Protein analogues and their applications

Abstract

A modified protein comprises a protein moiety and a modified moiety, wherein the protein moiety has a PAS optionally linked to its N-terminus, and the modified moiety is linked directly or via a linker to a cysteine ​​residue in the protein moiety or to a cysteine ​​residue in the optionally chosen PAS, the cysteine ​​residue being either naturally occurring or introduced by mutation. The modified protein not only maintains its original activity but also significantly improves its in vivo half-life. The preparation process for the modified protein is simple and is applicable to the development of a range of therapeutic agents for conditions such as non-alcoholic fatty liver disease, type 2 diabetes, hyperlipidemia, and atherosclerosis.

Description

[Technical Field]

[0001] This invention relates to the biomedical field. Specifically, it relates to modified proteins, particularly proteins modified with fatty acids, and their uses in the treatment of diseases. [Background technology]

[0002] Non-alcoholic steatohepatitis (NASH) is an inflammatory subtype of non-alcoholic fatty liver disease (NAFLD) characterized by inflammation and fibrosis of liver cells, and in some patients, it can progress to cirrhosis and liver cancer. Epidemiological studies indicate that 3–6% of the US adult population suffers from NASH, and it is one of the leading reasons for liver transplantation. In China, an estimated 30 million people are reported to have NASH. NASH is a chronic disease with diverse etiologies and complex pathologies, and is associated with multiple metabolic complications, making treatment complex. There are no NASH treatments that have undergone rigorous randomized controlled clinical trials.

[0003] Fibroblast growth factor 21 (FGF21) is a stress-induced hormone that regulates energy balance and glucose / lipid metabolism, and belongs to the endocrine type of FGFs. Preclinical studies have demonstrated that FGF21 has physiological and pharmacological activities such as weight loss, improvement of blood lipid abnormalities, increased insulin sensitivity, and reduction of hepatic steatosis and fibrosis, showing great potential in the treatment of NASH.

[0004] FGF21 has potential therapeutic effects against NASH, but its short half-life, low stability, and the need for frequent injections significantly impact patient compliance. Therefore, biopharmaceutical companies have employed various technological means to improve its drug potential. LY-2405319, developed by Lilly, enhances its stability by cleaving the N-terminal HPIP sequence and introducing a cysteine ​​mutation (L118C, A134C) to form an additional disulfide bond, and eliminates O-glycosylation generated by yeast expression by introducing the S167A mutation. BMS-986036 (pegbelfermin), a long-acting FGF21 once-weekly subcutaneous injection developed by Bristol-Myers Squibb, is obtained by inserting a non-natural amino acid pAcF (Q108pAcF) and modifying it with PEG containing an alkoxy-amino group via a bioorthogonal reaction, thereby extending the half-life and increasing solubility and stability. AKR-001, developed by Amgen, aims to reduce aggregation, decrease in vivo enzymatic degradation, and extend the half-life of human FGF21 by introducing three point mutations (L98R, P171G, and A180E) into human FGF21 and fusing and expressing it with an IgG1 Fc fragment. Pfizer modifies FGF21 in two ways. The first is to link FGF21 to a specific site (PF-05231023) of the framework antibody CVX-2000 Fab, thereby conferring pharmacokinetic properties similar to the antibody. The second is to mutate CHO cells to express the potential glycosylation site of FGF21 and fuse it with the Fc fragment (PF-06645849).

[0005] However, these methods for modifying FGF21 analogs still have many problems, such as the toxicity of PEG due to its failure to be metabolized in the body, the stability of Fc fusion proteins, and glycosylation issues. In recent years, in addition to PEG modification and Fc fusion technologies, various technologies have been used to modify polypeptides and protein drugs to have longer-acting effects, such as fusion with human serum albumin, fatty acid modification, and modification with PEG-like repeat polypeptide sequences. Human serum albumin (HSA) is the most abundant protein in human plasma, with a molecular weight of approximately 67 kDa, and its half-life in human plasma reaches 19 days. Similar to antibody Fc segments, HAS has a long half-life by evading the lysosomal degradation pathway by binding to the neonatal receptor (FcRn) in a pH-dependent manner. Therefore, fusion and expression with HAS is an ideal means for long-acting biopharmaceuticals; for example, the in vivo half-life of albiglutide reaches 4-7 days. However, due to the large molecular weight of HSA, fusion and expression with functional polypeptides or proteins can easily reduce biological activity and limit the in vivo distribution of the drug. As the shortcomings of PEG modification become increasingly apparent, various technologies that perform similar functions to PEG are emerging as alternative means to optimize the pharmacokinetic properties of biopharmaceuticals. German company XL Protein Biotechnology has discovered that a polypeptide chain consisting of three amino acids, Pro, Ala, and Ser (PAS), has similar properties to PEG, such as high hydrophilicity and high water solubility. Furthermore, fusion and expression with growth hormone, interferon, and Fab fragments can significantly improve in vivo activity and half-life. Compared to methods that require in vitro modifications such as PEG modification, PAS modification has the advantage of being fused and expressed in E. coli. [Overview of the Initiative] [Problems that the invention aims to solve]

[0006] The object of the present invention is to provide an active protein drug, such as a mutant of FGF21, which not only maintains its original activity but can also have an improved half-life. [Means for solving the problem]

[0007] In a first embodiment, the present invention provides a modified protein comprising a protein moiety and a modified moiety, wherein the protein moiety is optionally linked to the N-terminus of a PAS, and the modified moiety is linked directly or via a linker to a cysteine ​​residue in the protein moiety or to a cysteine ​​residue in the PAS, wherein the cysteine ​​residue is either naturally occurring or introduced by mutation.

[0008] In a preferred embodiment, the mutation is a substitution or insertion, preferably a substitution. In a preferred embodiment, the protein is a cell growth factor, growth hormone, interferon, enzyme, antibody, or an active fragment thereof.

[0009] In a preferred embodiment, the cell growth factor is epidermal fibroblast growth factor, platelet-derived fibroblast growth factor, fibroblast growth factor, insulin-like fibroblast growth factor, neurofibroblast growth factor, interleukin-like fibroblast growth factor, erythropoietin, or colony-stimulating factor. In a specific embodiment, the fibroblast growth factor is fibroblast growth factor 21.

[0010] In a preferred embodiment, the amino acid sequence of the fibroblast growth factor 21 is as shown in SEQ ID NO: 1 or 2. In a preferred embodiment, the amino acid sequence of the fibroblast growth factor 21 is as shown in SEQ ID NO:1 or 2, and a cysteine ​​residue is introduced therein by mutation. In a preferred embodiment, the amino acid sequence of the fibroblast growth factor 21 is as shown in SEQ ID NO:1 or 2, and the Q28, E30, D38, D46, K122, or H125 mutations are cysteine ​​residues.

[0011] In a preferred embodiment, the amino acid sequence of the fibroblast growth factor 21 is as shown in any of SEQ ID NO:4~9. In a preferred embodiment, the amino acid sequence of the fibroblast growth factor 21 is as shown in any of SEQ ID NO: 5 to 9.

[0012] In a preferred embodiment, the modified portion can extend the in vivo half-life of the protein portion. In a preferred embodiment, the modified portion can bind specifically or nonspecifically to proteins in plasma.

[0013] In a preferred embodiment, the protein in the plasma has a long half-life, for example, the half-life of the protein in the plasma is 10 days or more, preferably 15 days or more, and more preferably 19 days or more. In a preferred embodiment, the protein in the plasma is human serum albumin.

[0014] In a specific embodiment, the modified portion is an albumin binder. In a preferred embodiment, the albumin binder is a fatty acid. In a preferred embodiment, the fatty acid is a C18-22 fatty acid.

[0015] In a preferred embodiment, the binding affinity EC between the fatty acid and human serum albumin 50 The value is preferably less than about 10 μM, and preferably less than about 1 μM.

[0016] In a specific embodiment, the structure of the albumin binder is as shown in the following formula: R1-(X1) m -(X2) n -K(ε-R2) In the formula, K is lysine, R1 is a substituted or unsubstituted C 1-20 It is an acyl group, m is an integer between 0 and 5. n is an integer between 0 and 5. X1 and X2 are alanine (Ala), D-alanine (D-Ala), β-alanine (β-Ala), 4-aminobutyric acid (GABA), 2-aminoisobutyric acid (Aib), 2-aminobutyric acid (Abu), arginine (Arg), aspartic acid (Asp), asparagine (Asn), cysteine ​​(Cys), glutamic acid (Glu), D-glutamic acid (D-Glu), γ-glutamic acid (γ-Glu), glutamic acid Independently selected from the group consisting of glycine (Gln), glycine (Gly), histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys), proline (Pro), phenylalanine (Phe), serine (Ser), tyrosine (Tyr), threonine (Thr), tryptophan (Trp), valine (Val), methionine (Met), tranexamic acid (Trx), AEEA, and PEG, R2 is halogen-substituted C 1-6 Selected from an acyl group or H.

[0017] In a preferred embodiment, R1 is selected from heptanol, methylheptanoyl, octanoyl, methyloctanoyl, nonanoyl, methylnonanoyl, decanoyl, methyldecanoyl, lauroyl, myristoyl, palmitoyl, octadecanoyl, 17-carboxyheptadecanoyl, 15-carboxypentadecanoyl, 13-carboxytridecanoyl, and 11-carboxyundadecanoyl.

[0018] In a preferred embodiment, R1 is selected from lauroyl, myristoyl, palmitoyl, octadecanoyl, 17-carboxyheptadecanoyl, and 19-carboxynonadecanoyl. In a preferred embodiment, m is an integer between 1 and 3. In a preferred embodiment, n is an integer between 1 and 3.

[0019] In a preferred embodiment, X1 and X2 are independently selected from the group consisting of 4-aminobutyric acid (GABA), 2-aminoisobutyric acid (Aib), D-alanine (D-Ala), β-alanine (β-Ala), aspartic acid (Asp), cysteine (Cys), glutamic acid (Glu), γ-glutamic acid (γ-Glu), glycine (Gly), serine (Ser), tyrosine (Tyr).

[0020] In a preferred embodiment, X1 and X2 are independently selected from the group consisting of glutamic acid (Glu), γ-glutamic acid (γ-Glu), tyrosine (Tyr). In a preferred embodiment, R2 is selected from a halogen-substituted C 1-3 acyl group or H.

[0021] In a preferred embodiment, R2 is selected from an iodoacetyl group, a bromoacetyl group or H, more preferably a bromoacetyl group or H. In a specific embodiment, the albumin binder is selected from the group consisting of the following.

Chemical formula

[0022] In a specific embodiment, the PAS is PAS 15 -PAS 200 and preferably PAS 15 -PAS 100 and for example PAS 50 is. In a preferred embodiment, the PAS 50 is as shown in SEQ ID NO:11. In a preferred embodiment, a cysteine residue is inserted at the 3rd position from the N-terminus of the PAS 50 .

[0023] In a preferred embodiment, the PAS can be replaced with a linker sequence (GSSSS) p and p is selected from integers of 1 to 3. In a specific embodiment, the linker is a polyvalent linker, which can link one or more albumin binders and one or more protein moieties. In a preferred embodiment, the polyvalent linker is linked to one albumin binder and to multiple protein moieties.

[0024] In a specific embodiment, the structure of the polyvalent linker is as shown by the following formula: [ka] In the formula, LG1 is a leaving group that is reactive with primary amino groups. LG2 is a leaving group that is reactive with sulfidyl groups. In a preferred embodiment, LG1 is a leaving group that is reactive with respect to Lys. In a preferred embodiment, LG2 is a leaving group that is reactive with Cys.

[0025] In a preferred embodiment, LG1 is linked to the albumin binder, and LG2 is linked to the protein portion. In preferred embodiments, LG1 is selected from active ester groups commonly used in peptide synthesis and includes, but is not limited to, N-hydroxysuccinimide (NHS) ester groups, sulfo-NHS ester groups, pentafluorophenol (PFP) ester groups, p-nitrophenol (PNP) ester groups, hydroxybenzotriazole (HOBt) ester groups, and (hydroxyimino)cyanoacetate (Oxyma) groups.

[0026] In a preferred embodiment, LG1 is a carboxylic acid moiety or hydroxyl group activated by succinimidyl ester (OSu ester). In a preferred embodiment, LG2 is selected from halogens such as iodine, bromine, or chlorine.

[0027] In a preferred embodiment, the polyvalent linker is as shown by the following formula. [ka]

[0028] In a preferred embodiment, the albumin binder and the polyvalent linker form the following structure. [ka]

[0029] In a specific embodiment, the modified protein is characterized by being selected from compounds numbered 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 5.10, 5.11, 5.12, 5.13, 5.14, 5.15, 7.2, 7.3, 7.4, 7.5, 12.1, 12.2, 12.3, 12.4, 12.5, and 12.6. In a preferred embodiment, the modified protein is selected from compounds numbered 5.3, 5.4, 5.5, 5.15, 7.4, 7.5, 12.1, 12.2, 12.3, 12.4, 12.5, and 12.6.

[0030] In a second embodiment, the present invention provides a protein polymer comprising a polyvalent linker and a plurality of protein monomers, wherein the protein monomers are optionally linked to a PAS at their N-terminus, and the polyvalent linker is linked to a cysteine ​​residue in the protein monomer or a cysteine ​​residue in the PAS, wherein the cysteine ​​residue is either naturally occurring or introduced by mutation.

[0031] In a preferred embodiment, the protein polymer contains 2 to 3, preferably 2, protein monomers. In a preferred embodiment, the polyvalent linker in the protein polymer is linked to a cysteine ​​residue in the PAS.

[0032] In a specific embodiment, the PAS is 15 -PAS 200 And preferably PAS 15 -PAS 100 For example, PAS 50 That is the case. In a preferred embodiment, the PAS is a linker array (GSSSS) p It can be replaced with p, where p is selected from integers between 1 and 3. In a preferred embodiment, the mutation is a substitution or insertion, preferably a substitution.

[0033] In a preferred embodiment, the protein is a cell growth factor, growth hormone, interferon, enzyme, antibody, or an active fragment thereof. In a preferred embodiment, the cell growth factor is epidermal fibroblast growth factor, platelet-derived fibroblast growth factor, fibroblast growth factor, insulin-like fibroblast growth factor, neurofibroblast growth factor, interleukin-like fibroblast growth factor, erythropoietin, or colony-stimulating factor.

[0034] In a specific embodiment, the fibroblast growth factor is fibroblast growth factor 21. In a preferred embodiment, the amino acid sequence of the fibroblast growth factor 21 is as shown in SEQ ID NO: 1 or 2. In a preferred embodiment, the amino acid sequence of the fibroblast growth factor 21 is as shown in SEQ ID NO:1 or 2, and a cysteine ​​residue is introduced therein by mutation.

[0035] In a preferred embodiment, the amino acid sequence of the fibroblast growth factor 21 is as shown in SEQ ID NO:1 or 2, and the Q28, E30, D38, D46, K122, or H125 mutations are cysteine ​​residues. In a preferred embodiment, the amino acid sequence of the fibroblast growth factor 21 is as shown in any of SEQ ID NO:4~9. In a preferred embodiment, the amino acid sequence of the fibroblast growth factor 21 is as shown in any of SEQ ID NO: 5 to 9.

[0036] In a specific embodiment, the structure of the polyvalent linker is as shown by the following formula: [ka] In the formula, LG1 is a leaving group that is reactive with primary amino groups. LG2 is a leaving group that is reactive with sulfidyl groups.

[0037] In a preferred embodiment, LG1 is a leaving group that is reactive with respect to Lys. In a preferred embodiment, LG2 is a leaving group that is reactive with Cys. In a preferred embodiment, LG1 is linked to the albumin binder, and LG2 is linked to the protein portion.

[0038] In preferred embodiments, LG1 is selected from active ester groups commonly used in peptide synthesis and includes, but is not limited to, N-hydroxysuccinimide (NHS) ester groups, sulfo-NHS ester groups, pentafluorophenol (PFP) ester groups, p-nitrophenol (PNP) ester groups, hydroxybenzotriazole (HOBt) ester groups, and (hydroxyimino)cyanoacetate (Oxyma) groups.

[0039] In a preferred embodiment, LG1 is a carboxylic acid moiety or hydroxyl group activated by succinimidyl ester (OSu ester). In a preferred embodiment, LG2 is selected from halogens such as iodine, bromine, or chlorine.

[0040] In a preferred embodiment, the polyvalent linker is as shown by the following formula. [ka] In a preferred embodiment, the protein polymer is selected from compounds numbered 10.1, 10.2, 10.3, 10.4, and 10.5.

[0041] In a third aspect, the present invention provides a pharmaceutical composition comprising a modified protein as described in the first aspect or a protein polymer as described in the second aspect and a pharmaceutically acceptable excipient. In a preferred embodiment, the pharmaceutical composition is applied to non-alcoholic steatohepatitis, type 2 diabetes, hyperlipidemia, atherosclerosis, and preferably non-alcoholic steatohepatitis.

[0042] In a fourth embodiment, the present invention provides uses for the modified protein described in the first embodiment or the protein polymer described in the second embodiment in the preparation of drugs. In a preferred embodiment, the drug is applied to non-alcoholic steatohepatitis, type 2 diabetes, hyperlipidemia, atherosclerosis, and preferably non-alcoholic steatohepatitis.

[0043] In a fifth embodiment, the present invention provides an albumin binder, the structure of which is shown in the following formula: R1-(X1) m -(X2) n -K(ε-R2) In the formula, K is lysine, R1 is a substituted or unsubstituted C 1-20 It is an acyl group, m is an integer between 0 and 5. n is an integer between 0 and 5. X1 and X2 are alanine (Ala), D-alanine (D-Ala), β-alanine (β-Ala), 4-aminobutyric acid (GABA), 2-aminoisobutyric acid (Aib), 2-aminobutyric acid (Abu), arginine (Arg), aspartic acid (Asp), asparagine (Asn), cysteine ​​(Cys), glutamic acid (Glu), D-glutamic acid (D-Glu), γ-glutamic acid (γ-Glu), glutamic acid Independently selected from the group consisting of glycine (Gln), glycine (Gly), histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys), proline (Pro), phenylalanine (Phe), serine (Ser), tyrosine (Tyr), threonine (Thr), tryptophan (Trp), valine (Val), methionine (Met), tranexamic acid (Trx), AEEA, and PEG, R2 is halogen-substituted C 1-6 Selected from an acyl group or H.

[0044] In a preferred embodiment, R1 is selected from heptanol, methylheptanoyl, octanoyl, methyloctanoyl, nonanoyl, methylnonanoyl, decanoyl, methyldecanoyl, lauroyl, myristoyl, palmitoyl, octadecanoyl, 17-carboxyheptadecanoyl, 15-carboxypentadecanoyl, 13-carboxytridecanoyl, and 11-carboxyundadecanoyl.

[0045] In a preferred embodiment, R1 is selected from lauroyl, myristoyl, palmitoyl, octadecanoyl, 17-carboxyheptadecanoyl, and 19-carboxynonadecanoyl. In a preferred embodiment, m is an integer between 1 and 3. In a preferred embodiment, n is an integer between 1 and 3.

[0046] In a preferred embodiment, X1 and X2 are independently selected from the group consisting of 4-aminobutyric acid (GABA), 2-aminoisobutyric acid (Aib), D-alanine (D-Ala), β-alanine (β-Ala), aspartic acid (Asp), cysteine ​​(Cys), glutamic acid (Glu), gamma-glutamic acid (γ-Glu), glycine (Gly), serine (Ser), and tyrosine (Tyr).

[0047] In a preferred embodiment, X1 and X2 are independently selected from the group consisting of glutamic acid (Glu), gamma-glutamic acid (γ-Glu), and tyrosine (Tyr). In a preferred embodiment, R2 is a halogen-substituted C 1-3 Selected from an acyl group or H. In a preferred embodiment, R2 is selected from an iodoacetyl group, a bromoacetyl group, or H, more preferably from a bromoacetyl group or H.

[0048] In a specific embodiment, the albumin binder is selected from the group consisting of the following. [ka]

[0049] In a sixth embodiment, the present invention provides a polyvalent linker, the structure of which is shown in the following formula: [ka] In the formula, LG1 is a leaving group that is reactive with primary amino groups. LG2 is a leaving group that is reactive with sulfidyl groups.

[0050] In a preferred embodiment, LG1 is a leaving group that is reactive with respect to Lys. In a preferred embodiment, LG2 is a leaving group that is reactive with Cys. In a preferred embodiment, LG1 is linked to the albumin binder, and LG2 is linked to the protein portion.

[0051] In preferred embodiments, LG1 is selected from active ester groups commonly used in peptide synthesis and includes, but is not limited to, N-hydroxysuccinimide (NHS) ester groups, sulfo-NHS ester groups, pentafluorophenol (PFP) ester groups, p-nitrophenol (PNP) ester groups, hydroxybenzotriazole (HOBt) ester groups, and (hydroxyimino)cyanoacetate (Oxyma) groups.

[0052] In a preferred embodiment, LG1 is a carboxylic acid moiety or hydroxyl group activated by succinimidyl ester (OSu ester). In a preferred embodiment, LG2 is selected from halogens such as iodine, bromine, or chlorine.

[0053] In a preferred embodiment, the polyvalent linker is as shown by the following formula. [ka]

[0054] In a seventh aspect, the present invention provides a polypeptide comprising the amino acid sequence shown in SEQ ID NO:11. In the eighth aspect, the present invention provides a polypeptide whose amino acid sequence is as shown in SEQ ID NO:11, wherein a cysteine ​​residue is inserted from the N-terminus to the 3rd position.

[0055] In a ninth embodiment, the present invention provides a polypeptide comprising an amino acid sequence represented by any of SEQ ID NO: 4 to 9, preferably an amino acid sequence represented by any of SEQ ID NO: 5 to 9. In the tenth embodiment, the present invention provides an isolated nucleic acid molecule, the nucleic acid molecule encoding a polypeptide according to any of the seventh to ninth embodiments.

[0056] In the eleventh embodiment, the present invention provides an expression vector comprising an isolated nucleic acid molecule as described in the tenth embodiment. In a twelfth aspect, the present invention provides a host cell which contains the expression vector described in the eleventh aspect, or the genome of the host cell incorporates the nucleic acid molecule described in the tenth aspect.

[0057] In a thirteenth aspect, the present invention provides uses for the polypeptides described in the tenth to twelfth aspects in the preparation of the modified protein described in the first aspect, the protein polymer described in the second aspect, or the pharmaceutical composition described in the third aspect.

[0058] In a fourteenth embodiment, the present invention provides a therapeutic method comprising administering a therapeutically effective amount of a modified protein according to the first embodiment, a protein polymer according to the second embodiment, or a pharmaceutical composition according to the third embodiment to a subject in need. In a preferred embodiment, the treatment method is applied to non-alcoholic steatohepatitis, type 2 diabetes, hyperlipidemia, atherosclerosis, and preferably non-alcoholic steatohepatitis. [Effects of the Invention]

[0059] It should be understood that, within the scope of the present invention, new or preferred technical solutions can be constructed by combining the above-described technical features of the present invention with the technical features specifically described below (e.g., in the examples). Due to space limitations, this will not be repeated here. [Brief explanation of the drawing]

[0060] [Figure 1] The in vivo pharmacokinetic curve in mice is shown. [Figure 2]The in vivo pharmacokinetic curve in miniature pigs is shown. [Modes for carrying out the invention]

[0061] As a result of extensive and thorough research, the inventors unexpectedly discovered an analogue of the metabolic regulatory protein FGF21, thereby obtaining an FGF21 analogue with potential for treating NASH. Such an FGF21 analogue not only has excellent binding affinity to its receptor, but also a significantly improved in vivo half-life. Based on this, the present invention was completed.

[0062] Unless otherwise specified, the terms used herein have the meanings generally understood by those skilled in the art. Where trade names are mentioned herein, they are intended to refer to the corresponding products or their active ingredients. For clarity, some terms used herein are defined below.

[0063] As used herein, the term “isolated nucleic acid molecule” has the meaning commonly understood by those skilled in the art and means that 1) the total nucleic acid is isolated from at least about 50% of the naturally occurring proteins, lipids, carbohydrates or other materials when isolated from the source cell; 2) the “isolated nucleic acid molecule” is not linked to all or part of the naturally occurring polynucleotides; 3) it is effectively linked to polynucleotides not naturally occurring; or 4) it is naturally present as part of a larger polynucleotide sequence. Preferably, the isolated nucleic acid molecule is substantially free from any other contaminating nucleic acid molecules or other contaminants that could impair its use in the production of polypeptides present in its natural environment or its use in the treatment, diagnosis, prevention or research.

[0064] As used herein, the term “vector” has the meaning commonly understood by those skilled in the art and refers to any molecule (e.g., nucleic acid, plasmid, or virus) used to deliver coding information to a host cell.

[0065] As used herein, the term “expression vector” has the meaning generally understood by those skilled in the art and refers to a vector of nucleic acid sequences suitable for transforming host cells and for inducing and / or controlling the expression of an inserted heterologous nucleic acid sequence. Expression includes, but is not limited to, processes such as transcription, translation, and RNA splicing (if introns are present).

[0066] As used herein, the term “effectively concatenated” has the meaning generally understood by those skilled in the art and refers to a method of arranging a flanking sequence such that the flanking sequence described herein is configured or assembled to perform its normal function. Thus, a flanking sequence effectively concatenated to a coding sequence can influence the replication, transcription, and / or translation of the coding sequence. For example, if a promoter can induce the transcription of a coding sequence, then the coding sequence is effectively concatenated to the promoter. A flanking sequence does not need to be adjacent to a coding sequence as long as it functions correctly. Thus, for example, an intervening sequence that is not translated but is transcribed can exist between a promoter sequence and a coding sequence, and the promoter sequence can still be considered “effectively concatenated” to the coding sequence.

[0067] As used herein, the term “host cell” has the meaning commonly understood by those skilled in the art and refers to a cell that has been transformed with a nucleic acid sequence (e.g., the nucleic acids provided herein) or, after transformation with such nucleic acid sequence, is capable of expressing a selected target gene. The term includes offspring of a parent cell, regardless of whether the offspring are morphologically or genetically identical to the original parent.

[0068] In the specification of the present invention, the term "FGF21" as used herein refers to human fibroblast growth factor 21 and mutants of said FGF21. As used herein, "FGF21" and "human fibroblast growth factor 21" are used interchangeably. The mutant refers to a compound obtained by the steps of substituting one or more amino acid residues in the FGF21 sequence with another native or non-native amino acid, and / or adding one or more native or non-native amino acids to the FGF21 sequence, and / or deleting one or more amino acid residues from the FGF21 sequence, wherein any one of these steps may optionally involve further derivatization of one or more amino acid residues. Specifically, if one amino acid residue is substituted with another amino acid residue belonging to the same group (i.e., substituted with another amino acid residue having similar properties), such substitution is considered conservative. Based on these characteristics, amino acids can be appropriately classified into groups such as basic amino acids (e.g., arginine, lysine, histidine), acidic amino acids (e.g., glutamic acid and aspartic acid), polar amino acids (e.g., glutamine, cysteine, and asparagine), hydrophobic amino acids (e.g., leucine, isoleucine, proline, methionine, and valine), aromatic amino acids (e.g., phenylalanine, tryptophan, tyrosine), and low molecular weight amino acids (e.g., glycine, alanine, serine, and threonine). Typically, FGF21 mutants have at least 80% identity with human FGF21.

[0069] "Substantially" means almost completely or completely, satisfying one or more of the following conditions, for example, more than 50%, 51% or more, 75% or more, 80% or more, 90% or more, and 95% or more.

[0070] As used herein, “sequence identity” is determined by aligning the sequence of a reference DNA with another DNA sequence, thereby maximizing the overlap between the two sequences and simultaneously minimizing the sequence gap, where any highlighted sequences between the two sequences are ignored. For any sequence identity described herein, at least 80% sequence identity is preferred, 85% is more preferred, 90% is even more preferred, 95% is even more preferred, and 96%, 97%, 98%, and 99% are most preferred.

[0071] The abbreviations for amino acid residues in proteins are as follows: Phenylalanine is Phe or F, leucine is Leu or L, isoleucine is Ile or I, methionine is Met or M, valine is Val or V, serine is Ser or S, proline is Pro or P, threonine is Thr or T, alanine is Ala or A, tyrosine is Tyr or Y, histidine is His or H, glutamine is Gln or Q, asparagine is Asn or N, lysine is Lys or K, aspartic acid is Asp or D, glutamic acid is Glu or E, cysteine ​​is Cys or C, tryptophan is Trp or W, arginine is Arg or R, and glycine is Gly or G.

[0072] The terms “optionally” or “optionally” mean that the event or situation described thereafter may or may not occur, and the description includes both the occurrence and non-occurrence of such event or situation. For example, “optionally” substitution of an ethyl group with a halogen means that the ethyl group may be unsubstituted (CH2CH3), monosubstituted (e.g., CH2CH2F), polysubstituted (e.g., CHFCH2F, CH2CHF2, etc.), or fully substituted (CF2CF3). Those skilled in the art will understand that no group containing one or more substituents introduces a sterically impossible and / or unsynthetic substitution or substitution pattern.

[0073] C used in this specification m-n This refers to having m to n carbon atoms in that part. For example, "C 0-6 An "alkylene group" refers to an alkylene group having 0 to 6 carbon atoms. If an alkylene group has 0 carbon atoms, the group is a bond.

[0074] In this specification, a numerical range refers to each integer within a specified range. For example, "C 1-6 " " refers to the fact that the group may have one carbon atom, two carbon atoms, three carbon atoms, four carbon atoms, five carbon atoms, or six carbon atoms. If any variable (e.g., R) appears one or more times in the composition or structure of a compound, its definition under each of these circumstances is independent. Therefore, for example, if one group is substituted by two Rs, each R has an independent choice.

[0075] The term "substituted" refers to the substitution of any one or more hydrogen atoms on a particular atom by a substituent, provided that the valence of that atom is normal and the resulting compound is stable. When the substituent is oxo (i.e., =O), it means that two hydrogen atoms are substituted, and oxo atoms are not present in aromatic groups. As a noun, the term "conjugation" refers to a modified protein, that is, the portion of the protein to which it is bound in order to alter its properties. As a verb, the term refers to the process by which a portion of a protein binds to a protein and modifies its properties.

[0076] As used herein, the term “FGF21 fusion protein” refers to the fusion of one or more amino acid residues (e.g., heterologous protein or peptide) to the N-terminus or C-terminus of any FGF21 polypeptide mutant described herein. Heterogeneous peptides and polypeptides include, but are not limited to, epitopes for detecting and / or isolating FGF21 polypeptide mutants: transmembrane receptor proteins or their portions, such as extracellular domains or transmembrane and intracellular domains; ligands or their portions that bind to transmembrane receptor proteins; enzymes or their catalytically active portions; polypeptides or peptides that promote oligomerization, such as leucine zipper domains; stability-enhancing polypeptides or peptides, such as immunoglobulin constant regions (e.g., domains); half-life extension sequences containing two or more (e.g., 2, 5, 10, 15, 20, 25, etc.) combinations of naturally occurring or unnaturally occurring charged and / or uncharged amino acids (e.g., serine, glycine, glutamic acid, or aspartic acid) designed to form fusion partners with mainly hydrophilic or mainly hydrophobic mutants; functional or non-functional natural antibodies or their heavy or light chains; and polypeptides having different activity (e.g., therapeutic activity) from the FGF21 polypeptide mutants of the present invention. FGF21 fusion proteins can be prepared by fusing a heterologous sequence to the N-terminus or C-terminus of an FGF21 polypeptide mutant. The heterologous sequences described herein may be amino acid sequences or polymers containing non-amino acids. The heterologous sequences may be fused directly to the FGF21 polypeptide mutant or via a linker or adapter molecule. The linker or adapter molecule may consist of one or more amino acid residues (or amino acid polymers), for example, 1, 2, 3, 4, 5, 6, 7, 8, or 9 residues (or amino acid polymers), preferably 10 to 50 amino acid residues (or amino acid polymers), for example, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 residues (or amino acid polymers), more preferably 15 to 35 amino acid residues (or amino acid polymers).The linker or adapter molecule may also be designed to have a cleavage site for DNA restriction endonucleases or proteases in order to isolate the fusion region.

[0077] As used herein, “analogs” or “derivatives” of FGF21, i.e., FGF21 analogs or FGF21 derivatives, refer to polypeptides derived from or that can be derived from natural FGF21, particularly SEQ ID NO:1, i.e., polypeptides modified by modification of their amino acid sequence, such modification, amendment, or change may include substitution, deletion, and / or addition of one or more amino acids. For example, amino acids may be added and / or deleted at the C-terminus, N-terminus, or internally in the amino acid sequence. Preferably, amino acids are added and / or deleted at the C-terminus and / or N-terminus, more preferably at the N-terminus. An amino acid sequence having an amino acid deleted from the C-terminus or N-terminus may also be called a cleaved sequence, as is known in the art. Similarly, an amino acid added internally in a sequence may also be called an insertion.

[0078] A "side chain" is a chemical unit that does not possess any particular properties, especially biological activity, and in one or more embodiments of the present invention, the side chain includes an albumin-binding side chain and a polyvalent linker side chain.

[0079] The term "albumin conjugate" is not limiting in itself, but rather descriptive, as it reflects the overall goal or objective linked to FGF21, namely that the resulting conjugate (or analogue) is capable of binding to human serum albumin and providing sustained efficacy to the analogue of the present invention, or at least contributing to a sustained effect. Where necessary, this term may be replaced with other general chemical terms such as "compound."

[0080] "Polyvalent linker": In the context of the present invention, a linker is a chemical fraction or residue used to covalently bond to the protein being discussed. When a linker reacts with a protein, it forms a linker group. Thus, the term "-linker-" is intended to refer to a chemical unit of a conjugate that covalently bonds to an amino acid residue of each polypeptide in the protein conjugate. However, the present invention uses polyvalent linkers (including divalent and trivalent linkers) to obtain FGF21 dimers.

[0081] "PAS" is used to describe repeating amino acid sequences consisting of proline (P), alanine (A), and serine (S), as well as amino acid sequences consisting of 0 to approximately 400 amino acid residues, such as sequences consisting of approximately 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, and 400 amino acid residues. The number of PAS amino acids is indicated by a subscript; for example, PAS10 is a PAS consisting of 10 proline (P), alanine (A), and serine (S) residues. In some embodiments, the proline residues of the PAS account for 10% to 40% of the total amino group residues of the polypeptide PAS. In specific embodiments, the PAS is a PAS 15 -PAS 200 And preferably PAS 15 -PAS 100 For example, PAS 50 That is the case.

[0082] In the specification of this invention, the term "pharmaceutically acceptable salt" refers to a salt that is not harmful to the patient. The salt includes pharmaceutically acceptable acid addition salts, pharmaceutically acceptable metal salts, ammonium salts, and alkylated ammonium salts. Acid addition salts include inorganic salts and organic salts. Typical examples of suitable inorganic acids include hydrochloric acid, hydrobromic acid, hydroiodic acid, phosphoric acid, sulfuric acid, and nitric acid. Typical examples of suitable organic acids include formic acid, acetic acid, trichloroacetic acid, trifluoroacetic acid, propionic acid, benzoic acid, cinnamic acid, citric acid, fumaric acid, glycolic acid, lactic acid, maleic acid, malic acid, malonic acid, mandelic acid, oxalic acid, picric acid, pyruvic acid, salicylic acid, succinic acid, methanesulfonic acid, ethanesulfonic acid, tartaric acid, ascorbic acid, pamoic acid, bismethylenesalicylic acid, ethanedisulfonic acid, gluconic acid, citric acid, aspartic acid, stearic acid, palmitic acid, EDTA, glycolic acid, p-aminobenzoic acid, glutamic acid, benzenesulfonic acid, p-toluenesulfonic acid, etc. Other metal salts include lithium salts, sodium salts, potassium salts, magnesium salts, etc. Ammonium salts and alkylated ammonium salts include ammonium salts, methylammonium salts, dimethylammonium salts, trimethylammonium salts, ethylammonium salts, hydroxyethylammonium salts, diethylammonium salts, butylammonium salts, tetramethylammonium salts, and the like.

[0083] This application also includes compounds of the present application that are identical to the hail described herein, but in which one or more atoms are substituted with isotopes having atomic weights or mass numbers different from those normally found in nature. Examples of isotopes that bind to the compounds of the present application include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, iodine, and chlorine, for example, respectively 2 H, 3 H, 11 C, 13 C, 14 C, 13 N, 15 N, 15 O, 17 O, 18 O, 31 P, 32 P,35 S, 18 F, 123 I, 125 I and 36 Includes Cl, etc.

[0084] Specific compounds of this application labeled with isotopes (e.g.) 3 H and 14 Those labeled with C can be used for tissue distribution analysis of compounds and / or substrates. Tritiation (i.e.) 3 H) and carbon-14 (i.e.) 14 C) Isotopes are particularly preferred due to their ease of preparation and detection. Furthermore, heavier isotopes (e.g., deuterium (i.e.) 2 Substitution with H)) may result in therapeutic benefits (e.g., increased in vivo half-life or reduced dose demand) due to greater metabolic stability, and therefore may be preferable in certain situations. 15 O, 13 N, 11 C and 18 Positron-emitting isotopes such as 14F can be used in positron emission tomography (PET) studies to measure substrate occupancy. Typically, isotope-labeled compounds of this application can be prepared by substituting an isotope-labeled reagent with an isotope-labeled reagent by following a similar procedure to those disclosed in the following scheme and / or examples.

[0085] Therapeutic Uses of FGF21 Analogues: FGF21 analogues can be used to treat, diagnose, improve or prevent a variety of diseases, conditions, or pathologies, including but not limited to metabolic disorders. These include diabetes, dyslipidemia, hypertension, hepatic steatosis such as non-alcoholic steatohepatitis (NASH), and cardiovascular diseases such as atherosclerosis.

[0086] In applications, diseases or conditions such as diabetes or obesity can be treated by administering a therapeutically effective amount of the FGF21 mutant polypeptide described herein to patients in need. It can be administered according to the methods described herein, for example, by intravenous injection, intraperitoneal injection, intramuscular injection, or orally in the form of tablets or liquid formulations. In most cases, the desired dose is determined by the clinician described herein and also represents the therapeutically effective dose of the FGF21 mutant polypeptide. It will be apparent to those skilled in the art that the therapeutically effective dose of the FGF21 mutant polypeptide is determined in particular by the administration scheme, the unit dose of the administered substance (whether the nucleic acid molecule or polypeptide is used in combination with other therapeutic agents), the immune status, and the health status of the recipient. As used herein, the term “therapeutically effective dose” refers to the amount of FGF21 mutant polypeptide that elicits a biological or pharmacological response in a tissue system, animal, or human as requested by the researcher or other clinician, including the alleviation of symptoms of the disease or condition being treated.

[0087] In a specific embodiment, the FGF21 variant of the present invention contains an amino acid sequence represented by any of SEQ ID NO:4 to 9, preferably any of SEQ ID NO:5 to 9.

[0088] The modified protein of the present invention In the present invention, a protein drug can be coupled to a specific chemical molecule or domain (e.g., ABD) that can bind to a protein with a long in vivo half-life, thereby extending the in vivo half-life of the drug while minimizing its in vivo distribution. The protein with a long in vivo half-life is a protein in plasma, preferably human serum albumin.

[0089] When the protein with a long in vivo half-life is human serum albumin, the specific chemical molecule that can bind to the protein with a long in vivo half-life is an albumin binder. The albumin binder is a substance that can bind specifically or nonspecificly to human serum albumin, such as a fatty acid.

[0090] In a specific embodiment, the structure of the albumin binder of the present invention is as shown in the following formula: R1-(X1) m -(X2) n -K(ε-R2) In the formula, K, R1, m, n, X1, X2, and R2 are as described above.

[0091] Based on the above-described albumin-binding agent, the present invention provides such a modified protein in which the albumin-binding agent is linked to a cysteine ​​residue in the protein portion. Based on the disclosure of the present invention, those skilled in the art will understand that the cysteine ​​residue may be naturally occurring or introduced by mutation. The method of mutation may be substitution or insertion, but substitution is preferred.

[0092] In the modified protein of the present invention, a PAS may be ligated to the N-terminus of the protein portion. The meaning of "PAS" in this specification is as described above. In a specific embodiment, the PAS contained in the N-terminus of the modified protein of the present invention is PAS 50 The albumin binder can also bind to the protein portion of the modified protein via the PAS. Therefore, a cysteine ​​residue can be introduced into the PAS via a mutagenesis method (preferably an insertion method) in order to bind to the albumin binder. In a preferred embodiment, the PAS 50 Insert a cysteine ​​residue at the 3rd position of the N-terminus.

[0093] The inventors have further found that in the modified protein of the present invention, the modified portion can be linked via a linker to a cysteine ​​residue in the protein portion or to a cysteine ​​residue in the PAS, which is either naturally occurring or introduced by mutation. The linker is a polyvalent linker, which can link one or more albumin binders and one or more protein portions. Preferably, in the modified protein of the present invention, one albumin binder and multiple protein portions are linked via a polyvalent linker. The specific structure of the polyvalent linker is as shown above.

[0094] Based on the teachings of the present invention, those skilled in the art will understand how to obtain the modified protein of the present invention, for example by conventional synthetic techniques to obtain the albumin binder and linker of the present invention, and by conventional biotechnology to obtain the protein moiety and the protein moiety linked to the PAS, and then link the albumin binder to the protein moiety directly or via the linker. For example, those skilled in the art can use conventional means to obtain the protein moiety or the nucleic acid molecule encoding the PAS in the modified protein of the present invention, and then use conventional biotechnological means to obtain the protein moiety or the protein moiety linked to the PAS in a host cell. The present invention further includes such a nucleic acid molecule, or an expression vector containing the nucleic acid molecule and a host cell.

[0095] After preparing the modified protein of the present invention, the modified protein can be purified using techniques known in the art. These techniques include, but are not limited to, reverse-phase high-performance preparative liquid chromatography, ion-exchange chromatography, gel electrophoresis, affinity chromatography, and size exclusion chromatography. The actual conditions for purifying the protein depend in part on factors such as net charge, hydrophobicity, and hydrophilicity, and will be readily understood by those skilled in the art. The modified protein can be isolated using high-performance preparative liquid chromatography. The purity of the modified protein can be measured by any of several known analytical methods, including gel electrophoresis and high-performance liquid chromatography, and its molecular weight can be verified by mass spectrometry.

[0096] In further embodiments, the present invention relates to an N-terminal PAS 50 Provides a modified FGF21 fusion protein. N-terminal PAS 50 We successfully prepared a modified FGF21 fusion protein and measured its cell activity using the reporter gene principle. As is well known to those skilled in the art, compounds linked by disulfide bonds, such as Cys obtained by reacting with free Cys from the FGF21 fusion protein, reduced glutathione, and cysteamine, are also contained within the FGF21 fusion protein.

[0097] N-terminal PAS 50 Based on the modified FGF21 fusion protein, the inventors obtained an albumin-binding side-chain modified FGF21 conjugate. The albumin binder of the present invention connects to a protein moiety, such as PAS, via a linker, such as a polyvalent linker. 50 Modified FGF21 protein is obtained. In a specific embodiment, the albumin binder is N-terminal PAS via a polyvalent linker. 50 It is linked to the modified FGF21 fusion protein.

[0098] In specific embodiments, the modified protein of the present invention is selected from compounds numbered 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 5.10, 5.11, 5.12, 5.13, 5.14, 5.15, 7.2, 7.3, 7.4, 7.5, 12.1, 12.2, 12.3, 12.4, 12.5, and 12.6, preferably selected from compounds numbered 5.3, 5.4, 5.5, 5.15, 7.4, 7.5, 12.1, 12.2, 12.3, 12.4, 12.5, and 12.6, and its biological activity is identified by reporter gene method.

[0099] Using the polyvalent linker of the present invention, the inventors have further obtained a protein polymer comprising a plurality of protein monomers linked by the polyvalent linker. The protein monomers have a PAS optionally linked to their N-terminus, and the polyvalent linker is linked to a cysteine ​​residue in the protein portion or a cysteine ​​residue in the PAS, the cysteine ​​residue being either naturally occurring or introduced by mutation.

[0100] The number of protein monomers in the protein polymer can be determined by those skilled in the art based on actual circumstances. For example, the protein polymer of the present invention may contain 2 to 3 protein monomers, preferably 2. Those skilled in the art will know that PAS in protein polymers is also a linker sequence (GSSSS) p You will understand that this can be replaced with and that p is selected from integers between 1 and 3.

[0101] In a specific embodiment, the structure of the polyvalent linker is as shown in the following formula. [ka] In specific embodiments, the protein polymer of the present invention is selected from compounds numbered 10.1, 10.2, 10.3, 10.4, and 10.5.

[0102] The advantages of the present invention are as follows: 1. The present invention provides novel modified proteins or protein polymers. 2. The modified protein or protein polymer of the present invention maintains the intrinsic activity of the protein and significantly improves its in vivo half-life. 3. The modified proteins or protein polymers of the present invention have a simple preparation process and can be easily obtained by technical means such as biotechnology and chemical synthesis. 4. The modified proteins or protein polymers of the present invention provide a new material basis for the development of a range of therapeutic agents for non-alcoholic steatohepatitis, type 2 diabetes, hyperlipidemia, atherosclerosis, and other conditions.

[0103] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are used solely for illustrative purposes and do not limit the scope of the invention. Experimental methods in the following examples that do not specify concrete conditions generally follow general conditions, e.g., those described in (Sambrook and Russell et al., Molecular Cloning - A Laboratory Manual (3rd edition) (2001), CSHL Publishing) or those suggested by the manufacturer. Unless otherwise specified, percentages and quantities are calculated by weight. The experimental materials and reagents used in the following examples are available through commercial channels unless otherwise specified.

[0104] Examples The abbreviations used in the following examples have meanings that are generally understood by those skilled in the art. For clarity, these abbreviations are explained below. Tris-HCl: Trishydroxymethylaminomethane hydrochloride SDS: Sodium dodecyl sulfate DMF: N,N-dimethylformamide DTT: Dithiothreitol EDTA: Ethylenediaminetetraacetic acid TFA: Trifluoroacetic acid HSA: Human serum albumin PBS: Potassium dihydrogen phosphate (KH2PO4) 0.24 g / L, sodium dihydrogen phosphate (Na2HPO4) 1.44 g / L, sodium chloride (NaCl) 8 g / L, potassium chloride (KCl) 0.2 g / L, pH 7.4 TCEP: Tris(2-carboxyethyl)phosphine Tris: Tris(hydroxymethyl)aminomethane or 2-amino-2-hydroxymethylpropane-1,3-diol LCMS: Liquid Chromatography-Mass Spectrometry AEEA: 2-(2-(2-aminoethoxy)ethoxy)acetic acid RP-HPLC: Reverse-phase high-performance liquid chromatography DCM: Dichloromethane Fmoc:9-Fluorenylmethoxycarbonyl DIEA: N,N-diisopropylethylamine HOBt: 1-hydroxybenzotriazole DIC: N,N-diisopropylcarbodiimide PIP: Piperidine

[0105] General detection and characterization methods RP-HPLC [Table A]

[0106] LC-MS method [Table B]

[0107] Example 1: Preparation of FGF21 and its mutants The sequence of mature human FGF21 is disclosed and can be searched in the UniProt database using accession number Q9NSA1, as shown in SEQ ID NO:1. Literature such as PLoS One.2012, 7(11):e49345 and Endocrinology.2017, 158(5):1314-1327 has reported that L98R, P171G, and A180E mutations effectively improve the anti-aggregation and anti-degradation capabilities of FGF21. The procedure for preparing the mutant in question (SEQ ID NO:2), i.e., FGF21RGE, is as follows.

[0108] (1) Express FGF21RGE using the form of a tagged fusion protein. Construct an expression cassette containing an N-terminal 6-polyhistidine (His6) tag and a ubiquitin-like modified protein (SUMO) tag for FGF21RGE expression and insert it into a pET30 plasmid. Then transform competent E. coli BL21 (DE3) bacteria and screen to obtain a modified strain expressing the His6-SUMO-FGF21RGE (SEQ NO:3) fusion protein. Culture the modified strain at high density to express the His6-SUMO-FGF21RGE fusion protein in a soluble form at high concentrations in the cytoplasm.

[0109] (2) First, the soluble fusion protein His6-SUMO-FGF21RGE (SEQ NO:3) is purified, and the soluble FGF21RGE protein is obtained by enzymatic digestion, and the soluble FGF21RGE protein is obtained by purification. First, the soluble fusion protein expressed in cells is released by high-pressure homogenization. Then, the pH of the turbid homogenate is adjusted to 3.0, followed by centrifugation and then adjustment to 8.0. The fusion protein is concentrated by metal ion chelation chromatography (IMAC) using HisTrap Ni excel (5 ml, purchased from Cytiva). The fusion protein containing the His6-SUMO tag is eluted with 250 mM imidazole, and the elution peak is digested enzymatically by adding the ULP1 enzyme (which specifically cleaves the SUMO tag). The enzymatic digestion system contains 0.2 mM TCEP. The solution after enzymatic digestion is purified by anion exchange chromatography (AEX) using HiTrap Q HP (5 ml, purchased from Cytiva), maintaining the chromatographic pH at 8.0. Elution is performed with a linear salt gradient of 0-500 mM NaCl, and high-purity soluble FGF21RGE protein is obtained after SDS-PAGE and RP-HPLC analysis. Finally, the FGF21RGE protein is filtered through an MWCO 10kDa filter membrane into PBS. LC-MS analysis revealed that the actual molecular weight of FGF21RGE prepared by the above method was 19453.0 Da, which is consistent with the theoretical molecular weight (19453.8 Da).

[0110] In the subsequent examples, various FGF21 proteins, including FGF21RGE and FGF21 mutants, are expressed in bacterial expression systems such as E. coli BL21(DE3). Unless otherwise specified, expression and purification are performed according to the methods described in these examples.

[0111] Example 2: Synthesis of modified side chains 2.1. Synthesis of C18 diacid-AEEA-gGlu-gGlu-Lys(bromoacetyl)-COOH [ka] (1) Materials and reagents 2-CTC resin, substitution value 1.15 mmol / g The amino acids are Fmoc-Lys(Alloc)-OH, Fmoc-AEEA-OH, Fmoc-Glu-otBu, and octadecanediol mono-t-butyl ester. Synthetic reagents: HOBt, DIC, DMF, DCM, PIP, DIEA. (2)Equipment CS-BIO type polypeptide synthesis apparatus, Waters 600 semi-preparative high-performance liquid chromatograph, Beckman centrifuge, BUCHI rotary evaporator.

[0112] (3) Synthesis of brominated fatty acid side chains a. Solid-phase chemical synthesis of polypeptides Weigh 1.00 g of 2-CTC resin, place it in the polypeptide synthesis reactor, add 10 ml of DCM, and allow to swell for 1 hour. Weigh 2-3 times the amount of Fmoc-Lys(Alloc)-OH and 4-6 times the amount of DIEA, dissolve them in 10 ml of DMF, and place them in the reactor. React at room temperature for 2 hours, i.e., coupling the first amino acid to the resin. Then wash the resin 6 times with DCM, measure the substitution value (SD) of the resin in this case, and then 20% PIP / D Add 10 ml of MF solution and mix for 10 minutes to remove the amino protecting group Fmoc. Repeat this process once, then wash the resin six times with DCM to couple the second amino acid. Weigh three times the amount of Fmoc-Glu-otBu, HOBt, and DIC, dissolve them in 10 ml of a mixed solvent of DMF / DCM (1:1), and allow to react at room temperature. Monitor the progress of the reaction with ninhydrin; if colorless, it indicates that the reaction is complete. Wash the resin six times with DCM. Next, the coupling reaction of AEEA, glutamic acid, and octadecanediic acid can be continued according to the above coupling method, and repeat this cycle until all amino acids are coupled.

[0113] Next, the Alloc protecting group of the Fmoc-Lys(Alloc)-OH side chain is selectively removed by the orthogonal protection method. The specific method for removing the Alloc protecting group is as follows: Tetrakis(triphenylphosphine)palladium (0.1x amount) and phenylsilane (10x amount) are weighed, dissolved in 15 ml of DCM, and then added to the reactor and reacted with the resin peptide for 25 minutes. This process must be carried out in the dark and under nitrogen gas protection. After the reaction is complete, the resin is sequentially washed with DCM (15 ml x 6 times, 2 minutes each time), 0.02 mol / L N,N-diethyldithiocarbamic acid / DMF solution (15 ml x 3 times, 2 minutes each time), and DMF (15 ml x 6 times, 2 minutes each time). A small amount of resin is weighed and ninhydrin detection is performed. The resin granules appear purplish-black, indicating that deprotection is complete.

[0114] Finally, add 2 equivalents of acetyl bromide and 10 ml of DMF, and allow to react at room temperature for 1 hour. Monitor the progress of the reaction with ninhydrin; a colorless result indicates that the reaction is complete. Wash the resin 6 times with DCM, vacuum dry the resin peptide, and store it.

[0115] b. Pyrolysis and precipitation The pyrolysis reagent was added according to a ratio of 10 ml of pyrolysis reagent to 1 g of resin, with a reagent ratio of TFA:TIS:H2O = 95:2.5:2.5 (V:V). The mixture was reacted at room temperature for 1 hour, the resin was removed by filtration, and the filtrate was subjected to a rotary evaporator at 40°C to remove as much TFA as possible. The fatty acid side chains were precipitated with 7-10 times the amount of ice ether, placed in a refrigerator at -20°C for 20 minutes, centrifuged, and vacuum-dried to obtain the crude fatty acid side chain product. Purity: 66%, Theoretical molecular weight: 966.96 Da, Actual molecular weight will be verified by LC-MS.

[0116] 2.2. Synthesis of C18 diacitate-gGlu-AEEA-AEEA-Lys(bromoacetyl)-COOH [ka] It is synthesized according to 2.1 and conventional solid-phase synthesis methods. Purity: 57%, Theoretical molecular weight: 983.01 Da, Actual molecular weight will be verified for accuracy by LC-MS.

[0117] 2.3. Synthesis of C20 diacid-AEEA-gGlu-gGlu-Lys(bromoacetyl)-COOH [ka] It is synthesized according to 2.1 and conventional solid-phase synthesis methods. Purity: 44%, Theoretical molecular weight: 995.02 Da, Actual molecular weight will be verified by LC-MS.

[0118] 2.4. Synthesis of C20 diacitate-gGlu-AEEA-AEEA-Lys(bromoacetyl)-COOH [ka] It is synthesized according to Example 2.1 and conventional solid-phase synthesis methods. Purity: 59%, Theoretical molecular weight: 1011.06 Da, Actual molecular weight will be verified by LC-MS.

[0119] 2.5.C18 diacid-gGlu-AEEA-AEEA-Lys(3,5-bis[(2-bromoacetyl)amino]benzoyl)-COOH [ka] First, (2,5-dioxopyrrolidine-1-yl)-3,5-bis[(2-bromoacetyl)amino]benzoate is synthesized according to CN112839681A. C18 dioxide-gGlu-AEEA-AEEA-Lys-COOH is synthesized according to 2.2 and conventional solid-phase synthesis methods and dissolved in 2% triethylamine. (2,5-dioxopyrrolidine-1-yl)-3,5-bis[(2-bromoacetyl)amino]benzoate is dissolved in ethanol and added dropwise to C18 dioxide-gGlu-AEEA-AEEA-Lys-COOH in a molar ratio of 1:1.2. After reacting for 20-30 minutes, acetic acid is added to adjust the pH to 5.0, and a solid is precipitated using a rotary evaporator at 37°C. Water is added to obtain the desired product as a white solid. Purity: 40%, Theoretical molecular weight: 1238 Da, Actual molecular weight will be verified by LC-MS.

[0120] Example 3: Dual luciferase reporter gene method for measuring the in vitro activity of the measurement analogue iLite FGF21 assay-ready cells (purchased from SAVR) are genetically modified reporter cell lines that respond to FGF21-induced downstream signals by expressing firefly luciferase. The receptor FGFR1c and cofactor β-Klotho are co-overexpressed on the surface of iLite FGF21 assay-ready cells, and their sensitivity is enhanced through gene optimization, thereby increasing FGF21 luciferase expression in a dose-response manner. The luciferase activity detection kit is purchased from Promega (catalog number #E2980).

[0121] iLite FGF21 assay-ready cells are cultured in adherent culture medium (DMEM Gluta Max, purchased from Gibco) containing 10% fetal bovine serum (FBS, purchased from Gibco) and 1% penicillin / streptomycin (purchased from Gibco). For activity measurement experiments, 1 to 30,000 cells / well, along with detection samples such as FGF21 mutant protein and albumin-binding modified FGF21 conjugate molecules, are added to a 96-well all-white culture plate (purchased from Corning), and the chemiluminescence value is detected using a microplate reader according to the specifications of the luciferase activity detection kit. Finally, the semi-effective concentration (EC) of the detected sample is determined using the 4-parameter fitting method. 50 Calculate the value.

[0122] Example 4: In vitro activity of FGF21 mutant Select Q28 (SEQ ID NO: 4), E30 (SEQ ID NO: 5), D38 (SEQ ID NO: 6), D46 (SEQ ID NO: 7), K122 (SEQ ID NO: 8), and H125 (SEQ ID NO: 9) of FGF21RGE and perform cysteine ​​mutations. These FGF21 cysteine ​​mutants are subjected to the construction, fermentation, and purification of modified strains as described in Example 1. Except for the FGF21Q28C mutant, which is difficult to express and purify, the remaining mutants are prepared normally.

[0123] [Table C]

[0124] The in vitro activity of FGF21RGE and FGF21 cysteine ​​mutants was evaluated using the method of Example 3. The results are shown in the table below. All successfully expressed FGF21 mutants showed activity that stimulated downstream signal delivery of the FGFR1c receptor. Compared to the FGF21RGE control protein, FGF21Q28C activity was significantly reduced, and all remaining mutants showed in vitro activity equivalent to that of the control protein.

[0125] [Table D]

[0126] Example 5: Preparation of side-chain modified FGF21 analogs The preparation process for a typical compound is shown in Compound 5.1. Preparation of compound 5.1 [ka] The compound in question is an FGF21 analog obtained by modifying the FGF21E30C mutant of SEQ NO:5 (Example 4) with the albumin binder described in Example 2.1. The specific preparation process and identification are as follows.

[0127] The FGF21E30C mutant before the modification reaction was filtered to Tris-HCl buffer (pH 8.0) to a final concentration of 0.5-2 mg / ml. Simultaneously, 0.2 mM TCEP (tris(2-carboxyethyl)phosphine, dissolved in Tris-HCl buffer and adjusted to pH 8.0) and 2-2.5 mM EDTA·2Na were added. The side chain described in Example 2.1 was dissolved in a 5 M potassium iodide solution (dissolved in Tris-HCl buffer and adjusted to pH 8.0) (concentration 4-10 mg / ml), and the dissolved albumin binder solution was added to the FGF21E30C mutant in a molar ratio of FGF21E30C mutant:albumin binder 1:3, and the reaction was carried out overnight under stirring conditions. After the reaction was complete, the FGF21 analog modified with the albumin binder was purified by anion exchange. Purified water was added to the reaction solution, and the conductivity was 6 mS / cm. The sample was then collected in a pre-equalized HiTrap Q HP (purchased from Cytiva). Mobile phase A was 25 mM Tris-HCl (pH 8.0), and mobile phase B was 25 mM Tris-HCl 1 M NaCl (pH 8.0). The flow rate was 5 ml / min, the operating time was 40 minutes, and the gradient was 0-50%. By matching the target peaks using RP-HPLC and SDS-PAGE analysis, a high-purity FGF21E30C mutant (Example 4) with sequence number 4 was modified with the albumin binder described in Example 2.1 to obtain an FGF21 analog. Purity: >90%, Theoretical molecular weight: 20313.9 Da, Actual molecular weight: 20313.8 Da.

[0128] Preparation of compound 5.2 [ka] The compound in question is an FGF21 analog obtained by modifying the FGF21D38C mutant of SEQ NO:6 (Example 4) with the albumin binder described in Example 2.1. The preparation process and identification are as follows. Process: Prepare in the same manner as compound 5.1. Purity: >90%, Theoretical molecular weight: 20328 Da, Actual molecular weight: 20327.6 Da.

[0129] Preparation of compound 5.3 (Compound 5.3) [ka] The compound in question is an FGF21 analog obtained by modifying the FGF21D46C mutant of SEQ NO:7 (Example 4) with the albumin binder described in Example 2.1. The preparation process and identification are as follows. Process: Prepare in the same manner as compound 5.1. Purity: >90%, Theoretical molecular weight: 20328 Da, Actual molecular weight: 20327.6 Da.

[0130] Preparation of compound 5.4 (compound 5.4) [ka] The compound in question is an FGF21 analog obtained by modifying the FGF21K122C mutant of SEQ NO:8 (Example 4) with the albumin binder described in Example 2.1. The preparation process and identification are as follows. Process: Prepare in the same manner as compound 5.1. Purity: >90%, Theoretical molecular weight: 20314.9Da, Actual molecular weight: 20314.7Da.

[0131] Preparation of compound 5.5 [ka] The compound in question is an FGF21 analog obtained by modifying the FGF21H125C mutant of SEQ NO:9 (Example 4) with the albumin binder described in Example 2.1. The preparation process and identification are as follows. Process: Prepare in the same manner as compound 5.1. Purity: >90%, Theoretical molecular weight: 20305.9Da, Actual molecular weight: 20306.6Da.

[0132] Preparation of compound 5.6 (compound 5.6) [ka] The compound in question is an FGF21 analog obtained by modifying the FGF21D38C mutant (Example 4) with SEQ ID NO:6 using the albumin binder described in Example 2.2. The preparation process and identification are as follows. Process: Prepare in the same manner as compound 5.1. Purity: >90%, Theoretical molecular weight: 20344 Da, Actual molecular weight: 20343.5 Da.

[0133] Preparation of compound 5.7 (compound 5.7) [ka] The compound in question is an FGF21 analog obtained by modifying the FGF21D46C mutant (Example 4) with SEQ ID NO:7 using the albumin binder described in Example 2.2. The preparation process and identification are as follows. Process: Prepare in the same manner as compound 5.1. Purity: >90%, Theoretical molecular weight: 20344 Da, Actual molecular weight: 20343.1 Da.

[0134] Preparation of compound 5.8 (compound 5.8) [ka] The compound in question is an FGF21 analog obtained by modifying the FGF21K122C mutant of SEQ NO:8 (Example 4) with the albumin binder described in Example 2.2. The preparation process and identification are as follows. Process: Prepare in the same manner as compound 5.1. Purity: >90%, Theoretical molecular weight: 20330.9Da, Actual molecular weight: 20330.3Da.

[0135] Preparation of compound 5.9 (compound 5.9) [ka] The compound in question is an FGF21 analog obtained by modifying the FGF21H125C mutant of SEQ NO:9 (Example 4) with the albumin binder described in Example 2.2. The preparation process and identification are as follows. Process: Prepare in the same manner as compound 5.1. Purity: >90%, Theoretical molecular weight: 20321.9 Da, Actual molecular weight: 20321.4 Da.

[0136] Preparation of compound 5.10 (Compound 5.10) [ka] The compound in question is an FGF21 analog obtained by modifying the FGF21D46C mutant (Example 4) with SEQ ID NO:7 using the albumin binder described in Example 2.3. The preparation process and identification are as follows. Process: Prepare in the same manner as compound 5.1. Purity: >90%, Theoretical molecular weight: 20356 Da, Actual molecular weight: 20355.2 Da.

[0137] Preparation of compound 5.11 (compound 5.11) [ka] The compound in question is an FGF21 analog obtained by modifying the FGF21K122C mutant of SEQ NO:8 (Example 4) with the albumin binder described in Example 2.3. The preparation process and identification are as follows. Process: Prepare in the same manner as compound 5.1. Purity: >90%, Theoretical molecular weight: 20342.9 Da, Actual molecular weight: 20342.3 Da.

[0138] Preparation of compound 5.12 (compound 5.12) [ka] The compound in question is an FGF21 analog obtained by modifying the FGF21H125C mutant (Example 4) with SEQ ID NO:9 using the albumin binder described in Example 2.3. The preparation process and identification are as follows. Process: Prepare in the same manner as compound 5.1. Purity: >90%, Theoretical molecular weight: 20333.9 Da, Actual molecular weight: 20333.1 Da.

[0139] Preparation of compound 5.13 (compound 5.13) [ka] The compound in question is an FGF21 analog obtained by modifying the FGF21D46C mutant (Example 4) with SEQ ID NO:7 using the albumin binder described in Example 2.4. The preparation process and identification are as follows. Process: Prepare in the same manner as compound 5.1. Purity: >90%, Theoretical molecular weight: 20372 Da, Actual molecular weight: 20370.9 Da.

[0140] Preparation of compound 5.14 (compound 5.14) [ka] The compound in question is an FGF21 analog obtained by modifying the FGF21K122C mutant of SEQ NO:8 (Example 4) with the albumin binder described in Example 2.4. The preparation process and identification are as follows. Process: Prepare in the same manner as compound 5.1. Purity: >90%, Theoretical molecular weight: 20358.9 Da, Actual molecular weight: 20358.1 Da.

[0141] Preparation of compound 5.15 (compound 5.15) [ka] The compound in question is an FGF21 analog obtained by modifying the FGF21H125C mutant (Example 4) with SEQ ID NO:9 using the albumin binder described in Example 2.4. The preparation process and identification are as follows. Process: Prepare in the same manner as compound 5.1. Purity: >90%, Theoretical molecular weight: 20349.9 Da, Actual molecular weight: 20349.3 Da.

[0142] Example 6: In vitro activity of albumin-binding modified FGF21 analog The in vitro activity of FGF21RGE, FGF21 cysteine ​​mutants, and albumin-binding modified FGF21 analogs was evaluated using the method of Example 3. The results are shown in the table below. [Table E]

[0143] Example 7: Preparation of PAS50 and FGF21 analogs with side chain modifications 7.1. PAS 50 Modified FGF21 mutant (PAS 50 Preparation of FGF21) According to the method described in Example 1, a PAS as shown in SEQ ID NO:10 was produced in the bacterial expression system E. coli BL21(DE3) 50 Modified FGF21 fusion protein (PAS 50 Express FGF21. LC-MS analysis was performed to determine the PAS prepared according to the above method. 50 The actual molecular weight of FGF21 is 23556.9 Da, which is consistent with the theoretical molecular weight (23557.3 Da).

[0144] Preparation of compound 7.2 (Compound 7.2) [ka] The compound in question is PAS with SEQ ID NO:10. 50 Modified FGF21 fusion protein PAS 50FGF21 (Compound 7.1) is a FGF21 analog modified with the albumin binder described in Example 2.1, and the preparation process and identification are as follows. Process: Prepare in the same manner as Compound 5.1. Purity: >90%, Theoretical molecular weight: 24443.4 Da, Actual molecular weight: 24442.7 Da.

[0145] Preparation of 7.3 (Compound 7.3)

Chemical formula

[0146] Preparation of 7.4 (Compound 7.4)

Chemical formula

[0147] Preparation of 7.5 (Compound 7.5)

Chemical formula

[0148] 8.1. Synthesis of C18 diacitate-gGlu-AEEA-AEEA-Lys-OH [ka] (1) Materials and reagents The Fmoc-Lys(BOC)-Wang resin has a substitution value of 0.5 mmol / g. Amino acids: Fmoc-AEEA-OH, Fmoc-Glu-otBu, Octadecanediol mono-t-butyl ester Synthetic reagents: HOBt, DIC, DMF, DCM, PIP, DIEA. (2)Equipment CS-BIO type polypeptide synthesis apparatus, Waters 600 semi-preparative high-performance liquid chromatograph, Beckman centrifuge, BUCHI rotary evaporator.

[0149] (3) Synthesis of brominated fatty acid side chains a. Solid-phase chemical synthesis of polypeptides Weigh 1.00 g of Fmoc-Lys(BOC)-Wang resin and place it in a polypeptide synthesis reactor. Add 10 ml of DCM and allow to swell for 1 hour. Then, add 10 ml of 20% PIP / DMF solution and mix for 10 minutes to remove the amino protecting group Fmoc. Repeat this process once. After that, wash the resin 6 times with DCM to couple the second amino acid. Weigh three times the amount of resin containing Fmoc-AEEA-OH, HOBt, and DIC, dissolve them in 10 ml of a mixed solvent of DMF / DCM (1:1), and allow to react at room temperature. Monitor the progress of the reaction with ninhydrin. If it is colorless, the reaction is complete. Wash the resin 6 times with DCM. Next, the coupling reaction of AEEA, glutamic acid, and octadecanediic acid can be continued according to the above coupling method, and repeat this cycle until all amino acids are coupled.

[0150] b. Pyrolysis and precipitation The pyrolysis reagent was added according to a ratio of 10 ml of pyrolysis reagent to 1 g of resin, with the reagent ratio being TFA:TIS:H2O = 95:2.5:2.5 (V:V). The mixture was reacted at room temperature for 2 hours, the resin was removed by filtration, the fatty acid side chains were precipitated with 7-10 times the amount of ice isopropyl ether, the mixture was placed in a refrigerator at -20°C for 20 minutes, centrifuged, and vacuum-dried to obtain the crude peptide. Purity: 90%, Theoretical molecular weight: 862.07 Da, Actual molecular weight will be verified for accuracy by LC-MS.

[0151] 8.2. Synthesis of C18 diacid-gGlu-AEEA-AEEA-Lys-NH2 [ka] (1) Materials and reagents Rink Amide MBHA resin, substitution value 0.5 mmol / g. Amino acids: Fmoc-Lys(BOC)-OH, Fmoc-AEEA-OH, Fmoc-Glu-otBu, Octadecanediol mono-t-butyl ester Synthetic reagents: HOBt, DIC, DMF, DCM, PIP, DIEA. (2) Synthesized according to the solid-phase synthesis method described in 8.1, Purity: 91%, theoretical molecular weight: 861.07 Da, the actual molecular weight is verified for accuracy by LC-MS.

[0152] 8.3 Synthesis of 3,5-bis[(2-bromoacetyl)amino]benzoic acid

Chemical formula

[0153] 8.4 Synthesis of (2,5-dioxopyrrolidin-1-yl)-3,5-bis[(2-bromoacetyl)amino]benzoate

Chemical formula

[0154] 8.5 Synthesis of C18 diacid-gGlu-AEEA-AEEA-Lys(3,5-bis[(2-bromoacetyl)amino]benzoyl)-OH

Chemical formula

[0155] 8.6. Synthesis of C18 diacid-gGlu-AEEA-AEEA-Lys(3,5-bis[(2-bromoacetyl)amino]benzoyl)-NH2 [ka] The crude peptide from Example 8.2 is dissolved in 2% triethylamine, (2,5-dioxopyrrolidine-1-yl)-3,5-bis[(2-bromoacetyl)amino]benzoate is dissolved in ethanol and added dropwise to the crude peptide solution in a molar ratio of 1:1.2. After reacting for 1 to 3 hours, acetic acid is added to adjust the pH to 6.6, and the mixture is concentrated in a rotary evaporator until a solid precipitate forms. Water is then added to obtain the desired product as a white solid. Purity: 50%, Theoretical molecular weight: 1237 Da, Actual molecular weight will be verified by LC-MS.

[0156] Example 9: In vitro activity of FGF21 mutant These FGF21 mutants were constructed, fermented, and purified according to Example 1. All mutants were successfully prepared. [Table F]

[0157] The in vitro activity of FGF21 cysteine ​​mutants modified with FGF21RGE and PAS was evaluated according to the method of Example 3. The results are shown in the table below. Successfully expressed FGF21 mutants exhibit activity that stimulates downstream signaling of the FGFR1c receptor. Compared to the FGF21RGE control protein, PAS200FGF21 activity was significantly reduced, while the remaining mutants had in vitro activity equivalent to that of the control protein.

[0158] [Table G]

[0159] Example 10: Preparation of PAS-FGF21 dimer analog Preparation of compound 10.1 (compound 10.1) [ka] The compound in question is PAS with SEQ ID NO:13. 15 Modified FGF21 fusion protein PAS 15 The FGF21 is an FGF21 analog modified with the side chain described in Example 8.3, and the preparation process and identification are as follows.

[0160] Process: Before modification reaction, PAS 15The FGF21 mutant is filtered to Tris-HCl and 10 mM EDTA-2Na buffer (pH 7.5) to a final concentration of 1-4 mg / ml. Simultaneously, it is added to 0.1 mM TCEP (tris(2-carboxyethyl)phosphine, dissolved in Tris-HCl buffer and adjusted to pH 8.0). The side chain described in Example 8.3 is dissolved in ethanol (concentration of 4-10 mg / ml), with a molar ratio of side chain to protein of side chain:protein = 0.9:2. The side chain solution is added to the reaction in 10 portions at 5-minute intervals and reacted overnight under stirring conditions. After the reaction is complete, the PAS-FGF21 dimer analog is purified by anion exchange. Purified water was added to the reaction mixture, and the conductivity was 6 mS / cm. The sample was collected in a pre-equalized HiTrap Q HP (purchased from Cytiva). Mobile phase A was 25 mM Tris-HCl (pH 8.0), and mobile phase B was 25 mM Tris-HCl + 1 M NaCl (pH 8.0). The flow rate was 5 ml / min, the operating time was 40 minutes, and the gradient was 0-50%. The target peak was aligned by RP-HPLC and SDS-PAGE analysis, and high-purity PAS with SEQ ID NO: 13 was obtained. 15 An FGF21 mutant is modified with the albumin-binding agent described in Example 8.3 to obtain an FGF21 analog. Purity: >85%, Theoretical molecular weight: 41838.8Da, Actual molecular weight: Confirmed.

[0161] Preparation of compound 10.2 (compound 10.2) [ka] The compound is an FGF21 analog in which the FGF21 fusion protein PAS30FGF21, having SEQ ID NO:14 and PAS50 modification, is modified with the side chain described in Example 8.3. The preparation process and identification are as follows. Process: Prepared in the same manner as compound 10.1, with a molar ratio of side chain to protein of side chain:protein = 0.9:2, and the side chain is dissolved in ethanol. The side chain solution is added to the reaction in 10 fractions at 5-minute intervals. Purity: >85%, theoretical molecular weight: 44243.6 Da, actual molecular weight: confirmed.

[0162] Preparation of 10.3 (Compound 10.3)

Chem.

[0163] Preparation of 10.4 (Compound 10.4)

Chem.

[0164] Preparation of 10.5 (Compound 10.5)

Chem.

[0165] Example 11: In vitro activity of PAS-FGF21 dimer analog The in vitro activity of FGF21RGE, a series of PAS-FGF21 mutants, and PAS-FGF21 dimer analogs was evaluated using the method of Example 3. The results are shown in the table below. [Table H]

[0166] Example 12: Preparation of PAS-FGF21 dimer analogs with fatty acid modification Preparation of compound 12.1 (compound 12.1) [ka] The compound is an FGF21 fusion protein PAS with SEQ ID NO:13 PAS15 modification. 15 The FGF21 is an FGF21 analog modified with the side chain described in Example 8.5, and the preparation process and identification are as follows. Process: Prepared in the same manner as compound 10.1. Purity: >85%, Theoretical molecular weight: 42679.8Da, Actual molecular weight: Confirmed.

[0167] Preparation of compound 12.2 (compound 12.2) [ka] When the compound is an FGF21 fusion protein PAS having the PAS30 modification of SEQ ID NO:14 30 FGF21 is an FGF21 analog modified with the side chain described in Example 8.5, and the preparation process and identification are as follows. Process: Prepared in the same manner as Compound 10.1. Purity: >85%, theoretical molecular weight: 45084.6 Da, actual molecular weight: confirmed.

[0168] Preparation of 12.3 (Compound 12.3)

Chemical formula

[0169] Preparation of 12.4 (Compound 12.4)

Chemical formula

[0170] Preparation of 12.5 (Compound 12.5)

Chemical formula

[0171] Preparation of compound 12.6 (compound 12.6) [ka] The compound is an FGF21 analog in which the FGF21 fusion protein PAS200FGF21, having SEQ ID NO:17 and PAS200 modification, is modified with the side chain described in Example 8.6. The preparation process and identification are as follows. Process: Prepared in the same manner as compound 10.1. Purity: >85%, Theoretical molecular weight: 48186.8Da, Actual molecular weight: Confirmed.

[0172] Example 13: In vitro activity of PAS-FGF21 dimer analog with fatty acid modification The in vitro activity of FGF21RGE, a series of PAS-FGF21 mutants, and PAS-FGF21 dimer analogs with fatty acid modifications was evaluated using the method of Example 3. The results are shown in the table below. [Table I]

[0173] Example 14: Pharmacokinetic study The pharmacokinetic properties of protein analogs can be tested using mice or miniature pigs. Mice study (N=3): The injection dose was 1 mg / kg, and the injection method was subcutaneous. Plasma samples were collected at 0.08, 0.25, 0.5, 1, 2, 4, 6, 8, 12, 24, 48, and 72 hours. Blood was collected in EDTA-2K coated tubes (which needed to be stored on ice temporarily) and centrifuged at 1200 × g for 10 minutes at 4°C. The plasma was then transferred to a micronic tube and stored at -20°C. Plasma drug concentrations were monitored using the Biovendor Human FGF21 Detection ELISA Kit (RD191108200R).

[0174] Minipig study (N=3): The injection dose was 1 mg / kg, administered subcutaneously. Plasma samples were collected at 0.083, 0.25, 0.5, 1, 2, 4, 6, 8, 12, 24, 48, 72, 96, 120, 144, 168, 192, 216, 240, 264, and 288 hours. Blood was collected in EDTA-2K coated tubes (which needed to be stored on ice temporarily) and centrifuged at 1200 × g for 10 minutes at 4°C. The plasma was then transferred to Micronic tubes and stored at -20°C. Plasma drug concentrations were monitored using the Biovendor Human FGF21 Detection ELISA Kit (CAT: RD191108200R).

[0175] Figure 1 shows the in vivo pharmacokinetic curves in mice. The in vivo half-lives of compounds 5.7 and 5.15 in mice were 15.9 hours and 14.1 hours, respectively, while the measured half-life of FGF21RGE (SEQ ID NO:2) was only 0.5 hours. Figure 2 shows the in vivo pharmacokinetic curves in miniature pigs, with the in vivo half-lives of compounds 12.2, 12.4, and 12.6 in miniature pigs being 65.9 ± 3.8 hours, 75.4 ± 9.4 hours, and 61.3 ± 1.1 hours, respectively.

[0176] Example 10. Pharmacological study in high-fat diet-induced db / db mice Construction of a high-fat diet-induced db / db mouse model: Male db / db mice, 8 weeks old, are adapted to a standard diet for 2-3 days, then their diet is switched entirely to a 60% high-fat diet. After 3 weeks of feeding on the high-fat diet, a NASH model can be obtained. Administration: N=6, Vehicle and protein analogs (5 or 20 nmol / kg) are administered subcutaneously every 3 days for 6 weeks.

[0177] Liver tissue sections will be collected at the end of the administration period and stained with hematoxylin and eosin to detect the effect of protein analogs on the non-alcoholic fatty liver disease activity score (NAS score) in high-fat diet-induced db / db mice. [Table J]

[0178] Sequences involved in the examples: SEQ ID NO:1 >Human mature FGF21 HPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVGSSDPLSMVGPSQGRSPSYAS

[0179] SEQ ID NO:2 Human mature FGF21 mutant RGE (FGF21RGE) HPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRERLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVGGSQGRSPSYES

[0180] SEQ ID NO:3 Human mature FGF21 mutant RGE (His6-SUMO-FGF21RGE) containing an N-terminal 6-polyhistidine (His6) tag and a ubiquitin-like modified protein (SUMO) tag. MAHHHHHHSSSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGGHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIRE DGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRERLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPAPPPEPPGILAPQPPDVGSSDPLSMVGGSQGRSPSYES

[0181] SEQ ID NO:4 Human mature FGF21 mutant Q28C (FGF21Q28C) HPIPDSSPLLQFGGQVRQRYLYTDDAQCTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRERLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVGGSQGRSPSYES

[0182] SEQ ID NO:5 Human mature FGF21 mutant E30C (FGF21E30C) HPIPDSSPLLQFGGQVRQRYLYTDDAQQTCAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRERLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVGGSQGRSPSYES

[0183] SEQ ID NO:6 Human mature FGF21 mutant D38C (FGF21D38C) HPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIRECGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRERLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVGGSQGRSPSYES

[0184] SEQ ID NO:7 Human mature FGF21 mutant D46C (FGF21D46C) HPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAACQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRERLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVGGSQGRSPSYES

[0185] SEQ ID NO:8 Human mature FGF21 mutant K122C (FGF21K122C) HPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRERLLEDGYNVYQSEAHGLPLHLPGNCSPHRDPAPRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVGGSQGRSPSYES

[0186] SEQ ID NO:9 Human mature FGF21 mutant H125C (FGF21H125C) HPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRERLLEDGYNVYQSEAHGLPLHLPGNKS PCRDPAPRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVGGSQGRSPSYES

[0187] SEQ ID NO:10 >Human mature FGF21 fusion protein with N-terminal PAS modification (PAS 50 FGF21) SPCASPAAPAPASPAAPAPSAPAAASAAAPAAASAAAASAPSAASAAAASPAAHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQI LGVKTSRFLCQRPDGALYGSLHFDPEACSFRERLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVGGSQGRSPSYES

[0188] SEQ ID NO:11 >PAS 50 SPASPAAPAPASPAAPAPSAPAAASAAPAAASAAAASAPSAASAAASPAA SEQ ID NO:12 >Cysteine ​​was inserted in 3rd place in PAS 50 SPCASPAAPAPASPAAPAPSAPAAASAAPAAASAAAASAPSAASAAASPAA

[0189] SEQ ID NO:13 >Human mature FGF21 fusion protein with N-terminal PAS15 modification (PAS 15 FGF21) SPCASPAAPAPASPAAHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRERLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVGGSQGRSPSYES

[0190] SEQ ID NO:14 >Human mature FGF21 fusion protein with N-terminal PAS30 modification (PAS30FGF21) SPCASPAAPAPASPAAPAPSAPAAASAAAPAHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRERLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVGGSQGRSPSYES

[0191] SEQ ID NO:15 >N-terminal PAS 50 Modified human mature FGF21 fusion protein (PAS 50 FGF21) SPCASPAAPAPASPAAPAPSAPAAASAAAPAAASAAAASAPSAASAAAASPAAHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQI LGVKTSRFLCQRPDGALYGSLHFDPEACSFRERLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVGGSQGRSPSYES

[0192] SEQ ID NO:16 Human mature FGF21 fusion protein with N-terminal PAS100 modification (PAS100FGF21) SPCASPAAPAPASPAAPAPSAPAAASAAAPAAAASAAASAPSAASAAASPAAPSAPPAAASPAAPSAPPAAASPAAPAPASPAASAPSAPAAASPAAPAPASHPIPDSSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGT VGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRERLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPAPPPEPPGILAPQPPDVGSSDPLSMVGGSQGRSPSYES

[0193] SEQ ID NO:17 Human mature FGF21 fusion protein with N-terminal PAS200 modification (PAS200FGF21) SPCASPAAPAPASPAAPAPSAPAAASAAAPAAAASAAASAPSAASAAASPAAPSAPPAAASPAAPSAPPAAASPAAPAPASPASPAASAPSAPAAASPAAPAPASPAAAPAPSAPAAASAAAPAAASAAASAPSASAAASAAAPAAAASAAAPSAASSAASPAAPSAPPAAASPAAPSAPPAAASPAAPAPASPA APAPSAPAAAHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRERLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVGGSQGRSPSYES

[0194] All documents referenced in this invention are cited as references in this application, as if each document were cited individually. Furthermore, after reading the above teachings of this invention, those skilled in the art will understand that various changes or modifications can be made to the invention, and these equivalent forms also fall within the scope defined by the claims appended to this application.

Claims

1. Modified proteins, The modified protein comprises a protein portion and a modified portion, wherein the protein portion has a PAS optionally linked to its N-terminus, and the modified portion is linked directly or via a linker to a cysteine ​​residue in the protein portion or to a cysteine ​​residue in the PAS, and the cysteine ​​residue is either naturally occurring or introduced by mutation.

2. The protein is fibroblast growth factor 21. The modified protein according to claim 1.

3. The modified portion is an albumin binder. The modified protein according to claim 1.

4. The structure of the albumin binder is as shown in the following formula: R １ -(X １ ) ｍ -(X ２ ) ｎ -K(ε-R ２ ) In the formula, K is lysine, R １ C is either substituted or non-substituted. １－２０ It is an acyl group, m is an integer between 0 and 5. n is an integer between 0 and 5. X １ and X ２ is independently selected from the group consisting of alanine (Ala), D-alanine (D-Ala), β-alanine (β-Ala), 4-aminobutyric acid (GABA), 2-aminoisobutyric acid (Aib), 2-aminobutyric acid (Abu), arginine (Arg), aspartic acid (Asp), asparagine (Asn), cysteine (Cys), glutamic acid (Glu), D-glutamic acid (D-Glu), γ-glutamic acid (γ-Glu), glutamine (Gln), glycine (Gly), histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys), proline (Pro), phenylalanine (Phe), serine (Ser), tyrosine (Tyr), threonine (Thr), tryptophan (Trp), valine (Val), methionine (Met), tranexamic acid (Trx), AEEA, PEG; R ２ is halogen-substituted C １－６ Selected from an acyl group or H, The modified protein according to claim 3.

5. Albumin binders include the following: 【Chemistry 1】 Selected from the group consisting of, The modified protein according to claim 4.

6. PAS is PAS １５ - PAS ２００ And preferably PAS １５ - PAS １００ For example, PAS ５０ That is, The modified protein according to claim 1.

7. The linker is a polyvalent linker, which allows for the linking of one or more albumin binders and one or more protein moieties. The modified protein according to claim 1.

8. The structure of the polyvalent linker is as shown in the following equation: 【Chemistry 2】 In the formula, LG1 is a leaving group that is reactive with primary amino groups. LG2 is a leaving group that is reactive with the sulfidyl group. The modified protein according to claim 7.

9. The modified protein is selected from compounds numbered 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 5.10, 5.11, 5.12, 5.13, 5.14, 5.15, 7.2, 7.3, 7.4, 7.5, 12.1, 12.2, 12.3, 12.4, 12.5, and 12.

6. A modified protein according to any one of claims 1 to 8.

10. It is a protein polymer, The protein polymer comprises a polyvalent linker and a plurality of protein monomers, wherein the protein monomers are optionally linked to a PAS at their N-terminus, and the polyvalent linker is linked to a cysteine ​​residue in the protein monomer or a cysteine ​​residue in the PAS, wherein the cysteine ​​residue is naturally occurring or introduced by mutation.

11. PAS is PAS １５ - PAS ２００ And preferably PAS １５ - PAS １００ For example, PAS ５０ That is, The protein polymer according to claim 10.

12. The protein is fibroblast growth factor 21. The protein polymer according to claim 10.

13. The structure of a polyvalent linker is as shown in the following equation: The protein polymer according to claim 10. 【Transformation 3】

14. The protein polymer is selected from compounds numbered 10.1, 10.2, 10.3, 10.4, and 10.

5. The protein polymer according to claim 10.

15. A pharmaceutical composition, The pharmaceutical composition comprises a modified protein according to any one of claims 1 to 9 or a protein polymer according to any one of claims 10 to 15 and a pharmaceutically acceptable excipient.

16. Use of a modified protein according to any one of claims 1 to 9 or a protein polymer according to any one of claims 10 to 15 in the preparation of a drug.

17. Albumin binder, The structure of the albumin binder is as shown in the following formula: R １ -(X １ ) ｍ -(X ２ ) ｎ -K(ε-R ２ ) In the formula, K is lysine, R １ C is either substituted or non-substituted. １－２０ It is an acyl group, m is an integer between 0 and 5. n is an integer between 0 and 5. X １ and X ２ These include alanine (Ala), D-alanine (D-Ala), β-alanine (β-Ala), 4-aminobutyric acid (GABA), 2-aminoisobutyric acid (Aib), 2-aminobutyric acid (Abu), arginine (Arg), aspartic acid (Asp), asparagine (Asn), cysteine ​​(Cys), glutamic acid (Glu), D-glutamic acid (D-Glu), γ-glutamic acid (γ-Glu), and glutamine (G Independently selected from the group consisting of ln), glycine (Gly), histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys), proline (Pro), phenylalanine (Phe), serine (Ser), tyrosine (Tyr), threonine (Thr), tryptophan (Trp), valine (Val), methionine (Met), tranexamic acid (Trx), AEEA, and PEG, R ２ is halogen-substituted C １－６ The albumin binder selected from an acyl group or H.

18. The albumin binder is selected from the following group: The albumin binder according to claim 18. 【Chemistry 4】

19. It is a polyvalent linker, The structure of the aforementioned polyvalent linker is as shown by the following formula: 【Transformation 5】 In the formula, LG1 is a leaving group that is reactive with primary amino groups. LG2 is the polyvalent linker, which is a leaving group that is reactive with the sulfidyl group.

20. It is a polypeptide, The polypeptide comprises the amino acid sequence shown in SEQ ID NO:

11.

21. It is a polypeptide, The amino acid sequence of the polypeptide is as shown in SEQ ID NO: 11, wherein a cysteine ​​residue is inserted from the N-terminus to the 3rd position.

22. It is a polypeptide, The polypeptide comprises SEQ ID NO: 4 to 9, and preferably comprises an amino acid sequence shown in any of SEQ ID NO: 5 to 9.

23. Isolated nucleic acid molecules, The nucleic acid molecule is the isolated nucleic acid molecule which encodes the polypeptide described in any one of claims 21 to 23.

24. An expression vector, The expression vector comprises the nucleic acid molecule described in claim 24.

25. It is a host cell, The host cell comprises the expression vector described in claim 25, or the host cell's genome incorporates the nucleic acid molecule described in claim 24.

26. The use of a polypeptide according to any one of claims 21 to 23 in the preparation of a modified protein according to any one of claims 1 to 9, a protein polymer according to any one of claims 10 to 15, or a pharmaceutical composition according to claim 16.

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