A recombinant fusion protein with multiple activities and uses thereof

Abstract

The present application provides a recombinant fusion protein with multiple activities, its encoding nucleic acid, vector and host cell, and a method and use of the recombinant fusion protein for treating metabolic syndrome (such as diabetes, obesity, non-alcoholic fatty liver).

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to recombinant fusion proteins with multiple activities, their pharmaceutical compositions, preparation methods, and uses. Background Technology

[0002] Fibroblast growth factor 21 (FGF21) is a metabolic regulatory hormone primarily expressed in the liver. It functions by activating the FGF receptor (FGFR) and its co-receptor β-Klotho, improving insulin sensitivity, promoting glucose uptake and utilization, promoting fatty acid oxidation and lipolysis, reducing triglyceride and cholesterol levels, increasing energy expenditure, reducing body weight, and regulating appetite and food intake, thereby modulating glucose metabolism, lipid metabolism, and energy balance. FGF21 is closely associated with metabolic diseases such as obesity, type 2 diabetes, non-alcoholic fatty liver disease, and cardiovascular disease. Several long-acting FGF21 analogs have been developed; for example, efruxifermin has shown significant liver and metabolic improvements in clinical trials, particularly in reducing liver fat, inflammation, and fibrosis.

[0003] Glucagon-like peptide-1 (GLP-1) is an important incretin hormone, primarily secreted by intestinal L cells, with GLP-1(7-36) being its main active form. GLP-1 exerts its effects by binding to the GLP-1 receptor (GLP-1R), promoting insulin secretion from pancreatic β-cells, delaying gastric emptying, reducing postprandial blood glucose fluctuations, increasing satiety, reducing food intake, and lowering the risk of cardiovascular disease. It plays a crucial role in regulating blood glucose, appetite, and energy metabolism. Due to its multiple metabolic regulatory effects, GLP-1 has become an important target for the treatment of type 2 diabetes and obesity, leading to the development of various GLP-1R agonists, including GLP-1 analogs such as liraglutide and smegglutide.

[0004] Glucagon (GCG) is a polypeptide hormone secreted by pancreatic α cells that primarily functions in hypoglycemic states by raising blood glucose levels through promoting glycogenolysis and gluconeogenesis. GCG works by binding to the glucagon receptor (GCGR), maintaining glycemic homeostasis in hypoglycemic states, and can also influence body weight by promoting lipolysis and increasing energy expenditure.

[0005] How to rationally utilize the metabolic regulatory effects between various targets to achieve excellent disease treatment results remains a technical problem that urgently needs to be solved in this field. Summary of the Invention

[0006] This invention provides a recombinant fusion protein with multiple activities, which simultaneously exhibits FGF21, GLP-1 and GCG activities, thereby exerting drug effects by co-activating three targets: GLP-1R, GCGR and FGF21 receptor (FGF21R), and has an extended in vivo half-life to further improve the therapeutic effect.

[0007] In one aspect, the present invention provides a recombinant fusion protein with multiple activities, comprising a domain P1 and a domain P3, wherein P1 is a domain with dual GLP-1 / GCG activity and P3 is a domain with FGF21 activity; optionally, the recombinant fusion protein further comprises a half-life extension domain P2.

[0008] In some embodiments, P1 has dual agonist activity of GLP-1R / GCGR, and P3 has FGF21R ​​agonist activity.

[0009] In some embodiments, P1 comprises an amino acid sequence selected from (1) or (2): (1) HGQGTFTSDYSKYLDEKRAKEFVQWLX1X2X3X4X5X a (SEQ ID NO.1), X1 is selected from any amino acid of M, I, L, Q; X2 is selected from any amino acid of N, D, R; X3 is selected from any amino acid of T, S, Y, A or X3 is omitted; X4 is selected from any amino acid of A, V, S or X4 is omitted; X5 is selected from any amino acid of G, A, V, S or X5 is omitted, and, When X5 exists, X a The representative amino acid sequence is GPSSGAPPPS (SEQ ID NO.2); When X5 is missing, X a The representative amino acid sequence is GGPSSGGPSSGGPSS (SEQ ID NO.3) with X4 missing, or X... a And X4 is missing; (2) HGQGTFTSDYSKYLDERAAKDFIX6WLESA (SEQ ID NO.7), where X6 is selected from any one of the amino acids K, A, Q, and E.

[0010] In some specific embodiments, X1 is M, X2 is N, X3 is T, or X3 is missing. In some preferred embodiments, X1 is M, X2 is N, and X3 is missing.

[0011] In some specific implementations, X1 to X3 are selected from any of the 16 combinations listed in the table below, where "-" indicates that X3 is missing: , Preferably, it is selected from any one of combinations 1, 3, 4-8, 12-14, and more preferably from any one of combinations 3, 4, 6 or 8.

[0012] In some preferred embodiments, X6 is K.

[0013] In some specific implementations, X4 is selected from any one of amino acids A, V, and S, X5 is G, and X... a The representative amino acid sequence is GPSSGAPPPS (SEQ ID NO.2). In some specific embodiments, X4 is deleted, X5 is selected from any one of the amino acids G, A, V, and S, and X... a The representative amino acid sequence is GPSSGAPPPS (SEQ ID NO.2). In some specific embodiments, X5 and X4 are deleted, X... a The representative amino acid sequence is GGPSSGGPSSGGPSS (SEQ ID NO.3). In some preferred embodiments, X4 is A or X4 is deleted, X5 is G, and X... a The representative amino acid sequence is GPSSGAPPPS (SEQ ID NO.2).

[0014] In some specific embodiments, P1 is selected from any of the amino acid sequences listed in Table 1.1, preferably any of the amino acid sequences shown in P1-02, P1-03, P1-05, P1-17, P1-18, P1-20, and P1-22, and more preferably SEQ ID NO. 17, 18, 20, 32, 33, or 35.

[0015] In some embodiments, P3 is an FGF21 analog. In some embodiments, the amino acid sequence of P3, compared to the wild-type FGF21 protein, contains one or any combination of the following amino acid substitutions: R19V, L98R, P171G, A180E, N121Q, M168L.

[0016] In some embodiments, the amino acid sequence of P3, compared to the wild-type FGF21 protein, contains the following amino acid substitutions: R19V, P171G, and A180E, and optionally, further contains any one of L98R, N121Q, and M168L, or any combination thereof. In some embodiments, the amino acid sequence of P3, compared to the wild-type FGF21 protein, contains the following amino acid substitutions: R19V, L98R, P171G, and A180E, and optionally, further contains N121Q and / or M168L. In some embodiments, the amino acid sequence of P3, compared to the wild-type FGF21 protein, contains the following amino acid substitutions: R19V, P171G, and A180E, and optionally, further contains N121Q and / or M168L. The amino acid sequence of the wild-type FGF21 has the NCBI reference sequence number NP_061986.1. In one specific embodiment, the wild-type FGF21 is the amino acid sequence shown in P3-06 of Table 1.3. In this invention, all amino acid substitutions involving FGF21 are numbered based on their position in the wild-type FGF21 (e.g., P3-06). For example, the first histidine (H) in the amino acid sequence shown in P3-06 is numbered 1, the second is numbered 2, and so on until the last amino acid is numbered 181.

[0017] In some specific embodiments, P3 is selected from any of the amino acid sequences listed in Table 1.3 (including SEQ ID NO. 54-59), preferably the amino acid sequences shown in P3-01 or P3-02. In one specific embodiment, P3 is the amino acid sequence shown in SEQ ID NO. 55.

[0018] In some embodiments, P2 comprises an Fc fragment of a mammalian immunoglobulin, such as an Fc fragment of human IgG1, IgG2, IgG3, or IgG4. In some embodiments, P2 comprises a variant of the Fc fragment, preferably an Fc variant of human IgG1, which has altered effector functions mediated by the Fc fragment (e.g., ADCC and / or CDC activity, affinity for FcγRIIIa and / or C1q), prolonged half-life, promotes polypeptide dimerization, and / or increases therapeutic efficacy. In some embodiments, the Fc variant has at least one amino acid substitution compared to a wild-type human IgG1 or IgG4 Fc fragment, for example, about 1-10 amino acid substitutions, or about 2-7 amino acid substitutions, or about 3-5 amino acid substitutions in a wild-type human IgG1 or IgG4 Fc fragment. The Fc variant may have at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identity with the wild-type human IgG1 Fc fragment. Examples of amino acid substitutions in the Fc fragment can be found, for example, in International Publications WO 02 / 060919, WO 98 / 23289, and WO 97 / 34631, and in U.S. Patents 5,869,046, 6,121,022, 6,277,375, 7,658,921, and 6,165,745, the entire contents of which are incorporated herein by reference. In some embodiments, the Fc variant has one or more of the following amino acid substitutions compared to the wild-type human IgG1 Fc fragment from which it is derived: L234A, L235A, P329A, P329G, D265S, M252Y, S254T, T256E, M428L, N434S (according to their positions in the EU numbering system), for example, L234A, L235A, and P329G correspond to the 14th, 15th, and 109th positions of the amino acid sequence shown in P2-01 of Table 1.2, respectively; preferably one or more of the following combinations of amino acid substitutions: (1) L234A / L235A (LALA); (2) L234A / L235A / P329A (LALAPA); (3) L234A / L235A / D265S (LALADS); (4) L234A / L235A / P329G(LALAPG); (5) M252Y / S254T / T256E(YTE); (6) M428L / N434S(LS); More preferably, the amino acid substitutions include any one of LALA, LALAPA, LALADS, LALAPG and / or any one of YTE, LS.In some embodiments, the Fc variant comprises L234A, L235A, and P329G. In some embodiments, the Fc fragment comprises any of the amino acid sequences shown in Table 1.2 (including SEQ ID NO. 46-53), or a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with any of the amino acid sequences shown in Table 1.2. In some embodiments, the Fc fragment comprises an amino acid sequence that has undergone mutations of up to 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0 compared to any of the amino acid sequences shown in Table 1.2, and the Fc fragment comprises any of LALA, LALAPA, LALADS, and / or any of YTE, LS. In some embodiments, the Fc fragment comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% identity with the amino acid sequence shown in P2-01, and having L234A, L235A, and P329G. In one specific embodiment, the Fc fragment comprises the amino acid sequence shown in P2-02 (SEQ ID NO. 47) in Table 1.2.

[0019] In some implementations, P1, P2, and P3 have the following connection relationships, in order from N-terminal to C-terminal: (a) P1-L a -P2-L b -P3; or, (b) P3-L a -P2-L b -P1; Among them, L a and L b Independently representing a peptide linker, "-" represents a peptide bond; optionally, L a and / or L b It does not exist.

[0020] In some preferred embodiments, P1, P2, and P3 have the following connection relationship in order from N-terminus to C-terminus: P1-L a -P2-L b -P3; where, L a and L b Independently representing a peptide linker, "-" represents a peptide bond; optionally, L a and / or L b It does not exist.

[0021] In some embodiments, the peptide linker may be a flexible linker, for example, selected from amino acid sequences rich in glycine (G), serine (S), and / or alanine (A), or an amino acid sequence from the hinge region of the antibody heavy chain may be used as the linker. See, for example, WO1996 / 34103. In some embodiments, the peptide linker is a human IgG1, IgG2, IgG3, or IgG4 hinge or a variant thereof. In some embodiments, the peptide linker is rich in glycine-serine, typically selected from SEQ ID NO.4: (GGGGS) n Or SEQ ID NO.5: (GS) n The term n is an integer at least 1, such as n being 1, 2, 3, 4, 5, or 6, preferably n being 1, 2, or 3, more preferably n being 1 or 3, for example, SEQ ID NO. 6: (GGGGS)3 or GS. In some embodiments, the peptide linker is rich in glycine-alanine, typically selected from SEQ ID NO. 8: (GGGGA). n Where n is an integer at least 1, for example, n is 1, 2, 3, 4, 5, or 6, such as SEQ ID NO. 9: (GGGGA)3. In some embodiments, the peptide linker is rich in alanine-serine, or the peptide linker is selected from other flexible linkers known in the art, such as SEQ ID NO. 10: (G) 1-30 SEQ ID NO. 11: KESGSVSSEQLAQFRSLD, SEQ ID NO. 12: EGKSSGSGSESKST, SEQ ID NO. 13: GSAGSAAGSGEF. In some embodiments, the peptide linker is 2-26 amino acids long. In some embodiments, the peptide linker is 2-20 amino acids long. In some embodiments, the peptide linker is 2-15 amino acids long, or 3-15 amino acids long, or 5-15 amino acids long, preferably 15 amino acids long. In some embodiments, the peptide linker ensures that two adjacent domains do not affect each other spatially or functionally.

[0022] In some implementations, the L a and L b They can be the same, for example, both containing SEQ ID NO.4: (GGGGS) n Or SEQ ID NO.8: (GGGGA) n Preferably, n=3. In some embodiments, the L a and L b They can be different, where L a Includes SEQ ID NO.4: (GGGGS) n L bIncludes SEQ ID NO.5: (GS) n Or SEQ ID NO.8: (GGGGA) n In one specific implementation plan, L a Contains SEQ ID NO.6: (GGGGS)3,L b Includes GS. In one specific implementation, L a Contains SEQ ID NO.6: (GGGGS)3,L b Contains SEQ ID NO.9: (GGGGA)3. In one specific embodiment, L a L does not exist. b Includes SEQ ID NO.4: (GGGGS) n The preferred value is n=3.

[0023] In some embodiments, the recombinant fusion protein comprises a polypeptide chain having the structure shown in (a) above, the polypeptide chain being selected from any of the amino acid sequences shown in Table 2, preferably the amino acid sequences shown in FGG02, FGG03 or FGG05.

[0024] In some embodiments, the polypeptide chain comprises any one of the following amino acid sequences: HGQGTFTSDYSKYLDEKRAKEFVQWLX (1) X (2) X (3)GGPSSGAPPPSGGGGSGGGGSGGGGSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLT VLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSHPIPDSSPLLQFGGQVRQVYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTS RFLCQRPDGALYGSLHFDPEACSFRERLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVGGSQGRSPSYES(SEQ ID NO.14), where X (1) To X (3) Choose any one of the 16 combinations listed in the table below, where "-" represents X. (3) Missing: , Preferably, X (1) To X (3) Selected from any one of combinations 1, 3, 4-8, and 12-14. In some preferred embodiments, the polypeptide chain comprises any one of the amino acid sequences shown in Table 1.4.

[0025] In some embodiments, the polypeptide chain comprises any one of the following amino acid sequences: HGQGTFTSDYSKYLDEKRAKEFVQWLLDAGGPSSGAPPPSGGGGSGGGGSGGGGSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKX (5) HPIPDSSPLLQFGGQVRQVYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFREX (6) LLEDGYNVYQSEAHGLPLHLPGX (7) KSPHRDPAPRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSX (8) VGGSQGRSPSYES (SEQ ID NO.15), where X (5) To X (8) Choose any one of the 10 combinations listed in the table below: .

[0026] In some preferred embodiments, the polypeptide chain comprises any one of the amino acid sequences shown in Table 7, preferably SEQ ID NO. 80 or 81.

[0027] In some embodiments, the recombinant fusion protein is a homopolymer (e.g., a homodimer) consisting of at least two identical polypeptide chains. The polypeptide chains are linked to each other primarily through non-covalent interactions and / or disulfide bonds, for example, through disulfide bonds in the hinge region of the Fc fragment. In one specific embodiment, the recombinant fusion protein consists of two identical polypeptide chains dimerized via the Fc fragment.

[0028] In some embodiments, the recombinant fusion protein retains at least 0.001%, at least 0.01%, at least 0.1%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% of the wild-type GLP-1 activity or the wild-type GLP-1 agonistic activity against GLP-1R. In some embodiments, the recombinant fusion protein retains 0.1%-100%, 0.1%-50%, 0.1%-40%, 0.1%-35%, 0.1%-30%, 0.1%-25%, 0.1%-20%, 0.1%-15%, 0.1%-10%, 0.1%-5%, 1%-50%, 1%-40%, 1%-35%, or 1%-30% of the wild-type GLP-1 activity or the wild-type GLP-1's agonistic activity towards GLP-1R. 1%-25%, 1%-20%, 1%-15%, 1%-10%, 5%-50%, 5%-40%, 5%-35%, 5%-30%, 5%-25%, 5%-20%, 10%-50%, 10%-40%, 10%-35%, 10%-30%, 10%-25%, 10%-20%, 20%-50%, 20%-40%, 20%-35%, 20%-30%, 30%-50%, or 30%-40%. In some embodiments, the recombinant fusion protein has better GLP-1 activity or GLP-1R agonist activity than Survodutide.

[0029] In some embodiments, the recombinant fusion protein retains at least 0.001‰, at least 0.01‰, at least 0.1‰, at least 1‰, at least 0.01%, at least 0.1%, at least 0.5%, at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, or at least 50% of the wild-type GCG activity or the wild-type GCG agonistic activity against GCGR. In some embodiments, the recombinant fusion protein retains wild-type GCG activity or wild-type GCG agonistic activity against GCGR at 0.01‰-0.1‰, 0.1‰-1‰, 1‰-0.01%, 0.01%-0.1%, 0.1%-1%, 1%-10%, 10%-20%, 20%-30%, 30%-40%, 40%-50%, 0.1%-0.5%, 0.5%-1%, 1%-5%, 5%-10%, 10%-15%, 15%-20%, 20%-25%, or 25%-30%.

[0030] In some embodiments, the recombinant fusion protein retains at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 160%, at least 170%, at least 180%, at least 190%, at least 200%, at least 210%, at least 220%, at least 230%, or at least 240% of the wild-type FGF21 activity or the wild-type FGF21 agonistic activity against FGF21R. In some embodiments, the recombinant fusion protein retains 80%-100%, 100%-150%, 150%-200%, 200%-240%, 130%-160%, or 180%-210% of the wild-type FGF21 activity or the wild-type FGF21 agonist activity towards FGF21R. In some embodiments, the recombinant fusion protein has superior FGF21 activity or FGF21R ​​agonist activity compared to Efruxifermin.

[0031] In some embodiments, the GLP-1 / GCG activity ratio of the recombinant fusion protein or the GLP-1R agonistic activity of the recombinant fusion protein is at least 1, at least 5, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 200, at least 300, at least 400, or at least 500 times that of the GCGR agonistic activity. In some embodiments, the GLP-1 / GCG activity ratio of the recombinant fusion protein or the GLP-1R agonistic activity of the recombinant fusion protein is 1-10 times, 1-20 times, 1-50 times, 50-100 times, 50-150 times, 100-200 times, 150-250 times, 200-300 times, 250-350 times, 300-400 times, 350-450 times, or 400-500 times that of the GCGR agonistic activity.

[0032] In some embodiments, the recombinant fusion protein exhibits better activity compared to a reference, including but not limited to: (1) Any one or any combination of GLP-1R, GCGR, and FGF21R ​​agonist activities, preferably GLP-1R and / or FGF21R ​​agonist activities; or, any one or any combination of GLP-1, GCG, and FGF21 activities, preferably GLP-1 and / or FGF21 activities. (2) Lower blood sugar; (3) Reduce weight or decrease weight gain; (4) Reduce fat intake; (5) Reduce hepatic steatosis or lower triglyceride levels in the liver; (6) Reduce AST, ALT and / or ALP levels; (7) Reduce or alleviate liver fibrosis; (8) Reduce appetite or decrease food intake; (9) Increase insulin sensitivity or adiponectin; (10) Lower triglyceride and cholesterol levels.

[0033] In some embodiments, the reference material is selected from: Survodutide, Efruxifermin, DR10624, and Efinopegdutide. In some embodiments, the reference material is selected from the triple-active protein described in patent document CN109836504B, such as C382L. 13 F8L 10 M2, C462L 13 F8L 10 M2, C495L 13 F8L 10 M2.

[0034] In one aspect, the present invention provides a GLP-1 and GCG dual receptor agonist comprising the aforementioned domain P1; optionally, the dual receptor agonist further comprises the aforementioned half-life extension domain P2, and / or, the dual receptor agonist has the following structure from the N-terminus to the C-terminus: P1-L a -P2, where L a It is a peptide linker, selected from any of the aforementioned peptide linkers.

[0035] In some implementations, the L a Includes SEQ ID NO.4: (GGGGS) n Or SEQ ID NO.8: (GGGGA) n Preferably, n = 1, 2 or 3.

[0036] In some embodiments, P1 is selected from SEQ ID NO. 17, 18, 20, 32, 33 or 35, P2 is selected from IgG1 Fc or IgG4 Fc or their Fc variants, and L... a Selected from (GGGGS) n Or (GGGGA) n Or GS, where n = 1, 2 or 3.

[0037] In some embodiments, the dual receptor agonist comprises an amino acid sequence selected from SEQ ID NO. 91 or 92.

[0038] In some embodiments, the dual receptor agonist comprises a sequence selected from amino acid residues 1-282 of SEQ ID NO. 79, a sequence selected from amino acid residues 1-271 of SEQ ID NO. 80, a sequence selected from amino acid residues 1-281 of SEQ ID NO. 81, a sequence selected from amino acid residues 1-282 of SEQ ID NO. 82, a sequence selected from amino acid residues 1-282 of SEQ ID NO. 83, a sequence selected from amino acid residues 1-282 of SEQ ID NO. 84, a sequence selected from amino acid residues 1-281 of SEQ ID NO. 85, a sequence selected from amino acid residues 1-281 of SEQ ID NO. 86, a sequence selected from amino acid residues 1-281 of SEQ ID NO. 87, a sequence selected from amino acid residues 1-281 of SEQ ID NO. 88, a sequence selected from amino acid residues 1-281 of SEQ ID NO. 89, or a sequence selected from amino acid residues 1-281 of SEQ ID NO. 90.

[0039] In one aspect, the present invention provides an FGF21 analog comprising the aforementioned domain P3; optionally, the analog further comprises the aforementioned half-life extension domain P2, and / or, the analog has the following structure from the N-terminus to the C-terminus: P2-L b -P3, where L b It is a peptide linker, selected from any of the aforementioned peptide linkers.

[0040] In some implementations, the L b Includes SEQ ID NO.4: (GGGGS) n Or SEQ ID NO.8: (GGGGA) n Preferably, n = 1, 2 or 3.

[0041] In some embodiments, P3 is selected from the amino acid sequence shown in SEQ ID NO. 55, P2 is selected from IgG1 Fc or IgG4 Fc or their Fc variants, and L... a Selected from (GGGGS) n Or (GGGGA) n , where n = 1, 2 or 3.

[0042] In some embodiments, the FGF21 analog comprises a sequence consisting of amino acid residues 56-478 of SEQ ID NO.79.

[0043] In one aspect, the present invention provides a polynucleotide encoding the recombinant fusion protein of the present invention, or the GLP-1 and GCG dual receptor agonist of the present invention, or the FGF21 analog of the present invention. The polynucleotide of the present invention may be, for example, DNA or RNA, and may or may not contain intron sequences. In a preferred embodiment, the polynucleotide is a cDNA molecule. The polynucleotide of the present invention can be prepared or obtained by known means based on information of the amino acid sequence of the recombinant fusion protein of the present invention, for example by automated DNA synthesis and / or recombinant DNA technology.

[0044] As is well known in the art, multiple codons can encode the same amino acid. Therefore, nucleic acids encoding protein sequences include those with codon degeneracy. The amino acid sequences described in this invention can be encoded by a variety of nucleic acids. The genetic code is universal and well-known. Nucleic acids encoding any of the amino acid sequences described in this invention can be readily conceived based on common knowledge in the art and can be optimized for production. Although the number of possible nucleic acid sequences encoding a given amino acid is large, given a standard table of the genetic code and with the aid of a calculator, those skilled in the art can readily generate every possible combination of nucleic acid sequences encoding a given amino acid.

[0045] In one aspect, the present invention provides a vector comprising the polynucleotides of the present invention. The vector is capable of delivering and preferably expressing in a host cell a construct of one or more target genes or sequences. The vector includes eukaryotic expression vectors, prokaryotic expression vectors, viral vectors such as bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors.

[0046] In one aspect, the present invention provides a host cell comprising the polynucleotide or vector of the present invention. The host cell is capable of producing or secreting the complete recombinant fusion protein of the present invention, or the GLP-1 and GCG dual receptor agonist of the present invention, or the FGF21 analog of the present invention. The host cell comprises prokaryotic cells, fungal cells, or mammalian cells, such as CHO cells, 293 cells, NSO cells, or other mammalian cells, *Escherichia coli* or other prokaryotic cells, yeast cells, or other fungal cells.

[0047] In one aspect, the present invention provides a pharmaceutical composition comprising the recombinant fusion protein of the present invention or the GLP-1 and GCG dual receptor agonist of the present invention or the FGF21 analog of the present invention or the polynucleotide of the present invention or the vector of the present invention or the host cell of the present invention, and one or more pharmaceutically acceptable vectors.

[0048] In some preferred embodiments, the pharmaceutical composition contains a therapeutically effective amount of the fusion protein or polynucleotide of the present invention or the carrier or host cell of the present invention, as well as one or more pharmaceutically acceptable carriers, diluents, buffers or excipients.

[0049] In some embodiments, the pharmaceutical composition may contain any number of excipients. Excipients that may be used include carriers, surfactants, thickeners or emulsifiers, solid binders, dispersants or suspending agents, solubilizers, colorants, flavoring agents, coating agents, disintegrants, lubricants, sweeteners, preservatives, isotonic agents, or combinations thereof. The selection and use of appropriate excipients is taught in the following, Gennaro, ed., Remington: The Science and Practice of Pharmacy, 20th edition (Lippincott Williams & Wilkins 2003), the disclosure of which is incorporated herein by reference.

[0050] In one aspect, the present invention provides a kit comprising the recombinant fusion protein of the present invention, or the GLP-1 and GCG dual receptor agonist of the present invention, or the FGF21 analog of the present invention, or the polynucleotide of the present invention, or the vector of the present invention, or the host cell of the present invention, or the pharmaceutical composition of the present invention.

[0051] In one aspect, the present invention provides a method for preventing, treating, or alleviating a disease, comprising administering to a subject in need a therapeutically effective amount of the recombinant fusion protein of the present invention, or the GLP-1 and GCG dual receptor agonist of the present invention, or the FGF21 analog of the present invention, or the polynucleotide of the present invention, or the vector of the present invention, or the host cell of the present invention, or the pharmaceutical composition of the present invention.

[0052] In one aspect, the recombinant fusion protein of the present invention, or the GLP-1 and GCG dual receptor agonist of the present invention, or the FGF21 analog of the present invention, or the polynucleotide of the present invention, or the vector of the present invention, or the host cell of the present invention, or the pharmaceutical composition of the present invention, is provided for use in the preparation of a medicament. In some embodiments, the medicament is used as an agonist of FGF21R, GLP-1R, and / or GCGR, for example, a GLP-1 / GCG dual receptor agonist, or a GLP-1 / GCG / FGF21 triple receptor agonist, or an FGF21 single receptor agonist. In some embodiments, the medicament is used to treat a disease.

[0053] In some embodiments, the disease is selected from diseases caused by metabolic disorders of FGF21, GLP-1, and / or GCG, metabolic syndrome, fibrotic diseases, and cardiovascular diseases. In some embodiments, the disease is selected from diabetes, fatty liver, obesity, pancreatitis, dyslipidemia, hypertension, hepatic steatosis, non-alcoholic fatty liver disease (NASH), non-alcoholic fatty liver disease (NAFLD), metabolic dysfunction-associated steatohepatitis (MASH), atherosclerosis, arteriosclerosis, coronary heart disease, peripheral artery disease, and stroke.

[0054] In some embodiments, the drug is used in one or more of the following: (1) Lower blood glucose levels (e.g., fasting blood glucose); (2) Weight loss or reduction of weight gain or reduction of fat mass; (3) Improve liver weight and liver lipids, such as reducing liver weight, reducing hepatic steatosis, or reducing triglyceride levels in the liver; (4) Improve liver function, such as reducing AST, ALT and / or ALP levels; (5) Reduce or alleviate liver fibrosis; (6) Reduce appetite or decrease food intake; (7) Increase insulin sensitivity or adiponectin; (8) Improve blood lipids.

[0055] For clarity, this article defines the general terminology used in the description of compounds.

[0056] Unless otherwise stated, the following terms and phrases used herein are intended to have the following meanings. A particular term or phrase should not be considered uncertain or unclear unless specifically defined, but should be understood in its ordinary sense. When a trade name appears herein, it is intended to refer to the corresponding product or its active ingredient. The term "pharmaceutically acceptable" as used herein refers to compounds, materials, compositions, and / or dosage forms that, within the bounds of reliable medical judgment, are suitable for use in contact with human and animal tissues without undue toxicity, irritation, allergic reactions, or other problems or complications, in proportion to a reasonable benefit / risk ratio.

[0057] As used herein, the term “FGF21R” refers to a known or suspected multimeric receptor complex of FGF21 formed in vivo. In several embodiments, FGF21R ​​includes (i) FGFRs, such as FGFR1c, FGFR2c, FGFR3c, or FGFR4, and (ii) β-Klotho. FGF21 signaling is mediated by its interaction with receptor complexes comprising β-Klotho and FGFR (Ogawa et al. (2007), Proc. Natl. Acad. Sci. USA 104:7432-7437; Suzuki et al. (2008), Mol. Endocrinol. 22:1006-1014).

[0058] The term “FGF21 analogue” refers to the substitution of a portion of the amino acids in the wild-type FGF21 protein (e.g., SEQ ID NO. 59) that can activate the FGF21 receptor.

[0059] The term "GLP-1 / GCG dual receptor agonist" refers to a substance that can simultaneously activate both GLP-1 and GCG receptors.

[0060] As used herein, the term "half-life extension domain" refers to a pharmaceutically acceptable portion that is directly or via a linker to (fused to or conjugated to) the active domain. Mechanisms by which the half-life extension domain positively influences pharmacokinetic or pharmacodynamic behavior include, but are not limited to, (i) preventing or mitigating in vivo protease degradation or other chemical modifications that reduce the activity of the active domain, (ii) improving half-life or other pharmacokinetic properties by reducing renal filtration, decreasing receptor-mediated clearance, or increasing bioavailability, (iii) reducing toxicity, (iv) improving solubility, and (v) increasing the bioactivity and / or target selectivity of the active domain.

[0061] As used herein, the terms “Fc region,” “Fc fragment,” and “Fc” are used interchangeably to refer to a crystallizable fragment (Fc) of an immunoglobulin (e.g., IgG). An Fc fragment can be obtained by removing two antigen-binding regions (Fab fragments) from an immunoglobulin. One method of removing the Fab fragment is to digest the immunoglobulin with papain. Thus, the Fc region is formed by approximately identical fragments of constant regions (CH) of two heavy chains linked by non-covalent interactions and optionally disulfide bonds. The Fc fragment may include a hinge region and extend towards the C-terminus of the antibody via CH2 and CH3. In some embodiments, the Fc region is a human IgG1, IgG2, IgG3, or IgG4 Fc region. In some embodiments, the Fc region is an IgG1 Fc region.

[0062] As used herein, the term “peptide linker” refers to an amino acid or polypeptide that can be used to connect two protein domains to provide space and / or flexibility between the two protein domains.

[0063] As used herein, the term "dimer" or "dimerization" generally refers to a form of protein-protein interaction. For example, it can include a protein-protein complex composed of two related subunits. In this invention, the term "homodimer" generally refers to a dimer composed of two identical subunits (e.g., two identical polypeptide chains).

[0064] As used herein, the term "fusion protein" refers to a protein containing more than one domain, typically derived from different sources (e.g., different proteins, peptides, cells, etc.), said domains being covalently linked by peptide bonds. In some embodiments, recombination produces fusion proteins. In some embodiments, the domains of a fusion protein are covalently linked by linking gene sequences encoding each domain into a single nucleic acid molecule.

[0065] As used herein, the term "signal peptide" is defined as a peptide that is attached (fused) to the N-terminus of a biologically active polypeptide in a reading-frame manner and directs the polypeptide into the secretory pathway of the cell. Signal peptides may also be referred to as "lead peptides," "peptide precursors," "propeptides," etc. The use of signal peptides to direct protein secretion is known in the art (e.g., U.S. Patent US8658174, the contents of which are incorporated herein by reference in their entirety). The sequence of a signal peptide can be determined using techniques known in the art (see, e.g., Zhang and Henzel, 2004, Protein Science 13:2819-2824). Typically, signal peptides are about 10–60 amino acids long, more commonly 15–50 amino acids long.

[0066] As used herein, the terms "variant" and "mutant" are used interchangeably and refer to a polypeptide derived from a wild-type or parental polypeptide that contains at least one mutation (e.g., substitution, insertion, deletion) at one or more positions. Variants can be obtained using a variety of techniques known in the art. In particular, examples of techniques for altering the DNA sequence encoding a wild-type protein include, but are not limited to, site-directed mutagenesis, random mutagenesis, and synthetic oligonucleotide constructs. Therefore, as used herein, the term "mutation" relative to a specific position means that at least one amino acid at that specific position has been mutated (e.g., substituted) compared to the amino acid at that specific position in the wild-type protein or parental polypeptide.

[0067] As used herein, the term "substitution" refers to the replacement of an amino acid residue by another amino acid residue. Preferably, the term "substitution" refers to the replacement of an amino acid residue by another amino acid residue selected from the standard 20 naturally occurring amino acid residues, rare naturally occurring amino acid residues (e.g., hydroxyproline, hydroxylysine, allohydroxylysine, 6-N-methyllysine, N-ethylglycine, N-methylglycine, N-ethylasparagine, alloleucine, N-methylisoleucine, N-methylvaline, pyroglutamine, GABA, ornithine, ortholeucine, orthovaline), and non-naturally occurring amino acid residues that are usually synthesized (e.g., cyclohexylalanine). Preferably, the term "substitution" refers to the replacement of an amino acid residue by another amino acid residue selected from the standard 20 naturally occurring amino acid residues (G, P, A, V, L, I, M, C, F, Y, W, H, K, R, Q, N, E, D, S, and T). In this document, the following terminology is used to designate substitutions: R19V indicates that the amino acid residue arginine (R) at position 19 of the parental or wild-type sequence is replaced by valine (V). Substitutions can be conserved or non-conserved. Conservative substitutions refer to the mutual substitution of amino acids belonging to the same class or having similar characteristics (e.g., charge, side chain size, hydrophobicity, hydrophilicity, main chain conformation, and rigidity). Examples of conserved substitutions include basic amino acids (arginine, lysine, and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine, asparagine, and threonine), hydrophobic amino acids (methionine, leucine, isoleucine, cysteine, and valine), aromatic amino acids (phenylalanine, tryptophan, and tyrosine), and small amino acids (glycine, alanine, and serine).

[0068] As used in this article, the term "recombinant" refers to nucleic acid constructs, vectors, peptides, or cells prepared through genetic engineering.

[0069] As used herein, the term “expression” refers to any step involved in the preparation of a polypeptide, such as transcription, post-transcriptional modification, translation, post-translational modification, or secretion.

[0070] As used herein, the terms “peptide,” “polypeptide,” “protein,” and “protein” refer to a chain of amino acids linked by peptide bonds, regardless of the number of amino acids forming the chain. The terms “peptide,” “polypeptide,” “protein,” and “protein” are used interchangeably throughout this text.

[0071] As used herein, the term "amino acid sequence" refers to the order in which amino acids are linked together to form a peptide chain (or polypeptide). An amino acid sequence can only be read in one direction. There are over 100 different types of amino acids, of which 20 are commonly used. This invention does not exclude modifications to the amino acid chain by other substances, such as carbohydrates or lipids, nor is it limited to the 20 commonly used amino acids.

[0072] As used herein, the term “EU number” refers to the EU numbering rules for antibody constant regions, as described in Edelman, GM et al., Proc. Natl. Acad. USA, 63, 78-85 (1969) and Kabat et al., Sequences of Proteins of Immunological Interest, USDept. Health and Human Services, 5th ed., 1991, all of which are incorporated herein by reference in their entirety.

[0073] As used herein, the terms “nucleic acid,” “nucleic acid sequence,” “polynucleotide,” “oligonucleotide,” and “nucleotide sequence” are used interchangeably and refer to a sequence of deoxyribonucleotides and / or ribonucleotides. The nucleic acid may be DNA (cDNA or gDNA), RNA, or a mixture thereof. It may be in single-stranded or double-stranded form or a mixture thereof. It may be of recombinant, artificial, and / or synthetic origin and may contain modified nucleotides, including, for example, modified bonds, modified purine or pyrimidine bases, or modified sugars. The nucleic acids of the present invention may be in isolated or purified form and may be prepared, isolated, and / or manipulated by techniques known in the art, such as cloning and expression of cDNA libraries, amplification, enzymatic synthesis, or recombinant techniques. Nucleic acids may also be synthesized in vitro by known chemical synthesis techniques, such as those described in Belousov (1997) Nucleic Acids Res. 25:3440-3444.

[0074] As used herein, the terms “comprising,” “including,” and “having” are used interchangeably to indicate the inclusiveness of a scheme, meaning that the scheme may contain elements other than those listed. It should also be understood that the use of “comprising,” “including,” and “having” in this document also provides for schemes “consisting of…”.

[0075] The terms "identity" and "sequence consistency" used herein are interchangeable and are calculated as follows: To determine the percentage of "identity" between two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., vacancies may be introduced in one or both of the first and second amino acid sequences or nucleic acid sequences for optimal alignment, or non-homologous sequences may be discarded for comparison purposes). The amino acid residues or nucleotides at corresponding amino acid or nucleotide positions are then compared. The molecules are identical at that position when a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence.

[0076] The term "IC" 50"IC" refers to the half-maximal inhibitory concentration, which measures the efficacy of a substance (e.g., a fusion protein) in inhibiting a specific biological or biochemical reaction. 50 The smaller the value, the stronger the indicator effect.

[0077] The term "EC" 50 "EC" refers to the half-maximal effective concentration, which measures the efficacy of a substance (e.g., fusion) in inducing a specific biological or biochemical reaction. 50 The smaller the value, the stronger the indicator effect.

[0078] The term "subject" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, such as mammals and non-mammals, for example, non-human primates, rodents, rabbits, pigs, dogs, cats, chickens, amphibians, and reptiles, although mammals such as non-human primates and rodents are preferred.

[0079] The term "therapeutic effective amount" refers to an amount of the recombinant fusion protein of the present invention or a GLP-1 and GCG dual receptor agonist or FGF21 analog that is sufficient to prevent or improve symptoms associated with a disease or condition (e.g., obesity, MASH) and / or reduce the severity of the disease or condition. Therapeutic effective amount should be understood in the context of the condition being treated, where the actual effective amount can be readily identified by those skilled in the art.

[0080] The term "metabolic disorders" generally refers to diseases caused by disruptions in biochemical processes within the body, resulting in the accumulation or deficiency of certain metabolic substances (such as sugars, lipids, proteins, purines, etc.). Metabolic disorders can be caused by congenital or acquired factors. Congenital factors may include congenital genetic defects. Acquired factors may include pathological changes and functional disorders of internal organs, external factors including medications and food, such as obesity caused by consuming excessive amounts of fat and / or cholesterol.

[0081] The term "obesity" means the accumulation of excess adipose tissue in the body, and a body mass index (BMI) of 25 or higher is considered obese. Obesity is usually caused by an energy imbalance—when food intake consistently exceeds energy expenditure. Obesity is a metabolic disease affecting the whole body and increases the risk of diabetes, high cholesterol, sexual dysfunction, arthritis, and cardiovascular disease, and in some cases, it is associated with cancer.

[0082] The terms “activation” or “activation” are used to refer to a receptor or receptor complex to reflect a biological effect that arises directly and / or through participation in a multi-component signal transduction cascade, which is a ligand-binding response caused by the binding of an agonist ligand to the receptor.

[0083] The amino acid sequences of domain P1 used in this invention are listed in Table 1.1, the amino acid sequences of domain P2 are listed in Table 1.2, the amino acid sequences of domain P3 are listed in Table 1.3, and the amino acid sequences of some exemplary recombinant fusion proteins are listed in Table 1.4. In the tables, "-" indicates that there are no amino acid substitutions.

[0084] Table 1.1 Amino acid sequence of P1

[0085] Table 1.2 Amino acid sequence of P2

[0086] Table 1.3 Amino acid sequence of P3

[0087] Table 1.4 Amino acid sequences of exemplary recombinant fusion proteins

[0088] Other features and advantages of the present invention will become apparent from the following detailed description and examples, and should not be construed as limiting. All references, Genbank entries, patents, and published patent applications cited throughout this application are expressly incorporated herein by reference. Attached Figure Description

[0089] Figure 1 An exemplary structure of the recombinant fusion protein of the present invention is shown.

[0090] Figure 2 The FGF21 activity of a portion of the recombinant fusion protein of the present invention, as tested according to Example 2.3, is shown. nativeFGF21 represents wild-type FGF21, and EFX represents Efruxifermin, which was prepared according to SEQ ID NO.47 as described in patent document CN102655877B.

[0091] Figure 3 The GLP-1 activity of the recombinant fusion protein of the present invention, as tested according to Example 2.1, is shown, where native GLP1 represents wild-type GLP-1.

[0092] Figure 4 The GLP-1 activity of the recombinant fusion protein of the present invention was demonstrated, wherein, Figure 4 A and Figure 4 B represents the GLP-1 activity tested according to Example 2.1, and native GLP1 represents wild-type GLP-1.

[0093] Figure 5 The FGF21 and GLP-1 activities of the recombinant fusion protein of the present invention were demonstrated, wherein, Figure 5 A and Figure 5 B represents the FGF21 activity tested according to Example 2.3, and EFX stands for Efruxifermin; Figure 5 C represents the GLP-1 activity tested according to Example 2.1, and native GLP1 indicates wild-type GLP-1.

[0094] Figure 6 The FGF21 activity of the recombinant fusion protein of the present invention, as tested according to Example 2.3, is shown, where nativeFGF21 represents wild-type FGF21.

[0095] Figure 7 The GLP-1 activity of the recombinant fusion protein of the present invention, as tested according to Example 2.1, is shown, where native GLP1 represents wild-type GLP-1.

[0096] Figure 8 The GLP-1, GCG, and FGF21 activities of the recombinant fusion protein of the present invention were demonstrated.

[0097] Figure 9 The GLP-1 and GCG activities of the recombinant fusion protein were demonstrated.

[0098] Figure 10-11 The effects of the test substance in Example 4.1 on mouse body weight, liver function, and fasting blood glucose are shown sequentially.

[0099] Figure 12-15 The effects of the test substance in Example 4.2 on body weight, liver function, liver weight, liver lipids, and NAS score in NASH mice are shown sequentially.

[0100] Figure 16 The effects of the test substance in Example 4.3 on NAS score and degree of liver fibrosis are shown. Specific Implementation Example 1: Preparation of Recombinant Fusion Protein

[0101] A recombinant fusion protein is provided, which is a homodimer composed of two polypeptide chains, the polypeptide chains having the following structure in sequence from the N-terminus to the C-terminus: P1-L a -P2-L b -P3; where P1 represents a domain with dual GLP-1R / GCGR agonist activity; P2 represents a half-life extension domain containing an Fc fragment; P3 represents a domain with FGF21R ​​agonist activity; L a and L b Independently representing peptide linkers, optionally, La and / or L b No. A schematic diagram of an exemplary recombinant fusion protein of the present invention is shown below. Figure 1 The amino acid sequences of the polypeptide chains contained in the exemplary recombinant fusion protein of the present invention are shown in Table 2 (underlined portions indicate peptide linkers).

[0102] Table 2

[0103] Based on the amino acid sequences in Table 2 and the amino acid sequences of the desired signal peptide, the corresponding DNA sequences were designed. The corresponding fusion protein coding gene fragments containing the signal peptide were prepared using a whole-genome synthesis method. Each coding gene fragment was inserted into an expression vector using standard molecular biology techniques (e.g., see Sambrook, J., Fritsch, EF, and Maniais, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press). After verifying the correctness of the inserted sequences by sequencing, the constructed expression vectors were transfected into Expi293 or ExpiCHO-s expression systems using commercial transfection kits. The transfected Expi293 or ExpiCHO-s cells were cultured, and the cell culture medium was harvested after several days. The supernatant was collected by centrifugation, filtered, and concentrated. Subsequently, purification was performed using the standard Protein A procedure. The purified fusion proteins were harvested and identified by SDS-PAGE gel electrophoresis and SEC-HPLC purity testing to obtain the expected recombinant fusion proteins. Example 2 In vitro activity detection 2.1 GLP-1R cellular agonistic activity

[0104] A CHO cell line overexpressing the human GLP-1 receptor was used to evaluate the GLP-1 activity of the recombinant fusion protein. First, a cell line stably expressing human GLP-1R was constructed. The specific steps were as follows: the human GLP-1R gene was cloned into an expression vector, and CHO-K1 cells were transfected with this expression vector. After antibiotic selection, single clones were picked, and positive clones were identified and expanded to obtain a stable human GLP-1R-expressing cell line, named the CHO-K1 / GLP-1R cell line.

[0105] The CHO-K1 / GLP-1R cell line stably expresses the human GLP-1R gene. When a GLP-1R agonist binds to GLP-1R on the surface of CHO-K1 / GLP-1R cells, it activates adenylate cyclase through signal transduction systems such as G proteins. Under the action of adenylate cyclase, ATP loses one pyrophosphate to generate cAMP. Therefore, the activation effect of the recombinant fusion protein on the GLP-1R signaling pathway can be reflected by monitoring the cAMP concentration signal in the cells. The cAMP concentration signal was measured using a cAMP kit (Revvity, Product Number: 62AM4PEB). The specific experimental protocol is as follows: (1) Adjust the CHO-K1 / GLP-1R cell density to 8×10⁻⁶ cells / year. 5 (1) Seed 5 μL / well into a 96-well plate and culture; (2) Dilute each test sample and wild-type GLP-1 (R&D Systems, Catalog #: 5374) to the initial working concentration, and then serially dilute to several concentration gradients; (3) Add 5 μL of test sample diluent and wild-type GLP-1 diluent (positive control) at each concentration gradient to each cell culture well, and set blank control wells and negative control wells. After sealing the plate, incubate it in a 37℃, 5%CO2 incubator for 30 min; (4) Take out the 5×cAMP EU Cryptate antibody and 5×cAMP d2 reagent from the kit and dilute them to 1× according to the instructions. Add 5 μL of 1×cAMP d2 reagent and 1×cAMP EU Cryptate antibody to each cell culture well. After sealing the plate, place it at room temperature for 1 h; (5) Select the HTRF (Europium / Red) module of the PerkinElmer VICTOR Nivo multi-microplate reader and set it as follows: Dual Emission, Excitation filter: 355 / 40nm and Emission filter: 620 / 10nm and 665 / 8nm, readings; (6) HTRF Ratio = (665nm signal value / 620nm signal value) × 10000, HTRF Ratio and the concentration of the sample to be measured are processed by GraphPad Prism for curve fitting, and EC is calculated. 50 Value. All the above tests were performed with duplicate holes. 2.2 Activatory activity at the GCGR cell level 2.2.1 cAMP detection of cellular-level agonist activity

[0106] Cell lines overexpressing the human GCG receptor were used to evaluate the GCG activity of the recombinant fusion protein. The GCGR / CRE-luc / HEK293 cell line (Nanjing Kebai Biotechnology, CBP71345) stably expressed the human GCGR gene. When a GCGR agonist bound to GCGR on the surface of GCGR / CRE-luc / HEK293 cells, it activated adenylate cyclase via G protein signaling systems. Under the action of adenylate cyclase, ATP lost a pyrophosphate phosphate to generate cAMP. The cAMP content in cells was measured using a cAMP kit (Revvity, Product Number: 62AM4PEB). The specific experimental protocol was performed according to the steps in Example 2.1. The positive control was wild-type glucagon (aladdin, catalog number G118963). 2.2.2 Detection of cellular-level agonist activity using CRE-luc reporter gene assay

[0107] The density of GCGR / CRE-luc / HEK293 cells (Nanjing Kebai Biotechnology, CBP71345) was adjusted to 2×10⁻⁶. 5 Cells / mL, 100 μL / well were seeded into 96-well plates and incubated overnight at 37°C with 5% CO2. The test samples and wild-type glucagon (aladdin, catalog number G118963) were diluted to the initial working concentration using DMEM + 10% FBS, and then serially diluted to several concentration gradients. The culture medium was removed from the cell culture wells, and 100 μL / well of the test sample dilution and wild-type glucagon dilution (positive control) were added to each concentration gradient. The plates were sealed and incubated at 37°C with 5% CO2 for 6 hours. After incubation, the plates were removed, and 100 μL of Bio-Lite luciferase reagent (Novizan; DD1201) was added to each cell culture well. The fluorescence signal was detected using a microplate reader, and the EC50 was calculated by curve fitting using GraphPad Prism. 50 value. 2.3 Detection of FGF21R ​​cellular agonistic activity

[0108] H_FGF21 Reporter HEK-293 cells (Jiman Biotechnology; GM-C19834) were adjusted to a density of 1.3 × 10⁻⁶. 5Cells / mL, 100 μL / well were seeded into 96-well plates and incubated overnight at 37°C and 5% CO2. The test samples and wild-type FGF21 (Sinobiological, Cat: 10911-H07E) were diluted to the initial working concentration using DMEM + 1% FBS + 1% PS, and then serially diluted to several concentration gradients. The culture medium was removed from the cell culture wells, and 100 μL / well of the test sample dilution and wild-type FGF21 (positive control) were added to each concentration gradient. The plates were sealed and incubated at 37°C and 5% CO2 for 6 hours. After incubation, the plates were removed, and 100 μL of Bio-Lite luciferase reagent (Novazia; DD1201) was added to each cell culture well. The fluorescence signal was detected using a microplate reader, and the EC50 was calculated by curve fitting using GraphPad Prism. 50 value.

[0109] The activity of FGG01 to FGG14 against each target was tested using the method in Example 2, showing that all of FGG01 to FGG14 exhibited FGF21, GCG, and GLP-1 activity. The results for FGG01 to FGG14 indicate that changes in the P1 terminus of the domain and its linker La do not significantly affect the FGF21 activity at the P3 terminus. Figure 2 The results of FGF21 assays for some recombinant fusion proteins are presented, indicating that the recombinant fusion proteins have similar FGF21 activities; however, variations in the P1 domain lead to differences in GLP-1 and GCG activities. The GLP-1 activity results for each recombinant fusion protein are shown below. Figure 3 The GCG activity of each recombinant fusion protein is no higher than 50% of that of wild-type GCG. The differences in GLP-1 and GCG activity of some fusion proteins are summarized in Table A (GCG activity was tested by method 2.2.2).

[0110] Table A Example 3 Optimization of recombinant fusion protein

[0111] Using FGG03 as an example, optimization was performed. Specifically, amino acid substitutions were made for the P1 and P3 domains of FGG03, and the peptide linker was optimized, resulting in several protein variants, numbered FGG15 to FGG40. FGG15 to FGG40 were expressed and purified according to the method in Example 1, and their in vitro activity was evaluated according to the method in Example 2.

[0112] Among them, the amino acids (MN) at positions 27-28 of FGG03 were substituted as shown in Table 3 to obtain FGG15 to FGG30. The test results showed that FGG15 to FGG30 had triple activities of FGF21, GLP-1, and GCG. Among them, the FGF21 activity did not change significantly compared with its parent FGG03, and the GLP-1 activity was shown to be... Figure 4 In this study, the GCG activity of each recombinant fusion protein was no higher than 50% of that of wild-type GCG.

[0113] The general amino acid sequence structures of FGG15 to FGG30 are shown below, and the X of each fusion protein is shown below. (1) To X (3) The specific amino acids corresponding to each point are listed in Table 3: HGQGTFTSDYSKYLDEKRAKEFVQWLX (1) X (2) X (3) GGPSSGAPPPSGGGGSGGGGSGGGGSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLT VLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSHPIPDSSPLLQFGGQVRQVYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTS RFLCQRPDGALYGSLHFDPEACSFRERLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVGGSQGRSPSYES(SEQ ID NO.14) Table 3

[0114] The activities of some fusion proteins are summarized in Table B (GCG activity was tested using method 2.2.2).

[0115] Table B

[0116] Furthermore, peptide linker replacement and P3 domain amino acid substitution were performed based on FGG22 to obtain FGG31 to FGG40 and FGG33-1, FGG33-2. The results showed that FGG31 to FGG40 and FGG33-1, FGG33-2 possessed triple activities of FGF21, GLP-1, and GCG. The GLP-1 and GCG activities were similar to those of their parent FGG22 (Table C, GCG activity was tested using method 2.2.2). The GLP-1 activity of some fusion proteins was observed in... Figure 5 In C, the activity of FGF21 is demonstrated in Figure 5 In A-5B, the GCG activity of each recombinant fusion protein is no higher than 50% of that of wild-type GCG.

[0117] The general amino acid sequence structures of FGG31 to FGG40 are shown below, and the X fusion proteins are shown below. (5) To X (8) The specific amino acids corresponding to each point are listed in Table 5: HGQGTFTSDYSKYLDEKRAKEFVQWLLDAGGPSSGAPPPSGGGGSGGGGSGGGGSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL TVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKX (5) HPIPDSSPLLQFGGQVRQVYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFREX (6) LLEDGYNVYQSEAHGLPLHLPGX (7) KSPHRDPAPRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSX (8) VGGSQGRSPSYES (SEQ ID NO.15) Table 5

[0118] Table C

[0119] Amino acid substitutions of the P3 domain were performed on FGG02, FGG03, FGG05, FGG17, FGG18, and FGG20 to obtain FGG41 to FGG46, and further substitutions of the P2 domain were performed to obtain FGG47 to FGG52. The amino acid sequences of FGG41 to FGG52 are shown in Table 7. The GLP-1 and FGF21 activities of each fusion protein are as follows: Figure 6-8 As shown, the GCG activity of each recombinant fusion protein is no higher than 50% of that of wild-type GCG. According to... Figure 8 The results showed that various mutations in the amino acids of the Fc region did not significantly affect the FGF21, GCG, and GLP-1 activities of the fusion proteins. FGG47 to FGG52 had similar FGF21, GCG, and GLP-1 activities to their parent molecule FGG43.

[0120] Table 7

[0121] In addition, fusion proteins of P1-03 and Fc were prepared. The two domains were linked by (GGGGS)n of different lengths to obtain P1-03F1 and P1-03F2. The effect of linker changes on GLP-1 and GCG activities was tested. Results are shown below. Figure 9 It can be seen that the GLP-1 and GCG activities of P1-03F1 and P1-03F2 are comparable to those of FGG43. The linker changes and the presence or absence of the FGF21 domain do not significantly affect the activity of the P1 domain.

[0122] The triple-active protein C495L was prepared independently according to the sequence and method described in patent document CN109836504B. 13 F8L 10 M2, referred to as C495 in this paper, has the amino acid sequence shown in SEQ ID NO.93.

[0123] EFX stands for Efruxifermin, which is prepared independently according to the method described in patent document CN102655877B, and its amino acid sequence is shown in SEQ ID NO.94.

[0124] Table 8

[0125] Furthermore, the activity differences between the partial fusion protein of the present invention and C495, EFX or BI 456906 (i.e., Survodutide, derived from commercial products) were compared, and the results are shown in Table D (" / " indicates not tested).

[0126] Table D (GCG activity was tested using method 2.2.2) Example 4: In vivo efficacy 4.1 Pharmacodynamic evaluation of the test substance in the DIO model

[0127] This study aimed to evaluate the efficacy of the test substance in diet-induced obese (DIO) mice. Evaluation indicators included body weight, food intake, body composition, fasting blood glucose (FBG), blood lipids (total cholesterol (CHO), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C)), serum biochemistry (alanine aminotransferase (ALT), aspartate aminotransferase (AST)), and liver weight.

[0128] Five- to six-week-old male C57BL / 6J mice were used, including lean control mice (Normal, basal diet) or obese mice (D12492, Research diets) induced to become obese for 16 to 18 weeks. Mice were randomly assigned to groups (n=9 / group) based on baseline body weight, as shown in Table 9. Each group received an equimolar dose of the test substance at a volume of 5 mL / kg (2 nmol / kg).

[0129] Table 9

[0130] During the study, changes in mouse body weight, cumulative food intake, and blood glucose levels (random blood glucose, fasting blood glucose) were monitored. At the experimental endpoint (day 22), fasting blood glucose was measured, and mouse serum was collected for blood biochemical analysis (TC, TG, LDL-C, HDL-C, AST, and ALT) using a fully automated biochemical analyzer. Subsequently, the mice were euthanized, and liver tissue was dissected and harvested. The liver weight was measured, and 80-100 mg of tissue from the middle lobe of the liver was rapidly frozen in liquid nitrogen for biochemical analysis (TG, TC). The left lobe of the liver was fixed in fixative for pathological analysis (H&E staining to assess NAS score).

[0131] Data analysis was performed using Graphpad Prism statistical software. First, the data were tested for normality and homogeneity of variance. For data conforming to normality (P>0.05) and homogeneity of variance (P>0.05): one-way ANOVA with Dunnett's multiple comparison test was used for comparisons among multiple groups, with P<0.05 considered statistically significant. If variances were unequal (P<0.05), Brown-Forsythe and Welch ANOVA tests were used. If the data did not conform to normality: the non-parametric Kruskal-Wallis H method was used for multiple comparisons among multiple groups, with P<0.05 considered statistically significant.

[0132] See results Figure 10-11 The effects of each group of test substances on mouse body weight, liver function (ALT, AST) and fasting blood glucose were shown in turn. Figure 10 In the study, compared with the G2 group, groups G3 to G11 all significantly reduced the weight of obese mice (p < 0.001). Among them, FGG43 was slightly better than FGG42, and FGG43 and FGG42 had significantly better weight loss effects than C495. Figure 11 In A and 11B, G3 to G11 significantly improved liver function in mice. Compared with G2, G3 to G11 groups significantly reduced ALT levels (p < 0.001), and G3 to G5 and G7 to G9 groups significantly reduced AST levels compared with G2 (p < 0.001). The G3 to G5 and G7 to G9 groups were more effective than G6, G10, and G11 groups in reducing AST levels (G6 vs G2, p < 0.05; G10 or G11 vs G2, p < 0.01). Figure 11 In C, fasting blood glucose on day 10 showed that groups G3 to G10 significantly reduced fasting blood glucose in obese mice (vs G2, p<0.001), and the changes in fasting blood glucose were consistent with the changes in body weight. 4.2 Pharmacodynamic evaluation of the test substance in the NASH model

[0133] This study aimed to evaluate the efficacy of the test substance in a mouse NASH model induced by a high-fat, high-sugar, and high-cholesterol diet. Evaluation indicators included body weight, food intake, fasting blood glucose, blood lipids (CHO, TG, LDL-C, HDL-C), serum biochemistry (ALT, AST), liver weight, and liver pathological score (NAS score based on HE staining and fibrosis score based on Sirius red staining).

[0134] Five-week-old male C57BL / 6 mice were fed a high-fat diet (HFD) with free access to 15% fructose water (w / v) for 16 consecutive weeks. The HFD was then replaced with a modified high-fat diet (mHFD) containing high cholesterol, with free access to 15% fructose water (v / w) for 5 weeks. After 5 weeks, orbital blood samples were collected from all animals, and serum ALT levels were measured by centrifugation. Animals were randomly assigned to groups based on body weight and serum ALT levels for drug administration. During the drug administration period, the modified high-fat diet (mHFD) and free access to 15% fructose water (v / w) were maintained.

[0135] The study groups are shown in Table 10 below. Groups G3 to G6 were administered equimolar doses (3 nmol / kg): Table 10

[0136] During the study, mouse body weight changes and food intake were monitored (twice weekly), and serum ALT levels were measured regularly (weeks 25 and 28). At the experimental endpoint (week 28), fasted blood glucose levels were measured, and mouse serum was collected for blood biochemical analysis (TC, TG, LDL-C, HDL-C, AST, and ALT) using a fully automated biochemical analyzer. Mice were then euthanized, and liver tissue was dissected and harvested. The liver weight was measured, and 80-100 mg of tissue from the middle lobe was rapidly frozen in liquid nitrogen for biochemical analysis (TG, TC). The left lobe of the liver was fixed in fixative for pathological analysis (H&E staining to assess NAS score, SR staining to assess Fibrosis score and percentage). The NAS scoring criteria are shown in Table 11 below, and the Fibrosis scoring criteria are shown in Table 12 below.

[0137] Table 11 NAFLD activity score

[0138] Table 12 Fibrosis score

[0139] Data analysis was performed using Graphpad Prism statistical software. First, the data were tested for normality and homogeneity of variance. For data conforming to normality (P>0.05) and homogeneity of variance (P>0.05): one-way ANOVA with Dunnett's multiple comparison test was used for comparisons among multiple groups, with P<0.05 considered statistically significant. If variances were unequal (P<0.05), Brown-Forsythe and Welch ANOVA tests were used. If the data did not conform to normality: the non-parametric Kruskal-Wallis H method was used for multiple comparisons among multiple groups, with P<0.05 considered statistically significant.

[0140] See results Figure 12-15 The effects of each test substance on body weight, liver function, liver weight, liver lipids, and NAS score of NASH mice were shown sequentially. Figure 12 The results show that the recombinant fusion protein of the present invention also has a significant weight loss effect in NASH mice (vs G2, p < 0.0001), exhibiting a dose-dependent effect. Figure 13 The results showed that ALT levels improved with prolonged treatment cycles, and at the end of treatment, ALT levels in groups G3, G4, G6, and G7 decreased to levels comparable to those in normal mice. Figure 14 The results showed that, except for the EFX treatment group, the liver weight and liver lipids of the other treatment groups almost returned to the levels of the normal diet control group (G1). Figure 15 The results show that groups G3, G4, G6, and G7 significantly reduced NAS scores (NAS scores decreased by at least 2 points). This demonstrates that the recombinant fusion protein of this invention can effectively improve NASH. 4.3 Pharmacodynamic Evaluation in the NASH Model

[0141] This study aimed to evaluate the efficacy of the test substance in mice with NASH induced by a high-fat, high-sugar, and high-cholesterol diet. Evaluation indicators included body weight, food intake, fasting blood glucose, blood lipids (CHO, TG, LDL-C, HDL-C), serum biochemistry (ALT, AST), liver weight, and liver pathological score (NAS score based on HE staining and fibrosis score based on Sirius red staining).

[0142] Five-week-old male C57BL / 6 mice were fed a high-fat diet (HFD) with free access to 15% fructose water (w / v) for 16 consecutive weeks. The HFD was then replaced with a modified high-fat diet (mHFD) containing high cholesterol, with free access to 15% fructose water (v / w) for 4 weeks. After 4 weeks, orbital blood samples were collected from all animals, and serum ALT levels were measured by centrifugation. Animals were randomly assigned to groups based on body weight and serum ALT levels for drug administration. During the drug administration period, the modified high-fat diet (mHFD) and free access to 15% fructose water (v / w) were maintained.

[0143] The study groups are shown in Table 13 below. Among them, BI 456906 (also known as Survodutide) is a commercially available GCGR / GLP-1R dual agonist. Table 13 Animal grouping and administration regimen

[0144] During the study, mouse body weight changes and food intake were monitored (twice weekly). After 8 weeks of drug administration, the endpoint of fasting blood glucose was measured, and mouse serum was collected for blood biochemical analysis (TC, TG, LDL-C, HDL-C, AST, and ALT) using a fully automated biochemical analyzer. The mice were then euthanized, and liver tissue was dissected and harvested. The liver weight was measured, and 80-100 mg of tissue from the middle lobe was rapidly frozen in liquid nitrogen for biochemical analysis (TG, TC). The left lobe of the liver was fixed in fixative for pathological analysis (H&E staining to assess NAS score, SR staining to assess Fibrosis score and percentage). The NAS scoring criteria are shown in Table 11, and the Fibrosis scoring criteria are shown in Table 12.

[0145] Data analysis was performed using Graphpad Prism statistical software. First, the data were tested for normality and homogeneity of variance. For data conforming to normality (P>0.05) and homogeneity of variance (P>0.05): one-way ANOVA with Dunnett's multiple comparison test was used for comparisons among multiple groups, with P<0.05 considered statistically significant. If variances were unequal (P<0.05), Brown-Forsythe and Welch ANOVA tests were used. If the data did not conform to normality: the non-parametric Kruskal-Wallis H method was used for multiple comparisons among multiple groups, with P<0.05 considered statistically significant.

[0146] See results Figure 16 It is evident that the recombinant fusion protein of the present invention can effectively improve NAS scores and liver fibrosis, and its effect on improving NASH is significantly better than that of C495 and the single or combination therapy groups.

[0147] Although the invention has been described through one or more embodiments, it should be understood that the invention is not limited to these embodiments, and the specification is intended to cover all alternatives, modifications, and variations falling within the spirit and broad scope of the appended claims. All references cited in this invention are incorporated herein by reference in their entirety.

Claims

1. A recombinant fusion protein with multiple activities, characterized in that, The recombinant fusion protein comprises a domain P1 and a domain P3, wherein P1 is a domain with dual GLP-1 / GCG activity and P3 is a domain with FGF21 activity; optionally, the recombinant fusion protein further comprises a half-life extension domain P2. The P1 contains an amino acid sequence selected from either (1) or (2): (1) HGQGTFTSDYSKYLDEKRAKEFVQWLX1X2X3X4X5X a X1 is selected from any amino acid among M, I, L, and Q; X2 is selected from any amino acid among N, D, and R; X3 is selected from any amino acid among T, S, Y, and A, or X3 may be omitted; X4 is selected from any amino acid among A, V, and S, or X4 may be omitted; X5 is selected from any amino acid among G, A, V, and S, or X5 may be omitted. When X5 exists, X a The representative amino acid sequence is GPSSGAPPPS; When X5 is missing, X a The representative amino acid sequence is GGPSSGGPSSGGPSS with X4 missing, or X... a And X4 is missing; (2) HGQGTFTSDYSKYLDERAAKDFIX6WLESA, X6 is selected from any one of the amino acids K, A, Q and E.

2. The recombinant fusion protein according to claim 1, characterized in that, X1 is M, X2 is N, X3 is T or X3 is missing; or... X1 to X3 are selected from any of the 16 combinations in the table below, where "-" indicates that X3 is missing: 。 3. The recombinant fusion protein according to claim 2, characterized in that, X4 is selected from any amino acid among A, V, and S, X5 is G, and X... a The representative amino acid sequence is GPSSGAPPPS; or, X4 is missing, X5 is selected from any one of the amino acids G, A, V, and S, and X a The representative amino acid sequence is GPSSGAPPPS; or, X5 and X4 are missing, X a The representative amino acid sequence is GGPSSGGPSSGGPSS; or, X4 is either A or X4 is missing, X5 is G, and X a The representative amino acid sequence is GPSSGAPPPS.

4. The recombinant fusion protein according to claim 3, characterized in that, The P1 is selected from any one of SEQ ID NO.16-45; Alternatively, P1 may be selected from any one of SEQ ID NO. 17, 18, 20, 32, 33 or 35.

5. The recombinant fusion protein according to any one of claims 1-4, characterized in that, The P3 is an FGF21 analog; Alternatively, the amino acid sequence contained in P3, compared with that of wild-type FGF21 protein, contains any combination of one or more of the following amino acid substitutions: R19V, L98R, P171G, A180E, N121Q, M168L. Alternatively, the amino acid sequence contained in P3, compared with the wild-type FGF21 protein, has the following amino acid substitutions: R19V, P171G, and A180E, and optionally, further has any one of L98R, N121Q, M168L, or any combination thereof. Alternatively, P3 may be selected from any one of SEQ ID NO.54-59; Alternatively, P3 may be selected from SEQ ID NO.

55.

6. The recombinant fusion protein according to any one of claims 1-5, characterized in that, The P2 contains an Fc fragment of a mammalian immunoglobulin or a variant thereof; Alternatively, the P2 may be selected from IgG1 Fc or a variant thereof; Alternatively, the P2 is selected from IgG4 Fc or a variant thereof; Alternatively, P2 may be selected from any of SEQ ID NO.46-53.

7. The recombinant fusion protein according to any one of claims 1-6, characterized in that, Following the order from N to C, P1, P2, and P3 have the following connection relationships: P1-L a -P2-L b -P3; where, L a and L b Independently representing a peptide linker, "-" represents a peptide bond; optionally, L a and / or L b It does not exist.

8. The recombinant fusion protein according to claim 7, characterized in that, The peptide linker is selected from flexible linkers or from amino acid sequences rich in glycine (G), serine (S), and / or alanine (A), L a and L b Same or different; And / or, the peptide linker is selected from (GGGGS). n (GS) n Or (GGGGA) n n is 1, 2, 3, 4, 5 or 6; And / or, the length of the peptide linker is 2-20 amino acids; And / or, L a and L b All contain (GGGGS)3; Or, L a Includes (GGGGS)3, L b Includes GS; Or, L a Includes (GGGGS)3, L b Includes (GGGGA)3.

9. The recombinant fusion protein according to claim 1, characterized in that, The recombinant fusion protein comprises a polypeptide chain selected from any one of SEQ ID NO. 60-78; or, The polypeptide chain contains any one of the following amino acid sequences: HGQGTFTSDYSKYLDEKRAKEFVQWLX (1) X (2) X (3) GGPSSGAPPPSGGGGSGGGGSGGGGSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLT VLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSHPIPDSSPLLQFGGQVRQVYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTS RFLCQRPDGALYGSLHFDPEACSFRERLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVGGSQGRSPSYES, where X (1) To X (3) Choose any one of the 16 combinations listed in the table below, where "-" represents X. (3) Missing: ； or, The polypeptide chain contains any one of the following amino acid sequences: HGQGTFTSDYSKYLDEKRAKEFVQWLLDAGGPSSGAPPPSGGGGSGGGGSGGGGSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKX (5) HPIPDSSPLLQFGGQVRQVYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFREX (6) LLEDGYNVYQSEAHGLPLHLPGX (7) KSPHRDPAPRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSX (8) VGGSQGRSPSYES, where X (5) To X (8) Choose any one of the 10 combinations listed in the table below. ； or, The polypeptide chain comprises any one of SEQ ID NO.79-90.

10. The recombinant fusion protein according to any one of claims 1-9, characterized in that, The recombinant fusion protein is a homopolymer; Alternatively, the recombinant fusion protein is a homodimer.

11. The recombinant fusion protein according to any one of claims 1-10, characterized in that, The recombinant fusion protein exhibits better activity compared to the reference protein, and the activity includes one or more of the following: (1) Any one or any combination of GLP-1R, GCGR, and FGF21R ​​agonist activities; (2) Lower blood sugar; (3) Reduce weight or decrease weight gain; (4) Reduce fat intake; (5) Reduce hepatic steatosis or lower triglyceride levels in the liver; (6) Reduce AST, ALT and / or ALP levels; (7) Reduce or alleviate liver fibrosis; (8) Reduce appetite or decrease food intake; (9) Increase insulin sensitivity or adiponectin; (10) Lower triglyceride and cholesterol levels; And / or, the reference material is selected from: Survodutide, Efruxifermin, DR10624, Efinopegdutide, C382L 13 F8L 10 M2, C462L 13 F8L 10 M2, C495L 13 F8L 10 M2.

12. A GLP-1 and GCG dual receptor agonist comprising the domain P1 as described in any one of claims 1-4; optionally, the dual receptor agonist further comprises a half-life extension domain P2, and / or, the dual receptor agonist has the following structure from the N-terminus to the C-terminus: P1-L a -P2, where L a It is a peptide linker.

13. An FGF21 analog comprising the domain P3 of claim 5; optionally, the analog further comprising a half-life-extending domain P2, and / or, the analog having the following structure from the N-terminus to the C-terminus: P2-L b -P3, where L b It is a peptide linker.

14. A polynucleotide encoding the recombinant fusion protein of any one of claims 1-11, or the GLP-1 and GCG dual receptor agonist of claim 12, or the FGF21 analog of claim 13.

15. A vector comprising the polynucleotide of claim 14.

16. A host cell comprising the polynucleotide of claim 14 or the vector of claim 15.

17. A pharmaceutical composition or kit comprising the recombinant fusion protein of any one of claims 1-11, or the GLP-1 and GCG dual receptor agonist of claim 12, or the FGF21 analog of claim 13, or the polynucleotide of claim 14, or the vector of claim 15, or the host cell of claim 16.

18. The use of the recombinant fusion protein of any one of claims 1-11, or the GLP-1 and GCG dual receptor agonist of claim 12, or the FGF21 analog of claim 13, or the polynucleotide of claim 14, or the vector of claim 15, or the host cell of claim 16, or the pharmaceutical composition or kit of claim 17, in the preparation of a medicament, characterized in that, The drug acts as an agonist of FGF21R, GLP-1R, and / or GCGR; or, The drug is used to treat diseases selected from those caused by metabolic disorders of FGF21, GLP-1 and / or GCG, metabolic syndrome, fibrotic diseases, or cardiovascular diseases.

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