GDF15 fusion proteins and uses thereof

Abstract

GDF15 fusion proteins and uses thereof are provided. The fusion proteins include an Fc variant and a GDF15 active domain, where the Fc variant has an amino acid substitution at position 356 and / or 439 of IgG Fc according to EU numbering, and still has the ability to form homodimers. By fusing the Fc variant with the GDF15 active domain, the Fc-GDF15 fusion protein has significantly improved physicochemical properties and recombinant expression levels, has in vitro activity comparable to or better than the native GDF15 molecule, and has a significantly extended in vivo circulating half-life, which can support an administration frequency of once every two weeks or even once a month.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of Chinese Patent Application No. 202110977378.7, filed with the State Intellectual Property Office of China on August 24, 2021, which is incorporated by reference herein in its entirety.

[0002] The present invention relates to the field of biomedical technology, in particular to GDF15 fusion proteins and uses thereof. [Background technology]

[0003] Growth differentiation factor 15 (GDF15), also known as macrophage inhibitory cytokine-1 (MIC-1), placental transforming growth factor-β (PTGF-β), placental bone morphogenetic factor (PLAB), prostate-derived factor (PDF), and nonsteroidal anti-inflammatory drug-activated gene (NAG-1), is a distant member of the transforming growth factor (TGF-β) superfamily. While the mature GDF15 polypeptide contains 112 amino acids, the biologically active GDF15 circulating in vivo is a homodimeric protein (24.5 kD) formed by two polypeptides via interchain disulfide bonds.

[0004] It has been reported that elevated circulating levels of GDF15 are associated with reduced food intake and weight loss in patients with advanced cancer (Johnen H et al., Nat Med, 2007). Furthermore, both transgenic mice with high expression of GDF15 and mice administered recombinant GDF15 exhibited reduced body weight and food intake and had improved glucose tolerance (Xiong Y et al., Sci Transl Med, 2017), indicating that GDF15 has the potential to treat diseases such as obesity, type II diabetes (T2D), and nonalcoholic steatohepatitis (NASH).

[0005] The circulating half-life (t1 / 2) of the native GDF15 dimer in vivo is short, only about 2-3 hours, which significantly limits its clinical therapeutic application. Conventional methods for extending the circulating half-life of biopharmaceuticals include fusing active molecules with the Fc fragment of immunoglobulin G (IgG). However, due to the three-dimensional structural characteristics of the GDF15 dimer and Fc fragment dimer molecules, direct fusion of GDF15 and Fc fragment significantly affects the overall stability of the fusion molecule and the yield of recombinant expression.

[0006] Therefore, it is essential to develop stable therapeutic GDF15 molecules with longer half-life and best druggability for the treatment of metabolic-related diseases such as obesity, T2D, and NASH. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] WO2019195091 [Non-patent literature]

[0008] [Non-Patent Document 1] Johnen H et al., Nat Med, 2007 [Non-Patent Document 2] Xiong Y et al., Sci Transl Med, 2017 [Non-Patent Document 3] Edelman et al., The covalent structure of an entire γG immunoglobulin molecule. Proc. Natl. Acad. Sci., USA, 1969 Summary of the Invention [Problem to be solved by the invention]

[0009] The object of the present invention is to provide a GDF15 fusion protein and its use, which has the advantages of best physicochemical properties, long in vivo half-life and duration of efficacy, simple manufacturing process, etc., and can be used to treat metabolic diseases such as obesity, type II diabetes, NASH, dyslipidemia, etc. [Means for solving the problem]

[0010] To this end, in a first aspect, the present invention provides a fusion protein comprising a GDF15 active domain and an Fc variant, the C-terminus of the Fc variant being linked, either directly or via a peptide linker, to the N-terminus of the GDF15 active domain, the Fc variant comprises an amino acid substitution at position 356 and / or 439 of IgG Fc according to EU numbering, A fusion protein is provided.

[0011] In some embodiments, the Fc variants retain the ability to form homodimers.

[0012] In some embodiments, the IgG Fc is selected from one of an IgG1 Fc, an IgG2 Fc, an IgG3 Fc, and an IgG4 Fc.

[0013] In some embodiments, the IgG Fc is a human IgG1 Fc. In some embodiments, the IgG Fc is a human IgG4 Fc.

[0014] The Fc variant further comprises an amino acid substitution at position 356 of IgG Fc according to EU numbering with an amino acid other than D, E and C. For example, the amino acid at position 356 of IgG Fc is substituted with one of the following group: G, S, A, T, V, N, L, I, Q, Y, F, H, P, M, K, R. The Fc variant further comprises an amino acid substitution at position 439 of IgG Fc according to EU numbering with an amino acid other than R, H, K and C. For example, the amino acid at position 439 of IgG Fc is substituted with one of the following group: G, S, A, T, V, D, N, L, I, E, Q, Y, F, P, M.

[0015] In some embodiments, the Fc variant comprises one of the following mutations in IgG Fc according to EU numbering: E356G, E356S, E356A, E356T, E356V, E356N, E356L, E356I, E356Q, E356Y, E356F, E356H, E356P, E356M, E356K, E356R, and / or one of K439G, K439S, K439A, K439T, K439V, K439D, K439N, K439L, K439I, K439E, K439Q, K439Y, K439F, K439H, K439P, K439M.

[0016] In a preferred embodiment, the Fc variant comprises one of the following mutations in IgG Fc according to EU numbering: E356R, E356Q, E356A, E356N, and / or one of K439D, K439E, K439Q, K439A, K439N.

[0017] In some embodiments, the Fc variant comprises one of the following mutations in IgG Fc according to EU numbering: K439D, K439E, K439Q, K439A, K439N. In preferred embodiments, the Fc variant comprises one of the following mutations in IgG Fc according to EU numbering: K439D, K439E, K439Q.

[0018] In some embodiments, the Fc variant comprises one of the following mutations in IgG Fc according to EU numbering: E356R, E356Q, E356A, E356N. In a preferred embodiment, the Fc variant comprises the following mutation in IgG Fc according to EU numbering: E356R.

[0019] In some embodiments, the Fc variants further comprise the following mutations: amino acid substitutions at positions 234 and 235 of IgG Fc according to EU numbering to AA, and / or an amino acid deletion at position 447. For example, for an IgG1 Fc, the following mutations are also included: L234A and L235A, and / or an amino acid deletion at position 447, and for an IgG4 Fc, the following mutations are also included: F234A and L235A, and / or an amino acid deletion at position 447.

[0020] In certain embodiments, the Fc variant has only one of the following amino acid substitutions in an IgG Fc according to EU numbering: Amino acid substitution at position 356 according to EU numbering, Amino acid substitution at position 439 according to EU numbering, Amino acid substitutions at positions 356 and 439 according to EU numbering, Amino acid substitutions at positions 356, 234, and 235 according to EU numbering, Amino acid substitutions at positions 439, 234, and 235 according to EU numbering, Amino acid substitutions at positions 356, 439, 234, and 235 according to EU numbering, The amino acid substitutions described at each position have the meanings described in the present invention.

[0021] In certain embodiments, the amino acid sequence of the Fc variant comprises one of the following sequences: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45.

[0022] [Table 1A]

[0023] [Table 1B]

[0024] [Table 1C]

[0025] [Table 1D]

[0026] In another embodiment, the Fc variant comprises an amino acid sequence having at least 85%, 90%, 95% or 99% sequence identity to one of the following sequences: SEQ ID NOs: 1-22, SEQ ID NO: 27, SEQ ID NOs: 29-45.

[0027] Furthermore, the GDF15 active domain may be a full-length mature GDF15 protein, an N-terminally truncated GDF15 protein, or any variant that retains the biological activity of GDF15.

[0028] In another embodiment, the GDF15 activity domain is selected from the group consisting of: SEQ ID NO:46; 1-14 amino acid truncations at the N-terminus of SEQ ID NO:46 and / or 1-3 amino acid substitutions in SEQ ID NO:46 or having at least 85%, 90%, 95% or 99% sequence identity thereto.

[0029] In another embodiment, the GDF15 activity domain is selected from the group consisting of: SEQ ID NO:46; 1-14 amino acid truncations at the N-terminus of SEQ ID NO:46 and / or 1-3 amino acid substitutions in SEQ ID NO:46 The amino acid sequence is selected from one of:

[0030] Further, the number of amino acid truncations at the N-terminus of SEQ ID NO:46 is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14.

[0031] Furthermore, the positions of the amino acid substitutions in SEQ ID NO: 46 are selected from one, two or three of the following group: 5, 6, 21, 26, 30, 47, 54, 55, 57, 67, 69, 81, 94, 107.

[0032] Additionally, the amino acid substitutions in SEQ ID NO:46 are selected from one, any two, or any three of the following positions: D5E, H6D, H6E, R21Q, R21H, D26E, A30S, A47D, A54S, A55E, M57T, R67Q, K69R, A81S, T94E, K107Q.

[0033] In some embodiments, the GDF15 activity domain comprises the amino acid sequence set forth in SEQ ID NO:46.

[0034] In another embodiment, the GDF15 active domain comprises the following sequence: an amino acid sequence truncated by 1-14 amino acids at the N-terminus of SEQ ID NO:46.

[0035] In yet another embodiment, the GDF15 active domain comprises the following sequence: an amino acid sequence having one to three amino acid substitutions in SEQ ID NO: 46, wherein the amino acid substitutions in SEQ ID NO: 46 have the meanings described herein.

[0036] In yet another embodiment, the GDF15 active domain comprises the following sequence: an amino acid sequence having 1-14 amino acids truncated at the N-terminus of SEQ ID NO:46, and 1-3 amino acid substitutions in SEQ ID NO:46, wherein the amino acid substitutions in SEQ ID NO:46 have the meaning described in the present invention.

[0037] In certain embodiments, the GDF15 active domain comprises one of the following sequences: SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66.

[0038] [Table 2A]

[0039] [Table 2B]

[0040] In another embodiment, the GDF15 active domain comprises one of the amino acid sequences of the following group: SEQ ID NOs: 46 to 66, or an amino acid sequence having at least 95% sequence identity thereto, preferably the GDF15 active domain comprises one of the amino acid sequences of the following group: SEQ ID NOs: 46 to 66.

[0041] In another embodiment, the GDF15 active domain comprises an amino acid sequence having at least 85%, 90%, 95% or 99% sequence identity to one of the following sequences: SEQ ID NOs: 46-66.

[0042] In some embodiments, the C-terminus of the Fc variant is linked to the N-terminus of the GDF15 active domain via a peptide linker, and the peptide linker is selected from one of a flexible peptide linker, a rigid peptide linker, or a combination thereof. Any suitable linker can be used in the fusion protein of the present invention. Herein, the term "linker" refers to a linking moiety that contains a peptide linker. Preferably, the linker helps to ensure proper folding and minimize steric hindrance, and does not significantly interfere with the structure of each functional component in the fusion protein. More preferably, rigid peptide linkers and rigid / flexible hybrid peptide linkers are used in the present invention to obtain higher protein expression yields.

[0043] In some embodiments, the peptide linker is (G4X) n , (X'P) m , (EAAAK) p and G q or a combination of two or more thereof, wherein each of n, m, p, and q is independently selected from integers 1 to 10, or integers 11 to 20. X is serine (S) or alanine (A), and X' is alanine (A), lysine (K), or glutamic acid (E).

[0044] In certain embodiments, the peptide linker is (G4X) n and (X'P) m wherein n, m, X, and X' have the meanings described in the present invention.

[0045] In certain embodiments, the peptide linker is (G4X) n1 -(X'P) m -(G4X) n2 wherein each of n1, m, and n2 is independently selected from the integers 1 to 10, or the integers 11 to 20. X is S or A, and X′ is A, K, or E.

[0046] In certain embodiments, the peptide linker is (G4X) n1 -(X'P) m -(G4X) n2 wherein n1 is 2, m is 10, and n2 is 2.

[0047] In another embodiment, the peptide linker is q and (X'P) m wherein q, m, and X' have the meanings described herein.

[0048] In certain embodiments, the peptide linker is G q1 -(X'P) m -G q2 wherein each of q1, m, and q2 is independently selected from an integer 1 to 10, or an integer 11 to 20; and X′ is A, K, or E.

[0049] In certain embodiments, the peptide linker is G q1 -(X'P) m -G q2 wherein q1 is 4, m is 10, and q2 is 4.

[0050] In certain embodiments, the peptide linker is (G4X) n , (X'P) m and G q wherein n, m, q, X, and X' have the meanings described in the present invention.

[0051] In some embodiments, the peptide linker is (G4X) n1 -G q1 -(X'P) m -(G4X) n2 -G q2 wherein each of n1, q1, m, n2, and q2 is independently selected from an integer of 1 to 10, or an integer of 11 to 20. X is S or A, and X′ is A, K, or E.

[0052] In some embodiments, the peptide linker is (G4X) n1 -Gq1 -(X'P) m -(G4X) n2 -G q2 wherein n1 is 1, q1 is 4, m is 10, n2 is 1, and q2 is 4.

[0053] In some embodiments, the peptide linker comprises (G4S)5 (SEQ ID NO: 123). In some embodiments, the peptide linker comprises (G4S)8 (SEQ ID NO: 124). In some embodiments of the invention, the peptide linker comprises G4(AP) 10 G4 (SEQ ID NO: 125). In another embodiment of the invention, the peptide linker comprises (G4S)2(AP) 10 (G4S)2 (SEQ ID NO: 126). In yet another embodiment of the invention, the peptide linker comprises (G4S)2(EP) 10 (G4S)2 (SEQ ID NO: 127).

[0054] In some embodiments, the fusion protein comprises an amino acid sequence selected from one of the following groups: SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:111, SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:114, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118.

[0055] [Table 3A]

[0056] [Table 3B]

[0057] In another embodiment, the fusion protein comprises an amino acid sequence having at least 85%, 90%, 95% or 99% sequence identity to one of the following sequences: SEQ ID NOs: 67 to 78, 80 to 102, SEQ ID NOs: 105 to 118. In another embodiment, the fusion protein comprises an amino acid sequence of one of the following group: SEQ ID NOs: 67 to 78, 80 to 102, SEQ ID NOs: 105 to 118, or an amino acid sequence having at least 95% sequence identity thereto, preferably the fusion protein comprises an amino acid sequence of one of the following group: SEQ ID NOs: 67 to 78, 80 to 102, SEQ ID NOs: 105 to 118.

[0058] In a second aspect, the present invention provides a homodimeric fusion protein comprising a fusion protein according to the first aspect of the invention.

[0059] In a third aspect, the present invention provides a method for producing a medicament for the treatment of a disease comprising: (i), (ii), and (iii): (i) a nucleic acid comprising a nucleotide sequence encoding a fusion protein of the invention; (ii) a vector comprising the nucleic acid of (i); (iii) a host cell containing the nucleic acid of (i) and / or the vector of (ii). The biological material is provided as any one of the following:

[0060] In a fourth aspect, the present invention provides a pharmaceutical composition comprising as an active ingredient a homodimeric fusion protein of the present invention, wherein the homodimeric fusion protein is present in a therapeutically effective amount.

[0061] In addition, the pharmaceutical composition also includes a pharma- ceutically acceptable carrier.

[0062] In a fifth aspect, the present invention provides the use of the fusion protein, the homodimeric fusion protein, or the pharmaceutical composition in the manufacture of a medicament for treating a metabolic disease.

[0063] Further, metabolic diseases include type II diabetes, obesity, dyslipidemia, diabetic nephropathy, nonalcoholic steatohepatitis, nonalcoholic fatty liver disease, and the like.

[0064] In another aspect, the invention provides a method of treating a metabolic disorder in a subject, comprising administering to the subject a therapeutically effective amount of the fusion protein, homodimeric fusion protein, or pharmaceutical composition.

[0065] Further, metabolic diseases include type II diabetes, obesity, dyslipidemia, diabetic nephropathy, nonalcoholic steatohepatitis, nonalcoholic fatty liver disease, and the like.

[0066] In another aspect, the invention provides a fusion protein, a homodimeric fusion protein, or a pharmaceutical composition for use in treating a metabolic disease in a subject.

[0067] Further, metabolic diseases include type II diabetes, obesity, dyslipidemia, diabetic nephropathy, nonalcoholic steatohepatitis, nonalcoholic fatty liver disease, and the like.

[0068] In a sixth aspect, the present invention provides the use of the fusion protein, homodimeric fusion protein, or pharmaceutical composition in the manufacture of a medicament for reducing food intake, body weight, insulin levels, triglyceride levels, cholesterol levels or glucose levels in a subject.

[0069] In another aspect, the invention provides a method of reducing food intake, body weight, insulin levels, triglyceride levels, cholesterol levels, or glucose levels in a subject, comprising administering to the subject a therapeutically effective amount of the fusion protein, homodimeric fusion protein, or pharmaceutical composition.

[0070] In another aspect, the invention provides a fusion protein, homodimeric fusion protein, or pharmaceutical composition for use in reducing a subject's food intake, body weight, insulin levels, triglyceride levels, cholesterol levels, or glucose levels in a subject.

[0071] In a seventh aspect, the present invention provides a method of treating a metabolic disease comprising administering to a subject the fusion protein, homodimeric fusion protein, or pharmaceutical composition of the present invention.

[0072] Furthermore, metabolic disorders have the meaning described in the present invention.

[0073] Compared with the prior art, the present invention has the following advances: by fusing the Fc variants provided by the present invention with any GDF15 domain (mature GDF15, truncated GDF15, or variants thereof) that maintains biological activity, compared with the fusion proteins of unmutated or monomeric or heterodimeric IgG Fc and GDF15 active domain, the Fc-GDF15 fusion proteins have significantly improved physicochemical properties and recombinant expression levels, have in vitro activity comparable to or better than that of the native GDF15 molecule, and have significantly extended in vivo circulatory half-life, which can support administration frequencies of once every two weeks or even once a month. In addition, the Fc-GDF15 fusion proteins of the present invention have simpler manufacturing processes and lower production costs than the fusion proteins formed by heterodimers in the prior art.

[0074] Various other advantages and benefits will become apparent to those skilled in the art upon reading the following detailed description of the preferred embodiments. The figures are only for purposes of illustrating the preferred embodiments and are not to be construed as limiting the invention. [Brief description of the drawings]

[0075] [Figure 1]FIG. 1 is a schematic diagram of homodimers formed by Fc-GDF15 fusion proteins. [Diagram 2] Graph showing the effect of a single dose of Fc-GDF15 fusion protein on body weight in normal mice (monitored for 14 days). [Diagram 3] FIG. 1 is a graph showing the effect of a single dose of Fc-GDF15 fusion protein on food intake in normal mice (monitored for 14 days). [Figure 4] Graph showing the effect of a single dose of Fc-GDF15 fusion protein on body weight in normal mice (monitored for 30 days). [Diagram 5] FIG. 1 is a graph showing the effect of a single dose of Fc-GDF15 fusion protein on food intake in normal mice (monitored for 30 days). [Figure 6] Graph showing the effect of a single dose of Fc-GDF15 fusion protein on body weight in normal mice (monitored for 56 days). [Figure 7] FIG. 1 is a graph showing the effect of a single dose of Fc-GDF15 fusion protein on food intake in normal mice (monitored for 56 days). [Figure 8] FIG. 1 is a graph showing the effect of repeated doses of Fc-GDF15 fusion protein on body weight in diet-induced obese mice (monitored for 42 days). [Figure 9] 1 is a graph showing the results of a glucose tolerance test in diet-induced obese mice following administration of multiple doses of Fc-GDF15 fusion protein. [Figure 10] FIG. 13 is a graph showing the effect of repeated dosing of M39 constructs at various dose levels on body weight in diet-induced obese mice (monitored for 49 days). [Figure 11] FIG. 13 is a graph showing fasting blood glucose levels in diet-induced obese mice (day 46) following repeated administration of various doses of M39 constructs. [Figure 12a]FIG. 13 is a graph showing the results of a glucose tolerance test in diet-induced obese mice (day 46) following repeated dose administration of M39 constructs at various dose levels. [Figure 12b] Graph showing statistics of area under the curve of glucose tolerance test (**-p<0.01, ***-p<0.001 vs. vehicle. Statistical analysis method: One-way ANOVA followed by Dunnett's). [Figure 13] FIG. 13 is a graph showing adiposity index in diet-induced obese mice after repeated administration of various doses of M39 constructs (**-p<0.01, ***-p<0.001, #-p<0.05, ##-p<0.01, ###-p<0.001 vs. vehicle. Statistical analysis method: One-way ANOVA followed by Dunnett). [Figure 14] FIG. 13 is a graph showing alanine transaminase levels in diet-induced obese mice after repeated administration of various doses of M39 constructs (***-p<0.001 vs. vehicle. Statistical analysis method: One-way ANOVA followed by Dunnett). [Figure 15] FIG. 13 is a graph showing aspartate transaminase levels in diet-induced obese mice after repeated administration of various doses of M39 constructs (***-p<0.001 vs. vehicle. Statistical analysis method: One-way ANOVA followed by Dunnett). [Figure 16a] Graph showing the effect of repeated dosing of various doses of M6 and M39 constructs on body weight in ob / ob obese mice (monitored for 52 days) Body weight change relative to baseline. [Figure 16b] Graph showing the effect of repeated dosing of various doses of M6 and M39 constructs on body weight in ob / ob obese mice (monitored for 52 days). Body weight change relative to vehicle control. [Figure 17] FIG. 13 is a graph showing the effect of repeated dosing of various doses of M6 and M39 constructs on food intake in ob / ob obese mice (monitored for 52 days). [Figure 18]Graph showing liver index in ob / ob obese mice after repeated dose administration of various doses of M6 and M39 constructs (***-p<0.001 vs. vehicle, ###-p<0.001 vs. semaglutide. Statistical analysis method: One-way ANOVA followed by Dunnett). [Figure 19] Graph showing alanine transaminase levels in ob / ob obese mice after repeated administration of various doses of M6 and M39 constructs (***-p<0.001 vs. vehicle, ##-p<0.01 vs. semaglutide. Statistical analysis method: One-way ANOVA followed by Dunnett). [Figure 20] Graph showing aspartate transaminase levels in ob / ob obese mice after repeated administration of various doses of M6 and M39 constructs (*-p<0.05, **-p<0.01, ***-p<0.001 vs. vehicle, #-p<0.05 vs. semaglutide. Statistical analysis method: One-way ANOVA followed by Dunnett). [Figure 21] Graph showing amelioration of liver pathology in ob / ob obese mice after repeated administration of various doses of M6 and M39 constructs (***-p<0.001 vs. vehicle, ###-p<0.001 vs. semaglutide. Statistical analysis method: One-way ANOVA followed by Dunnett). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS EXAMPLES

[0076] Exemplary embodiments of the present disclosure are described in more detail below with reference to the accompanying drawings. Although exemplary embodiments of the present disclosure are illustrated in the drawings, it should be understood that the present disclosure may be embodied in various forms and should not be limited to the embodiments illustrated herein. On the contrary, these embodiments are provided so that the present disclosure may be more fully understood and the scope of the present disclosure may be fully conveyed to those skilled in the art.

[0077] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly understood by those skilled in the art. In addition to the specific methods, devices, and materials used in the embodiments, any methods, devices, and materials in the prior art that are similar or equivalent to the methods, devices, and materials described in the embodiments of the present invention may be used to practice the present invention, according to the skill of the art and the description of the present invention by those skilled in the art.

[0078] As used herein, the terms "native," "wild-type," or "WT" refer to a protein or polypeptide that does not contain genetically engineered mutations and that occurs in nature or isolatable from the environment.

[0079] As used herein, an amino acid "substitution" or "replacement" refers to the replacement of one amino acid in a polypeptide with another amino acid.

[0080] As used herein, amino acid substitutions are designated by a first letter followed by a number followed by a second letter, where the first letter refers to the amino acid in the wild-type protein, the number refers to the amino acid position to which it is substituted, and the second letter refers to the amino acid used to replace the wild-type amino acid.

[0081] As used herein, deletions at the amino terminus of a protein or polypeptide are designated by ΔN followed by a number, which represents the number of amino acids deleted at the amino terminus.

[0082] As used herein, "IgG" or "IgG antibody" refers to an antibody having the structure of a naturally occurring immunoglobulin G molecule. IgG antibodies include, for example, IgG1, IgG2, IgG3, and IgG4. IgG1 Fc, IgG2 Fc, IgG3 Fc, and IgG4 Fc represent the Fc or Fc region of IgG1, IgG2, IgG3, and IgG4, respectively. The IgG Fc in the fusion protein herein may be IgG1 Fc, IgG2 Fc, IgG3 Fc, or IgG4 Fc, preferably IgG1 Fc or IgG4 Fc.

[0083] As used herein, a "peptide linker" refers to a single amino acid or polypeptide sequence that joins two proteins. The length of a peptide linker can be, for example, about 1 to 40 amino acids, including, for example, repeated alanine, glycine, and serine. Common peptide linkers include: flexible peptide linkers, such as combinations of glycine and serine, such as (GGGGS) n It is possible to adjust the distance between the linked proteins by changing the number of repeat units in the peptide linker; a peptide linker consisting of only glycine, e.g., G n , common ones include G6, G8, etc.; rigid peptide linkers, e.g., (EAAAK) n An α-helical peptide linker with the sequence: proline-rich (XP) n where X can designate any amino acid, common amino acids include alanine, lysine, or glutamic acid; (XP) n The sequence does not have a helical structure, and the presence of proline in the peptide linker increases the rigidity of the backbone, allowing for effective separation of the domains. Structures with proline-rich sequences are widely distributed in the body, such as the (AP)7 structure at the N-terminus of skeletal muscle proteins. The choice of peptide linker can be determined by one skilled in the art based on routine experimentation, for example, using only flexible peptide linkers, using only rigid peptide linkers, using a combination of flexible and rigid peptide linkers, and the like.

[0084] As used herein, "Fc" or "Fc region" is used to define the C-terminal region of the heavy chain of an antibody, which contains at least a portion of the constant region. The Fc region may be a native sequence Fc region or a variant Fc region. Those skilled in the art are aware that the Fc region generally comprises two constant domains: CH2 and CH3, and that the extent of the Fc region may vary. In this context, the C-terminus of the Fc region is the C-terminus of the heavy chain of the antibody, while the N-terminus of the Fc region may vary. In some embodiments, the N-terminus of the Fc region may, for example, start at position 231 (EU numbering), or 233 (EU numbering), or 236 (EU numbering), or 237 (EU numbering), in other embodiments, the N-terminus of the Fc region of the invention does not include a hinge region, and in yet other embodiments, the N-terminus of the Fc region of the invention includes a hinge region. As used herein, the numbering of amino acid residues in the Fc region is according to the EU numbering system, also known as the EU index, as described in Edelman et al., The covalent structure of an entire γG immunoglobulin molecule. Proc. Natl. Acad. Sci., USA, 1969.

[0085] GDF15 polypeptide needs to form a dimer to exert its activity. During the research process of the present invention, it was found that although IgG Fc can naturally form a dimer, the spatial distance between the carboxyl termini of two monomeric Fc in an IgG Fc dimer is significantly different from the spatial distance between the amino termini of a GDF15 monomer, and even if a flexible or rigid peptide linker is used, the fusion protein formed by the C-terminus of IgG Fc and the N-terminus of GDF15 still has problems such as instability during construction and low expression yield. The Fc variant provided by the present invention significantly improves the stability of the Fc-GDF15 molecule by introducing a mutation at a specific Fc site, for example, at position 439 or 356, thereby realizing efficient construction of Fc-GDF15 fusion protein and increasing expression yield. The production process is simple, and the fusion protein retains the ability to form a homodimer. In addition, the Fc variants of the present invention are applicable to all GDF15 proteins or variants thereof that maintain biological activity, whether they be mature GDF15 proteins, truncated GDF15 proteins, or any variants thereof, which can form fusion proteins with the Fc variants of the present invention and function.

[0086] As used herein, "dimer" refers to a protein dimer that is composed of two protein polypeptides and is a quaternary structure of a protein. The two protein polypeptides that compose a dimer may be referred to as a monomer molecule of a dimer or a monomer protein. Dimers may include both "homodimers" and "heterodimers", where homodimers are formed by the combination of two identical monomer molecules, and heterodimers are formed by the combination of two different monomer molecules. Homodimers are generally considered to be simpler to prepare than heterodimers, since only one peptide needs to be encoded. In the present invention, the Fc variants provided by the present invention have the ability to form homodimers, and the fusion proteins of the Fc variants and GDF15 active domains provided by the present invention also have the ability to form homodimers. Referring to FIG. 1, in the homodimer formed by the fusion protein, there is homodimer formation between two monomeric Fc variants and two monomeric GDF15 active domains, respectively.

[0087] As used herein, "growth differentiation factor 15," "GDF15," or "GDF15 active domain" refers in some embodiments to naturally occurring mature human GDF15 or a domain thereof, and in other embodiments to variants of GDF15 or a domain thereof. Mature GDF15 is comprised of amino acids 197(a)-308(I) (GDF15(197-308) (SEQ ID NO: 46)) of a total of 308 amino acids (UniProtq99988), excluding the signal peptide and leader peptide, or a polypeptide having at least 85%, 90%, 95%, and 99% sequence identity to the amino acid sequence within the range that maintains the unique activity and structure of GDF15. Variants include truncations and / or one or more amino acid mutations. In some embodiments, truncated GDF15 may be an N-terminal deletion variant, such as a sequence with 1-14 amino acids truncated at the N-terminus, or a polypeptide having at least 85%, 90%, 95%, and 99% sequence identity to the truncated amino acid sequence, and in some embodiments, GDF15 variant refers to the substitution, insertion, or deletion of at least one amino acid site compared to mature GDF15 or truncated GDF15. As long as the GDF15 variant maintains at least one of the biological activities of the GDF15 protein, such as its effect on food intake, blood glucose levels, insulin resistance, and body weight, it is within the scope of protection of the present invention.

[0088] GDF15 variants can also be generated by introducing one or more conservative or non-conservative amino acid substitutions at specific positions in the GDF15 polypeptide, using naturally occurring or non-naturally occurring amino acids, or deleting specific residues or segments thereof.

[0089] In this application, the term "conservative amino acid substitution" may involve the replacement of a naturally occurring amino acid residue (i.e., a residue found at a given position in the wild-type GDF15 polypeptide sequence) with a non-naturally occurring residue (i.e., a residue not found at that same position in the wild-type GDF15 polypeptide sequence) such that there is little or no effect on the polarity or charge of the amino acid residue at that position. Conservative amino acid substitutions also encompass non-naturally occurring amino acid residues, which are generally incorporated by chemical peptide synthesis rather than synthesis in a biological system. These include peptidomimetics and other inverted or inverted forms of amino acid moieties.

[0090] Naturally occurring amino acid residues can be grouped into the following classes based on common side chain properties: (1) Hydrophobic residues: M, A, V, L, I, (2) Neutral hydrophilic residues: C, S, T, N, Q, (3) Acid residues: D, E, (4) Alkaline residues: H, K, R, (5) Residues that affect chain orientation: G, P, and (6) Aromatic residues: W, Y, F.

[0091] Conservative substitutions may involve exchanging a member of one of these categories for another member of the same category.

[0092] Some conservative amino acid substitutions are shown in the table below:

[0093] [Table 4]

[0094] Conservative substitutions may involve the exchange of a member of one of these categories for another member of the same category. Non-conservative substitutions may involve the exchange of a member of one of these categories for a member of another category.

[0095] As used herein, "identity" or "homology" generally refers to the sequence similarity between two peptides or proteins, or between two nucleic acid molecules. Percent "identity" or "homology" refers to the percentage of identical residues between amino acids or nucleotides in the compared molecules, and is calculated based on the size of the smallest of the molecules being compared.

[0096] A fusion protein formed by the Fc variant of the present invention and native mature human GDF15 or a variant thereof is capable of achieving the objectives of the present invention.

[0097] As used herein, "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked, including vectors that are autonomously replicating nucleic acid structures and vectors that integrate into the genome of a host cell into which they are introduced. Certain vectors are capable of directing the expression of a nucleic acid to which they are operably linked, and such vectors are referred to herein as "expression vectors."

[0098] As used herein, "host cell" refers to a cell into which exogenous nucleic acid has been introduced, including the progeny of such a cell. Host cells include the originally transformed cell and the progeny resulting therefrom (regardless of the number of passages). The progeny may not be identical to the parent cell in nucleic acid content and may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected in the originally transformed cell are included herein. Host cells are any type of cell line that can be used to produce the fusion protein of the invention. Host cells include cultured mammalian cells, such as CHO cells, BHK cells, NS0 cells, SP2 / 0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells or hybridoma cells, yeast cells, bacterial cells, such as E. coli, insect cells and plant cells, as well as transgenic animals, transgenic plants, or cells contained in cultured plants or animal tissues.

[0099] As used herein, a "pharmaceutical composition" refers to a formulation in a form that allows the biological activity of the active ingredients contained therein to be effective and free of additional ingredients that are unacceptably toxic to the subject to which the pharmaceutical composition is administered.

[0100] As used herein, a "therapeutically effective amount" refers to an amount effective to achieve a desired therapeutic or prophylactic result, including, for example, eliminating, reducing, delaying, minimizing or preventing adverse effects of a disease.

[0101] As used herein, a "pharmaceutically acceptable carrier" refers to an ingredient, other than an active ingredient, in a pharmaceutical composition that is not toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0102] As used herein, "treatment" refers to an attempt to change the natural course of a disease in a treated individual, and may be for prophylaxis or clinical intervention performed during the course of clinical pathology. The desired effects of treatment include, but are not limited to, preventing the onset or recurrence of a disease, alleviating symptoms, reducing any direct or indirect pathological consequences of a disease, slowing the rate of disease progression, ameliorating or eliminating the pathology, and reversing or improving prognosis.

[0103] As used herein, an "individual" or "subject" is a mammal. Mammals include, but are not limited to, primates (e.g., humans and non-human primates, such as monkeys) or other mammals (e.g., cows, sheep, cats, dogs, horses, rabbits, and rodents, such as mice and rats). In particular, an individual or subject is a human.

[0104] The conventional one-letter or three-letter symbols are used herein for the naturally occurring amino acids:

[0105] [Table 5]

[0106] Example 1 Design of Fc variants In this example, the effect of introducing various mutations on the structural properties of Fc was tested, and the test results for some mutants are shown in Table 4. The test procedure included the following:

[0107] The Fc nucleic acid sequence was inserted into a mammalian cell expression vector using molecular cloning methods, and the ExpiCHO Fectamine™ CHO Transfection Kit (ThermoFisher Scientific) was used for transient transfection and expression in CHO-S cells, and the target protein was obtained after purification by Protein A column, and the target protein was analyzed by size-exclusion high performance liquid chromatography (SEC).

[0108] The main principle of SEC analysis is based on the molecular weight and three-dimensional structure of an analyte (e.g., a protein). In general, analytes with a larger molecular weight pass through a chromatography column more quickly and therefore have a shorter column retention time. Conversely, analytes with a smaller molecular weight have a longer retention time. Therefore, SEC can be used to analyze the aggregation / oligomerization (e.g., dimerization) of protein molecules.

[0109] Natural immunoglobulin Fc fragments naturally form homodimer structures. To improve the stability of the fusion protein, Fc molecules need to be mutated, which may disrupt the structure of Fc homodimers. However, the inventors have surprisingly found that Fc molecules after the introduction of mutations at position 439 (K439D or K439E) have the same retention time in SEC analysis as Fc molecules without mutations, indicating that the introduced mutations do not interfere with the formation of Fc dimers. As a control, this example also tested an Fc molecule with the amino acid sequence of SEQ ID NO: 24 (containing F405Q / Y407E mutations). According to previous patent document WO2019195091, F405Q / Y407E mutations can disrupt Fc dimers to form monomeric Fc molecules. In this experiment, it was found that this mutation has a significantly extended retention time, indicating that the Fc molecule exists in a monomeric form.

[0110] [Table 6]

[0111] Example 2 Preparation of Fc-GDF15 construct Construction of expression vector: The Fc-GDF15 molecules shown in Table 3 were inserted into the polyclonal restriction site of mammalian cell vector pXC17.4 to construct expression vectors expressing Fc-GDF15 shown in Table 3, and the Fc-GDF15 expression vectors were extracted with an endotoxin depletion plasmid extraction kit (OMEGA) for cell transfection.

[0112] Cell transfection and culture: CHO-S cells were recovered and subcultured, and the cells were cultured at a density of approximately 6×10 for cell transfection. 6Cells were transfected using ExpiCHO Fectamine™ CHO Transfection Kit (ThermoFisher Scientific), in which the final concentration of Fc-GDF15 expression vector was 1 μg / ml. Approximately 20 hours after transfection, ExpiCHO Fectamine CHO Enhancer and ExpiCHO Feed were added to maintain the growth of the transfected cells. Cell culture medium was harvested when cell viability decreased to approximately 80%.

[0113] Protein purification: After the cell culture medium was centrifuged, the supernatant was collected and filtered through a 0.22 μm filter to obtain the cell culture supernatant. The cell culture supernatant was purified using a two-step chromatography method. First step of chromatography: The cell culture supernatant was applied to an AT Protein A Diamond column (BestChrom) equilibrated with phosphate buffered saline (PBS). After the target protein was bound, the mixture was washed with more than 3 CV (column volume) of equilibration solution to remove unbound impurities and eluted with 100 mM acetic acid-sodium acetate (pH 3.0) buffer. The target protein was collected, and then the pH of the collected sample was quickly adjusted to 7.2-7.6 with 1.0 M Tris-HCl (pH 8.0) solution, and the pH-adjusted sample was diluted with purified water until its conductivity value was less than 5 ms / cm to obtain the first sample. Second step of chromatography: The first sample obtained by Protein A was further purified using a Bestarose Q HP column equilibrated with 20 mM Tris-HCl buffer. The first sample was loaded and washed with 3CV of 20mM Tris-HCl buffer and 3CV more of 20mM PB buffer to replace the buffer system, and finally eluted with PBS. The elution fractions were collected to obtain the target protein sample. The quality and purity of the target protein sample were evaluated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometry. The target protein sample was quantified by a micro nucleic acid protein analyzer (NanoDrop2000 / 2000c spectrophotometer).

[0114] Example 3 Effect of Fc variants on expression yield of Fc-GDF15 fusion protein The Fc-GDF15 fusion proteins described in Table 3 were expressed by the method described in Example 2, and the effect of applying different Fc variants on the expression yield of the Fc-GDF15 fusion proteins was compared. The results of the study are shown in Table 5.

[0115] According to the test results, Fc-GDF15 fusion proteins that are not mutated at K439 (EU numbering) or E356 (EU numbering) of Fc, such as M1 (homodimer having a monomer having the sequence of SEQ ID NO: 119), M14 (homodimer having a monomer having the sequence of SEQ ID NO: 79), have a smaller expression yield, while Fc-GDF15 fusion proteins mutated at K439 and / or E356 of Fc, such as M2 (homodimer having a monomer having the sequence of SEQ ID NO: 67), M3 (homodimer having a monomer having the sequence of SEQ ID NO: 68), M4 (homodimer having a monomer having the sequence of SEQ ID NO: 69), M5 (homodimer having a monomer having the sequence of SEQ ID NO: 70), M6 (homodimer having a monomer having the sequence of SEQ ID NO: 71), M7 (homodimer having a monomer having the sequence of SEQ ID NO: 72), M8 (homodimer having a monomer having the sequence of SEQ ID NO: 73), M9 (homodimer having a monomer having the sequence of SEQ ID NO: 74), M10 (homodimer having a monomer having the sequence of SEQ ID NO: 75), M11 (homodimer having a monomer having the sequence of SEQ ID NO: 76), M12 (homodimer having a monomer having the sequence of SEQ ID NO: 77), M13 (homodimer having a monomer having the sequence of SEQ ID NO: 78), M14 (homodimer having a monomer having the sequence of SEQ ID NO: 79), M15 (homodimer having a monomer having the sequence of SEQ ID NO: 79), M16 (homodimer having a monomer having the sequence of SEQ ID NO: 79), M17 (homodimer having a monomer having the sequence of SEQ ID NO: 79), M It was found that the homodimers M1 (having a monomer having a sequence of SEQ ID NO: 2), M8 (having a monomer having a sequence of SEQ ID NO: 73), M9 (having a monomer having a sequence of SEQ ID NO: 74), M10 (having a monomer having a sequence of SEQ ID NO: 75), M11 (having a monomer having a sequence of SEQ ID NO: 76), M15 (having a monomer having a sequence of SEQ ID NO: 80), M39 (having a monomer having a sequence of SEQ ID NO: 105), M50 (having a monomer having a sequence of SEQ ID NO: 116) had improved fusion protein expression yields, indicating that mutation of the amino acid at position 356 (EU numbering) or 439 (EU numbering) is more advantageous for organized expression of the Fc-GDF15 fusion protein.

[0116] [Table 7]

[0117] Example 4 Physicochemical properties of Fc-GDF15 fusion protein The physicochemical properties of protein molecules, such as their tendency to aggregate and degrade, affect the druggability of the molecule and therefore an ideal drug molecule should have a stable single component, i.e., be unlikely to aggregate or degrade.

[0118] To test the physicochemical properties of Fc-GDF15 fusion protein, Fc-GDF15 fusion protein was expressed and purified by the method described in Example 2, and then the fusion protein was analyzed by size exclusion chromatography (SEC). Specifically, a Waters Xbridge BEH 200A, 3.5 μm (7.8×300 mm) chromatography column was used for SEC analysis, the mobile phase was 150 mM phosphate buffer / acetonitrile (9:1, pH 6.5), and the flow rate was 0.5 ml / min. In the test results, the percentage of high molecular weight (HMW) components represents the percentage of aggregation, and the percentage of low molecular weight (LMW) components represents the percentage of degradation. The test results are shown in Table 6 (Table 8).

[0119] [Table 8A]

[0120] [Table 8B]

[0121] The results in Table 6 (Table 8) showed that Fc-GDF15 fusion proteins without mutations at K439 (EU numbering) or E356 (EU numbering), such as M1 and M14, had 50.2% and 67.6% high molecular weight (HMW) aggregation, respectively, while Fc-GDF15 fusion proteins mutated at K439 (EU numbering) and / or E356 (EU numbering) of Fc all had significantly reduced polymer aggregation ratios, with most fusion proteins having less than 5% polymer aggregation, indicating that mutations of Fc at K439 (EU numbering) or E356 (EU numbering) may result in better druggability of Fc-GDF15 fusion proteins.

[0122] Example 5 In vitro activity of Fc-GDF15 fusion proteins Previous studies have shown that the function of GDF15 in vivo requires signaling through its specific receptor GFRAL and co-receptor RET. Binding of GDF15 to its receptor on the cell surface can activate downstream signaling pathways, one of which is ERK1 / 2 phosphorylation. Therefore, the in vitro activity and efficacy of GDF15 can be evaluated by detecting the phosphorylation level of ERK1 / 2 in cells.

[0123] The gene sequences of human GFRAL (UniProtKB-Q6UXV0) and human RET (UniProtKB-P07949) were linked by an IRES sequence and placed downstream of the CMV promoter, then transfected into HEK293T cells (ATCC). Puromycin (Gibco) was added to screen the positive cells stably expressing the two receptors, which were shortly called receptor-expressing cells, and these were used for in vitro GDF15 activity analysis. The phosphorylation level of ERK1 / 2 in the cells was detected using the Advanced phospho-ERK1 / 2 (Thr202 / Tyr204) HTRF (homogeneous time-resolved fluorescence) kit (Cisbio) according to the manufacturer's instructions.

[0124] Briefly, receptor-expressing cells were seeded in 384-well plates at a density of 16,000 cells / well. After starvation, cells were stimulated with Fc-GDF15 fusion protein or native GDF15 (ACROBiosystems, GD5-H5149). After stimulation, cells were lysed and incubated with labeled antibodies (supplied with the assay kit) at room temperature overnight. A multifunctional microplate reader (SpectraMaxi3X, Molecular devices) was used to detect the fluorescence values ​​at 665 nm and 620 nm with an excitation wavelength of 337 nm. The signal intensity was calculated by the ratio of the fluorescence values ​​at 665 nm and 620 nm, and the formula is as follows:

[0125]

number

[0126] Among these: A665 is the fluorescence value detected at 665 nm wavelength, and A620 is the fluorescence value detected at 620 nm wavelength. The intensity of the response value corresponds to the phosphorylation intensity of ERK1 / 2 in the cells.

[0127] EC for each construct 50 is determined using a variable slope sigmoidal dose-response curve with four-parameter logistic regression in GraphPad Prism. Relative activity ratios (native GDF15 EC50:Fc-GDF15 construct EC50) were calculated. Results are shown in Table 7.

[0128] [Table 9A]

[0129] [Table 9B]

[0130] The results show that, except for M35, which had significantly reduced in vitro activity, the remaining Fc-GDF15 fusion proteins all had in vitro activity comparable to or better than that of native GDF15.

[0131] Example 6 Thermal stability of Fc-GDF15 fusion protein In this example, differential scanning calorimetry (DSC) was used to test the thermal stability of various Fc-GDF15 fusion proteins. The endothermic transition curve of the DSC spectrum represents the denaturation and unfolding process of protein molecules, and protein thermal stability can be characterized by the thermal transition midpoint (Tm) value. Specifically, the samples were heated from 30°C to 110°C at a heating rate of 120°C / h, and the Tm values ​​of various Fc-GDF15 fusion proteins were determined. The results are shown in Table 8. The experimental results show that the Fc-GDF15 fusion protein has good thermal stability and is suitable for drug development.

[0132] [Table 10]

[0133] Example 7 In vivo efficacy of a single dose of Fc-GDF15 fusion protein in normal mice GDF15 can inhibit food intake and reduce body weight, and has the potential to treat obesity and other related metabolic diseases.However, the natural GDF15 molecule has a serum half-life of only about 3 hours, which greatly limits its direct therapeutic application.The Fc-GDF15 fusion protein provided by the present invention can extend the half-life of drug molecules.In this example, the effect of single dose administration of various Fc-GDF15 fusion proteins on food intake and body weight in normal mice was tested to evaluate the in vivo efficacy and duration of action of various Fc-GDF15 fusion proteins.

[0134] Specifically, 8-9 week old C57BL / 6 mice (Hunan SJA Laboratory Animal Co., Ltd.) were weighed on day 0 and administered a single subcutaneous dose of the tested Fc-GDF15 fusion protein or vehicle (PBS), and food intake and body weight were continuously monitored after administration. The percentage of body weight change was calculated as follows: Body weight change (%) = (measured body weight - initial body weight) / initial body weight x 100%.

[0135] The results for food intake and body weight of normal mice treated with a single dose for 14 days are shown in Table 9, Figures 2 and 3.

[0136] [Table 11]

[0137] The results show that a single administration of Fc-GDF15 fusion protein at a dose of 3 nmol / kg in normal mice suppressed food intake and significantly reduced body weight compared to vehicle on days 8 and 14. Among them, the GDF15 fusion protein monoFc-GDF15 containing monomeric Fc ("Compound 2" disclosed in Patent No. WO2019195091, monomer has sequence of SEQ ID NO: 104, Fc has sequence of SEQ ID NO: 18, F405Q, Y407E) reached the best efficacy on day 8, and surprisingly, Fc-GDF15 fusion proteins mutated at K439 of Fc, such as M3 (homodimer having monomer with sequence of SEQ ID NO: 68) and M15 (homodimer having monomer with sequence of SEQ ID NO: 80) reached the best efficacy on day 8. As well as Fc-GDF15 fusion proteins further comprising mutations at various amino acid sites of GDF15, such as M20 (homodimer having a monomer having the sequence of SEQ ID NO:85), M23 (homodimer having a monomer having the sequence of SEQ ID NO:88), M24 (homodimer having a monomer having the sequence of SEQ ID NO:89) and M35 (homodimer having a monomer having the sequence of SEQ ID NO:100), further showed a trend towards reduced body weight in mice on day 14 post-administration, and the weight loss was significantly greater than the effect of the mono Fc-GDF15 fusion protein.

[0138] The results for food intake and body weight of normal mice treated with a single dose for 30 days are shown in Table 10, Figures 4 and 5.

[0139] [Table 12]

[0140] The results show that a single administration of Fc-GDF15 fusion protein at a dose of 1 nmol / kg in normal mice can significantly reduce the body weight of the mice. Among them, the GDF15 fusion protein monoFc-GDF15 containing monomeric Fc had a weaker body weight reducing effect, and the mice recovered to their initial body weight on day 14. However, surprisingly, the Fc-GDF15 constructs containing various amino acid mutations at K439 position of Fc, such as M3 (homodimer having a monomer with a sequence of SEQ ID NO: 68), M6 (homodimer having a monomer with a sequence of SEQ ID NO: 71), M7 (homodimer having a monomer with a sequence of SEQ ID NO: 72), M16 (homodimer having a monomer with a sequence of SEQ ID NO: 81) and M51 (homodimer having a monomer with a sequence of SEQ ID NO: 117) had a significantly stronger body weight reducing effect, and could continuously reduce the body weight of the mice up to 28 days, suggesting a significantly extended drug effect maintenance time.

[0141] The results for food intake and body weight of normal mice treated with a single dose for 56 days are shown in Table 11, Figures 6 and 7.

[0142] [Table 13]

[0143] The results show that a single dose treatment of 1 nmol / kg M9 (homodimer having a monomer with a sequence of SEQ ID NO:74), M18 (homodimer having a monomer with a sequence of SEQ ID NO:83), M22 (homodimer having a monomer with a sequence of SEQ ID NO:87) or M35 (homodimer having a monomer with a sequence of SEQ ID NO:100) could inhibit food intake and significantly reduce body weight in normal mice. Furthermore, the body weight of the mice continued to decrease until about day 21 before rebounding and returning to baseline at about day 56, indicating that Fc-GDF15 fusion proteins mutated at FcK439 or E356, such as M9 and M18, as well as fusion proteins further containing mutations at various amino acid sites of GDF15, such as M22 and M35, all have an ultra-long duration of efficacy. The body weight reducing efficacy could be maintained for about one month after administration of a single dose. At the same time, the inventors surprisingly found that construct M35 has a significantly reduced in vitro activity, yet still maintains an in vivo efficacy similar to the other constructs.

[0144] Example 8 In vivo efficacy of repeated doses of Fc-GDF15 fusion protein in diet-induced obese (DIO) mice This example examines the effect of repeated dose administration of Fc-GDF15 fusion protein on appetite suppression, weight loss, and improvement of various metabolic indices in DIO mice. After 4 months of induction with a high-fat diet, C57BL / 6 mice were randomly grouped according to body weight, and then subcutaneously administered vehicle (PBS, once every 2 weeks), various Fc-GDF15 fusion proteins (1 nmol / kg, once every 2 weeks), or semaglutide (3 nmol / kg, once daily) for 49 days. The changes in mouse body weight and food intake were continuously monitored throughout the experiment. An intraperitoneal glucose tolerance test (IPGTT) was performed 45 days after the first administration, and the mice were sacrificed on the 49th day to analyze fat content and various serum biochemical variables.

[0145] The results for mouse body weight change and food intake are shown in Table 12, the body weight change curves are shown in FIG. 8, the IPGTT data are shown in FIG. 9, and the variables area under the glucose tolerance curve, adiposity index, serum total cholesterol (TC), triglycerides (TG), alanine transaminase (ALT), aspartate transaminase (AST) and low density lipoprotein (LDL) are shown in Table 13.

[0146] [Table 14]

[0147] [Table 15]

[0148] The results show that administration of Fc-GDF15 fusion proteins M4 (homodimer with monomer having sequence SEQ ID NO: 69), M5 (homodimer with monomer having sequence SEQ ID NO: 70) or M9 (homodimer with monomer having sequence SEQ ID NO: 74) at a dose of 1 nmol / kg every 2 weeks can inhibit food intake and significantly reduce body weight in DIO mice with efficacy comparable to daily administration of 3 nmol / kg semaglutide, which can bring the body weight of the mice to normal levels; M4 and M5 can also significantly improve the glucose tolerance level of DIO mice; M4, M5 and M9 can significantly reduce fat content and blood lipids including total cholesterol, triglycerides and low density lipoprotein; M4, M5 and M9 can also improve liver function, for example significantly reduce ALT and AST levels. It is shown that the Fc-GDF15 construct of the present invention is applicable to the treatment of obesity, diabetes, hyperlipidemia or fatty liver / steatohepatitis.

[0149] In another experiment, the effect of repeated administration of various doses of construct M39 on suppressing appetite, reducing body weight, and improving various metabolic variables in DIO mice was tested. DIO mice were randomly divided into groups (10-12 mice per group) and subcutaneously administered vehicle (PBS, once a week), various doses of M39 (0.03 nmol / kg, 0.1 nmol / kg, and 1 nmol / kg, once a week; 3 nmol / kg, once every 2 weeks) or semaglutide (3 nmol / kg, once a day). Changes in mouse body weight and food intake were continuously monitored. Fasting blood glucose and glucose tolerance were measured 46 days after the first administration, and mice were sacrificed on day 49 to analyze fat content and various serum biochemical indices.

[0150] The body weight changes of the mice at the end of the treatment are shown in Table 14, and the body weight change curves, fasting blood glucose levels, IPGTT (day 46) and area under the curve, fat index, and transaminase levels are shown in Figures 10 to 15, respectively.

[0151] [Table 16]

[0152] The above results show that subcutaneous injection of M39 once a week or once every two weeks can dose-dependently reduce the body weight of DIO mice, and the weight reduction effect of 1 nmol / kg once a week is significantly stronger than that of 3 nmol / kg semaglutide daily administration. At the same time, M39 can also significantly reduce the fat content of mice and improve fasting blood glucose level, glucose tolerance and liver function biomarkers in mice.

[0153] Example 9 In vivo efficacy of Fc-GDF15 fusion protein in an ob / ob obese mouse model This example tests the effect of constructs M6 and M39 on suppressing appetite, reducing body weight, and improving various metabolic indices in an ob / ob obese mouse model. After acclimation, 6-7 week old ob / ob mice (GemPharmatech) were randomly divided into groups according to body weight and subcutaneously administered vehicle (PBS), various doses of M6 or M39 (0.1 nmol / kg, dosed on days 0 and 24; 1 nmol / kg, 10 nmol / kg, dosed on days 0 and 36) or semaglutide (3 nmol / kg, once daily). The changes in body weight and food intake of the mice were continuously monitored. The mice were sacrificed on day 52 and various serum biochemical indices and liver pathology were analyzed.

[0154] The body weight changes of the mice at the end of the treatment are shown in Table 15, and the body weight change curves, cumulative food intake, liver index, transaminase levels, and liver pathology results are shown in Figures 16 to 21, respectively.

[0155] [Table 17]

[0156] The above results show that the Fc-GDF15 construct designed by the present invention had an ultra-long duration of drug efficacy. M6 and M39 administered at low doses once on days 0 and 24, and at medium and high doses once on days 0 and 36, could significantly reduce food intake in ob / ob mice. The improvement of body weight was significantly stronger than that of semaglutide administered daily, and M6 and M39 could also significantly improve liver function and fatty liver in ob / ob mice, the effect of which was also significantly better than that of semaglutide.

[0157] Example 10 Pharmacokinetics of Fc-GDF15 fusion protein in mice This example tested the pharmacokinetic properties of constructs M38 (homodimer with monomer having sequence of SEQ ID NO: 103), M3 (homodimer with monomer having sequence of SEQ ID NO: 68), M15 (homodimer with monomer having sequence of SEQ ID NO: 80) in mice. The above fusion proteins were diluted to 0.05 mg / ml in 10 mM PBS, and then subcutaneously injected into C57BL / 6 mice at a dose of 0.25 mg / kg, with 3 mice per dose group. Blood was collected from each animal before administration and 2 h, 7 h, 24 h, 48 h, 96 h, 168 h, 356 h, 504 h, and 672 h after administration, respectively, and plasma Fc-GDF15 concentrations were determined by sandwich ELISA. Specifically, mouse anti-human GDF15 monoclonal antibody (Sino Biological Inc.) was used to coat plates, mouse anti-human IgG4 Fc-HRP was used to detect the target protein, and then pharmacokinetic data was calculated. The results are shown in Table 16.

[0158] [Table 18]

[0159] The results showed that the fusion protein M38 containing monomeric Fc had a half-life of 58 h in mice, and surprisingly, it was found that the half-lives of the Fc-GDF15 fusion proteins M15 and M3 with Fc variants in mice reached 244 h and 306 h, respectively, which is much longer than the half-life of conventional Fc fusion proteins in mice.

[0160] Example 11 Pharmacokinetics of Fc-GDF15 fusion protein in cynomolgus monkeys This example tested the pharmacokinetic parameters of construct M39 in cynomolgus monkeys. M39 was administered subcutaneously to cynomolgus monkeys at doses of 1 mg / kg and 2 mg / kg, respectively, with 3 animals per gender. Blood was collected from each animal before administration and 0.5 h, 2 h, 6 h, 48 h, 168 h, 356 h, 504 h, and 672 h after administration, and plasma Fc-GDF15 concentrations were determined by sandwich ELISA. Specifically, mouse anti-human GDF15 monoclonal antibody (Sino Biological Inc.) was used to coat plates, and mouse anti-human IgG4 Fc-HRP was used to detect the target protein, and then pharmacokinetic data was calculated. The results are shown in Table 17.

[0161] [Table 19]

[0162] The inventors were surprised to find that construct M39 also exhibited an ultra-long half-life in cynomolgus monkeys, reaching 200-300 hours, much longer than the half-life of conventional non-antibody Fc fusion proteins in cynomolgus monkeys, which was approximately 100 hours.

[0163] The above description is merely a preferred embodiment of the present invention, and the protection scope of the present invention is not limited thereto. Any modifications or replacements that can be easily conceived by those skilled in the art within the technical scope disclosed by the present invention shall be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Claims

1. A fusion protein comprising a GDF15 active domain and an Fc variant, wherein the C-terminus of the Fc variant is linked to the N-terminus of the GDF15 active domain directly or via a peptide linker, and the Fc variant comprises an amino acid substitution at positions 356 and / or 439 of IgG Fc according to EU numbering.

2. The fusion protein of claim 1, wherein the Fc variant has the ability to form homodimers.

3. the Fc variant comprises an amino acid substitution at position 356 of IgG Fc according to EU numbering with an amino acid other than aspartic acid (D), glutamic acid (E) and cysteine (C), and / or the Fc variant comprises an amino acid substitution at position 439 of IgG Fc according to EU numbering with an amino acid other than arginine (R), histidine (H), lysine (K) and cysteine (C), Preferably, the Fc variant comprises an amino acid substitution at position 356 of IgG Fc according to EU numbering with one of glycine (G), serine (S), alanine (A), threonine (T), valine (V), asparagine (N), leucine (L), isoleucine (I), glutamine (Q), tyrosine (Y), phenylalanine (F), histidine (H), proline (P), methionine (M), lysine (K) and arginine (R), and / or the Fc variant comprises an amino acid substitution at position 356 of IgG Fc according to EU numbering with one of glycine (G), serine (S), alanine (A), threonine (T), valine (V), asparagine (N), leucine (L), isoleucine (I), glutamine (Q), tyrosine (Y), phenylalanine (F), histidine (H), proline (P), methionine (M), lysine (K) and arginine (R). an amino acid substitution at position 439 of Fc with one of glycine (G), serine (S), alanine (A), threonine (T), valine (V), aspartic acid (D), asparagine (N), leucine (L), isoleucine (I), glutamic acid (E), glutamine (Q), tyrosine (Y), phenylalanine (F), proline (P), and methionine (M); More preferably, the Fc variant comprises one of the following mutations: E356R, E356Q, E356A, E356N, and / or one of K439D, K439E, K439Q, K439A, K439N.

3. The fusion protein of claim 1 or 2.

4. the Fc variant comprises one of the following mutations: K439D, K439E, K439Q, K439A, K439N; Preferably, the Fc variant comprises one of the following mutations: K439D, K439E, K439Q, A fusion protein according to any one of claims 1 to 3.

5. the Fc variant comprises one of the following mutations: E356R, E356Q, E356A, E356N; Preferably, the Fc variant comprises the E356R mutation. A fusion protein according to any one of claims 1 to 3.

6. 6. The fusion protein of any one of claims 1 to 5, wherein the Fc variant further comprises the following mutations: amino acid substitutions with alanine (AA) at positions 234 and 235 of IgG Fc according to EU numbering, and / or an amino acid deletion at position 447 of IgG Fc.

7. 7. The fusion protein of any one of claims 1 to 6, wherein the Fc variant comprises an amino acid sequence of one of the following group: SEQ ID NO: 1 to 22, SEQ ID NO: 27, SEQ ID NO: 29 to 45, or an amino acid sequence having at least 85%, 90%, 95% or 99% sequence identity thereto.

8. 3. The fusion protein of claim 1, wherein the GDF15 active domain is a full-length mature GDF15 protein, an N-terminally truncated GDF15 protein, or any variant that retains the biological activity of GDF15.

9. The GDF15 active domain is selected from the group consisting of: SEQ ID NO: 46, A 1-14 amino acid truncation at the N-terminus of SEQ ID NO:46 and / or a 1-3 amino acid substitution in SEQ ID NO:46 or having at least 85%, 90%, 95%, or 99% sequence identity thereto, 3. The fusion protein of claim 1 or 2.

10. The GDF15 active domain is selected from the group consisting of: SEQ ID NO: 46, A 1-14 amino acid truncation at the N-terminus of SEQ ID NO:46 and / or a 1-3 amino acid substitution in SEQ ID NO:46 10. The fusion protein of claim 9, comprising an amino acid sequence selected from one of:

11. the positions of the amino acid substitutions in SEQ ID NO: 46 are selected from one, two or three of the following group: 5, 6, 21, 26, 30, 47, 54, 55, 57, 67, 69, 81, 94, 107; Preferably, the amino acid substitutions in SEQ ID NO: 46 are selected from one, any two, or any three of the following positions: D5E, H6D, H6E, R21Q, R21H, D26E, A30S, A47D, A54S, A55E, M57T, R67Q, K69R, A81S, T94E, K107Q. The fusion protein of claim 10.

12. 11. The fusion protein of claim 10, wherein the N-terminus of SEQ ID NO: 46 is truncated by 3, 4, or 14 amino acids.

13. The fusion protein of claim 10, wherein the GDF15 active domain comprises an amino acid sequence of one of the following group: SEQ ID NOs: 46-66, or an amino acid sequence having at least 85%, 90%, 95% or 99% sequence identity thereto.

14. the GDF15 active domain comprises one of the following group of amino acid sequences: SEQ ID NOs: 46-66, or an amino acid sequence having at least 95% sequence identity thereto; Preferably, the GDF15 active domain comprises an amino acid sequence of one of the following group: SEQ ID NOs: 46-66; The fusion protein of claim 13.

15. 15. The fusion protein of any one of claims 1 to 14, comprising an amino acid sequence of one of the following group: SEQ ID NOs: 67 to 78, 80 to 102, 105 to 118, or an amino acid sequence having at least 85%, 90%, 95% or 99% sequence identity thereto.

16. comprising an amino acid sequence of one of the following groups: SEQ ID NOs: 67-78, 80-102, 105-118, or an amino acid sequence having at least 95% sequence identity thereto; Preferably, it comprises an amino acid sequence of one of the following groups: SEQ ID NOs: 67 to 78, 80 to 102, 105 to 118, The fusion protein of claim 15.

17. 17. A homodimeric fusion protein comprising the fusion protein of any one of claims 1 to 16.

18. (i), (ii), and (iii) below: (i) a nucleic acid comprising a nucleotide sequence encoding the fusion protein of any one of claims 1 to 16, (ii) a vector comprising the nucleic acid of (i); (iii) a host cell containing the nucleic acid of (i) and / or the vector of (ii). A biological material that is one of the following:

19. 14. A pharmaceutical composition comprising as an active ingredient the homodimeric fusion protein of claim 13, wherein the homodimeric fusion protein is present in a therapeutically effective amount; Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

20. (iv) and (v) of the following in the subject: (iv) treating metabolic diseases; preferably, metabolic diseases include type II diabetes, obesity, dyslipidemia, diabetic nephropathy, nonalcoholic steatohepatitis, and nonalcoholic fatty liver disease; (v) reducing food intake, body weight, insulin levels, triglyceride levels, cholesterol levels, or glucose levels in a subject 20. The fusion protein of any one of claims 1 to 16, the homodimeric fusion protein of claim 17, or the pharmaceutical composition of claim 19, for use in any one of

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