WO2023227015A1 - 具有延长的半衰期的fviii融合蛋白缀合物及其应用 - Google Patents

具有延长的半衰期的fviii融合蛋白缀合物及其应用 Download PDF

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WO2023227015A1
WO2023227015A1 PCT/CN2023/095973 CN2023095973W WO2023227015A1 WO 2023227015 A1 WO2023227015 A1 WO 2023227015A1 CN 2023095973 W CN2023095973 W CN 2023095973W WO 2023227015 A1 WO2023227015 A1 WO 2023227015A1
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seq
amino acid
acid sequence
sequence shown
peg
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PCT/CN2023/095973
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English (en)
French (fr)
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王亚里
高洁
陈宪
莫炜川
苏鸿声
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江苏晟斯生物制药有限公司
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Priority to CN202380009524.4A priority Critical patent/CN117337307A/zh
Publication of WO2023227015A1 publication Critical patent/WO2023227015A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/36—Blood coagulation or fibrinolysis factors
    • A61K38/37—Factors VIII
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/04—Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/745—Blood coagulation or fibrinolysis factors
    • C07K14/755—Factors VIII, e.g. factor VIII C (AHF), factor VIII Ag (VWF)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes

Definitions

  • the present invention relates to the field of biomedicine, and in particular to a recombinant human coagulation factor VIII fusion protein conjugate with extended half-life and its preparation method and application.
  • Hemophilia A is an inherited bleeding disorder caused by a lack or dysfunction of clotting factor VIII (FVIII) activity. Supplementation with active FVIII is an effective treatment for hemophilia A.
  • the FVIII molecule is one of the longest gene fragments cloned to date and is the largest molecular weight protein drug used clinically. Due to the short half-life of FVIII in the blood, only 8-12 hours, patients with severe hemophilia A must undergo intravenous injections of FVIII approximately three times a week when undergoing preventive treatment.
  • polymers with high solubility such as polyethylene glycol (PEG) are routinely used to chemically modify the surface of protein drugs.
  • PEG polyethylene glycol
  • the higher the modification rate the more obvious the reduction in protein antigenicity and the greater the loss of activity.
  • PEG polyethylene glycol
  • Hemophilia patients require lifelong infusions of coagulation factors to stop bleeding and prevent bleeding. Therefore, there is a need to develop coagulation factors with longer half-lives to reduce the frequency of administration. In addition, how to maintain good biological activity while extending half-life is also a problem that needs to be solved.
  • WO2019219049A1 discloses a PEG-modified FVIII-Fc fusion protein.
  • the PEGylated fusion protein can reduce the severe bleeding rate and 12h recurrence rate of the model animal in the hemophilia A mouse tail vein transection model, and increase the model's 48h survival rate of animals.
  • the inventor unexpectedly discovered that not any Fc region and any number of PEG modifications have similar effects on extending the half-life of FVIII-Fc fusion proteins.
  • the Fc region from IgG and a specific number of PEG modifications can lead to an unexpectedly significant increase in half-life.
  • Clinical data shows that the half-life of the PEGylated fusion protein involved in the present invention is 30.87 hours to 31.91 hours, which is 2.10-2.25 times higher than that of the PEGylated fusion protein and 2.61-2.80 times longer than that of the natural protein. It can be administered once a week.
  • a first aspect of the present invention provides a conjugate of coagulation factor VIII-Fc fusion protein and polyethylene glycol (PEG), wherein the coagulation factor VIII-Fc fusion protein comprises the active part of coagulation factor VIII (FVIII) and Fc fragment, the active part of coagulation factor VIII (FVIII) is directly connected to the Fc fragment or indirectly connected through a linker (Linker) to form the fusion protein, and the average number of PEG modifications in the conjugate is 3- 8, 3-7, 3-6, 4-8, 4-7, 4-6 or 4.2-5.1, preferably 4-6, more preferably 4.2-5.1.
  • PEG polyethylene glycol
  • the average number of PEG modifications of the conjugate is the molar ratio of the PEG to the fusion protein, which can also be understood as the modified number of each fusion protein in the sample (containing more than 1 fusion protein).
  • the average number of PEGs Since the number of PEGs attached to each modified fusion protein may be different, the average value may be an integer or a non-integer.
  • the modifying agent used for PEG modification has the structural formula shown in formula (2):
  • 0 ⁇ m2 ⁇ 6, m2 is preferably 2; 0 ⁇ m3 ⁇ 6, m3 is preferably 1; mPEG- represents a methoxy single-capped polyethylene glycol group;
  • the molecular weight of the PEG is 30kD-50kD, preferably 40kD.
  • the active portion of factor VIII is full-length or truncated human factor VIII, preferably human factor VIII with a truncated B region, more preferably comprising SEQ ID NO: 1 or SEQ ID A polypeptide having the amino acid sequence shown in NO: 2, or a polypeptide having at least 90%, 95% or higher identity with the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 and still having FVIII activity.
  • the Fc fragment is an Fc fragment derived from IgG, preferably an Fc fragment from IgG1, IgG2 or IgG4, more preferably an Fc fragment from IgG2.
  • amino acid sequence of the Fc fragment comprises a sequence selected from:
  • the conjugation of the fusion protein to PEG is random or site-directed, and the conjugation position is selected from free amino groups, sulfhydryl groups, glycosyl groups and/or carboxyl groups, preferably free amino groups.
  • the linker includes a flexible unit and a rigid unit.
  • the flexible unit contains 2 or more amino acid residues selected from glycine, serine, alanine and threonine.
  • the flexible peptide linker has the general sequence formula (GS ) a (GGS) b (GGGS) c (GGGGS) d , where a, b, c, and d are integers greater than or equal to 0, and a+b+c+d ⁇ 1.
  • the flexible unit comprises an amino acid sequence selected from:
  • the rigid unit comprises a carboxy-terminal peptide of human chorionic gonadotropin beta subunit, or the rigid unit has an amino acid sequence of 70% with the carboxy-terminal peptide of human chorionic gonadotropin beta subunit %, 80%, 90%, 95% or higher identity; the rigid unit may contain 1, 2 or more glycosylation sites.
  • the rigid unit comprises an amino acid sequence selected from:
  • the factor VIII-Fc fusion protein includes human factor VIII with a truncated B region, a flexible unit, a rigid unit and an Fc fragment in order from the nitrogen terminus to the carbon terminus, wherein:
  • the human coagulation factor VIII of the truncated B region includes the amino acid sequence shown in SEQ ID NO: 2, the flexible unit includes the amino acid sequence shown in SEQ ID NO: 7, and the rigid unit includes the amino acid sequence shown in SEQ ID NO: 12 Sequence, the Fc fragment includes the amino acid sequence shown in SEQ ID NO: 4;
  • the human coagulation factor VIII of the truncated B region includes the amino acid sequence shown in SEQ ID NO: 2, the flexible unit includes the amino acid sequence shown in SEQ ID NO: 6, and the rigid unit includes the amino acid sequence shown in SEQ ID NO: 11 Sequence, the Fc fragment includes the amino acid sequence shown in SEQ ID NO: 4;
  • the human coagulation factor VIII of the truncated B region includes the amino acid sequence shown in SEQ ID NO: 2, the flexible unit includes the amino acid sequence shown in SEQ ID NO: 7, and the rigid unit includes the amino acid sequence shown in SEQ ID NO: 12 Sequence, the Fc fragment includes the amino acid sequence shown in SEQ ID NO: 5;
  • the human coagulation factor VIII of the truncated B region includes the amino acid sequence shown in SEQ ID NO: 2
  • the flexible unit includes the amino acid sequence shown in SEQ ID NO: 6
  • the rigid unit includes the amino acid sequence shown in SEQ ID NO: 11
  • the Fc fragment includes the amino acid sequence shown in SEQ ID NO: 5.
  • the PEG molecule is connected to the primary amine (-NH 2 ) group on the lysine residue in the factor VIII-Fc fusion protein through an active linker succinimidyl ester (SCM);
  • the modifier used for PEG modification has the structural formula shown in formula (2):
  • m2 is 2; m3 is 1; the molecular weight of the PEG is 40kD.
  • a second aspect of the present invention provides a pharmaceutical composition comprising the aforementioned conjugate and a pharmaceutically acceptable carrier.
  • a third aspect of the present invention provides the use of the conjugate in the preparation of medicaments for preventing and/or treating bleeding disorders.
  • the bleeding disorder is a bleeding disorder in patients with congenital or acquired deficiency of FVIII or spontaneous or surgical bleeding in patients with hemophilia A.
  • a fourth aspect of the present invention provides a method for preventing and/or treating a bleeding disorder, comprising administering the conjugate to a subject in need thereof.
  • the bleeding disorder is selected from the group consisting of FVIII congenital or bleeding disorders in patients with acquired deficiencies and spontaneous or surgical bleeding in patients with hemophilia A.
  • the present invention also provides a method for preparing the conjugate of the first aspect, comprising:
  • step 2) React the fusion protein obtained in step 1) with PEG, wherein the molar ratio of PEG to the fusion protein is (50-120):1, preferably 100:1, and the molecular weight of PEG is 30-50kD, preferably 40kD.
  • the molar ratio of PEG to the fusion protein is (50-120):1, preferably 100:1, and the molecular weight of PEG is 30-50kD, preferably 40kD.
  • the reaction conditions include 20°C ⁇ 5°C, reaction for 1-3 hours, preferably 2 hours.
  • Figure 1 shows the SEC-HPLC purity chart of FL1G2-40Y.
  • Figure 2 shows the RI chromatogram of hFVIII fusion protein at a concentration of 0.20 mg/mL.
  • Figure 3 shows the RI chromatogram of a PEG standard solution with a concentration of 0.20 mg/mL.
  • Figure 4 shows the UV chromatogram of hFVIII fusion protein at a concentration of 0.20 mg/mL.
  • Figure 5 shows the RI chromatogram of the purified conjugate at a concentration of 0.20 mg/mL.
  • Figure 6 shows the UV chromatogram of the purified conjugate at a concentration of 0.20 mg/mL.
  • factor VIII also called factor VIII, or FVIII
  • coagulation factor VIII active part refers to the part that causes the fusion protein of the present invention to exhibit FVIII activity.
  • Native human FVIII consists of 2351 amino acids, including the signal peptide, and contains several distinct domains defined by homology. There are 3 A domains, 1 unique B domain and 2 C domains. The sequence of domains may be listed as NH2- A1-A2-B-A3-C1-C2-COOH. FVIII circulates in plasma as two chains separated at the B-A3 boundary. this The two chains are connected by binding of divalent metal ions.
  • the A1-A2-B chain is called the heavy chain (HC), while the A3-C1-C2 chain is called the light chain (LC).
  • Endogenous Factor VIII molecules circulate in the body as a pool of molecules with B domains of varying sizes. What may occur in vivo is a stepwise enzymatic excision of the B domain, generating a library of molecules with B domains of varying sizes. It is generally thought that cleavage at position 740 (where the last part of the B domain is excised) occurs in association with thrombin activation.
  • the "coagulation factor VIII” described in the present invention can refer to its natural wild-type sequence (such as SEQ ID NO: 1), and also covers its variant forms, such as one or more amino acid substitutions, deletions Or a variant protein obtained after insertion while retaining the activity of coagulation factor VIII.
  • coagulation factor VIII is a molecule with a truncated B domain, wherein the remaining domain substantially corresponds to the sequence set forth in amino acid numbers 1-745 and 1640-2332 in SEQ ID NO: 1.
  • the B domain truncated molecule of the invention may have slight differences from the sequence shown in SEQ ID NO: 2, meaning that the remaining domains (i.e., 3 A domains and 2 C domains) are identical to SEQ ID NO:
  • the amino acid sequence shown in 2 may have one or more amino acid substitutions, additions or deletions, for example, 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, altering the interaction between Factor VIII and various other components (e.g. LRP, various receptors, other coagulation factors, cell surface) while retaining the essential activity of Factor VIII Binding ability, introduction and/or elimination of glycosylation sites, etc.
  • various other components e.g. LRP,
  • the Fc fragment can 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 and also May result in reduced binding to Fc ⁇ RIIa and Fc ⁇ RIII receptors. These mutations do not alter binding to the FcRn receptor, which promotes long circulating half-life via the endocytic recycling pathway.
  • the modified IgG Fc domain in the fusion protein of the invention contains one or more of the following mutations, which will result in reduced affinity to certain Fc receptors (L234A, L235E and G237A) and C1q- Mediated complement fixation is reduced (A330S and P331S).
  • Polyethylene glycol (PEG) in the present invention can be linear or branched.
  • the backbones of branched polymers are well known in the art and generally branched polymers have a central branched core portion and one or more linear polymer chains connected to the central branched core.
  • Preferably branched forms of PEG are used in the present invention.
  • branched polyethylene glycol can be represented by the general formula R(-PEG-OH) m , where R represents the core moiety, such as glycerol or pentaerythritol, and m represents the number of arms.
  • the number of branches in a branched PEG is 2, which is also called a "Y-type" PEG (such as mPEG), that is, it contains two PEGs or straight Branched chain PEG of chain methoxy PEG.
  • PEG modification i.e., conjugation
  • mPEG modification is more preferably used, wherein the modification is random modification or site-directed modification, and the positions of the modification include free amino groups, sulfhydryl groups, and sugar groups. and/or carboxyl groups, such as free amino groups.
  • the PEG molecules used for cross-linking reactions with proteins are also called modifiers. It is usually activated polyethylene glycol (also known as polyethylene glycol modifier or PEG modifier), that is, polyethylene glycol with functional groups (such as active linkers).
  • polyethylene glycol also known as polyethylene glycol modifier or PEG modifier
  • PEG modifier polyethylene glycol with functional groups (such as active linkers).
  • the modifying agent used for random modification of mPEG of free amino groups can be selected from: mPEG-SS (methoxypolyethylene glycol-succinimide succinate), mPEG-SC (methoxypolyethylene glycol-succinimide carbonate), mPEG-SPA (methoxypolyethylene glycol-succinimide propionate) and mPEG-SG (methoxypolyethylene glycol-succinimide propionate) Succinimide glutarate) and other one of them.
  • mPEG-SS methoxypolyethylene glycol-succinimide succinate
  • mPEG-SC methoxypolyethylene glycol-succinimide carbonate
  • mPEG-SPA methoxypolyethylene glycol-succinimide propionate
  • mPEG-SG methoxypolyethylene glycol-succinimide propionate Succinimide glutarate
  • the N-terminal modifiers are: mPEG-ALD (methoxypolyethylene glycol-acetaldehyde), mPEG-pALD (methoxypolyethylene glycol-propionaldehyde) and mPEG-bALD (methoxypolyethylene glycol) -butyraldehyde) and other one of them.
  • the shape of the modifying agents mPEG-SS, mPEG-SC, mPEG-SPA, mPEG-SG, mPEG-ALD, mPEG-pALD, and mPEG-bALD is linear or branched.
  • the modifier used for random modification of free thiol groups is mPEG-mal (methoxypolyethylene glycol-maleimide), mPEG-OPSS (methoxypolyethylene glycol-maleimide), Alcohol - o-dithiopyridine), mPEG-Vinylsulfone (methoxy polyethylene glycol - vinyl sulfone) and mPEG-Thiol (methoxy polyethylene glycol - thiol), etc.
  • mPEG-mal methoxypolyethylene glycol-maleimide
  • mPEG-OPSS methoxypolyethylene glycol-maleimide
  • Alcohol - o-dithiopyridine Alcohol - o-dithiopyridine
  • mPEG-Vinylsulfone methoxy polyethylene glycol - vinyl sulfone
  • mPEG-Thiol methoxy polyethylene glycol - thiol
  • the modifying agent used for random modification of the sugar group and/or carboxyl group is mPEG-ZH (methoxypolyethylene glycol-hydrazide).
  • the structure of the mPEG-modified modifier is shown in formula (1):
  • m1 is preferably 5; mPEG- represents a methoxy single-terminated polyethylene glycol group, and the molecular weight of the modifier shown in formula (1) is 5kD-60kD (kD, kilodal) Dayton), preferably 40kD.
  • the modifier represented by formula (1) is used to randomly modify mPEG of free amino groups.
  • the structure of the mPEG-modified modifier is shown in formula (2):
  • 0 ⁇ m2 ⁇ 6, m2 is preferably 2; 0 ⁇ m3 ⁇ 6, m3 is preferably 1; mPEG- represents a methoxy single-terminated polyethylene glycol group, and the molecular weight of the modifier represented by formula (2) It is 5kD-60kD, preferably: 40kD.
  • the modifier represented by formula (2) is used to perform random modification of mPEG with free amino groups.
  • the size of the polymer backbone can vary, but polymers such as 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 conjugated hydrophilic polymer of the invention varies primarily within the following ranges: 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, from about 20kD to about 50kD; about 30kD to about 50kD or about 30kD to 40kD. It should be understood that these sizes represent approximations and are not exact measurements, as is accepted practice in the art.
  • the size of PEG or mPEG used in the present invention is above 35kD (ie not less than 35kD), preferably not less than 40kD, not less than 45kD, not less than 50kD, not less than 55kD, not less than 60kD, Not less than 65kD or not less than 70kD, for example, the molecular weight is specifically 40kD, 50kD, 60kD, 70kD, 80kD, 90kD, 100kD, 110kD, 120kD, 130kD, 140kD, 150kD or 160kD.
  • the coagulation factor VIII-Fc fusion protein includes human coagulation factor VIII with a truncated B region, a flexible unit, a rigid unit and an Fc fragment in order from the nitrogen terminus to the carbon terminus, wherein:
  • the human coagulation factor VIII of the truncated B region includes the amino acid sequence shown in SEQ ID NO: 2, the flexible unit includes the amino acid sequence shown in SEQ ID NO: 7, and the rigid unit includes the amino acid sequence shown in SEQ ID NO: 12 Sequence, the Fc fragment includes the amino acid sequence (FL1G2-0) shown in SEQ ID NO: 4;
  • the human coagulation factor VIII of the truncated B region includes the amino acid sequence shown in SEQ ID NO: 2, the flexible unit includes the amino acid sequence shown in SEQ ID NO: 6, and the rigid unit includes the amino acid sequence shown in SEQ ID NO: 11 Sequence, the Fc fragment includes the amino acid sequence shown in SEQ ID NO: 4 (FL2G2-0);
  • the human coagulation factor VIII of the truncated B region includes the amino acid sequence shown in SEQ ID NO: 2, the flexible unit includes the amino acid sequence shown in SEQ ID NO: 7, and the rigid unit includes the amino acid sequence shown in SEQ ID NO: 12 Sequence, the Fc fragment includes the amino acid sequence (FL1G4-0) shown in SEQ ID NO: 5;
  • the human coagulation factor VIII with the truncated B region includes the amino acid sequence shown in SEQ ID NO: 2
  • the flexible unit includes the amino acid sequence shown in SEQ ID NO: 6
  • the rigid unit includes the amino acid sequence shown in SEQ ID NO: 11
  • the Fc fragment includes the amino acid sequence shown in SEQ ID NO: 5 (FL2G4-0).
  • the conjugate is PEG randomly modified FL1G2-0, FL2G2-0, FL1G4-0 or FL2G4-0.
  • the random modification includes connecting the PEG molecule to a primary amine (-NH 2 ) on a lysine residue in the fusion protein through an active linker succinimide ester (SCM). ) group.
  • SCM succinimide ester
  • the PEG is a branched PEG containing two PEGs or a linear methoxy PEG (Y-PEG).
  • the PEG has the structural formula shown in formula (2).
  • m2 is 2; m3 is 1; and the molecular weight of the PEG is 40kD.
  • the PEG modifier represented by formula (2) connects the PEG molecule to the fusion protein molecule by forming an amide bond with the primary amine on the lysine residue (as shown in formula 4). .
  • the PEG in the conjugate has the structure represented by formula (5):
  • a molecule of the invention has an altered, preferably an increased circulating half-life compared to a wild-type Factor VIII molecule.
  • the circulating 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 a circulating half-life increased by at least 400%, 500%, 600%, or even 700%.
  • pharmaceutically acceptable carrier includes, but is not limited to: saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof.
  • pharmaceutical preparations should match the mode of administration.
  • the pharmaceutical composition of the present invention can be made into an injection form, for example, prepared by conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants.
  • the pharmaceutical composition is preferably manufactured under sterile conditions.
  • the active ingredients are administered in amounts that are therapeutically effective.
  • the pharmaceutical preparation of the present invention can also be made into a sustained-release preparation.
  • hFVIII fusion protein expression plasmids were constructed according to molecular cloning techniques well known to those skilled in the art. The expression plasmids were transfected into DHFR-deficient CHO cells (see U.S. Pat. No. 4,818,679) to express each hFVIII fusion protein (Table 1). For specific preparation and purification steps of the fusion protein, see Chinese Patent ZL201610692838.0 and WO2019219049A1, which are incorporated herein by reference in their entirety.
  • hFVIII with truncated B domain is also called BDD FVIII, which consists of 90kD A1-A2 heavy chain and 80kD light chain.
  • *SEQ ID NO:7-SEQ ID NO:12 means that the connector is formed by connecting the rigid unit shown in SEQ ID NO:12 to the C end of the flexible unit shown in SEQ ID NO:7; **SEQ ID NO:6- SEQ ID NO: 11 indicates that the connector is formed by connecting the rigid unit shown in SEQ ID NO: 11 to the C end of the flexible unit shown in SEQ ID NO: 6.
  • hFVIII-L1-G1 and hFVIII-L2-G1 fusion proteins have been disclosed in WO2019219049A1, corresponding to hFVIII-L1-Fc (FL1F-0) and hFVIII-L2-Fc (FL2F-0) respectively, and are used as controls in this application.
  • 40kD PEG-modified FL1G1-40Y (40kD, Y-type PEG-modified FL1G1-0, subsequent naming rules are the same) was prepared and purified according to the description of WO2019219049A1.
  • the 40kD PEG-modified hFVIII fusion protein FL1G2-40Y was prepared and purified according to the following method.
  • Y-SCM-40K PEG with Y type, molecular weight of 40kD and active linker SCM (the molecular formula is as shown in formula (2), where m2 is 2 and m3 is 1, purchased from Beijing Jiankai Technology Co., Ltd., Y- NHS-40K) and hFVIII fusion protein for cross-linking reaction.
  • the active linker of PEG is succinimide ester, which easily reacts with the primary amine (-NH 2 ) group on the lysine residue in the protein to form a stable amide bond, thereby obtaining a protein-PEG cross-linked product.
  • conjugates according to different molar ratios of PEG and fusion protein, such as 30:1, 50:1, 100:1, 120:1 .
  • the conjugate prepared according to the ratio of 100:1 showed excellent half-life extension effect in subsequent tests.
  • binding buffer binding buffer: 20mM His-HCl, 0.1M NaCl, 5mM CaCl 2 , 0.02% Tween 80, pH 6.8-7.2
  • binding buffer 20mM His-HCl, 0.1M NaCl, 5mM CaCl 2 , 0.02% Tween 80, pH 6.8-7.2
  • the second step uses Source 15Q (GE healthcare) anion chromatography column for separation.
  • Use binding buffer binding buffer: 20mM His-HCl, 0.1M NaCl, 5mM CaCl2, 0.02% Tween 80, pH6.8-7.2
  • binding buffer binding buffer: 20mM His-HCl, 0.1M NaCl, 5mM CaCl2, 0.02% Tween 80, pH6.8-7.2
  • the sample obtained after the first step of molecular sieve chromatography was loaded at a linear flow rate of 150cm/h
  • after the loading was completed use equilibrium buffer (20mM His-HCl, 0.1M NaCl, 5mM CaCl 2 , 0.02% Tween 80, pH6.8-7.2), flush the chromatography column for 3-5 column volumes (CV) at a linear flow rate of 150cm/h, and balance until the pH and conductivity are consistent with the buffer; use elution buffer (20mM His-HCl, 2M NaCl, 5mM CaC
  • TSKgel UltraSW Aggregate chromatographic column (7.8mm ⁇ 300mm, 3 ⁇ m) was used for detection.
  • the mobile phase was (0.3mol/L arginine, 0.2mol/L sodium chloride, 0.01mol/L anhydrous calcium chloride, 0.02mol/L L histidine, 0.02% poloxamer 188, pH 7.0)-10% acetonitrile), isocratic elution, flow rate is 0.5mL/min, UV detection wavelength is 280nm.
  • Dilute the sample to be tested with dilution buffer to a concentration of approximately 0.30 mg/mL take 100 ⁇ L of the test solution and inject it into the liquid chromatograph, and calculate the purity of the test product according to the area normalization method. As shown in Figure 1, the purity of the sample to be tested was 99.5%.
  • TSKgel UltraSW Aggregate chromatographic column (7.8mm ⁇ 300mm, 3 ⁇ m) was used for detection.
  • the mobile phase was (0.3mol/L arginine, 0.2mol/L sodium chloride, 0.01mol/L anhydrous calcium chloride, 0.02mol/L L histidine, 0.02% poloxamer 188, pH 7.0)-10% acetonitrile, isocratic elution, flow rate is 0.5mL/min, UV detection wavelength is 280nm, column temperature is 25°C, differential refractive index detector temperature is 30°C.
  • the UV absorption value of the conjugate with the same protein content is the same as the UV absorption value of the fusion protein before modification.
  • the purified conjugate was calculated by the standard curve method.
  • the content of the fusion protein part in the conjugate 2) Since the RI absorption value of the fusion protein solution has a linear relationship with the corresponding concentration, the RI value provided by the fusion protein part in the conjugate can be calculated through the standard curve method; 3) Since the RI absorption value of the conjugate is the sum of the RI absorption value of the PEG part in the conjugate and the RI absorption value of the fusion protein part, the RI absorption value of the PEG part in the conjugate can be calculated; 4) Because of the RI absorption value of PEG It has a linear relationship with the corresponding concentration, and the content of the PEG part in the conjugate can be calculated through the standard curve method; 5) The ratio of the number of moles of the PEG part in the conjugate to the number of moles of the fusion protein part is the ratio of the number of moles in the conjugate to the number of moles in the fusion protein part. The number of PEG modifications.
  • Example 3 Direct determination of biological activity of fusion protein by one-stage method
  • the human coagulation factor VIII titer determination method used in this example is also called the one-phase method.
  • the first-phase method determines the biological activity of FVIII by correcting the prolongation of clotting time caused by factor FVIII-deficient plasma.
  • the test kit Coagulation Factor VIII Deficient Plasma (Cat. No. OTXW17) produced by Siemens Company of Germany was used.
  • the detection method includes: diluting the WHO FVIII activity standard with 5% FVIII-deficient plasma to 1IU/ml, and then diluting the above solution 10 times, 20 times, 40 times, and 80 times with 5% FVIII-deficient plasma, respectively, for measurement. Setting time.
  • the test sample is diluted to about 1IU/ml with 5% FVIII-deficient plasma, and then the above solution is diluted 10 times and 20 times with 5% FVIII-deficient plasma respectively and the coagulation time is measured in the same way.
  • the FVIII of the sample to be tested is What is the potency?
  • the specific activity of FVIII of the sample to be tested can be calculated, and the unit is IU/mg.
  • the results showed that the corresponding specific activities of FL1G1-40Y and FL1G2-40Y were 1210IU/mg and 1300IU/mg respectively, and the specific activity of FL1G2-40Y was stronger than FL1G1-40Y.
  • HA mice 18 HA mice were randomly divided into 3 groups, namely FL1G1-40Y, FL1G2-40Y and recombinant human coagulation factor VIII (Xyntha) for injection as a control, 6 mice/group, male, all single dose Intravenous injection of 200IU/kg.
  • Blood samples (approximately 0.12 mL) were collected from non-administration sites in the subcutaneous veins of the animals' hind limbs into 1.5 mL sodium citrate blood collection tubes (1:9).
  • the blood collection time points were: 0 h before administration of animals in each group and 10 min after the start of administration. , 1h, 2h, 6h, 12h, 24h, 32h, 48h, 72h and 96h.
  • cynomolgus monkeys were randomly divided into 4 groups (6/group, half male and half female). Groups 1-3 were given a single intravenous injection of 50, 125, and 300IU/kg FL1G2-40Y respectively, and group 4 was given 125IU/kg of Renjie. Blood samples (approximately 1.8 mL) were collected from non-administration sites in the subcutaneous veins of the animals' hind limbs into 2 mL sodium citrate blood collection tubes (1:9). The blood collection time points were: 0 h before administration of animals in each group, 10 min, and 10 min after the start of administration.
  • Eligible subjects must be: 1) 12 years old or younger, male; 2) Clinically diagnosed patients with severe hemophilia A (coagulation factor VIII ⁇ 1%), with previous medical records confirming that they have received Coagulation factor VIII treatment (EDs ⁇ 150); 4) Non-acute bleeding state; 5) Previous negative coagulation factor VIII inhibitors ( ⁇ 0.6BU), no family history of inhibitors; 6) Platelet count >100,000 cells/ ⁇ L; 7) Normal prothrombin time or INR ⁇ 1.3; 8 ) Thrombin time (TT) is normal; 9) Previous vWF antigen test results are normal; 10) Lupus anticoagulant is negative.
  • Clinical design The trial is set up in 2 dose groups, 25IU/kg and 50IU/kg respectively, with no less than 6 effective cases in each group.
  • the subjects were first given a single dose of the control drug Baiyinzhi (recombinant human coagulation factor VIII for injection, ADVATE), and then a single dose of the test drug.
  • the subjects in each group were admitted to the clinical trial center the day before the administration (day -1). On the morning of the administration, the subjects were given intravenous bolus on an empty stomach.
  • the validated ELISA method was used to detect and analyze the concentration of the drug in plasma, and the non-compartmental model method (NCA) of Phoenix WinNonlin software (Certara L.P., version 8.2) was used to calculate the kinetic parameters of each administration group.
  • NCA non-compartmental model method
  • the results of pharmacokinetic parameters are shown in Table 8.

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Abstract

本公开提供了一种具有延长的循环半衰期的与聚乙二醇缀合的凝血因子VIII-Fc融合蛋白及其制备方法和应用。

Description

具有延长的半衰期的FVIII融合蛋白缀合物及其应用
本申请要求于2022年05月25日提交中国专利局、申请号为202210577314.2的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明涉及生物医药领域，特别涉及一种具有延长的半衰期的重组人凝血因子VIII融合蛋白缀合物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。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时，必须每周静脉内注射FVIII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，其在人体内半衰期为18.8小时，仅比天然蛋白延长了1.5～1.7倍(Dumont J A等,Blood,2012,119:3024–3030；Powell JS等,Blood,2012,119:3031-3037)，仍需每3～5天注射1次。而Bioverativ公司构建的rFVIII Fc与Fc的双表达载体转染HEK-293细胞后，未能在其表达产物中检测出预期的rFVIIIFc同源二聚体形式的融合蛋白，仅表达出单体-二聚体杂合体型rFVIIIFc融合蛋白和Fc二聚体。
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。目前已经报道的通过聚乙二醇(PEG)延长FVIII半衰期的有：Novonordisk(N8-GP)，Bayer(BAY94-9027)和Baxter(Bax 855)，这些公司均开发了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)。
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，需要开发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也是需要解决的问题。
发明内容
WO2019219049A1公开了一种PEG修饰的FVIII-Fc融合蛋白，所述PEG化融合蛋白在血友病A小鼠尾静脉横切模型中能够降低模型动物的严重出血率和12h复出血率，并增加模型动物的48h存活率。
发明人在进一步研究中意外发现，并非任意Fc区以及任意数量的PEG修饰都具有相近的延长FVIII-Fc融合蛋白半衰期的作用，对于特定的FVIII片段，来自lgG的Fc区和特定数量的PEG修饰能带来出人意料的显著延长的半衰期。临床数据表明本发明涉及的PEG化融合蛋白半衰期为30.87小时-31.91小时，相对于百因止半衰期提高了2.10-2.25倍，相对于天然蛋白延长了2.61-2.80倍，可以达到一周给药一次的频率，大大降低药物的注射频率，使需长期给药的患者实现在工作日能正常的工作学习，只需在周末给药即可，极大地提高了患者的依从性，显著提升病人的生活质量。基于此，完成了本发明。
本发明提供如下技术方案：
本发明的第一个方面，提供一种凝血因子VIII-Fc融合蛋白与聚乙二醇(PEG)的缀合物，其中所述凝血因子VIII-Fc融合蛋白包含凝血因子VIII活性部分(FVIII)和Fc片段，所述凝血因子VIII活性部分(FVIII)与所述Fc片段直接连接或者通过连接子(Linker)间接连接形成所述融合蛋白，所述缀合物中PEG平均修饰个数为3-8、3-7、3-6、4-8、4-7、4-6或4.2-5.1，优选为4-6，更优选为4.2-5.1。本文中，缀合物的PEG平均修饰个数为所述PEG与所述融合蛋白的摩尔比，也可以理解为样品(含有的融合蛋白个数多于1个)中每个融合蛋白被修饰后带有的PEG个数的平均值。由于每个融合蛋白被修饰后带有的PEG个数可能不同，其平均值可能是整数也可能是非整数。
在一些实施方案中，用于PEG修饰的修饰剂具有式(2)所示的结构式：
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；
优选地，所述PEG的分子量为30kD-50kD，优选为40kD。
在一些实施方案中，所述凝血因子VIII活性部分为全长或截短的人凝血因子VIII，优选为截短的B区域的人凝血因子VIII，更优选为包含SEQ ID NO：1或SEQ ID NO：2所示氨基酸序列的多肽，或者与SEQ ID NO：1或SEQ ID NO：2所示氨基酸序列具有至少90％、95％或更高的同一性且仍然具有FVIII活性的多肽。
在另一些实施方案中，所述Fc片段为源自IgG的Fc片段，优选为IgG1、IgG2或IgG4的Fc片段，更优选为IgG2的Fc片段。
在另一些实施方案中，所述Fc片段的氨基酸序列包含选自以下的序列：
(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；和
(iii)SEQ ID NO：5所示的氨基酸序列。
在另一些实施方案中，所述融合蛋白与PEG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。
在另一些实施方案中，所述连接子(Linker)包括柔性单元和刚性单元。
在另一些实施方案中，所述柔性单元含有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。
优选地，所述柔性单元包含选自以下的氨基酸序列：
(i)GSGGGSGGGGSGGGGS(SEQ ID NO:6)，
(ii)GSGGGGSGGGGSGGGGSGGGGSGGGGS(SEQ ID NO:7)，
(iii)GGGGSGGGGSGGGGSGGGGS(SEQ ID NO:8)，
(iv)GSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS(SEQ ID NO:9)，和
(v)GGGSGGGSGGGSGGGSGGGS(SEQ ID NO:10)。
在另一些实施方案中，所述刚性单元包含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)，
(iv)SRLPGPSDTPILPQ(SEQ ID NO:14)，和
(v)GSGGGGSGGGGSGGGGSGGGGSGGGGSSSSSKAPPPSLPSPSRLPGPSDTPILPQ(SEQ ID NO:15)。
在另一些实施方案中，所述凝血因子VIII-Fc融合蛋白自氮末端至碳末端依次包括截短B区域的人凝血因子VIII、柔性单元、刚性单元和Fc片段，其中：
所述截短B区域的人凝血因子VIII包含SEQ ID NO：2所示氨基酸序列，所述柔性单元包含SEQ ID NO:7所示氨基酸序列，所述刚性单元包含SEQ ID NO:12所示氨基酸序列，所述Fc片段包含SEQ ID NO：4所示氨基酸序列；
所述截短B区域的人凝血因子VIII包含SEQ ID NO：2所示氨基酸序列，所述柔性单元包含SEQ ID NO:6所示氨基酸序列，所述刚性单元包含SEQ ID NO:11所示氨基酸序列，所述Fc片段包含SEQ ID NO：4所示氨基酸序列；
所述截短B区域的人凝血因子VIII包含SEQ ID NO：2所示氨基酸序列，所述柔性单元包含SEQ ID NO:7所示氨基酸序列，所述刚性单元包含SEQ ID NO:12所示氨基酸序列，所述Fc片段包含SEQ ID NO：5所示氨基酸序列；
或
所述截短B区域的人凝血因子VIII包含SEQ ID NO：2所示氨基酸序列，所述柔性单元包含SEQ ID NO:6所示氨基酸序列，所述刚性单元包含SEQ ID NO:11所示氨基酸序列，所述Fc片段包含SEQ ID NO：5所示氨基酸序列。
在一些实施方式中，所述PEG分子通过活性接头琥珀酰亚胺酯(SCM)连接于所述凝血因子VIII-Fc融合蛋白中赖氨酸残基上的伯胺(-NH2)基团；
优选地，用于PEG修饰的修饰剂具有式(2)所示的结构式：
其中，m2为2；m3为1；所述PEG的分子量为40kD。
本发明的第二个方面，提供一种药物组合物，其包含前述的缀合物，及药学上可接受的载体。
本发明的第三个方面，提供所述的缀合物在制备用于预防和/或治疗出血性疾病的药物中的用途。优选地，所述出血性疾病为FVIII先天性或获得性缺乏症患者的出血性疾病或血友病A患者的自发或手术性出血。
本发明的第四个方面，提供一种预防和/或治疗出血性疾病的方法，其包括向有此需要的对象施用所述缀合物，优选地，所述出血性疾病选自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。
本发明还提供制备第一方面所述缀合物的方法，包括：
1)制备所述凝血因子VIII-Fc融合蛋白；
2)将步骤1)获得的融合蛋白与PEG反应，其中，PEG与所述融合蛋白的摩尔比为(50-120):1，优选为100：1，PEG分子量为30-50kD，优选为40kD支链PEG；和
3)纯化步骤2)中获得的缀合物。
在一些实施方式中，反应条件包括20℃±5℃，反应1-3小时，优选为2小时。
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1示FL1G2-40Y的SEC-HPLC纯度图谱。
图2示浓度为0.20mg/mL的hFVIII融合蛋白的RI色谱图。
图3示浓度为0.20mg/mL的PEG标准品溶液的RI色谱图。
图4示浓度为0.20mg/mL的hFVIII融合蛋白的UV色谱图。
图5示浓度为0.20mg/mL的纯化后缀合物的RI色谱图。
图6示浓度为0.20mg/mL的纯化后缀合物的UV色谱图。
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。
Fc片段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)。
本发明中的聚乙二醇(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,也称methoxy-PEG)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。
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。
本文中，用于与蛋白进行交联反应的PEG分子也称为修饰剂。其通常为活化的聚乙二醇(又称为聚乙二醇修饰剂或PEG修饰剂)，即带有官能团(如活性接头)的聚乙二醇。本文中，术语“修饰剂”、“用于PEG修饰的修饰剂”、“聚乙二醇修饰剂”和“PEG修饰剂”在本文中可替换使用。
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：
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：
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。
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。
在一个具体的实施方案中，所述凝血因子VIII-Fc融合蛋白自氮末端至碳末端依次包括截短B区域的人凝血因子VIII、柔性单元、刚性单元和Fc片段，其中：
所述截短B区域的人凝血因子VIII包含SEQ ID NO：2所示氨基酸序列，所述柔性单元包含SEQ ID NO:7所示氨基酸序列，所述刚性单元包含SEQ ID NO:12所示氨基酸序列，所述Fc片段包含SEQ ID NO：4所示氨基酸序列(FL1G2-0)；
所述截短B区域的人凝血因子VIII包含SEQ ID NO：2所示氨基酸序列，所述柔性单元包含SEQ ID NO:6所示氨基酸序列，所述刚性单元包含SEQ ID NO:11所示氨基酸序列，所述Fc片段包含SEQ ID NO：4所示氨基酸序列(FL2G2-0)；
所述截短B区域的人凝血因子VIII包含SEQ ID NO：2所示氨基酸序列，所述柔性单元包含SEQ ID NO:7所示氨基酸序列，所述刚性单元包含SEQ ID NO:12所示氨基酸序列，所述Fc片段包含SEQ ID NO：5所示氨基酸序列(FL1G4-0)；
或
所述截短B区域的人凝血因子VIII包含SEQ ID NO：2所示氨基酸序列，所述柔性单元包含SEQ ID NO:6所示氨基酸序列，所述刚性单元包含SEQ ID NO:11所示氨基酸序列，所述Fc片段包含SEQ ID NO：5所示氨基酸序列(FL2G4-0)。
在另一个具体的实施方案中，所述缀合物为PEG随机修饰的FL1G2-0、FL2G2-0、FL1G4-0或FL2G4-0。
在另一个具体的实施方案中，所述随机修饰包括将所述PEG分子通过活性接头琥珀酰亚胺酯(SCM)连接于所述融合蛋白中赖氨酸残基上的伯胺(-NH2)基团。
在另一个具体的实施方案中，所述PEG为包含两个PEG或直链甲氧基PEG的支链型PEG(Y型PEG)。
在另一个具体的实施方案中，所述PEG具有式(2)所示的结构式，优选的，式(2)中m2为2；m3为1；所述PEG的分子量为40kD。
在另一个具体的实施方案中，式(2)所示的PEG修饰剂通过与赖氨酸残基上的伯胺形成酰胺键(如式4所示)，将PEG分子连接于融合蛋白分子上。
在另一个具体的实施方案中，所述缀合物中的PEG具有式(5)所示的结构：
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 hFVIII融合蛋白的制备和纯化
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 4818679)，表达各个hFVIII融合蛋白(表1)。融合蛋白的具体制备和纯化步骤参见中国专利ZL201610692838.0和WO2019219049A1，其通过引用其全文并入本文中。
表1融合蛋白的组成和序列
注：B结构域截短的hFVIII也称BDD FVIII，由90kD的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所示的柔性单元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所示的柔性单元C末端形成。
hFVIII-L1-G1和hFVIII-L2-G1融合蛋白已在WO2019219049A1公开，分别对应于hFVIII-L1-Fc(FL1F-0)和hFVIII-L2-Fc(FL2F-0)，在本申请中作为对照。
实施例2 mPEG修饰的hFVIII融合蛋白的制备及纯化
按照WO2019219049A1的记载制备和纯化40kD PEG修饰的FL1G1-40Y(40kD，Y型PEG修饰的FL1G1-0，后续命名规则相同)。
在本实施例中，按照以下方法制备和纯化40kD PEG修饰的hFVIII融合蛋白FL1G2-40Y。
采用Y型、分子量为40kD、活性接头为SCM的Y-SCM-40K PEG(分子式如式(2)所示，其中，m2为2，m3为1，购自北京键凯科技有限公司，Y-NHS-40K)与hFVIII融合蛋白进行交联反应。PEG的活性接头为琥珀酰亚胺酯，易与蛋白质中赖氨酸残基上的伯胺(-NH2)基团发生反应形成稳定的酰胺键，从而得到蛋白质-PEG交联产物。称取Y-SCM-40K PEG和滤浓缩后的hFVIII融合蛋白FL1G2-0，按照不同PEG与融合蛋白的摩尔比制备缀合物，例如30：1、50：1、100：1、120：1。其中，按照100：1比例制备的缀合物(本实施例的缀合物)在之后的检测中显示优异的延长半衰期效果，其制备方法如下：按照摩尔比PEG∶蛋白＝100：1(质量比10.26:1)进行投料，于20℃±5℃反应2小时，交联后样品0.2μm滤膜过滤后，样品2-8℃暂存待进行纯化。
纯化：首先使用S200(GE healthcare)分子筛层析进行分离。使用结合缓冲液(binding buffer：20mM His-HCl,0.1M NaCl,5mM CaCl2,0.02％Tween 80,pH6.8-7.2)，以150cm/h的线性流速平衡层析柱3-5个柱体积(CV)；以150cm/h的线性流速进行上样；上样完毕后，使用平衡buffer(20mM His-HCl,200mM NaCl,5mM CaCl2,0.02％Tween 80,pH6.8-7.2)，以150cm/h的线性流速冲洗层析柱子3-5柱体积(CV)，平衡至pH及电导同缓冲液一致；用缓冲液(20mM His-HCl,0.1M NaCl,5mM CaCl2,0.02％Tween 80,pH6.8-7.2)进行洗脱，收集A280/260大于1.8的峰。第二步采用Source 15Q(GE healthcare)阴离子层析柱分离。使用结合缓冲液(binding buffer：20mM His-HCl，0.1M NaCl,5mM CaCl2,0.02％Tween 80,pH6.8-7.2)，以150cm/h的线性流速平衡层析柱3-5个柱体积(CV)；经第一步分子筛层析后得到的样品以150cm/h的线性流速进行上样；上样完毕后，使用平衡buffer(20mM His-HCl,0.1M NaCl,5mM CaCl2,0.02％Tween 80,pH6.8-7.2)，以150cm/h的线性流速冲洗层析柱子3-5柱体积(CV)，平衡至pH及电导同缓冲液一致；用洗脱缓冲液(20mM His-HCl,2M NaCl, 5mM CaCl2,0.02％Tween 80,pH6.8-7.2)按照0-100％以100cm/h线性速度进行洗脱，分管收集A280/260大于1.8的洗脱峰，分别进行纯度检测和PEG平均修饰数确认。
SEC-HPLC法测定FL1G2-40Y纯度
采用TSKgel UltraSW Aggregate色谱柱(7.8mm×300mm，3μm)进行检测，流动相为(0.3mol/L精氨酸、0.2mol/L氯化钠、0.01mol/L无水氯化钙、0.02mol/L组氨酸、0.02％泊洛沙姆188，pH 7.0)-10％乙腈)，等度洗脱，流速为0.5mL/min，紫外检测波长为280nm。用稀释缓冲液将待测样品稀释至浓度约为0.30mg/mL，取供试品溶液100μL注入液相色谱仪，按面积归一化法计算供试品纯度。如图1所示，待测样品纯度为99.5％。
SEC-HPLC-UV-RID联用方法测定获得的FL1G2-40Y的PEG平均修饰数
采用TSKgel UltraSW Aggregate色谱柱(7.8mm×300mm，3μm)进行检测，流动相为(0.3mol/L精氨酸、0.2mol/L氯化钠、0.01mol/L无水氯化钙、0.02mol/L组氨酸、0.02％泊洛沙姆188，pH 7.0)-10％乙腈，等度洗脱，流速为0.5mL/min，紫外检测波长为280nm，柱温为25℃，示差折光检测器温度为30℃。测定并记录hFVIII融合蛋白UV峰面积和RI峰面积，纯化后缀合物溶液UV峰面积和RI峰面积，以及PEG标准品溶液的RI峰面积，分别用hFVIII融合蛋白和PEG标准品建立标准曲线。FL1G2-40Y用稀释缓冲液稀释至浓度约为0.20mg/mL，取100μL注入液相色谱仪，按外标法计算供试品PEG平均修饰数。
修饰数结果推算如下：
1)因缀合物中的PEG部分在波长280nm处没有UV吸收，相同蛋白含量的缀合物的UV吸收值与修饰前融合蛋白的UV吸收值相同，通过标准曲线法计算出纯化后缀合物中的融合蛋白部分的含量；2)因融合蛋白溶液的RI吸收值与相对应的浓度均呈线性关系，通过标准曲线法可计算出缀合物中的融合蛋白部分提供的RI值；3)因缀合物的RI吸收值为缀合物中PEG部分RI吸收值和融合蛋白部分RI吸收值之和，可计算出缀合物中的PEG部分RI吸收值；4)因PEG的RI吸收值与相对应的浓度呈线性关系，通过标准曲线法可计算出缀合物中PEG部分的含量；5)缀合物中PEG部分的摩尔数与融合蛋白部分的摩尔数的比率即为单个蛋白中PEG的修饰个数。
示例性的RI色谱图和UV色谱图参见图2-6。检测数据参见表2-4。hFVIII融合蛋白和PEG标准品RI峰面积与浓度的线性方程分别为Y＝2E+06X-197.15(R2＝0.9998)和Y＝1E+06X+1960.1(R2＝0.9998)；rhFVIII融合蛋白UV峰面积与浓度的线性方程为Y＝17590X-13.542(R2＝0.9999)；不同批次0.20mg/mL纯化后缀合物对应的RI峰面积和UV峰面积均值分别为579850.509和3483.775，通过计算得到待测缀合物中PEG平均修饰个数约为4.6。
表2.hFVIII融合蛋白RI峰面积与浓度的线性关系
表3.PEG标准品溶液RI峰面积与浓度的线性关系
表4.hFVIII融合蛋白UV峰面积与浓度的线性关系
对不同批次制备得到的FL1G2-40Y的PEG平均修饰数进行检测，结果类似，PEG平均修饰数在4.2-5.1之间，参见表5。
表5不同批次FL1G2-40Y中PEG平均修饰数检测结果

实施例3 一期法直接测定融合蛋白的生物学活性
本实施例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2020版三部通则。一期法测定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公司生产的试剂盒Coagulation Factor VIII Deficient Plasma(Cat.No.OTXW17)。检测方法包括：WHO FVIII活性标准品用5％乏FVIII血浆将其稀释至1IU/ml，然后再用5％乏FVIII血浆将上述溶液分别稀释10倍、20倍、40倍、80倍，分别测定凝固时间。将已知标准品的活性与测得的凝固时间分别取对数，再进行线性拟合绘制标准曲线。供试品用5％乏FVIII血浆稀释至约1IU/ml，然后再用5％乏FVIII血浆将上述溶液分别稀释10倍和20倍同法测定凝固时间，通过代入标准曲线，可知待测样品FVIII的效价为多少，据此可求算出待测样品FVIII的比活性大小，单位为IU/mg。结果表明FL1G1-40Y和FL1G2-40Y对应的比活分别为1210IU/mg和1300IU/mg，FL1G2-40Y比活强于FL1G1-40Y。
实施例4 PEG修饰的hFVIII融合蛋白在HA小鼠中的药代动力学试验
HA小鼠18只，随机分为3组，分别为FL1G1-40Y、FL1G2-40Y和作为对照的注射用重组人凝血因子VIII(任捷，Xyntha)，6只/组，雄性，均为单次静脉注射200IU/kg。从动物后肢皮下静脉非给药部位采集血样(约0.12mL)至1.5mL枸橼酸钠采血管(1:9)中，采血时间点为：各组动物给药前0h，给药开始后10min、1h、2h、6h、12h、24h、32h、48h、72h及96h。采血后30min内2～8℃离心(约5000rpm，5min)。分离血浆样本后分装2分，保存于超低温冰箱(-70℃～-90℃)。血样采集至离心完成需在2小时内完成。采用经过验证的ELISA方法检测分析血浆中药物的浓度，采用Phoenix WinNonlin软件(CertaraL.P.，8.2版本)软件的非房室模型法对各给药组的动力学参数进行计算，各组药代动力学参数结果见表6。结果显示，在同等剂量下，对照药物任捷、FL1G1-40Y和FL1G2-40Y对应的半衰期分别为9.46h、15.53h和20h。这表明经PEG修饰后，FL1G2-40Y相对于任捷和FL1G1-40Y，半衰期分别提高了2.11和1.29倍，表现出显著延长的半衰期，是现有技术中没有报道的。
表6.在HA小鼠中的PK相关参数

实施例5 PEG修饰的hFVIII融合蛋白在食蟹猴中的药代动力学试验
食蟹猴24只，随机分为4组(6只/组，雌雄各半)。组1-3分别单次静脉注射给予50、125、300IU/kg的FL1G2-40Y，组4给予125IU/kg的任捷。从动物后肢皮下静脉非给药部位采集血样(约1.8mL)至2mL枸橼酸钠采血管(1:9)中，采血时间点为：各组动物给药前0h，给药开始后10min、1h、2h、6h、12h、24h、32h、48h、72h、96h、120h及168h。采血后30min内2～8℃离心(约4000rpm，5min)。分离血浆样本后分装2分，保存于超低温冰箱(-70℃～-90℃)。血样采集至离心完成需在2小时内完成。采用经过验证的ELISA方法检测分析血浆中药物的浓度，采用Phoenix WinNonlin软件(CertaraL.P.，8.2版本)软件的非房室模型法对各给药组的动力学参数进行计算，各组药代动力学参数结果见表7。结果表明，在同等剂量125IU/kg下，对照药物任捷和FL1G2-40Y对应的半衰期分别为7.57h和27.8h；相对于任捷，FL1G2-40Y半衰期提高了3.67倍，是现有技术中没有报道的。
表7.在食蟹猴中的PK相关参数
实施例6 PEG修饰的hFVIII融合蛋白在重症血友病A患者中的药代动力学试验
入组标准：符合条件的受试者必须：1)12岁≤年龄<60岁，男性；2)临床确诊为重型血友病A患者(凝血因子Ⅷ<1％)，既往医疗记录证实接受过凝血因子Ⅷ治疗(EDs≥150)；4) 非急性出血状态；5)既往凝血因子Ⅷ抑制物阴性(<0.6BU)，家族无抑制物史；6)血小板计数>100,000个细胞/μL；7)凝血酶原时间正常或INR<1.3；8)凝血酶时间(TT)正常；9)既往vWF抗原检查结果正常；10)狼疮抗凝物阴性。
临床设计：试验设置2个剂量组，分别为25IU/kg和50IU/kg，每组有效病例不少于6例。受试者首先单次给予对照药百因止(注射用重组人凝血因子Ⅷ，ADVATE)，后单次给予试验药。每组受试者于给药前一天(第-1天)入住临床试验中心，给药当日早晨空腹，静脉推注给药。
采用经过验证的ELISA方法检测分析血浆中药物的浓度，采用Phoenix WinNonlin软件(CertaraL.P.，8.2版本)的非房室模型法(NCA)对各给药组的动力学参数进行计算，各组药代动力学参数结果见表8。
表8.在重症血友病A患者中的PK相关参数
结果表明，百因止在25IU/kg和50IU/kg剂量下半衰期分别为14.18h和14.68h；FL1G2-40Y在25IU/kg和50IU/kg剂量下半衰期分别为31.91h和30.87h，相对于百因止半衰期分别提高了2.25倍(25IU/kg)和2.10倍(50IU/kg)。已上市药物任捷在50IU/kg剂量下，在≥12岁患者体内的半衰期为13.76h，与同等剂量下百因止相当，可以合理推测，FL1G2-40Y的体内半衰期也明显高于任捷，具有显著延长的半衰期。
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 (15)

  1. 一种凝血因子VIII-Fc融合蛋白与聚乙二醇(PEG)的缀合物，其中所述凝血因子VIII-Fc融合蛋白包含凝血因子VIII活性部分(FVIII)和Fc片段，所述凝血因子VIII活性部分(FVIII)与所述Fc片段直接连接或者通过连接子(Linker)间接连接形成所述融合蛋白，所述缀合物中PEG平均修饰个数为3-8、3-7、3-6、4-8、4-7、4-6或4.2-5.1，优选为4.2-5.1，所述平均修饰个数为所述PEG与所述融合蛋白的摩尔比。
  2. 根据权利要求1所述的缀合物，其中用于PEG修饰的修饰剂具有式(2)所示的结构式：
    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；
    优选地，所述PEG的分子量为30kD-50kD，优选为40kD。
  3. 根据权利要求1或2所述的缀合物，其中所述凝血因子VIII活性部分为全长或截短的人凝血因子VIII，优选为截短B区域的人凝血因子VIII，更优选为包含SEQ ID NO：1或SEQ ID NO：2所示氨基酸序列的多肽，或者与SEQ ID NO：1或SEQ ID NO：2所示氨基酸序列具有至少90％、95％或更高的同一性且仍然具有FVIII活性的多肽。
  4. 根据权利要求1至3任一项所述的缀合物，其中所述Fc片段为源自IgG的Fc片段，优选为IgG1、IgG2或IgG4的Fc片段，更优选为IgG2的Fc片段。
  5. 根据权利要求4所述的缀合物，其中所述Fc片段的氨基酸序列包含选自以下的序列：
    (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；和
    (iii)SEQ ID NO：5所示的氨基酸序列。
  6. 根据权利要求1至5任一项所述的缀合物，其中所述连接子(Linker)包括柔性单元和刚性单元。
  7. 根据权利要求6所述的缀合物，其中所述柔性单元具有序列通式(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， 优选地，所述柔性单元包含选自以下的氨基酸序列：
    (i)GSGGGSGGGGSGGGGS(SEQ ID NO:6)，
    (ii)GSGGGGSGGGGSGGGGSGGGGSGGGGS(SEQ ID NO:7)，
    (iii)GGGGSGGGGSGGGGSGGGGS(SEQ ID NO:8)，
    (iv)GSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS(SEQ ID NO:9)，和
    (v)GGGSGGGSGGGSGGGSGGGS(SEQ ID NO:10)。
  8. 根据权利要求6所述的缀合物，其中所述刚性单元包含人绒毛膜促性腺激素β亚基的羧基末端肽，
    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)，
    (iv)SRLPGPSDTPILPQ(SEQ ID NO:14)，和
    (v)GSGGGGSGGGGSGGGGSGGGGSGGGGSSSSSKAPPPSLPSPSRLPGPSDTPILPQ(SEQ ID NO:15)。
  9. 根据权利要求1至8任一项所述的缀合物，其中，所述凝血因子VIII-Fc融合蛋白自氮末端至碳末端依次包括截短B区域的人凝血因子VIII、柔性单元、刚性单元和Fc片段，其中：
    所述截短B区域的人凝血因子VIII包含SEQ ID NO：2所示氨基酸序列，所述柔性单元包含SEQ ID NO:7所示氨基酸序列，所述刚性单元包含SEQ ID NO:12所示氨基酸序列，所述Fc片段包含SEQ ID NO：4所示氨基酸序列；
    所述截短B区域的人凝血因子VIII包含SEQ ID NO：2所示氨基酸序列，所述柔性接头包含SEQ ID NO:6所示氨基酸序列，所述刚性单元包含SEQ ID NO:11所示氨基酸序列，所述Fc片段包含SEQ ID NO：4所示氨基酸序列；
    所述截短B区域的人凝血因子VIII包含SEQ ID NO：2所示氨基酸序列，所述柔性单元包含SEQ ID NO:7所示氨基酸序列，所述刚性单元包含SEQ ID NO:12所示氨基酸序列，所述Fc片段包含SEQ ID NO：5所示氨基酸序列；
    或
    所述截短B区域的人凝血因子VIII包含SEQ ID NO：2所示氨基酸序列，所述柔性单 元包含SEQ ID NO:6所示氨基酸序列，所述刚性单元包含SEQ ID NO:11所示氨基酸序列，所述Fc片段包含SEQ ID NO：5所示氨基酸序列。
  10. 根据权利要求9所述的缀合物，其中：
    所述截短B区域的人凝血因子VIII包含SEQ ID NO：2所示氨基酸序列，所述柔性单元包含SEQ ID NO:7所示氨基酸序列，所述刚性单元包含SEQ ID NO:12所示氨基酸序列，所述Fc片段包含SEQ ID NO：4所示氨基酸序列；
    所述PEG分子通过活性接头琥珀酰亚胺酯(SCM)连接于所述凝血因子VIII-Fc融合蛋白中赖氨酸残基上的伯胺(-NH2)基团；
    用于PEG修饰的修饰剂具有式(2)所示的结构式：
    其中，m2为2；m3为1；所述PEG的分子量为40kD。
  11. 一种药物组合物，其包含权利要求1至10任一项所述的缀合物，及药学上可接受的载体。
  12. 权利要求1至10任一项所述的缀合物在制备用于预防和/或治疗出血性疾病的药物中的用途。
  13. 根据权利要求12所述的用途，其中所述出血性疾病为FVIII先天性或获得性缺乏症患者的出血性疾病或血友病A患者的自发或手术性出血。
  14. 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10任一项所述的缀合物，优选地，所述出血性疾病选自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。
  15. 制备权利要求1至10任一项所述的缀合物的方法，包括：
    1)制备所述凝血因子VIII-Fc融合蛋白；
    2)将步骤1)获得的融合蛋白与PEG反应，其中，PEG与所述融合蛋白的摩尔比为(50-120):1，优选为100:1，PEG分子量为30-50kD，优选为40kD支链PEG；和
    3)纯化步骤2)中获得的缀合物。
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CN102770450A (zh) * 2010-02-16 2012-11-07 诺沃—诺迪斯克有限公司 因子viii融合蛋白
CN106279437A (zh) * 2016-08-19 2017-01-04 安源医药科技（上海）有限公司 高糖基化人凝血因子viii融合蛋白及其制备方法与用途
WO2019219049A1 (zh) * 2018-05-18 2019-11-21 北京辅仁瑞辉生物医药研究院有限公司 改进的fviii融合蛋白及其应用
WO2019219048A1 (zh) * 2018-05-18 2019-11-21 北京辅仁瑞辉生物医药研究院有限公司 具有延长半衰期的融合多肽缀合物
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CN102770450A (zh) * 2010-02-16 2012-11-07 诺沃—诺迪斯克有限公司 因子viii融合蛋白
CN106279437A (zh) * 2016-08-19 2017-01-04 安源医药科技（上海）有限公司 高糖基化人凝血因子viii融合蛋白及其制备方法与用途
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