WO2018032637A1 - 高糖基化人凝血因子viii融合蛋白及其制备方法与用途 - Google Patents

高糖基化人凝血因子viii融合蛋白及其制备方法与用途 Download PDF

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WO2018032637A1
WO2018032637A1 PCT/CN2016/106010 CN2016106010W WO2018032637A1 WO 2018032637 A1 WO2018032637 A1 WO 2018032637A1 CN 2016106010 W CN2016106010 W CN 2016106010W WO 2018032637 A1 WO2018032637 A1 WO 2018032637A1
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fusion protein
human
fviii
amino acid
ctp
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PCT/CN2016/106010
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English (en)
French (fr)
Inventor
李强
朱文臣
李媛丽
朱成功
高永娟
任子甲
朱鹿燕
孙乃超
王晓山
刘宾
李智
王文文
姜明
王齐磊
王莉蕊
王淑亚
朱松林
高洁
苏鸿声
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安源医药科技（上海）有限公司
辅仁药业集团有限公司
旭华（上海）生物研发中心有限公司
开封制药(集团)有限公司
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Priority to US16/479,494 priority Critical patent/US11471513B2/en
Priority to BR112019003302A priority patent/BR112019003302A2/pt
Priority to RU2019106765A priority patent/RU2722374C1/ru
Priority to JP2019530527A priority patent/JP6923115B2/ja
Priority to MX2019001925A priority patent/MX2019001925A/es
Priority to KR1020197007918A priority patent/KR102276157B1/ko
Priority to GB1903581.5A priority patent/GB2568624B/en
Publication of WO2018032637A1 publication Critical patent/WO2018032637A1/zh

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    • 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
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    • A61K47/65—Peptidic linkers, binders or spacers, e.g. peptidic enzyme-labile linkers
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    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
    • A—HUMAN NECESSITIES
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    • 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]
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    • C07K14/745—Blood coagulation or fibrinolysis factors
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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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 VIII (FVIII), a preparation method and use thereof, and particularly to the use of various coagulation-related diseases.
  • FVIII human coagulation factor VIII
  • Factor VIII also known as anti-hemophilia factor, plays an important role in endogenous coagulation systems. According to a large number of studies on molecular genetics of FVIII, it is indicated that the lack of FVIII in the sex chromosome X-linked gene leads to hemophilia A. According to statistics, the prevalence of hemophilia A in the male population is 1 in 5,000, accounting for more than 80% of the total number of hemophilia. The currently used treatment for hemophilia A is replacement therapy, which is to supplement factor VIII that is lacking in patients with hemophilia.
  • FVIII is a multi-structured macromolecular glycoprotein divided into six domains: three A domains (A1, A2, A3), one carbohydrate-rich and non-essential central domain (B-domain) and two C domain (C1, C2).
  • the mature protein consists of a light chain and a heavy chain of approximately 280 kDa.
  • the light chain has a molecular weight of about 80 kDa, and the structure includes A3, C1 and C2 in a connection mode of A3-C1-C2.
  • the heavy chain has a molecular weight of about 90 to 200 kDa, and the structure includes A1, A2 and B in the form of A1-A2-B.
  • Heavy and light chain linkages are metal ion dependent.
  • FVIII is activated by amino acid Arg372 and Arg740 in the heavy chain, and proteolytic cleavage by activated clotting factor FX (FXa) and thrombin at Arg1689 in the light chain, resulting in the release of vWF factor and production of activated FVIII dimer ( FVIIIa), in the presence of Ca2+, forms a tight complex with activated coagulation factors FIX (FIXa) and FX on the phospholipid surface, FX is then activated by FIXa, and activated FX is dissociated from the complex, which in turn condenses The zymogen is converted to thrombin, which converts fibrinogen directly into fibrin.
  • FVIIIa activated clotting factor
  • thrombin thrombin at Arg1689 in the light chain
  • the FVIII molecule is one of the longest gene fragments cloned to date, and is the most abundant protein drug used in clinical practice. Mammalian cells are optimal for recombinant proteins with high molecular weight and high glycosylation Expression system. However, the amount of recombinant expression of FVIII in vitro was significantly lower than that of other genes, such as FVIII expression level of only 1% of FIX. This may be a reflection of the body's need for FVIII, but this is undoubtedly a major obstacle to recombinant expression of FVIII in vitro. In addition, since FVIII has a short half-life in the blood, only 8-12 hours, patients with severe hemophilia A undergo prophylactic treatment, and must be injected intravenously (i.v.) about 3 times a week.
  • FVIII FVIII to extended half-life moieties such as PEG, human serum albumin (HSA), transferrin or IgG Fc.
  • extended half-life moieties such as PEG, human serum albumin (HSA), transferrin or IgG Fc.
  • PEG polyethylene glycol
  • HSA human serum albumin
  • transferrin transferrin
  • IgG Fc extended half-life moieties
  • N8-GP Novo Nordisk
  • Bayer BAY94-9027
  • Baxter BAX 855
  • the additional step of chemical conjugation of PEG to FVIII reduces the final yield and increases the cost of preparation.
  • PEGylated FVIII did not achieve a significantly prolonged half-life, such as N8-GP has a circulating half-life of about 18 hours in patients with hemophilia A (Tiede A et al, J Thromb Haemost, 2013, 11). :670-678).
  • a clinical phase I study of BAY94-9027 showed a half-life of about 18.2 hours in healthy individuals, about 1.4 times longer than wild-type FVIII (Coyle T et al, Haemophilia, 2012, 18 (Suppl 3): 22).
  • the half-life of Bax 855 is about 18 hours (Turecek PL et al, Hamostaseologie, 2012, 32 Suppl 1: S29-38).
  • a monomer-dimer hybrid FVIIIFc fusion protein (monomer-dimer hybrid FVIII-Fc) developed by Biogen Idec, USA: It was approved by the US FDA in June 2014. Clinical data show that in the human body The half-life is extended by only 1.5 to 1.7 times (Dumont J A et al, Blood, 2012, 119: 3024-3030; Powell JS et al, Blood, 2 012, 119: 3031-3037), and is required to be injected once every 3 to 5 days.
  • CTP human chorionic gonadotropin
  • CTP acts as a linker between the beta subunit and the alpha subunit of follicle stimulating hormone; among the fusion proteins disclosed in WO2005058953A2, CTP is used as a linker for Linking the beta and alpha subunits of the glycoprotein hormone.
  • the inventors did not use CTP as a linker or as an extended half-life part according to the prior art, but instead linked it to a flexible peptide linker (eg (GGGGS)n) to form a new linker sequence, placed between FVIII and the extended half-life portion (eg (GGGGS)n) to form a new linker sequence, placed between FVIII and the extended half-life portion (
  • a flexible peptide linker eg (GGGGS)n
  • an immunoglobulin Fc fragment but not including the CTP suggested by the prior art, constitutes a novel FVIII fusion protein, thereby further extending the half-life and maintaining good biological activity and function.
  • the present invention provides an Fc fusion protein of a hyperglycosylated homodimeric type factor VIII having an extended in vivo half-life of activity and similar biological activity as recombinant FVIII. Furthermore, the present invention provides a method for efficiently and stably expressing the fusion protein, which expresses a fusion protein having high yield, good stability during preparation and storage, and its biological activity and marketed recombinant FVIII factor. Similar advantages.
  • a highly glycosylated FVIII fusion protein comprising human factor VIII (hFVIII) and a flexible peptide linker (Linker, L) from N-terminal to C-terminally
  • a carboxy terminal peptide rigid unit of at least one human chorionic gonadotropin beta subunit hereinafter referred to as a CTP rigid unit, denoted as (CTP)n, preferably, n is 1, 2, 3, 4, or 5
  • CTP carboxy terminal peptide rigid unit of at least one human chorionic gonadotropin beta subunit
  • CTP CTP rigid unit
  • n is 1, 2, 3, 4, or 5
  • prolonging the half-life portion eg, immunoglobulin Fc fragment, albumin, transferrin or PEG, preferably human IgG Fc variant (denoted as vFc)
  • the fusion protein is represented by hFVIII-L-CTPn-vFc.
  • the hFVIII is wild type or a mutant thereof; further, the wild type hFVIII has an amino acid sequence as shown in SEQ ID NO: 1; preferably, the mutant hFVIII is represented by SEQ ID NO: The amino acid sequence is at least 85% homologous; more preferably, the mutant hFVIII and SEQ ID The amino acid sequence of NO: 1 is at least 90% homologous. Most preferably, the mutant hFVIII 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 produces a sufficient spatial distance between hFVIII 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:
  • L2 GSGGGGSGGGGSGGGGSGGGGSGGGGS (as shown in SEQ ID NO: 3);
  • L4 GSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS (as shown in SEQ ID NO: 5);
  • 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 7 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 FVIII 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: 7, ie SSSS*KAPPPS*; or the CTP rigid unit comprises 14 amino acids of SEQ ID NO: 7C, ie S*RLPGPS*DTPILPQ; as another example
  • the CTP rigid unit comprises three glycosylation sites, exemplarily, said The CTP rigid unit comprises 16 amino acids of the N-terminus of SEQ ID NO: 7, ie SSSS*KAPPPS*LPSPS*R; as in other embodiments, the CTP rigid unit comprises 4 glycosylation sites, exemplarily,
  • the CTP rigid unit comprises 28, 29, 30, 31, 32 or 33 amino acids and begins at position 113, 114, 115, 116, 117 or 118 of the human chorionic gonadotropin beta subunit, terminating at position
  • the CTP rigid unit comprises 28 amino acids of the N-terminus of SEQ ID NO: 7, namely SSSS*KAPPPS*LPSPS*RLPGPS*DTPILPQ.
  • * represents a glycosylation site.
  • 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.
  • CTP 1 PRFQDSSSSKAPPPSLPSPSRLPGPSDTPILPQ (as shown in SEQ ID NO: 7);
  • CTP 2 SSSSKAPPPSLPSPSRLPGPSDTPILPQ (as shown in SEQ ID NO: 8);
  • CTP 4 SRLPGPSDTPILPQ (shown as SEQ ID NO: 10).
  • the fusion protein comprises one of the above CTP rigid units.
  • the fusion protein comprises one or more of the above CTP rigid units, preferably 2, 3, 4 or 5 of the above CTP rigid units, for example, in an embodiment of the present 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: 11);
  • 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: 12);
  • 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: 13);
  • 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: 14);
  • (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: 15).
  • the IgG Fc variants provided by the present invention include, but are not limited to, the five variants described in (i) to (v), and may also be a combination or superposition of two types of functional variant mutation sites between IgG isotypes.
  • the variant as described in (iv) above is a novel variant of the novel IgG2Fc obtained by superimposing the mutation sites in (ii) and (iii).
  • An Fc variant (vFc) in a fusion protein of the invention which comprises a hinge region, a CH2 and a CH3 region of human IgG such as human IgG1, IgG2 and IgG4.
  • This CH2 region contains amino acid mutations at positions 228, 234, 235 and 331 (as determined by the EU counting system). It is believed that these amino acid mutations reduce the effector function of Fc.
  • Human IgG2 does not bind to FcyR but shows very weak complement activity.
  • An Fc[gamma]2 variant with a Pro331Ser mutation should have a lower complement activity than native Fc[gamma]2 and is still an Fc[gamma]R non-binding element.
  • IgG4Fc is defective in the activation of the complement cascade and its binding affinity to 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 FVIII 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 as shown in SEQ ID NO:
  • a DNA encoding the above fusion protein is provided.
  • the DNA sequence of the fusion protein is set forth in SEQ ID NO: 17.
  • 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 derived cell line DG44 of CHO.
  • a pharmaceutical composition comprising a pharmaceutically acceptable 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 DG44.
  • 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 activity of the fusion protein prepared by the above method is > 6000 IU/mg.
  • the 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 a malfunction of FVIII is provided.
  • the disease includes type A (or type A) hemophilia.
  • type A or type A hemophilia.
  • the fusion protein of the present invention plays Control or prevent the effects of bleeding.
  • the FVIII fusion protein constructed by the present invention, wherein the Fc fragment is non-lytic, that is, by mutating the complement and receptor binding domains of the Fc fragment, regulating the binding affinity of the Fc to the corresponding receptor, and reducing or eliminating the ADCC and CDC effects. While retaining only the Fc segment to prolong the half-life of the active protein in vivo, it does not produce cytotoxicity.
  • the FVIII fusion protein developed by Biogen whose Fc segment is of natural origin, predicts that Fc-mediated adverse effector functions will necessarily increase the patient's therapeutic risk.
  • the present invention uses CHO cells to express the fusion protein and the expression product only contains the homodimeric FVIII Fc fusion protein, and the purification step is simple and efficient.
  • Biogen constructed a double expression vector expressing rFVIIIFc and Fc, which was transfected into HEK-293 cells (U.S. Patent No. US20130274194A1).
  • the fusion protein fermentation broth expressed by it is expected to contain three forms of products, namely FVIII-Fc: FVIII-Fc homodimeric (Dimeric) fusion protein, FVIII-Fc: Fc monomer-dimer miscellaneous Monomericic fusion protein and Fc: Fc dimer.
  • the host cells need to simultaneously express two single-stranded molecules of FVIII-Fc and Fc, and then polymerize separately to form the above three products, thereby greatly reducing the expression efficiency of the final target product;
  • the other two forms of impurities must also be removed, which complicates the purification process, reduces production efficiency, and greatly increases production costs. Therefore, the preparation method of the invention has certain technical advantages and price advantages over the Monomeric rFVIIIFc fusion protein developed by Biogen, and the expression and purification processes are simpler, more efficient, and the production cost is lower.
  • HemA mice were administered FVIII fusion protein FP-B according to the doses of 30IU/kg, 90IU/kg and 270IU/kg.
  • the medium and high dose groups can effectively control the acute hemorrhage of HemA mice, and the dose of FP-B.
  • the survival rate of the mice in the group was higher than that in the recombinant FVIII (Xyntha, Pfizer) administration group, reflecting that the fusion protein FP-B has a longer lasting pharmacological effect than Xyntha.
  • the high-dose and low-dose groups of FP-B showed dose-related effects in terms of bleeding time and amount of bleeding.
  • 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 prepared by the invention maintains high biological activity, and the purified batches of the fusion egg White activity is in the range of about 6000-10000 IU/mg, converted to a molar activity of about 2340-3900 IU/nM (each fusion protein contains 2 FVIII, equivalent to 1170-1950 IU/nM FVIII); there are also some batches, purified fusion
  • the activity of the protein was even more than 12,000 IU/mg (converted to a molar activity of about 4680 IU/nM, which corresponds to 2340 IU/nM FVIII).
  • the fusion protein prepared by the present invention and the monomer-dimer hybrid rFVIIIFc fusion protein (activity 1660-1770 IU/nM) developed by Biogen (J. McCue et al., Biologicals, 2015, 43: 213-219).
  • the activity of the recombinant FVIII ReFacto (activity 1521-2287 IU/nM) (US Patent No. US20130274194A1) which has been marketed is quite or even higher, indicating that the fusion protein provided by the present invention has a C-terminally fusiond Fc activity against FVIII. Almost no effect.
  • the fusion protein provided by the present invention comprises a rigid CTP polypeptide having a plurality of glycosyl side chains, which can form a stable stereo conformation with respect to the random coiling of a flexible linker peptide such as (GGGGS)n.
  • the action causes the FVIII and Fc segments to fold independently to form the correct three-dimensional conformation without affecting each other's biological activity.
  • 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 fusion protein of the invention has good stability in fermentation, purification process and storage process.
  • the method for preparing 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 be at least 150 mg/L, which can be scaled up for large-scale industrial production.
  • 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. Compared with the other three hormones, the half-life of hCG is significantly prolonged, which is mainly derived from its ⁇ -subunit.
  • CTP carboxy terminal peptides
  • the native CTP contains 37 amino acid residues with four O-glycosylation sites and a terminal 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).
  • the inventors creatively linked at least one CTP polypeptide to a flexible linker of appropriate length, collectively as a linker peptide, for ligation of FVIII with an extended half-life portion (eg, an immunoglobulin Fc fragment).
  • the inventors have found that by adding a CTP peptide between FVIII and an Fc variant, it corresponds to the addition of a rigid linker peptide.
  • This aspect ensures that the N-terminally fused FVIII does not affect the binding site of the Fc variant to FcRn, thereby affecting the half-life; in addition, the Fc-ProteinA binding site is important for the purification step in the preparation process, and the CTP is bonded to ensure the N-terminus.
  • the fused FVIII also does not "cover" its binding site to protein A, thus allowing 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 FVIII, resulting in a decrease or loss of its biological activity/function.
  • 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, While IgG4 does not bind to C1q (Jefferis R et al, Immunol Rev, 1998, 163: 59-76), 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, calcium phosphate precipitation, lipofection, electroporation, microinjection, viral infection, alkali metal ion methods.
  • 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 present invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising an effective amount (preferably about 2 to 10 ⁇ g/kg) of the fusion protein of the present invention, and a pharmaceutically acceptable carrier.
  • an effective amount of a fusion protein of the invention can be formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium wherein the pH is usually from about 5 to about 8, preferably, the pH is from about 6 to about 8.
  • the term "effective amount” or “effective amount” refers to an amount that is functional or active to a human and/or animal and that is acceptable to humans and/or animals.
  • 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 and the like. The selection of a preferred effective amount can be determined by one of ordinary skill in the art based on various factors (e.g., by clinical trials). The factors include, but are not limited to, the pharmacokinetic parameters of the fusion protein such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated by the patient, the patient's weight, the patient's immune status, the route of administration, etc. .
  • Figure 1 Nucleotide sequence and deduced amino acid sequence of FP-B of a fragment of SpeI-EcoRI (labeled with a label) in a pcDNA3 expression vector according to an embodiment of the present invention.
  • Human FVIII consists of a signal peptide (1-19, underlined Marked) and mature FVIII protein (20-1457).
  • the mature fusion protein contains hFVIII (20-1457), flexible peptide linker (1458-1484, underlined __), CTP rigid unit (1485-1512, underlined Marked) and vFc ⁇ 2-3 variants (1513-1735).
  • Figure 4 The amount of bleeding ( ⁇ l) per mouse after tail-breaking. Note: *p ⁇ 0.05, **p ⁇ 0.01.
  • FIG. 5 Bleeding time (s) of each mouse after tail-breaking. Note: *p ⁇ 0.05, **p ⁇ 0.01.
  • the gene sequences encoding the FVIII leader peptide, the mature protein, the flexible peptide linker, the CTP rigid unit, and the human IgG vFc variant are all artificially optimized CHO cell-preferred codons and are obtained by artificial synthesis.
  • the 5' and 3' ends of the full-length DNA fragment of the fusion protein have a restriction endonuclease site, respectively SpeI and BamHI, and the full-length DNA fragment is inserted into the corresponding cleavage site of the pUC57 transfer vector, and DNA sequencing verification 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 expression plasmid PTY1A1 which was transformed with pcDNA3.1 as a template to obtain a fusion protein high expression plasmid.
  • the PTY1A1 plasmid includes, but is not limited to, the following important expression components: 1) human cytomegalovirus early promoter and mammalian cells are required for exogenous high expression; 2) dual screening markers with kanamycin resistance in bacteria sexually, has G418 resistance in mammalian cells; 3) murine dihydrofolate reductase (DHFR) gene expression cassette, when the host cell is deficient in the DHFR gene, 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 medium was changed to a screening medium containing 0.6 mg/mL G418, and the cells were planted in a 96-well culture plate at a concentration (5000-10000 viable cells/well) for 10-14 days until large. Discrete cell clones appear. Transfectants resistant to the selected drug were screened by ELISA assay. Subclones were generated to produce high levels of fusion protein wells by limiting dilution of 96-well plates.
  • the present invention constructs a series of hFVIII fusion proteins comprising peptide linkers of different lengths (Linker), CTP rigid units of different compositions, and IgG Fc variant (vFc) elements of several different subtypes.
  • Linker peptide linkers of different lengths
  • vFc IgG Fc variant
  • the eight expression plasmids obtained in Example 1 were transfected into 3 ⁇ 107 CHO-K1 cells in a 30 ml shake flask using DNAFect LT ReagentTM (ATGCell), and the transfected cells contained 1000 ng/ml.
  • the vitamin K1 was grown in serum-free growth medium for 5 days, the concentration of the fusion protein in the supernatant was measured, and its activity was measured by the method described in Example 6 or 7.
  • the ELISA results showed that the transient expression levels of the eight plasmids under these conditions were similar, but their coagulation activities showed large differences.
  • FP-A the molar activity of FP-A as 100%.
  • the fusion protein in the FP-G cell culture supernatant was mostly secreted in the form of an inactive form of the polymer; while the fusion protein expressed by the FP-F and FP-H plasmids was less active, about 20.5% of the FP-A and 15.2%, the same is mostly expressed in the form of a polymer; in addition, the fusion proteins FP-F, FP-G and FP-H are less stable and prone to degradation.
  • a rigid peptide linker at one end is added to keep away from each other, and more importantly, the CTP rigid unit contains a plurality of sugar-based side chains, and the CTP rigid unit can form a fixed spatial conformation with respect to the random coil form of the flexible peptide linker. It can effectively separate the different functional regions of the fusion protein, which is more conducive to the two parts of independent folding to form the correct three-dimensional conformation, maintaining a high activity.
  • We verified the validity of this speculation by comparing the activity of FP-B and FP-F, that is, the activity of FP-F of CTP rigid unit placed at Fc C end is less than 20% of FP-B of CTP rigid unit placed at Fc N end. .
  • the above results confirmed that the CTP rigid unit is critical for the activity of the fusion protein, and that the CTP rigid unit is placed at the N-terminus of the Fc to effectively increase the activity of the fusion protein.
  • the expression plasmids of FP-A, FP-B, FP-C, FP-D and FP-E described above were transfected into a mammalian host cell line to express a FVIII fusion protein.
  • Preferred host cells for maintaining stable high levels of expression It is a DHFR-deficient CHO cell (U.S. Patent No. 4,818,679).
  • a preferred method of transfection is electroporation, and other methods can be used, including calcium phosphate co-precipitation, lipofection, and microinjection.
  • Electroporation method Using a Gene Pulser Electroporator (Bio-Rad Laboratories) set to 300 V voltage and 1050 ⁇ Fd capacitance, 50 ⁇ g of PvuI linearized expression plasmid was added to 2 to 3 ⁇ 107 cells placed in a cuvette, and electroporation was performed. The resulting cells were transferred to shake flasks containing 30 ml of growth medium. Two days after transfection, the medium was changed to a growth medium containing 0.6 mg/mL G418, and the cells were seeded in a 96-well culture plate at a concentration for 10-12 days until large discrete cell clones appeared.
  • a Gene Pulser Electroporator Bio-Rad Laboratories
  • the anti-human IgG Fc ELISA method is used to screen the transfectants that are resistant to the selected drugs, and the anti-FVIII ELISA method can also be used for the quantitative determination of the fusion protein expression, and then the high-level expression fusion is produced by sub-cloning by limiting dilution method.
  • the pores of the protein are used to screen the transfectants that are resistant to the selected drugs, and the anti-FVIII ELISA method can also be used for the quantitative determination of the fusion protein expression, and then the high-level expression fusion is produced by sub-cloning by limiting dilution method. The pores of the protein.
  • 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 3) IU/106 (ie, millions) of cells per 24 hours is subjected to adaptive suspension culture using a serum-free medium, and then the fusion protein is purified using a conditioned medium. .
  • FP-B is taken as an example to specifically describe the fermentation, purification steps and methods of several fusion proteins screened in the above examples, and the FP-A, FP-C, FP-D and FP-E methods are the same, in the examples. No longer.
  • 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 FP-B 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 15 ⁇ 106 / 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 20 mg/L per day, and is lived in a shake flask.
  • the cell density can reach up to 30 x 106 cells/mL.
  • the biological activities of the assays for recombinant fusion proteins produced by the above two methods are comparable.
  • the present invention mainly uses a four-step chromatography method to purify the fusion protein FP-B.
  • Affinity chromatography, hydrophobic chromatography, anion exchange chromatography and molecular sieve chromatography respectively the protein purification instrument used in this example is AKTA pure 25M from GE Corporation of the United States.
  • the reagents used in this example are all purchased from Sinopharm Chemicals). Reagent Co., Ltd., the purity is analytical grade).
  • affinity chromatography using euros's alkali-resistant Protein A Diamond or other commercially available recombinant proteinA affinity chromatography media (eg GE's Mabselect, Mabselect Sure, TOSOH's Toyopearl AF-rProtein A-650F, Tiandi Renhe's rProtein A Bead, Safran's MabPurix, and Pall's ProteinACeramic HyperD) perform sample capture, concentration, and removal of some contaminants.
  • recombinant proteinA affinity chromatography media eg GE's Mabselect, Mabselect Sure, TOSOH's Toyopearl AF-rProtein A-650F, Tiandi Renhe's rProtein A Bead, Safran's MabPurix, and Pall's ProteinACeramic HyperD
  • the clarified fermentation broth was loaded at a linear flow rate of 50-100 cm/h, and the loading was not higher than 50,000 IU/ml; after the loading was completed, the equilibrium buffer was used: 20 mM His-HCl, 150 mM NaCl, 5 mM CaCl2, 0.02 %Tween 80, pH 6.8-7.2, equilibrate the column at 3-5 column volumes (CV) at a linear flow rate of 50-100 cm/h, flush unbound components; use decontamination buffer 1:20 mM His-HCl 2M NaCl, 4M urea, 5mM CaCl2, 0.02% Tween 80, pH 6.8-7.2, rinse the column at 3-5 column volumes at a linear flow rate of 50-100 cm/h to remove some of the contaminants; use a balanced buffer: 20 mM His-HCl, 150 mM NaCl, 5 mM CaCl 2 , 0.02% Tween 80, pH 6.8-7.2, equilibrate the column at
  • the second step, hydrophobic chromatography use Boglon's Butyl HP or other commercially available hydrophobic chromatography media (For example, GE's Butyl HP, TOSOH's Toyopearl Butyl-650, Tiandiren and Butyl Beads4FF, Seiko's Generik MC30-HIC Butyl, Merck's Fractogel EMD Propyl) for intermediate purification to reduce polymer content.
  • the first step of the affinity eluent still contains a certain proportion of the polymer, because the formation of the polymer is diverse, including unstructured polymerization and structural changes, their biological activities are quite different, so for the organism Analysis of the activity of the study brings greater interference.
  • the polymer is first removed.
  • the target protein is polymerized, there are differences in properties between the polymer and the monomer, including charge characteristics and hydrophobicity, and we use the difference in hydrophobicity to separate the two.
  • the final step of purification is molecular sieve chromatography, the fusion protein captured by the first affinity chromatography is followed by Butyl HP for the second purification step, with the goal of partially removing the polymer to a level below 10%.
  • the affinity-captured sample was diluted 2-fold with a balanced buffer to reduce the organic solvent content, and then equal volume was added to the concentrated buffer: 20 mM His-HCl, 3 M NaCl, 5 mM CaCl 2 , 0.02% Tween 80, pH 6.8-7.2, Then load, the load is controlled at ⁇ 20000 IU/ml; after loading, use balanced buffer: 20 mM His-HCl, 1.5 M NaCl, 5 mM CaCl2, 0.02% Tween 80, pH 6.8-7.2, 50-100 cm/ Linear flow rate of h is flushed to 3-5 column volumes (CV); then wash buffer: 20 mM His-HCl, 1.5 M NaCl, 5 mM CaCl2, 0.02% Tween 80, 20% ethylene glycol, pH 6.8- 7.2, rinse the column with 3-5 column volumes (CV) at a linear flow rate of 50-100 cm/h to remove part of the polymer; finally, elute the target protein using an el
  • the third step, anion exchange chromatography use Q-HP from Bogron or other commercially available anion exchange chromatography media (such as GE's Q HP, TOSOH's Toyopearl GigaCap Q-650, Tiandiren and DEAE Beads 6FF). , Seike Technology's Generik MC-Q, Merck's Fractogel EMD TMAE, Pall's Q Ceramic HyperD F) for intermediate purification, separation of structural variants, further removal of HCP, DNA and other contaminants.
  • anion exchange chromatography media such as GE's Q HP, TOSOH's Toyopearl GigaCap Q-650, Tiandiren and DEAE Beads 6FF.
  • the target protein separated by the second step of hydrophobic chromatography is diluted 2 times, the concentration of organic matter is decreased, and the loading is controlled at 5000-10000 IU/ml; after the loading is completed, the equilibrium buffer is used: 20 mM His-HCl, 200 mM NaCl, 5 mM.
  • Step 4 Molecular sieve chromatography: Separation using Bodlon's Chromdex 200prep grade or other commercially available molecular sieve media (eg GE's superdex 200) with the goal of reducing the polymer content to ⁇ 5% and further reducing critical contamination The content of the substance. Flush the column with 2 column volumes (CV) at a linear flow rate of 20-40 cm/h using a balanced buffer: 20 mM His-HCl, 200 mM NaCl, 5 mM CaCl2, 0.02% Tween 80, pH 6.8-7.2; No more than 3% of the column volume, flushed at a linear flow rate of 20 cm/h, and the fractions were collected in turn, detected by SEC, and combined.
  • CV column volumes
  • the non-reducing electrophoresis contains the unprocessed fusion protein (390KDa), and the fragment of HC-LC-L-CTP-Fc:LC-L-CTP-Fc (300KDa) which sheds a heavy chain of FVIII during electrophoresis, shedding two weights Post-chain (LC-L-CTP-Fc) 2 dimerization fragment (210KDa) and HC (90KDa); clear HC-LC-L-CTP-Fc (190kDa), LC-L-CTP- Fc (105KDa) and HC (90KDa) single-stranded bands.
  • Example 6 Indirect determination of in vitro activity of fusion protein by chromogenic substrate method
  • the activity of the FVIII fusion protein can be determined using a chromogenic substrate method.
  • This example was measured using a Chromogenix Coatest SP FVIII kit (Chromogenix, Ref. K824086).
  • the detection principle is as follows: When activated by thrombin, FVIIIa binds to FIXa in the presence of phospholipids and calcium ions to form an enzyme complex, which in turn Activation factor X is converted to its active form Xa. Activation of the formed factor Xa can then cleave its specific chromogenic substrate (S-2765), releasing the chromophoric group pNA.
  • the activity of FXa directly proportional to its amount is known, wherein the content of factor IXa and factor X is constant and excessive in the system, and the activity of FXa is only related to the content of FVIIIa.
  • the specific activity of the FVIII fusion protein determined by this method is about 6000-10000 IU/mg.
  • the coagulation method for determining the biological activity of FVIII was obtained by correcting the ability of the FVIII factor-deficient plasma to cause prolonged clotting time.
  • the method for detecting FVIII activity using the Cogulation Factor VIII Deficient Plasma (Cat. No. OTXW17) kit from Siemens, Germany, is as follows: First, the known potency of the F1 active standard of the Chinese Academy of Hospitals is diluted to 10 IU with 5% spent FVIII plasma. /ml, and then diluted 10 times, 20 times, 40 times and 80 times. The activated partial thromboplastin time (APTT) was measured by a fully automated blood coagulation analyzer (CA500, SYSMEX).
  • the logarithm of the FVIII activity standard solution titer (IU/ml) was linearly regressed corresponding to the logarithm of its corresponding clotting time (s), and a standard curve was established using the FVIII standard.
  • the sample to be tested is then diluted moderately and mixed with spent FVIII matrix plasma for APTT assay. Substituting into the standard curve equation, the titer of the sample FVIII can be calculated, and the specific activity of the sample FVIII to be tested can be calculated according to this, and the unit is IU/mg.
  • the specific activity of the FVIII fusion protein determined by this method is about 6000-10000 IU/mg.
  • mice were evaluated the hemostatic activity of the fusion protein FP-B prepared in Example 5 in HemA mice by the VIII factor gene knockout homozygous HemA mouse tail bleeding model. 8-12 weeks old male HemA mice (purchased from Shanghai Southern Model Bio-Limited Co., Ltd.) were selected. After adaptive feeding for one week, the mice were randomly divided into 6 groups according to body weight. Another group of HemA mice negative control group and one The control group of normal C57 mice was given a single active dose of fusion protein FP-B or the control drug Xyntha (Pfizer) in a single tail vein injection. The animal groupings are shown in Table 2.
  • mice Prior to administration, mice were first anesthetized with 1.0% pentobarbital sodium (Sigma) at a dose of 0.1 ml/10 g, and then the mice were placed on a heating pad at 37 ° C to maintain their body temperature.
  • the tail of the mouse was immersed in warm water of 37 ° C for 10 minutes to dilate the tail vein, and then administered at the dose shown in Table 2. After 10 minutes of administration, it was cut at a distance of 1.5 cm from the tail end of the mouse, and the tail was quickly immersed in a centrifuge tube containing about 13 ml of preheated physiological saline, and the timing was started. If the bleeding stops within 30 minutes, record the bleeding time and the amount of bleeding. If the bleeding time exceeds 30 minutes, it is recorded as 30 minutes.
  • the amount of bleeding (ml) (weight of centrifuge tube after blood collection (g) - weight of centrifuge tube (g) before blood collection) / 1.05. After 30 minutes, the rat tail was taken out from the saline tube. The rebleeding was recorded every 10 minutes within 24 hours, and the number of mice survived was recorded. All data in mean ⁇ standard error T-test analysis was used for comparison between the experimental groups. The analysis software used Graphpad Prism 5.0, p ⁇ 0.05 was considered statistically significant.
  • FP-B can be used as an effective clotting agent for acute bleeding in clotting factors such as hemophilia; after administration of FP-B 90IU/kg, the mice are similar to the C57 control group in bleeding time and bleeding volume. .
  • the bleeding time is more than 30min, recorded as 1800 seconds;

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Abstract

一种高糖基化的重组人凝血因子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，从而改善药代动力学和药效。

Description

高糖基化人凝血因子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。据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时会导致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凝血因子VIII。
FVIII是多结构的大分子糖蛋白，分为6个结构域：三个A结构域(A1、A2、A3)，一个富碳水化合物且非必需的中央结构域(B-结构域)和两个C结构域(C1、C2)。成熟的蛋白质由大约分子量为280kDa的轻链和重链组成。轻链分子量约为80kDa，结构包括A3，C1和C2，连接方式为A3-C1-C2。重链分子量约90至200kDa，结构包括A1，A2和B，连接方式为A1-A2-B。重链和轻链连接是金属离子依赖性的。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在重链内的氨基酸Arg372和Arg740，及在轻链内的Arg1689处被活化的凝血因子FX(FXa)及凝血酶蛋白水解切割而活化，导致vWF因子释放并产生活化的FVIII二聚体(FVIIIa)，在Ca2+存在下，它在磷脂表面上与活化凝血因子FIX(FIXa)及FX形成紧密复合物，FX接着被FIXa激活，活化的FX被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达几个数量级。
FVIII分子是迄今克隆的最长的基因片段之一，且是应用于临床的分子量最大的蛋白药物。对于分子量大、糖基化程度高的重组蛋白，哺乳动物细胞是最优 的表达系统。然而，FVIII在体外重组表达量明显低于与其它性质类似的基因，如FVIII表达水平只有FIX的1％。这可能是机体对FVIII需求的反映，但这对体外重组表达FVIII而言无疑是一大障碍。此外，由于FVIII在血液中半衰期较短，仅为8-12小时，严重血友病A患者进行预防性治疗，必须每周静脉内(i.v.)注射约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。例如，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，但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。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，如N8-GP在A型血友病患者体内的循环半衰期约18小时(Tiede A等，J Thromb Haemost，2013，11：670-678)。BAY94-9027的临床I期研究显示其在健康人体内半衰期约为18.2小时，较野生型FVIII延长了约1.4倍(Coyle T等，Haemophilia，2012，18(Suppl 3)：22)。Bax 855的半衰期约18小时(Turecek PL等，Hamostaseologie，2012，32Suppl 1：S29-38)。
由美国Biogen Idec公司开发的单体-二聚体杂合体型重组FVIIIFc融合蛋白(monomer-dimer hybrid FVIII-Fc)：
Figure PCTCN2016106010-appb-000001
于2014年6月被美国FDA批准上市。临床数据显示，在人体内
Figure PCTCN2016106010-appb-000002
半衰期延长了仅1.5～1.7倍(Dumont J A等，Blood，2012，119：3024-3030；Powell JS等，Blood，2 012，119：3031-3037)，需每3～5天注射1次。据报道，Biogen公司构建的rFVIIIFc与Fc的双表达载体转染HEK-293细胞，在其表达的产物中并未预期检测出rFVIIIFc同源二聚体形式的融合体，仅表达出单体-二聚体杂合体型rFVIIIFc融合蛋白和Fc二聚体，该公司研究人员推测可能原因是所采用的表达系统因同源二聚体型分子量过大，宿主细胞未能成功分泌分子量约400KDa的rFVIIIFc同源二聚体蛋白，或因rFVIIIFc单体间的空间位阻效应，而未能发生聚合(Peters RT等，J Thromb Haemost，2013，11(1)：132-41)，由此可见，同源二聚体型FVIII融合蛋白的表达是相当困难的。
人绒毛膜促性腺激素(hCG)β链的羧基末端肽(以下称其为CTP)也具有 延长某些蛋白质体内半衰期的作用，因此一些专利文献公开的融合蛋白中包含的延长半衰期部分可以选择使用免疫球蛋白Fc片段、HSA、CTP或其他能延长半衰期的融合配体。另外，CTP也可以作为接头，主要用于连接同一个蛋白质的不同亚基。例如，中国专利CN103539860A、CN103539861A、CN103539868A和CN103539869A公开的融合蛋白中，CTP作为接头，位于促卵泡激素的beta亚基和alpha亚基之间；专利WO2005058953A2公开的融合蛋白中，CTP作为接头，用于连接糖蛋白激素的beta亚基和alpha亚基。
本发明人没有根据现有技术将CTP作为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)连接组成新的接头序列，设置于FVIII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FVIII融合蛋白，从而进一步延长了半衰期，并且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。
本发明第一方面，提供一种高糖基化FVIII融合蛋白(以下简称融合蛋白)，所述融合蛋白从N端至C端依次含有人凝血因子VIII(hFVIII)、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hFVIII-L-CTPn-vFc。
其中，所述hFVIII为野生型或其突变型；进一步地，所述野生型hFVIII具有如SEQ ID NO：1所示的氨基酸序列；优选地，所述突变型hFVIII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hFVIII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hFVIII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hFVIII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(如SEQ ID NO：2所示)；
(ii)L2：GSGGGGSGGGGSGGGGSGGGGSGGGGS(如SEQ ID NO：3所示)；
(iii)L3：GGGGSGGGGSGGGGSGGGGS(如SEQ ID NO：4所示)；
(iv)L4：GSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS(如SEQ ID NO：5所示)；
(v)L5：GGGSGGGSGGGSGGGSGGGS(如SEQ ID NO：6所示)；
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：7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FVIII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：7N端的10个氨基酸，即SSSS*KAPPPS*；或所述CTP刚性单元包含SEQ ID NO：7C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：7N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：7N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(如SEQ ID NO：7所示)；
(ii)CTP2：SSSSKAPPPSLPSPSRLPGPSDTPILPQ(如SEQ ID NO：8所示)；
(iii)CTP3：SSSSKAPPPS(如SEQ ID NO：9所示)；
(iv)CTP4：SRLPGPSDTPILPQ(如SEQ ID NO：10所示)。
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1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：11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：12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：13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：14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：15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。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：16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：17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DG44。
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/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DG44。
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、本发明构建的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。Biogen公司开发的FVIII融合蛋白，其Fc段是天然来源的，可以预测由Fc介导的不良效应子功能必将会增加患者的治疗风险。
2、本发明采用CHO细胞表达融合蛋白且表达产物中仅含有同源二聚体型FVIII Fc融合蛋白，其纯化步骤简单、高效。而Biogen公司为了表达单体-二聚体杂合体(Monomeric)FVIII融合蛋白，构建了一种表达rFVIIIFc与Fc的双表达载体，转染HEK-293细胞(美国专利，公开号：US20130274194A1)。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FVIII-Fc：FVIII-Fc同源二聚体型(Dimeric)融合蛋白、FVIII-Fc：Fc单体-二聚体杂合体(Monomeric)融合蛋白以及Fc：Fc二聚体。在融合蛋白表达过程中，因宿主细胞需同时表达FVIII-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；同时，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，生产效率降低、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 rFVIIIFc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。
3、HemA小鼠按30IU/kg、90IU/kg和270IU/kg剂量给予本发明FVIII融合蛋白FP-B，中、高剂量组能有效地控制HemA小鼠急性出血状况，且FP-B各剂量组小鼠的存活率较重组FVIII(Xyntha，辉瑞)给药组均有所提高，反映出融合蛋白FP-B比Xyntha具有更持久的药效作用。同时，FP-B高剂量和低剂量组无论在出血时间和出血量等结果均表现出剂量相关性。
4、较重组FVIII，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。
5、本发明制备的融合蛋白保持了较高的生物学活性，纯化的各批次融合蛋 白活性大约在6000-10000IU/mg范围，换算为摩尔比活性约2340-3900IU/nM(每个融合蛋白含有2个FVIII，相当于1170-1950IU/nM FVIII)；还有一些批次，纯化融合蛋白的活性甚至超过12000IU/mg(换算为摩尔比活性约4680IU/nM，相当于2340IU/nM FVIII)。因而，本发明制备的融合蛋白与Biogen公司所开发的单体-二聚体杂合体rFVIIIFc融合蛋白(活性为1660-1770IU/nM)(J.McCue等，Biologicals，2015，43：213-219)以及已上市的重组FVIII ReFacto(活性为1521-2287IU/nM)(美国专利，公开号：US20130274194A1)的活性相当甚至更高，说明本发明提供的融合蛋白，其C端融合的Fc对FVIII的活性几乎没有影响。
6、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。
7、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。
8、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150mg/L，可进行工艺放大，实现大规模工业化生产。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。
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FVIII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ProteinA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(较佳约2～10μg/kg)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、根据本发明实施例的在pcDNA3表达载体内SpeI-EcoRI(酶切位点以标注)片段的FP-B的核苷酸序列及推导的氨基酸序列。人FVIII由信号肽(1-19，以下划线
Figure PCTCN2016106010-appb-000003
标注)和成熟FVIII蛋白(20-1457)构成。成熟的融合蛋白含有hFVIII(20-1457)、柔性肽接头(1458-1484，以下划线__标注)、CTP刚性单元(1485-1512，以下划线
Figure PCTCN2016106010-appb-000004
标注)和vFcγ2-3变体(1513-1735)。
图2、纯化后FP-B蛋白的SEC-HPLC峰谱图。
图3、纯化后FP-B蛋白的SDS-PAGE电泳图。
图4：断尾后每只小鼠出血量(μl)。注：*p＜0.05，**p＜0.01。
图5：断尾后每只小鼠出血时间(s)。注：*p＜0.05，**p＜0.01。
具体实施方式
实施例1.构建编码FVIII融合蛋白的表达质粒
编码FVIII前导肽、成熟蛋白、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BamHI，全长DNA片段插入至pUC57转移载体相应酶切位点间，并由DNA测序验证序列。
然后再将上述获得的融合蛋白全长基因片段从中间载体转移到以pcDNA3.1为模板并改造的表达质粒PTY1A1的相应酶切位点间，得到融合蛋白高表达质粒。PTY1A1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。
如下表所示，本发明构建了一系列hFVIII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。为了验证含有至少1个，并且不同长度的CTP刚性单元均能显著提高融合蛋白的活性，我们构建了融合蛋白FP-A、FP-B、FP-C、FP-D和FP-E；其中，FP-B的氨基酸及编码核苷酸如图1所示。为了验证CTP刚性单元对融合蛋白活性的重要性，我们还同时构建了不含CTP刚性单元的Fc融合蛋白FP-G和FP-H，表达质粒构建方法同上。此外，我们还构建了CTP位于Fc C端的FP-F，以验证CTP刚性单元位置的重要性。详见表1。各组成元件的氨基酸序列见序列表。
表1、各种FVIII融合蛋白组成
Figure PCTCN2016106010-appb-000005
实施例2.瞬时表达各融合蛋白和活性测定
将实施例1得到的8种表达质粒，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或7中描述的方法测定其活性。ELISA结果显示8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FP-A的摩尔比活性定义为100％。FP-G细胞培养上清液中的融合蛋白多以无活性形式的聚合体形式分泌；而FP-F和FP-H质粒表达的融合蛋白活性较低，分别约为FP-A的20.5％和15.2％，同样大部分以聚合体形式表达；另外，融合蛋白FP-F、FP-G和FP-H的稳定性较差，易发生降解。据报道FVIII 2303-2332区域的脂质结合区(Lipid binding region)对其功能至关重要，该区域极微小的构象变化即会引起蛋白的聚合，导致活性的丧失(Gilbert GE等，Biochemistry，1993，32(37)：9577-9585)。因而我们推测培养上清中的FVIII融合蛋白FP-F、FP-G和FP-H因脂质结合区构象受C端Fc配体的影响发生改变，引起了蛋白的聚集，使活性大大降低。而含有CTP的FP-B、FP-C、FP-D和FP-E的活性分别为FP-A的113.4％、96.0％、87.4％和93.7％。
从FP-B和FP-F、FP-H的活性差异可知仅靠延长肽接头并不能有效改善融合蛋白的活性，也无法解决融合蛋白易发生聚合及降解的问题。而CTP的加入使得融合蛋白FP-B的活性显著增加。我们推测原因，过长的柔性肽接头，给了FVIII更高的灵活度，使其能相对Fc自由转动，这可能使FVIII的立体结构更加靠近Fc区，而在二者之间加入CTP刚性单元，一方面相当增加了一端刚性肽接头，使彼此远离，更重要的是CTP刚性单元含多个糖基侧链，相对于柔性肽接头的无规则卷曲形态，CTP刚性单元可以形成固定的空间构象，能够有效的分开融合蛋白的不同功能区，这更有利于两部分独立折叠形成正确的三维构象，保持了较高的活性。我们通过FP-B和FP-F的活性比较验证了这种推测的正确性，即CTP刚性单元置于Fc C端的FP-F的活性不足CTP刚性单元置于Fc N端的FP-B的20％。以上结果证实CTP刚性单元对融合蛋白的活性极为关键，并且CTP刚性单元置于Fc的N端能有效提高融合蛋白的活性。
实施例3.筛选高表达融合蛋白的稳定转染细胞系
将上述FP-A、FP-B、FP-C、FP-D和FP-E的表达质粒转染入哺乳动物宿主细胞系，以表达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(美国专利NO.4818679)。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2～3×107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，也可用抗FVIII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3)IU/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。
以下实施例中以FP-B为例，具体描述上述实施例筛选的几种融合蛋白发酵、纯化步骤及方法，FP-A、FP-C、FP-D和FP-E方法相同，实施例中不再赘述。
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FP-B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15×106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20mg/L，在摇瓶中活 细胞密度最高可达到30×106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
本发明主要采用四步层析法对融合蛋白FP-B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：采用博格隆公司的耐碱Protein A Diamond或其它市售的重组proteinA亲和层析介质(例如GE的Mabselect、Mabselect Sure、TOSOH的Toyopearl AF-rProteinA-650F、天地人和的rProtein A Bead，赛分科技的MabPurix、Pall的ProteinACeramic HyperD)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：20mM His-HCl，150mM NaCl，5mM CaCl2，0.02％Tween-80，pH6.8-7.2，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：20mM His-HCl，150mM NaCl，5mM CaCl2，0.02％Tween 80，pH6.8-7.2，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 1：20mM His-HCl，2M NaCl，4M urea，5mM CaCl2，0.02％Tween 80，pH6.8-7.2，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：20mM His-HCl，150mM NaCl，5mM CaCl2，0.02％Tween 80，pH6.8-7.2，以50-100cm/h的线性流速平衡层析柱3-5个柱体积(CV)；然后使用去污buffer 2：20mM His-HCl，5mM EDTA，150mM NaCl，30％乙二醇、5mM CaCl2，0.02％Tween 80，pH6.8-7.2，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：20mM His-HCl，150mM NaCl，5mM CaCl2，0.02％Tween 80，pH6.8-7.2，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，5mM CaCl2，0.02％Tween 80，50％乙二醇，pH5.0，以不高于50cm/h的线性流速洗脱目标产物，收集目标峰，用2M Tris，pH9.0中合pH值至中性(7.0-8.0)。
第二步，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 Beads4FF，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。第一步亲和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。因此，在完成捕获以后，首先去除聚合体。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，所以经第一步亲和层析捕获的融合蛋白再使用Butyl HP进行第二步纯化，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 CaCl2，0.02％Tween 80，pH6.8-7.2，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2倍，用于降低有机溶剂含量，然后等体积加入浓缩buffer：20mM His-HCl，3M NaCl，5mM CaCl2，0.02％Tween 80，pH6.8-7.2，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 CaCl2，0.02％Tween 80，pH6.8-7.2，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 CaCl2，0.02％Tween 80，20％乙二醇，pH6.8-7.2，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 CaCl2，0.02％Tween 80，50％乙二醇，pH6.8-7.2，以不高于60cm/h的线性流速洗脱，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。
第三步，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，200mM NaCl，5mM CaCl2，0.02％Tween 80，pH6.8-7.2，以50-100cm/h的线性流速冲洗层析柱3-5个柱体积(CV)；经第二步疏水层析分离得到的目标蛋白稀释2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，200mM NaCl，5mM  CaCl2，0.02％Tween 80，pH6.8-7.2，以50-100cm/h的线性流速冲洗层析柱3-5个柱体积(CV)；之后采用线性梯度的盐浓度进行洗脱，洗脱buffer：20mM His-HCl，1M NaCl，5mM CaCl2，0.02％Tween 80，pH6.8-7.2，条件为洗脱buffer从0-100％，用时2小时，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10000IU/mg。
第四步，分子筛层析：使用博格隆公司的Chromdex 200prep grade或其它市售的分子筛介质(例如GE的superdex 200)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：20mM His-HCl，200mM NaCl，5mM CaCl2，0.02％Tween 80，pH6.8-7.2，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2和图3，其中SEC-HPLC结果显示，纯化后融合蛋白FP-B的主峰纯度达97％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(390KDa)，电泳时脱落FVIII的一条重链后的片段HC-LC-L-CTP-Fc：LC-L-CTP-Fc(300KDa)，脱落两条重链后的(LC-L-CTP-Fc)2二聚片段(210KDa)和HC(90KDa)；还原后可得清晰的HC-LC-L-CTP-Fc(190kDa)、LC-L-CTP-Fc(105KDa)和HC(90KDa)单链条带。
实施例6.发色底物法间接测定融合蛋白体外活性
FVIII融合蛋白的活性可采用发色底物法测定。本实施例采用Chromogenix Coatest SP FVIII试剂盒(Chromogenix，Ref.K824086)测定，其检测原理如下：当被凝血酶激活后，FVIIIa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(S-2765)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FVIII融合蛋白的比活性约为6000-10000IU/mg。
实施例7.凝血法直接测定融合蛋白的生物学活性
凝固法测定FVIII生物学活性是通过纠正FVIII因子缺失血浆所导致凝固时间延长的能力而获得的。采用德国Siemens公司Coagulation Factor VIII Deficient Plasma(Cat.No.OTXW17)试剂盒检测FVIII活性的方法如下：首先将已知效价的中检院FVIII活性标准品用5％乏FVIII血浆将其稀释至10IU/ml，然后再分别稀释10倍、20倍、40倍和80倍。以全自动血凝分析仪(CA500，SYSMEX公司)测定活化部分凝血活酶时间(APTT)。以FVIII活性标准品溶液效价(IU/ml)的对数对应其相应的凝固时间(s)的对数作线性回归，以FVIII标准品建立标准曲线。然后将待测样本经适度稀释后与乏FVIII基质血浆混合，进行APTT测定。代入标准曲线方程，可计算得出待测样品FVIII的效价，据此可求算出待测样品FVIII的比活性大小，单位为IU/mg。以本法测定FVIII融合蛋白的比活性约为6000-10000IU/mg。
实施例8.融合蛋白对血友病A小鼠急性出血的止血作用
我们以VIII因子基因剔除纯合子HemA小鼠断尾出血模型(tail clip bleeding model)评估实施例5中所制备的融合蛋白FP-B在HemA小鼠体内的止血活性。选取8-12周龄雄性HemA小鼠(购自上海南方模式生物责任有限公司)，适应性饲养一周后将小鼠按体重随机分为6组，另设一组HemA小鼠阴性对照组和一组正常C57小鼠的对照组，分别单次尾静脉注射给予不同活性剂量的融合蛋白FP-B或对照药物任捷(Xyntha，辉瑞)，动物分组情况见表2。
表2：融合蛋白对HemA小鼠止血效果实验动物分组情况
Figure PCTCN2016106010-appb-000006
给药前，首先以1.0％戊巴比妥钠(Sigma公司)按照0.1ml/10g剂量腹腔注射麻醉小鼠，然后将小鼠置于37℃的加热垫上以保持其体温。将小鼠尾巴浸入37℃的温水中10分钟，使尾静脉扩张，然后按照表2所示剂量进行给药。给药10分钟后，在距小鼠尾末端1.5cm处剪断，迅速将尾端浸入装有约13ml预热生理盐水的离心管中，并开始计时。如果出血在30分钟内停止，记录出血时间和出血量。如果出血时间超过30分钟，则记为30分钟。出血量(ml)＝(采血后离心管重量(g)-采血前离心管重量(g))/1.05。30分钟后，将鼠尾从生盐水管中取出。在24小时内，每隔10min观察记录复出血情况，并记录小鼠存活数量。所有数据以均数±标准误差
Figure PCTCN2016106010-appb-000007
表示，各实验组间比较采用t-test检验分析，分析软件采用Graphpad Prism 5.0，p＜0.05认为有统计学意义。
从图4和图5中各组动物的出血时间和出血量的统计结果分析，HemA小鼠给予FP-B 270IU/kg 10分钟后，其出血时间和出血量与C57对照组接近，促凝血效果明显，说明FP-B可以作为血友病等凝血因子缺乏症发生急性出血情况的有效凝血剂；小鼠给药FP-B 90IU/kg后，在出血时间和出血量上同样与C57对照组接近。分别给予同等活性剂量的FP-B和任捷，HemA小鼠出血量无显著差异，但各个剂量下，FP-B组的出血时间均略少于任捷组，显示FP-B比任捷可能具有一定的药效优势。与FP-B 30IU/kg给药组比较，FP-B 90IU/kg组的出血时间显著缩短(p＜0.05)，FP-B 270IU/kg组的出血时间显著缩短(p＜0.05)，出血量也显著减少(p＜0.05)，说明融合蛋白FP-B对HemA小鼠急性出血的止血作用具有一定的剂量-效应关系(详细结果见表3)。
从术后恢复情况来看，给予相同活性剂量的FP-B和任捷，FP-B各剂量组的小鼠存活率均优于同等活性剂量的任捷组，反映出融合蛋白FP-B比任捷具有更持久的药效作用(见表3)。
表3：各组HemA小鼠断尾试验的出血时间、出血量、复出血次数和存活率统计
Figure PCTCN2016106010-appb-000008
注：a，24-48h内又有小鼠死亡，故统计48h死亡率；
b，出血时间超过30min，记做1800秒；
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，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落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VIII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，延长半衰期部分选自人免疫球蛋白Fc片段、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VIII包含如SEQ ID NO：1所示的氨基酸序列，或者所述人凝血因子VIII的氨基酸序列与如SEQ ID NO：1所示的氨基酸序列具有至少90％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，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：7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1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：16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＞6000IU/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：17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1IU/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，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。
  25. 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。
  26. 如权利要求25所述的应用，包括用于制备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。
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KR1020197007918A KR102276157B1 (ko) 2016-08-19 2016-11-16 고글리코실화 인간 혈액응고인자 viii 융합 단백질 및 그 제조방법과 용도
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BR112019003302A2 (pt) 2019-10-15
RU2722374C1 (ru) 2020-05-29
GB2568624B (en) 2022-05-25
JP6923115B2 (ja) 2021-08-18
KR20190042629A (ko) 2019-04-24
GB2568624A (en) 2019-05-22
CN106279437B (zh) 2017-10-31
CN106279437A (zh) 2017-01-04
MX2019001925A (es) 2019-10-02
GB201903581D0 (en) 2019-05-01
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