CN113166271A - Fusion polypeptide conjugates with extended half-life - Google Patents

Fusion polypeptide conjugates with extended half-life Download PDF

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CN113166271A
CN113166271A CN201980033065.7A CN201980033065A CN113166271A CN 113166271 A CN113166271 A CN 113166271A CN 201980033065 A CN201980033065 A CN 201980033065A CN 113166271 A CN113166271 A CN 113166271A
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fusion protein
seq
group
equal
mpeg
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王亚里
刘宾
王晓山
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
苏鸿声
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Zhengzhou Shengsi Biotechnology Co ltd
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Zhengzhou Shengsi Biotechnology Co ltd
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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
    • 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

Abstract

The present invention relates to a polyalkylene glycol conjugated biologically active polypeptide fusion protein with an extended circulating half-life, wherein the biologically active polypeptide moiety is directly linked to a fusion partner or indirectly linked with a peptide linker, and said fusion protein is further conjugated with polyalkylene glycol, and methods for the preparation and use thereof. Compared with the biologically active polypeptide fusion protein which is not modified by the polyalkylene glycol, the half life period of the biologically active polypeptide fusion protein is obviously improved.

Description

Fusion polypeptide conjugates with extended half-life
Cross-referencing
The present application claims priority of chinese patent application 201810483378.X, entitled "fusion polypeptide conjugate with extended half-life" filed by 2018 on month 5 and 18 to the chinese patent office, the contents of which are incorporated herein by reference in their entirety.
Technical Field
The invention relates to the field of protein engineering, in particular to a fusion polypeptide conjugate with a prolonged half-life period, a preparation method and application thereof.
Background
Recombinant protein drugs are an important class of biopharmaceuticals and are often administered clinically by intravenous or subcutaneous injection. However, after administration, protein drugs often degrade, resulting in reduced activity and bioavailability, often requiring repeated and multiple administrations to achieve the desired blood levels and therapeutic effect, reducing patient compliance. Therefore, the development of long-acting protein drugs is clinically needed.
Common principles of methods for increasing the half-life of protein drugs include increasing the molecular weight of the protein drug, decreasing glomerular filtration rate or decreasing the in vivo clearance of the protein. Conventional strategies for achieving long-term drug effect include glycosylation modification, pegylation, albumin fusion, transferrin fusion, Fc fusion, and the like. However, such modifications or alterations may have an effect on the activity of the protein itself, and such modifications/alterations often fail to achieve the intended effect.
Therefore, there is an urgent need in the art for new means for increasing the half-life of protein drugs and corresponding protein products.
Disclosure of Invention
The present inventors have conducted many years of studies and long-term experiments to find that a means of fusing a polypeptide (protein) to a fusion partner (e.g., Fc fragment, albumin, XTEN, or transferrin) capable of extending the half-life and further conjugating the polypeptide to a hydrophilic polymer (e.g., polyalkylene glycol, more e.g., PEG, including mPEG) is effective in improving the in vivo stability of a bioactive polypeptide, particularly when the hydrophilic polymer (e.g., PEG) has a branched structure (or a so-called branched structure), and particularly when the molecular weight of the hydrophilic polymer is within a certain range, e.g., equal to or greater than a specific value, the in vivo stability of the bioactive polypeptide can be significantly improved, thereby completing the present invention.
The invention provides the following technical scheme:
1. a biologically active polypeptide fusion protein conjugated to a polyalkylene glycol, wherein the biologically active polypeptide moiety is directly linked or indirectly linked with a peptide linker to a half-life extending fusion partner, and the fusion protein is further conjugated to a polyalkylene glycol.
2. The fusion protein of embodiment 1, wherein the biologically active polypeptide moiety can confer an activity on the fusion protein selected from the group consisting of: hormones, cytokines, coagulation factors, enzymes, receptor extracellular regions, immunomodulatory factors, interleukins, interferons, tumor necrosis factors, transforming growth factors, colony stimulating factors, chemokines, neuropeptides, insulin, GLP-1 receptor agonists, growth hormones, erythropoietin, G-CSF and GM-CSF.
3. The fusion protein of embodiment 1 or 2, wherein the fusion partner is: an immunoglobulin Fc fragment, albumin, XTEN, or transferrin, said fusion partner being derived, for example, from a human; preferably an IgG Fc fragment; for example, the IgG Fc fragment has reduced ADCC effect and/or CDC effect and/or enhanced binding affinity to FcRn receptor; more preferably, the IgG Fc fragment has an amino acid sequence selected from the group consisting of:
(i) SEQ ID NO: 3;
(ii) SEQ ID NO: 4; or
(iii) SEQ ID NO: 5.
4. The fusion protein according to any one of embodiments 1 to 3, wherein the polyalkylene glycol is polypropylene glycol or polyethylene glycol; the polyalkylene glycol may be terminally capped, for example, with alkoxy groups such as methoxy groups; and/or the polyalkylene glycol is linear or branched; preferably the polyalkylene glycol is branched, for example a branched polyethylene glycol, especially a methoxy-terminated branched polyethylene glycol; the molecular weight of the polyalkylene glycol may be > 1, > < 10, > < 20, > < 30, > < 40, > < 50, > < 60, > < 70, > < 80, > < 90, > < 100, > < 110, > < 120, > < 130, > < 140, > < 150 or > -160 kDa, such as 5, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100kDa, or any number therebetween.
5. The fusion protein according to any of embodiments 1 to 4, wherein the conjugation of the fusion protein to the polyalkylene glycol is random or site-directed, the conjugation position being selected from free amino, thiol, glycosyl and/or carboxyl, preferably free amino.
6. The fusion protein according to embodiment 5, wherein the conjugation is achieved using a modifying agent, which may preferably be in the form of an activated ester, e.g. selected from the following formulae (1), (2) or (3):
Figure PCTCN2019087144-APPB-000001
wherein m1 is more than or equal to 0 and less than or equal to 6, and m1 is preferably 5; mPEG represents methoxy single-end-capped polyethylene glycol group with the molecular weight of 5KD to 60 KD;
Figure PCTCN2019087144-APPB-000002
wherein m2 is more than or equal to 0 and less than or equal to 6, and m2 is preferably 2; 0 is more than or equal to m3 is less than or equal to 6, m3 is preferably 1; mPEG represents methoxy single-end-capped polyethylene glycol group with molecular weight of 5KD-100KD dalton, preferably 40KD, 50KD, 60KD, most preferably 40 KD; or
Figure PCTCN2019087144-APPB-000003
Wherein m4 is more than or equal to 0 and less than or equal to 6, and m4 is preferably 2; mPEG represents methoxy single-end-capped polyethylene glycol group with molecular weight of 5KD-100 KD.
7. The fusion protein according to any one of embodiments 1 to 6, wherein the biologically active polypeptide moiety is linked to the fusion partner by a peptide linker comprising a flexible peptide linker and/or a rigid unit, e.g. may comprise 1, 2, 3, 4, 5 or more of the rigid units.
8. The fusion protein of embodiment 7, wherein the flexible peptide linker contains 2 or more amino acid residues selected from the group consisting of glycine, serine, alanine, and threonine,
preferably, the flexible peptide linker has the General Sequence (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,
more preferably, the flexible peptide linker has a sequence selected from the group consisting of:
(i)GSGGGSGGGGSGGGGS(SEQ ID NO:6)；
(ii)GSGGGGSGGGGSGGGGSGGGGSGGGGS(SEQ ID NO:7)；
(iii)GGGGSGGGGSGGGGSGGGGS(SEQ ID NO:8)；
(iv) GSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 9); or
(v)GGGSGGGSGGGSGGGSGGGS(SEQ ID NO:10)。
9. The fusion protein of embodiment 7 or 8, wherein said rigid unit is a carboxy terminal peptide of a human chorionic gonadotrophin β subunit, or said rigid unit has 70%, 80%, 90%, 95% or more identity to an amino acid sequence of a carboxy terminal peptide of a human chorionic gonadotrophin β subunit; the rigid unit may comprise 1, 2 or more glycosylation sites;
preferably, the rigid unit comprises an amino acid sequence selected from the group consisting of:
(i)PRFQDSSSSKAPPPSLPSPSRLPGPSDTPILPQ(SEQ ID NO:11)；
(ii)SSSSKAPPPSLPSPSRLPGPSDTPILPQ(SEQ ID NO:12)；
(iii) SSSSKAPPPS (SEQ ID NO: 13); or
(iv)SRLPGPSDTPILPQ(SEQ ID NO:14)；
More preferably, the peptide linker comprises the sequence shown in SEQ ID NO 15.
10. A pharmaceutical composition comprising an effective amount of the fusion protein of any one of embodiments 1-9, and a pharmaceutically acceptable carrier.
11. A method for preventing and/or treating a disease that can be prevented and/or treated with the activity of a biologically active polypeptide, comprising administering to a subject in need thereof a fusion protein according to any one of embodiments 1 to 9 or a pharmaceutical composition according to embodiment 10.
12. The fusion polypeptide according to any one of embodiments 1 to 9, wherein the fusion protein is glycosylated, preferably by expression in a mammalian cell, preferably a chinese hamster ovary cell.
13. The fusion polypeptide according to any one of embodiments 1 to 9, wherein the fusion polypeptide has an extended half-life, in particular an extended circulating half-life, compared to not being conjugated with a polyalkylene glycol.
14. A method of improving the half-life of a biologically active polypeptide wherein a biologically active polypeptide moiety is linked directly or indirectly with a peptide linker to a half-life enhancing fusion partner and further conjugated to a polyalkylene glycol.
15. The method of embodiment 14, wherein the biologically active polypeptide moiety can confer an activity on the fusion protein selected from the group consisting of: hormones, cytokines, coagulation factors, enzymes, receptor extracellular regions, immunomodulatory factors, interleukins, interferons, tumor necrosis factors, transforming growth factors, colony stimulating factors, chemokines, neuropeptides, insulin, GLP-1 receptor agonists, growth hormones, erythropoietin, G-CSF and GM-CSF.
16. A method according to embodiment 14 or 15 wherein the fusion partner is: immunoglobulin Fc fragment, albumin, XTEN or transferrin, these fusion partners being derived from, for example, human; preferably an IgG Fc fragment; for example, the IgG Fc fragment has reduced ADCC effect and/or CDC effect and/or enhanced binding affinity to FcRn receptor; more preferably, the IgG Fc fragment has an amino acid sequence selected from the group consisting of:
(i) SEQ ID NO: 3;
(ii) SEQ ID NO: 4; or
(iii) SEQ ID NO: 5.
17. The method of any one of embodiments 14 to 16, wherein the polyalkylene glycol is polypropylene glycol or polyethylene glycol; the polyalkylene glycol may be terminally capped, for example, with alkoxy groups such as methoxy groups; and/or the polyalkylene glycol is linear or branched; preferably the polyalkylene glycol is branched, for example a branched polyethylene glycol, especially a methoxy-terminated branched polyethylene glycol; the molecular weight of the polyalkylene glycol may be > 1, > < 10, > < 20, > < 30, > < 40, > < 50, > < 60, > < 70, > < 80, > < 90, > < 100, > < 110, > < 120, > < 130, > < 140, > < 150 or > -160 kDa, such as 5, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100kDa, or any number therebetween.
18. The method according to any one of embodiments 14 to 17, wherein the conjugation of the fusion protein to the polyalkylene glycol is random or site-directed, the conjugation position being selected from free amino, thiol, glycosyl and/or carboxyl groups, preferably free amino groups.
19. The method according to any one of embodiments 14 to 18, wherein said conjugation is effected using a modifying agent, preferably said modifying agent may be in the form of an activated ester and other types of modifying agents, more preferably a modifying agent selected from the following formulae (1), (2) or (3):
Figure PCTCN2019087144-APPB-000004
wherein m1 is more than or equal to 0 and less than or equal to 6, and m1 is preferably 5; mPEG-represents a methoxy single-end-capped polyethylene glycol group, and the molecular weight of the modifier shown in the formula (1) is between 5KD and 60 KD;
Figure PCTCN2019087144-APPB-000005
wherein m2 is more than or equal to 0 and less than or equal to 6, and m2 is preferably 2; 0 is more than or equal to m3 is less than or equal to 6, m3 is preferably 1; mPEG-represents methoxy single-end-capped polyethylene glycol group, and the molecular weight of the modifier shown in the formula (2) is 5KD-100KD, preferably 40KD, 50KD and 60KD, and most preferably 40 KD;
Figure PCTCN2019087144-APPB-000006
wherein m4 is more than or equal to 0 and less than or equal to 6, and m4 is preferably 2; mPEG-represents methoxy single-end-capped polyethylene glycol group, and the molecular weight of the modifier shown in the formula (3) is 5KD-100 KD.
20. The method according to any one of embodiments 14 to 19, wherein the biologically active polypeptide and the fusion partner are linked by a peptide linker comprising a flexible peptide linker and/or a rigid unit, e.g. may comprise 1, 2, 3, 4, 5 or more of the rigid units.
21. The method of embodiment 20, wherein the flexible peptide linker contains 2 or more amino acid residues selected from the group consisting of glycine, serine, alanine, and threonine,
preferably, the flexible peptide linker has the General Sequence (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,
more preferably, the flexible peptide linker has a sequence selected from the group consisting of:
(i)GSGGGSGGGGSGGGGS(SEQ ID NO:6)；
(ii)GSGGGGSGGGGSGGGGSGGGGSGGGGS(SEQ ID NO:7)；
(iii)GGGGSGGGGSGGGGSGGGGS(SEQ ID NO:8)；
(iv) GSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 9); or
(v)GGGSGGGSGGGSGGGSGGGS(SEQ ID NO:10)。
22. The fusion protein of embodiment 20 or 21, wherein said rigid unit is a carboxy terminal peptide of a human chorionic gonadotrophin β subunit, or said rigid unit has 70%, 80%, 90%, 95% or more identity to an amino acid sequence of a carboxy terminal peptide of a human chorionic gonadotrophin β subunit; the rigid unit may comprise 1, 2 or more glycosylation sites;
preferably, the rigid unit comprises an amino acid sequence selected from the group consisting of:
(i)PRFQDSSSSKAPPPSLPSPSRLPGPSDTPILPQ(SEQ ID NO:11)；
(ii)SSSSKAPPPSLPSPSRLPGPSDTPILPQ(SEQ ID NO:12)；
(iii) SSSSKAPPPS (SEQ ID NO: 13); or
(iv)SRLPGPSDTPILPQ(SEQ ID NO:14)；
More preferably, the peptide linker comprises the sequence shown in SEQ ID NO 15.
Drawings
In order to more clearly illustrate the embodiments of the present application and the technical solutions of the prior art, the following briefly introduces the drawings required for the embodiments and the prior art, and obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1a shows the SEC-HPLC detection result of FVIII-Fc (FF-0) without mPEG modification.
FIG. 1b shows the SEC-HPLC detection result of 5K molecular weight mPEG modified FVIII-Fc (FF-5L).
FIG. 1c shows the SEC-HPLC detection of 10K molecular weight mPEG modified FVIII-Fc (FF-10L).
FIG. 1d shows the SEC-HPLC detection of 20K molecular weight mPEG modified FVIII-Fc (FF-20L).
FIG. 1e shows the SEC-HPLC detection of 30K molecular weight mPEG modified FVIII-Fc (FF-30L).
FIG. 1f shows the SEC-HPLC detection of 40K molecular weight mPEG modified FVIII-Fc (FF-40L).
FIG. 2a shows the SEC-HPLC detection results (purity > 99%, polymer < 1%) of FVIII-Linker1-Fc (FL1F-0) without mPEG modification.
FIG. 2b shows the SEC-HPLC assay of 20K molecular weight mPEG modified FVIII-L1-Fc (FL1F-20L) (> 95% purity, < 5% polymer, < 1% uncrosslinked).
FIG. 2c shows SEC-HPLC assay results (purity > 95%, polymer < 5%, uncrosslinked < 1%) for linear, 30K molecular weight mPEG modified FVIII-L1-Fc (FL 1F-30L).
FIG. 2d shows SEC-HPLC assay results for linear, 40K molecular weight mPEG modified FVIII-L1-Fc (FL1F-40L) (> 95% purity, < 5% polymer, < 1% uncrosslinked).
FIG. 2e shows the SEC-HPLC assay of linear, 50K molecular weight mPEG modified FVIII-L1-Fc (FL1F-50L) (> 95% purity, < 5% aggregates, < 1% uncrosslinked).
FIG. 2f shows SEC-HPLC assay results (purity > 95%, polymer < 5%, uncrosslinked < 1%) for type Y-40K molecular weight mPEG modified FVIII-L1-Fc (FL 1F-40Y).
FIG. 3a shows SDS-PAGE results before and after changing stock G25 of hFVIII-Fc (FF-0) without mPEG modification (H represents reduction, F represents non-reduction).
FIG. 3b shows the result of SDS-PAGE of hFVIII-Fc cross-linked with mPEG of different molecular weights (FF-5L to FF-40L) (non-reduced).
FIG. 3c shows the SDS-PAGE detection (reduction) of hFVIII-Fc cross-linked to mPEG of different molecular weights (FF-5L to FF-40L).
Detailed Description
In order to make the objects, technical solutions and advantages of the present application more apparent, the present application is further described in detail below by referring to the accompanying drawings and examples. It is to be understood that the embodiments described are only a few embodiments of the present application and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present application.
The term "polypeptide" refers to a compound formed by the dehydration condensation of a plurality of amino acid molecules, and in the present application, a "polypeptide" has the same meaning as a "protein", or "peptide", and may be used interchangeably;
the term "biologically active polypeptide" refers to a protein capable of acting on a specific physiological or pathological process of an organism, including: growth, development, apoptosis, death, catalysis, angiogenesis, pathology, tumorigenesis, metastasis, signaling, coagulation, blood glucose and lipid regulation, etc., including but not limited to activities selected from the group consisting of: hormones, cytokines, coagulation factors, enzymes, receptor extracellular regions, immunomodulatory factors, interleukins, interferons, tumor necrosis factors, transforming growth factors, colony stimulating factors, chemokines, neuropeptides, insulin, GLP-1 receptor agonists, growth hormones, erythropoietin, G-CSF and GM-CSF.
The term "fusion protein" is also referred to as a fusion polypeptide, a chimeric polypeptide, or a chimeric protein, and is a single protein having functional properties derived from each original protein, which is obtained by directly linking two or more genes each independently encoding a different protein or by linking them via a linker and then expressing and translating the resulting protein.
The term "fusion partner" refers to another polypeptide and its effect-enhancing variants fused to a polypeptide of interest (i.e., a polypeptide whose half-life is intended to be extended), which fusion partner is capable of altering the half-life of the fusion protein by a number of different mechanisms.
In one embodiment, the fusion partner delays in vivo clearance of the polypeptide of interest by interacting with the neonatal Fc receptor (FcRn). In one embodiment, the fusion partner is the Fc domain (Fc region) of an immunoglobulin, albumin, XTEN, or transferrin, or a portion thereof. In a preferred embodiment, IgG Fc domains are preferred because of their longer half-life.
Fc domains may also be modified to improve other functions, for example 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 Fc γ RI receptors and possibly also to Fc γ RIIa and Fc γ RIII receptors. These mutations do not alter binding to the FcRn receptor, which facilitates a long circulating half-life through an endocytic recycling pathway. Preferably, the modified IgG Fc domain of the fusion protein of the invention comprises one or more mutations that will result in decreased affinity for certain Fc receptors (L234A, L235E and G237A) and C1 q-mediated decrease in complement binding (a330S and P331S), respectively.
The term "polyalkylene glycol" is a hydrophilic polymer which in the present invention is conjugated to a biologically active polypeptide and/or to a specific position on the fusion partner, and may be straight or branched chain and may contain one or more independently selected polymeric moieties, preferably polyethylene glycol including m-PEG, polypropylene glycol including m-PPG, and the like.
The polyalkylene glycol of the present invention may be polyethylene glycol (PEG), and the main chain may be linear or branched. Branched polymer backbones are well known in the art and, in general, branched polymers have a central branched core portion and one or more linear polymer chains attached to the central branched core. The present invention preferably uses PEG in branched form. In one example, the branched polyethylene glycol may be represented by the general formula R (-PEG-OH) m, where R represents a core moiety, such as glycerol or pentaerythritol, and m represents the number of arms.
In one embodiment, the number of branches in the branched PEG or mPEG is 2, also referred to herein as "Y-type" PEG or mPEG, i.e., a branched PEG comprising two PEGs or linear methoxy PEGs.
Examples of other suitable polymers include, but are not limited to, other polyalkylene glycols (e.g., polypropylene glycol (PPG), copolymers of ethylene glycol and propylene glycol, and the like), polyoxyethylated polyols, polyalkenol (olefmic alcohol), polyvinylpyrrolidone, polyhydroxypropylmethacrylamide, poly ([ alpha ] -hydroxy acid), polyvinyl alcohol, polyphosphazene, polyoxazoline, poly N-acryloylmorpholine, and copolymers, terpolymers, and mixtures thereof.
In one embodiment of the present application, PEG modification is used, more preferably mPEG modification is used, wherein the modification is random or site-directed, and the position of the modification comprises a free amino group, a thiol group, a sugar group and/or a carboxyl group.
In a particular embodiment of the present application, the modifier used for the random modification of mPEG of the free amino groups may be selected from: one of mPEG-SS (methoxy polyethylene glycol-succinimide succinate), mPEG-SC (methoxy polyethylene glycol-succinimide carbonate), mPEG-SPA (methoxy polyethylene glycol-succinimide propionate) and mPEG-SG (methoxy polyethylene glycol-succinimide glutarate). The N-terminal modifier is: one of mPEG-ALD (methoxy polyethylene glycol-acetaldehyde), mPEG-pALD (methoxy polyethylene glycol-propionaldehyde), mPEG-bALD (methoxy polyethylene glycol-butyraldehyde) and the like. The modifier mPEG-SS, mPEG-SC, mPEG-SPA, mPEG-SG, mPEG-ALD, mPEG-pALD and mPEG-bALD are linear or branched.
In a specific embodiment of the present application, the modifying agent used for the random modification of free Thiol groups is one of mPEG-mal (methoxy polyethylene glycol-maleimide), mPEG-OPSS (methoxy polyethylene glycol-o-dithiopyridine), mPEG-vinylilsulfone (methoxy polyethylene glycol-vinyl sulfone), mPEG-Thiol (methoxy polyethylene glycol-Thiol), and the like.
In a specific embodiment of the present application, the modifying agent used for the random modification of the sugar and/or carboxyl groups is mPEG-ZH (methoxypolyethylene glycol-hydrazide).
In one embodiment of the present application, the mPEG modified modifier has the structure shown in formula (1):
Figure PCTCN2019087144-APPB-000007
wherein m1 is more than or equal to 0 and less than or equal to 6, and m1 is preferably 5; mPEG-represents a methoxy single-terminated polyethylene glycol group, and the molecular weight of the modifier represented by formula (1) is 5kD to 60kD (kD, kilodalton), preferably 40 kD. Preferably, in one embodiment of the present application, the random modification of the mPEG of the free amino group is performed with a modifier represented by formula (1).
In one embodiment of the present application, the mPEG modified modifier has the structure shown in formula (2):
Figure PCTCN2019087144-APPB-000008
wherein m2 is more than or equal to 0 and less than or equal to 6, and m2 is preferably 2; 0 is more than or equal to m3 is less than or equal to 6, m3 is preferably 1; mPEG-represents a methoxy single-terminated polyethylene glycol group, and the molecular weight of the modifier represented by the formula (2) is 5kD to 60kD, and preferably: 40 kD. Preferably, in one embodiment of the present application, the random modification of the mPEG of the free amino group is performed with a modifier represented by formula (2).
In one embodiment of the present application, the mPEG modified modifier has the structure shown in formula (3):
Figure PCTCN2019087144-APPB-000009
wherein m4 is more than or equal to 0 and less than or equal to 6, and m4 is preferably 2; mPEG-represents a methoxy single-terminated polyethylene glycol group, and the molecular weight of the modifier represented by formula (3) is 5kD to 60kD, preferably 40 kD. In one embodiment of the present application, random modification of the mPEG of the free thiol group is performed with a modifier represented by formula (3).
The size of the polymer backbone can vary, but the polymer (e.g., PEG, mPEG, PPG, or mPG) typically ranges from about 0.5KD to about 160KD, such as from about 1KD to about 100 KD. More specifically, the size of each of the presently conjugated hydrophilic polymers varies primarily within the following ranges: about 1KD to about 80KD, about 2KD to about 70 KD; about 5KD to about 70 KD; about 10KD to about 60KD, about 20KD to about 50 KD; about 30 KD-to about 50KD or about 30KD-40 KD. It should be understood that these sizes represent approximate values, and not exact measures, as is generally accepted in the art.
In a specific embodiment, the PEG or mPEG used in the present invention has a size of above 35KD (i.e., 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 term "improved circulation half-life": the molecules of the invention have an altered circulating half-life, preferably an increased circulating half-life, compared to the wild-type factor biologically active polypeptide. The circulation 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 increased circulatory half-life of at least 400%, 500%, 600%, or even 700%.
The term "pharmaceutically acceptable carrier" includes, but is not limited to: saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical preparations should generally be adapted to the mode of administration, and the pharmaceutical compositions of the present application may be prepared in the form of injections, for example, 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 amount of active ingredient administered is a therapeutically effective amount. The pharmaceutical formulations of the present application may also be formulated as sustained release formulations.
Examples
Example 1 preparation and purification of mPEG-modified hFVIII fusion proteins
1.1 preparation of mPEG modified hFVIII fusion protein
1.1.1 first a series of hFVIII fusion protein expression plasmids were constructed according to molecular cloning techniques well known to those skilled in the art, and the expression plasmids were transfected into DHFR deficient CHO cells (see U.S. Pat. No. 4,818,679) separately to express the respective hFVIII fusion proteins (Table 1). Specific preparation steps for fusion proteins are described in chinese patent ZL201610692838.0, which is incorporated herein by reference in its entirety.
TABLE 1 composition and Structure of fusion proteins
Figure PCTCN2019087144-APPB-000010
Note: the hFVIII with the deleted B domain is called BDD FVIII for short, and consists of a heavy chain of 90kD A1-A2 and a light chain of 80 kD;
SEQ ID NO.7-SEQ ID NO.12 indicates that the linker is formed by the rigid unit shown in SEQ ID NO.12 linked to the C-terminus of the flexible peptide linker shown in SEQ ID NO. 7; SEQ ID NO.6 to SEQ ID NO.11 show that the linker is formed by the rigid unit shown in SEQ ID NO.11 linked to the C-terminus of the flexible peptide linker shown in SEQ ID NO. 6.
1.1.2, respectively centrifuging and filtering the fermentation liquor of each fusion protein of 1.1.1, and sequentially carrying out Affinity Chromatography (Affinity Chromatography)/Hydrophobic Chromatography (hydrophic Interaction Chromatography)/Ion-Exchange Chromatography (Ion-Exchange Chromatography)/molecular Size exclusion Chromatography (Size exclusion Chromatography) to respectively obtain five hFVIII fusion proteins FF-0, FL1F-0, FL2F-0, F (full length) L1F '-0 and F (full length) L2F' -0, wherein the polymer is detected by SEC-HPLC to be less than 5%. Preparing five hFVIII fusion proteins into hFVIII fusion protein stock solutions with the protein concentration of 0.95 mg/ml;
1.1.3, 5ml of each of the five hFVIII fusion protein stock solutions prepared in 1.1.2 are subjected to G25(GE Healthcare) molecular sieve chromatography. The method comprises the following specific steps:
preparing a buffer solution: 20mM Hepes, 0.1M NaCl, 5.0mM CaCl2, 0.02% Tween8.0, pH 7.0;
the chromatography process comprises the following steps:
(1) balancing: balancing the chromatographic column with 3 times of column volume of Binding Buffer solution (Binding Buffer) until the pH and the electrical conductivity are consistent with those of the Buffer solution, wherein the flow rate is 150 cm/h;
(2) loading: the sample loading flow rate is unified to 150 cm/h;
(3) balancing: balancing the chromatographic column by using buffer solution with 3 times of column volume until the pH and the electrical conductivity are consistent with those of the buffer solution, wherein the flow rate is unified to 150 cm/h;
(4) balancing: equilibrating the chromatographic column with buffer solution, and collecting the peak with A280/A260 greater than 1.8;
(5) in-situ cleaning of the chromatographic column: reverse washing with 0.2M NaOH at a flow rate of 60cm/h for 1.5 column volumes and neutralizing with buffer;
(6) and (3) storage of the chromatographic column: after the experiment was completed, the column was washed with 3 column volumes of purified water at a flow rate of 100cm/h, and then stored with 2 column volumes of 20% ethanol.
And (3) ultrafiltration concentration: five stock solutions of hFVIII fusion proteins (FF-0, FL1F-0, FL2F-0, F (full length) L1F '-0 and F (full length) L1F' -0) which are subjected to G25 liquid exchange are ultrafiltered and concentrated by a 50kD ultrafiltration tube, and are centrifugally concentrated under the conditions of 4 ℃ and 3800rpm until the protein concentration is preferably 1.5 mg/ml.
1.1.4, weighing mPEG-SC (the molecular weight of which is respectively 5kD, 10kD, 20kD, 30kD and 40kD and the structure of which is shown as a formula (1)) according to the molar (mol) ratio of the hFVIII fusion protein to mPEG-SC (purchased from Beijing Kekeh science and technology Co., Ltd.) of 1:10-1:100, adding 1.1.3 of the ultra-filtered and concentrated hFVIII fusion protein, reacting for 4 hours, adding histidine with the molar (mol) ratio being 10 times that of the substrate hFVIII fusion protein to stop the reaction, and obtaining the mPEG-SC modified hFVIII fusion proteins with different molecular weights; the partial modification product numbers and their compositions are shown in Table 2 below.
TABLE 2
Figure PCTCN2019087144-APPB-000011
Figure PCTCN2019087144-APPB-000012
1.2 purification of mPEG-modified hFVIII fusion proteins
1.2.1 Each mPEG-modified hFVIII fusion protein prepared under section "1.1.4" of example 1 was subjected to S200(GE Healthcare) molecular sieve chromatography. The method comprises the following specific steps:
preparing a buffer solution: 20mM histidine, 0.1M NaCl, 5.0mM CaCl2，0.02％Tween8.0，pH 7.0；
The chromatography process comprises the following steps:
(1) balancing: balancing the chromatographic column with 3 times of column volume of Binding Buffer solution (Binding Buffer) until the pH and the electrical conductivity are consistent with those of the Buffer solution, wherein the flow rate is 150 cm/h;
(2) loading: the sample loading flow rate is unified to 150 cm/h;
(3) balancing: balancing the chromatographic column by using buffer solution with 3 times of column volume until the pH and the electrical conductivity are consistent with those of the buffer solution, wherein the flow rate is unified to 150 cm/h;
(4) balancing: equilibrating the chromatographic column with buffer solution, and collecting the peak with A280/A260 greater than 1.8;
(5) in-situ cleaning of the chromatographic column: reverse washing with 0.2M NaOH at a flow rate of 60cm/h for 1.5 column volumes and neutralizing with buffer;
(6) and (3) storage of the chromatographic column: after the experiment was completed, the column was washed with 3 column volumes of purified water at a flow rate of 100cm/h, and then stored with 2 column volumes of 20% ethanol.
1.2.2, subjecting the chromatography product obtained in item "1.2.1" to Source 15Q (GE healthcare) anion chromatography:
preparing a buffer solution:
binding buffer: 20mM histidine, 0.1M NaCl, 5.0mM CaCl20.02% tween8.0, pH 7.0; elution buffer: 20mM histidine, 2.0M NaCl, 5.0mM CaCl2，0.02％Tween8.0，pH 7.0；CIP：0.5M NaOH；
The chromatography process comprises the following steps:
(1) balancing: balancing the chromatographic column by using a binding buffer solution with 3 times of column volume until the pH and the electrical conductivity are consistent with those of the buffer solution, wherein the flow rate is unified to 150 cm/h;
(2) loading: the sample loading flow rate is unified to 150 cm/h;
(3) balancing: balancing the chromatographic column by using a binding buffer solution with 3 times of column volume until the pH and the electrical conductivity are consistent with those of the buffer solution, wherein the flow rate is unified to 150 cm/h;
(4) and (3) elution: eluting the samples by a column with 20 times of linear gradient of 0-100% buffer solution B, wherein the elution flow rates are unified to be 100cm/h, collecting elution peaks with A280/A260 being more than 1.8 in different tubes, and performing SEC-HPLC detection on each tube of samples;
(5) in-situ cleaning of the chromatographic column: reverse washing with 0.5M NaOH at a flow rate of 60cm/h for 1.5 column volumes and neutralization with binding buffer;
(6) and (3) storage of the chromatographic column: after the experiment was completed, the column was washed with 3 column volumes of purified water at a flow rate of 100cm/h, and then stored with 2 column volumes of 20% ethanol.
1.2.3, SEC-HPLC detection:
subjecting the chromatography product obtained in item "1.2.2" to SEC-HPLC detection, wherein:
a chromatographic column: G3000/G4000; flow rate: 0.5 mL/min; detection wavelength: 280 nm; column temperature: 25 ℃; sample introduction volume: 100 μ L (sample size 20 μ g); mobile phase: 0.30M sodium chloride; 0.02M imidazole; 0.01M calcium chloride; 25ppm Tween 80; 10% ethanol; pH 7.0; operating time: 35-50 min;
the results of the detection of FF-0 to FF-40L are shown in FIGS. 1a to 1 f. The results of FL1F-0 to FL1F-50L and FL1F-40Y are shown in FIGS. 2a-2f, and show that FL1F-0 to FL1F-60L is > 95% pure, polymeric < 5% and uncrosslinked < 1%.
1.2.4, protein gel electrophoresis detection by SDS-PAGE:
the product obtained in item "1.2.2" was subjected to SDS-PAGE, comprising the following steps:
(1) preparing glue: 1 × Tris-glycine electrophoresis buffer: 0.4g of SDS, 1.21g of Tris base, 7.5g of glycine and double distilled water to reach the constant volume of 400 mL.
5% concentrated gum: double distilled water 4.1mL, 1M Tris-HCl (pH 6.8)0.75mL, 30% (w/v) polyacrylamide 1mL, 10% (w/v) ammonium persulfate 60 μ L, 10% (w/v) SDS60 μ L, TEMED 6 μ L.
6% separation gel: double distilled water 4.9mL, 1.5M Tris-HCl (pH 8.8)3.8mL, 30% (w/v) polyacrylamide 6mL, 10% (w/v) ammonium persulfate 150 μ L, 10% (w/v) SDS 150 μ L, TEMED 6 μ L.
(2)5 × protein loading buffer: 5mL of glycerol, 2.5mL of 1M Tris-HCl (pH 6.8), 0.05g of bromophenol blue, 1g of SDS, and double distilled water with a constant volume of 10mL, storing at 4 ℃, and adding 0.5mL of beta-mercaptoethanol before use.
(3) Preparing a sample: mixing a sample to be detected with an equal-volume loading buffer solution, reducing SDS-PAGE, and adding 0.1mg/mL of 2-mercaptoethanol with the equal volume with the sample; non-reducing SDS-PAGE without 2-mercaptoethanol. After mixing the sample with the loading buffer, the sample was water-washed in boiling water for 10 minutes.
(4) Electrophoresis: and (3) sequentially adding 10 mu l of the boiled sample to be detected and the protein Marker into a sample application hole, carrying out concentration electrophoresis at the voltage of 60V, observing that a display agent bromophenol blue dye is concentrated into the separation gel, increasing the voltage to 120V, carrying out separation electrophoresis until the bromophenol blue dye reaches the bottom of the separation gel, and turning off the power supply.
(5) Dyeing: the SDS-PAGE gel was carefully removed and placed in a plastic box containing Coomassie Brilliant blue staining solution and heated in a microwave oven for 1 minute.
(6) And (3) decoloring: and taking out the dyed SDS-PAGE gel, placing the SDS-PAGE gel in a decoloring solution, shaking for decoloring, replacing the decoloring solution once after 2 hours, and stopping after a band which is visible and clear by naked eyes is observed.
(7) Recording: the finished SDS-PAGE gels were either photographically recorded or stored dry. FIGS. 3a-3c show the SDS-PAGE detection of FF-5L to FF-40L.
Example 2 Indirect assay of the in vitro Activity of mPEG-modified hFVIII fusion proteins by chromogenic substrate method
The activity of the mPEG modified hFVIII fusion protein prepared in example 1 was determined using chromogenic substrate assay (chromogenic substrate assay). The detection principle is as follows by adopting ChromogenixCoatest SP FVIII kit (Chromogenix, Ref.K824086): 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 the conversion of factor X to its active form Xa. Activation of the formed factor Xa in turn leads to cleavage of its specific chromogenic substrate (S-2765) and release of the chromophoric group pNA. The amount of pNA produced is measured at 405nm, and the activity of FXa which is directly proportional to the amount thereof is known, wherein the amounts of factor IXa and factor X in the system are constant and excessive, and the activity of FXa is directly related to the FVIIIa content only. Results of indirect measurement of FVIII biological activity by chromogenic substrate method are shown in table 3.
TABLE 3 Indirect measurement of FVIII biological Activity by chromogenic substrate method
Figure PCTCN2019087144-APPB-000013
Figure PCTCN2019087144-APPB-000014
Note: elockate is a recombinant factor VIII Fc fusion protein that has been marketed by Bioverativ, which has not been modified by mPEG.
Example 3 human coagulation factor VIII potency assay
The method for measuring the titer of the human blood coagulation factor VIII adopted by the invention is also called a first-stage method, and the specific steps are shown in the three parts of the Chinese pharmacopoeia 2010 edition. One-phase assays for FVIII biological activity were performed by correcting the ability of FVIII deprived plasma to prolong clotting time. A kit, Cooperation Factor VIII purification plant (Cat. No. OTXW17), was used, which was manufactured by Siemens, Germany. The method comprises the following steps: first, FVIII activity Standard WHO International Standard 8th International Standard Factor VIII Concentrate (Cat. No.07/350) with known titer was diluted to 4IU/ml, and then gradient-diluted to different titers (IU/ml), and mixed with FVIII-deficient matrix plasma, partial thromboplastin time (APTT) was determined, and a Standard curve was established by linear regression of the logarithm of the FVIII activity Standard solution titer (IU/ml) corresponding to the logarithm of the corresponding clotting time(s). And mixing the sample to be tested with FVIII-deficient matrix plasma after being diluted appropriately, and carrying out APTT determination. By substituting the standard curve, the titer of the FVIII sample to be detected can be known, and the specific activity of the FVIII sample to be detected can be calculated according to the titer, wherein the unit is IU/mg. The results are shown in Table 4.
TABLE 4 direct determination of biological Activity by one-stage method
Figure PCTCN2019087144-APPB-000015
Figure PCTCN2019087144-APPB-000016
Shown in tables 3 and 4: although FL1F-40Y, FL2F-40Y, F (full length) L1F '-40Y and F (full length) L2F' -40Y measured by the chromogenic substrate method and the first-stage method have lower biological activity than that of an Elocate/FL 1F-0/FL2F-0 experimental group of uncrosslinked mPEG, the biological activity is caused by the influence on the spatial structure of the modified protein after the mPEG is modified. Similar phenomena occur in other mPEG modified proteins of the prior art (e.g., PEG-INTRON; Pegfilgrastim). Surprisingly, the fusion protein of the present application can still maintain relatively high activity after being modified by mPEG of 40kD or higher, and meanwhile, the half-life period is greatly prolonged as further verified by subsequent experiments.
Example 4 prophylactic pharmacodynamic experiments on hemophilia a mouse tail vein transection model
This example compares the activity half-life of each mPEG-modified hFVIII fusion protein in hemophilia a mice (HA mice) by Tail Vein Transection (TVT) experiments.
4.1 Male HA mice (purchased from the Central laboratory of Motif, Shanghai) of 10-12 weeks old were selected according to the literature, randomly grouped into 12 mice/group, administered at a dose of 15IU/kg, and each mPEG-modified fusion protein or positive control drug Elockate of the present application was administered via the tail vein. At 48h after administration, the tail was measured and marked with a cannula 2.7mm in inside diameter, the unilateral tail vein was transected with a 11 gauge straight blade, after transection, the tail was quickly placed into a pre-warmed saline tube containing about 13ml, and the bleeding time was recorded. After bleeding had ceased (no significant blood flow through the incision), the rat tails were removed from the raw saline tubes and the mice were then placed on a 37 ℃ heating pad to maintain their body temperature without touching the wound. After the mice revived, the mice were placed in a mouse cage padded with A4 white paper, and were raised in a single cage, and the white paper was replaced or the mouse cage was replaced after each observation, so that the bleeding degree was judged. The survival rate of the mice within 48h after tail-breaking and the number of recurrent bleeding within 12h after tail-breaking were counted (12 hours in total, the number of bleeding within one hour is counted as 1), and the results are shown in table 5.
The rate of the repeated bleeding is counted, namely the proportion of mice with repeated bleeding during the counting period, and the rate of the severe bleeding is counted, namely the proportion of mice with severe bleeding (+++) phenomenon or multiple moderate bleeding (++) phenomena in the 12-time repeated bleeding. Of these, moderate bleeding (++) refers to: a4 white paper has multiple bloodstains, the coverage area is not less than 30%, the color of the bloodstains is medium, but no large-area bloodbank (the area is more than 3cm 2); severe bleeding (+++) refers to: a4 white paper has a large amount of blood traces, the coverage area is not less than 30%, the color of blood trace is heavy, and the white paper has large area blood bank; even if the coverage area is less than small, severe bleeding can be seen (mice lose a lot of blood, range of motion is reduced, blood heavily soaks white paper).
TABLE 5 survival at 48h and 12h rebleeding rates of TVT 48h post-dose
Name of fusion protein Bleeding rate after 12h Rate of severe bleeding Survival rate of 48h
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 (full Length) L1F' -40L 75.0％(9/12) 16.7％(2/12) 66.7％(8/12)
F (full Length) L1F' -40Y 66.7％(8/12) 8.3％(1/12) 83.3％(10/12)
F (full Length) L1F' -50L 66.7％(8/12) 25.0％(3/12) 50.0％(6/12)
F (full Length) L2F "-40L 75.0％(9/12) 8.3％(1/12) 58.3％(7/12)
F (full Length) L2F' -40Y 58.3％(7/12) 8.3％(1/12) 83.3％(10/12)
F (full Length) L2F "-50L 75.0％(9/12) 16.7％(2/12) 58.3％(7/12)
The results show that: compared with an Elocate/FL 1F-0/FL2F-0 experimental group without mPEG modification, the survival rate of 83.3 percent of the FL1F-40Y experimental group, the survival rate of 75.0 percent of the FL2F-40Y experimental group, the survival rate of 83.3 percent of the F (full-length) L1F '-40Y experimental group and the survival rate of 83.3 percent of the F (full-length) L2F' -40Y experimental group are all obviously improved compared with other groups. The 12h bleeding rate and severe bleeding rate of these experimental groups were significantly reduced compared to the other groups. FL1F-40Y, FL2F-40Y, F (full length) L1F' -40Y, F (full length) L2F "-40Y possesses a longer duration of protection in prophylactic pharmacodynamics in a tail vein transection model of hemophilia a mice.
4.2 Male HA mice 10-12 weeks old, 12/group, were subjected to TVT experiment at 84h after administration, in the same manner as 4.1, and the results are shown in Table 6.
TABLE 6 survival at 48h and rebound at 12h for TVT after 84h dosing
Name of fusion protein Survival rate of 48h Bleeding rate after 12h
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 (full Length) L1F' -40L 66.7％(8/12) 75.0％(10/12)
F (full Length) L1F' -40Y 75.0％(9/12) 66.7％(8/12)
F (full Length) L1F' -50L 58.3％(7/12) 91.7％(11/12)
F (full Length) L2F "-40L 41.7％(5/12) 91.7％(11/12)
F (full Length) L2F' -40Y 75.0％(9/12) 75.0％(9/12)
F (full Length) L2F "-50L 58.3％(7/12) 91.7％(11/12)
The results show that: compared with the Elockate without cross-linked PEG and other experimental groups, the survival rate of 66.7 percent of the FL1F-40Y experimental group, the survival rate of 75.0 percent of the FL2F-40Y experimental group, the survival rate of 75.0 percent of the F (full-length) L1F '-40Y experimental group and the survival rate of 75.0 percent of the F (full-length) L2F' -40Y experimental group are all obviously improved, and the corresponding recurrent blood rate is obviously reduced. FL1F-40Y, FL2F-40Y, F (full length) L1F '-40Y, F (full length) L2F' -40Y has certain advantages in preventive pharmacodynamics on a haemophilia A mouse tail vein transection model after 84h after administration.
4.3 the same method as 4.1 was used to perform TVT test on 10-12 week old HA mice, 10 male and female, 20/group at 90h after administration, and the results are shown in Table 7.
TABLE 7 survival at 48h and 12h relapse rates of TVT at 90h post-dose
Name of fusion protein Survival rate of 48h Bleeding rate after 12h
Eloctate 70％ 80％
FL1F-40L 55％) 70％
FL2F-40L 63.2％ 73.68％
FL1F-50L 84.2％ 68.4％
FL2F-50L 40％ 90％
The results show that: FL1F-50L has similar survival rate and recurrent bleeding rate with the Elockate experimental group of the uncrosslinked PEG; because the experimental animal is not single sex, the experimental result is not compared with other single sex experimental results.
4.4 in the same manner as in 4.1, TVT test was carried out on 10-12 weeks old male HA mice, 12 mice/group, 96h after the administration, and the results are shown in Table 8.
TABLE 8 survival at 48h and 12h relapse rates of TVT 96h post-dose
Name of fusion protein Survival rate of 48h Bleeding rate after 12h
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％
The results show that: compared with an Elockate experimental group without cross-linked PEG, FL1F-40Y/FL2F-40Y has a small advantage in survival rate, but has a significantly reduced 12h bleeding rate. The survival rate of FL1F-40Y/FL2F-40Y is obviously improved compared with other groups; the re-bleeding rate after 12h is obviously reduced compared with other groups. FL1F-40Y/FL2F-40Y possessed a longer period of prophylaxis than the other groups on the tail vein transection model in hemophilia A mice.
The above description is only exemplary of the present application and should not be taken as limiting the present application, as any modification, equivalent replacement, or improvement made within the spirit and principle of the present application should be included in the scope of protection of the present application.

Claims (11)

  1. A biologically active polypeptide fusion protein conjugated to a polyalkylene glycol, wherein the biologically active polypeptide moiety is directly linked or indirectly linked with a peptide linker to a half-life enhancing fusion partner, and the fusion protein is further conjugated to a polyalkylene glycol.
  2. The fusion protein of claim 1, wherein the biologically active polypeptide moiety can confer a biological activity to the fusion protein selected from the group consisting of: hormones, cytokines, coagulation factors, enzymes, receptor extracellular regions, immunomodulatory factors, interleukins, interferons, tumor necrosis factors, transforming growth factors, colony stimulating factors, chemokines, neuropeptides, insulin, GLP-1 receptor agonists, growth hormones, erythropoietin, G-CSF and GM-CSF.
  3. The fusion protein of claim 1 or 2, wherein the fusion partner is: an immunoglobulin Fc fragment, albumin, XTEN, or transferrin, said fusion partner being derived, for example, from a human; preferably an IgG Fc fragment; for example, the IgG Fc fragment has reduced ADCC effect and/or CDC effect and/or enhanced binding affinity to FcRn receptor; more preferably, the IgG Fc fragment has an amino acid sequence selected from the group consisting of:
    (i) SEQ ID NO: 3;
    (ii) SEQ ID NO: 4; or
    (iii) SEQ ID NO: 5.
  4. The fusion protein according to any one of claims 1 to 3, wherein the polyalkylene glycol is polypropylene glycol or polyethylene glycol; the polyalkylene glycol may be terminally capped, for example, with alkoxy groups such as methoxy groups; and/or the polyalkylene glycol is linear or branched; preferably the polyalkylene glycol is branched, for example a branched polyethylene glycol, especially a methoxy-terminated branched polyethylene glycol; the molecular weight of the polyalkylene glycol may be > 1, > < 10, > < 20, > < 30, > < 40, > < 50, > < 60, > < 70, > < 80, > < 90, > < 100, > < 110, > < 120, > < 130, > < 140, > < 150 or > -160 kDa, such as 5, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100kDa, or any number therebetween.
  5. The fusion protein according to any one of claims 1 to 4, wherein the conjugation of the fusion protein to the polyalkylene glycol is random or site-directed, the conjugation position being selected from free amino, thiol, glycosyl and/or carboxyl groups, preferably free amino groups.
  6. The fusion protein of claim 5, wherein the conjugation is achieved using any suitable modifier, which may be an activated ester form or other principle type modifier, for example selected from the group consisting of the modifiers represented by the following formulae (1), (2) or (3):
    Figure PCTCN2019087144-APPB-100001
    wherein m1 is more than or equal to 0 and less than or equal to 6, and m1 is preferably 5; mPEG represents a methoxy single-capped polyethylene glycol group;
    Figure PCTCN2019087144-APPB-100002
    wherein m2 is more than or equal to 0 and less than or equal to 6, and m2 is preferably 2; 0 is more than or equal to m3 is less than or equal to 6, m3 is preferably 1; mPEG represents a methoxy single-capped polyethylene glycol group; or
    Figure PCTCN2019087144-APPB-100003
    Wherein m4 is more than or equal to 0 and less than or equal to 6, and m4 is preferably 2; mPEG represents a methoxy single-capped polyethylene glycol group.
  7. The fusion protein of any one of claims 1 to 6, wherein the biologically active polypeptide and the fusion partner are linked by a peptide linker comprising a flexible peptide linker and/or a rigid unit, e.g. may comprise 1, 2, 3, 4, 5 or more of the rigid units.
  8. The fusion protein of claim 7, wherein the flexible peptide linker contains 2 or more amino acid residues selected from the group consisting of glycine, serine, alanine, and threonine,
    preferably, the flexible peptide linker has the General Sequence (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,
    more preferably, the flexible peptide linker has a sequence selected from the group consisting of:
    (i)GSGGGSGGGGSGGGGS(SEQ ID NO:6)；
    (ii)GSGGGGSGGGGSGGGGSGGGGSGGGGS(SEQ ID NO:7)；
    (iii)GGGGSGGGGSGGGGSGGGGS(SEQ ID NO:8)；
    (iv) GSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 9); or
    (v)GGGSGGGSGGGSGGGSGGGS(SEQ ID NO:10)。
  9. The fusion protein of claim 7 or 8, wherein the rigid unit is a carboxy terminal peptide of a human chorionic gonadotrophin β subunit, or the rigid unit has 70%, 80%, 90%, 95% or more identity to an amino acid sequence of a carboxy terminal peptide of a human chorionic gonadotrophin β subunit; the rigid unit may comprise 1, 2 or more glycosylation sites;
    preferably, the rigid unit comprises an amino acid sequence selected from the group consisting of:
    (i)PRFQDSSSSKAPPPSLPSPSRLPGPSDTPILPQ(SEQ ID NO:11)；
    (ii)SSSSKAPPPSLPSPSRLPGPSDTPILPQ(SEQ ID NO:12)；
    (iii) SSSSKAPPPS (SEQ ID NO: 13); or
    (iv)SRLPGPSDTPILPQ(SEQ ID NO:14)；
    More preferably, the peptide linker comprises the amino acid sequence shown in SEQ ID NO 15.
  10. A pharmaceutical composition comprising an effective amount of the fusion protein of any one of claims 1-9, and a pharmaceutically acceptable carrier.
  11. A method of preventing and/or treating a disease that can be prevented and/or treated with the activity of a biologically active polypeptide, comprising administering the fusion protein of any one of claims 1 to 9 or the pharmaceutical composition of claim 10 to a subject in need thereof.
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