be expected to have reduced immunogenicity and reduce the production of neutralizing antibodies in a patient.
  • the fusion protein of the invention has good stability in fermentation, purification process and storage process.
  • the fusion protein provided by the present invention comprises a rigid CTP polypeptide having a plurality of glycosyl side chains which form a stable stereo conformation with respect to the random coiling of a flexible linker peptide such as (GGGGS)n.
  • the "barrier" effect causes the FIX and Fc segments to fold independently to form the correct three-dimensional conformation without affecting their respective biological activities.
  • CTP contains a glycosyl group, and the negatively charged, highly sialylated CTP can resist the clearance of the kidney and further prolong the half-life of the fusion protein.
  • the protective effect of the CTP glycosyl side chain can reduce the sensitivity of the linker to protease.
  • the fusion protein is not easily degraded in the junction region.
  • the preparation method of the fusion protein provided by the invention has high yield and is cultured in a 300 ml shake flask for 14 days, and the cumulative yield can reach at least 200 mg/L, which can be scaled up for large-scale industrial production.
  • the fusion protein constructed by the present invention is more efficient and convenient than the Monomeric FIX fusion protein constructed by Biogen, and the production cost can be greatly reduced.
  • Biogen Corporation constructed a dual expression vector for rFIXFc and Fc, wherein the Fc molecule was labeled with Flag (European Patent, Publication No. EP1624891B1).
  • the fusion protein fermentation broth expressed by it is expected to contain three forms of products, namely FIX-Fc: FIX-Fc homodimeric (Dimeric) fusion protein, FIX-Fc: FLAG-Fc monomer-dimerization Monomeric fusion protein and FLAG-Fc: FLAG-Fc dimer three products.
  • the preparation method of the invention has certain technical advantages and price advantages over the Monomeric rFIXFc fusion protein developed by Biogen, and the expression and purification processes are simpler, more efficient, and the production cost is lower.
  • CTP is a short peptide derived from the carboxy terminus of the ⁇ -subunit of human chorionic gonadotropin (hCG).
  • hCG human chorionic gonadotropin
  • reproductive-related peptide hormones follicle stimulating hormone (FSH), luteinizing hormone (LH), thyrotropin (TSH), and chorionic gonadotropin (hCG) contain the same alpha-subunit and their respective specific beta - Yaki.
  • FSH follicle stimulating hormone
  • LH luteinizing hormone
  • TSH thyrotropin
  • hCG chorionic gonadotropin
  • the half-life of hCG is significantly prolonged, mainly due to the unique carboxy terminal peptide (CTP) on its ⁇ -subunit (Fares FA et al, Proc Natl Acad Sci USA, 1992, 89 (10): 4304-4308).
  • Natural CTP contains 37 amino acid residues and it has 4 O-glycosylation site, the terminal is a sialic acid residue. Negatively charged, highly sialylated CTP is resistant to the clearance of the kidneys, thereby prolonging the half-life of the protein in vivo (Fares F A et al, Proc Natl Acad Sci USA, 1992, 89(10): 4304-4308). However, the inventors creatively linked at least one CTP polypeptide to a flexible linker of appropriate length, collectively as a linker peptide, for ligation of FIX with an extended half-life portion (eg, an immunoglobulin Fc fragment).
  • a linker peptide eg, an immunoglobulin Fc fragment
  • the inventors have found that by adding a CTP peptide between FIX and an Fc variant, it is equivalent to adding a stretch of a rigid linker peptide.
  • This aspect ensures that the N-terminally fused FIX does not affect the binding site of the Fc variant to FcRn, thereby affecting the half-life; in addition, the Fc Protein A binding site is important for the purification step in the preparation process, and the CTP is guaranteed to N-
  • the end-fused FIX also does 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, reducing the cost of purification.
  • CTP also allows the Fc fragment of about 25 kDa size to not interfere with the correct folding of the N-terminally fused FIX, resulting in a decrease or loss of its biological activity/function.
  • a rigid CTP polypeptide having multiple glycosyl side chains which forms a stable stereoconfiguration relative to the random coiling of a flexible linker such as (GGGGS)n, which causes the FIX and Fc segments to fold independently.
  • the correct three-dimensional conformation does not affect each other's biological activity.
  • the protective effect of the CTP glycosyl side chain can reduce the sensitivity of the linker peptide to proteases, making the fusion protein less susceptible to degradation in the junction region.
  • the Fc element is derived from the constant region Fc fragment of immunoglobulin IgG, which plays an important role in eradicating the immune defense of pathogens.
  • the effector function of Fc-mediated IgG is exerted through two mechanisms: (1) binding to cell surface Fc receptors (Fc ⁇ Rs), digestion of pathogens by phagocytosis or cleavage or killer cells via antibody-dependent cellular cytotoxicity (ADCC) pathway , or (2) binding to C1q of the first complement component C1, eliciting a complement-dependent cytotoxicity (CDC) pathway, thereby lysing the pathogen.
  • Fc ⁇ Rs cell surface Fc receptors
  • ADCC antibody-dependent cellular cytotoxicity
  • CDC complement-dependent cytotoxicity
  • IgG1 and IgG3 efficiently bind to Fc ⁇ Rs, and the binding affinity of IgG4 to Fc ⁇ Rs is low, and the binding of IgG2 to Fc ⁇ Rs is too low to be determined, so human IgG2 has almost no ADCC effect.
  • human IgG1 and IgG3 can also efficiently bind to C1q to activate the complement cascade.
  • Human IgG2 binds relatively weakly to C1q, whereas IgG4 does not bind to C1q (Jefferis R et al, Immunol Rev, 1998, 163: 59-76), so the human IgG2 CDC effect is also weak.
  • Fc variant with enhanced binding affinity to neonatal receptor (FcRn)
  • the plasma half-life of IgG depends on its binding to FcRn, which typically binds at pH 6.0 and dissociates at pH 7.4 (plasma pH). By studying the binding sites of the two, the site of binding to FcRn on IgG was engineered to increase the binding ability at pH 6.0. Mutations in some residues of the human Fc ⁇ domain important for binding to FcRn have been shown to increase serum half-life. Mutations in T250, M252, S254, T256, V308, E380, M428 and N434 have been reported to increase or decrease FcRn binding affinity (Roopenian et al, Nat. Rview Immunology 7: 715-725, 2007). Korean Patent No.
  • KR 10-1027427 discloses variants of trastuzumab (Herceptin, Genentech) having increased FcRn binding affinity, and these variants are selected from the group consisting of 257C, 257M, 257L, 257N, 257Y, 279Q, One or more amino acid modifications of 279Y, 308F and 308Y.
  • Korean Patent Publication No. KR 2010-0099179 provides variants of bevacizumab (Avastin, Genentech) and these variants show increased in vivo by amino acid modifications contained in N434S, M252Y/M428L, M252Y/N434S and M428L/N434S half life.
  • 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 synthetic or traditional subcloning, and the specific method can be found in J. Sambrook, Molecular Cloning Experiment Guide.
  • the nucleic acid sequence of the present invention is constructed by subcloning a nucleotide sequence and then subcloning.
  • the invention also provides an expression vector for a mammalian cell comprising a fusion protein sequence encoding the invention and an expression control sequence operably linked thereto.
  • operably linked or “operably linked” is meant a condition in which portions of a linear DNA sequence are capable of modulating or controlling the activity of other portions of the same linear DNA sequence. For example, if a promoter controls the transcription of a sequence, then it is operably linked to the coding sequence.
  • the mammalian cell expression vector can be commercially available, for example, but not limited to, pcDNA3, pIRES, pDR, pBK, pSPORT, etc., which can be used for expression in eukaryotic cell systems.
  • 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 skilled person can prepare the present invention by inserting the coding sequence of the fusion protein of the present invention into a suitable restriction site by restriction enzyme cleavage and splicing according to a conventional method. Recombinant expression vector.
  • the invention also provides a host cell expressing a fusion protein of the invention comprising a coding sequence for a fusion protein of the invention.
  • the host cell is preferably a eukaryotic cell such as, but not limited to, a CHO cell, a COS cell, a 293 cell, an RSF cell, and the like.
  • the cell is a CHO cell which can preferably express the fusion protein of the present invention, and a fusion protein having good activity and good stability can be obtained.
  • the invention also provides a method for preparing a fusion protein of the invention by recombinant DNA technology, the steps of which comprise:
  • 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, In: calcium phosphate precipitation, lipofection, electroporation, microinjection, viral infection, alkali metal ion method.
  • the fusion protein obtained as described above can be purified to a substantially uniform property, such as a single or specific band on SDS-PAGE electrophoresis.
  • the supernatant is first concentrated, and the concentrate can be further purified by gel chromatography or by ion exchange chromatography.
  • ion exchange chromatography For example, anion exchange chromatography or cation exchange chromatography.
  • the gel matrix may be a medium commonly used for protein purification such as agarose, dextran, polyamide, and the like.
  • the Q- or SP- group is a preferred ion exchange group.
  • 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 using conventional methods such as high salt buffer, pH change, and the like.
  • the invention also provides a pharmaceutical composition comprising an effective amount of a fusion protein of the 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.
  • a "pharmaceutically acceptable” ingredient is one which is suitable for use in humans and/or mammals without excessive adverse side effects (such as toxicity, irritation, and allergies), i.e., materials having a reasonable benefit/risk ratio.
  • pharmaceutically acceptable carrier refers to a carrier for the administration of a therapeutic agent, including 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 into a sustained release preparation.
  • the effective amount of the fusion protein of the present invention may vary depending on the mode of administration and the severity of the disease to be treated. Degree changes. The selection of a preferred effective amount can be determined by one of ordinary skill in the art based on various factors (e.g., by clinical trials). The factors include, but are not limited to, the pharmacokinetic parameters of the fusion protein such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated by the patient, the patient's weight, the patient's immune status, the route of administration, etc. .
  • Figure 1 shows the nucleotide sequence and deduced amino acid sequence of the fusion protein of the Spe I-EcoR I fragment in the pF9-5B expression vector.
  • Mature fusion protein contains hFIX, flexible peptide linker (underlined), CTP rigid unit (underlined Marked) and vFc ⁇ 2-3 variants.
  • the gene sequences encoding the full-length FIX as well as the different lengths of flexible peptide linkers, CTP rigid peptides of different lengths and different IgG Fc variants are artificially optimized CHO cell-preferred codons obtained by chemical synthesis.
  • a restriction endonuclease site at the 5' and 3' ends of the synthesized fragment, respectively SpeI and EcoRI.
  • the fusion-purified fusion gene was digested with SpeI and EcoRI, and inserted into the corresponding restriction sites of the expression plasmid PXY1A1 which was modified with PCDNA3.1 as a template to obtain a fusion gene expression plasmid pF9-5.
  • the PXY1A1 plasmid includes, but is not limited to, the following important expression components: 1) human cytomegalovirus early promoter and mammalian cells with high exogenous expression of the desired enhancer; 2) dual screening marker with kanamycin resistance in bacteria sexuality, G418 resistance in mammalian cells; 3) Murine dihydrofolate reductase (DHFR) gene expression cassette, when the host cell is DHFR gene-deficient, methotrexate (MTX) can amplify the fusion gene And the DHFR gene (see U.S. Patent 4,399,216).
  • DHFR Murine dihydrofolate reductase
  • the fusion protein expression plasmid is then transfected into a mammalian host cell line, and in order to obtain stable high levels of expression, the preferred host cell line is a DHFR enzyme deficient CHO-cell (see U.S. Patent 4,818,679).
  • the present invention constructs a series of hFIX fusion proteins having flexible peptide linkers of different lengths, CTP rigid units of different compositions, and IgG Fc (vFc) variant elements of several different subtypes. Pieces.
  • the nucleotide sequence and translated amino acid sequence of F9-5B are shown in FIG. 1 .
  • Example 2 transient expression of different fusion proteins and in vitro activity assay
  • Example 1 of the embodiment A series of expression plasmids obtained in Example 1 of the embodiment, the reagent used DNAFect LT TM (ATGCell Company) in 30ml shake bottle transfection 3 ⁇ 10 7 CHO-K1 cells, the transfected cells were cultured at 1000ng / ml vitamin K1 The growth was carried out for 5 days in serum-free growth medium, and the concentration of the fusion protein in the supernatant was measured, and its activity was measured by the method described in Example 6. The ELISA results showed that the transient expression levels of several plasmids under these conditions were similar, but their coagulation activities showed large differences.
  • F9-5F activity is only about 30% of F9-5B, which may be that the CTP rigid unit is placed at the N-terminus of Fc, which can form a fixed spatial conformation, which can effectively separate the different functional regions of the fusion protein, which is more conducive to FIX and The Fc portions are independently folded to form the correct three-dimensional conformation, thereby maintaining a high activity.
  • the fusion protein in the supernatant of F9-5G cell culture mostly exists in the form of inactive polymer. This may be because the long peptide linker can not only increase the activity of the fusion protein, but will cause the protein to fold incorrectly and become inactive.
  • the form of the polymer exists.
  • the expression plasmid for the above fusion protein is transfected into a mammalian host cell line to express the FIX fusion protein.
  • a preferred host cell is a DHFR deficient CHO cell (U.S. Patent 4,818,679).
  • a preferred method of transfection is electroporation, and other methods can be used, including calcium phosphate co-precipitation, lipofection, and microinjection.
  • Electroporation method Using a Gene Pulser Electroporator (Bio-Rad Laboratories) set to 300 V voltage and 1050 ⁇ Fd capacitance, 50 ⁇ g of PvuI linearized expression plasmid was added to 3 ⁇ 10 7 cells placed in a cuvette, after electroporation The cells were transferred to shake flasks containing 30 ml of growth medium. Two days after transfection, the medium was changed to a growth medium containing 0.6 mg/mL G418, and the cells were seeded in a 96-well culture plate at a concentration for 12-15 days until large discrete cell clones appeared.
  • the anti-human IgG Fc ELISA method is used to screen the transfectants that are resistant to the selected drugs.
  • the anti-FIX ELISA method can also be used to quantitatively determine the expression of the fusion protein, and then the sub-cloning by the limiting dilution method to generate high-level expression fusion. The pores of the protein.
  • the transfected fusion protein gene was co-amplified with the DHFR gene in growth medium containing increasing concentrations of MTX.
  • Subclones with positive dilution DHFR expression were gradually pressurized, and transfectants capable of growing in up to 6 ⁇ M MTX medium were screened, the secretion rate was determined, and a cell line highly expressing the foreign protein was selected.
  • a cell line having a secretion rate of more than about 1 (preferably about 2) mg/10 6 (i.e., millions) of cells per 24 hours is subjected to adaptive suspension culture using a serum-free medium, and then purified by conditioned medium. protein.
  • the high-yield cell line preferably obtained in Example 3 was first subjected to serum-free domestication culture in a culture dish, and then transferred to a shake flask for suspension and domestication culture. After the cells were adapted to these culture conditions, supplemental flow culture was then carried out in a 300 ml shake flask or perfusion culture was simulated by changing the medium daily.
  • the CHO-derived cell line produced by the screening of the fusion protein F9-5B obtained in Example 3 was fed and cultured for 14 days in a 300 ml volume shake flask, and the cumulative yield of the expressed recombinant fusion protein reached 200 mg/L, and the viable cell density was obtained. Up to 18 ⁇ 10 6 / mL.
  • 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 30 mg/L per day, and the viable cell density can reach up to 35 in the shake flask. ⁇ 10 6 / mL.
  • the biological activities of the assays for recombinant fusion proteins produced by the above two methods are comparable.
  • the invention mainly uses the affinity chromatography method to purify the FIX fusion protein F9-5B (the protein purification instrument used in the embodiment is the AKTA Explorer 100 of the American GE company; the reagents in the embodiment are all purchased from the country Pharmaceutical Group Chemical Reagent Co., Ltd., the purity is analytical grade).
  • affinity chromatography using Mabselect Sure from GE or other commercially available recombinant protein A affinity chromatography media (eg GE's Mabselect, Mabselect Sure LX, Bogron alkaloid Protein A Diamond, TOSOH's Toyopearl Sample capture, concentration, and partial contaminant removal were performed on AF-rProtein A-650F, Tiandiren and rProtein A Bead, Sabre Technology's MabPurix, Pall's KANEKA KanCapA, and Merck's Eshumono A).
  • the second step, anion chromatography use GE's Q Sepharase FF or other commercially available anion chromatography media (such as GE's DEAE Sepharose FF, Q Sepharose HP, Capto Q, Capto DEAE, TOSOH's Toyopearl GigaCap Q-650, Tiandi Renhe's DEAE Beads 6FF, Saizhi Technology's Generik MC-Q, Merck's Fractogel EMD TMAE, and Pall's Q Ceramic HyperD F) were used for intermediate purification to reduce HCP, residual DNA, and shed ProteinA.
  • GE's Q Sepharase FF or other commercially available anion chromatography media (such as GE's DEAE Sepharose FF, Q Sepharose HP, Capto Q, Capto DEAE, TOSOH's Toyopearl GigaCap Q-650, Tiandi Renhe's DEAE Beads 6FF, Saizhi Technology's Gene
  • the first step of the affinity eluate still contains a certain proportion of HCP, residual DNA, endotoxin and other contaminants, so we must find ways to remove these pollutants.
  • the sample was diluted 1 time with a balanced buffer and then loaded. Under this condition, the target protein flowed through. When A 280 was raised to 100 mAU, the flow-through was started.
  • the equilibrium buffer was used: 40 mM Na 2 PO 4 - Citric, 0.1 M NaCl, pH 4.8-5.2, continue to rinse the column at a linear flow rate of 50-100 cm/h and continue to collect the flow peak to A 280 down to 100 mAU, at which point the collection is stopped; then regenerating buffer: 1 M is used. NaCl, 1M NaOH, 3-5 column volumes of the column were washed at a linear flow rate of 50-100 cm/h. The column was regenerated and the collected samples were sent for HCP, DNA, ProteinA, and SEC-HPLC.
  • the third step affinity chromatography: using JNC's Cellufine Sulfate or other commercially available affinity chromatography media (such as GE's Heparin FF, Heparin HP) for final purification, removal of the polymer, further removal of HCP, DNA, etc. Contaminants.
  • affinity chromatography using JNC's Cellufine Sulfate or other commercially available affinity chromatography media (such as GE's Heparin FF, Heparin HP) for final purification, removal of the polymer, further removal of HCP, DNA, etc. Contaminants.
  • a balanced buffer 20 mM PB, 100 mM NaCl, pH 7.0-7.4, and rinse the column at 3-5 column volumes at a linear flow rate of 50-100 cm/h; the target protein is separated by the second step of anion chromatography.
  • Example 6 Determination of in vitro activity of fusion protein by chromogenic substrate method
  • the activity of the FIX-Fc fusion protein can be determined by the chromogenic substrate method. This example was measured using the BIOPHEN Factor IX kit (HYPHEN BioMed, Ref. A221802).
  • the detection principle is as follows: Factor XIa provided in the kit activates Factor IX in the test sample to FIXa, and activated FIXa in thrombin, In the presence of phospholipids (PLPs) and calcium ions (Ca 2+ ), thrombin-activated FVIII:C, PLPs, and Ca 2+ form a thrombin complex, which in turn converts factor X in the assay system to an activated form of Xa .
  • PLPs phospholipids
  • Ca 2+ calcium ions
  • the activation activity of factor X by thrombin complex was positively correlated with the content of factor IX in the test sample.
  • the activated factor Xa activity can be detected by its specific cleavage of the chromogenic substrate (SXa-11), that is, the absorbance of the cleavage product pNA is detected at 405 nm, and the pNA absorbance is directly proportional to the FIXa activity.
  • the specific activity of the purified FIX fusion protein F9-5B can be determined by the method to be 200 IU/mg or more.
  • mice Male SPF SD rats (purchased from Shanghai Bikai Experimental Animal Co., Ltd.) were randomly divided into 2 groups, 2 rats in each group, given a single intravenous injection of 4.5 mg/kg (high dose group) and 1.5. Mg/kg
  • the F9-5B fusion protein (low dose group) was examined for changes in plasma concentration and time.
  • the control group and the administration group were bled by eyelids at 0, 1, 3, 6, 24, 48, 72, 96, 120, 144 and 168 hours after administration, respectively, about 0.3 ml each time. After taking blood, it was allowed to stand, and the blood was allowed to stand at room temperature for 30 minutes, centrifuged at 5000 rpm for 10 minutes, and the serum was separated and stored at -20 °C.
  • the amount of fusion protein in the serum at each time point was determined by an ELISA method specific for FIX.
  • the main pharmacokinetic parameters of each group were calculated by the software PKSOLVER. The results are shown in Table 2.
  • the in vivo half-lives of the high and low dose F9-5B fusion proteins were 31 and 30 hours, respectively, and the T 1/2 ⁇ value was increased by 8 times compared with rhFIX (Chinese patent CN104427994).
  • the F9-5B fusion protein showed an improved half-life compared to rhFIX, demonstrating that the addition of a linker peptide and an Fc variant at the C-terminus of FIX did not interfere with the activity of the fusion protein, but instead produced an unexpected technique for the activity and half-life of the FIX fusion protein.
  • the effect presumably, is due to the FIX and Fc variants linked by a flexible peptide linker and CTP.
  • the CTP rigid peptide not only further prolongs its half-life in vivo, but also increases the space between the fusion protein molecules by blocking the multiple glycosylated side chains.
  • the distance causes the FIX and Fc segments to each fold to form the correct three-dimensional conformation without affecting their respective biological activities. It can be seen that F9-5B exhibits superior performance in terms of bioavailability and pharmacokinetics compared to rhFIX.

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Abstract

一种高糖基化的重组人凝血因子IX(FIX)融合蛋白、其制备方法及用途。所述融合蛋白从N端到C端依次包含人FIX、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。该融合蛋白具有与重组FIX类似的生物学活性及延长的体内活性半衰期，降低的免疫原性，从而改善药代动力学和药效。

Description

人凝血因子IX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，涉及一种人凝血因子IX(FIX)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
血友病B是一种连锁隐性遗传病，其发病机制是位于X染色体上的人凝血因子IX(FIX)基因发生了突变，导致血浆中该凝血因子含量或活性大幅下降，从而使得内源性凝血途径受到阻碍，无法进行正常凝血。据估计我国血友病B患者总数约20000名，占血友病的15％-20％，血友病B在男性中的发病率为1/30000，而在女性中则非常罕见。目前最常用的治疗方式是FIX药物替代治疗，包括血浆富集来源FIX和重组细胞表达来源FIX。
人凝血因子IX(Factor IX，FIX)是一种丝氨酸蛋白酶的酶原，包含461个氨基酸，是凝血级联内源性途径的一个重要组分，主要在肝脏中合成并分泌至血浆中。FIX由多个分开的功能域组成，包括信号肽、前肽区、Gla结构域、两个类表皮生长因子(EGF)结构域、激活肽和类胰蛋白酶催化域(丝氨酸蛋白酶域)。该蛋白酶原进一步被加工成活性形式，通过二硫键连接的轻链和重链构成异质二聚体。FIX在内源性凝血途径中发挥着重要作用，活化的FIXa才能与活化的FVIII(FVIIIa)、磷脂和Ca2+一起将FX激活为FXa，启动共同凝血途径，发挥止凝血作用。目前研究已记载了超过100个FIX的突变；其中一些不引起任何临床症状，但其它则导致明显的出血性疾病。如果不及时治疗，血友病B会引起损伤后无法控制的肌肉、关节和体腔内出血，并可能导致死亡。过去该疾病治疗主要是施用来自人血浆制备的FIX。然而，一方面这具有随之而来的感染血源性病毒风险，包括人类免疫缺陷病毒(HIV)和丙型肝炎病毒(HCV)。另一方面天然FIX在人体内半衰期很短，约18～24小时，患者需要反复输血或血液制品，这样不仅费用昂贵，还可能引起严重的输血反应，凝血酶原复合物中微量的激活因子还可能激活凝血级联反应，引起血栓形成和栓塞。目前市售重组FIX的半衰期相对较短，只有18小时，这使得血友病人在发生出血后应急式按需治疗过程中抑或是出血发生前预防治疗应用中都需要接受频繁静脉注射给药。B型血友 病人推荐每周接受2～3次，每次40～100IU/kg剂量注射FIX以预防出血事件发生。因而开发长效重组FIX制剂，具有延长的血浆半衰期不仅能减少用药次数、又可减轻患者身心负担，大大提高患者依从性。
为了延长FIX的体内功能半衰期，现有技术将FIX与PEG、人血清白蛋白(HSA)、XTEN、CTP或IgG Fc等延长半衰期部分连接。例如，Novo Nordisk公司的N9-GP(PEG化)、CSL Behring公司的FIX-FP(HSA融合蛋白)和OPKO/Prolor公司的长效FIX-CTP(CTP融合蛋白)均进入了临床研究阶段。N9-GP临床试验结果显示，累计给药3次可使FIX半衰期延长5倍(平均半衰期为110h)，然而试验观测到1例严重的超敏反应，3例产生了非抑制性抗体。N9-GP的免疫原性还有待进一步研究(Collins PW等，Blood，2014，124(26)：3880-3886)。FIX-FP临床试验结果显示，FIX-FP半衰期为89-96小时，且病人没有出现特殊免疫反应。FIX-CTP在血友病B小鼠模型结果显示，FIX半衰期延长了4倍，出血频率和时间降低，但FIX活性也被降低(Hart G等,Haemophilia,2012,18:32.)。首个FIX和Fc融合蛋白(FIX-Fc)与2014年3月通过美国FDA批准上市，商品名为Alprolix(Biogen Idec公司)，是目前唯一获批的重组长效FIX药物。Alprolix是人IgG1的双链Fc片段N末端共价结合单个FIX分子形成的融合蛋白，由HEK-293H细胞重组表达。临床研究显示，Alprolix半衰期在57-86小时，预防性用药时用药频次可达7或10天给药一次。目前，Alprolix已在全球多个国家获批上市。但同样Fc融合后也不可避免地发生了比活性降低问题，体外活性检测证实FIX-Fc的摩尔比活性(IU/nmol)仅为FIX
Figure PCTCN2017079872-appb-000001
的50％(Peters RT等,Blood,2010,115(10):2057-64)。
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，研究表明它具有延长体内半衰期作用。中国专利CN103539860A和CN103539861A公开了一种融合蛋白，以CTP作为接头连接FSH的β亚基和α亚基，延长融合蛋白体内半衰期。专利WO2013121416公开了一种包含至少一个CTP的长效凝血因子IX，所述CTP连接至凝血因子IX的羧基端。相对于rhFIX、FIX-CTP或FIX-CTP-CTP而言，包含3个串联CTP的FIX-(CTP)3表现出改善的药代动力学性能，FIX-CTP与rhFIX具有相当的体外活性和半衰期，FIX-CTP-CTP在大鼠和FIX-缺陷型小鼠中的半衰期是rhFIX的3倍，FIX-(CTP)3是rhFIX的2.5-4倍。 然而，体内凝血试验中FIX-(CTP)3表现出了降低的凝血活性，此外，与BeneFIX相比，FIX-(CTP)3的凝血活性会延迟至1小时后开始，这可能是由于添加三个串联的CTP可能掩蔽FIX的活化位点，延迟级联开始。
本发明人没有根据现有技术将CTP作为单独接头或者作为延长半衰期部分，而是将它与柔性肽接头(例如(GGGGS)n)连接组成含有GS的柔性肽接头与含有多个糖基侧链的刚性CTP肽接头的混合型连接肽，设置于FIX和延长半衰期部分之间(如，免疫球蛋白Fc片段，但不包括现有技术所提示的CTP)之间，组成新的FIX融合蛋白，不仅进一步延长了半衰期，并且降低了免疫原性，提高了生物利用度，大大降低了融合配体Fc对FIX的位阻效应，保持了良好的生物学活性和功能。
发明内容
本发明提供一种高糖基化的同源二聚体型凝血因子IX(FIX)的Fc融合蛋白，具有延长的体内活性半衰期，较低的免疫原性且与重组FIX相似的生物学活性。同时，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FIX相似的优点。
在本发明第一方面，提供一种高糖基化FIX融合蛋白，所述融合蛋白从N端至C端依次含有人凝血因子IX(hFIX)、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(CTP)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hFIX-L-CTP-vFc。
其中，所述hFIX为野生型或其突变型；进一步地，所述野生型hFIX具有如SEQ ID NO：1所示的氨基酸序列；优选地，所述突变型hFIX与SEQ ID NO：1所示氨基酸序列至少85％同源；更优选地，所述突变型hFIX与SEQ ID NO：1所示氨基酸序列至少90％同源。最优选地，所述突变型hFIX与SEQ ID NO：1所示氨基酸序列至少95％同源。
其中，所述柔性肽接头优选非免疫原性的，并且在hFIX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)。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刚性单元选自由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：2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FIX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：2N端的10个氨基酸，即SSSS*KAPPPS*；或所述CTP刚性单元包含SEQ ID NO：2C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：2N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：2N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)CTP1：PRFQDSSSSKAPPPSLPSPSRLPGPSDTPILPQ；
(ii)CTP2：SSSSKAPPPSLPSPSRLPGPSDTPILPQ；
(iii)CTP3：SSSSKAPPPS；
(iv)CTP4：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，例如，本发明的一实施例中，所述融合蛋白包含2个CTP3刚性单元：SSSSKAPPPSSSSSKAPPPS(CTP3-CTP3，或表示为(CTP3)2)。
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)vFcγ1：含有Leu234Val、Leu235Ala和Pro331Ser突变的人IgG1绞链区、CH2和CH3区域(如SEQ ID NO：3所示氨基酸序列)；
(ii)vFcγ2-1：含有Pro331Ser突变的人IgG2绞链区、CH2和CH3区域(如SEQ ID NO：4所示氨基酸序列)；
(iii)vFcγ2-2：含有Thr250Gln和Met428Leu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)；
(iv)vFcγ2-3：含有Pro331Ser、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)；
(v)vFcγ4：含有Ser228Pro和Leu235Ala突变的人IgG4绞链区、CH2和CH3区域(如SEQ ID NO：7所示氨基酸序列)。
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。IgG4Fc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FIX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：8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：9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1mg/106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>200IU/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FIX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，包括用于制备FIX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B患者的自发或手术性出血的预防或治疗的药物中应用。
本发明人发现，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、融合蛋白采用的融合配体人IgG Fc变体是非裂解性的，降低了与FcγRs及Clq结合而触发的效应子功能。
2、较重组FIX，本发明所述融合蛋白可以预期具有降低的免疫原性，降低患者体内中和抗体的产生。
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稳定性。
4、本发明提供的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FIX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。
5、本发明提供的所述融合蛋白的制备方法，产量高，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。
6、本发明构建的融合蛋白相对于Biogen公司构建的单体-二聚体杂合型(Monomeric)FIX融合蛋白，其表达、纯化步骤更加高效便捷、可大大降低生产成本。Biogen公司构建了rFIXFc与Fc的双表达载体，其中Fc分子以Flag标记(欧洲专利，公开号：EP1624891B1)。其所表达的融合蛋白发酵液中预期应含有三种形式的产物，分别是FIX-Fc:FIX-Fc同源二聚体型(Dimeric)融合蛋白、FIX-Fc:FLAG-Fc单体-二聚体杂合体(Monomeric)融合蛋白以及FLAG-Fc:FLAG-Fc二聚体三种产物。一方面，在融合蛋白表达过程中，因宿主细胞需同时表达FIX-Fc和Fc两种单链分子，再分别两两聚合形成上述三种产物，因而使最终目的产物的表达效率大大减弱；再者，在纯化过程中还必须去除另外两种形式的杂质，这使其纯化过程也更为复杂、生产效率低下，其生产成本也大大增加。因此，本发明的制备方法相对于Biogen公司开发的Monomeric rFIXFc融合蛋白具有一定的技术优势和价格优势，其表达、纯化工艺都更简单、高效，生产成本也更低。
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)。天然的CTP含有37个氨基酸残基，它具有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，从而延长蛋白在体内的半衰期(Fares F A等,Proc Natl Acad Sci USA,1992,89(10):4304-4308)。然而，本发明人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FIX与延长半衰期部分(如，免疫球蛋白Fc片段)。
本发明人发现，通过在FIX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FIX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FIX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FIX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FIX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FIX-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pH6.0时结合，在pH7.4(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pH6.0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，可获得活性良好，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、显示了在pF9-5B表达载体内Spe I-EcoR I片段的融合蛋白的核苷酸序列及推导的氨基酸序列。成熟的融合蛋白含有hFIX、柔性肽接头(以下划线标注)、CTP刚性单元(以下划线
Figure PCTCN2017079872-appb-000002
标注)和vFcγ2-3变体。
图2、纯化后融合蛋白F9-5B的SEC-HPLC峰谱图。
图3、纯化后融合蛋白F9-5B的SDS-PAGE电泳图。
具体实施方式
实施例1构建编码FIX融合蛋白的表达质粒
编码全长FIX的基因序列以及不同长度柔性肽接头、不同长度CTP刚性肽和不同IgG Fc变体的基因序列都是人工优化的CHO细胞偏爱密码子，经化学合成方法获得。为了便于将目的片段插入表达载体的特定位点，在所合成片段5’和3’端各有一个限制性酶内切位点，分别为SpeI和EcoRI。测序验证后的融合基因用SpeI和EcoRI酶切，然后插入到以PCDNA3.1为模板并改造后的表达质粒PXY1A1的相应酶切位点间，得到融合基因表达质粒pF9-5。PXY1A1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高外源表达所需增强子；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)。
如表1所示，本发明构建了一系列hFIX融合蛋白，它们具有不同长度的柔性肽接头、不同组成的CTP刚性单元以及几种不同亚型的IgG Fc(vFc)变体元 件。其中，F9-5B的核苷酸序列及翻译的氨基酸序列如图1所示。
表1、构建的几种FIX融合蛋白组成
Figure PCTCN2017079872-appb-000003
实施例2瞬时表达不同融合蛋白和体外活性测定
将实施例1中得到的一系列表达质粒，在30ml的摇瓶里使用DNAFect LT试剂TM(ATGCell公司)转染3×107CHO-K1细胞，经转染的细胞在含有1000ng/ml维生素K1的无血清生长培养基中生长5天，测定上清液中的融合蛋白浓度，并用实施例6中描述的方法测定其活性。ELISA结果显示几种质粒在该条件下的瞬时表达量相似，但是它们凝血活性却显示出较大差别。其中，我们将F9-5A的摩尔比活性定义为100％,F9-5B、F9-5C、F9-5D和F9-5E的活性分别为F9-5A的119.5％、104.2％、83.9％和94.7％。F9-5F活性仅为F9-5B的30％左右，这可能是CTP刚性单元置于Fc的N端，可以形成固定空间构象，能够有效的分开融合蛋白的不同功能区，这更有利于FIX和Fc部分独立折叠形成正确三维构象，从而保持较高活性。F9-5G细胞培养上清液中的融合蛋白多以无活性聚合体形式存在，这可能是由于过长的肽接头不仅不能提高融合蛋白的活性，反而会使蛋白发生错误折叠，以无活性多聚体形式存在。
实施例3融合蛋白在转染细胞系中的表达
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FIX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CHO细胞(美国专利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3×107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12-15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，也可用抗FIX的ELISA分析方法进行融合蛋白表达的定量测定，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2)mg/106(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4.生产融合蛋白
将实施例3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3筛选得到的生产融合蛋白F9-5B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18×106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30mg/L，在摇瓶中活细胞密度最高可达到35×106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5.纯化与定性融合蛋白
本发明主要采用亲和层析法对FIX融合蛋白F9-5B进行纯化(本实施例采用的蛋白纯化仪为美国GE公司的AKTA Explorer 100；本实施例中试剂均购自国 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Mabselect Sure或其它市售的重组protein A亲和层析介质(例如GE的Mabselect、Mabselect Sure LX、博格隆耐碱Protein A Diamond、TOSOH的Toyopearl AF-rProteinA-650F、天地人和的rProtein A Bead，赛分科技的MabPurix、Pall的KANEKA KanCapA、Merck的Eshumono A)进行样品捕获、浓缩以及部分污染物去除。首先使用平衡buffer：20mM PB，140mM NaCl，pH6.8-7.4，以50-100cm/h的线性流速平衡层析柱3-5个柱体积；将经过澄清后的发酵液以50-100cm/h的线性流速上样；上样完毕后，使用平衡buffer：20mM PB，140mM NaCl，pH6.8-7.4，以50-100cm/h的线性流速平衡层析柱3-5个柱体积，冲洗未结合的组份；使用去污buffer 1：20mM Citric-Citrate，0.5M NaCl，pH4.8-5.2，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；使用去污buffer 2：20mM Citric-Citrate，pH4.8-5.2，以50-100cm/h的线性流速平衡层析柱3-5个柱体积；之后使用洗脱buffer：50mM NaAc-HAc，1.0M Urea，pH3.0-4.0，以不高于60cm/h的线性流速洗脱目标产物，收集目标峰，用1M Tris，pH9.0中合pH值至中性偏酸环境(pH4.8-5.2)。
第二步，阴离子层析：使用GE公司的Q Sepharase FF或其它市售的阴离子层析介质(例如GE的DEAE Sepharose FF、Q Sepharose HP、Capto Q、Capto DEAE、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，用于降低HCP、残留DNA、脱落ProteinA等。第一步亲和洗脱液中仍含有一定比例的HCP、残留DNA、内毒素等污染物，因此要想办法对这些污染物进行去除。首先，使用平衡buffer：40mM Na2PO4-Citric，0.1M NaCl，pH4.8-5.2，以50-100cm/h的线性流速平衡层析柱3-5个柱体积(CV)；亲和捕获的样品使用平衡buffer稀释1倍，然后上样，在此条件下目的蛋白流穿，待A280上升至100mAU时开始收集流穿样；上样完毕后，使用平衡buffer：40mM Na2PO4-Citric，0.1M NaCl，pH4.8-5.2，以50-100cm/h的线性流速继续冲洗层析柱并继续收集流穿峰至A280下降至100mAU，此时停止收集；之后使用再生buffer：1M NaCl，1M NaOH，以50-100cm/h的线性流速冲洗层析柱3-5个柱体积，对层析柱进行再生处理，收集的样品送检HCP、DNA、ProteinA、SEC-HPLC。
第三步，亲和层析：使用JNC公司的Cellufine Sulfate或其它市售的亲和层析介质(例如GE的Heparin FF、Heparin HP)进行最后精纯，去除聚体、进一步去除HCP、DNA等污染物。首先使用平衡buffer：20mM PB，100mM NaCl，pH7.0-7.4，以50-100cm/h的线性流速冲洗层析柱3-5个柱体积；经第二步阴离子层析分离得到的目标蛋白用平衡buffer稀释1倍，降低有机物浓度后上样；上样完毕，使用平衡buffer：20mM PB，100mM NaCl，pH7.0-7.4，以50-100cm/h的线性流速冲洗层析柱3-5个柱体积；之后采用线性梯度的盐浓度进行洗脱，洗脱buffer：20mM PB，1M NaCl，pH7.0-7.4，条件为洗脱buffer从0-100％，15个柱体积，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，计算出蛋白比活力约为200IU/mg。
样品SEC-HPLC纯度和SDS-PAGE电泳结果分别见图2和图3。SEC-HPLC结果显示，纯化后融合蛋白的主峰纯度大于90％，SDS-PAGE电泳带型符合预期，非还原电泳包含融合蛋白，还原后可得清晰的单链条带。
实施例6.发色底物法测定融合蛋白体外活性
FIX-Fc融合蛋白的活性可采用发色底物法测定。本实施例采用BIOPHEN Factor IX试剂盒(HYPHEN BioMed，Ref.A221802)测定，其检测原理如下:试剂盒中提供的因子XIa会将测试样品中的因子IX活化为FIXa，活化的FIXa在凝血酶、磷脂(PLPs)和钙离子(Ca2+)的存在下，与凝血酶激活的FVIII:C、PLPs和Ca2+形成凝血酶复合物，继而将测定系统中的因子X转变为激活形式的Xa。凝血酶复合物对因子X的激活活性与测试样品中因子IX的含量呈正相关关系。激活的因子Xa活性可通过其对发色底物(SXa-11)特异性裂解进行检测，即在405nm下检测其裂解产物pNA的吸光值，pNA吸光值与FIXa活性成正比。
以本方法测定纯化的FIX融合蛋白F9-5B的比活性可达200IU/mg以上。
实施例7融合蛋白的药代动力学测定
雄性SPF级SD大鼠(购自上海必凯实验动物有限公司)，预饲一周后随机分为2组，每组2只，分别单次给予静脉注射4.5mg/kg(高剂量组)和1.5mg/kg (低剂量组)的F9-5B融合蛋白，考察血药浓度和时间的变化规律。对照组和给药组分别在给药后0、1、3、6、24、48、72、96、120、144和168小时经眼眶采血，每次约0.3ml。取血后静置，血液在室温放置30min后5000rpm离心10min，分离出血清，-20℃保存。用针对FIX特异的ELISA方法测定各时间点血清中融合蛋白的含量。通过软件PKSOLVER，计算各组主要药代动力学参数，结果见表2。
表2、FIX融合蛋白在SD大鼠中的药代动力学参数
Figure PCTCN2017079872-appb-000004
根据药代动力学数据可以看出，高、低剂量F9-5B融合蛋白的体内半衰期分别为31和30小时，与rhFIX相比T1/2β值增加了8倍(中国专利CN104427994)。F9-5B融合蛋白表现出了与rhFIX相比改善的半衰期，证明在FIX C端添加连接肽和Fc变体并没有干扰融合蛋白的活性，反而对FIX融合蛋白的活性和半衰期产生了意外的技术效果，推测这是由于通过柔性肽接头和CTP连接FIX与Fc变体，CTP刚性肽不仅能进一步延长其体内半衰期，同时借助多个糖基化侧链的阻隔作用，增加融合蛋白分子间的空间距离，促使FIX和Fc段各自折叠形成正确的三维构象而互不影响各自的生物活性。由此可见，相比rhFIX，F9-5B在生物利用度和药代动力学等方面表现出更为优异的性能。
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 (26)

  1. 一种人凝血因子IX的融合蛋白，所述融合蛋白从N端至C端依次包含人凝血因子IX、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，所述延长半衰期部分包含免疫球蛋白Fc段、白蛋白、转铁蛋白或PEG。
  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。
  3. 如权利要求2所述的融合蛋白，其特征在于，所述融合蛋白是通过在哺乳动物细胞中表达而糖基化的。
  4. 如权利要求3所述的融合蛋白，其特征在于，所述融合蛋白是通过在中国仓鼠卵巢细胞中表达而糖基化的。
  5. 如权利要求1所述的融合蛋白，其特征在于，所述人凝血因子IX包含如SEQ ID NO：1所示的氨基酸序列，或者所述人凝血因子IX的氨基酸序列与SEQ ID NO：1所示的氨基酸序列具有至少85％、90％或95％的同一性。
  6. 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。
  7. 如权利要求6所述的融合蛋白，其特征在于，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。
  8. 如权利要求7所述的融合蛋白，其特征在于，所述柔性肽接头优选自下组：
    (i)GSGGGSGGGGSGGGGS；
    (ii)GSGGGGSGGGGSGGGGSGGGGSGGGGS；
    (iii)GGGGSGGGGSGGGGSGGGGS；
    (iv)GSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS；
    (v)GGGSGGGSGGGSGGGSGGGS。
  9. 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：2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。
  10. 如权利要求9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以下氨基酸序列：
    (i)PRFQDSSSSKAPPPSLPSPSRLPGPSDTPILPQ；
    (ii)SSSSKAPPPSLPSPSRLPGPSDTPILPQ；
    (iii)SSSSKAPPPS；
    (iv)SRLPGPSDTPILPQ。
  11. 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9或10所述刚性单元的氨基酸序列至少具有70％，80％，90％或95％的同一性。
  12. 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。
  13. 如权利要求1所述的融合蛋白，其特征在于，所述融合蛋白的延长半衰期部分为人免疫球蛋白Fc变体。
  14. 如权利要求13所述的融合蛋白，其特征在于，所述人免疫球蛋白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。
  15. 如权利要求14所述的融合蛋白，其特征在于，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。
  16. 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：8所示。
  17. 如权利要求1-16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>200IU/mg。
  18. 编码如权利要求1-17中任一项所述的融合蛋白的DNA分子。
  19. 如权利要求18所述的DNA分子，其特征在于，包含如SEQ ID NO：9所示序列。
  20. 一种载体，其特征在于，包含如权利要求18或19所述的DNA分子。
  21. 一种宿主细胞，其特征在于，包含如权利要求20所述的载体，或者转染了权利要求20所述的载体。
  22. 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1-17中任一项所述的融合蛋白。
  23. 一种如权利要求1-17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18或19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1mg/106(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。
  24. 如权利要求23所述的方法，其特征在于，步骤(a)中的CHO衍生细胞系为DXB-11。
  25. 如权利要求23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和阴离子交换层析。
  26. 一种如权利要求1-17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的药物中应用，包括用于制备FIX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B患者的自发或手术性出血的预防或治疗的药物中应用。
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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2020204470B1 (en) * 2020-07-03 2021-05-27 Ampsource Biopharma Shanghai Inc. Dual-function protein for lipid and blood glucose regulation
US11471513B2 (en) 2016-08-19 2022-10-18 Ampsource Biopharma Shanghai Inc. Highly glycosylated human blood-clotting factor VIII fusion protein, and manufacturing method and application of same
US11472863B2 (en) 2016-08-19 2022-10-18 Ampsource Biopharma Shanghai Inc. Human coagulation factor IX (FIX) fusion protein, preparation method therefor, and use thereof
US11981718B2 (en) 2020-05-27 2024-05-14 Ampsource Biopharma Shanghai Inc. Dual-function protein for lipid and blood glucose regulation

Families Citing this family (49)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106256835A (zh) * 2016-08-19 2016-12-28 安源医药科技（上海）有限公司 高糖基化人生长激素融合蛋白及其制备方法与用途
EP3502143A4 (en) 2016-08-19 2020-07-15 Ampsource Biopharma Shanghai Inc. BINDING PEPTIDE FOR THE CONSTRUCTION OF A FUSION PROTEIN
CN108440668A (zh) * 2017-02-16 2018-08-24 瑞阳(苏州)生物科技有限公司 Fgf21与igf-1的融合蛋白及其应用
CN108623693B (zh) * 2017-03-20 2022-03-25 徐寒梅 一种融合蛋白及其制备方法和其在制备治疗眼科疾病、抗炎、抗肿瘤药物中的应用
CN107012186A (zh) * 2017-03-29 2017-08-04 江南大学 生产低聚果糖的方法
CN107082815A (zh) * 2017-06-28 2017-08-22 杭州皓阳生物技术有限公司 一种fsh‑hsa融合蛋白及其制备方法
CN107312797B (zh) * 2017-07-28 2021-06-18 广州中科蓝华生物科技有限公司 一种蛋白调控系统及其制备方法和应用
CN109553684A (zh) * 2017-09-25 2019-04-02 中国科学院过程工程研究所 一种纳米载体蛋白及其制备方法和应用
CN113603794B (zh) * 2018-01-11 2024-01-16 安源医药科技（上海）有限公司 用于调节血糖和脂质的增效型双功能蛋白
WO2020113403A1 (en) * 2018-12-04 2020-06-11 Beijing Percans Oncology Co. Ltd. Cytokine fusion proteins
CN108383912A (zh) * 2018-02-06 2018-08-10 清华大学 人工融合蛋白及其用途
CN108404119B (zh) * 2018-05-07 2020-09-25 江苏康缘瑞翱生物医药科技有限公司 Fgf-21类似物的制备及在血栓治疗中的应用
AU2019271149B2 (en) * 2018-05-14 2023-07-13 Werewolf Therapeutics, Inc. Activatable interleukin 12 polypeptides and methods of use thereof
JP7460609B2 (ja) * 2018-05-14 2024-04-02 ウェアウルフ セラピューティクス， インコーポレイテッド 活性化可能なインターロイキン－２ポリペプチド及びその使用方法
CN109251896A (zh) * 2018-08-13 2019-01-22 中山大学 一株高表达hKLs-his蛋白的细胞株及其制备方法和应用
CN110950964B (zh) * 2018-09-26 2021-06-18 安源医药科技（上海）有限公司 突变型单链人凝血因子viii融合蛋白及其制备方法与用途
CN109439679A (zh) * 2018-10-09 2019-03-08 上海市儿童医院 一种高活性重组人凝血因子ix融合蛋白及其制备方法
WO2020088164A1 (zh) * 2018-11-01 2020-05-07 山东新时代药业有限公司 双特异性抗体及其用途
CN111195234B (zh) * 2018-11-16 2022-08-26 鲁南制药集团股份有限公司 一种重组FGF21-Fc融合蛋白冻干粉制剂
CN111378045B (zh) * 2018-12-28 2022-08-02 长春金赛药业有限责任公司 二价双特异性抗体及其制备方法、编码基因、宿主细胞、组合物
BR112021012472A2 (pt) * 2019-01-28 2021-11-30 Toray Industries Forma modificada por polietileno glicol de um fator de crescimento de hepatócito ou um fragmento ativo do mesmo, e, medicamento
US20220064244A1 (en) * 2019-03-05 2022-03-03 Sunshine Lake Pharma Co., Ltd. A polypeptide molecule and application thereof
CN111944035B (zh) * 2019-05-14 2022-08-12 温州医科大学 Fgf4及其应用
CN111944055B (zh) * 2019-05-16 2022-08-02 浙江道尔生物科技有限公司 一种治疗代谢疾病的融合蛋白
CN112138149A (zh) * 2019-06-28 2020-12-29 北京基科晟斯医药科技有限公司 重组凝血因子viii制剂
CN112175088B (zh) * 2019-07-02 2023-03-28 江苏晟斯生物制药有限公司 改进的fix融合蛋白、缀合物及其应用
CN112279920B (zh) 2019-07-25 2024-01-16 安源医药科技（上海）有限公司 FGF21 Fc融合蛋白、GLP-1 Fc融合蛋白及它们的组合治疗剂和用途
WO2021043127A1 (zh) * 2019-09-02 2021-03-11 甘李药业股份有限公司 嵌合蛋白
CN110713545B (zh) * 2019-09-11 2021-10-22 潍坊医学院 一种人源程序性细胞死亡因子受体蛋白-1及其生产方法和应用
CN110846297B (zh) * 2019-11-26 2023-04-07 温氏食品集团股份有限公司 一种多功能融合酶和多功能融合酶真核表达载体及其构建方法
CN115379850A (zh) * 2019-12-24 2022-11-22 裘美娜治疗公司 再生性多肽及其用途
CN113121696A (zh) * 2019-12-31 2021-07-16 周易 Fab改造诱导形成的双特异性抗体及其制备方法和用途
CN113444710A (zh) * 2020-03-25 2021-09-28 上海英脉德医疗科技有限公司 ACE2-Fc融合蛋白及其用途
CN113461827A (zh) * 2020-03-31 2021-10-01 安源医药科技（上海）有限公司 高效分离纯化重组人凝血因子VIII Fc融合蛋白的方法
CN111499764B (zh) * 2020-04-02 2022-02-08 北京翼方生物科技有限责任公司 一种具有促红细胞生成素活性的长效融合蛋白
CN111635461A (zh) * 2020-05-07 2020-09-08 乾元康安(苏州)生物科技有限公司 一种ACE2-Albumin重组蛋白及其制备方法和应用
CN112375149B (zh) * 2020-10-30 2023-04-18 沣潮医药科技（上海）有限公司 Ace2免疫融合蛋白及其应用
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CN113880954B (zh) * 2021-09-29 2024-05-14 江苏大学 一种重组人生长激素及其构建方法和应用
WO2023122257A2 (en) * 2021-12-22 2023-06-29 La Jolla Institute For Immunology Coronavirus spike glycoprotein with improved expression and stability
CN114426569A (zh) * 2022-01-25 2022-05-03 宁波甲贝医药科技有限公司 一种用于构建融合蛋白的连接肽
CN115141283A (zh) * 2022-03-10 2022-10-04 厦门柏慈生物科技有限公司 一种融合蛋白、制备方法及其应用
CN114807265A (zh) * 2022-03-31 2022-07-29 上海锐康生物技术研发有限公司 一种s-烟碱的合成方法
WO2023245543A1 (en) * 2022-06-23 2023-12-28 Ampsource Biopharma Shanghai Inc. Uses of fgf21 fusion proteins
CN117126832B (zh) * 2023-10-26 2024-01-30 四川维亚本苑生物科技有限公司 一种重组人凝血因子ix融合蛋白及其制备方法

Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102639144A (zh) * 2009-07-09 2012-08-15 波洛尔生物科技有限公司 长效凝固因子及产生其的方法
CN103140237A (zh) * 2010-07-09 2013-06-05 比奥根艾迪克依蒙菲利亚公司 因子ix多肽及其使用方法
CN103539860A (zh) * 2013-11-01 2014-01-29 广州优联康医药科技有限公司 一种长效重组人促卵泡激素融合蛋白
CN104114183A (zh) * 2011-12-20 2014-10-22 印第安纳大学研究及科技有限公司 用于治疗糖尿病的基于ctp的胰岛素类似物
CN104693270A (zh) * 2013-12-10 2015-06-10 清华大学 一种用于融合蛋白的连接肽
CN106117370A (zh) * 2016-08-19 2016-11-16 安源医药科技（上海）有限公司 高糖基化Exendin‑4及其类似物的融合蛋白、其制备方法和用途
CN106256835A (zh) * 2016-08-19 2016-12-28 安源医药科技（上海）有限公司 高糖基化人生长激素融合蛋白及其制备方法与用途
CN106279436A (zh) * 2016-08-19 2017-01-04 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
CN106279437A (zh) * 2016-08-19 2017-01-04 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
CN106317226A (zh) * 2016-08-19 2017-01-11 安源医药科技（上海）有限公司 用于构建融合蛋白的连接肽

Family Cites Families (91)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4399216A (en) 1980-02-25 1983-08-16 The Trustees Of Columbia University Processes for inserting DNA into eucaryotic cells and for producing proteinaceous materials
US4818679A (en) 1985-02-19 1989-04-04 The Trustees Of Columbia University In The City Of New York Method for recovering mutant cells
US6225449B1 (en) 1991-10-04 2001-05-01 Washington University Hormone analogs with multiple CTP extensions
US6103501A (en) * 1997-11-17 2000-08-15 Washington University Single chain glycoprotein hormones comprising two β and one α subunits and recombinant production thereof
US6660843B1 (en) 1998-10-23 2003-12-09 Amgen Inc. Modified peptides as therapeutic agents
WO2001032678A1 (en) 1999-11-05 2001-05-10 Smithkline Beecham Corporation sbgFGF-19a
US20040259780A1 (en) 2001-07-30 2004-12-23 Glasebrook Andrew Lawrence Method for treating diabetes and obesity
EP1329227A1 (en) 2002-01-22 2003-07-23 Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts Diagnostic conjugate useful for intercellular imaging and for differentiating between tumor- and non-tumor cells
US7081446B2 (en) * 2002-01-31 2006-07-25 The Trustees Of Columbia University In The City Of New York Long-acting follicle stimulating hormone analogues and uses thereof
US20050176108A1 (en) 2003-03-13 2005-08-11 Young-Min Kim Physiologically active polypeptide conjugate having prolonged in vivo half-life
ES2558102T3 (es) 2003-05-06 2016-02-02 Biogen Hemophilia Inc. Proteínas quiméricas del factor de coagulación para el tratamiento de un trastorno hemostático
KR100758755B1 (ko) 2003-06-12 2007-09-14 일라이 릴리 앤드 캄파니 Ｇｌｐ-1 유사체 융합 단백질
DE602004011770T2 (de) 2003-06-12 2009-02-05 Eli Lilly And Co., Indianapolis Fusionsproteine
CN1889937B (zh) 2003-12-03 2011-02-09 诺和诺德公司 糖基聚乙二醇化的因子ⅸ肽
WO2005058953A2 (en) * 2003-12-12 2005-06-30 Regeneron Pharmaceuticals, Inc. Ogh fusion polypeptides and therapeutic uses thereof
US20050250185A1 (en) 2003-12-12 2005-11-10 Murphy Andrew J OGH fusion polypeptides and therapeutic uses thereof
US20070265200A1 (en) * 2004-03-17 2007-11-15 Eli Lilly And Company Glycol Linked Fgf-21 Compounds
SI1751184T1 (sl) 2004-05-13 2010-01-29 Lilly Co Eli Fgf-21 fuzijski proteini
EP1789443A1 (en) 2004-09-02 2007-05-30 Eli Lilly And Company Muteins of fibroblast growth factor 21
DK1789442T3 (da) 2004-09-02 2009-11-16 Lilly Co Eli Muteiner af fibroblastvækstfaktor 21
CN102718859A (zh) 2004-09-29 2012-10-10 诺和诺德医疗保健公司 通过分级洗脱从阴离子交换材料中纯化批量凝血因子vii多肽
JP5948627B2 (ja) 2004-10-29 2016-07-20 レイショファーム ゲーエムベーハー 線維芽細胞成長因子（ｆｇｆ）のリモデリングと糖質ペグ化
EP2332985A3 (en) 2004-11-12 2012-01-25 Xencor, Inc. Fc variants with altered binding to FcRn
JP2008522617A (ja) 2004-12-14 2008-07-03 イーライ リリー アンド カンパニー 線維芽細胞成長因子２１の突然変異タンパク質
CN1331888C (zh) * 2005-03-02 2007-08-15 中国人民解放军军事医学科学院生物工程研究所 一种抗人b细胞淋巴瘤的单链双特异性抗体
US7989193B2 (en) 2005-04-13 2011-08-02 Medimmune Limited Compositions and methods for producing gamma-carboxylated proteins
EP1746161A1 (en) * 2005-07-20 2007-01-24 Cytheris Glycosylated IL-7, preparation and uses
ES2398574T3 (es) 2005-07-22 2013-03-20 Bayer Healthcare Llc Activación en solución de factor VII
US8048849B2 (en) * 2006-02-03 2011-11-01 Modigene, Inc. Long-acting polypeptides and methods of producing same
US8304386B2 (en) 2006-02-03 2012-11-06 Prolor Biotech, Inc. Long-acting growth hormone and methods of producing same
EP1816201A1 (en) 2006-02-06 2007-08-08 CSL Behring GmbH Modified coagulation factor VIIa with extended half-life
BRPI0809583B1 (pt) 2007-03-30 2022-02-22 Ambrx, Inc Polipeptídeo fgf-21 modificado, composição compreendendo o mesmo, método para produzir o referido polipetídeo fgf-21 e célula compreendendo um polinucleotídeo
SI2235059T1 (sl) 2007-12-26 2015-06-30 Xencor, Inc. Fc variante s spremenjeno vezjo na fcrn
US8420088B2 (en) * 2008-01-28 2013-04-16 Novartis Ag Methods and compositions using FGF23 fusion polypeptides
JOP20190083A1 (ar) 2008-06-04 2017-06-16 Amgen Inc بولي ببتيدات اندماجية طافرة لـfgf21 واستخداماتها
MX341149B (es) * 2008-10-10 2016-08-09 Amgen Inc Mutantes fgf21 y uso de los mismos.
CA2748593A1 (en) 2009-01-23 2010-07-29 Novo Nordisk A/S Fgf21 derivatives with albumin binder a-b-c-d-e- and their use
PE20120358A1 (es) * 2009-05-05 2012-04-26 Amgen Inc Mutantes fgf21 y usos de los mismos
CA2760674A1 (en) 2009-05-05 2010-11-11 Amgen Inc. Fgf21 mutants and uses thereof
EP2440235A1 (en) 2009-06-11 2012-04-18 Novo Nordisk A/S Glp-1 and fgf21 combinations for treatment of diabetes type 2
BR112012013502A2 (pt) 2009-12-06 2017-01-10 Biogen Idec Hemophilia Inc "polipeptídeos quiméricos e híbridos de fator viii-fc, e métodos de uso dos mesmos".
UA109888C2 (uk) 2009-12-07 2015-10-26 ІЗОЛЬОВАНЕ АНТИТІЛО АБО ЙОГО ФРАГМЕНТ, ЩО ЗВ'ЯЗУЄТЬСЯ З β-КЛОТО, РЕЦЕПТОРАМИ FGF І ЇХНІМИ КОМПЛЕКСАМИ
US9493543B2 (en) 2010-02-16 2016-11-15 Novo Nordisk A/S Factor VIII fusion protein
MX2012011986A (es) 2010-04-15 2013-03-05 Amgen Inc RECEPTOR FGF HUMANO Y PROTEINAS ENLAZADAS A ß-KLOTHO.
DK2591099T3 (da) 2010-07-09 2021-02-15 Bioverativ Therapeutics Inc Kimære koagulationsfaktorer
EP3508573A1 (en) 2010-07-09 2019-07-10 Bioverativ Therapeutics Inc. Systems for factor viii processing and methods thereof
JP6069198B2 (ja) 2010-07-20 2017-02-01 ノヴォ ノルディスク アー／エス Ｎ末端が修飾されたｆｇｆ２１化合物
US9023791B2 (en) 2010-11-19 2015-05-05 Novartis Ag Fibroblast growth factor 21 mutations
ES2628385T3 (es) 2011-05-16 2017-08-02 F. Hoffmann-La Roche Ag Agonistas de FGFR1 y procedimientos de uso
US9574002B2 (en) 2011-06-06 2017-02-21 Amgen Inc. Human antigen binding proteins that bind to a complex comprising β-Klotho and an FGF receptor
EP2717898B1 (en) 2011-06-10 2018-12-19 Bioverativ Therapeutics Inc. Pro-coagulant compounds and methods of use thereof
EP2537864B1 (en) 2011-06-24 2019-08-07 Laboratoire Français du Fractionnement et des Biotechnologies Fc variants with reduced effector functions
PE20141727A1 (es) 2011-07-01 2014-11-26 Ngm Biopharmaceuticals Inc Composiciones, usos y metodos para tratamiento de trastornos y enfermedades metabolicas
EA029045B1 (ru) 2011-07-08 2018-02-28 Байоджен Хемофилия Инк. Химерные и гибридные полипептиды фактора viii и способы их применения
EA028914B1 (ru) 2011-07-25 2018-01-31 Байоджен Хемофилия Инк. Исследования для мониторинга нарушений свертываемости крови
UY34346A (es) * 2011-09-26 2013-04-30 Novartis Ag Proteínas de fusión para tratar trastornos metabólicos
TW201315742A (zh) 2011-09-26 2013-04-16 Novartis Ag 治療代謝病症之雙功能蛋白質
KR102041412B1 (ko) 2011-12-30 2019-11-11 한미사이언스 주식회사 면역글로불린 Ｆｃ 단편 유도체
ES2700583T3 (es) 2012-01-12 2019-02-18 Bioverativ Therapeutics Inc Procedimientos para reducir la inmunogenicidad contra el Factor VIII en individuos sometidos a terapia con Factor VIII
MY172539A (en) 2012-02-14 2019-11-30 Opko Biologics Ltd Long-acting coagulation factors and methods of producing same
WO2013152351A2 (en) 2012-04-06 2013-10-10 The Trustees Of Columbia University In The City Of New York Fusion polypeptides and methods of use thereof
CA2875247A1 (en) * 2012-06-08 2013-12-12 Biogen Idec Ma Inc. Chimeric clotting factors
AU2013270682A1 (en) 2012-06-08 2014-12-11 Biogen Ma Inc. Procoagulant compounds
TWI513705B (zh) 2012-06-11 2015-12-21 Lilly Co Eli 纖維母細胞生長因子２１蛋白質
EP2870250B2 (en) 2012-07-06 2022-06-29 Bioverativ Therapeutics Inc. Cell line expressing single chain factor viii polypeptides and uses thereof
SG11201500045RA (en) 2012-07-11 2015-02-27 Amunix Operating Inc Factor viii complex with xten and von willebrand factor protein, and uses thereof
WO2014018777A2 (en) 2012-07-25 2014-01-30 Biogen Idec Ma Inc. Blood factor monitoring assay and uses thereof
EP2882457A1 (en) 2012-08-13 2015-06-17 Novo Nordisk A/S Liquid factor viii formulations
BR112015004734A2 (pt) 2012-09-07 2017-11-21 Sanofi Sa proteínas de fusão para tratar uma síndrome metabólica
JOP20200236A1 (ar) * 2012-09-21 2017-06-16 Regeneron Pharma الأجسام المضادة لمضاد cd3 وجزيئات ربط الأنتيجين ثنائية التحديد التي تربط cd3 وcd20 واستخداماتها
WO2014052490A1 (en) * 2012-09-25 2014-04-03 Biogen Idec Ma Inc. Methods of using fix polypeptides
EP2908847B1 (en) 2012-10-18 2022-03-30 Bioverativ Therapeutics Inc. Methods of using a fixed dose of a clotting factor
BR122020018510B1 (pt) * 2012-11-20 2023-03-14 Opko Biologics Ltd Método para aumentar incrementalmente o tamanho hidrodinâmico de um fator de coagulação ativado viia
EP2938636A2 (en) * 2012-12-28 2015-11-04 AbbVie Inc. High-throughput system and method for identifying antibodies having specific antigen binding activities
EA201890671A1 (ru) 2013-03-15 2019-01-31 Биовератив Терапьютикс Инк. Препараты полипептида фактора viii
TW201536811A (zh) 2013-05-31 2015-10-01 Biogen Idec Inc 嵌合ｆｖｉｉ－ｘｔｅｎ分子及其用途
CN103599527A (zh) * 2013-08-16 2014-02-26 安源生物科技(上海)有限公司 包含改良型人凝血因子FVII-Fc融合蛋白的药物组合物
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
CN103539869B (zh) 2013-10-11 2015-11-18 浙江凯胜畜产品加工有限公司 一种提高粗肝素钠提取工艺酶解效率的方法
CN103554268B (zh) * 2013-11-01 2015-04-01 广州联康生物科技有限公司 长效重组促卵泡激素及其应用
CN103539861B (zh) 2013-11-01 2015-02-18 广州联康生物科技有限公司 长效重组人促卵泡激素融合蛋白
CN103539862B (zh) 2013-11-01 2015-04-01 广州联康生物科技有限公司 一种长效重组促卵泡激素及其应用
CN103539868B (zh) 2013-11-01 2016-05-04 上海春露生物化学有限公司 一种制备羧甲基壳聚糖的方法
CN103897064B (zh) 2013-11-29 2016-05-11 广州优联康医药科技有限公司 长效重组人绒促性素融合蛋白
CA2934081A1 (en) * 2013-12-23 2015-07-02 Csl Limited Fusion proteins comprising factor ix for prophylactic treatment of hemophilia and methods thereof
CN105753945B (zh) * 2014-12-15 2019-04-09 清华大学无锡应用技术研究院 一种连接肽及其应用
KR20160088656A (ko) 2015-01-16 2016-07-26 주식회사유한양행 지속형 fgf21 융합 단백질 및 이를 포함하는 약학적 조성물
CN104558191B (zh) * 2015-01-21 2020-08-21 武汉友芝友生物制药有限公司 一种双特异性抗体cd20×cd3的构建及应用
AU2016346870B2 (en) 2015-10-28 2021-04-22 Yuhan Corporation Dual function proteins and pharmaceutical composition comprising same
EP3502143A4 (en) 2016-08-19 2020-07-15 Ampsource Biopharma Shanghai Inc. BINDING PEPTIDE FOR THE CONSTRUCTION OF A FUSION PROTEIN
CN113603794B (zh) 2018-01-11 2024-01-16 安源医药科技（上海）有限公司 用于调节血糖和脂质的增效型双功能蛋白

Patent Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102639144A (zh) * 2009-07-09 2012-08-15 波洛尔生物科技有限公司 长效凝固因子及产生其的方法
CN103140237A (zh) * 2010-07-09 2013-06-05 比奥根艾迪克依蒙菲利亚公司 因子ix多肽及其使用方法
CN104114183A (zh) * 2011-12-20 2014-10-22 印第安纳大学研究及科技有限公司 用于治疗糖尿病的基于ctp的胰岛素类似物
CN103539860A (zh) * 2013-11-01 2014-01-29 广州优联康医药科技有限公司 一种长效重组人促卵泡激素融合蛋白
CN104693270A (zh) * 2013-12-10 2015-06-10 清华大学 一种用于融合蛋白的连接肽
CN106117370A (zh) * 2016-08-19 2016-11-16 安源医药科技（上海）有限公司 高糖基化Exendin‑4及其类似物的融合蛋白、其制备方法和用途
CN106256835A (zh) * 2016-08-19 2016-12-28 安源医药科技（上海）有限公司 高糖基化人生长激素融合蛋白及其制备方法与用途
CN106279436A (zh) * 2016-08-19 2017-01-04 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
CN106279437A (zh) * 2016-08-19 2017-01-04 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
CN106317226A (zh) * 2016-08-19 2017-01-11 安源医药科技（上海）有限公司 用于构建融合蛋白的连接肽

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11471513B2 (en) 2016-08-19 2022-10-18 Ampsource Biopharma Shanghai Inc. Highly glycosylated human blood-clotting factor VIII fusion protein, and manufacturing method and application of same
US11472863B2 (en) 2016-08-19 2022-10-18 Ampsource Biopharma Shanghai Inc. Human coagulation factor IX (FIX) fusion protein, preparation method therefor, and use thereof
US11981718B2 (en) 2020-05-27 2024-05-14 Ampsource Biopharma Shanghai Inc. Dual-function protein for lipid and blood glucose regulation
AU2020204470B1 (en) * 2020-07-03 2021-05-27 Ampsource Biopharma Shanghai Inc. Dual-function protein for lipid and blood glucose regulation

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