FGF21 derivative and use thereof

Abstract

A long-acting FGF21 derivative, specifically relating to an FGF21 derivative comprising an FGF21 mutant protein and a fatty acid chain. The FGF21 derivative, which is obtained by coupling the fatty acid chain to a cysteine residue at position 172 of an FGF21 protein by means of a linker, has relatively high FGF21 activity, and has a better weight loss effect in model mice. The FGF21 mutant protein not only retains the activity of mature FGF21, but also has significantly improved stability in serum.

Description

FGF21 derivatives and their uses Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a long-acting FGF21 derivative, its preparation method, and its uses. Background Technology

[0002] FGF21 (Fibroblast Growth Factor 21), along with FGF15 / 19 and FGF23, belongs to the FGF family of endocrine hormones. These are members of a polypeptide family widely expressed in developmental and adult tissues, playing important roles in various physiological functions. The human FGF21 protein consists of 209 amino acids; the mature FGF21 protein is obtained by cleaving the N-terminal 28-amino acid signal peptide. FGF21 is mainly expressed in pancreatic β-cells, liver, WATS, and skeletal muscle. Recent studies have also found lower expression in the thymus, vascular endothelium, kidney, and testicular tissues, demonstrating significant tissue specificity. In the presence of the co-receptor β-Klotho, FGF21 can bind to and activate three FGF receptor subtypes (FGFR1c, FGFR2c, and FGFR3c). The tissue-specific expression of β-Klotho in the pancreas, liver, and adipose tissue determines the metabolic activity of FGF21 (Kurosu H, et al., J Biol Chem 282:26687-26695, 2007; Kharitonenkov A, et al., J Cell Physiol 215:1-7, 2008). FGF21 is an important hormone regulating glucose and lipid metabolism and has the potential to treat diseases such as diabetes, obesity, dyslipidemia, and metabolic syndrome.

[0003] The N and C terminals of the FGF21 protein are crucial to its biological activity, with the N-terminus binding to FGFR and the C-terminus binding to β-Klotho. Human FGF21 is readily degraded in vivo by prolyl peptidase (FAP, a serine protease), with a half-life of approximately 30 min in mice and 2 h in monkeys. The main sites of FAP degradation of FGF21 are Pro2, Pro4, and Pro171, with degradation of Pro171 inactivating FGF21. In addition, studies by Amgen and Lilly have shown that the deletion of 5-6 or more amino acids at the N-terminus and 2-3 or more amino acids at the C-terminus of FGF21 significantly affects its activity (Yie, J., et al, FEBS letters, 583(1), 19-24, 2009; Micanovic R, et al, J Cell Physiol 219:227-234, 2009).

[0004] Studies have shown that modifying the C-terminal region of FGF21 can reduce degradation. Different modification methods have been applied, including FGF21 mutants, Fc fusion, and PEG conjugation. The mutation sites of clinically available FGF21 products are mainly concentrated at positions 98, 121, 167, 170, 171, 175, and 180. Among them, mutations at positions 171 and 180 can largely prevent degradation. However, simple mutants cannot solve the problem of short clinical half-life. They often need to be fused with long-acting units or chemically cross-linked. However, the biological activity of this long-acting form of FGF21 molecule may be reduced or even completely lost. For example, the FGF21-Fc fusion protein obtained by fusing Fc to the C-terminus of FGF21 has much lower activity than natural FGF21 and Fc-FGF21 (Fc fused to the N-terminus of FGF21) (Hecht, R., et al., PloS one, 7(11), e49345, 2012). Besides product heterogeneity, the PEG-conjugated product of FGF21 also carries the risk of tissue vacuolation, particularly in the renal tubular epithelium, where it accumulates and is difficult to metabolize and remove. For drugs treating metabolic diseases such as diabetes and obesity, safety is paramount. Therefore, developing a new drug that is long-acting, highly active, stable, homogeneous, and easy to manufacture has significant clinical value for patients with metabolic diseases. Summary of the Invention

[0005] In view of the shortcomings of the prior art described above, the object of the present invention is to provide a new FGF21 mutant and its derivatives. A method for their preparation and uses are also provided to address the problems in the prior art.

[0006] A first aspect of the present invention provides an FGF21 derivative comprising an FGF21 mutant and a fatty acid chain HOOC-(CH2)a-CO-. The amino acid sequence of the FGF21 mutant, compared to the sequence shown in SEQ ID NO. 16 (human naturally mature FGF21), includes an S172C mutation (serine at position 172 is replaced by cysteine). The fatty acid chain HOOC-(CH2)a-CO- is linked via a linker to the cysteine ​​residue at position 172 of the FGF21 mutant. The linker is selected from one of the following: -γGlu-2×OEG-NH-(CH2)2-NH-CO-CH2-, -γGlu-2×OEG-γGlu-NH-(CH2)2-NH-CO-CH2-, -γGlu-OEG-γGlu-OEG-NH-(CH2)2-NH-CO-CH2-, or -γGlu-OEG-2×γGlu-OEG-NH-(CH2)2-NH-CO-CH2-, wherein the -γGlu end is attached to the CO-end of the fatty acid chain HOOC-(CH2)a-CO-, and the CH2-end is attached to the cysteine ​​residue at position 172 of the FGF21 mutant. Furthermore, a is an integer greater than or equal to 12 and less than or equal to 20, for example, a is 12, 13, 14, 15, 16, 17, 18, 19, or 20.

[0007] In this invention, the amino acid sequence of the FGF21 mutant, compared to the sequence shown in SEQ ID NO. 16, further includes one or more amino acid mutations selected from the following: Q108A, P124A, P143L, A144A, A154G. In some preferred embodiments, the amino acid sequence of the FGF21 mutant, compared to the sequence shown in SEQ ID NO. 16, includes any one of the following amino acid mutations: Q108A, P124A, P143L, A144A, A154G. In some preferred embodiments, the amino acid sequence of the FGF21 mutant is as shown in any one of SEQ ID NO. 1 to 5.

[0008] In the embodiments of this invention, *E. coli* was used as the expression system for the FGF21 mutant. *E. coli* is known for its ease of operation, low cost, high expression levels, and short fermentation cycle. However, it is well known that proteins expressed in the *E. coli* system theoretically have their N-terminal methionine (Met) encoded by the start codon cleaved by intracellular proteases. However, this cleavage efficiency can vary significantly depending on the nature of the first amino acid following Met. For example, the presence of the first amino acid His in the FGF21 protein or the FGF21 mutant often leads to extremely low cleavage efficiency, resulting in Met residue and reduced product homogeneity. Adding alanine (Ala) to the N-terminus of the FGF21 mutant protein can improve the cleavage efficiency of the protease. The FGF21 mutant of this invention can also be prepared and expressed in eukaryotic expression systems, such as *Pichia pastoris*, mammalian CHO cells, or HEK293 cells. In these recombinant protein expression systems with secretory signal peptides, the signal peptide is completely cleaved before secretion, thus eliminating the need to consider the problem of N-terminal Met residue.

[0009] In other embodiments of this invention, a histidine His tag (6xHis) is added to the N-terminus of the FGF21 mutant. The His tag can bind affinity to the nickel ion medium on the nickel chromatography column, improving sample purity and facilitating preparation.

[0010] In this invention, adding alanine (Ala) or a His tag (6xHis) to the N-terminus of the FGF21 mutant is merely a means to improve product purity and should not be regarded as a limitation of this invention.

[0011] More specifically, in some embodiments, the amino acid sequence of the FGF21 mutant is shown in any one of SEQ ID NO. 6 to 15.

[0012] In this invention, the fatty acid chain is a dicarboxylic acid with the structural formula HOOC-(CH2)a-CO-. The fatty acid chain can non-covalently and reversibly bind to albumin in serum, utilizing the mechanism of albumin-FcRN cycling to prolong the half-life of the protein drug.

[0013] In some embodiments, a is 16, and the fatty acid chain is HOOC-(CH2). 16 -CO-. In some embodiments, a is 17, and the fatty acid chain is HOOC-(CH2). 17 -CO-. In some embodiments, a is 18, and the fatty acid chain is HOOC-(CH2). 18 -CO-.

[0014] In some embodiments, the linker is -γGlu-2×OEG-γGlu-NH-(CH2)2-NH-CO-CH2-.

[0015] In some embodiments, the fatty acid chain is HOOC-(CH2). 16 -CO-, the linker is -γGlu-2×OEG-γGlu-NH-(CH2)2-NH-CO-CH2-. HOOC-(CH2) 16 -CO- is linked via the linker -γGlu-2×OEG-γGlu-NH-(CH2)2-NH-CO-CH2- to the cysteine ​​residue at position 172 of the FGF21 mutant, with its -γGlu terminus linked to the fatty acid chain HOOC-(CH2). 16 The CO-terminus of -CO- is attached to the CH2-terminus of the cysteine ​​residue at position 172 of the FGF21 mutant.

[0016] In some embodiments, the fatty acid chain is HOOC-(CH2). 16 -CO-, the linker is -γGlu-2×OEG-NH-(CH2)2-NH-CO-CH2-. HOOC-(CH2) 16 -CO- is linked via the linker -γGlu-2×OEG-NH-(CH2)2-NH-CO-CH2- to the cysteine ​​residue at position 172 of the FGF21 mutant, with its -γGlu terminus linked to the fatty acid chain HOOC-(CH2). 16 The CO-terminus of -CO- is attached to the CH2-terminus of the cysteine ​​residue at position 172 of the FGF21 mutant.

[0017] In some embodiments, the fatty acid chain is HOOC-(CH2). 16 -CO-, the linker is -γGlu-OEG-γGlu-OEG-NH-(CH2)2-NH-CO-CH2-. HOOC-(CH2) 16 -CO- is linked via the linker -γGlu-OEG-γGlu-OEG-NH-(CH2)2-NH-CO-CH2- to the cysteine ​​residue at position 172 of the FGF21 mutant, with its -γGlu terminus linked to the fatty acid chain HOOC-(CH2). 16 The CO-terminus of -CO- is attached to the CH2-terminus of the cysteine ​​residue at position 172 of the FGF21 mutant.

[0018] In some embodiments, the fatty acid chain is HOOC-(CH2). 16-CO-, the linker is -γGlu-OEG-2×γGlu-OEG-NH-(CH2)2-NH-CO-CH2-. HOOC-(CH2) 16 -CO- is linked via the linker -γGlu-OEG-2×γGlu-OEG-NH-(CH2)2-NH-CO-CH2- to the cysteine ​​residue at position 172 of the FGF21 mutant, with its -γGlu terminus linked to the fatty acid chain HOOC-(CH2). 16 The CO-terminus of -CO- is attached to the CH2-terminus of the cysteine ​​residue at position 172 of the FGF21 mutant.

[0019] In some embodiments of the present invention, the FGF21 derivative is selected from one of the following compounds or a pharmaceutically acceptable salt thereof:

[0020] In some specific embodiments of the present invention, the FGF21 derivative is selected from one of the following compounds or a pharmaceutically acceptable salt thereof:

[0021] In some specific embodiments of the present invention, the FGF21 derivative is selected from one of the following compounds or a pharmaceutically acceptable salt thereof:

[0022] A second aspect of this invention provides a method for preparing the FGF21 derivative described in the first aspect. The method includes preparing the FGF21 derivative via a coupling reaction. In some embodiments, the method includes: culturing suitable host cells under appropriate conditions to express the aforementioned FGF21 mutant; isolating and purifying the FGF21 mutant; and then chemically cross-linking a fatty acid chain onto the FGF21 mutant to provide the aforementioned long-acting FGF21 derivative. For example, the host cell typically contains a construct encoding the aforementioned FGF21 mutant polynucleotide, or its genome integrates an exogenous polynucleotide encoding the aforementioned FGF21 mutant.

[0023] A third aspect of the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of the FGF21 derivative described in the first aspect of the present invention. The pharmaceutical composition may further comprise a pharmaceutically acceptable carrier. These carriers may include various excipients and diluents, which are generally not essential active ingredients themselves and do not cause excessive toxicity upon administration. Suitable carriers should be well known to those skilled in the art; for example, a thorough discussion of pharmaceutically acceptable carriers can be found in Remington's Pharmaceutical Sciences (Mack Pub. Co., NJ, 1991).

[0024] In some embodiments of the present invention, the FGF21 derivative or its pharmaceutical composition may be used as a single active ingredient or in combination with other active ingredients.

[0025] A fourth aspect of this invention provides the use of the FGF21 derivative described in the first aspect or the pharmaceutical composition described in the third aspect in the preparation of a medicament. The FGF21 derivative provided by this invention exhibits activity advantages comparable to or even higher than that of the naturally occurring mature FGF21 protein in in vitro cell activity experiments. Correspondingly, in in vivo experiments, it can effectively reduce the body weight of test subjects (e.g., the disease model DIO mouse), thereby enabling its use in the preparation of a medicament.

[0026] In some embodiments of the present invention, the above-described drug can be used to prepare a drug for treating metabolic diseases. For example, metabolic diseases may be selected from diabetes, obesity, dyslipidemia, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), other metabolic syndromes associated with diabetes, high triglycerides, low HDL cholesterol and high LDL cholesterol, insulin resistance, obesity, or impaired glucose tolerance.

[0027] In some embodiments, diabetes is type I or type II diabetes; other metabolic syndromes associated with diabetes include glucose intolerance, elevated fasting blood glucose, arteriosclerosis, coronary heart disease, hypertension, etc.

[0028] A fifth aspect of the present invention provides a treatment method comprising: administering to an individual a therapeutically effective amount of the FGF21 derivative provided in the first aspect of the present invention, or a composition provided in the third aspect of the present invention. The treatment method provided by the present invention can be used to treat metabolic diseases, etc.

[0029] In this invention, "individual" generally includes humans and non-human primates, such as mammals, dogs, cats, horses, sheep, pigs, cattle, etc., who can benefit from treatment using the aforementioned drugs, compositions, formulations, kits, or combination formulations.

[0030] In this invention, "therapeutic effective dose" generally refers to a dose that, after an appropriate period of administration, can achieve the effect of treating or alleviating the diseases listed above.

[0031] The long-acting FGF21 derivative provided by this invention possesses high FGF21 biological activity, exhibits strong weight-loss activity in DIO mice, and demonstrates high bioavailability upon subcutaneous administration, showing promising prospects for industrialization. The dosing frequency is determined based on the half-life in mice. Theoretically, a long half-life combined with high biological activity results in a better therapeutic effect.

[0032] A sixth aspect of the present invention also provides an FGF21 mutant with enhanced activity, wherein the amino acid sequence of the FGF21 mutant contains one or more amino acid mutations selected from the following, compared with the natural human FGF21 sequence (SEQ ID NO. 16): Q108A, P124A, P143L, A144A, A154G. The FGF21 mutant exhibits higher biological activity than natural FGF21.

[0033] In some embodiments, the amino acid sequence of the FGF21 mutant contains an S172C mutation compared to the natural FGF21 sequence (SEQ ID NO.16).

[0034] In some embodiments of the present invention, the FGF21 mutant comprises an amino acid sequence as shown in any of SEQ ID NO. 1 to 5. More specifically, in some embodiments, the amino acid sequence of the FGF21 mutant is shown in any of SEQ ID NO. 6 to 15.

[0035] A seventh aspect of the present invention provides a polynucleotide or an expression vector containing the polynucleotide, wherein the polynucleotide encodes the FGF21 mutant described in the sixth aspect of the present invention; the expression system contains the expression vector or its genome having an exogenous polynucleotide integrated therein.

[0036] The eighth aspect of the present invention provides a method for preparing the FGF21 mutant according to the sixth aspect of the present invention, comprising the following steps: culturing the expression system according to the seventh aspect of the present invention under conditions suitable for expressing the FGF21 mutant, thereby expressing the FGF21 mutant; preferably, the method further comprises purifying and isolating the FGF21 mutant.

[0037] A ninth aspect of the present invention also provides an FGF21 derivative modified based on the FGF21 mutant described in the sixth aspect of the present invention. The FGF21 derivative comprises the FGF21 mutant and the fatty acid chain HOOC-(CH2). b -CO-. The fatty acid chain HOOC-(CH2) bThe -CO- terminus is linked via a linker to a cysteine ​​residue at position 172 of the FGF21 mutant. The linker is selected from one of the following: -γGlu-2×OEG-NH-(CH2)2-NH-CO-CH2-, -γGlu-2×OEG-γGlu-NH-(CH2)2-NH-CO-CH2-, -γGlu-OEG-γGlu-OEG-NH-(CH2)2-NH-CO-CH2-, or -γGlu-OEG-2×γGlu-OEG-NH-(CH2)2-NH-CO-CH2-, wherein the -γGlu terminus is linked to the fatty acid chain HOOC-(CH2). b The CO-terminus of -CO- is attached to the CH2-terminus of the FGF21 mutant at position 172. Furthermore, b is an integer greater than or equal to 12 and less than or equal to 20, for example, a is 12, 13, 14, 15, 16, 17, 18, 19, or 20.

[0038] In some embodiments, b is 16, and the fatty acid chain is HOOC-(CH2). 16 -CO-; or b is 17, and the fatty acid chain is HOOC-(CH2). 17 -CO-; or b is 18, and the fatty acid chain is HOOC-(CH2). 18 -CO-.

[0039] In some embodiments, the linker is -γGlu-2×OEG-γGlu-NH-(CH2)2-NH-CO-CH2-.

[0040] In some embodiments, the fatty acid chain is HOOC-(CH2). 16 -CO-, the linker is -γGlu-2×OEG-γGlu-NH-(CH2)2-NH-CO-CH2-, fatty acid chain HOOC-(CH2) 16 -CO- is linked via the linker -γGlu-2×OEG-γGlu-NH-(CH2)2-NH-CO-CH2- to the cysteine ​​residue at position 172 of the FGF21 mutant, with its -γGlu terminus linked to HOOC-(CH2). 16 The CO-terminus of -CO- is attached to the CH2-terminus of the cysteine ​​residue at position 172 of the FGF21 mutant.

[0041] In some embodiments, the fatty acid chain is HOOC-(CH2). 16 -CO-, the linker is -γGlu-2×OEG-NH-(CH2)2-NH-CO-CH2-. HOOC-(CH2)16 -CO- is linked via the linker -γGlu-2×OEG-NH-(CH2)2-NH-CO-CH2- to the cysteine ​​residue at position 172 of the FGF21 mutant, with its -γGlu terminus linked to the fatty acid chain HOOC-(CH2). 16 The CO-terminus of -CO- is attached to the CH2-terminus of the cysteine ​​residue at position 172 of the FGF21 mutant.

[0042] In some embodiments, the fatty acid chain is HOOC-(CH2). 16 -CO-, the linker is -γGlu-OEG-γGlu-OEG-NH-(CH2)2-NH-CO-CH2-. HOOC-(CH2) 16 -CO- is linked via the linker -γGlu-OEG-γGlu-OEG-NH-(CH2)2-NH-CO-CH2- to the cysteine ​​residue at position 172 of the FGF21 mutant, with its -γGlu terminus linked to the fatty acid chain HOOC-(CH2). 16 The CO-terminus of -CO- is attached to the CH2-terminus of the cysteine ​​residue at position 172 of the FGF21 mutant.

[0043] In some embodiments, the fatty acid chain is HOOC-(CH2). 16 -CO-, the linker is -γGlu-OEG-2×γGlu-OEG-NH-(CH2)2-NH-CO-CH2-. HOOC-(CH2) 16 -CO- is linked via the linker -γGlu-OEG-2×γGlu-OEG-NH-(CH2)2-NH-CO-CH2- to the cysteine ​​residue at position 172 of the FGF21 mutant, with its -γGlu terminus linked to the fatty acid chain HOOC-(CH2). 16 The CO-terminus of -CO- is attached to the CH2-terminus of the cysteine ​​residue at position 172 of the FGF21 mutant.

[0044] In some embodiments, the FGF21 derivative is selected from one of the following compounds or a pharmaceutically acceptable salt thereof:

[0045] In some specific embodiments of the present invention, the FGF21 derivative is selected from one of the following compounds or a pharmaceutically acceptable salt thereof:

[0046] In some specific embodiments of the present invention, the FGF21 derivative is selected from one of the following compounds or a pharmaceutically acceptable salt thereof:

[0047] The present invention also provides a method for preparing the above-mentioned FGF21 derivative, comprising: preparing the FGF21 derivative by coupling reaction.

[0048] The present invention also provides a pharmaceutical composition comprising the FGF21 mutant described in the sixth aspect or an FGF21 derivative thereof. The pharmaceutical composition may further comprise a pharmaceutically acceptable carrier. These carriers may include various excipients and diluents, which are generally not essential active ingredients themselves and do not cause excessive toxicity upon administration. Suitable carriers should be well known to those skilled in the art; for example, a thorough discussion of pharmaceutically acceptable carriers can be found in Remington's Pharmaceutical Sciences (Mack Pub. Co., NJ, 1991).

[0049] In some embodiments of the present invention, the FGF21 derivative or its pharmaceutical composition may be used as a single active ingredient or in combination with other active ingredients.

[0050] This invention also provides the use of the FGF21 mutant described in the sixth aspect of this invention, the FGF21 derivative described in the ninth aspect of this invention, or the above-described pharmaceutical compositions in the preparation of a drug. The long-acting FGF21 derivative provided by this invention has shown comparable or even higher activity advantages than mature FGF21 in in vitro cell activity experiments. Correspondingly, in in vivo experiments, it can effectively reduce the body weight of test subjects (e.g., the disease model DIO mouse), and thus can be used in the preparation of a drug.

[0051] In some embodiments of the present invention, the above-described drug can be used to prepare a drug for treating metabolic diseases. For example, metabolic diseases may be selected from diabetes, obesity, dyslipidemia, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), other metabolic syndromes associated with diabetes, high triglycerides, low HDL cholesterol and high LDL cholesterol, insulin resistance, obesity, or impaired glucose tolerance.

[0052] In some embodiments, diabetes is type I or type II diabetes; other metabolic syndromes associated with diabetes include glucose intolerance, elevated fasting blood glucose, arteriosclerosis, coronary heart disease, hypertension, etc. Attached Figure Description

[0053] Figure 1 shows a schematic diagram of the stability results of the FGF21 derivative in human serum in Example 6 of the present invention.

[0054] Figure 2 shows a schematic diagram of the weight change results of the FGF21 derivative in DIO mice in Example 7 of the present invention.

[0055] Figure 3 shows a schematic diagram of the cumulative feeding results of the FGF21 derivative in DIO mice in Example 7 of the present invention.

[0056] Figure 4 shows a schematic diagram of the weight change results of the FGF21 derivative in DIO mice in Example 8 of the present invention.

[0057] Figure 5 shows a schematic diagram of the cumulative feeding results of the FGF21 derivative in DIO mice in Example 8 of the present invention.

[0058] Figure 6 shows a schematic diagram of the pharmacokinetic (PK) results of the FGF21 derivative in SD rats in Example 9 of the present invention. Detailed Implementation

[0059] Through in-depth research, the applicant has provided an FGF21 derivative comprising an FGF21 mutant and a fatty acid chain. This FGF21 derivative exhibits excellent weight-loss effects in mice. The invention further provides a method for preparing the FGF21 derivative, compositions of the FGF21 derivative, and the use of the FGF21 derivative and its compositions in pharmaceutical applications.

[0060] In this document, the terms "FGF21 mutant," "FGF21 protein," "FGF21 mutant protein," or "FGF21 analog" are used interchangeably to refer to an analog or variant of FGF21, and more specifically, an analog or variant of human FGF21 (181aa) that has undergone one or more amino acid substitutions or mutations compared to mature human FGF21 (181aa). The mature human FGF21 sequence, compared to the full-length natural human FGF21 sequence, does not contain a 28-aa signal peptide, as shown in SEQ ID NO. 16. In the embodiments of this invention, *Escherichia coli* (E. coli) was used as the expression system for the FGF21 mutant. *E. coli* is known for its ease of operation, low cost, high expression levels, and short fermentation cycle. However, as mentioned above, the recombinant mature FGF21 mutant expressed in *E. coli* exhibits extremely low N-terminal methionine (Met) cleavage efficiency (due to the first amino acid at the N-terminus being His), and because most of the Met is retained, it is referred to as MetFGF21 (182aa), as shown in SEQ ID NO. 17. In some embodiments of the present invention, the N-terminus of the FGF21 mutant carries a His tag to improve purification efficiency, namely MHHHHHH (SEQ ID NO. 18). In other embodiments of the present invention, the FGF21 mutant can also use AlaFGF21 (i.e., Ala is uniformly added to the N-terminus of the FGF21 sequence), which results in the protein sequence expressed in *E. coli* not having Met at the N-terminus (automatically cleaved during in vivo expression), thus enhancing the uniformity of the expression product. It should be emphasized that the FGF21 mutant of the present invention can also be prepared and expressed in eukaryotic expression systems, such as mammalian cell CHO or HEK293 cells. Recombinant proteins expressed in these mammalian cells have had their signal peptides completely cleaved before being secreted extracellularly, thus eliminating the need to consider the issue of residual Met at the N-terminus. Therefore, the method used for preparation or the form of the mutant protein used in this invention should not be considered a limitation of the invention. To ensure a one-to-one correspondence with the natural mature human FGF21 sequence, the amino acid sequence of the AlaFGF21 analogue is numbered without the N-terminal Ala, starting from the second histidine (His), designated as 1, and the C-terminal serine (Ser) designated as 181.

[0061] The following are some examples of naming FGF21 mutants in this invention.

[0062] M-His6(Q108A, S172C)FGF21 represents a mutant of FGF21. At position 108 corresponding to mature human FGF21 (181aa, SEQ ID NO.16), Q at position 108 is replaced by A, resulting in the Q108A mutation; at position 172 corresponding to mature human FGF21 (181aa, SEQ ID NO.16), S at position 172 is replaced by C, resulting in the S172C mutation; and the N-terminus contains MHHHHHH. Therefore, the amino acid sequence of M-His6(Q108A, S172C)FGF21 is the N-terminus sequence of SEQ ID NO.1 with MHHHHHH, as shown in SEQ ID NO.6.

[0063] M-His6(P124A, S172C)FGF21 represents a mutant of FGF21. At position 124, corresponding to position 124 of mature human FGF21 (181aa, SEQ ID NO.16), P is replaced by A, resulting in the P124A mutation; at position 172, corresponding to position 172 of mature human FGF21 (181aa, SEQ ID NO.16), S is replaced by C, resulting in the S172C mutation; and the N-terminus contains MHHHHHH. Therefore, the amino acid sequence of M-His6(P124A, S172C)FGF21 is the N-terminus sequence of SEQ ID NO.2 with MHHHHHH, as shown in SEQ ID NO.7.

[0064] A(P144A, S172C)FGF21 represents a mutant of FGF21. At position 124 corresponding to mature human FGF21 (181aa, SEQ ID NO.16), P at position 144 is replaced by A, resulting in the P143A mutation; at position 172 corresponding to mature human FGF21 (181aa, SEQ ID NO.16), S at position 172 is replaced by C, resulting in the S172C mutation; the N-terminus contains alanine Ala. Therefore, the amino acid sequence of A(P144A, S172C)FGF21 is the N-terminus of SEQ ID NO.4 containing an Ala sequence, as shown in SEQ ID NO.14.

[0065] A(A154G, S172C)FGF21 represents a mutant of FGF21. At position 154, corresponding to position 154 of mature human FGF21 (181aa, SEQ ID NO.16), the A at position 154 is replaced by G, resulting in the A154G mutation. At position 172, corresponding to position 172 of mature human FGF21 (181aa, SEQ ID NO.16), the S at position 172 is replaced by C, resulting in the S172C mutation. The N-terminus contains alanine (Ala). Therefore, the amino acid sequence of A(A154G, S172C)FGF21 is the N-terminus of SEQ ID NO.5 containing an Ala sequence, as shown in SEQ ID NO.15.

[0066] Table 1. FGF21 mutant sequence

[0067] The term "FGF21 derivative" refers to a chemically modified FGF21 mutant in which one or more chemical substituents are covalently attached to one or more specific amino acid residues of the FGF21 mutant via linkers or adapters. This substituent may be referred to as a side chain. In this invention, the substituent is a fatty acid chain.

[0068] In this invention, the term "linker" refers to the portion between the FGF21 mutant and the fatty acid chain, also known as a connector. In this invention, one end of the linker is a -γGlu terminus, which connects to the fatty acid chain; the other end is a CH2- terminus, which connects to the thiol group on the cysteine ​​residue of the FGF21 mutant (e.g., via a bromination reaction (using a Br-substitute as the reaction reagent)), thereby coupling the fatty acid chain to the FGF21 mutant.

[0069] The term "pharmaceutical composition" refers to a formulation in which the biological activity of the active ingredient contained therein is effective and which does not contain any other ingredients that would have unacceptable toxicity to a subject who would receive the composition.

[0070] The term "bioavailability" refers to the fraction of an administered drug dose that reaches systemic circulation without alteration. When administered intravenously, the bioavailability of a drug is considered 100%. When a drug is administered via other routes (e.g., oral or subcutaneous), its bioavailability is generally reduced (due to incomplete absorption and metabolism). Therefore, bioavailability is considered an important parameter in pharmacokinetics. Specifically, bioavailability is typically calculated by dividing the area under the curve (AUC) of the drug at administration by the area under the curve of the drug at administration (AUC) administered intravenously; a higher ratio indicates higher bioavailability.

[0071] FGF21 derivatives

[0072] This invention provides an FGF21 derivative comprising an FGF21 mutant and a fatty acid chain. The amino acid sequence of the FGF21 mutant is further subjected to an S172C mutation compared to the sequence shown in SEQ ID NO.16 (mature FGF21) to crosslink the fatty acid. The fatty acid chain HOOC-(CH2)a-CO- is linked via a linker to the cysteine ​​thiol group at position 172 of the FGF21 mutant. The linker is selected from one of the following: -γGlu-2×OEG-NH-(CH2)2-NH-CO-CH2-, -γGlu-2×OEG-γGlu-NH-(CH2)2-NH-CO-CH2-, -γGlu-OEG-γGlu-OEG-NH-(CH2)2-NH-CO-CH2-, or -γGlu-OEG-2×γGlu-OEG-NH-(CH2)2-NH-CO-CH2-, with the -γGlu end of the linker connected to HOOC-(CH2). a -CO-'s CO- end, the CH2- end of the linker is attached to the thiol group of S172C of the FGF21 mutant; a is an integer greater than or equal to 12 and less than or equal to 20.

[0073] The chemical structural formula of OEG(-2-(2-(2-aminoethoxy)ethoxy)acetyl-) is shown below.

[0074] The 2×OEG indicates that two -OEGs are connected.

[0075] The chemical structural formula of γGlu(-γ-glutamyl-) is shown below.

[0076] The 2×γGlu represents two -γGlu molecules linked together.

[0077] Compared with the sequence shown in SEQ ID NO.16 (mature FGF21), the FGF21 mutant may further contain one or more amino acid mutations selected from the following: Q108A, P124A, P143L, A144A, A154G.

[0078] Preferably, in one embodiment of the present invention, the amino acid sequence of the FGF21 mutant is shown in SEQ ID NO. 6; in another embodiment of the present invention, the amino acid sequence of the FGF21 mutant is shown in SEQ ID NO. 7; in another embodiment of the present invention, the amino acid sequence of the FGF21 mutant is shown in SEQ ID NO. 8; in another embodiment of the present invention, the amino acid sequence of the FGF21 mutant is shown in SEQ ID NO. 9; and in another embodiment of the present invention, the amino acid sequence of the FGF21 mutant is shown in SEQ ID NO. 10.

[0079] Preferably, in one embodiment of the present invention, the amino acid sequence of the FGF21 mutant is shown in SEQ ID NO.11; in another embodiment of the present invention, the amino acid sequence of the FGF21 mutant is shown in SEQ ID NO.12; in another embodiment of the present invention, the amino acid sequence of the FGF21 mutant is shown in SEQ ID NO.13; in another embodiment of the present invention, the amino acid sequence of the FGF21 mutant is shown in SEQ ID NO.14; and in another embodiment of the present invention, the amino acid sequence of the FGF21 mutant is shown in SEQ ID NO.15.

[0080] In a specific embodiment of the present invention, the fatty acid chain adopts HOOC-(CH2). 16 The -CO- linker is -γGlu-2×OEG-γGlu-NH-(CH2)2-NH-CO-CH2-, with the following structural formula, where the -γGlu end is attached to the fatty acid chain HOOC-(CH2). 16 The CO-terminus of -CO- is attached to the CH2-terminus of the S172C group of the FGF21 mutant, and its CH2-terminus is attached to the thiol group.

[0081] In specific embodiments of the present invention, the FGF21 derivative is selected from one of the following compounds or a pharmaceutically acceptable salt thereof: formula (M-His6-I), formula (M-His6-II), formula (M-His6-III), formula (M-His6-IV), formula (M-His6-V), formula (M-His6-VI), formula (M-His6-VII), formula (M-His6-VIII), or formula (M-His6-IX). Alternatively, the FGF21 derivative is selected from one of the following compounds or a pharmaceutically acceptable salt thereof: formula (A1), formula (A-II), formula (A-III), formula (A-IV), formula (A-AV), formula (A-VI), formula (A-VII), formula (A-VIII), or formula (A-IX).

[0082] In specific embodiments of the present invention, some examples of the FGF21 derivative are shown in Table 2.

[0083] Table 2 FGF21 derivatives

[0084] The preparation methods for derivatives #6 and #7 are referenced in patent CN107108709B. Their structures are shown as formulas (M-His6-VI) and (M-His6-VII), respectively.

[0085] Pharmaceutical Composition

[0086] This invention provides a pharmaceutical composition containing an effective amount of the FGF21 derivative described herein and a carrier; the carrier is pharmaceutically, food-wise, or health-product-wise acceptable. The composition includes, but is not limited to, pharmaceutical compositions, food compositions, or health-product compositions.

[0087] As used herein, “effective amount” or “effective dose” means an amount that is functional or active in humans and / or animals and is acceptable to humans and / or animals, as used herein.

[0088] The dosage form of the pharmaceutical composition described in this invention can be diverse, as long as it enables the active ingredient to effectively reach the mammalian body. Examples include: gels, aerosols, tablets, capsules, powders, granules, syrups, solutions, or suspensions. Those skilled in the art can choose a convenient dosage form to treat the type of disease treated by the compounds according to this invention.

[0089] Suitable pharmaceutically acceptable carriers are well known to those skilled in the art. A thorough description of pharmaceutically acceptable carriers can be found in Remington's Pharmaceutical Sciences (Mack Pub. Co., NJ 1991). Pharmaceutically acceptable carriers in compositions may contain liquids such as water, phosphate buffer, Ringer's solution, physiological saline, balanced salt solution, glycerol, or sorbitol. Additionally, these carriers may contain auxiliary substances such as lubricants, flow aids, wetting agents or emulsifiers, pH buffers, and stabilizers such as albumin.

[0090] From the standpoint of ease of preparation and administration, preferred pharmaceutical compositions are solid compositions, especially tablets and solid-filled or liquid-filled capsules. The compounds of the present invention or pharmaceutical compositions thereof may also be stored in sterile instruments suitable for injection or infusion.

[0091] Those skilled in the art can select an appropriate therapeutically effective dose based on the actual situation, such as the size of the subject, the severity of the subject's symptoms, and the specific composition or route of administration chosen. The prescription for treatment (e.g., the determination of dosage) can be determined by a physician, typically considering factors including, but not limited to, the disease being treated, the individual patient's condition, the site of delivery, the method of administration, and other factors.

[0092] use

[0093] This invention provides the use of the above-mentioned FGF21 derivatives or pharmaceutical compositions in the preparation of drugs for treating metabolic diseases.

[0094] Examples of metabolic diseases that can be treated in this invention may include, but are not limited to, diabetes, obesity, dyslipidemia, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, other metabolic syndromes associated with diabetes, high triglycerides, low HDL cholesterol and high LDL cholesterol, insulin resistance, obesity, or impaired glucose tolerance.

[0095] This invention also provides a treatment method for a disease, comprising the steps of administering to an individual the FGF21 mutant provided in the first aspect of this invention, or the FGF21 derivative provided in the second aspect. In a randomized blood glucose testing experiment, DIO model mice administered the FGF21 derivative of this invention showed significantly better blood glucose lowering and weight loss effects than the control group.

[0096] Besides its applications in disease prevention, relief, or treatment, the FGF21 derivatives of this invention can also be applied to some non-therapeutic uses. As seen in some results from embodiments of the invention, the FGF21 derivatives can significantly reduce food intake, lower fat, reduce weight, or lower blood sugar. Therefore, the FGF21 derivatives of this invention can also be applied to subjects who do not yet exhibit disease characteristics but require control of food intake, reduction of fat, and weight loss.

[0097] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0098] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention.

[0099] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, apparatus, and materials used in the embodiments, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description of this invention, any prior art methods, apparatus, and materials similar to or equivalent to those described, apparatus, and materials in the embodiments of this invention may be used to implement the present invention.

[0100] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in this invention all employ conventional techniques in molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields. These techniques have been well described in existing literature; see Sambrook et al., *MOLECULAR CLONING: A LABORATORY MANUAL*, Second edition, Cold Spring Harbor Laboratory Press, 1989 and Third edition, 2001; Ausubel et al., *CURRENT PROTOCOLS IN MOLECULAR BIOLOGY*, John Wiley & Sons, New York, 1987 and periodic updates; the series *METHODS IN ENZYMOLOGY*, Academic Press, San Diego; Wolffe, *CHROMATIN STRUCTURE AND FUNCTION*, Third edition, Academic Press, San Diego, 1998; *METHODS IN ENZYMOLOGY*, Vol. 304, Chromatin (PM Wassarman and AP Wolffe, eds.), Academic Press, San Diego, 1999; and *METHODS IN MOLECULAR*. BIOLOGY, Vol. 119, Chromatin Protocols (PBBecker, ed.) Humana Press, Totowa, 1999, etc.

[0101] The abbreviations used in the examples have the following specific meanings:

[0102] Table 3

[0103] The manufacturers and product models of the commercially available amino acids, amino acid fragments, and resins involved in the examples are as follows:

[0104] Fmoc protected amino acid raw material, 2-CTC resin and Wang resin are all conventional commercially available reagents (protected amino acid manufacturer: Chengdu Zhengyuan Biochemical Technology Co., Ltd., resin manufacturer: Tianjin Nankai Hecheng Technology Co., Ltd.);

[0105] The organic solvents and other raw materials were all commercially available products (manufacturer: Sinopharm Chemical Reagent Co., Ltd.; chemically pure).

[0106] In addition, the conditions, equipment models, and manufacturers for HPLC and mass spectrometry are as follows:

[0107] Instrument: HPLC UltiMate3000 (Thermo Fisher); detection conditions are shown in Table 4 below.

[0108] Table 4

[0109] Preparative liquid phase: Beijing Innovation Tongheng, LC3000.

[0110] Mass spectrometry: The instrument model was 5800MALDI-TOF-TOF (AB SCIEX), and the analysis software was TOF / TOF Explorer and Data Explorer. The MS parameters were: Reflector Positive: CID (OFF), mass range (700-6500 Da), Focus Mass (1200 Da), Fixed laser intensity (5600), and Digitizer: Bin Size (0.5 ns).

[0111] A positive control, NNC0194-0499 analog, was prepared using the method described in CN107108709B (see compound 16). NNC0194-0499 analog is an FGF21 derivative.

[0112] A positive control, AKR-001analog, was prepared according to the method described in EP2427207B (see SEQ ID NO:47). AKR-001analog is an Fc-FGF21 fusion protein.

[0113] Example 1: Expression of mature human FGF21 and FGF21 mutant

[0114] The DNA and amino acid sequences of natural human FGF21 are available from public databases via accession number UNIPROT:Q9NSA1.

[0115] The gene sequence encoding mature human FGF21 or an FGF21 mutant was codon-optimized for expression in *E. coli* and cloned into the NdeI and EcoRI sites of the vector pET41a. This placed the FGF21 gene under the control of the phage T7 promoter. The expression construct was transformed into *E. coli* BL21(DE3). Single colonies were picked and grown in LB+Amp 100 μg / mL until an OD 450 of 0.5 was reached. Expression was induced at 37°C with 0.3 mM IPTG for 4 hours. A crude extract of the cells was prepared by sonication for analysis of FGF21 expression. Coomassie blue stained SDS-PAGE showed successful expression of FGF21, which was mainly identified in the precipitate.

[0116] In this application, MetFGF21 (SEQ ID NO. 17) is used as a reference compound. As previously mentioned, when mature FGF21 is produced via an E. coli expression system, the N-terminus of FGF21 retains methionine. However, the retention of Met is not considered to affect biological activity, therefore FGF21 (SEQ ID NO. 16) and MetFGF21 (SEQ ID NO. 17) are generally considered as reference compounds with equivalent biological activity.

[0117] Example 2: Purification of mature human FGF21 and FGF21 mutant

[0118] 2.1 Purification of FGF21 mutants (mutants corresponding to derivatives #1-7):

[0119] E. coli cell precipitate was resuspended at a 1:10 m / v ratio in a solution of 20 mM Tris, 2 M urea, and 5 mM cysteine ​​at pH 8.0, followed by sonication to lyse the cells (40% power, 3 s / 3 s, 1 g cells = 20 min). The supernatant was separated by centrifugation (10,000 × g, 30 min) and used for column purification. The supernatant was added to a Ni column (Ni-TED, Borglon) and eluted with 100 mM imidazole. The conductivity of the eluted sample was adjusted to approximately 4 ms / cm and added to an anion exchange column (Q Bestarose Fast Flow resin, Borglon). The equilibration buffer was 20 mM Tris at pH 8.0; the eluent was 20 mM Tris, 0.5 M NaCl, pH 8.0. After reequilibration, elution was performed with 50% eluent. The final product was analyzed by SDS-PAGE. The FGF21 mutants M-His6(Q108A, S172C)FGF21, M-His6(P124A, S172C)FGF21, M-His6(P143L, S172C)FGF21, M-His6(P144A, S172C)FGF21, and M-His6(A154G, S172C)FGF21 were finally obtained.

[0120] E. coli cell precipitate was resuspended at a 1:10 m / v ratio in a solution of 20 mM Tris, 2 M urea, and / or 5 mM cysteine ​​at pH 8.0, followed by sonication to disrupt the cells (40% power, 3 s / 3 s, 1 g cells for 20 min). The supernatant was separated by centrifugation (10,000 × g, 30 min) and used for column purification. The supernatant was added to an anion exchange column (Q Bestarose Fast Flow resin, Borglon) and eluted with 50–250 mM NaCl, collecting the FGF21 target peak. Ammonium sulfate was added to the target peak to a concentration of 1 mol / L, and the eluent was then added to a phenyl HP column (GE Healthcare) equilibrated at pH 8.0 with 20 mM Tris and 1 M ammonium sulfate. The column was washed with 20 mM Tris and 1.5 M sodium chloride at pH 8.0, followed by elution with 10% Tris-chloride buffer (20 mM Tris and 1.5 M sodium chloride at pH 8.0). The final product was analyzed by SDS-PAGE. The FGF21 mutants A(Q108A, S172C)FGF21, A(P124A, S172C)FGF21, A(P143L, S172C)FGF21, A(P144A, S172C)FGF21, and A(A154G, S172C)FGF21 were finally obtained.

[0121] 2.2 FGF21 mutant (the mutant corresponding to #8) and purification of mature FGF21:

[0122] E. coli cell precipitate was resuspended at a m / v ratio of 1:10 in 20 mM Tris, pH 8.0 solution, followed by sonication to disrupt the cells (40% power, 3 s / 3 s, 1 g cells for 20 min). Inclusion bodies were obtained by centrifugation (10,000 × g, 30 min) and dissolved in 20 mM Tris, 2 M Urea, 5 mM cysteine, pH 8.0. After complete dissolution, the supernatant was separated by centrifugation and loaded onto an anion exchange column (Q Bestarose Fast Flow resin, Borglon) and eluted with 50–250 mM NaCl. 1 M ammonium sulfate was added to the eluent, which was then loaded onto a phenyl HP column (GE Healthcare) equilibrated in 20 mM Tris and 1 M ammonium sulfate at pH 8.0. The column was washed with 20 mM Tris and 1.5 M sodium chloride at pH 8.0, followed by elution with 10% Tris-chloride buffer (20 mM Tris and 1.5 M sodium chloride at pH 8.0). The final product was analyzed by SDS-PAGE. The FGF21 mutant A (A154G, S172C)FGF21 was finally obtained.

[0123] Example 3: Preparation of reagents for deriving FGF21 mutants

[0124] Example 3.1 Preparation of CPK24

[0125] The steps are as follows:

[0126] 1. Attach mono-Fmoc ethylenediamine to 2-chlorotriphenylmethyl chloride resin. Mix mono-Fmoc ethylenediamine (23 mg, 100 μmol), 2-chlorotriphenylmethyl chloride resin (100 mg, 80 μmol), and 5 mL of DMF. Add DIPEA (70 μL, 400 μmol). Wash the resin three times each with DMF and DCM, and treat with CH3OH / DCM / DIPEA (8:1:1) for 10 min to block unreacted triphenylmethyl chloride sites. After vacuum drying, store the resin in a desiccator.

[0127] 2. Removal of Fmoc protecting groups. Take 50 mg of the resin (40 μmol), add 5 mL of 20% piperidine DMF solution, mix well, and shake for 1 min. Discard excess liquid. Add another 5 mL of 20% piperidine DMF solution, mix well, and shake for 15 min. After the reaction is complete, filter and wash the resin three times each with DMF and DCM.

[0128] 3. Dissolve the coupling reagents HATU (76 mg, 200 μmol) and DIPEA (35 μL, 200 μmol) in 5 mL of DMF. Then add the resin from the previous reaction, followed by N-Fmoc-L-glutamic acid α-tert-butyl ester (85 mg, 200 μmol). Mix well and react for 2 hours. Then wash the resin according to step 2 to remove the Fmoc protecting group.

[0129] 4. Dissolve the coupling reagents HATU (76 mg, 200 μmol) and DIPEA (35 μL, 200 μmol) in 5 mL of DMF. Then add the resin from the previous reaction, followed by [2-[2-(Fmoc-amino)ethoxy]ethoxy]acetic acid (77 mg, 200 μmol). Mix well and react for 2 hours. Then wash the resin as described above to remove the protecting group. Repeat the above steps. After both reactions are complete, wash the resin again to remove the protecting group.

[0130] 5. Dissolve the coupling reagents HATU (76 mg, 200 μmol) and DIPEA (35 μL, 200 μmol) in 5 mL of DMF. Then add the resin from the previous reaction, followed by N-Fmoc-L-glutamic acid α-tert-butyl ester (85 mg, 200 μmol). Mix well and react for 2 hours. Then wash the resin as described above to remove the Fmoc protecting group.

[0131] 6. Dissolve the coupling reagents HATU (76 mg, 200 μmol) and DIPEA (35 μL, 200 μmol) in 5 mL of DMF. Then add the resin from the previous reaction, followed by tert-butyl octadecanoate (74 mg, 200 μmol). Mix well and react for 2 hours. Filter and wash the resin with DMF and DCM.

[0132] 7. Treat the resin with 5 mL of 10% TFA in DCM solution (containing 10% H2O and 10% triisopropylsilane) for 1 h to cleave the reaction product from the previous step from the resin. Dissolve bromoacetic anhydride (55 mg, 200 μmol) and DIPEA (35 μL, 200 μmol) in 2 mL of DCM, then add the reaction product from the previous step, mix well, and stir for 30 min. Subsequently, remove TFA by drying under reduced pressure. Wash the resulting yellow residue several times with cold diethyl ether, and finally dry under a nitrogen stream to obtain a crude product as a yellow powder.

[0133] 8. Purify the crude product from the previous step using preparative chromatography. Dissolve the crude product (50 mg) in DMSO (0.1 mL) and dilute to 0.5 mL with 50% CH3CN- aqueous solution. Filter. Equilibrate the HPLC column (Phenomenex, PrepC18, 300A, 50 × 250 mm) with 10% CH3CN- aqueous solution (containing 0.1% TFA), load the filtrate, and elute with 10% CH3CN- aqueous solution (containing 0.1% TFA) to remove DMSO. Subsequently, increase the eluent composition from 10% CH3CN-water (containing 0.1% TFA) to 35% CH3CN-water (containing 0.1% TFA) within 1 min, perform linear elution, and increase the proportion of CH3CN in the eluent at a rate of 0.5% / min for 50 min. The purity of the eluted fractions was determined on an analytical reversed-phase C18 column (Phenomenex, C18, 120A, 4.6 × 50 mm). Fractions with a purity >95% were combined and freeze-dried to obtain CPK24 (17 mg, yield 16.6%).

[0134] 9. Molecular weight of the product was analyzed by ESI-MS: Calculated MW 1024.5, 1026.0; Measured value 1025.8 [M+1] + 1027.7 [M+1] + .

[0135] Example 3.2 Preparation of CPK25

[0136] The steps are as follows:

[0137] 1. Attach mono-Fmoc ethylenediamine to 2-chlorotriphenylmethyl chloride resin. Mix mono-Fmoc ethylenediamine (23 mg, 100 μmol), 2-chlorotriphenylmethyl chloride resin (100 mg, 80 μmol), and 5 mL of DMF. Add DIPEA (70 μL, 400 μmol). Wash the resin three times each with DMF and DCM, and treat with CH3OH / DCM / DIPEA (8:1:1) for 10 min to block unreacted triphenylmethyl chloride sites. After vacuum drying, store the resin in a desiccator.

[0138] 2. Removal of Fmoc protecting groups. Take 50 mg of the resin (40 μmol), add 5 mL of 20% piperidine DMF solution, mix well, and shake for 1 min. Discard excess liquid. Add another 5 mL of 20% piperidine DMF solution, mix well, and shake for 15 min. After the reaction is complete, filter and wash the resin three times each with DMF and DCM.

[0139] 3. Dissolve the coupling reagents HATU (76 mg, 200 μmol) and DIPEA (35 μL, 200 μmol) in 5 mL of DMF. Then add the resin from the previous reaction, followed by [2-[2-(Fmoc-amino)ethoxy]ethoxy]acetic acid (77 mg, 200 μmol). Mix well and react for 2 hours. Then wash the resin as described above to remove the Fmoc protecting group.

[0140] 4. Dissolve the coupling reagents HATU (76 mg, 200 μmol) and DIPEA (35 μL, 200 μmol) in 5 mL of DMF. Then add the resin from the previous reaction, followed by N-Fmoc-L-glutamic acid α-tert-butyl ester (85 mg, 200 μmol). Mix well and react for 2 hours. Then wash the resin as described above to remove the Fmoc protecting group.

[0141] 5. Dissolve the coupling reagents HATU (76 mg, 200 μmol) and DIPEA (35 μL, 200 μmol) in 5 mL of DMF. Then add the resin from the previous reaction, followed by [2-[2-(Fmoc-amino)ethoxy]ethoxy]acetic acid (77 mg, 200 μmol). Mix well and react for 2 hours. Then wash the resin as described above to remove the Fmoc protecting group.

[0142] 6. Dissolve the coupling reagents HATU (76 mg, 200 μmol) and DIPEA (35 μL, 200 μmol) in 5 mL of DMF. Then add the resin from the previous reaction, followed by N-Fmoc-L-glutamic acid α-tert-butyl ester (85 mg, 200 μmol). Mix well and react for 2 hours. Then wash the resin as described above to remove the Fmoc protecting group.

[0143] 7. Dissolve the coupling reagents HATU (76 mg, 200 μmol) and DIPEA (35 μL, 200 μmol) in 5 mL of DMF. Then add the resin from the previous reaction, followed by tert-butyl octadecanoate (74 mg, 200 μmol). Mix well and react for 2 hours. Wash the resin with DMF and DCM.

[0144] 8. Treat the resin with 5 mL of 10% TFA in DCM solution (containing 10% H2O and 10% triisopropylsilane) for 1 h to cleave the reaction product from the previous step from the resin. Dissolve bromoacetic anhydride (55 mg, 200 μmol) and DIPEA (35 μL, 200 μmol) in 2 mL of DCM, then add the reaction product from the previous step, mix well, and stir for 30 min. Subsequently, remove TFA by drying under reduced pressure. Wash the resulting yellow residue several times with cold diethyl ether, and finally dry under a nitrogen stream to obtain a crude product as a yellow powder.

[0145] 9. Purify the crude product from the previous step using preparative chromatography. Dissolve the crude product (50 mg) in DMSO (0.1 mL) and dilute to 0.5 mL with 50% CH3CN- aqueous solution. Filter. Equilibrate the HPLC column (Phenomenex, PrepC18, 300A, 50 × 250 mm) with 10% CH3CN- aqueous solution (containing 0.1% TFA), load the filtrate, and elute with 10% CH3CN- aqueous solution (containing 0.1% TFA) to remove DMSO. Subsequently, increase the eluent composition from 10% CH3CN-water (containing 0.1% TFA) to 35% CH3CN-water (containing 0.1% TFA) within 1 min, perform linear elution, and increase the proportion of CH3CN in the eluent at a rate of 0.5% / min for 50 min. The purity of the eluted fractions was determined on an analytical reversed-phase C18 column (Phenomenex, C18, 120A, 4.6 × 50 mm). Fractions with a purity >95% were combined and freeze-dried to obtain CPK25 (13 mg, yield 12.7%).

[0146] 10. Molecular weight of the product was analyzed by ESI-MS: calculated MW 1024.5, 1026.0; measured value 1025.8 [M+1] + 1027.7 [M+1] + .

[0147] Example 3.3 Preparation of CPK26

[0148] The steps are as follows:

[0149] 1. Attach mono-Fmoc ethylenediamine to 2-chlorotriphenylmethyl chloride resin. Mix mono-Fmoc ethylenediamine (23 mg, 100 μmol), 2-chlorotriphenylmethyl chloride resin (100 mg, 80 μmol), and 5 mL of DMF. Add DIPEA (70 μL, 400 μmol). Wash the resin three times each with DMF and DCM, and treat with CH3OH / DCM / DIPEA (8:1:1) for 10 min to block unreacted triphenylmethyl chloride sites. After vacuum drying, store the resin in a desiccator.

[0150] 2. Removal of Fmoc protecting groups. Take 50 mg of the resin (40 μmol), add 5 mL of 20% piperidine DMF solution, mix well, and shake for 1 min. Discard excess liquid. Add another 5 mL of 20% piperidine DMF solution, mix well, and shake for 15 min. After the reaction is complete, filter and wash the resin three times each with DMF and DCM.

[0151] 3. Dissolve the coupling reagents HATU (76 mg, 200 μmol) and DIPEA (35 μL, 200 μmol) in 5 mL of DMF. Then add the resin from the previous reaction, followed by [2-[2-(Fmoc-amino)ethoxy]ethoxy]acetic acid (77 mg, 200 μmol). Mix well and react for 2 hours. Then wash the resin as described above to remove the Fmoc protecting group.

[0152] 4. Dissolve the coupling reagents HATU (76 mg, 200 μmol) and DIPEA (35 μL, 200 μmol) in 5 mL of DMF. Then add the resin from the previous reaction, followed by N-Fmoc-L-glutamic acid α-tert-butyl ester (85 mg, 200 μmol). Mix well and react for 2 hours. Then wash the resin as described above to remove the Fmoc protecting group. Repeat the above steps. After both reactions are complete, wash the resin again to remove the protecting group.

[0153] 5. Dissolve the coupling reagents HATU (76 mg, 200 μmol) and DIPEA (35 μL, 200 μmol) in 5 mL of DMF. Then add the resin from the previous reaction, followed by [2-[2-(Fmoc-amino)ethoxy]ethoxy]acetic acid (77 mg, 200 μmol). Mix well and react for 2 hours. Then wash the resin as described above to remove the Fmoc protecting group.

[0154] 6. Dissolve the coupling reagents HATU (76 mg, 200 μmol) and DIPEA (35 μL, 200 μmol) in 5 mL of DMF. Then add the resin from the previous reaction, followed by N-Fmoc-L-glutamic acid α-tert-butyl ester (85 mg, 200 μmol). Mix well and react for 2 hours. Then wash the resin as described above to remove the Fmoc protecting group.

[0155] 7. Dissolve the coupling reagents HATU (76 mg, 200 μmol) and DIPEA (35 μL, 200 μmol) in 5 mL of DMF. Then add the resin from the previous reaction, followed by tert-butyl octadecanoate (74 mg, 200 μmol). Mix well and react for 2 hours. Filter and wash the resin with DMF and DCM.

[0156] 8. Treat the resin with 5 mL of 10% TFA in DCM solution (containing 10% H2O and 10% triisopropylsilane) for 1 h to cleave the reaction product from the previous step from the resin. Dissolve bromoacetic anhydride (55 mg, 200 μmol) and DIPEA (35 μL, 200 μmol) in 2 mL of DCM, then add the reaction product from the previous step, mix well, and stir for 30 min. Subsequently, remove TFA by drying under reduced pressure. Wash the resulting yellow residue several times with cold diethyl ether, and finally dry under a nitrogen stream to obtain a crude product as a yellow powder.

[0157] 9. Purify the crude product from the previous step using preparative chromatography. Dissolve the crude product (50 mg) in DMSO (0.1 mL) and dilute to 0.5 mL with 50% CH3CN- aqueous solution. Filter. Equilibrate the HPLC column (Phenomenex, PrepC18, 300A, 50 × 250 mm) with 10% CH3CN- aqueous solution (containing 0.1% TFA), load the filtrate, and elute with 10% CH3CN- aqueous solution (containing 0.1% TFA) to remove DMSO. Subsequently, increase the eluent composition from 10% CH3CN-water (containing 0.1% TFA) to 35% CH3CN-water (containing 0.1% TFA) within 1 min, perform linear elution, and increase the proportion of CH3CN in the eluent at a rate of 0.5% / min for 50 min. The purity of the eluted fractions was determined on an analytical reversed-phase C18 column (Phenomenex, C18, 120A, 4.6 × 50 mm). Fractions with a purity >95% were combined and freeze-dried to obtain CPK26 (10 mg, yield 8.7%).

[0158] 10. Molecular weight of the product was analyzed by ESI-MS: calculated MW 1153.5, 1155.2; measured value 1154.9 [M+1] + 1156.7 [M+1] + .

[0159] Example 4 Preparation of FGF21 derivatives

[0160] The preparation of representative FGF21 derivatives is given in Example 4.1 (Derivative #1). The FGF21 derivatives (compounds #2 to #5) of Examples 4.2-4.5 were prepared according to the method provided in Example 4.1.

[0161] Example 4.1: Derivative #1

[0162] This compound is a derivative of M-His6(Q108A,S172C)FGF21.

[0163] M-His6(Q108A, S172C)FGF21 was prepared according to the general description in Examples 1-3, and the thiol group of the Cys residue at position 172C was modified with the reagent CPK24 prepared in Example 3, as follows:

[0164] 1) TCEP reduction: Add 1 mM TCEP to the purified sample M(A108, C172)FGF21 (approximately 0.5 mg / ml, buffer: 20 mM Tris, 150 mM NaCl, pH 8.0) and react at 37 degrees Celsius for 0.5 h.

[0165] 2) TCEP removal: Ultrafiltration to remove TCEP (using a buffer of 20mM Tris, 150mM NaCl, pH 8.0, the reduced mixture is ultrafiltered and the buffer is changed approximately 400 times, with the final protein concentration controlled at around 1mg / ml).

[0166] 3) Coupling: Add 5 eq. (mol equivalent) of CPK24 to the sample from the previous step, and react with shaking at 25 degrees Celsius for 4 hours (protein concentration around 1 mg / ml, system buffer: 20 mM Tris, 150 mM NaCl, pH 8.0).

[0167] 4) Purification: An appropriate volume of deionized water was added to reduce the conductivity to approximately 4 mS / cm. The mixture was then purified using anion exchange on a Q Bestarose Fast Flow column. The buffer used was A-buffer: 20 mM Tris, pH 8.0; B-buffer: 20 mM Tris, 500 mM NaCl, pH 8.0, at a flow rate of 10 ml / min and a gradient of 0-80% B. The column was subjected to 30 CV cycles. Recovery: 38%.

[0168] LCMS: Theoretical mass: 21272.17 Da; Measured mass: 21272.60 Da.

[0169] Example 4.2: Derivative #2

[0170] This compound is a derivative of M-His6(P124A, S172C)FGF21 prepared using reagent CPK24 from Example 3.1 by the method described in Example 4.1.

[0171] LCMS: Theoretical mass: 21315.19 Da; Actual mass: 21315.45 Da.

[0172] Example 4.3: Derivative #3

[0173] This compound is a derivative of M-His6(P143L, S172C)FGF21 prepared using reagent CPK24 from Example 3.1 by the method described in Example 4.1.

[0174] LCMS: Theoretical mass: 21327.20 Da; Measured mass: 21326.92 Da.

[0175] Example 4.4: Derivative #4

[0176] This compound is a derivative of M-His6(P144A, S172C)FGF21 prepared using reagent CPK24 from Example 3.1 by the method described in Example 4.1.

[0177] LCMS: Theoretical mass: 21297.12 Da; Measured mass: 21297.04 Da.

[0178] Example 4.5: Derivative #5

[0179] This compound is a derivative of M-His6(A154G, S172C)FGF21 prepared using reagent CPK24 from Example 3.1 by the method described in Example 4.1.

[0180] LCMS: Theoretical mass: 21309.14 Da; Measured mass: 21307.55 Da

[0181] Example 4.6: Derivative #8

[0182] This compound is a derivative of A(A154G, S172C)FGF21 prepared using reagent CPK24 from Example 3.1 by the method described in Example 4.1.

[0183] LCMS: Theoretical mass: 20426.42 Da; Measured mass: 20426.26 Da

[0184] Example 4.7: Derivative #9

[0185] This compound is a derivative of A(P144A, S172C)FGF21 prepared using reagent CPK25 from Example 3.1 by the method described in Example 4.1.

[0186] LCMS: Theoretical mass: 21297.13 Da; Measured mass: 21297.56 Da.

[0187] Example 4.8: Derivative #10

[0188] This compound is a derivative of A(P144A, S172C)FGF21 prepared using reagent CPK26 from Example 3.1 by the method described in Example 4.1.

[0189] LCMS: Theoretical mass: 21426.17 Da; Measured mass: 21426.03 Da.

[0190] Example 5: In vitro cell viability assay of FGF21 mutants and derivatives

[0191] hKLB / hFGFR1c agonist activity was detected using the luciferase reporter gene assay (Jonathan W Day et al.: Nat Chem Biol. 2009 Oct; 5(10):749-57).

[0192] The puromycin resistance genes pac, GAL4DBD-ELK1, IRES, and KLB (β-klotho) were sequentially cloned into the mammalian cell expression plasmid pCDNA3.1 to construct the recombinant expression plasmid pcDNA-GAL4DBD-ELK1-IRES-KLB-Puro. 6 μL of the recombinant plasmid and 4.6 μg of 5XUAS-luc (Agilent) plasmid were transfected into HEK293T cells, and stable expression lines were selected.

[0193] Cells were cultured in T75 culture flasks in high-glucose DMEM medium (complete medium) containing 10% FBS. Once confluence was >90%, the culture supernatant was discarded, and 5 ml of Accutase was added for digestion at 37°C. The cells were then neutralized with 10 ml of complete medium, centrifuged, and the supernatant was discarded. The cells were resuspended in 4 ml of high-glucose DMEM medium (analytical medium) containing 5% FBS and counted. Cells were diluted to 4 × 10⁶ cells / mL with analytical medium. 5 / ml, seeded into 96-well plates, 100μL per well, and incubated overnight.

[0194] To compare the activity of the FGF21 mutant, samples were serially diluted using DMEM containing 5% FBS. When loading the sample, 50 μl of supernatant per well of a 96-well plate containing cultured cells was discarded, and 50 μl of sample per well was added. The plate was stimulated for 6 hours before detection. The results are summarized in Table 5.

[0195] Table 5 Cell viability of FGF21 mutants

[0196] The activity of FGF21 derivatives was compared using two detection systems. System 1: Samples were serially diluted with FBS-free DMEM. During sample loading, 90 μl of supernatant per well of the empty 96-well plate containing cultured cells was discarded, and 90 μl of sample per well (equivalent to a 0.5% FBS system) was added. After stimulation for 6 hours, the Bright-Globe assay was performed. TM The Luciferase Assay System (Promega, E2620) was used for testing according to its instructions. System Two: Samples were serially diluted with DMEM containing 5% FBS. When adding the sample, 50 μl of supernatant per well of a 96-well plate containing cultured cells was discarded, and 50 μl of sample per well was added. The plate was stimulated for 6 hours before testing. The results are summarized in Table 6.

[0197] Table 6. Cellular activities of FGF21 derivatives

[0198] The results showed that the FGF21 mutants generally retained all or higher levels of FGF21 activity. M-His6(P143L, S172C)FGF21 and M-His6(P144A, S172C)FGF21 exhibited the highest activity, indicating that the introduction of the mutation site did not affect activity. The activities of A(A154G, S172C)FGF21 and M-His6(A154G, S172C)FGF21 were similar, indicating that the addition of A at the N-terminus did not affect the overall activity. In the 0.5% FBS system (where the albumin concentration was low and its effect on activity was not significant), a comparison of the activities of the FGF21 derivatives and FGF21 mutants revealed that the cys-coupled fatty acid chain at position 172 did not cause a loss of activity; on the contrary, they generally retained high activity, except for a slight decrease in activity in #2. In the 5% FBS system, the binding of fatty acid chains to albumin in the system generally caused a decrease in activity, at least 10 times, indicating that each derivative has the ability to bind albumin.

[0199] Example 6: Stability test in human serum

[0200] Take the FGF21 derivatives corresponding to Table 2 and natural human mature FGF21 (SEQ ID. NO. 17), dilute 10-fold with human serum to a final protein concentration of approximately 50 μg / ml, filter sterilize, and aliquot into sterile centrifuge tubes, 100 μL per tube. One tube of each sample was frozen at -20°C as a control, and the remaining tubes were incubated at 37°C. Samples were taken at 3 and 7 days to detect the relative activity of the ELISA. Residual activity: The activity value at 0 days was set at 100%, and the values ​​measured at subsequent time points were compared to this value.

[0201] Figure 1 shows the change in relative activity over time. The results indicate that FGF21 derivatives #1 to #5 retained 30-40% of their activity after being stored at 37℃ for 7 days. In contrast, mature FGF21 exhibited the worst stability, retaining only 6% of its activity after 7 days. This suggests that the serum stability of FGF21 derivatives is significantly improved compared to mature FGF21.

[0202] Example 7: Weight Loss Experiment in Diet-Induced Obesity (DIO) Mice 1

[0203] Preparation of the DIO mouse model: Approximately 7-week-old male C57BL / 6J mice were fed a high-fat diet (60% kcal from fat) for approximately 16 weeks (total 23 weeks), until their body weight reached approximately 45g, at which point the experiment was conducted. The DIO mice were randomly divided into 7 groups of 5-6 mice each, with no difference in baseline body weight, and were weighed daily. Grouping, statistical analysis, and drug administration regimens are shown in Table 7.

[0204] Table 7 Note: SC is for subcutaneous injection; Q4D is for administration every four days; QD is for administration once daily.

[0205] During the experiment, #7, #4, #5, the positive control AKR-001analog, or the negative control saline were administered subcutaneously. The day of grouping was designated D0, and medication began on the following day (D1). Body weight was measured on the first day of administration and continued until the end of the experiment on D35, with daily recording of weight and food intake. The administration was divided into two phases: D1 to D24 and D25 to D35, as shown in Tables 7 and 8.

[0206] The results of the weight change measurement in the test animals are shown in Figure 2. The results show that FGF21 derivatives #4, #5 and #7 can significantly reduce the weight of the test animals, and the high dose of #4 shows a dose-dependent weight loss effect. The 35-day weight loss effect shows that the low dose of FGF21 derivative #5 is more effective than the high dose of AKR-001analog.

[0207] The results of the feeding assay in the test animals are shown in Figure 3. The results show that the feeding inhibition and weight loss of FGF21 derivatives #4, #5, #7 and AKR-001analog are positively correlated.

[0208] Table 8

[0209] Example 8: Weight Loss Experiment in Diet-Induced Obesity (DIO) Mice 2

[0210] Preparation of the DIO mouse model: Male C57BL / 6J mice, approximately 7 weeks old, were fed a high-fat diet (60% kcal from fat) for approximately 16 weeks (total 23 weeks) until they reached a weight of approximately 45g for the experiment. The DIO mice were randomly divided into 5 groups of 5-6 mice each, with no difference in baseline weight, and were weighed daily.

[0211] DIO mice were grouped as follows: negative control (0.9% saline, sc, QD), FGF21 derivative #5 (30 nmol / kg, sc, QD), FGF21 derivative #5 (30 nmol / kg, sc, Q3D), and NNC0194-0499 analog (30 nmol / kg, sc, Q3D). A normal mouse group (0.9% saline, sc, QD) was also included. Q3D was administered every 3 days.

[0212] During the experiment, #5, #6, or saline were administered subcutaneously. The day of grouping was recorded as D0, and the medication was started on the day following grouping (D1). Body weight was measured on the first day of medication and continued until the end of the experiment on D42. Body weight and food intake were recorded daily.

[0213] The results of the weight change measurement of the test animals are shown in Figure 4. The results show that the weight loss effect of FGF21 derivative #5 with Q3D administration is significantly better than that of NNC0194-0499 analog. #5 with QD administration has better weight loss, with a 40% reduction in weight after 42 days of administration.

[0214] The results of the feeding measurement of the test animals are shown in Figure 5. The results show that the drug administration group showed different degrees of feeding inhibition, and all of them showed suppression of appetite in the early stage (e.g., 7-28 days) and gradually recovered appetite in the later stage (after 28 days).

[0215] Example 9 Metabolism in SD rats

[0216] Twelve male SD rats, 6-7 weeks old and weighing 240-250g, were selected. Grouping is shown in Table 9 below:

[0217] Table 9 Note: IV (intravenous injection), SC (subcutaneous injection).

[0218] The pharmacokinetic (PK) parameters of the test animals are compared in Figure 6 and Table 10. The results show that after a single dose, FGF21 derivative #4 and FGF21 derivative #6 have similar half-lives, but FGF21 derivative #4 has higher bioavailability than FGF21 derivative #6. Higher bioavailability is beneficial for drug absorption and distribution in the body, thus improving therapeutic efficacy.

[0219] Table 10

Claims

1. A compound containing an FGF21 mutant and a fatty acid chain HOOC-(CH2) a -CO- FGF21 derivatives, wherein the amino acid sequence of the FGF21 mutant includes the S172C mutation compared to the sequence shown in SEQ ID NO.16; the fatty acid chain HOOC-(CH2) a -CO- is linked via a linker to a cysteine ​​residue at position 172 of the FGF21 mutant, where a is an integer greater than or equal to 12 and less than or equal to 20; the linker is selected from one of the following: -γGlu-2×OEG-NH-(CH2)2-NH-CO-CH2-, -γGlu-2×OEG-γGlu-NH-(CH2)2-NH-CO-CH2-, -γGlu-OEG-γGlu-OEG-NH-(CH2)2-NH-CO-CH2-, or -γGlu-OEG-2×γGlu-OEG-NH-(CH2)2-NH-CO-CH2-, wherein the -γGlu terminus is linked to the fatty acid chain HOOC-(CH2). a The CO-terminus of -CO- is attached to the CH2-terminus of the cysteine ​​residue at position 172 of the FGF21 mutant.

2. The FGF21 derivative as described in claim 1, characterized in that, The amino acid sequence of the FGF21 mutant, compared with the sequence shown in SEQ ID NO.16, further includes one or more amino acid mutations selected from the following: Q108A, P124A, P143L, A144A, A154G.

3. The FGF21 derivative as described in claim 2, characterized in that, The FGF21 mutant includes an amino acid sequence as shown in any of SEQ ID NO.1 to 5.

4. The FGF21 derivative as described in claim 1, characterized in that, a is 16, and the fatty acid chain is HOOC-(CH2). 16 -CO-; or a is 17, and the fatty acid chain is HOOC-(CH2). 17 -CO-; or a is 18, and the fatty acid chain is HOOC-(CH2). 18 -CO-.

5. The FGF21 derivative as described in claim 1, characterized in that, The linker is -γGlu-2×OEG-γGlu-NH-(CH2)2-NH-CO-CH2-.

6. The FGF21 derivative as described in claim 1, characterized in that, The fatty acid chain is HOOC-(CH2). 16 -CO-, the linker is -γGlu-2×OEG-γGlu-NH-(CH2)2-NH-CO-CH2-, HOOC-(CH2) 16 -CO- is linked to the cysteine ​​residue at position 172 of the FGF21 protein via -γGlu-2×OEG-γGlu-NH-(CH2)2-NH-CO-CH2-, with its -γGlu terminus linked to HOOC-(CH2). 16 The CO-terminus of -CO- is attached to the CH2-terminus of the cysteine ​​residue at position 172 of the FGF21 mutant.

7. The FGF21 derivative as described in claim 1, characterized in that, The FGF21 derivative is selected from one of the following compounds or a pharmaceutically acceptable salt thereof:

8. A method for preparing the FGF21 derivative according to any one of claims 1-7, comprising: The FGF21 derivative was prepared by a coupling reaction.

9. A pharmaceutical composition comprising the FGF21 derivative according to any one of claims 1 to 7 and a pharmaceutically acceptable carrier.

10. Use of the FGF21 derivative of any one of claims 1-7 or the pharmaceutical composition of claim 9 in the preparation of a medicament for treating metabolic diseases.

11. The use as described in claim 10, characterized in that, The metabolic diseases mentioned are selected from diabetes, obesity, dyslipidemia, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, other metabolic syndromes associated with diabetes, high triglycerides, low HDL cholesterol and high LDL cholesterol, insulin resistance, or impaired glucose tolerance.

12. An FGF21 mutant, wherein the amino acid sequence of the FGF21 mutant contains one or more amino acid mutations selected from the following, compared with the sequence shown in SEQ ID NO.16: Q108A, P124A, P143L, A144A, A154G.

13. The FGF21 mutant of claim 12, wherein the amino acid sequence of the FGF21 mutant further contains the S172C mutation compared with the sequence shown in SEQ ID NO.

16.

14. The FGF21 mutant of claim 13, wherein the FGF21 mutant comprises an amino acid sequence as shown in any one of SEQ ID NO. 1 to 5.

15. A polynucleotide or an expression vector containing the polynucleotide, characterized in that, The polynucleotide encodes the FGF21 mutant as described in any one of claims 12 to 14.

16. An expression system comprising the expression vector of claim 15 or the genome thereof having an exogenous polynucleotide of claim 15 integrated therein.

17. A method for preparing the FGF21 mutant according to any one of claims 12 to 14, comprising the following steps: culturing the expression system as described in claim 16 under conditions suitable for expressing the FGF21 mutant, thereby expressing the FGF21 mutant; preferably, the preparation method further comprises purifying and isolating the FGF21 mutant.

18. An FGF21 derivative comprising the FGF21 mutant as described in any one of claims 12 to 14, and further comprising a fatty acid chain and a linker attached to the FGF21 mutant.

19. The FGF21 derivative of claim 18, wherein the fatty acid chain is HOOC-(CH2). b -CO-, b is an integer greater than or equal to 12 and less than or equal to 20, the fatty acid chain is linked via a linker to a cysteine ​​residue at position 172 of the FGF21 mutant, the linker being selected from one of the following: -γGlu-2×OEG-NH-(CH2)2-NH-CO-CH2-, -γGlu-2×OEG-γGlu-NH-(CH2)2-NH-CO-CH2-, -γGlu-OEG-γGlu-OEG-NH-(CH2)2-NH-CO-CH2-, or -γGlu-OEG-2×γGlu-OEG-NH-(CH2)2-NH-CO-CH2-, the -γGlu end of the linker being connected to HOOC-(CH2). b The CO-terminus of the linker is connected to the CH2-terminus of the linker at position 172 of the FGF21 mutant.

20. The FGF21 derivative of claim 19, wherein b is 16, and the fatty acid chain is HOOC-(CH2). 16 -CO-; or b is 17, and the fatty acid chain is HOOC-(CH2). 17 -CO-; or b is 18, and the fatty acid chain is HOOC-(CH2). 18 -CO-.

21. The FGF21 derivative of claim 19, wherein the linker is -γGlu-2×OEG-γGlu-NH-(CH2)2-NH-CO-CH2-.

22. The FGF21 derivative as described in claim 19, characterized in that, The fatty acid chain is HOOC-(CH2). 16 -CO-, the linker is -γGlu-2×OEG-γGlu-NH-(CH2)2-NH-CO-CH2-, HOOC-(CH2) 16 -CO- is linked to the cysteine ​​residue at position 172 of the FGF21 mutant via -γGlu-2×OEG-γGlu-NH-(CH2)2-NH-CO-CH2-, with its -γGlu terminus linked to HOOC-(CH2). 16 The CO-terminus of the -CO- protein has its CH2-terminus attached to a cysteine ​​residue at position 172 of the FGF21 protein.

23. The FGF21 derivative as described in claim 18, characterized in that, The FGF21 derivative is selected from one of the following compounds or a pharmaceutically acceptable salt thereof:

24. A method for preparing the FGF21 derivative according to any one of claims 18 to 23, comprising: The FGF21 derivative was prepared by a coupling reaction.

25. A pharmaceutical composition comprising the FGF21 mutant of any one of claims 12 to 14 or the FGF21 derivative of any one of claims 18 to 23, and a pharmaceutically acceptable carrier.

26. Use of the FGF21 mutant of any one of claims 12 to 14, the FGF21 derivative of any one of claims 18 to 23, or the pharmaceutical composition of claim 25 in the preparation of a medicament for treating metabolic diseases.

27. The use as described in claim 26, characterized in that, The metabolic diseases mentioned are selected from diabetes, obesity, dyslipidemia, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, other metabolic syndromes associated with diabetes, high triglycerides, low HDL cholesterol and high LDL cholesterol, insulin resistance, or impaired glucose tolerance.

Images