WO2019219048A1 - Fused polypeptide conjugate having extended half-life period - Google Patents

Abstract

The present invention relates to a biologically active polypeptide fused protein which is conjugated to a polyalkylene glycol and has an extended cyclic half-life period, a preparation method therefor and use thereof. A biologically active polypeptide moiety and a fusion partner is directly linked or indirectly linked by a peptide linker, and said fused protein is further conjugated to a polyalkylene glycol. The half-life period is significantly increased compared with a biologically active polypeptide fused protein that has not been modified with a polyalkylene glycol.

Description

Fusion polypeptide conjugate with extended half-life

cross reference

The present application claims priority to the Chinese Patent Application No. 201810483378.X, the disclosure of which is incorporated herein by reference.

Technical field

The invention relates to the field of protein engineering, in particular to a fusion polypeptide conjugate having an extended half-life and a preparation method and application thereof.

Background technique

Recombinant protein drugs are important types in biopharmaceuticals and are often administered clinically by intravenous or subcutaneous injection. However, after administration, protein drugs often degrade, resulting in decreased activity and reduced bioavailability. Repeated and multiple doses are often required to achieve the desired blood concentration and efficacy, reducing patient compliance. Therefore, it is clinically necessary to develop long-acting protein drugs.

The principles of the common method of increasing the half-life of a protein drug include increasing the molecular weight of the protein drug, reducing the glomerular filtration rate, or reducing the in vivo clearance of the protein. Strategies for the implementation of conventional protein drug long-acting include glycosylation, PEGylation, albumin fusion, transferrin fusion, Fc fusion, and the like. However, the above modifications or modifications have an effect on the activity of the protein itself, and such modification/modification often fails to achieve the desired effect.

Therefore, there is an urgent need in the art for new means of increasing the half-life of protein drugs and corresponding protein products.

Summary of the invention

After years of research and long-term experiments, the inventors have discovered that a polypeptide (protein) is fused to a fusion partner capable of prolonging half-life (eg, Fc fragment, albumin, XTEN, or transferrin), and further with a hydrophilic polymer (eg, poly. Alkyl diols, more particularly PEG, including mPEG) conjugates, are effective to increase the in vivo stability of biologically active polypeptides, particularly when hydrophilic polymers (eg, PEG) have a branched structure (or branched structure) In particular, when the molecular weight of the hydrophilic polymer is still within a certain range, for example, greater than or equal to a specific value, the in vivo stability of the biologically active polypeptide can be remarkably improved, thereby completing the present invention.

The invention provides the following technical solutions:

A biologically active polypeptide fusion protein conjugated to a polyalkylene glycol, wherein the biologically active polypeptide moiety is directly linked to an extended half-life fusion partner or indirectly linked by a peptide linker, and the fusion protein is further The polyalkylene glycol is conjugated.

2. The fusion protein according to embodiment 1, wherein the biologically active polypeptide moiety confers the fusion protein an activity selected from the group consisting of hormones, cytokines, coagulation factors, enzymes, receptor extracellular regions, immunoregulatory factors , interleukins, interferons, tumor necrosis factor, transforming growth factor, growth factor, colony stimulating factor, chemokine, neuropeptide, insulin, GLP-1, GLP-1 receptor agonist, growth hormone, erythropoiesis , G-CSF and GM-CSF.

3. The fusion protein according to embodiment 1 or 2, wherein the fusion partner is: an immunoglobulin Fc fragment, albumin, XTEN or transferrin, the fusion partner is for example derived from a human; preferably an IgG Fc fragment For example, the IgG Fc fragment has a reduced ADCC effect and/or CDC effect and/or enhanced binding affinity to the FcRn receptor; more preferably, the IgG Fc fragment has an amino acid sequence selected from the group consisting of:

(i) the amino acid sequence of SEQ ID NO: 3;

(ii) the amino acid sequence set forth in SEQ ID NO: 4; or

(iii) the amino acid sequence shown in SEQ ID NO: 5.

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 end-capped, for example And/or the polyalkylene glycol is linear or branched; preferably the polyalkylene glycol is branched, for example branched Polyethylene glycol, especially methoxy-terminated branched polyethylene glycol; the molecular weight of the polyalkylene glycol can be >=1, >=10, >=20, >=30, > =40, >=50, >=60, >=70, >=80, >=90, >=100, >=110, >=120, >=130, >=140, >=150 or >=160kDa For example, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 kDa, or a value between any two of the above values.

5. The fusion protein according to any one of embodiments 1 to 4, wherein the fusion protein is conjugated to a polyalkylene glycol which is random or site-directed, the conjugation position being selected from the group consisting of a free amino group and a thiol group. , a glycosyl group and/or a carboxyl group, preferably a free amino group.

6. The fusion protein according to embodiment 5, wherein the conjugation is achieved using a modifying agent, preferably, the modifying agent may be a modified ester in the form of an activated ester, for example, the modifying agent is selected from the following formula (1) , (2) or (3):

Wherein, 0≤m1≤6, m1 is preferably 5; mPEG represents a mono-terminated polyethylene glycol group having a molecular weight of between 5 KD and 60 KD;

Wherein 0≤m2≤6, m2 is preferably 2; 0≤m3≤6, m3 is preferably 1; mPEG represents a mono-terminated polyethylene glycol group having a molecular weight of 5KD-100KD Dalton, preferably 40KD , 50KD, 60KD, most preferably 40KD; or

Wherein, 0≤m4≤6, and m4 is preferably 2; mPEG represents a mono-terminated polyethylene glycol group having a molecular weight of 5KD-100KD.

The fusion protein according to any one of embodiments 1 to 6, wherein the biologically active polypeptide moiety is linked to the fusion partner via a peptide linker comprising a flexible peptide linker and/or a rigid unit, For example, 1, 2, 3, 4, 5 or more of the rigid units may be included.

The fusion protein according to embodiment 7, wherein the flexible peptide linker comprises two or more amino acid residues selected from the group consisting of glycine, serine, alanine and threonine,

Preferably, the flexible peptide linker has the sequence formula (GS) a(GGS)b(GGGS)c(GGGGS)d, wherein a, b, c and d are integers greater than or equal to 0, and a+b+ c+d≥1,

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).

The fusion protein according to embodiment 7 or 8, wherein the rigid unit is a carboxy terminal peptide of human chorionic gonadotropin β subunit, or the rigid unit and a carboxyl group of human chorionic gonadotropin β subunit The amino acid sequence of the terminal peptide has a consistency of 70%, 80%, 90%, 95% or higher; the rigid unit may comprise 1, 2 or more glycosylation sites;

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 set forth in SEQ ID NO: 15.

A pharmaceutical composition comprising an effective amount of the fusion protein of any one of embodiments 1 to 9, and a pharmaceutically acceptable carrier.

A method for preventing and/or treating a disease which can be prevented and/or treated by the activity of a biologically active polypeptide, which comprises administering the fusion protein according to any one of embodiments 1 to 9 or administering to a subject in need thereof. The pharmaceutical composition of claim 10.

The fusion polypeptide of any one of embodiments 1 to 9, wherein the fusion protein is glycosylated, preferably glycosylated by expression in a mammalian cell, the mammalian cell preferably For Chinese hamster ovary cells.

The fusion polypeptide of any of embodiments 1 to 9, wherein the fusion polypeptide has an extended half-life, particularly an extended cyclic half-life, compared to the non-polyalkylene glycol.

14. A method of improving the half-life of a biologically active polypeptide, wherein the biologically active polypeptide moiety is directly linked to a half-life enhancing fusion partner or indirectly linked to a peptide linker and further conjugated to a polyalkylene glycol.

15. The method of embodiment 14, wherein the biologically active polypeptide moiety is capable of conferring the fusion protein an activity selected from the group consisting of hormones, cytokines, coagulation factors, enzymes, receptor extracellular regions, immunoregulatory factors, and leukocyte mediators. , interferon, tumor necrosis factor, transforming growth factor, growth factor, colony stimulating factor, chemokine, neuropeptide, insulin, GLP-1, GLP-1 receptor agonist, growth hormone, erythropoietin, G - CSF and GM-CSF.

16. The method according to embodiment 14 or 15, wherein the fusion partner is: an immunoglobulin Fc fragment, albumin, XTEN or transferrin, said fusion partner being for example derived from a human; preferably an IgG Fc fragment; The IgG Fc fragment has a reduced ADCC effect and/or CDC effect and/or enhanced binding affinity to the FcRn receptor; more preferably, the IgG Fc fragment has an amino acid sequence selected from the group consisting of:

(i) the amino acid sequence of SEQ ID NO: 3;

(ii) the amino acid sequence set forth in SEQ ID NO: 4; or

(iii) the amino acid sequence shown in SEQ ID NO: 5.

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 end-capped, for example And/or the polyalkylene glycol is linear or branched; preferably the polyalkylene glycol is branched, for example branched Polyethylene glycol, especially methoxy-terminated branched polyethylene glycol; the molecular weight of the polyalkylene glycol can be >=1, >=10, >=20, >=30, > =40, >=50, >=60, >=70, >=80, >=90, >=100, >=110, >=120, >=130, >=140, >=150 or >=160kDa For example, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 kDa, or a value between any two of the above values.

The method of 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 is selected from the group consisting of a free amino group, a thiol group , a glycosyl group and/or a carboxyl group, preferably a free amino group.

The method of any of embodiments 14 to 18, wherein the conjugation is achieved using a modifying agent, preferably the modifying agent may be a modified ester in the form of an activated ester and other types of modifying agents, more preferably The modifier is selected from the following formula (1), (2) or (3):

Wherein, 0≤m1≤6, m1 is preferably 5; mPEG- represents a mono-terminated polyethylene glycol group, and the molecular weight of the modifier represented by formula (1) is between 5KD and 60KD;

Wherein 0≤m2≤6, m2 is preferably 2; 0≤m3≤6, m3 is preferably 1; mPEG- represents a methoxy-terminated polyethylene glycol group, and the molecular weight of the modifier represented by formula (2) 5KD-100KD, preferably 40KD, 50KD, 60KD, most preferably 40KD;

Wherein, 0≤m4≤6, m4 is preferably 2; mPEG- represents a methoxy-mono-terminated polyethylene glycol group, and the modifier represented by formula (3) has a molecular weight of 5KD-100KD.

The method of any one of embodiments 14 to 19, wherein the biologically active polypeptide is linked to the fusion partner via a peptide linker comprising a flexible peptide linker and/or a rigid unit, for example Includes 1, 2, 3, 4, 5 or more of the rigid units.

The method of embodiment 20, wherein the flexible peptide linker comprises two or more amino acid residues selected from the group consisting of glycine, serine, alanine, and threonine,

Preferably, the flexible peptide linker has the sequence formula (GS) a(GGS)b(GGGS)c(GGGGS)d, wherein a, b, c and d are integers greater than or equal to 0, and a+b+ c+d≥1,

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).

The fusion protein according to embodiment 20 or 21, wherein the rigid unit is a carboxy terminal peptide of human chorionic gonadotropin β subunit, or the rigid unit and a carboxyl group of human chorionic gonadotropin β subunit The amino acid sequence of the terminal peptide has a consistency of 70%, 80%, 90%, 95% or higher; the rigid unit may comprise 1, 2 or more glycosylation sites;

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 set forth 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 description of the embodiments and the drawings used in the prior art will be briefly introduced. Obviously, the drawings in the following description are only Some embodiments of the application may also be used to obtain other figures from those of ordinary skill in the art without departing from the scope of the invention.

Figure 1a shows the results of SEC-HPLC of FVIII-Fc (FF-0) without mPEG modification.

Figure 1b shows the results of SEC-HPLC of 5K molecular weight mPEG modified FVIII-Fc (FF-5L).

Figure 1c shows the results of SEC-HPLC of 10K molecular weight mPEG modified FVIII-Fc (FF-10L).

Figure 1d shows the results of SEC-HPLC of 20K molecular weight mPEG modified FVIII-Fc (FF-20L).

Figure 1e shows the results of SEC-HPLC of 30K molecular weight mPEG modified FVIII-Fc (FF-30L).

Figure 1f shows the results of SEC-HPLC of 40K molecular weight mPEG modified FVIII-Fc (FF-40L).

Figure 2a shows the results of SEC-HPLC of the non-mPEG-modified FVIII-Linker1-Fc (FL1F-0) (purity >99%, polymer <1%).

Figure 2b shows the results of SEC-HPLC of 20K molecular weight mPEG modified FVIII-L1-Fc (FL1F-20L) (purity >95%, polymer <5%, uncrosslinked <1%).

Figure 2c is a SEC-HPLC assay of linear, 30K molecular weight mPEG modified FVIII-L1-Fc (FL1F-30L) (purity >95%, polymer <5%, uncrosslinked <1%).

Figure 2d is a SEC-HPLC assay of linear, 40K molecular weight mPEG modified FVIII-L1-Fc (FL1F-40L) (purity >95%, polymer <5%, uncrosslinked <1%).

Figure 2e is a SEC-HPLC assay of linear 50 g molecular weight mPEG modified FVIII-L1-Fc (FL1F-50L) (purity >95%, polymer <5%, uncrosslinked <1%).

Figure 2f shows the results of SEC-HPLC of Y--40K molecular weight mPEG modified FVIII-L1-Fc (FL1F-40Y) (purity >95%, polymer <5%, uncrosslinked <1%).

Figure 3a shows the results of SDS-PAGE before and after liquid exchange of hFVIII-Fc (FF-0) stock solution G25 without modification of mPEG (H stands for reduction and F stands for non-reduction).

Figure 3b shows the results of SDS-PAGE (non-reduction) of hFVIII-Fc cross-linking with different molecular weights of mPEG (FF-5L to FF-40L).

Figure 3c shows the results of SDS-PAGE detection (reduction) of hFVIII-Fc cross-linked with different molecular weights mPEG (FF-5L to FF-40L).

Detailed ways

In order to make the objects, technical solutions, and advantages of the present application more comprehensible, the present application will be further described in detail below with reference to the accompanying drawings. It is apparent that the described embodiments are only a part of the embodiments of the present application, and not all of them. All other embodiments obtained by a person of ordinary skill in the art based on the embodiments of the present application without departing from the inventive scope are the scope of the present application.

The term "polypeptide" refers to a compound formed by dehydration condensation of a plurality of amino acid molecules. In the present application, "polypeptide" has the same meaning as "protein", "protein" or "peptide" and can be used interchangeably;

The term "biologically active polypeptide" refers to a protein that is capable of exerting an effect on a particular physiological or pathological process of an organism, including: growth, development, apoptosis, death, catalysis, angiogenesis, pathology, tumorigenesis, Metastasis, signal transduction, 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, immunoregulatory factors, interleukins, interferons , tumor necrosis factor, transforming growth factor, growth factor, colony stimulating factor, chemokine, neuropeptide, insulin, GLP-1, GLP-1 receptor agonist, growth hormone, 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 obtained by directly or two or more genes which independently encode different proteins, and which are obtained by translation and translation. A single protein of the functional properties of an original protein.

The term "fusion partner" refers to another polypeptide that is fused to a polypeptide of interest (ie, a polypeptide that is desired to extend its half-life) and an effect-enhancing variant thereof that is capable of altering the half-life of the fusion protein by a variety of different mechanisms.

In one embodiment, the fusion partner delays in vivo clearance of the polypeptide of interest by interacting with a 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, the IgG Fc domain is preferred due to the longer half-life of the IgG antibody.

The Fc domain may also be modified to improve other functions, such as complement binding and/or binding to certain Fc receptors. Mutations at positions 234, 235 and 237 in the IgG Fc domain will generally result in reduced binding to the FcγRI receptor. It may also result in reduced binding to the FcγRIIa and FcγRIII receptors. These mutations do not alter binding to the FcRn receptor, which promotes long circulating half-life through the endocytic recycling pathway. Preferably, the modified IgG Fc domain of the fusion protein of the invention comprises one or more of the following mutations which will result in decreased affinity for certain Fc receptors (L234A, L235E and G237A) and C1q-mediated, respectively. The guided complement binding is reduced (A330S and P331S).

The term "polyalkylene glycol" is a hydrophilic polymer which is conjugated to a specific position on a biologically active polypeptide and/or fusion partner in the present invention, and the polyalkylene glycol may be linear or Branched and may comprise one or more independently selected polymeric moieties. Preferably, the polyalkylene glycol is polyethylene glycol including m-PEG, polypropylene glycol including m-PPG, and the like.

The polyalkylene glycol in 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. Typically, a branched polymer has a central branched core portion and one or more linear polymer chains attached to the central branched core. It is preferred in the present invention to use PEG in branched form. In one example, the branched polyethylene glycol can be represented by the formula R(-PEG-OH)m, wherein R represents a core moiety, such as glycerol or pentaerythritol, and m represents the number of arms.

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, ie, a branched PEG comprising two PEG or linear methoxy PEG .

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, etc.), polyoxyethylated polyols, olefmic alcohols , polyvinylpyrrolidone, polyhydroxypropylmethacrylamide, poly([α]-hydroxy acid), polyvinyl alcohol, polyphosphazene, polyoxazoline, poly-N-acryloylmorpholine and copolymers thereof, Meta-copolymers and mixtures.

In one embodiment of the present application, PEG modification is used, more preferably mPEG modification, wherein the modification is a random modification or a site-directed modification, the position of the modification comprising a free amino group, a thiol group, a glycosyl group, and/or a carboxyl group.

In a specific embodiment of the present application, the modifier for the random modification of the free amino group of mPEG may be selected from the group consisting of: mPEG-SS (methoxypolyethylene glycol-succinimide succinate), mPEG-SC (methoxypolyethylene glycol-succinimide carbonate), mPEG-SPA (methoxypolyethylene glycol-succinimidyl propionate) and mPEG-SG (methoxypolyethylene glycol- Succinimide glutarate) and the like. The N-terminal modifiers are: mPEG-ALD (methoxy polyethylene glycol-acetaldehyde), mPEG-pALD (methoxy polyethylene glycol-propionaldehyde) and mPEG-bALD (methoxy polyethylene glycol) -butyraldehyde) and the like. The modifiers mPEG-SS, mPEG-SC, mPEG-SPA, mPEG-SG, mPEG-ALD, mPEG-pALD, mPEG-bALD are linear or branched in shape.

In a specific embodiment of the present application, the modifier used in the random thiol-based modification is mPEG-mal (methoxypolyethylene glycol-maleimide), mPEG-OPSS (methoxypolyethylene) One of alcohol-o-dithiopyridine), mPEG-Vinylsulfone (methoxypolyethylene glycol-vinylsulfone), and mPEG-Thiol (methoxypolyethylene glycol-thiol).

In a specific embodiment of the present application, the modifier used in the random modification of the glycosyl and/or carboxyl groups is mPEG-ZH (methoxypolyethylene glycol-hydrazide).

In one embodiment of the present application, the structure of the mPEG-modified modifier is as shown in formula (1):

Wherein, 0≤m1≤6, m1 is preferably 5; mPEG- represents a mono-terminated polyethylene glycol group, and the modifier represented by formula (1) has a molecular weight of 5 kD-60 kD (kD, thousand Daoer) Preferably, it is 40 kD. Preferably, in one embodiment of the present application, the mPEG random modification of the free amino group is carried out using the modifying agent represented by the formula (1).

In one embodiment of the present application, the structure of the mPEG-modified modifier is as shown in formula (2):

Wherein 0≤m2≤6, m2 is preferably 2; 0≤m3≤6, m3 is preferably 1; mPEG- represents a methoxy-terminated polyethylene glycol group, and the molecular weight of the modifier represented by formula (2) It is 5 kD to 60 kD, preferably 40 kD. Preferably, in one embodiment of the present application, the mPEG random modification of the free amino group is carried out using the modifier shown in formula (2).

In one embodiment of the present application, the structure of the mPEG-modified modifier is as shown in formula (3):

Wherein, 0 ≤ m4 ≤ 6, m4 is preferably 2; mPEG- represents a methoxy-mono-terminated polyethylene glycol group, and the modifier represented by the formula (3) has a molecular weight of 5 kD to 60 kD, preferably 40 kD. In one embodiment of the present application, a free thiol-based mPEG random modification is carried out using a modifying agent represented by formula (3).

The size of the polymer backbone can vary, but polymers (e.g., PEG, mPEG, PPG, or mPPG) typically range from about 0.5 KD to about 160 KD, such as from about 1 KD to about 100 KD. More specifically, the size of each of the conjugated hydrophilic polymers of the present invention varies primarily in the range of from about 1 KD to about 80 KD, from about 2 KD to about 70 KD; from about 5 KD to about 70 KD; from about 10 KD to about 60 KD, about 20KD to about 50KD; about 30KD to about 50KD or about 30KD-40KD. It should be understood that these sizes represent approximate values and are not accurate measurements, which is recognized in the art.

In a specific embodiment, the size of the PEG or mPEG used in the present invention is 35 KD or more (ie, not less than 35 KD), preferably not less than 40 KD, not less than 45 KD, not less than 50 KD, not less than 55 KD, not less than 60 KD, Not less than 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 "modified circulating half-life": The molecule of the invention has an altered circulating half-life, preferably an increased circulating half-life, as compared to a wild-type factor biologically active polypeptide. The cyclic half-life is preferably increased by at least 10%, preferably at least 15%, preferably at least 20%, preferably at least 25%, preferably at least 30%, preferably at least 35%, preferably at least 40%, preferably at least 45%, preferably at least 50%, preferably at least 55%, preferably at least 60%, preferably at least 65%, preferably at least 70%, preferably at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 100%, more preferably At least 125%, more preferably at least 150%, more preferably at least 175%, more preferably at least 200%, and most preferably at least 250% or 300%. Even more preferably, the molecule has an increase in circulating half-life of at least 400%, 500%, 600%, or even 700%.

The term "pharmaceutically acceptable carrier" includes, but is not limited to, saline, buffer, dextrose, water, glycerol, ethanol, and combinations thereof. In general, the pharmaceutical preparation should be matched to the mode of administration, and the pharmaceutical composition of the present application can be prepared in the form of an injection, for example, by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants. The pharmaceutical composition is preferably manufactured under sterile conditions. The amount of active ingredient administered is a therapeutically effective amount. The pharmaceutical preparation of the present application can also be formulated into a sustained release preparation.

Example

Example 1 Preparation and Purification of mPEG Modified hFVIII Fusion Protein

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, respectively (see U.S. Patent No. 4,818,679), to express each hFVIII. Fusion protein (Table 1). The specific preparation procedure for the fusion protein is described in Chinese Patent No. ZL201610692838.0, which is incorporated herein in its entirety by reference.

Table 1 Composition and structure of the fusion protein

Note: *B domain deleted hFVIII is abbreviated as BDD FVIII, consisting of 90kD A1-A2 heavy chain and 80kD light chain;

**SEQ ID NO. 7 - SEQ ID NO. 12 indicates that the adaptor is ligated from the rigid unit set forth in SEQ ID NO. 12 to the C-terminus of the flexible peptide linker set forth in SEQ ID NO. 7; SEQ ID NO. - SEQ ID NO. 11 shows that the linker is formed by the rigid unit shown by SEQ ID NO. 11 linked to the C-terminus of the flexible peptide linker shown by SEQ ID NO.

1.1.2. The fermentation broth of each 1.1.1 fusion protein is subjected to centrifugation and filtration, followed by Affinity chromatography/Hydrophobic Interaction Chromatography/Ion-Exchange chromatography. / Size exclusion chromatography, respectively obtained five hFVIII fusion proteins FF-0, FL1F-0, FL2F-0, F (full length) L1F'-0 and F (full length) L2F" -0, SEC-HPLC detected the polymer <5%. The five hFVIII fusion proteins were separately prepared as a protein concentration of 0.95mg / ml hFVIII fusion protein stock;

1.1.3. Take 5 ml of each of the five hFVIII fusion protein stocks prepared in 1.1.2, and perform G25 (GE Healthcare) molecular sieve chromatography. Specific steps are as follows:

Buffer preparation: 20 mM Hepes, 0.1 M NaCl, 5.0 mM CaCl 2 , 0.02% Tween 8.0, pH 7.0;

Chromatography process:

(1) Balance: equilibrate the column with 3 column volumes of Binding Buffer, equilibrate to pH and the conductance is consistent with the buffer, and the flow rate is uniform to 150 cm/h;

(2) Loading: The loading flow rate is unified to 150cm/h;

(3) Balance: equilibrate the column with 3 column volumes of buffer, balance to pH and conductance is consistent with the buffer, the flow rate is uniform 150cm / h;

(4) Balance: equilibrate the column with a buffer to collect a peak of A280/A260 greater than 1.8;

(5) In-situ cleaning of the chromatography column: reverse-cleaning 1.5 column volumes with 0.2 M NaOH at a flow rate of 60 cm/h, and neutralizing with a buffer;

(6) Preservation of the column: After the end of the experiment, the column was washed with 3 column volumes of purified water at a flow rate of 100 cm/h, and then the column was stored with 2 column volumes of 20% ethanol.

Ultrafiltration Concentration: Five hFVIII fusion proteins (FF-0, FL1F-0, FL2F-0, F (full length) L1F'-0 and F (full length) L1F"-0) stock solution after G25 exchange It was concentrated by ultrafiltration using a 50 kD ultrafiltration tube, concentrated by centrifugation at 4 ° C, 3800 rpm, and concentrated to a protein concentration of preferably 1.5 mg/ml.

1.1.4, weigh mPEG-SC according to the ratio of hFVIII fusion protein and mPEG-SC (purchased from Beijing Keykai Technology Co., Ltd.) molar ratio (1:1):100 (molecular weight 5kD, 10kD respectively) , 20kD, 30kD, 40kD structure such as the linear L-shaped mPEG-SC shown in formula (1), and the molecular weight of 40kD structure as shown in formula (2), branched-chain Y-shaped mPEG-SC), added 1.1. 3 ultrafiltration concentrated hFVIII fusion protein, reaction for 4 hours, plus the substrate hFVIII fusion protein 10-fold molar (mol) ratio of histidine to terminate the reaction, to obtain mPEG-SC modified hFVIII fusion protein of different molecular weight; partial modification The product numbers and their composition are shown in Table 2 below.

Table 2

Purification of 1.2 mPEG-modified hFVIII fusion protein

1.2.1. Each mPEG-modified hFVIII fusion protein prepared in Example 1 "1.1.4" was subjected to S200 (GE Healthcare) molecular sieve chromatography. details as follows:

Buffer preparation: 20 mM histidine, 0.1 M NaCl, 5.0 mM CaCl ₂ , 0.02% Tween 8.0, pH 7.0;

Chromatography process:

(1) Balance: equilibrate the column with 3 column volumes of Binding Buffer, equilibrate to pH and the conductance is consistent with the buffer, and the flow rate is uniform to 150 cm/h;

(2) Loading: The loading flow rate is unified to 150cm/h;

(3) Balance: equilibrate the column with 3 column volumes of buffer, balance to pH and conductance is consistent with the buffer, the flow rate is uniform 150cm / h;

(4) Balance: equilibrate the column with a buffer to collect a peak of A280/A260 greater than 1.8;

(5) In-situ cleaning of the chromatography column: reverse-cleaning 1.5 column volumes with 0.2 M NaOH at a flow rate of 60 cm/h, and neutralizing with a buffer;

(6) Preservation of the column: After the end of the experiment, the column was washed with 3 column volumes of purified water at a flow rate of 100 cm/h, and then the column was stored with 2 column volumes of 20% ethanol.

1.2.2. Perform the chromatographic products obtained in “1.2.1” for Source 15Q (GE Healthcare) anion chromatography:

Buffer preparation:

Binding buffer: 20 mM histidine, 0.1 M NaCl, 5.0 mM CaCl ₂ , 0.02% Tween 8.0, pH 7.0; elution buffer: 20 mM histidine, 2.0 M NaCl, 5.0 mM CaCl ₂ , 0.02% Tween 8. 0, pH 7.0; CIP: 0.5 M NaOH;

Chromatography process:

(1) Balance: equilibrate the column with 3 times column volume of binding buffer, balance to pH and conductance is consistent with buffer, the flow rate is uniform 150cm / h;

(2) Loading: The loading flow rate is unified to 150cm/h;

(3) Balance: equilibrate the column with 3 times column volume of binding buffer, balance to pH and conductance is consistent with the buffer, the flow rate is unified to 150cm / h;

(4) Elution: The sample was eluted according to a linear gradient of 0-100% buffer B 20 times. The elution flow rate was 100 cm/h, and the elution peaks of A280/A260 greater than 1.8 were collected in tubes. The samples were all tested by SEC-HPLC;

(5) In-situ cleaning of the column: 1.5 column volume was reverse-washed with 0.5 M NaOH at a flow rate of 60 cm/h, and neutralized with a binding buffer;

(6) Preservation of the column: After the end of the experiment, the column was washed with 3 column volumes of purified water at a flow rate of 100 cm/h, and then the column was stored with 2 column volumes of 20% ethanol.

1.2.3, SEC-HPLC detection:

The chromatographic products obtained in "1.2.2" were subjected to SEC-HPLC, wherein:

Column: G3000/G4000; Flow rate: 0.5 mL/min; Detection wavelength: 280 nm; Column temperature: 25 ° C; Injection volume: 100 μL (injection amount 20 μg); Mobile phase: 0.30 M sodium chloride; 0.02 M imidazole; 0.01M calcium chloride; 25ppm Tween 80; 10% ethanol; pH 7.0; running time: 35-50min;

The test results of FF-0 to FF-40L are shown in Figures 1a-1f. The results of FL1F-0 to FL1F-50L and FL1F-40Y are shown in Figures 2a-2f. The results show that the purity of FL1F-0 to FL1F-60L is >95%, the polymer is <5%, and the uncrosslinked <1%.

1.2.4, SDS-PAGE for protein gel electrophoresis detection:

The product obtained in the item "1.2.2" is subjected to SDS-PAGE detection, and includes the following steps:

(1) Glue: 1×Tris-glycine electrophoresis buffer: SDS 0.4 g, Tris base 1.21 g, glycine 7.5 g, and double distilled water to 400 mL.

5% concentrated gel: double distilled water 4.1 mL, 1 M Tris-HCl (pH 6.8) 0.75 mL, 30% (w/v) polyacrylamide 1 mL, 10% (w/v) ammonium persulfate 60 μL, 10% (w /v) SDS 60 μL, TEMED 6 μL.

6% separation gel: 4.9 mL of double distilled water, 3.8 mL of 1.5 M Tris-HCl (pH 8.8), 6 mL of 30% (w/v) polyacrylamide, 10% (w/v) ammonium persulfate 150 μL, 10% ( w/v) SDS 150 μL, TEMED 6 μL.

(2) 5× protein loading buffer: glycerol 5 mL, 1 M Tris-HCl (pH 6.8) 2.5 mL, bromophenol blue 0.05 g, SDS 1 g, double distilled water to a volume of 10 mL, stored at 4 ° C, added β before use - mercaptoethanol 0.5 mL.

(3) Sample preparation: the sample to be tested is mixed with an equal volume of loading buffer, and an equal volume of 0.1 mg/mL of 2-mercaptoethanol is added to the sample by reduction SDS-PAGE; non-reducing SDS-PAGE is not added 2- Mercaptoethanol. After the sample was mixed with the loading buffer, it was bathed in boiling water for 10 minutes.

(4) Electrophoresis: 10 μl of the sample to be tested and the protein Marker were sequentially added to the spotting hole, and concentrated electrophoresis was carried out at a voltage of 60 V. It was observed that the bromophenol blue dye was concentrated to the separation gel and the voltage was increased to 120 V. Separate and electrophoresis until the bromophenol blue dye reaches the bottom of the separation gel and turn off the power.

(5) Staining: The SDS-PAGE gel was carefully taken out and placed in a plastic box containing Coomassie Brilliant Blue staining solution, and placed in a microwave oven for 1 minute.

(6) Decolorization: The dyed SDS-PAGE gel was taken out and placed in a decolorizing solution, and the mixture was shaken and decolorized. The decolorizing solution was replaced once every 2 hours, and it was observed that a clear band was visible to the naked eye.

(7) Recording: The completed SDS-PAGE gel recording was recorded or dried. Figures 3a-3c show the results of SDS-PAGE detection of FF-5L to FF-40L.

Example 2 Indirect determination of in vitro activity of mPEG-modified hFVIII fusion protein by chromogenic substrate method

The activity of the mPEG-modified hFVIII fusion protein prepared in Example 1 was determined using a chromogenic substrate assay. The Chromogenix Coatest SP FVIII kit (Chromogenix, Ref. K824086) was used to determine the principle: when activated by thrombin, FVIIIa binds to FIXa in the presence of phospholipids and calcium ions to form an enzyme complex, which in turn activates factor X transformation. Into its active form Xa. Activation of the formed factor Xa can then cleave its specific chromogenic substrate (S-2765), releasing the chromophoric group pNA. By measuring the amount of pNA produced at 405 nm, the activity of FXa directly proportional to its amount can be known. The content of factor IXa and factor X in the system is constant and excessive, and the activity of FXa is only directly related to the content of FVIIIa. Related. The indirect determination of the biological activity of FVIII by the chromogenic substrate method is shown in Table 3.

Table 3 chromogenic substrate method for indirect determination of FVIII biological activity

Note: Eloctate is a recombinant Factor VIII Fc fusion protein already marketed by Bioverativ, which has not been modified by mPEG.

Example 3 Human Coagulation Factor VIII Titer Assay

The human coagulation factor VIII titer used in the present invention is also referred to as the first-stage method. For specific steps, see the Chinese Pharmacopoeia 2010 edition. One-stage assay for FVIII biological activity was performed by correcting the ability of FVIII-deficient plasma to cause prolonged clotting time. A kit Coagulation Factor VIII Deficient Plasma (Cat. No. OTXW17) manufactured by the German company Siemens was used. The method comprises: first, diluting a known potency FVIII activity standard, WHO International Standard 8th International Standard Factor VIII Concentrate (Cat. No. 07/350) to 4 IU/ml, and then performing a gradient dilution to different titers (IU/ Ml) and mixed with spent FVIII matrix plasma to determine partial thromboplastin time (APTT), the logarithm of FVIII activity standard solution titer (IU/ml) corresponding to the logarithm of its corresponding clotting time (s) Linear regression, establishing a standard curve. The sample to be tested is then diluted moderately and mixed with the spent FVIII matrix plasma for APTT assay. By substituting the standard curve, the titer of the sample to be tested FVIII can be known, and accordingly, the specific activity of the sample to be tested FVIII can be calculated, and the unit is IU/mg. The results are shown in Table 4.

Table 4 Direct Determination of Biological Activity by Phase I Method

Shown in Tables 3 and 4: FL1F-40Y, FL2F-40Y, F (full length) L1F'-40Y and F (full length) L2F"-40Y organisms measured by the chromogenic substrate method and the one-stage method The activity was lower than that of the Eloctate/FL1F-0/FL2F-0 experimental group without cross-linking mPEG, which was caused by the influence of mPEG modification on the spatial structure of the modified protein. Other mPEG-modified proteins in the prior art ( A similar phenomenon is observed, for example, in PEG-INTRON; Pegfilgrastim. Surprisingly, the fusion protein of the present application maintains relatively high activity after modification with mPEG of 40 kD or higher, and subsequent experiments further verify Its half-life is greatly extended.

Example 4 Prophylactic pharmacodynamics experiment on cerebral vein transection model of hemophilia A mice

In this example, the activity half-life of each mPEG-modified hFVIII fusion protein in hemophilia A mice (HA mice) was compared by tail vein transection (TVT) experiments.

4.1. According to the method reported in the literature, 10-12 weeks old male HA mice (purchased from Shanghai Southern Model Biological Research Center) were randomly divided into 12 groups/groups, and the dose was 15 IU/kg, respectively. The mPEG-modified fusion protein or the positive control drug Eloctate was administered via the tail vein. 48 hours after the administration, the tail was taken and marked with a 2.7 mm inner diameter cannula, and the unilateral tail vein was cross-cut with a No. 11 straight-blade surgical blade. After cross-cutting, the rat tail was quickly placed into a pre-filled with about 13 ml. Heat the saline tube and record the bleeding time. After stopping bleeding (no significant blood outflow at the incision), the rat tail was removed from the saline tube, and the mouse was placed on a 37 ° C heating pad to maintain its body temperature without touching the wound. After the mice were awakened, they were placed in a rat cage padded with A4 white paper, kept in a single cage, and replaced with white paper or a mouse cage after each observation to determine the degree of bleeding. After the tail was counted, the survival rate of the mice within 48 hours and the number of rebleeding within 12 hours after the tailings were counted (12 hours in total, and the number of bleedings within one hour was counted once). The results are shown in Table 5.

The rate of re-bleeding rate is the proportion of mice with re-bleeding during the statistical period. The severe bleeding rate is the phenomenon of severe bleeding (+++) or multiple moderate hemorrhage (++) in the re-bleeding statistics within 12. The proportion of mice. Among them, moderate bleeding (++) means: A4 white paper has a lot of blood, covering an area of not less than 30%, and the blood mark is medium in color, but there is no large area of blood beach (area > 3cm2); severe bleeding (++ +) means: A4 white paper has a lot of blood on the surface, the coverage area is not less than 30%, the blood mark is heavy, and there is a large area of blood beach; even if the coverage area is less than small, it can be regarded as severe bleeding (large blood loss in mice) The range of activity is reduced, and the blood is heavily wetted with white paper).

Table 5 48h survival rate and 12h complex bleeding rate of TVT 48h after administration

Fusion protein name	12h recurrent bleeding rate	Severe bleeding rate	48h survival rate
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 showed that 83.3% survival rate of FL1F-40Y experimental group, 75.0% survival rate of FL2F-40Y experimental group, F (full length) compared with the experimental group of Eloctate/FL1F-0/FL2F-0 without mPEG modification The 83.3% survival rate of the L1F'-40Y experimental group and the 83.3% survival rate of the F (full length) L2F"-40Y experimental group were significantly higher than those of the other groups. The corresponding 12h recurrence rate and severe bleeding rate of these experimental groups were higher than those of the other groups. There was a significant decrease in the group. The preventive efficacy of FL1F-40Y, FL2F-40Y, F (full length) L1F'-40Y, F (full length) L2F"-40Y in the cerebral vein transection model of hemophilia A mice Learn to have longer protection time.

4.2. In the same manner as in 4.1, 10T/week male HA mice, 12/group, were subjected to TVT test at 84 h after administration, and the results are shown in Table 6.

Table 6 48h survival rate and 12h recurrent bleeding rate of TVT after 84h administration

Fusion protein name	48h survival rate	12h recurrent bleeding rate
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 showed that 66.7% of the survival rate of the FL1F-40Y experimental group, 75.0% of the survival rate of the FL2F-40Y experimental group, and the F (full length) L1F'-40Y experimental group compared with the Eloctate without the cross-linked PEG and other experimental groups. 75.0% survival rate and F (full length) L2F"-40Y experimental group 75.0% survival rate were significantly higher than other groups, the corresponding complex bleeding rate was significantly reduced. FL1F-40Y, FL2F-40Y, F (full length) L1F '-40Y, F (full length) L2F"-40Y had certain advantages in the prophylactic pharmacodynamics of the hemophilia A mouse tail vein transection model 84 h after administration.

4.3. Using the same method as 4.1, 10-12 weeks old HA mice, male and female, 10, 20/group, TVT experiments were performed 90 hours after administration, and the results are shown in Table 7.

Table 7 48h survival rate and 12h recurrent bleeding rate of TVT at 90h after administration

Fusion protein name	48h survival rate	12h recurrent bleeding rate
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 showed that FL1F-50L and the unconjugated PEG Eloctate experimental group had similar survival rate and re-bleeding rate; because the experimental animals were not single sex, the experimental results were not compared with other single-sex experimental results.

4.4. In the same manner as in 4.1, 10T/week male HA mice, 12/group, were subjected to TVT test 96 h after administration, and the results are shown in Table 8.

Table 8 48h survival rate and 12h complex bleeding rate of TVT at 96h after administration

Fusion protein name	48h survival rate	12h recurrent bleeding rate
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 showed that: FL1F-40Y/FL2F-40Y compared with the Eloctate experimental group without cross-linked PEG, although the survival rate was small, the 12h re-bleed rate was significantly reduced. The survival rate of FL1F-40Y/FL2F-40Y was significantly higher than that of the other groups; the 12h recurrence rate was significantly lower than the other groups. FL1F-40Y/FL2F-40Y has a longer prophylaxis time than the other groups in the hemophilia A mouse tail vein cross-cut model.

The above is only the preferred embodiment of the present application, and is not intended to limit the present application. Any modifications, equivalent substitutions, improvements, etc., which are made within the spirit and principles of the present application, should be included in the present application. Within the scope of protection.

Claims (11)

1. A biologically active polypeptide fusion protein conjugated to a polyalkylene glycol, wherein the biologically active polypeptide moiety is directly linked to a half-life enhancing fusion partner or indirectly linked by a peptide linker, and the fusion protein is further associated with a poly The alkyl diol is conjugated.
2. The fusion protein according to claim 1, wherein the biologically active polypeptide moiety confers the fusion protein a biological activity selected from the group consisting of hormones, cytokines, coagulation factors, enzymes, receptor extracellular regions, immunoregulatory factors, Interleukins, interferons, tumor necrosis factor, transforming growth factor, growth factor, colony stimulating factor, chemokine, neuropeptide, insulin, GLP-1, GLP-1 receptor agonist, growth hormone, erythropoietin , G-CSF and GM-CSF.
3. The fusion protein according to claim 1 or 2, wherein the fusion partner is: an immunoglobulin Fc fragment, albumin, XTEN or transferrin, the fusion partner is for example derived from a human; preferably an IgG Fc fragment; The IgG Fc fragment has a reduced ADCC effect and/or CDC effect and/or enhanced binding affinity to the FcRn receptor; more preferably, the IgG Fc fragment has an amino acid sequence selected from the group consisting of:

   (i) the amino acid sequence of SEQ ID NO: 3;

   (ii) the amino acid sequence set forth in SEQ ID NO: 4; or

   (iii) the amino acid sequence shown in 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, via an alkane An oxy group such as a methoxy group; and/or the polyalkylene diol is linear or branched; preferably the polyalkylene diol is branched, for example, branched Ethylene glycol, especially a methoxy-terminated branched polyethylene glycol; the molecular weight of the polyalkylene glycol can be >=1, >=10, >=20, >=30, >=40 , >=50, >=60, >=70, >=80, >=90, >=100, >=110, >=120, >=130, >=140, >=150 or >=160kDa, for example Is 5, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 kDa, or a value between any two of the above values.
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, and the conjugation position is selected from the group consisting of a free amino group, a thiol group, and a sugar. Base and/or carboxyl group, preferably free amino group.
6. The fusion protein according to claim 5, wherein the conjugation is effected using any suitable modifying agent, which may be a modified ester or other principle type modifying agent, for example, the modifying agent is selected from the following A modifier represented by formula (1), (2) or (3):

   

   Wherein, 0≤m1≤6, m1 is preferably 5; mPEG represents a mono-terminated polyethylene glycol group;

   

   Wherein 0≤m2≤6, m2 is preferably 2; 0≤m3≤6, m3 is preferably 1; mPEG represents a mono-terminated polyethylene glycol group; or

   

   Wherein, 0 ≤ m4 ≤ 6, and m4 is preferably 2; mPEG represents a methoxy-mono-terminated polyethylene glycol group.
7. The fusion protein according to any one of claims 1 to 6, wherein the biologically active polypeptide is linked to the fusion partner via a peptide linker comprising a flexible peptide linker and/or a rigid unit, for example, which may comprise 1, 2, 3, 4, 5 or more of said rigid units.
8. The fusion protein according to claim 7, wherein said flexible peptide linker comprises two or more amino acid residues selected from the group consisting of glycine, serine, alanine and threonine,

   Preferably, the flexible peptide linker has the sequence formula (GS) a(GGS)b(GGGS)c(GGGGS)d, wherein a, b, c and d are integers greater than or equal to 0, and a+b+ c+d≥1,

   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 according to claim 7 or 8, wherein the rigid unit is a carboxy terminal peptide of human chorionic gonadotropin β subunit, or a carboxy terminal peptide of the rigid unit and human chorionic gonadotropin β subunit The amino acid sequence has a consistency of 70%, 80%, 90%, 95% or higher; 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 set forth in SEQ ID NO: 15.
10. A pharmaceutical composition comprising an effective amount of the fusion protein of any one of claims 1 to 9, and a pharmaceutically acceptable carrier.
11. A method of preventing and/or treating a disease which can be prevented and/or treated by the activity of a biologically active polypeptide, which comprises administering the fusion protein of any one of claims 1 to 9 or claim 10 to a subject in need thereof. Said pharmaceutical composition.

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