WO2019219049A1 - 改进的fviii融合蛋白及其应用 - Google Patents

改进的fviii融合蛋白及其应用 Download PDF

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WO2019219049A1
WO2019219049A1 PCT/CN2019/087156 CN2019087156W WO2019219049A1 WO 2019219049 A1 WO2019219049 A1 WO 2019219049A1 CN 2019087156 W CN2019087156 W CN 2019087156W WO 2019219049 A1 WO2019219049 A1 WO 2019219049A1
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fusion protein
seq
group
amino acid
factor viii
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PCT/CN2019/087156
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English (en)
French (fr)
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苏鸿声
王晓山
刘宾
陈宪
李相�
朱鹿燕
王淑亚
王双
王文文
黄灵丽
王齐磊
胡海涛
张莉莉
高洁
任子甲
肖春峰
王亚里
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北京辅仁瑞辉生物医药研究院有限公司
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Application filed by 北京辅仁瑞辉生物医药研究院有限公司 filed Critical 北京辅仁瑞辉生物医药研究院有限公司
Priority to EP19804136.0A priority Critical patent/EP3816181A4/en
Priority to US17/055,403 priority patent/US20210361775A1/en
Priority to BR112020023168-1A priority patent/BR112020023168A2/pt
Priority to MX2020012103A priority patent/MX2020012103A/es
Priority to CN202310860049.3A priority patent/CN117467019A/zh
Priority to CN201980033170.0A priority patent/CN113039200B/zh
Priority to KR1020207034852A priority patent/KR102575788B1/ko
Priority to JP2020564649A priority patent/JP2021530437A/ja
Publication of WO2019219049A1 publication Critical patent/WO2019219049A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/04—Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/36—Blood coagulation or fibrinolysis factors
    • A61K38/37—Factors VIII
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/545—Heterocyclic compounds
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/745—Blood coagulation or fibrinolysis factors
    • C07K14/755—Factors VIII, e.g. factor VIII C (AHF), factor VIII Ag (VWF)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/31—Fusion polypeptide fusions, other than Fc, for prolonged plasma life, e.g. albumin

Definitions

  • the invention relates to the field of recombinant human factor VIII fusion proteins, in particular to a fusion polypeptide having an extended half-life and a preparation method and application thereof.
  • Hemophilia A is an inherited bleeding disorder caused by a deficiency or dysfunction of factor VIII (FVIII) activity. Supplementation with active FVIII is an effective measure for the treatment of hemophilia A.
  • the FVIII molecule is one of the longest gene fragments cloned to date, and is the most abundant protein drug used in clinical practice. Divided into six domains: three A domains (A1, A2, A3), one non-essential central domain (B-domain) and two C domains (C1, C2).
  • A1, A2, A3 three A domains
  • B-domain non-essential central domain
  • C1, C2 two C domains
  • 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.
  • 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.
  • the double expression vector of rFVIII Fc and Fc constructed by Bioverativ was transfected into HEK-293 cells, and the fusion of rFVIIIFc homodimer form was not expected to be detected in the expressed product, only monomer-di Polyheteromeric rFVIIIFc fusion protein and Fc dimer, the researchers speculate that the possible reason is that the expression system used is too large in molecular weight of the homodimer type, and the host cell fails to successfully secrete rFVIIIFc homologous with a molecular weight of about 400 kDa.
  • polymers having high solubility such as polyethylene glycol (PEG) are conventionally used to chemically modify the surface of protein drugs.
  • PEG polyethylene glycol
  • the higher the modification rate the more pronounced the decrease in protein antigenicity and the greater the loss of activity.
  • Polyethylene glycol (PEG) has been reported to extend the half-life of FVIII: Novonordisk (N8-GP), Bayer (BAY94-9027) and Baxter (Bax 855) have developed PEGylated long-acting FVIII products. Enter the clinical study.
  • pharmacokinetic studies have shown that PEGylated FVIII does not achieve a significantly extended half-life (Tiede A et al, J Thromb Haemost.
  • clotting factor VIII is fused to a fusion partner (such as Fc segment, albumin, XTEN or transferrin) while binding to a conjugate (eg polyalkylene glycol,
  • a conjugate eg polyalkylene glycol,
  • PEG including mPEG
  • the in vivo stability of the protein can be remarkably improved, thereby completing the present invention.
  • a factor VIII fusion protein conjugated to a polyalkylene glycol wherein a factor VIII active moiety (FVIII) is directly linked to a half-life extending fusion partner or indirectly linked by a peptide linker to form the fusion protein, And the fusion protein is further conjugated to a polyalkylene glycol.
  • a factor VIII active moiety FVIII
  • fusion protein wherein the fusion partner is: an immunoglobulin Fc fragment, albumin, transferrin or XTEN, such fusion partners are for example derived from humans; preferably IgG Fc a fragment; for example, the IgG Fc fragment has a reduced ADCC effect and/or a CDC effect and/or enhanced binding affinity to an FcRn receptor; more preferably, the IgG Fc fragment has an amino acid sequence selected from the group consisting of:
  • fusion protein according to any one of embodiments 1 to 4, wherein the fusion protein is conjugated to a polyalkylene glycol which is random or site-directed, and the conjugation position is selected from the group consisting of a free amino group and a thiol group. , a glycosyl group and/or a carboxyl group, preferably a free amino group.
  • conjugation is effected using any activated ester form modifying agent, for example, using a modifying agent selected from the following formula (1), (2) or (3).
  • m1 is preferably 5;
  • mPEG represents a mono-terminated polyethylene glycol group;
  • 0 ⁇ m2 ⁇ 6, m2 is preferably 2; 0 ⁇ m3 ⁇ 6, m3 is preferably 1; mPEG represents a mono-terminated polyethylene glycol group; or
  • 0 ⁇ m4 ⁇ 6, and m4 is preferably 2; mPEG represents a methoxy-mono-terminated polyethylene glycol group.
  • fusion protein according to any one of embodiments 1 to 6, wherein the factor VIII active moiety is linked to the fusion partner via a peptide linker comprising a flexible peptide linker and/or a rigid unit
  • a peptide linker comprising a flexible peptide linker and/or a rigid unit
  • 1, 2, 3, 4, 5 or more of the rigid units may be included.
  • the flexible peptide linker comprises two or more amino acid residues selected from the group consisting of glycine, serine, alanine and threonine,
  • the flexible peptide linker has the sequence formula (GS) a(GGS)b(GGGS)c(GGGGS)d, wherein a, b, c and d are integers greater than or equal to 0, and a+b+ c+d ⁇ 1,
  • the flexible peptide linker has a sequence selected from the group consisting of:
  • the amino acid sequence of the terminal peptide has a consistency of 70%, 80%, 90%, 95% or higher; the rigid unit may comprise 1, 2 or more glycosylation sites;
  • the rigid unit comprises an amino acid sequence selected from the group consisting of:
  • SRLPGPSDTPILPQ SEQ ID NO: 14
  • the peptide linker comprises the amino acid sequence set forth in SEQ ID NO: 15.
  • a pharmaceutical composition comprising an effective amount of the fusion protein of any one of embodiments 1 to 9, and a pharmaceutically acceptable carrier.
  • a method for preventing and/or treating a bleeding disorder which comprises administering the fusion protein of any one of embodiments 1 to 9 or the pharmaceutical composition of embodiment 10 to a subject in need thereof,
  • the bleeding disorder is preferably selected from a hemorrhagic disease in a patient with FVIII congenital or acquired deficiency and spontaneous or surgical bleeding in a patient with hemophilia A.
  • a method of improving the half-life of factor VIII wherein the factor VIII active moiety is directly linked to a half-life enhancing fusion partner or indirectly linked to a peptide linker and further conjugated to a polyalkylene glycol.
  • the fusion partner is: an immunoglobulin Fc fragment, albumin, XTEN or transferrin, such fusion partners are for example derived from a human; preferably an IgG Fc fragment;
  • the IgG Fc fragment has a reduced ADCC effect and/or CDC effect and/or enhanced binding affinity to the FcRn receptor; more preferably, the IgG Fc fragment has an amino acid sequence selected from the group consisting of:
  • the modifying agent may be a modifying agent in the form of an activated ester, more preferably, the modifying agent is selected From the following formula (1), (2) or (3):
  • m1 is preferably 5;
  • mPEG- represents a mono-terminated polyethylene glycol group, and the molecular weight of the modifier represented by formula (1) is between 5KD and 60KD;
  • 0 ⁇ m2 ⁇ 6, m2 is preferably 2; 0 ⁇ m3 ⁇ 6, m3 is preferably 1; mPEG- represents a methoxy-terminated polyethylene glycol group, and the molecular weight of the modifier represented by formula (2) 5KD-100KD, preferably 40KD, 50KD, 60KD, most preferably 40KD;
  • m4 is preferably 2; mPEG- represents a methoxy-mono-terminated polyethylene glycol group, and the modifier represented by formula (3) has a molecular weight of 5KD-100KD.
  • the flexible peptide linker comprises two or more amino acid residues selected from the group consisting of glycine, serine, alanine, and threonine,
  • the flexible peptide linker has the sequence formula (GS) a(GGS)b(GGGS)c(GGGGS)d, wherein a, b, c and d are integers greater than or equal to 0, and a+b+ c+d ⁇ 1,
  • the flexible peptide linker has a sequence selected from the group consisting of:
  • the amino acid sequence of the terminal peptide has a consistency of 70%, 80%, 90%, 95% or higher; the rigid unit may comprise 1, 2 or more glycosylation sites;
  • the rigid unit comprises an amino acid sequence selected from the group consisting of:
  • SRLPGPSDTPILPQ SEQ ID NO: 14
  • the peptide linker comprises the amino acid sequence set forth in SEQ ID NO: 15.
  • Figure 1a shows the results of SEC-HPLC of FVIII-Fc (FF-0) without mPEG modification.
  • Figure 1b shows the results of SEC-HPLC of 5K molecular weight mPEG modified FVIII-Fc (FF-5L).
  • Figure 1c shows the results of SEC-HPLC of 10K molecular weight mPEG modified FVIII-Fc (FF-10L).
  • Figure 1d shows the results of SEC-HPLC of 20K molecular weight mPEG modified FVIII-Fc (FF-20L).
  • Figure 1e shows the results of SEC-HPLC of 30K molecular weight mPEG modified FVIII-Fc (FF-30L).
  • Figure 1f shows the results of SEC-HPLC of 40K molecular weight mPEG modified FVIII-Fc (FF-40L).
  • Figure 2a shows the results of SEC-HPLC of the non-mPEG-modified FVIII-Linker1-Fc (FL1F-0) (purity >99%, polymer ⁇ 1%).
  • Figure 2b shows the results of SEC-HPLC of 20K molecular weight mPEG modified FVIII-L1-Fc (FL1F-20L) (purity >95%, polymer ⁇ 5%, uncrosslinked ⁇ 1%).
  • Figure 2c is a SEC-HPLC assay of linear, 30K molecular weight mPEG modified FVIII-L1-Fc (FL1F-30L) (purity >95%, polymer ⁇ 5%, uncrosslinked ⁇ 1%).
  • Figure 2d is a SEC-HPLC assay of linear, 40K molecular weight mPEG modified FVIII-L1-Fc (FL1F-40L) (purity >95%, polymer ⁇ 5%, uncrosslinked ⁇ 1%).
  • Figure 2e is a SEC-HPLC assay of linear 50 g molecular weight mPEG modified FVIII-L1-Fc (FL1F-50L) (purity >95%, polymer ⁇ 5%, uncrosslinked ⁇ 1%).
  • Figure 2f shows the results of SEC-HPLC of Y--40K molecular weight mPEG modified FVIII-L1-Fc (FL1F-40Y) (purity >95%, polymer ⁇ 5%, uncrosslinked ⁇ 1%).
  • Figure 3a shows the results of SDS-PAGE before and after liquid exchange of hFVIII-Fc (FF-0) stock solution G25 without modification of mPEG (H stands for reduction and F stands for non-reduction).
  • Figure 3b shows the results of SDS-PAGE (non-reduction) of hFVIII-Fc cross-linking with different molecular weights of mPEG (FF-5L to FF-40L).
  • Figure 3c shows the results of SDS-PAGE detection (reduction) of hFVIII-Fc cross-linked with different molecular weights mPEG (FF-5L to FF-40L).
  • factor VIII also known as factor VIII, or FVIII
  • factor VIII active moiety refers to a moiety which renders the fusion protein of the invention exhibit FVIII activity.
  • Native human FVIII consists of 2351 amino acids, including signal peptides, and contains several different domains defined by homology. There are 3 A domains, 1 unique B domain and 2 C domains. The order of the domains can be listed as NH2-A1-A2-B-A3-C1-C2-COOH. FVIII circulates in plasma with two chains separated at the B-A3 border. The two chains are joined by a combination of divalent metal ions.
  • the A1-A2-B chain is referred to as the heavy chain (HC)
  • A3-C1-C2 is referred to as the light chain (LC).
  • Endogenous Factor VIII molecules circulate in vivo as a library of molecules with different sizes of B domains. What may occur in vivo is a stepwise enzymatic cleavage of the B domain, resulting in a library of molecules with different sizes of the B domain. It is generally believed that the occurrence of cleavage at position 740 (the last part of the B domain is thus excised) is associated with thrombin activation.
  • Vector VIII as used in the present invention may refer to both its native wild-type sequence (such as SEQ ID NO: 1) and its variant forms, for example, after one or more amino acid substitutions, deletions. Or the variant protein obtained after insertion, while retaining the activity of factor VIII.
  • Factor VIII is a B domain truncated molecule, wherein the remaining domains substantially correspond to the sequences set forth in amino acid numbers 1-745 and 1640-2332 of SEQ ID NO:1.
  • the B domain truncated molecule of the invention may differ slightly from the sequence set forth in SEQ ID NO: 2, meaning that the remaining domains (ie, 3 A domains and 2 C domains) and SEQ ID NO:
  • the amino acid sequence shown in 2 may have one or more amino acid substitutions, additions or deletions, for example, having a difference of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids or about 1 %, 2%, 3%, 4% or 5% difference, changing Factor VIII and various other components (eg LRP, multiple receptors, other coagulation factors, cell surface) while retaining the basic activity of Factor VIII The ability to bind, introduce and/or eliminate glycosylation sites, and the like.
  • fusion partner refers to another polypeptide that is fused to a polypeptide of interest (ie, a polypeptide that is expected to extend its half-life) that is capable of altering the function of the fusion protein by a variety of different mechanisms, such as extending the in vivo half-life of the polypeptide of interest. .
  • the fusion partner delays in vivo clearance of FVIII by interacting with a neonatal Fc receptor (FcRn).
  • the fusion partner is an immunoglobulin Fc region (Fc region), albumin, transferrin, XTEN or a portion thereof.
  • the IgG Fc domain is preferred due to the longer half-life of the IgG antibody.
  • the Fc domain may also be modified to improve other functions, such as complement binding and/or binding to certain Fc receptors. Mutations at positions 234, 235 and 237 in the IgG Fc domain will generally result in reduced binding to the Fc ⁇ RI receptor. It may also result in reduced binding to the Fc ⁇ RIIa and Fc ⁇ RIII receptors. These mutations do not alter binding to the FcRn receptor, which promotes long circulating half-life through the endocytic recycling pathway.
  • the modified IgG Fc domain of the fusion protein of the invention comprises one or more of the following mutations which will result in decreased affinity for certain Fc receptors (L234A, L235E and G237A) and C1q-, respectively. Mediated reduction in complement fixation (A330S and P331S).
  • polyalkylene glycol is a hydrophilic polymer which is conjugated to a specific position on Factor VIII and/or a fusion partner in the present invention, and the polyalkylene glycol may be linear or Branched and may comprise one or more independently selected polymeric moieties.
  • the polyalkylene glycol is polyethylene glycol (including its methoxy-terminated form m-PEG), polypropylene glycol (including mPPG), and the like. .
  • the polyalkylene glycol in the present invention may be polyethylene glycol (PEG), which may be linear or branched.
  • PEG polyethylene glycol
  • the backbone of the branched polymer is well known in the art, and typically the branched polymer has a central branch core portion and one or more linear polymer chains attached to the central branch core. It is preferred in the present invention to use PEG in branched form.
  • the branched polyethylene glycol can be represented by the formula R(-PEG-OH)m, wherein R represents a core moiety, such as glycerol or pentaerythritol, and m represents the number of arms.
  • the number of branches in a branched PEG is 2, also referred to herein as a "Y-type" PEG (eg, mPEG), ie, comprising two PEG or linear methoxy PEG.
  • Y-type PEG eg, mPEG
  • polystyrene resin examples include, but are not limited to, other polyalkylene glycols (e.g., polypropylene glycol (PPG), copolymers of ethylene glycol and propylene glycol, etc.), polyoxyethylated polyols, polyenols (olefmic alcoh0l) , polyvinylpyrrolidone, polyhydroxypropylmethacrylamide, poly([ ⁇ ]-hydroxy acid), polyvinyl alcohol, polyphosphazene, polyoxazoline, poly-N-acryloylmorpholine and copolymers thereof, Meta-copolymers and mixtures.
  • PPG polypropylene glycol
  • copolymers of ethylene glycol and propylene glycol etc.
  • polyoxyethylated polyols examples include, but are not limited to, other polyalkylene glycols (e.g., polypropylene glycol (PPG), copolymers of ethylene glycol and propylene glyco
  • PEG modification ie, conjugation
  • the modification is a random modification or a site-directed modification
  • the position of the modification includes a free amino group, a thiol group, a glycosyl group And / or a carboxyl group, such as a free amino group.
  • the modifier for the random modification of the mPEG of the free amino group may be selected from the group consisting of: mPEG-SS (methoxypolyethylene glycol-succinimide succinate), mPEG-SC (methoxypolyethylene glycol-succinimide carbonate), mPEG-SPA (methoxypolyethylene glycol-succinimidyl propionate) and mPEG-SG (methoxypolyethylene glycol- Succinimide glutarate) and the like.
  • mPEG-SS methoxypolyethylene glycol-succinimide succinate
  • mPEG-SC methoxypolyethylene glycol-succinimide carbonate
  • mPEG-SPA methoxypolyethylene glycol-succinimidyl propionate
  • mPEG-SG methoxypolyethylene glycol- Succinimide glutarate
  • the N-terminal modifiers are: mPEG-ALD (methoxy polyethylene glycol-acetaldehyde), mPEG-pALD (methoxy polyethylene glycol-propionaldehyde) and mPEG-bALD (methoxy polyethylene glycol) -butyraldehyde) and the like.
  • the modifiers mPEG-SS, mPEG-SC, mPEG-SPA, mPEG-SG, mPEG-ALD, mPEG-pALD, mPEG-bALD are linear or branched.
  • the modifier used in the random modification of the free thiol group is mPEG-mal (methoxypolyethylene glycol-maleimide), mPEG-OPSS (methoxypolyethylene) One of alcohol-o-dithiopyridine), mPEG-Vinylsulfone (methoxypolyethylene glycol-vinylsulfone), and mPEG-Thiol (methoxypolyethylene glycol-thiol).
  • the modifier used in the random modification of the glycosyl and/or carboxyl groups is mPEG-ZH (methoxypolyethylene glycol-hydrazide).
  • the mPEG modified modifier has the structure shown in formula (1):
  • m1 is preferably 5; mPEG- represents a mono-terminated polyethylene glycol group, and the modifier represented by formula (1) has a molecular weight of 5 kD-60 kD (kD, thousand Daoer) Preferably, it is 40 kD.
  • the mPEG of the free amino group is randomly modified with the modifying agent represented by the formula (1).
  • the structure of the mPEG-modified modifier is as shown in formula (2):
  • m2 is preferably 2; 0 ⁇ m3 ⁇ 6, m3 is preferably 1; mPEG- represents a methoxy-terminated polyethylene glycol group, and the molecular weight of the modifier represented by formula (2) It is 5 kD to 60 kD, preferably 40 kD.
  • the mPEG random modification of the free amino group is carried out using the modifying agent represented by the formula (2).
  • the structure of the mPEG modified modifier is as shown in formula (3):
  • m4 is preferably 2; mPEG- represents a methoxy-mono-terminated polyethylene glycol group, and the modifier represented by the formula (3) has a molecular weight of 5 kD to 60 kD, preferably 40 kD.
  • the free thiol-based mPEG random modification is carried out using a modifying agent of formula (3).
  • the size of the polymer backbone can vary, but polymers (e.g., PEG, mPEG, PPG, or mPPG) typically range from about 0.5 KD to about 160 KD, such as from about 1 KD to about 100 KD. More specifically, the size of each of the conjugated hydrophilic polymers of the present invention varies primarily in the range of from about 1 KD to about 80 KD, from about 2 KD to about 70 KD; from about 5 KD to about 70 KD; from about 10 KD to about 60 KD, about 20KD to about 50KD; about 30KD to about 50KD or about 30KD-40KD. It should be understood that these sizes represent approximate values and are not accurate measurements, which is recognized in the art.
  • the size of the PEG or mPEG used in the present invention is 35 KD or more (ie, not less than 35 KD), preferably not less than 40 KD, not less than 45 KD, not less than 50 KD, not less than 55 KD, not less than 60 KD, Not less than 65 KD or not less than 70 KD, for example, the molecular weight is specifically 40 KD, 50 KD, 60 KD, 70 KD, 80 KD, 90 KD, 100 KD, 110 KD, 120 KD, 130 KD, 140 KD, 150 KD or 160 KD.
  • modified circulating half-life The molecule of the invention has an altered circulating half-life, preferably an increased circulating half-life, compared to a wild-type Factor VIII molecule.
  • the cyclic half-life is preferably increased by at least 10%, preferably at least 15%, preferably at least 20%, preferably at least 25%, preferably at least 30%, preferably at least 35%, preferably at least 40%, preferably at least 45%, preferably at least 50%, preferably at least 55%, preferably at least 60%, preferably at least 65%, preferably at least 70%, preferably at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 100%, more preferably At least 125%, more preferably at least 150%, more preferably at least 175%, more preferably at least 200%, and most preferably at least 250% or 300%. Even more preferably, the molecule has an increase in circulating half-life of at least 400%, 500%, 600%
  • pharmaceutically acceptable carrier includes, but is 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.
  • BDD FVIII BDD FVIII, consisting of 90kD A1-A2 heavy chain and 80kD light chain;
  • **SEQ ID NO. 7 - SEQ ID NO. 12 indicates that the adaptor is ligated from the rigid unit set forth in SEQ ID NO. 12 to the C-terminus of the flexible peptide linker set forth in SEQ ID NO. 7;
  • SEQ ID NO. - SEQ ID NO. 11 shows that the linker is formed by the rigid unit shown by SEQ ID NO. 11 linked to the C-terminus of the flexible peptide linker shown by SEQ ID NO.
  • each 1.1.1 fusion protein is subjected to centrifugation and filtration, followed by Affinity chromatography/Hydrophobic Interaction Chromatography/Ion-Exchange chromatography. / Size exclusion chromatography, respectively obtained five hFVIII fusion proteins FF-0, FL1F-0, FL2F-0, F (full length) L1F'-0 and F (full length) L2F" -0, SEC-HPLC detected the polymer ⁇ 5%.
  • the five hFVIII fusion proteins were separately prepared as a protein concentration of 0.95mg / ml hFVIII fusion protein stock;
  • Buffer preparation 20 mM Hepes, 0.1 M NaCl, 5.0 mM CaCl 2 , 0.02% Tween 8.0, pH 7.0;
  • mPEG-SC was weighed according to the ratio of hFVIII fusion protein to mPEG-SC (purchased from Beijing Keykai Technology Co., Ltd.) in a molar ratio of 1:10-1:100 (molecular weight 5kD, 10kD, respectively) , 20kD, 30kD, 40kD structure such as the linear L-shaped mPEG-SC shown in formula (1), and the molecular weight of 40kD structure as shown in formula (2), branched-chain Y-shaped mPEG-SC), added 1.1.
  • Buffer preparation 20 mM histidine, 0.1 M NaCl, 5.0 mM CaCl 2 , 0.02% Tween 8.0, pH 7.0;
  • Binding buffer 20 mM histidine, 0.1 M NaCl, 5.0 mM CaCl 2 , 0.02% Tween 8.0, pH 7.0; elution buffer: 20 mM histidine, 2.0 M NaCl, 5.0 mM CaCl 2 , 0.02% Tween 8. 0, pH 7.0; CIP: 0.5 M NaOH;
  • test results of FF-0 to FF-40L are shown in Figures 1a-1f.
  • results of FL1F-0 to FL1F-50L and FL1F-40Y are shown in Figures 2a-2f.
  • the results show that the purity of FL1F-0 to FL1F-60L is >95%, the polymer is ⁇ 5%, and the uncrosslinked ⁇ 1%.
  • the product obtained in the item "1.2.2” is subjected to SDS-PAGE detection, and includes the following steps:
  • Glue 1 ⁇ Tris-glycine electrophoresis buffer: SDS 0.4 g, Tris base 1.21 g, glycine 7.5 g, and double distilled water to 400 mL.
  • sample preparation the sample to be tested is mixed with an equal volume of loading buffer, and an equal volume of 0.1 mg/mL of 2-mercaptoethanol is added to the sample by reduction SDS-PAGE; non-reducing SDS-PAGE is not added 2- Mercaptoethanol. After the sample was mixed with the loading buffer, it was bathed in boiling water for 10 minutes.
  • Electrophoresis 10 ⁇ l of the sample to be tested and the protein Marker were sequentially added to the spotting hole, and concentrated electrophoresis was carried out at a voltage of 60 V. It was observed that the bromophenol blue dye was concentrated to the separation gel and the voltage was increased to 120 V. Separate and electrophoresis until the bromophenol blue dye reaches the bottom of the separation gel and turn off the power.
  • Example 2 Indirect determination of in vitro activity of mPEG-modified hFVIII fusion protein by chromogenic substrate method
  • the activity of the mPEG-modified hFVIII fusion protein prepared in Example 1 was determined using a chromogenic substrate assay.
  • the Chromogenix Coatest SP FVIII kit (Chromogenix, Ref. K824086) was used to determine the principle: when activated by thrombin, FVIIIa binds to FIXa in the presence of phospholipids and calcium ions to form an enzyme complex, which in turn activates factor X transformation. Into its active form Xa. Activation of the formed factor Xa can then cleave its specific chromogenic substrate (S-2765), releasing the chromophoric group pNA.
  • Eloctate is a recombinant Factor VIII Fc fusion protein already marketed by Bioverativ, which has not been modified by mPEG.
  • the human coagulation factor VIII titer used in the present invention is also referred to as the first-stage method.
  • One-stage assay for FVIII biological activity was performed by correcting the ability of FVIII-deficient plasma to cause prolonged clotting time.
  • a kit Coagulation Factor VIII Deficient Plasma (Cat. No. OTXW17) manufactured by the German company Siemens was used. The method comprises: first, diluting a known potency FVIII activity standard, WHO International Standard 8th International Standard Factor VIII Concentrate (Cat. No.
  • mice After stopping bleeding (no significant blood outflow at the incision), the rat tail was removed from the saline tube, and the mouse was placed on a 37 ° C heating pad to maintain its body temperature without touching the wound. After the mice were awakened, they were placed in a rat cage padded with A4 white paper, kept in a single cage, and replaced with white paper or a mouse cage after each observation to determine the degree of bleeding. After the tail was counted, the survival rate of the mice within 48 hours and the number of rebleeding within 12 hours after the tailings were counted (12 hours in total, and the number of bleedings within one hour was counted once). The results are shown in Table 5.
  • the rate of re-bleeding rate is the proportion of mice with re-bleeding during the statistical period.
  • the severe bleeding rate is the phenomenon of severe bleeding (+++) or multiple moderate hemorrhage (++) in the re-bleeding statistics within 12. The proportion of mice.
  • moderate bleeding means: A4 white paper has a lot of blood, covering an area of not less than 30%, and the blood mark is medium in color, but there is no large area of blood beach (area > 3cm2); severe bleeding (++ +) means: A4 white paper has a lot of blood on the surface, the coverage area is not less than 30%, the blood mark is heavy, and there is a large area of blood beach; even if the coverage area is less than small, it can be regarded as severe bleeding (large blood loss in mice) The range of activity is reduced, and the blood is heavily wetted with white paper).

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Abstract

一种具有延长的循环半衰期的与聚亚烷基二醇缀合的凝血因子VIII融合蛋白及其制备方法和应用，其中凝血因子VIII活性部分与融合伴侣直接连接或以肽连接子间接连接，以及所述融合蛋白进一步与聚亚烷基二醇相缀合。与未经聚亚烷基二醇修饰的FVIII融合蛋白相比，其半衰期显著提高。

Description

改进的FVIII融合蛋白及其应用
交叉引用
本申请要求发明名称为“改进的FVIII融合蛋白及其应用”于2018年5月18日提交到中国专利局的中国专利申请201810481941.X的优先权，其内容通过引用以整体并入本文。
技术领域
本发明涉及重组人凝血因子VIII融合蛋白领域，特别涉及一种具有延长的半衰期的融合多肽及其制备方法和应用。
背景技术
A型血友病是由凝血因子VIII(FVIII)活性的缺乏或功能障碍造成的一种遗传性出血障碍。补充有活性的FVIII是治疗A型血友病的有效措施。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。分为6个结构域：三个A结构域(A1、A2、A3)，一个非必需的中央结构域(B-结构域)和两个C结构域(C1、C2)。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％。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。
由美国Bioverativ公司开发的单体-二聚体杂合体型重组FVIII-Fc融合蛋白(Eloctate)：于2014年6月被美国FDA批准上市。临床数据显示，在人体内半衰期延长了仅1.5～1.7倍(Dumont J A等，Blood，2012，119：3024-3030；Powell JS等，Blood，2012，119：3031-3037)，需每3～5天注射1次。然而，Bioverativ公司构建的rFVIII Fc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。
对于制备蛋白类药物的长效制剂而言，常规使用具有高溶解度的聚合物(如聚乙二醇(PEG))来化学修饰蛋白类药物的表面。一般说来，修饰率越高，蛋白质抗原性降低越明显，活性损失也越大。目前已经报道的聚乙二醇(PEG)延长FVIII半衰期的有：Novonordisk(N8-GP)，Bayer(BAY94-9027)和Baxter(Bax 855)公司均开发了PEG化的长效FVIII产品，并已进入临床研究。然而，药代动力学研究数据显示，PEG化的FVIII并未获得显著延长的半衰期(Tiede A等，J Thromb Haemost.2013；11：670-678)；(Coyle T等，Haemophilia.2012；18(Suppl 3)：22)；(Turecek PL等，Hamostaseologie，2012，32 Suppl 1：S29-38)。
蛋白药物长效化的实现策略包括糖基化改造、PEG化、白蛋白融合、转铁蛋白融合、Fc融合、XTEN融合等。已上市的长效药物仅采用上述策略中的一种用于延长半衰期。没有文献报道将两种以上的上述策略进行组合，特别是PEG化与融合蛋白的组合，也没有文献报道将两种以上策略能够使半衰期比采用单一策略时进一步延长。
血友病患者需要终生输注凝血因子来止血和预防出血，因此，研发人员需要不断地寻求半衰期更长的凝血因子来减少给药次数。另外，如何在延长半衰期的同时保持良好生物学活性是研发人员面临的难题。
发明内容
发明人经过多年的研究和长期的试验发现，将凝血因子VIII与融合伴侣(例如Fc段、白蛋白、XTEN或转铁蛋白)融合，同时结合缀合物(例如聚亚烷基二醇，更例如PEG，包括mPEG)修饰的手段，可以有效地提高多肽的体内稳定性，特别是当缀合部分具有支链结构(或者叫分支结构)时，尤其是在缀合部分的分子量还大于等于35KD，例如40KD时，能够显著提高蛋白的体内稳定性，从而完成了本发明。
本发明提供如下技术方案：
1、一种与聚亚烷基二醇缀合的凝血因子VIII融合蛋白，其中凝血因子VIII活性部分(FVIII)与延长半衰期的融合伴侣直接连接或者以肽连接子间接连接形成所述融合蛋白，以及所述融合蛋白进一步与聚亚烷基二 醇相缀合。
2、根据实施方案1所述的融合蛋白，其中所述凝血因子VIII活性部分源自人，例如全长或截短的人凝血因子VIII，例如缺失B区域的人凝血因子VIII；所述全长或截短的人凝血因子VIII可包含1或更多个氨基酸突变，条件是仍保留其FVIII活性；例如所述凝血因子VIII活性部分包含SEQ ID NO：1或2所示的氨基酸序列，或者与SEQ ID NO：1或2所示氨基酸序列具有至少90％、95％或更高的一致性。
3、根据实施方案1或2所述的融合蛋白，其中所述融合伴侣为：免疫球蛋白Fc片段、白蛋白、转铁蛋白或XTEN，这些所述融合伴侣例如源自人；优选为IgG Fc片段；例如所述IgG Fc片段具有降低的ADCC效应和/或CDC效应和/或与FcRn受体增强的结合亲和力；更优选地，所述IgG Fc片段具有选自以下的氨基酸序列：
(i)SEQ ID NO：3所示的氨基酸序列；
(ii)SEQ ID NO：4所示的氨基酸序列；或
(iii)SEQ ID NO：5所示的氨基酸序列。
4、根据实施方案1至3任一项所述的融合蛋白，其中所述聚亚烷基二醇是聚丙二醇或聚乙二醇；所述聚亚烷基二醇可以是末端封端的，例如经烷氧基如甲氧基封端的；和/或所述聚亚烷基二醇是直链的或支链的；优选地所述聚亚烷基二醇是支链的，例如是支链的聚乙二醇，尤其是经甲氧基封端的支链聚乙二醇；所述聚亚烷基二醇的分子量可以是＞＝1、＞＝10、＞＝20、＞＝30、＞＝40、＞＝50、＞＝60、＞＝70、＞＝80、＞＝90、＞＝100、＞＝110、＞＝120、＞＝130、＞＝140、＞＝150或＞＝160kDa，例如是5、10、20、30、40、50、60、70、80、90或100kDa，或者以上任意二数值之间的值。
5、根据实施方案1至4任一项所述的融合蛋白，其中所述融合蛋白与聚亚烷基二醇的缀合是随机的或定点的，所述缀合位置选自游离氨基、巯基、糖基和/或羧基，优选地游离氨基。
6、根据权利要求5所述的融合蛋白，其中使用任意活化酯形式修饰剂实现所述缀合，例如使用选自如下式(1)、(2)或(3)所述修饰剂实现所述缀合：
Figure PCTCN2019087156-appb-000001
其中，0≤m1≤6，m1优选为5；mPEG表示甲氧基单封端的聚乙二醇基团；
Figure PCTCN2019087156-appb-000002
其中0≤m2≤6，m2优选为2；0≤m3≤6，m3优选为1；mPEG表示甲氧基单封端的聚乙二醇基团；或
Figure PCTCN2019087156-appb-000003
其中，0≤m4≤6，m4优选为2；mPEG表示甲氧基单封端的聚乙二醇基团。
7、根据实施方案1至6任一项所述的融合蛋白，其中所述凝血因子VIII活性部分与所述融合伴侣通过肽连接子连接，所述肽连接子包括柔性肽接头和/或刚性单元，例如可以包括1、2、3、4、5或更多个所述刚性单元。
8、根据实施方案7所述的融合蛋白，其中所述柔性肽接头含有2个或更多个选自甘氨酸、丝氨酸、丙氨酸和苏氨酸的氨基酸残基，
优选地，所述柔性肽接头具有序列通式(GS)a(GGS)b(GGGS)c(GGGGS)d，其中a、b、c和d是大于或等于0的整数，且a+b+c+d≥1，
更优选地，所述柔性肽接头具有选自下组的序列：
(i)GSGGGSGGGGSGGGGS(SEQ ID NO：6)；
(ii)GSGGGGSGGGGSGGGGSGGGGSGGGGS(SEQ ID NO：7)；
(iii)GGGGSGGGGSGGGGSGGGGS(SEQ ID NO：8)；
(iv)GSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS(SEQ ID NO：9)；或
(v)GGGSGGGSGGGSGGGSGGGS(SEQ ID NO：10)。
9、根据实施方案7或8所述的融合蛋白，其中所述刚性单元为人绒毛膜促性腺激素β亚基的羧基末端肽，或所述刚性单元与人绒毛膜促性腺激素β亚基的羧基末端肽的氨基酸序列具有70％、80％、90％、95％或更高的一致性；所述刚性单元可以包含1个、2个或更多个糖基化位点；
优选地，所述刚性单元包含选自以下的氨基酸序列：
(i)PRFQDSSSSKAPPPSLPSPSRLPGPSDTPILPQ(SEQ ID NO：11)；
(ii)SSSSKAPPPSLPSPSRLPGPSDTPILPQ(SEQ ID NO：12)；
(iii)SSSSKAPPPS(SEQ ID NO：13)；或
(iv)SRLPGPSDTPILPQ(SEQ ID NO：14)；
更优选地，所述肽连接子包含SEQ ID NO：15所示的氨基酸序列。
10、一种药物组合物，其包含有效量的实施方案1至9中任一项所述的融合蛋白，及药学上可接受的载体。
11、一种预防和/或治疗出血性疾病的方法，其包括向有此需要的对象施用实施方案1至9中任一项所述的融合蛋白或实施方案10所述的药物组合物，所述出血性疾病优选地选自FVIII先天性或获得性缺乏症患者的出血性疾病和血友病A患者的自发或手术性出血。
12、一种改良凝血因子VIII的半衰期的方法，其中将凝血因子VIII活性部分与增加半衰期的融合伴侣直接连接或者以肽连接子间接连接，并进一步与聚亚烷基二醇相缀合。
13.根据实施方案12的方法，其中所述融合伴侣为：免疫球蛋白Fc片段、白蛋白、XTEN或转铁蛋白，这些所述融合伴侣例如源自人；优选为IgG Fc片段；例如所述IgG Fc片段具有降低的ADCC效应和/或CDC效应和/或与FcRn受体增强的结合亲和力；更优选地，所述IgG Fc片段具有选自以下的氨基酸序列：
(i)SEQ ID NO：3所示的氨基酸序列；
(ii)SEQ ID NO：4所示的氨基酸序列；或
(iii)SEQ ID NO：5所示的氨基酸序列。
14.根据实施方案12或13所述的方法，其中所述聚亚烷基二醇是聚丙二醇或聚乙二醇；所述聚亚烷基二醇可以是末端封端的，例如经烷氧基如甲氧基封端的；和/或所述聚亚烷基二醇是直链的或支链的；优选地所述聚亚烷基二醇是支链的，例如是支链的聚乙二醇，尤其是经甲氧基封端的支链聚乙二醇；所述聚亚烷基二醇的分子量可以是＞＝1、＞＝10、＞＝20、＞＝30、＞＝40、＞＝50、＞＝60、＞＝70、＞＝80、＞＝90、＞＝100、＞＝110、＞＝120、＞＝130、＞＝140、＞＝150或＞＝160kDa，例如是5、10、20、30、40、50、60、70、80、90或100kDa，或者以上任意二数值之间的值。
15.根据实施方案12至14中任一项所述的方法，其中所述融合蛋白与聚亚烷基二醇相缀合是随机的或定点的，所述缀合位置选自游离氨基、巯基、糖基和/或羧基，例如游离氨基。
16.根据实施方案12至15中任一项所述的方法，其中使用修饰剂实现所述缀合，优选地，所述修饰剂可以是活化酯形式的修饰剂，更优选地，修饰剂选自如下式(1)、(2)或(3)：
Figure PCTCN2019087156-appb-000004
其中，0≤m1≤6，m1优选为5；mPEG-表示甲氧基单封端的聚乙二醇基团，式(1)所示的修饰剂的分子量为5KD至60KD之间；
Figure PCTCN2019087156-appb-000005
其中0≤m2≤6，m2优选为2；0≤m3≤6，m3优选为1；mPEG-表示甲氧基单封端的聚乙二醇基团，式(2)所示的修饰剂的分子量为 5KD-100KD，优选为40KD、50KD、60KD，最优选为40KD；
Figure PCTCN2019087156-appb-000006
其中，0≤m4≤6，m4优选为2；mPEG-表示甲氧基单封端的聚乙二醇基团，式(3)所示的修饰剂的分子量为5KD-100KD。
17、根据实施方案12至16任一项所述的方法，其中所述凝血因子VIII与所述融合伴侣通过肽连接子连接，所述肽连接子包括柔性肽接头和/或刚性单元，例如可以包括1、2、3、4、5或更多个所述刚性单元。
18、根据实施方案17所述的方法，其中所述柔性肽接头含有2个或更多个选自甘氨酸、丝氨酸、丙氨酸和苏氨酸的氨基酸残基，
优选地，所述柔性肽接头具有序列通式(GS)a(GGS)b(GGGS)c(GGGGS)d，其中a、b、c和d是大于或等于0的整数，且a+b+c+d≥1，
更优选地，所述柔性肽接头具有选自下组的序列：
(i)GSGGGSGGGGSGGGGS(SEQ ID NO：6)；
(ii)GSGGGGSGGGGSGGGGSGGGGSGGGGS(SEQ ID NO：7)；
(iii)GGGGSGGGGSGGGGSGGGGS(SEQ ID NO：8)；
(iv)GSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS(SEQ ID NO：9)；或
(v)GGGSGGGSGGGSGGGSGGGS(SEQ ID NO：10)。
19、根据实施方案17或18所述的融合蛋白，其中所述刚性单元为人绒毛膜促性腺激素β亚基的羧基末端肽，或所述刚性单元与人绒毛膜促性腺激素β亚基的羧基末端肽的氨基酸序列具有70％、80％、90％、95％或更高的一致性；所述刚性单元可以包含1个、2个或更多个糖基化位点；
优选地，所述刚性单元包含选自以下的氨基酸序列：
(i)PRFQDSSSSKAPPPSLPSPSRLPGPSDTPILPQ(SEQ ID NO：11)；
(ii)SSSSKAPPPSLPSPSRLPGPSDTPILPQ(SEQ ID NO：12)；
(iii)SSSSKAPPPS(SEQ ID NO：13)；或
(iv)SRLPGPSDTPILPQ(SEQ ID NO：14)；
更优选地，所述肽连接子包含SEQ ID NO：15所示的氨基酸序列。
附图说明
为了更清楚地说明本发明实施例和现有技术的技术方案，下面对实施例和现有技术中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的实施方案。
图1a为未mPEG修饰的FVIII-Fc(FF-0)的SEC-HPLC检测结果。
图1b为5K分子量mPEG修饰的FVIII-Fc(FF-5L)的SEC-HPLC检测结果。
图1c为10K分子量mPEG修饰的FVIII-Fc(FF-10L)的SEC-HPLC检测结果。
图1d为20K分子量mPEG修饰的FVIII-Fc(FF-20L)的SEC-HPLC检测结果。
图1e为30K分子量mPEG修饰的FVIII-Fc(FF-30L)的SEC-HPLC检测结果。
图1f为40K分子量mPEG修饰的FVIII-Fc(FF-40L)的SEC-HPLC检测结果。
图2a为未mPEG修饰的FVIII-Linker1-Fc(FL1F-0)的SEC-HPLC检测结果(纯度＞99％，聚合体＜1％)。
图2b为20K分子量mPEG修饰的FVIII-L1-Fc(FL1F-20L)的SEC-HPLC检测结果(纯度＞95％，聚合体＜5％，未交联的＜1％)。
图2c为直链形，30K分子量mPEG修饰的FVIII-L1-Fc(FL1F-30L)的SEC-HPLC检测结果(纯度＞95％，聚合体＜5％，未交联的＜1％)。
图2d为直链形，40K分子量mPEG修饰的FVIII-L1-Fc(FL1F-40L)的SEC-HPLC检测结果(纯度＞95％，聚合体＜5％，未交联的＜1％)。
图2e为直链形，50K分子量mPEG修饰的FVIII-L1-Fc(FL1F-50L)的SEC-HPLC检测结果(纯度＞95％，聚合体＜5％，未交联的＜1％)。
图2f为Y型-40K分子量mPEG修饰的FVIII-L1-Fc(FL1F-40Y)的SEC-HPLC检测结果(纯度＞95％，聚合体＜5％，未交联的＜1％)。
图3a为未mPEG修饰的hFVIII-Fc(FF-0)原液G25换液前后，SDS-PAGE检测结果(H代表还原，F代表非还原)。
图3b为hFVIII-Fc与不同分子量mPEG交联(FF-5L至FF-40L)的SDS-PAGE检测结果(非还原)。
图3c为hFVIII-Fc与不同分子量mPEG交联(FF-5L至FF-40L)的SDS-PAGE检测结果(还原)。
具体实施方式
为使本发明的目的、技术方案、及优点更加清楚明白，以下参照附图并举实施例，对本发明进一步详细说明。显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施方案，都属于本发明保护的范围。
术语“凝血因子VIII”，也叫做因子VIII，或者FVIII，是主要由肝细胞产生的一种大而复杂糖蛋白。术语“凝血因子VIII活性部分”是指使本发明的融合蛋白表现出FVIII活性的部分。天然人FVIII由2351个氨基酸组成，包括信号肽，并含有由同源性限定的若干不同结构域。有3个A结构域、1个独特的B结构域和2个C结构域。结构域的顺序可列为NH2-A1-A2-B-A3-C1-C2-COOH。FVIII在血浆内以在B-A3边界分开的2条链而循环。这两条链通过二价金属离子结合而连接。A1-A2-B链称为重链(HC)，而A3-C1-C2称为轻链(LC)。
内源因子VIII分子在体内作为具有不同大小的B结构域的分子库而循环。在体内可能发生的是对B结构域的逐步酶切除，产生具有不同大小的B结构域的分子库。通常认为在740位的切割(B结构域的最后部分正是因此而被切除)的发生与凝血酶激活相关。
本发明中所述的“凝血因子VIII”既可以指代其天然的野生型序列(如SEQ ID NO：1)，也涵盖了其变体形式，例如经过1个或更多个氨基酸替 换、缺失或插入后得到的变体蛋白，同时保留凝血因子VIII的活性。
在一个实施方案中，凝血因子VIII是B结构域截短的分子，其中剩余结构域基本对应于SEQ ID NO：1中氨基酸编号1-745和1640-2332所示序列。此外，本发明的B结构域截短的分子可与SEQ ID NO：2所示序列具有微小差异，意即剩余结构域(即3个A结构域和2个C结构域)与SEQ ID NO：2所示氨基酸序列可具有一个或更多个氨基酸替换、添加或缺失，例如具有1、2、3、4、5、6、7、8、9、10或更多个氨基酸的差异或约1％、2％、3％、4％或5％差异，在保留因子VIII基本活性的情况下，改变因子VIII与多种其它组分(例如LRP、多种受体、其它凝血因子、细胞表面)的结合能力，引入和/或消除糖基化位点等。
术语“融合伴侣”是指与目标多肽(即希望延长其半衰期的多肽)进行融合的另一个多肽，所述融合伴侣能够通过多种不同的机制改变融合蛋白的功能，例如延长目标多肽的体内半衰期。
在一个实施方案中，融合伴侣通过与新生儿Fc受体(FcRn)相互作用而延迟FVIII的体内清除。在一个实施方案中，融合伴侣是免疫球蛋白Fc区(Fc区)、白蛋白、转铁蛋白、XTEN或其部分。在一个优选实施方案中，由于IgG抗体具有较长的半衰期从而优选IgG Fc结构域。
Fc结构域还可经修饰以改进其它功能，例如补体结合和/或与某些Fc受体结合在IgG Fc结构域中234、235和237位的突变通常将会导致与FcγRI受体的结合减少，还可能导致与FcγRIIa和FcγRIII受体的结合减少。这些突变不改变与FcRn受体的结合，其通过内吞再循环途径而促进了长循环半衰期。优选地，本发明的融合蛋白中经修饰的IgG Fc结构域包含一个或多个以下突变，所述突变将会分别导致对某些Fc受体的亲和力下降(L234A、L235E和G237A)以及C1q-介导的补体结合降低(A330S和P331S)。
术语“聚亚烷基二醇”是一种亲水性聚合物，在本发明中与凝血因子VIII和/或融合伴侣上的特定位置发生缀合，聚亚烷基二醇可以是直链或支链并且可包含一个或多个独立选择的聚合部分，优选地，聚亚烷基二醇是聚乙二醇(包括其甲氧基封端形式m-PEG)、聚丙二醇(包括mPPG)等。
本发明中的聚亚烷基二醇可以是聚乙二醇(PEG)，其可以是直链或支链的。支链聚合物的主链是本领域公知的，通常支链聚合物具有中央分 支核心部分和与该中央分支核心连接的一个或多个直链聚合物链。本发明优选使用支链形式的PEG。在一个实例中，支链聚乙二醇可以通式表示为R(-PEG-OH)m，其中R代表核心部分，例如甘油或季戊四醇，m代表臂的数目。
在一个实施方案中，支链PEG(如mPEG)中支链的数量为2，此时也被称为“Y型”PEG(如mPEG)，即包含两个PEG或直链甲氧基PEG的支链型PEG。
其他合适聚合物的实例包括但不限于其它聚亚烷基二醇(例如聚丙二醇(PPG)、乙二醇和丙二醇等的共聚物)、聚氧乙基化多元醇、聚烯醇(olefmic alcoh0l)、聚乙烯吡咯烷酮、聚羟丙基甲基丙烯酰胺、聚([α]-羟基酸)、聚乙烯醇、聚磷腈、聚噁唑啉、聚N-丙烯酰吗啉及其共聚物、三元共聚物和混合物。
在本发明的一种实施方案中，使用PEG修饰(即缀合)，更优选的采用mPEG修饰，其中所述修饰是随机修饰或者定点修饰，所述修饰的位置包括游离氨基、巯基、糖基和/或羧基，例如游离氨基。
在本发明的一种具体实施方案中，游离氨基的mPEG随机修饰所采用的修饰剂可以选自：mPEG-SS(甲氧基聚乙二醇-琥珀酰亚胺琥珀酸酯)、mPEG-SC(甲氧基聚乙二醇-琥珀酰亚胺碳酸酯)、mPEG-SPA(甲氧基聚乙二醇-琥珀酰亚胺丙酸酯)和mPEG-SG(甲氧基聚乙二醇-琥珀酰亚胺戊二酸酯)等其中的一种。N末端的修饰剂为：mPEG-ALD(甲氧基聚乙二醇-乙醛)、mPEG-pALD(甲氧基聚乙二醇-丙醛)和mPEG-bALD(甲氧基聚乙二醇-丁醛)等其中的一种。所述修饰剂mPEG-SS、mPEG-SC、mPEG-SPA、mPEG-SG、mPEG-ALD、mPEG-pALD、mPEG-bALD的形状为直链或分枝状。
在本发明的一种具体实施方案中，游离巯基随机修饰所采用的修饰剂为mPEG-mal(甲氧基聚乙二醇-马来酰亚胺)、mPEG-OPSS(甲氧基聚乙二醇-邻二硫吡啶)、mPEG-Vinylsulfone(甲氧基聚乙二醇-乙烯砜)和mPEG-Thiol(甲氧基聚乙二醇-硫醇)等其中的一种。
在本发明的一种具体实施方案中，所述糖基和/或羧基随机修饰所采用的修饰剂为mPEG-ZH(甲氧基聚乙二醇-酰肼)。
在本发明的一种实施方案中，所述mPEG修饰的修饰剂的结构如式(1)所示：
Figure PCTCN2019087156-appb-000007
其中，0≤m1≤6，m1优选为5；mPEG-表示甲氧基单封端的聚乙二醇基团，式(1)所示的修饰剂的分子量为5kD-60kD(kD，千道尔顿)，优选为40kD。较佳地，本发明的一种实施方案中，用式(1)所示的修饰剂进行游离氨基的mPEG随机修饰。
在本发明的一种实施方案中，所述mPEG修饰的修饰剂的结构如式(2)所示：
Figure PCTCN2019087156-appb-000008
其中0≤m2≤6，m2优选为2；0≤m3≤6，m3优选为1；mPEG-表示甲氧基单封端的聚乙二醇基团，式(2)所示的修饰剂的分子量为5kD-60kD，优选为：40kD。较佳地，本发明的一种实施方案中，用式(2)所示的修饰剂进行自由氨基的mPEG随机修饰。
在本发明的一种实施方案中，所述mPEG修饰的修饰剂的结构如式(3)所示：
Figure PCTCN2019087156-appb-000009
其中，0≤m4≤6，m4优选为2；mPEG-表示甲氧基单封端的聚乙二醇基团，式(3)所示的修饰剂的分子量为5kD-60kD，优选为40kD。本发明的一种实施方案中，用式(3)所示的修饰剂进行自由巯基的mPEG随机修饰。
聚合物主链的大小可以发生改变，但聚合物(例如PEG、mPEG、PPG或mPPG)典型的范围为约0.5KD至约160KD，例如约1KD至约100KD。更具体地讲，每种本发明缀合的亲水性聚合物的大小主要以下范围内变化：约1KD至约80KD，约2KD至约70KD；约5KD至约70KD；约10KD至约60KD，约20KD至约50KD；约30KD-至约50KD或约30KD-40KD。应当知道，这些大小代表约值，而并非精确测量值，这是本领域所公认的做法。
在一个具体的实施方案中，本发明所使用的PEG或mPEG的大小在35KD以上(即不低于35KD)，优选不小于40KD、不小于45KD、不小于50KD、不小于55KD、不小于60KD、不小于65KD或不小于70KD，例如，分子量具体为40KD、50KD、60KD、70KD、80KD、90KD、100KD、110KD、120KD、130KD、140KD、150KD或160KD。
术语“改良的循环半衰期”：与野生型因子VIII分子相比，本发明的分子具有改变的循环半衰期，优选增加的循环半衰期。循环半衰期优选增加至少10％，优选至少15％，优选至少20％，优选至少25％，优选至少30％，优选至少35％，优选至少40％，优选至少45％，优选至少50％，优选至少55％，优选至少60％，优选至少65％，优选至少70％，优选至少75％，优选至少80％，优选至少85％，优选至少90％，优选至少95％，优选至少100％，更优选至少125％，更优选至少150％，更优选至少175％，更优选至少200％，和最优选至少250％或300％。甚至更优选地，所述分子具有的循环半衰期增加至少400％，500％，600％，或甚至700％。
术语“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。
实施例
实施例1 mPEG修饰的hFVIII融合蛋白的制备及纯化
1.1、mPEG修饰的hFVIII融合蛋白的制备
1.1.1首先根据本领域技术人员熟知的分子克隆技术进行一系列hFVIII融合蛋白表达质粒构建，将表达质粒分别转染DHFR缺陷型CHO细胞(参见美国专利US 4,818,679)，表达各个hFVIII融合蛋白(表1)。融合蛋白的具体制备步骤参见中国专利ZL201610692838.0，该专利通过引用其全文并入本文中。
表1融合蛋白的组成和结构
Figure PCTCN2019087156-appb-000010
注：＊B结构域缺失的hFVIII简称BDD FVIII，由90kD A1-A2重链和80kD轻链组成；
＊＊SEQ ID NO.7-SEQ ID NO.12表示连接头由SEQ ID NO.12所示的刚性单元连接至SEQ ID NO.7所示的柔性肽接头的C末端形成；SEQ ID NO.6-SEQ ID NO.11表示连接头由SEQ ID NO.11所示的刚性单元连接至SEQ ID NO.6所示的柔性肽接头的C末端形成。
1.1.2、将1.1.1各个融合蛋白的发酵液，分别经过离心、过滤，依次经过亲和层析(Affinity chromatography)/疏水层析(Hydrophobic Interaction Chromatography)/离子交换层析(Ion-Exchange chromatography)/分子大小排阻层析(Size exclusion chromatography)，分别得到五种hFVIII融合蛋白FF-0、FL1F-0、FL2F-0、F(全长)L1F’-0和F(全长)L2F”-0，SEC-HPLC检测出聚合体＜5％。将五种hFVIII融合蛋白分别配制为蛋白质浓度为0.95mg/ml hFVIII融合蛋白原液；
1.1.3、分别取1.1.2制备的五种hFVIII融合蛋白原液各5ml，进行G25(GE Healthcare)分子筛层析。具体步骤如下：
缓冲液配制：20mM Hepes，0.1M NaCl，5.0mM CaCl2，0.02％Tween8.0，pH 7.0；
层析过程：
(1)平衡：用3倍柱体积的结合缓冲液(Binding Buffer)平衡层析柱，平衡至pH及电导同缓冲液一致即可，流速统一为150cm/h；
(2)上样：上样流速统一为150cm/h；
(3)平衡：用3倍柱体积的缓冲液平衡层析柱，平衡至pH及电导同缓冲液一致即可，流速统一为150cm/h；
(4)平衡：用缓冲液平衡层析柱，收集A280/A260大于1.8的峰；
(5)层析柱的在位清洗：用0.2M NaOH以60cm/h的流速进行反向清洗1.5柱体积，并用缓冲液进行中和；
(6)层析柱的保存：实验结束后，用3倍柱体积纯化水以100cm/h的流速清洗层析柱之后，用2倍柱体积20％乙醇对层析柱进行保存。
超滤浓缩：将经过G25换液后的五种hFVIII融合蛋白(FF-0、FL1F-0、FL2F-0、F(全长)L1F’-0和F(全长)L1F”-0)原液，用50kD的超滤管超滤浓缩，在4℃，3800rpm的条件下离心浓缩，浓缩至蛋白浓度优选为1.5mg/ml。
1.1.4、按hFVIII融合蛋白与mPEG-SC(购自于北京键凯科技有限公司)摩尔(mol)比为1∶10-1∶100的比例称取mPEG-SC(分子量分别为5kD、10kD、20kD、30kD、40kD的结构如式(1)所示的直链L形mPEG-SC，和分子量为40kD的结构如式(2)所示的支链Y形mPEG-SC)，加入1.1.3超滤浓缩后的hFVIII融合蛋白，反应4小时，加底物hFVIII融合蛋白10倍摩尔(mol)比例的组氨酸终止该反应，得到不同分子量的mPEG-SC修饰的hFVIII融合蛋白；部分修饰产物编号及其组成见下表2。
表2
Figure PCTCN2019087156-appb-000011
Figure PCTCN2019087156-appb-000012
1.2 mPEG修饰的hFVIII融合蛋白的纯化
1.2.1、将实施例1“1.1.4”项下制备的各mPEG修饰的hFVIII融合蛋白分别进行S200(GE Healthcare)分子筛层析。具体如下：
缓冲液配制：20mM组氨酸，0.1M NaCl，5.0mM CaCl 2，0.02％Tween8.0，pH 7.0；
层析过程：
(1)平衡：用3倍柱体积的结合缓冲液(Binding Buffer)平衡层析柱，平衡至pH及电导同缓冲液一致即可，流速统一为150cm/h；
(2)上样：上样流速统一为150cm/h；
(3)平衡：用3倍柱体积的缓冲液平衡层析柱，平衡至pH及电导同缓冲液一致即可，流速统一为150cm/h；
(4)平衡：用缓冲液平衡层析柱，收集A280/A260大于1.8的峰；
(5)层析柱的在位清洗：用0.2M NaOH以60cm/h的流速进行反向清洗1.5柱体积，并用缓冲液进行中和；
(6)层析柱的保存：实验结束后，用3倍柱体积纯化水以100cm/h的流速清洗层析柱之后，用2倍柱体积20％乙醇对层析柱进行保存。
1.2.2、将“1.2.1”项所得到的层析产物进行Source 15Q(GE Healthcare)阴离子层析：
缓冲液配制：
结合缓冲液：20mM组氨酸，0.1M NaCl，5.0mM CaCl 2，0.02％Tween8.0，pH 7.0；洗脱缓冲液：20mM组氨酸，2.0M NaCl，5.0mM CaCl 2，0.02％Tween8.0，pH 7.0；CIP：0.5M NaOH；
层析过程：
(1)平衡：用3倍柱体积的结合缓冲液平衡层析柱，平衡至pH及电导同缓冲液一致即可，流速统一为150cm/h；
(2)上样：上样流速统一为150cm/h；
(3)平衡：用3倍柱体积的结合缓冲液平衡层析柱，平衡至pH及电导同缓冲液一致即可，流速统一为150cm/h；
(4)洗脱：按照0-100％缓冲液B线性梯度20倍柱体对样品进行洗脱，洗脱流速统一为100cm/h，分管收集A280/A260大于1.8的洗脱峰，每一管样品均进行SEC-HPLC检测；
(5)层析柱的在位清洗：用0.5M NaOH以60cm/h的流速进行反向清洗1.5柱体积，并用结合缓冲液进行中和；
(6)层析柱的保存：实验结束后，用3倍柱体积纯化水以100cm/h的流速清洗层析柱之后，用2倍柱体积20％乙醇对层析柱进行保存。
1.2.3、SEC-HPLC检测：
将“1.2.2”项所得到的层析产物进行SEC-HPLC检测，其中：
色谱柱：G3000/G4000；流速：0.5mL/min；检测波长：280nm；柱温：25℃；进样体积：100μL(进样量20μg)；流动相：0.30M氯化钠；0.02M咪唑；0.01M氯化钙；25ppm吐温80；10％乙醇；pH 7.0；运行时间：35-50min；
FF-0至FF-40L的检测结果见图1a-1f。FL1F-0至FL1F-50L及FL1F-40Y的检测结果见图2a-2f，结果显示，FL1F-0至FL1F-60L的纯度＞95％，聚合体＜5％，未交联的＜1％。
1.2.4、SDS-PAGE进行蛋白质凝胶电泳检测：
将“1.2.2”项所得到的产物进行SDS-PAGE检测，包括以下步骤：
(1)制胶：1×Tris-甘氨酸电泳缓冲液：SDS 0.4g，Tris base 1.21g，甘氨酸7.5g，双蒸水定容至400mL。
5％浓缩胶：双蒸水4.1mL，1M Tris-HCl(pH 6.8)0.75mL，30％(w/v)聚丙烯酰胺1mL，10％(w/v)过硫酸铵60μL，10％(w/v)SDS 60μL，TEMED 6μL。
6％分离胶：双蒸水4.9mL，1.5M Tris-HCl(pH 8.8)3.8mL，30％(w/v)聚丙烯酰胺6mL，10％(w/v)过硫酸铵150μL，10％(w/v)SDS 150μL，TEMED 6μL。
(2)5×蛋白上样缓冲液：甘油5mL，1M Tris-HCl(pH 6.8)2.5mL，溴酚蓝0.05g，SDS 1g，双蒸水定容至10mL，4℃保存，使用前加入β-巯基乙醇0.5mL。
(3)制样：将待检测样品与等体积上样缓冲液混合，还原SDS-PAGE加入与样品等体积的0.1mg/mL的2-巯基乙醇；非还原性的SDS-PAGE不加2-巯基乙醇。样品与上样缓冲液混合后，在沸水中水浴10分钟。
(4)电泳：将煮好的待检测样品和蛋白质Marker分别取10μl依次加入到点样孔，于60V的电压下进行浓缩电泳，观察显示剂溴酚蓝染料集中至分离胶后增加电压至120V进行分离电泳，直到溴酚蓝染料达到分离胶的底部，关闭电源。
(5)染色：小心取出SDS-PAGE凝胶放置于装有考马斯亮蓝染色液的塑料盒中，放置于微波炉加热1分钟。
(6)脱色：取出染好的SDS-PAGE凝胶放置于脱色液中，震荡脱色，2小时更换一次脱色液，观察到出现肉眼可见清晰的条带后停止。
(7)记录：将完成的SDS-PAGE凝胶照相记录或者干燥保存。图3a-3c示出了FF-5L至FF-40L的SDS-PAGE检测结果。
实施例2发色底物法间接测定mPEG修饰的hFVIII融合蛋白的体外活性
采用发色底物法(chromogenic substrate assay)测定实施例1制备的mPEG修饰的hFVIII融合蛋白的活性。采用ChromogenixCoatest 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的含量直接相关。发色底物法间接测定FVIII生物学活性结果见表3。
表3发色底物法间接测定FVIII生物学活性
Figure PCTCN2019087156-appb-000013
Figure PCTCN2019087156-appb-000014
Figure PCTCN2019087156-appb-000015
注：Eloctate是Bioverativ公司已经上市的重组因子VIII Fc融合蛋白，其未经mPEG修饰。
实施例3人凝血因子VIII效价测定法
本发明所采用的人凝血因子VIII效价测定法，也称为一期法，具体步骤参见中国药典2010版三部。一期法测定FVIII生物学活性是通过纠正FVIII缺失血浆所导致凝固时间延长的能力而进行的。采用德国Siemens公司生产的试剂盒Coagulation Factor VIII Deficient Plasma(Cat.No.OTXW17)。方法包括：首先，将已知效价的FVIII活性标准品WHO International Standard 8th International Standard Factor VIII Concentrate(Cat.No.07/350)稀释至4IU/ml，再进行梯度稀释至不同效价(IU/ml)，并与乏FVIII基质血浆混合，测定部分凝血活酶时间(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)的对数作直线回归，建立标准曲线。再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。通过代入标准曲线，可知待测样品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。结果见表4。
表4一期法直接测定生物学活性
Figure PCTCN2019087156-appb-000016
Figure PCTCN2019087156-appb-000017
Figure PCTCN2019087156-appb-000018
在表3和表4中显示：虽然发色底物法和一期法测得的FL1F-40Y、FL2F-40Y、F(全长)L1F’-40Y和F(全长)L2F”-40Y生物学活性较未交联mPEG的Eloctate/FL1F-0/FL2F-0实验组低，这是由于mPEG修饰后，对被修饰蛋白的空间结构产生影响造成的。在现有技术的其他mPEG修饰蛋白(例如PEG-INTRON；Pegfilgrastim)中均有类似现象。出人意料地是，本发明的融合蛋白在经40kD或更高的mPEG修饰后仍能保持相对较高的活性，与此同时，后续实验进一步验证了其半衰期被大大延长。
实施例4血友病A小鼠尾静脉横切模型上的预防性药效学实验
本实施例通过尾静脉横切(tail vein transection，简称TVT)实验比较了各个mPEG修饰的hFVIII融合蛋白在血友病A小鼠(HA小鼠)体内的活性半衰期。
4.1、依文献报道的方法，选10-12周龄雄性HA小鼠(购自上海南方模式生物研究中心)，随机分组，12只/组，给药剂量15IU/kg，分别将本发明的各mPEG修饰的融合蛋白或阳性对照药物Eloctate经尾静脉给药。给药后48h，用内径2.7mm套管量取尾巴并做标记，用11号直刃手术刀片对单侧尾静脉进行横切，横切后，迅速将鼠尾放进装有约13ml的预热的生理盐水管中，并记录出血时间。停止出血(切口处无明显血液流出)后将鼠尾从生盐水管中取出，然后将小鼠置于37℃的加热垫上以保持其体温，不要碰触伤口。待小鼠苏醒后，将其放入垫有A4白纸的鼠笼里，单笼饲养，并于每次观察后更换白纸或更换鼠笼，以便判断出血程度。统计断尾后，48h内的小鼠的存活率和断尾后12h内的复出血次数(共统计12小时，一个小时之内出血多次的算是1次)，结果如表5所示。
复出血率统计即在统计期间发生复出血小鼠所占比例，严重出血率即在12内的复出血统计中有严重出血(+++)现象或者多次中等程度出血(++)现象的小鼠所占比例。其中，中等程度出血(++)是指：A4白纸上有多量血迹，覆盖面积不低于30％，且血痕颜色中等，但无大面积血滩(面积＞3cm2)；严重出血(+++)是指：A4白纸上有大量血迹，覆盖面积不低于30％，血痕颜色偏重，有大面积血滩者；即使覆盖面积低于小，也可看做重度出血(小鼠大量失血，活动范围减小，血液严重浸湿白纸)。
表5给药后48h进行TVT的48h存活率和12h复出血率
融合蛋白名称 12h复出血率 严重出血率 48h存活率
Eloctate 66.7％(8/12) 16.7％(2/12) 75.0％(9/12)
FL1F-0 83.3％(10/12) 33.3％(4/12) 25.0％(3/12)
FL1F-20L 83.3％(10/12) 33.3％(4/12) 41.7％(5/12)
FL1F-40L 66.7％(8/12) 16.7％(2/12) 75.0％(9/12)
FL1F-40Y 58.3％(7/12) 8.3％(1/12) 83.3％(10/12)
FL1F-50L 75.0％(9/12) 25.0％(3/12) 58.3％(7/12)
FL1F-60L 66.7％(8/12) 25.0％(3/12) 66.7％(8/12)
FL2F-0 83.3％(10/12) 33.3％(4/12) 33.3％(4/12)
FL2F-20L 75.0％(9/12) 25.0％(3/12) 41.6％(5/12)
FL2F-40L 75.0％(9/12) 16.7％(2/12) 66.7％(8/12)
FL2F-40Y 63.3％(7/11) 9.1％(1/11) 81.8％(9/11)
FL2F-50L 66.7％(8/12) 16.7％(2/12) 58.3％(7/12)
FL2F-60L 66.7％(8/12) 25.0％(3/12) 50.0％(6/12)
F(全长)L1F’-40L 75.0％(9/12) 16.7％(2/12) 66.7％(8/12)
F(全长)L1F’-40Y 66.7％(8/12) 8.3％(1/12) 83.3％(10/12)
F(全长)L1F’-50L 66.7％(8/12) 25.0％(3/12) 50.0％(6/12)
F(全长)L2F”-40L 75.0％(9/12) 8.3％(1/12) 58.3％(7/12)
F(全长)L2F”-40Y 58.3％(7/12) 8.3％(1/12) 83.3％(10/12)
F(全长)L2F”-50L 75.0％(9/12) 16.7％(2/12) 58.3％(7/12)
结果显示：与未经mPEG修饰的Eloctate/FL1F-0/FL2F-0实验组相比，FL1F-40Y实验组的83.3％存活率、FL2F-40Y实验组的75.0％存活率、F(全长)L1F’-40Y实验组83.3％存活率和F(全长)L2F”-40Y实验组83.3％存活率均较其他组有显著提高。这些实验组对应的12h复出血率和严重出血率，较其他组有显著降低。FL1F-40Y、FL2F-40Y、F(全长)L1F’-40Y、F(全长)L2F”-40Y在血友病A小鼠尾静脉横切模型上的预防性药效学上拥有更长的保护时间。
4.2、采用与4.1相同的方法，对10-12周龄雄性HA小鼠，12只/组，在给药后84h进行TVT实验，结果见表6。
表6给药84h后进行TVT的48h存活率和12h复出血率
融合蛋白名称 48h存活率 12h复出血率
Eloctate 66.7％(8/12) 83.3％(10/12)
FL1F-40L 50％(6/12) 83.3％(10/12)
FL2F-40L 58.3％(7/12) 91.7％(11/12)
FL1F-40Y 66.7％(8/12) 75.0％(9/12)
FL2F-40Y 75.0％(9/12) 75.0％(9/12)
FL1F-50L 50.0％(6/12) 100％(12/12)
FL2F-50L 33.3％(4/12) 100％(12/12)
FL1F-60L 58.3％(7/12) 83.3％(10/12)
FL2F-60L 41.7％(5/12) 83.3％(10/12)
F(全长)L1F’-40L 66.7％(8/12) 75.0％(10/12)
F(全长)L1F’-40Y 75.0％(9/12) 66.7％(8/12)
F(全长)L1F’-50L 58.3％(7/12) 91.7％(11/12)
F(全长)L2F”-40L 41.7％(5/12) 91.7％(11/12)
F(全长)L2F”-40Y 75.0％(9/12) 75.0％(9/12)
F(全长)L2F”-50L 58.3％(7/12) 91.7％(11/12)
结果显示：与未交联PEG的Eloctate以及其他实验组相比，FL1F-40Y实验组的66.7％存活率、FL2F-40Y实验组的75.0％存活率、F(全长)L1F’-40Y实验组75.0％存活率和F(全长)L2F”-40Y实验组75.0％存活率均较其他组有显著提高，相应的复出血率明显降低。FL1F-40Y、FL2F-40Y、F(全长)L1F’-40Y、F(全长)L2F”-40Y在给药后84h后血友病A小鼠尾静脉横切模型上的预防性药效学上存在一定优势。
4.3、采用与4.1相同的方法，对10-12周龄HA小鼠，雄性和雌性各10只，20只/组，在给药后90h进行TVT实验，结果见表7。
表7给药后90h进行TVT的48h存活率和12h复出血率
融合蛋白名称 48h存活率 12h复出血率
Eloctate 70％ 80％
FL1F-40L 55％) 70％
FL2F-40L 63.2％ 73.68％
FL1F-50L 84.2％ 68.4％
FL2F-50L 40％ 90％
结果显示：FL1F-50L与未交联PEG的Eloctate实验组拥有相近的存活率和复出血率；由于本实验动物非单一性别，实验结果不与其他单一性别实验结果作比较。
4.4、采用与4.1相同的方法，对10-12周龄雄性HA小鼠，12只/组，在给药后96h进行TVT实验，结果见表8。
表8给药后96h进行TVT的48h存活率和12h复出血率
融合蛋白名称 48h存活率 12h复出血率
Eloctate 50.0％ 91.7％
FL1F-30Y 8.3％ 91.7％
FL2F-30Y 16.7％ 83.3％
FL1F-40Y 63.6％ 54.5％
FL2F-40Y 54.5％ 54.5％
FL1F-50L 16.7％ 91.7％
FL2F-50L 25.0％ 83.3％
结果显示：FL1F-40Y/FL2F-40Y与未交联PEG的Eloctate实验组相比，虽然在存活率上优势小，但12h复出血率显著降低。在存活率上FL1F-40Y/FL2F-40Y均较其他组有显著提高；12h复出血率较其他组有显著降低。FL1F-40Y/FL2F-40Y较其他组在血友病A小鼠尾静脉横切模型上的拥有更长的预防时间。
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内，所做的任何修改、等同替换、改进等，均应包含在本发明保护的范围之内。

Claims (11)

  1. 一种与聚亚烷基二醇缀合的凝血因子VIII融合蛋白，其中凝血因子VIII活性部分(FVIII)与延长半衰期的融合伴侣直接连接或者以肽连接子间接连接形成所述融合蛋白，以及所述融合蛋白进一步与聚亚烷基二醇相缀合。
  2. 根据权利要求1所述的融合蛋白，其中所述凝血因子VIII活性部分源自人，例如全长或截短的人凝血因子VIII，例如缺失B区域的人凝血因子VIII；所述全长或截短的人凝血因子VIII可包含1或更多个氨基酸突变，条件是仍保留其FVIII活性；例如所述凝血因子VIII活性部分包含SEQ ID NO：1或2所示的氨基酸序列，或者与SEQ ID NO：1或2所示氨基酸序列具有至少90％、95％或更高的一致性。
  3. 根据权利要求1或2所述的融合蛋白，其中所述融合伴侣为：免疫球蛋白Fc片段、白蛋白、转铁蛋白或XTEN，这些所述融合伴侣例如源自人；优选为IgG Fc片段；例如所述IgG Fc片段具有降低的ADCC效应和/或CDC效应和/或与FcRn受体增强的结合亲和力；更优选地，所述IgG Fc片段具有选自以下的氨基酸序列：
    (i)SEQ ID NO：3所示的氨基酸序列；
    (ii)SEQ ID NO：4所示的氨基酸序列；或
    (iii)SEQ ID NO：5所示的氨基酸序列。
  4. 根据权利要求1至3任一项所述的融合蛋白，其中所述聚亚烷基二醇是聚丙二醇或聚乙二醇；所述聚亚烷基二醇可以是末端封端的，例如经烷氧基如甲氧基封端的；和/或所述聚亚烷基二醇是直链的或支链的；优选地所述聚亚烷基二醇是支链的，例如是支链的聚乙二醇，尤其是经甲氧基封端的支链聚乙二醇；所述聚亚烷基二醇的分子量可以是＞＝1、＞＝10、＞＝20、＞＝30、＞＝40、＞＝50、＞＝60、＞＝70、＞＝80、＞＝90、＞＝100、＞＝110、＞＝120、＞＝130、＞＝140、＞＝150或＞＝160kDa，例如是5、10、20、30、40、50、60、70、80、90或100kDa，或者以上任意二 数值之间的值。
  5. 根据权利要求1至4任一项所述的融合蛋白，其中所述融合蛋白与聚亚烷基二醇的缀合是随机的或定点的，所述缀合位置选自游离氨基、巯基、糖基和/或羧基，优选地游离氨基。
  6. 根据权利要求5所述的融合蛋白，其中使用任意合适的修饰剂实现所述缀合，所述修饰剂可以是活化酯形式的修饰剂或其它原理类型修饰剂，例如所述修饰剂选自如下式(1)、(2)或(3)所示的修饰剂：
    Figure PCTCN2019087156-appb-100001
    其中，0≤m1≤6，m1优选为5；mPEG表示甲氧基单封端的聚乙二醇基团；
    Figure PCTCN2019087156-appb-100002
    其中0≤m2≤6，m2优选为2；0≤m3≤6，m3优选为1；mPEG表示甲氧基单封端的聚乙二醇基团；或
    Figure PCTCN2019087156-appb-100003
    其中，0≤m4≤6，m4优选为2；mPEG表示甲氧基单封端的聚乙二醇基团。
  7. 根据权利要求1至6任一项所述的融合蛋白，其中所述凝血因子VIII活性部分与所述融合伴侣通过肽连接子连接，所述肽连接子包括柔性肽接头和/或刚性单元，例如可以包括1、2、3、4、5或更多个所述刚性单元。
  8. 根据权利要求7所述的融合蛋白，其中所述柔性肽接头含有2个或更多个选自甘氨酸、丝氨酸、丙氨酸和苏氨酸的氨基酸残基，
    优选地，所述柔性肽接头具有序列通式(GS)a(GGS)b(GGGS)c(GGGGS)d，其中a、b、c和d是大于或等于0的整数，且a+b+c+d≥1，
    更优选地，所述柔性肽接头具有选自下组的序列：
    (i)GSGGGSGGGGSGGGGS(SEQ ID NO：6)；
    (ii)GSGGGGSGGGGSGGGGSGGGGSGGGGS(SEQ ID NO：7)；
    (iii)GGGGSGGGGSGGGGSGGGGS(SEQ ID NO：8)；
    (iv)GSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS(SEQ ID NO：9)；或
    (v)GGGSGGGSGGGSGGGSGGGS(SEQ ID NO：10)。
  9. 根据权利要求7或8所述的融合蛋白，其中所述刚性单元为人绒毛膜促性腺激素β亚基的羧基末端肽，或所述刚性单元与人绒毛膜促性腺激素β亚基的羧基末端肽的氨基酸序列具有70％、80％、90％、95％或更高的一致性；所述刚性单元可以包含1个、2个或更多个糖基化位点；
    优选地，所述刚性单元包含选自以下的氨基酸序列：
    (i)PRFQDSSSSKAPPPSLPSPSRLPGPSDTPILPQ(SEQ ID NO：11)；
    (ii)SSSSKAPPPSLPSPSRLPGPSDTPILPQ(SEQ ID NO：12)；
    (iii)SSSSKAPPPS(SEQ ID NO：13)；或
    (iv)SRLPGPSDTPILPQ(SEQ ID NO：14)；
    更优选地，所述肽连接子包含SEQ ID NO：15所示的氨基酸序列。
  10. 一种药物组合物，其包含有效量的权利要求1至9中任一项所述的融合蛋白，及药学上可接受的载体。
  11. 一种预防和/或治疗出血性疾病的方法，其包括向有此需要的对象施用权利要求1至9中任一项所述的融合蛋白或权利要求10所述的药物组合物，所述出血性疾病优选地选自FVIII先天性或获得性缺乏症患者的出血性疾病和血友病A患者的自发或手术性出血。
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