WO2023025129A1 - Gdf15融合蛋白及其用途 - Google Patents

Gdf15融合蛋白及其用途 Download PDF

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WO2023025129A1
WO2023025129A1 PCT/CN2022/114143 CN2022114143W WO2023025129A1 WO 2023025129 A1 WO2023025129 A1 WO 2023025129A1 CN 2022114143 W CN2022114143 W CN 2022114143W WO 2023025129 A1 WO2023025129 A1 WO 2023025129A1
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seq
amino acid
fusion protein
gdf15
variant
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PCT/CN2022/114143
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French (fr)
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燕江雨
操蓉波
肖�琳
曾君南
黄丽玫
危喜玲
李钊锋
龚庆伟
闫兴国
李静
黄丹霞
陈小锋
李文佳
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广东东阳光药业有限公司
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Priority to EP22860475.7A priority Critical patent/EP4393940A1/en
Publication of WO2023025129A1 publication Critical patent/WO2023025129A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/06—Antihyperlipidemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Definitions

  • the invention relates to the technical field of biomedicine, in particular to a GDF15 fusion protein and a method for treating obesity.
  • Obesity refers to a chronic metabolic disease caused by the increase in the volume and number of fat cells in the body and the abnormal distribution of adipose tissue. At least 2.8 million deaths per year can be attributed to overweight or obesity.
  • AMA American Medical Association
  • AACE American Association of Clinical Endocrinologists
  • Obesity management includes lifestyle changes, medication, and bariatric surgery. Lifestyle changes often require protocol and maintenance by a professional team, but are often associated with high recurrence rates; bariatric surgery is associated with risks of death and surgical complications, is costly, and is generally reserved for severe obesity. Given the current limitations of lifestyle modifications and bariatric surgery, the use of medications to treat obesity is critical. Authoritative guidelines in the United States suggest that anti-obesity drugs can be used for treatment when the body mass index is ⁇ 30kg/m 2 , or when the body mass index is ⁇ 27kg/m 2 and is accompanied by at least one weight-related complication.
  • GDF15 Growth differentiation factor 15
  • MIC-1 macrophage inhibitory cytokine-1
  • PTGF- ⁇ placental transforming growth factor- ⁇
  • PLAB placental bone morphogenetic factor
  • PDF prostate-derived factor
  • NAG-1 non-steroidal anti-inflammatory drug activating gene
  • GDF15 Elevated circulating levels of GDF15 have been reported to be associated with decreased food intake and body weight in advanced cancer patients (Johnen H et al., Nat Med, 2007). Moreover, mice transgenic for high expression of GDF15 as well as mice administered with recombinant GDF15 showed decreased body weight and food intake, and had improved glucose tolerance levels (Xiong Y et al., Sci Transl Med, 2017), indicating that GDF15 has the potential to treat obesity, type 2 diabetes (T2D), nonalcoholic steatohepatitis (NASH) and other diseases.
  • T2D type 2 diabetes
  • NASH nonalcoholic steatohepatitis
  • the circulating half-life (t1/2) of the natural GDF15 dimer in the body is relatively short, only about 2-3 hours, which greatly limits its clinical therapeutic application.
  • Conventional methods to prolong the circulating half-life of macromolecular drugs include fusing the drug molecule to the Fc fragment of an antibody.
  • the purpose of the present invention is to provide a method for treating obesity-related diseases with Fc-GDF15 fusion protein.
  • the GDF15 fusion protein of the present invention has the advantages of stable physical and chemical properties, long half-life, long-acting effect, simple preparation process, etc., and can be used to treat metabolic related diseases, such as obesity, type 2 diabetes, NASH, dyslipidemia, etc., especially obesity.
  • the present invention provides a method for treating obesity-related diseases with an Fc-GDF15 fusion protein.
  • the present invention provides a fusion protein.
  • the fusion protein includes a GDF15 active domain and an Fc variant; the C-terminus of the Fc variant is connected to the GDF15 active domain directly or through a peptide linker. the N-terminus of the domain;
  • the Fc variants have an amino acid substitution at position 356 and/or 439 of IgG Fc according to EU numbering.
  • the Fc variant has the ability to form homodimers.
  • the IgG Fc is selected from one of IgG1 Fc, IgG2 Fc, IgG3 Fc, and IgG4 Fc.
  • the IgG Fc is human IgG1 Fc. In some embodiments, the IgG Fc is human IgG4 Fc.
  • the Fc variant replaces the 356th amino acid of IgG Fc with amino acids other than D, E and C according to the EU numbering.
  • the 356th amino acid of IgG Fc is replaced with one of the following groups: G, S, A, T, V, N, L, I, Q, Y, F, H, P, M, K, R.
  • the Fc variant replaces the 439th amino acid of IgG Fc with amino acids other than R, H, K and C according to the EU numbering.
  • the amino acid at position 439 of IgG Fc is substituted with one of the following groups: G, S, A, T, V, D, N, L, I, E, Q, Y, F, P, M.
  • the Fc variant contains the following mutations in IgG Fc according to EU numbering: E356G, E356S, E356A, E356T, E356V, E356N, E356L, E356I, E356Q, E356Y, E356F, E356H, E356P, E356M, One of E356K, E356R, and/or one of K439G, K439S, K439A, K439T, K439V, K439D, K439N, K439L, K439I, K439E, K439Q, K439Y, K439F, K439H, K439P, K439M.
  • the Fc variant contains the following mutations in IgG Fc according to EU numbering: one of E356R, E356Q, E356A, E356N, and/or one of K439D, K439E, K439Q, K439A, K439N kind.
  • the Fc variant contains the following mutations in IgG Fc according to EU numbering: one of K439D, K439E, K439Q, K439A, K439N. In a preferred embodiment, the Fc variant contains the following mutations in IgG Fc according to EU numbering: one of K439D, K439E, and K439Q.
  • the Fc variant contains the following mutations in IgG Fc according to EU numbering: one of E356R, E356Q, E356A, E356N. In a preferred embodiment, said Fc variant contains the following mutation in IgG Fc according to EU numbering: E356R.
  • the Fc variant further contains the following mutations: the amino acid at position 234 and position 235 of IgG Fc is replaced by AA according to EU numbering, and/or the amino acid at position 447 is deleted.
  • the amino acid at position 234 and position 235 of IgG Fc is replaced by AA according to EU numbering, and/or the amino acid at position 447 is deleted.
  • the amino acid at position 234 and position 235 of IgG Fc is replaced by AA according to EU numbering, and/or the amino acid at position 447 is deleted.
  • IgG1 Fc also contain the following mutations: L234A and L235A, and/or deletion of amino acid at position 447
  • IgG4 Fc also contain the following mutations: F234A and L235A, and/or deletion of amino acid at position 447.
  • amino acid substitution in the IgG Fc of the Fc variant according to EU numbering is only one of the following group:
  • amino acid substitutions stated at each position have the meanings described in the present invention.
  • the amino acid sequence of the Fc variant includes 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:
  • 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 NO: 1-22, SEQ ID NO : 27, SEQ ID NO: 29-45.
  • the GDF15 active domain is full-length mature GDF15 protein, mature GDF15 protein, N-terminal truncated GDF15 protein, or any variant thereof that retains GDF15 biological activity.
  • the GDF15 active domain comprises an amino acid sequence selected from one of the following groups or an amino acid sequence having at least 85%, 90%, 95% or 99% sequence identity with an amino acid sequence of one of the following groups :
  • the GDF15 active domain comprises an amino acid sequence selected from one of the following groups:
  • the number of amino acid truncations at the N-terminal of said SEQ ID NO: 46 is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 1, 12, 13 or 14.
  • the position of the amino acid substitution in said SEQ ID NO: 46 is selected from one, two, or three of the following group: the 5th, the 6th, the 21st, the 26th, the 30th, 47th, 54th, 55th, 57th, 67th, 69th, 81st, 94th, 107th.
  • amino acid substitution in said SEQ ID NO: 46 is selected from one of the following groups, any two of different positions, or any three of different positions: D5E, H6D, H6E, R21Q, R21H, D26E, A30S, A47D, A54S, A55E, M57T, R67Q, K69R, A81S, T94E, K107Q.
  • the GDF15 active domain comprises the amino acid sequence shown in SEQ ID NO: 46.
  • the GDF15 active domain comprises the following sequence: an amino acid sequence with 1-14 amino acid truncated at the N-terminal of SEQ ID NO:46.
  • the GDF15 active domain comprises the following sequence: an amino acid sequence containing 1-3 amino acid substitutions in SEQ ID NO: 46; the amino acid substitutions in SEQ ID NO: 46 have the following sequence: the stated meaning.
  • the GDF15 active domain comprises the following sequence: 1-14 amino acid truncated at the N-terminal of SEQ ID NO: 46, and 1-3 amino acid substitutions in SEQ ID NO: 46 Amino acid sequence; said amino acid substitution in SEQ ID NO: 46 has the meaning described in the present invention.
  • 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: 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.
  • the numbering method for the position of the amino acid is as follows: the first amino acid at the N-terminal of SEQ ID NO: 46 is the first, and the numbers are numbered in the direction of the C-terminus.
  • 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 NO: 46-66.
  • the GDF15 active domain comprises an amino acid sequence of one of the following groups, or an amino acid sequence having at least 95% identity with one of the following groups: SEQ ID NO: 46-66; preferably, the The GDF15 active domain comprises an amino acid sequence of one of the following groups: SEQ ID NO: 46-66.
  • the C-terminus of the Fc variant is connected to the N-terminus of the GDF15 active domain through a peptide linker; the peptide linker is selected from one or both of a flexible peptide linker and a rigid peptide linker. combination.
  • Any suitable linker may be used in the fusion proteins of the embodiments of the invention.
  • the term "linker” refers to a linking moiety comprising a peptide linker.
  • the linker helps to ensure correct folding, minimizes steric hindrance, and does not significantly disturb the structure of each functional component within the fusion protein. More preferably, in order to obtain higher protein expression, the present application can use rigid peptide linkers and rigid/flexible hybrid peptide linkers.
  • the peptide linker includes (G 4 X) n , (X'P) m , (EAAAK) p , G q or a combination of two or more, wherein n, m, p, q are each independently selected from an integer of 1-10, X is serine (S) or alanine (A), X' is alanine (A), lysine (K) or glutamic acid (E).
  • the peptide linker includes a combination of (G 4 X) n and (X'P) m , and the n, m, X, and X' have the meanings described in the present invention.
  • the peptide linker comprises (G 4 X) n1 -(X'P) m -(G 4 X) n2 , wherein n1, m, and n2 are each independently selected from an integer of 1-10, and X is S or A, and X' is A, K or E.
  • the peptide linker comprises (G 4 X) n1 -(X′P) m -(G 4 X) n2 , wherein n 1 is 2, m is 10, and n 2 is 2.
  • said peptide linker comprises a combination of G q and (X'P) m , said q, m, X' having the meanings described in the present invention.
  • the peptide linker includes G q1 -(X'P) m -G q2 , q1, m, and q2 are each independently selected from an integer of 1-10, and X' is A, K or E.
  • the peptide linker comprises G q1 -(X'P) m -G q2 , wherein q 1 is 4, m is 10, and q 2 is 4.
  • the peptide linker comprises a combination of (G 4 X) n , (X'P) m and G q , and said n, m, q, X, X' have the meaning.
  • the peptide linker comprises (G 4 X) n1 -G q1 -(X'P) m -(G 4 X) n2 -G q2 , n1, q1, m, n2, q2 are independently An integer selected from 1-10, X is S or A, and X' is A, K or E.
  • the peptide linker comprises (G 4 X) n1 -G q1 -(X'P) m -(G 4 X) n2 -G q2 , wherein n1 is 1, q1 is 4, and m is 10, n2 is 1, q2 is 4.
  • the peptide linker comprises (G 4 S) 5 (SEQ ID NO: 123); in some embodiments of the present invention, the peptide linker comprises (G 4 S) 8 (SEQ ID NO: 124); in some embodiments of the present invention, the peptide linker comprises G 4 (AP) 10 G 4 (SEQ ID NO: 125); in other embodiments of the present invention, the The peptide linker comprises (G 4 S) 2 (AP) 10 (G 4 S) 2 (SEQ ID NO: 126); in some other embodiments of the present invention, the peptide linker comprises (G 4 S) 2 (EP) 10 ( G4S ) 2 (SEQ ID NO: 127).
  • 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: 94, SEQ
  • the numbering of the amino acid position in the mutation annotation of the Fc variant is according to the EU numbering; the numbering of the amino acid position in the GDF15 active domain is: the first amino acid at the N-terminal of SEQ ID NO:46 The first digit is numbered sequentially toward the C-terminus.
  • 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 NO: 67-78, SEQ ID NO: 80-102, SEQ ID NOS: 105-118.
  • the fusion protein comprises an amino acid sequence of one of the following groups, or an amino acid sequence having at least 95% sequence identity with one of the following groups: SEQ ID NO: 67-78, SEQ ID NO: 80 -102, SEQ ID NO: 105-118;
  • the fusion protein comprises an amino acid sequence of one of the following groups: SEQ ID NO: 67-78, SEQ ID NO: 80-102, SEQ ID NO: 105-118 .
  • the second aspect of the present invention provides a method for treating obesity-related diseases with a homodimeric fusion protein, which is composed of the fusion protein described in the first aspect of the present invention.
  • a third aspect of the present invention provides a method for treating obesity-related diseases with a biological material, the biological material being any one of (i), (ii), and (iii):
  • nucleic acid described nucleic acid comprises the nucleotide sequence of encoding fusion protein of the present invention
  • the fourth aspect of the present invention provides a method for treating obesity-related diseases with a pharmaceutical composition, the pharmaceutical composition comprising the homodimer fusion protein of the present invention as an active ingredient, the homodimer The fusion protein is present in the pharmaceutical composition in a therapeutically effective amount.
  • the pharmaceutical composition also includes a pharmaceutically acceptable carrier.
  • the present invention has the following progress: relative to the fusion protein of unmutated IgG Fc and GDF15 active domain, by combining the Fc variant provided by the present invention with any GDF15 domain retaining biological activity (mature GDF15 , truncated GDF15, or its variants) fusion, so that the Fc-GDF15 fusion protein has significantly improved physicochemical properties and recombinant expression, has comparable or higher in vitro activity than natural GDF15 molecules, and has significantly longer in vivo Circulatory half-life, in the treatment of obesity, could support biweekly or even monthly dosing frequency.
  • the Fc-GDF15 fusion protein of the present invention has simpler preparation process, lower cost and better patient compliance.
  • Figure 1 Schematic diagram of the homodimer formed by the Fc-GDF15 fusion protein
  • Figure 2 Effect of single administration of Fc-GDF15 fusion protein on body weight of normal mice (monitored for 14 days);
  • FIG. 3 Effect of single administration of Fc-GDF15 fusion protein on food intake of normal mice (monitored for 14 days);
  • Figure 4 Effect of single administration of Fc-GDF15 fusion protein on body weight of normal mice (monitored for 30 days);
  • FIG. 5 Effect of single administration of Fc-GDF15 fusion protein on food intake of normal mice (monitored for 30 days);
  • Figure 6 Effect of single administration of Fc-GDF15 fusion protein on body weight of normal mice (monitored for 56 days);
  • Figure 7 The effect of a single administration of Fc-GDF15 fusion protein on the food intake of normal mice (monitored for 56 days);
  • Figure 8 Effect of repeated administration of Fc-GDF15 fusion protein on body weight of diet-induced obese mice (monitored for 42 days);
  • Figure 9 Glucose tolerance test results of diet-induced obese mice receiving repeated administration of Fc-GDF15 fusion protein
  • FIG. 10 Effects of repeated administration of M39 constructs at different doses on the body weight of diet-induced obese mice (monitored for 49 days);
  • Figure 11 Fasting blood glucose levels in diet-induced obese mice after repeated administration of different doses of M39 constructs (Day 46) (***-p ⁇ 0.001 vs. vehicle; statistical analysis method: one-way ANOVA, followed by Dunnett's );
  • Figure 12 a. Glucose tolerance test results of diet-induced obese mice after repeated administration of different doses of M39 constructs (Day 46); b. 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 Fat index levels in diet-induced obese mice after repeated administration of different 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's);
  • Figure 14 Alanine aminotransferase levels in diet-induced obese mice after repeated administration of different doses of M39 constructs (***-p ⁇ 0.001 relative to vehicle; statistical analysis method: one-way ANOVA, followed by Dunnett's);
  • Figure 15 Aspartate aminotransferase levels in diet-induced obese mice after repeated administration of different doses of M39 constructs (***-p ⁇ 0.001 relative to vehicle; statistical analysis method: one-way ANOVA, followed by Dunnett's);
  • Figure 16 Effect of repeated administration of M6 and M39 constructs at different doses on body weight in ob/ob obese mice (monitored for 52 days). a. Body weight change relative to baseline; b. Body weight change relative to vehicle control;
  • FIG. 18 Ob/ob obese mice liver index levels after repeated administration of different 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's);
  • FIG 19 Alanine aminotransferase levels in ob/ob obese mice after repeated administration of different 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's);
  • Figure 20 Aspartate aminotransferase levels in ob/ob obese mice after repeated administration of different doses of M6 and M39 constructs (*-p ⁇ 0.05, **-p ⁇ 0.01, ***-p ⁇ 0.001 relative to vehicle, # -p ⁇ 0.05 vs. semaglutide; statistical analysis method: one-way ANOVA, followed by Dunnett's);
  • Figure 21 Improvement of liver pathology in ob/ob obese mice after repeated administration of different doses of M6 and M39 constructs (***-p ⁇ 0.001 relative to vehicle, ###-p ⁇ 0.001 relative to semaglutide peptide; statistical analysis method: one-way ANOVA, followed by Dunnett's).
  • mutant refers to a protein or polypeptide that does not contain any genetic engineering mutations, occurs naturally or can be isolated from the environment.
  • amino acid substitution or “replacement” refers to the replacement of one amino acid in a polypeptide with another amino acid.
  • amino acid substitutions are indicated by a first letter followed by a number followed by a second letter.
  • the first letter indicates the amino acid in the wild-type protein; the number indicates the amino acid position in which it was substituted; the second letter indicates the amino acid used to replace the wild-type amino acid.
  • a deletion at the amino terminus of a protein or polypeptide is indicated by ⁇ N followed by a number, where the number indicates the number of amino acids deleted at the amino terminus.
  • IgG or "IgG antibody” refers to an antibody having the structure of a naturally occurring immunoglobulin G molecule.
  • IgG antibodies include, for example, IgGl, IgG2, IgG3, and IgG4.
  • IgG1 Fc, IgG2 Fc, IgG3 Fc, and IgG4 Fc represent Fc or Fc regions of IgG1, IgG2, IgG3, and IgG4, respectively.
  • the IgG Fc in the fusion protein herein can be IgG1 Fc, IgG2 Fc, IgG3 Fc, IgG4 Fc, preferably IgG1 Fc, IgG4 Fc.
  • peptide linker refers to a single amino acid or polypeptide sequence that links two proteins together.
  • a peptide linker can be, for example, about 1-40 amino acids in length, comprising, for example, repeats of alanine, glycine and serine.
  • Common peptide linkers include flexible peptide linkers, such as a combination of glycine and serine, such as (GGGGS) n , the distance between the connected proteins can be adjusted by the number of repeating units in the peptide linker; peptide linkers composed only of glycine, such as G n , common ones include G 6 , G 8 , etc.; rigid peptide linkers, such as ⁇ -helical peptide linkers with (EAAAK) n sequence; and proline-rich peptide linkers (XP) n , where X can be specified Any amino acid, commonly including alanine, lysine, or glutamic acid, (XP) n -sequence has no helical structure, and the presence of proline in the peptide linker can increase the backbone stiffness and effectively separate the domains.
  • flexible peptide linkers such as a combination of glycine and serine, such as (GGGGS) n , the distance between
  • peptide linker can be determined by those skilled in the art according to common experiments, such as using only flexible peptide linker, using only rigid peptide linker, using a combination of flexible peptide linker and rigid peptide linker, etc.
  • Fc or “Fc region” is used to define the C-terminal region of an antibody heavy chain that contains at least a portion of the constant region.
  • the Fc region may be a native sequence Fc region or a variant Fc region.
  • the Fc region generally includes two constant domains: CH2 and CH3, and the boundaries of the Fc region can vary.
  • the C-terminal of the Fc region is the C-terminal of the heavy chain of the antibody; while the N-terminal of the Fc region can vary, and in some embodiments, the N-terminal of the Fc region can start, for example, from position 231 (EU numbering), Or start at position 233 (EU numbering), or start at position 236 (EU numbering), or start at position 237 (EU numbering); in other embodiments, the N-terminus of the Fc region of the present invention does not include a hinge region ; In still other embodiments, the N-terminus of the Fc region of the present invention comprises a hinge region.
  • the numbering of amino acid residues in the Fc region follows the EU numbering system, also known as the EU index, as described in Edelman et al., The covalent structure of an entire ⁇ G immunoglobulin molecule.Proc.Natl.Acad.Sci., USA ,1969.
  • GDF15 needs to form a dimer to exercise its activity.
  • the spatial distance between the carboxyl terminals of the two Fc monomers in the IgG Fc dimer is the same as that of the amino group of the GDF15 monomer.
  • the spatial distance between the ends 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 and low expression yield during assembly.
  • 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 such as 439 or 356, realizes the efficient assembly of the Fc-GDF15 fusion protein, and improves the fusion protein.
  • the yield is expressed, the preparation process is simple, and the formation of homodimers is not destroyed.
  • the Fc variant of the present invention can be applied to all GDF15 proteins or variants thereof that retain biological activity, whether it is a mature GDF15 protein, or a truncated GDF15, or a variant thereof, can be mutated with the Fc of the present invention body to form a fusion protein.
  • dimer refers to a protein dimer, which is composed of two protein molecules and is a quaternary structure of a protein.
  • the two protein molecules that make up a dimer can be referred to as monomeric molecules or monomeric proteins of the dimer.
  • Dimers can include "homodimers” and "heterodimers". Among them, homodimers are composed of two identical monomer molecules; heterodimers are composed of two A combination of different monomer molecules. It is generally believed that homodimers are simpler to prepare than heterodimers because only one peptide needs to be synthesized.
  • the Fc variant provided by the present invention has the ability to form homodimers; the fusion protein of the Fc variant provided by the present invention and the GDF15 active domain also has the ability to form homodimers, refer to Figure 1.
  • homodimerization exists between two monomeric Fc variants and between two monomeric GDF15 active domains, respectively.
  • growth differentiation factor 15 is in some embodiments native mature human GDF15 or its domain, and in some embodiments refers to GDF15 or its domain mutant.
  • Mature GDF15 consists of amino acids 197 (A) to 308 (I) in a total of 308 amino acids (UniProt Q99988) except signal peptide and propeptide (GDF15(197-308)(SEQ ID NO:46)) , or a polypeptide having at least 85%, 90%, 95%, 99% sequence identity with said amino acid sequence within the scope of maintaining the unique activity and structure of GDF15.
  • the mutants include truncation and/or one or more amino acid mutations.
  • the truncated GDF15 can be an N-terminal deletion variant, such as a sequence truncated by 1-14 amino acids at the N-terminus, or at least 85%, 90%, 95%, 99% identical to the truncated amino acid sequence
  • the GDF15 variant refers to a substitution, insertion or deletion of at least one amino acid site compared to mature GDF15 or truncated GDF15, as long as it maintains GDF15
  • At least one of the biological activities of the protein is within the scope of the present invention.
  • the GDF15 variant can also be introduced by introducing one or more conservative or non-conservative amino acid substitutions at specific positions in the GDF15 polypeptide and using naturally occurring or non-naturally occurring amino acids or by deleting specific residues or segments of residues And produced.
  • Constant amino acid substitutions may involve the replacement of natural amino acid residues (i.e., residues found at a given position in the wild-type GDF15 polypeptide sequence) with unnatural residues (i.e., not at a given position in the wild-type GDF15 polypeptide sequence The residue found) is substituted so as to have 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 (as defined herein) that are typically incorporated by chemical peptide synthesis rather than by synthesis in biological systems. These include peptidomimetics and other inverted or inverted forms of amino acid moieties.
  • Naturally occurring residues can be divided into the following categories based on common side chain properties:
  • Conservative substitutions may involve the exchange of a member of one of these classes for another member of the same class.
  • Non-conservative substitutions may involve the exchange of a member of one of these classes for a member of another class.
  • identity 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 residues that are identical between amino acids or nucleotides in the molecules being compared and is calculated based on the size of the smallest molecule among the molecules being compared.
  • vector refers to a nucleic acid molecule capable of propagating another nucleic acid linked thereto, including a vector as a self-replicating nucleic acid structure and a vector integrated into the genome of a host cell into which it is introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operably linked, and such vectors are referred to herein as "expression vectors.”
  • host cell refers to a cell into which exogenous nucleic acid has been introduced, including the progeny of such cells.
  • a host cell includes both the initially transformed cell and the progeny derived therefrom (regardless of passage number). Progeny may not be identical to the parental cell in nucleic acid content, but may contain mutations. Mutant progeny having the same function or biological activity as screened or selected for in the original transformed cell are included herein.
  • a host cell is any type of cellular system that can be used to produce a fusion protein of the invention.
  • Host cells include mammalian cultured cells such as CHO cells, BHK cells, NSO cells, SP2/0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells or hybridoma cells, yeast cells, bacteria Cells such as Escherichia coli, insect cells, and plant cells, etc., but also cells contained in transgenic animals, transgenic plants, or cultured plant or animal tissues.
  • mammalian cultured cells such as CHO cells, BHK cells, NSO cells, SP2/0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells or hybridoma cells, yeast cells, bacteria Cells such as Escherichia coli, insect cells, and plant cells, etc., but also cells contained in transgenic animals, transgenic plants, or cultured plant or animal tissues.
  • pharmaceutical composition refers to a preparation in such a form as to allow the biological activity of the active ingredients contained therein to be effective and free of additional ingredients that would be unacceptably toxic to a subject to whom the pharmaceutical composition would be administered.
  • 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.
  • pharmaceutically acceptable carrier refers to ingredients other than the active ingredient in the pharmaceutical composition that are non-toxic to the subject.
  • Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers or preservatives.
  • treatment refers to an attempt to alter the natural course of disease in a treated individual, and may be clinical intervention for prevention or implemented during the course of clinical pathology. Desired effects of treatment include, but are not limited to, prevention of occurrence or recurrence of disease, alleviation of symptoms, reduction of any direct or indirect pathological consequences of disease, slowing of the rate of disease progression, amelioration or elimination of the disease state, and regression or improved prognosis.
  • an "individual” or “subject” is a mammal.
  • Mammals include, but are not limited to, primates (eg, humans and non-human primates such as monkeys) or other mammals (eg, cattle, sheep, cats, dogs, horses, rabbits, and rodents such as mice and rats).
  • said individual or subject is a human.
  • test steps include:
  • the molecular cloning method was used to insert the Fc nucleic acid molecular sequence into the mammalian cell expression vector, and the ExpiCHO Fectamine TM CHO Transfection Kit (ThermoFisher Scientific) was used to perform transient transfection and expression in CHO-S cells; after purification by Protein A chromatography column , the target protein was obtained, and the target protein was analyzed by size exclusion high performance liquid chromatography (SEC).
  • SEC size exclusion high performance liquid chromatography
  • the main principle of SEC analysis is based on the molecular weight and three-dimensional structure of the analyte (such as protein).
  • the analyte such as protein
  • the analyte with large molecular weight will pass through the chromatographic column more quickly, so it has a shorter retention time in the chromatographic column.
  • the molecular weight is smaller.
  • the analyte has a longer retention time, so SEC can be used to analyze the aggregation of protein molecules.
  • the natural immunoglobulin Fc fragment will naturally form a homodimer structure, and in order to improve the stability of the fusion protein, Fc needs to be mutated, but the mutation may destroy the Fc homodimer structure.
  • the inventors were surprised to find that the Fc molecule (K439D or K439E) after the mutation was introduced at position 439 had the same retention time in SEC analysis as the Fc molecule without the mutation, indicating that the introduced mutation would not destroy the Fc dimer formation .
  • this example also tested the Fc molecule (including the F405Q/Y407E mutation) with the amino acid sequence of SEQ ID NO: 24.
  • the F405Q/Y407E mutation can destroy the Fc dimer and form a monomeric Fc Molecule, the mutation was found to have a significantly prolonged retention time in this experiment, indicating that the Fc molecule does exist in the form of a monomer.
  • sequence Fc variant keep time Aggregate state SEQ ID NO: 23 IgG4 Fc(AA) 13.341min dimer SEQ ID NO: 3 IgG4 Fc(AA,K439E) 13.156min dimer SEQ ID NO: 7 IgG4 Fc(AA,K439D) 13.093min dimer SEQ ID NO: 24 IgG4 Fc(AA,F405Q/Y407E) 15.076min monomer
  • CHO-S cells were subcultured after thawing, and the cells were collected when the density was about 6 ⁇ 10 6 cells/mL for cell transfection.
  • ExpiCHO Fectamine TM CHO Transfection Kit (ThermoFisher Scientific) was used, and the final concentration of the Fc-GDF15 expression vector was 1 ⁇ g/ml.
  • Protein purification after centrifuging the cell culture medium, the supernatant was collected and filtered with a 0.22 ⁇ m filter membrane to obtain the cell culture supernatant.
  • the cell culture supernatant was purified by two-step chromatography: the first chromatography: the cell culture supernatant was applied to an AT Protein A Diamond chromatography column (Boger) equilibrated with phosphate buffered saline (PBS) Long Company), after the target protein is combined, wash with equilibrium solution for more than 3CV (column volume, column volume) to remove unbound impurities, elute with 100mM acetic acid-sodium acetate (pH 3.0) buffer and collect the target protein, and then Quickly use 1.0M Tris-HCl pH 8.0 solution to adjust the elution and collect the sample pH to 7.2-7.6, dilute the adjusted pH sample with purified water until its conductivity value is lower than 5ms/cm, and obtain the first sample; the second step Chromatography: Use
  • target protein samples were evaluated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometry analysis.
  • Target protein samples were quantified by a micronucleic acid protein analyzer (NanoDrop 2000/2000c Spectrophotometer).
  • the Fc-GDF15 fusion protein described in Table 3 was expressed by the method described in Example 2, and the effects of using different Fc variants on the expression yield of the Fc-GDF15 fusion protein were compared. The results of the tests are shown in Table 5.
  • the Fc-GDF15 fusion protein that does not mutate the K439 (EU numbering) or E356 (EU numbering) of Fc such as M1 (a homodimer with a monomer having the sequence of SEQ ID NO: 119), M14 (The monomer has the homologous dimer of SEQ ID NO:79 sequence) expression amount is lower, and the Fc-GDF15 fusion protein that Fc K439 and/or E356 is mutated, such as M2 (the monomer has the SEQ ID NO:67 homologous dimer of sequence), M3 (monomer has homologous dimer of SEQ ID NO:68 sequence), M4 (monomer has homologous dimer of SEQ ID NO:69 sequence), M5 (monomer has homologous dimer of SEQ ID NO:69 sequence) Body has the homologous dimer of SEQ ID NO:70 sequence), M6 (monomer has the homologous dimer of SEQ ID NO:71 sequence
  • Embodiment 4 Physicochemical properties of Fc-GDF15 fusion protein
  • HMW high molecular weight
  • LMW low molecular weight
  • GDF15 in vivo requires signal transduction through its specific receptor GFRAL and co-receptor RET.
  • GDF15 can activate downstream signaling pathways after binding to cell surface receptors, one of which is phosphorylation of ERK1/2. Therefore, the in vitro activity of GDF15 can be evaluated by detecting the phosphorylation level of ERK1/2 in cells.
  • the expression genes of human GFRAL (UniProtKB-Q6UXV0) and human RET (UniProtKB-P07949) were linked by IRES elements and placed downstream of the CMV promoter, then transfected into HEK293T cells (ATCC), and added puromycin (Gibco) to select for stable expression Positive cells for both receptors, referred to as receptor expressing cells for short, were used in the phosphorylation level detection assay.
  • the phosphorylation level of ERK1/2 in cells was detected by Advanced phospho-ERK1/2 (Thr202/Tyr204) kit (cisbio) based on HTRF (Homogeneous Time-Resolved Fluorescence) technology, and the operation was performed according to the kit manual .
  • the receptor expressing cells spread the receptor expressing cells on a 384-well plate at a density of 16,000 cells/well, stimulate the cells with the Fc-GDF15 fusion protein to be tested or natural GDF15 (ACROBiosystems, GD5-H5149) after starvation culture, add the kit after the cells are lysed After the labeled antibody was incubated at room temperature overnight, a multifunctional microplate reader (SpectraMax i3X, Molecular devices) was used as the excitation wavelength of 337nm to detect the fluorescence values of 665nm and 620nm. The response value is calculated by the ratio Ratio of the fluorescence values at 665nm and 620nm, the formula is as follows:
  • A665 is the fluorescence value detected at 665nm wavelength
  • A620 is the fluorescence value detected at 620nm wavelength.
  • the intensity of the response value corresponds to the phosphorylation intensity of ERK1/2 in the cell.
  • DSC differential scanning calorimetry
  • GDF15 can inhibit food intake, reduce body weight, and has the potential to treat obesity and other related metabolic diseases.
  • the natural GDF15 molecule has a half-life of only about 3 hours in the human body, which is not suitable for direct application in drug therapy.
  • the Fc-GDF15 fusion protein provided by the present invention can prolong the half-life of drug molecules.
  • the effects of a single administration of different Fc-GDF15 fusion proteins on the food intake and body weight of normal mice were tested, so as to evaluate the in vivo activity and drug effect maintenance time of different Fc-GDF15 fusion proteins.
  • weight change (%) (measured weight - initial weight) / initial weight ⁇ 100%
  • the GDF15 fusion protein monoFc-GDF15 containing monomer Fc (“Compound 2" disclosed in patent WO2019195091, the monomer has the sequence of SEQ ID NO: 104; the Fc has the sequence of SEQ ID NO: 18, wherein F405Q, Y407E) at The drug effect reaches the maximum in about 8 days; the Fc-GDF15 fusion protein that mutates Fc K439, such as M3 (a monomer with a homologous dimer of SEQ ID NO: 68) and M15 (a monomer with a sequence of SEQ ID NO: 80 sequence); and Fc-GDF15 fusion proteins further comprising mutations at different amino acid sites of GDF15, such as M20 (a homodimer with a sequence of SEQ ID NO: 85), M23
  • the GDF15 fusion protein MonoFc-GDF15 containing monomeric Fc has a weak effect on reducing body weight, and the mice have recovered to the initial body weight at 14 days.
  • the Fc-GDF15 constructs comprising different amino acid mutations at the Fc K439 position, such as M3 (a homologous dimer with a monomer of SEQ ID NO: 68 sequence), M6 (a homologous dimer with a monomer of SEQ ID NO: 71 sequence), etc.
  • M7 (monomer has a homodimer of SEQ ID NO: 72 sequence)
  • M16 (monomer has a homodimer of SEQ ID NO: 81 sequence)
  • M51 (monomer has a homodimer of SEQ ID NO: 81 sequence) : homodimer of 117 sequence)
  • M7 monomer has a homodimer of SEQ ID NO: 72 sequence
  • M16 monomer has a homodimer of SEQ ID NO: 81 sequence
  • M51 (monomer has a homodimer of SEQ ID NO: 81 sequence) : homodimer of 117 sequence)
  • has a more significant weight-lowering effect and can continuously reduce the body weight of mice within 21-28 days, and has a significantly prolonged duration of drug effect.
  • mice were single-administered M9 (the homodimer with the sequence of SEQ ID NO: 74), M18 (the homodimer with the sequence of SEQ ID NO: 83) with a dose of 1 nmol/kg. ), M22 (the homodimer with the sequence of SEQ ID NO: 87) or M35 (the homodimer with the sequence of SEQ ID NO: 100), which can inhibit the food intake of mice and significantly reduce weight.
  • M9 the homodimer with the sequence of SEQ ID NO: 74
  • M18 the homodimer with the sequence of SEQ ID NO: 83
  • M22 the homodimer with the sequence of SEQ ID NO: 87
  • M35 the homodimer with the sequence of SEQ ID NO: 100
  • the body weight of the mice continued to decrease until the 21st day before it tended to be stable and recovered, and it basically returned to the initial body weight on the 56th day, indicating that Fc-GDF15 fusion proteins that mutate Fc K439 or E356, such as M9 and M18, and further Fusion proteins containing mutations at different amino acid sites of GDF15, such as M22 and M35, have a long duration of drug effect, and the drug effect can be maintained for about 1 month after a single dose of treatment.
  • the inventors surprisingly found that although the construct M35 had significantly reduced in vitro activity, it still maintained similar in vivo drug efficacy to other constructs.
  • Example 8 Drug effect of repeated administration of Fc-GDF15 fusion protein in diet-induced obese (DIO) mice
  • This embodiment tests the effect of repeated administration of Fc-GDF15 fusion protein on appetite suppression, weight loss and improvement of various metabolic indicators in DIO mice.
  • C57BL/6 mice were randomly divided into groups according to body weight, and then subcutaneously administered vehicle (PBS, once every two weeks) and different Fc-GDF15 fusion proteins (1nmol/kg, once every two weeks) Or semaglutide (3nmol/kg, once a day), the test lasted for 49 days.
  • PBS subcutaneously administered vehicle
  • Fc-GDF15 fusion proteins 1nmol/kg, once every two weeks
  • semaglutide 3nmol/kg, once a day
  • mice body weight and food intake are shown in Table 12, and the body weight change curve is shown in Figure 8; the glucose tolerance curve is shown in Figure 9, the area under the glucose tolerance curve, fat index, serum total cholesterol, triglyceride, ALT, AST and LDL parameters are shown in Table 13.
  • treatment of DIO mice can inhibit food intake and significantly reduce the body weight of the mice, and the weight loss effect is the same as that given 3nmol/kg every day Semaglutide has the same effect, and both can make the weight of mice reach normal level; M4 and M5 can also significantly improve the glucose tolerance level of DIO mice; M4, M5 and M9 can significantly reduce the fat content of DIO mice, and can also Improve blood lipids such as significantly reducing total cholesterol, triglycerides and low-density lipoprotein; M4, M5 and M9 can also improve liver function such as significantly reducing ALT and AST levels. It shows that the Fc-GDF15 construct of the present invention can be applied to the treatment of obesity, diabetes, hyperlipidemia or fatty liver/steatohepatitis.
  • DIO mice were randomly divided into groups (10-12 mice per group), and subcutaneously administered vehicle (PBS, once a week), different doses of M39 (0.03nmol/kg, 0.1nmol/kg and 1nmol/kg, once a week) ; 3 nmol/kg, once every two weeks) or semaglutide (3 nmol/kg, once daily).
  • PBS subcutaneously administered vehicle
  • M39 0.03nmol/kg, 0.1nmol/kg and 1nmol/kg, once a week
  • semaglutide 3 nmol/kg, once daily
  • the body weight change of the mice at the end of treatment is shown in Table 14, and the body weight change curve, fasting blood glucose level and IPGTT after 46 days of treatment and the area under the curve, fat index, and transaminase level are shown in Figures 10 to 15, respectively.
  • M39 can also significantly reduce the fat content of mice, improve the fasting blood glucose level, glucose tolerance, and liver function indicators of mice.
  • mice The body weight changes of the mice at the end of treatment are shown in Table 15, and the body weight change curves, cumulative food intake, liver to body ratio, transaminase levels, and liver pathological results are shown in Figures 16 to 21, respectively.
  • the Fc-GDF15 construct designed by the present invention has an ultra-long duration of drug effect, M6 and M39 at low doses on day 0 and day 24, and at medium and high doses on day 0 and day 36, respectively
  • M6 and M39 at low doses on day 0 and day 24, and at medium and high doses on day 0 and day 36, respectively
  • the improvement of body weight is significantly stronger than that of semaglutide administered every day.
  • it can also significantly improve the liver function and hepatic steatosis of ob/ob mice.
  • the effect was also significantly better than semaglutide.
  • This embodiment has tested construct M38 (monomer has the homologous dimer of SEQ ID NO:103 sequence), M3 (monomer has the homologous dimer of SEQ ID NO:68 sequence), M15 (monomer has The pharmacokinetic performance of the homologous dimer of SEQ ID NO:80 sequence) in mice.
  • the above fusion protein was diluted to 0.05 mg/ml with 10 mM PBS, and then subcutaneously injected into C57BL/6 mice at a dose of 0.25 mg/kg, and 3 mice were injected with each fusion protein.
  • AUC INF_obs represents the area under the curve from the administration time to the theoretically extrapolated infinity time; C max represents the highest detected plasma concentration; T 1/2 represents the half-life; T max represents the time to reach the maximum plasma concentration.
  • This example tested the pharmacokinetic performance of construct M39 in cynomolgus monkeys.
  • Cynomolgus monkeys were given subcutaneous injection of M39 at doses of 1 mg/kg and 2 mg/kg, respectively, with three animals for each dose, male and female. Blood was collected from each animal before administration and at 0.5h, 2h, 6h, 48h, 168h, 356h, 504h, and 672h after administration, and the concentration of plasma Fc-GDF15 was determined by sandwich ELISA method.
  • mice anti-human GDF15 monoclonal antibody (Shenzhou Yiqiao Co., Ltd.) was used to coat the plate, and then the target protein was detected with mouse anti-human IgG4 Fc-HRP, and the pharmacokinetic data was calculated.
  • the results are shown in Table 17.

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Abstract

一种GDF15融合蛋白及其用于治疗肥胖的方法，该融合蛋白包括Fc变体和GDF15活性结构域，所述Fc变体依照EU编号在IgG Fc的第356位和/或第439位处具有氨基酸取代并且仍具有形成同源二聚体的能力。将该Fc变体与GDF15活性结构域融合，使得Fc-GDF15融合蛋白具有显著提高的理化性质和重组表达量，具有与天然GDF15分子相当或更优的体外活性，并且具有显著延长的体内循环半衰期，可以支持两周一次甚至一个月一次的给药频率。

Description

GDF15融合蛋白及其用途
本申请要求申请日为201.08.24的中国专利申请202110977378.7的优先权。本申请引用上述中国专利申请的全文。
技术领域
本发明涉及生物医药技术领域，具体涉及一种GDF15融合蛋白及其用于治疗肥胖的方法。
背景技术
肥胖是指由于体内脂肪细胞体积和数量增加、脂肪组织分布异常而形成的一种由多种因素相互作用的慢性代谢性疾病，每年至少有280万人死亡可归咎于超重或肥胖。美国医学会(AMA)的主要政策制订机构明确表示，肥胖不仅应被视为疾病危险因素，而是一种真实的疾病，美国临床内分泌医师协会(AACE)指出，超重或肥胖可增加罹患乳腺癌、冠心病、2型糖尿病、胆囊疾病、骨关节炎、结肠癌、高血压和卒中等风险。
肥胖管理包括生活方式改变、药物治疗和减肥手术。改变生活方式通常需要专业团队制定方案并进行维护，但经常与高复发率相关；减肥手术伴随着死亡和手术并发症的风险，且成本高昂，一般用于严重肥胖症。鉴于目前改变生活方式和减肥手术的局限性，使用药物治疗肥胖至关重要。美国权威指南建议，当体重指数≥30kg/m 2，或体重指数≥27kg/m 2并伴有至少一种体重相关的并发症时，可应用抗肥胖药物进行治疗。
生长分化因子15(GDF15)，又名巨噬细胞抑制细胞因子-1(MIC-1)、胎盘转化生长因子-β(PTGF-β)、胎盘骨形态发生因子(PLAB)、前列腺衍生因子(PDF)、非甾体抗炎药活化基因(NAG-1)，是转化生长因子(TGF-β)超家族分支成员。成熟的GDF15多肽包含112个氨基酸，而体内循环的具有生物活性的GDF15则是两条多肽通过链间二硫键形成的同源二聚体蛋白(24.5kD)。
已报道GDF15循环水平升高与晚期癌症患者食物摄入量减少和体重降低相关(Johnen H等人，Nat Med,2007)。并且，转基因高表达GDF15的小鼠以及施用重组GDF15的小鼠均表现出体重减低和食物摄入量减少，并且具有改善 的糖耐受水平(Xiong Y等人，Sci Transl Med，2017)，表明GDF15具有治疗肥胖症、2型糖尿病(T2D)、非酒精性脂肪肝炎(NASH)等疾病的潜力。
天然GDF15二聚体在体内的循环半衰期(t1/2)较短，只有大约2-3小时，极大限制了其在临床上的治疗应用。常规延长大分子药物循环半衰期的方法包括将药物分子与抗体的Fc片段融合，但是由于GDF15二聚体和Fc片段二聚体分子三维结构的特性，直接将GDF15与Fc片段融合会极大影响分子整体的稳定性和重组表达产量。
因此，开发稳定、半衰期更长并且工艺简单的GDF15融合蛋白分子，对于治疗肥胖，是非常必要的。
发明内容
本发明的目的在于提供一种Fc-GDF15融合蛋白用于治疗与肥胖相关的疾病的方法。，本发明所述的GDF15融合蛋白具有理化性质稳定、半衰期长、作用效果长效、制备工艺简单等优点，可用于治疗代谢相关疾病，例如肥胖、2型糖尿病、NASH、血脂异常等，尤其是肥胖。
第一方面，本发明提供一种Fc-GDF15融合蛋白用于治疗治疗与肥胖相关疾病的方法。
为此，本发明提供一种融合蛋白，在一些实施方案中，所述融合蛋白包括GDF15活性结构域和Fc变体；所述Fc变体的C末端直接或者通过肽接头连接至所述GDF15活性结构域的N末端；
所述Fc变体依照EU编号在IgG Fc的第356位和/或第439位处具有氨基酸取代。
在一些实施方式中，所述Fc变体具有形成同源二聚体的能力。
在一些实施方式中，所述IgG Fc选自IgG1 Fc、IgG2 Fc、IgG3 Fc、IgG4 Fc中的一种。
在一些实施方式中，所述IgG Fc为人IgG1 Fc。在一些实施方式中，所述IgG Fc为人IgG4 Fc。
进一步，所述Fc变体依照EU编号将IgG Fc的第356位的氨基酸取代为：除D、E和C以外的氨基酸。例如，将IgG Fc的第356位的氨基酸取代为下组中的一种：G、S、A、T、V、N、L、I、Q、Y、F、H、P、M、K、R。进一步， 所述Fc变体依照EU编号将IgG Fc的第439位的氨基酸取代为：除R、H、K和C以外的氨基酸。例如，将IgG Fc的第439位的氨基酸取代为下组中的一种：G、S、A、T、V、D、N、L、I、E、Q、Y、F、P、M。
在一些实施方式中，所述Fc变体依照EU编号在IgG Fc中含有以下突变：E356G、E356S、E356A、E356T、E356V、E356N、E356L、E356I、E356Q、E356Y、E356F、E356H、E356P、E356M、E356K、E356R中的一种，和/或K439G、K439S、K439A、K439T、K439V、K439D、K439N、K439L、K439I、K439E、K439Q、K439Y、K439F、K439H、K439P、K439M中的一种。
在优选的实施方式中，所述Fc变体依照EU编号在IgG Fc中含有以下突变：E356R、E356Q、E356A、E356N中的一种，和/或K439D、K439E、K439Q、K439A、K439N中的一种。
在一些实施方式中，所述Fc变体依照EU编号在IgG Fc中含有以下突变：K439D、K439E、K439Q、K439A、K439N中的一种。在优选的实施方式中，所述Fc变体依照EU编号在IgG Fc中含有以下突变：K439D、K439E、K439Q、中的一种。
在一些实施方式中，所述Fc变体依照EU编号在IgG Fc中含有以下突变：E356R、E356Q、E356A、E356N中的一种。在优选的实施方式中，所述Fc变体依照EU编号在IgG Fc中含有以下突变：E356R。
在一些实施方式中，所述Fc变体还含有以下突变：依照EU编号将IgG Fc的第234位和第235位的氨基酸取代为AA，和/或将第447位的氨基酸删除。例如，对于IgG1 Fc，还含有以下突变：L234A和L235A，和/或第447位的氨基酸删除；对于IgG4 Fc，还含有以下突变：F234A和L235A，和/或将第447位的氨基酸删除。
在某实施方式中，所述Fc变体依照EU编号在IgG Fc中的氨基酸取代仅为下组中的一种：
依照EU编号第356位的氨基酸取代；
依照EU编号第439位的氨基酸取代；
依照EU编号第356位和第439位的氨基酸取代；
依照EU编号第356位、第234位和第235位的氨基酸取代；
依照EU编号第439位、第234位和第235位的氨基酸取代；
依照EU编号第356位、第439位、第234位和第235位的氨基酸取代；
在各位置所述的氨基酸取代具有本发明所述的含义。
在某实施方式中，所述Fc变体的氨基酸序列包括下组序列之一：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。
表1 Fc变体
Figure PCTCN2022114143-appb-000001
Figure PCTCN2022114143-appb-000002
Figure PCTCN2022114143-appb-000003
Figure PCTCN2022114143-appb-000004
注：表1中，表示氨基酸位置的数字编号方式依照EU编号系统。
在另一实施方式中，所述Fc变体包含与下组序列之一具有至少85％、90％、95％或99％序列同一性的氨基酸序列：SEQ ID NO：1-22，SEQ ID NO：27，SEQ  ID NO：29-45。
进一步，所述GDF15活性结构域为全长成熟GDF15蛋白、成熟GDF15蛋白，N端截短的GDF15蛋白、或它们保留GDF15生物学活性的任意变体。
在一实施方式中，所述GDF15活性结构域包含选自下组之一的氨基酸序列或与下组之一的氨基酸序列具有至少85％、90％、95％或99％序列同一性的氨基酸序列：
SEQ ID NO：46；
在SEQ ID NO：46的N末端有1-14个氨基酸截短，和/或在SEQ ID NO：46中含有1-3个氨基酸取代。
在一实施方式中，所述GDF15活性结构域包含选自下组之一的氨基酸序列：
SEQ ID NO：46；
在SEQ ID NO：46的N末端有1-14个氨基酸截短，和/或在SEQ ID NO：46中含有1-3个氨基酸取代。
进一步，所述SEQ ID NO：46的N末端的氨基酸截短的数量为1个、2个、3个、4个、5个、6个、7个、8个、9个、10个、11个、12个、13个或14个。
进一步，所述SEQ ID NO：46中的氨基酸取代的位置选自下组中的一个、两个、或三个：第5位、第6位、第21位、第26位、第30位、第47位、第54位、第55位、第57位、第67位、第69位、第81位、第94位、第107位。
进一步，所述SEQ ID NO：46中的氨基酸取代选自下组中的一个、不同位置的任两个、或者不同位置的任三个：D5E、H6D、H6E、R21Q、R21H、D26E、A30S、A47D、A54S、A55E、M57T、R67Q、K69R、A81S、T94E、K107Q。
在一些实施方式中，所述GDF15活性结构域包含SEQ ID NO：46所示的氨基酸序列。
在另一些实施方式中，所述GDF15活性结构域包含以下序列：在SEQ ID NO：46的N末端有1-14个氨基酸截短的氨基酸序列。
在又一些实施方式中，所述GDF15活性结构域包含以下序列：在SEQ ID NO：46中含有1-3个氨基酸取代的氨基酸序列；所述在SEQ ID NO：46中的 氨基酸取代具有本发明所述的含义。
在再一些实施方式中，所述GDF15活性结构域包含以下序列：在SEQ ID NO：46的N末端有1-14个氨基酸截短，且在SEQ ID NO：46中含有1-3个氨基酸取代的氨基酸序列；所述在SEQ ID NO：46中的氨基酸取代具有本发明所述的含义。
在某实施方式中，所述GDF15活性结构域包含下组序列之一：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。
表2 GDF15活性结构域
Figure PCTCN2022114143-appb-000005
Figure PCTCN2022114143-appb-000006
注：表2中，表示氨基酸位置的数字编号方式为：以SEQ ID NO：46的N末端第一个氨基酸为第一位，依次向C末端方向进行编号。
在另一实施方式中，所述GDF15活性结构域包含与下组序列之一具有至少85％、90％、95％或99％序列同一性的氨基酸序列：SEQ ID NO：46-66。在另一实施方式中，所述GDF15活性结构域包含下组之一的氨基酸序列，或者与下组之一具有至少95％同一性的氨基酸序列：SEQ ID NO：46-66；优选地，所述GDF15活性结构域包含下组之一的氨基酸序列：SEQ ID NO：46-66。
在一些实施方式中，所述Fc变体的C末端通过肽接头连接至所述GDF15活性结构域的N末端；所述肽接头选自柔性肽接头、刚性肽接头中的一种或两种的组合。任何合适的接头均可用于本发明的实施方案的融合蛋白中。如本文，术语“接头”是指包含肽接头的连接部分。优选地，接头有助于确保正确的折叠，最小化空间位阻，并且不会显著地干扰融合蛋白内每个功能组分的结构。更优选地，为了获得较高的蛋白表达量，本申请可以采用刚性肽接头、刚性/柔性杂合肽接头。
在一些实施方式中，所述肽接头包括(G 4X) n、(X’P) m、(EAAAK) p、G q中的一种或两种以上的组合，其中n、m、p、q各自独立地选自1-10的整数，X为丝氨酸(S)或丙氨酸(A)，X’为丙氨酸(A)、赖氨酸(K)或谷氨酸(E)。
在某实施方式中，所述肽接头包括(G 4X) n和(X’P) m的组合，所述n、m、X、X’具有本发明所述的含义。
在某实施方式中，所述肽接头包括(G 4X) n1-(X’P) m-(G 4X) n2，其中n1、m、n2各自独立地选自1-10的整数，X为S或A，X’为A、K或E。
在某实施方式中，所述肽接头包括(G 4X) n1-(X’P) m-(G 4X) n2，其中n 1为2，m为10，n 2为2。
在另一实施方式中，所述肽接头包括G q和(X’P) m的组合，所述q、m、X’具有本发明所述的含义。
在某实施方式中，所述肽接头包括G q1-(X’P) m-G q2，q1、m、q2各自独立地选自1-10的整数，X’为A、K或E。
在某实施方式中，所述肽接头包括G q1-(X’P) m-G q2，其中q 1为4，m为10，q 2为4。
在另一实施方式中，所述的肽接头包括(G 4X) n、(X’P) m和G q的组合，所述n、m、q、X、X’具有本发明所述的含义。
在某些实施方式中，所述肽接头包括(G 4X) n1-G q1-(X’P) m-(G 4X) n2-G q2，n1、q1、m、n2、q2各自独立地选自1-10的整数，X为S或A，X’为A、K或E。
在某些实施方式中，所述肽接头包括(G 4X) n1-G q1-(X’P) m-(G 4X) n2-G q2，其中n1为1，q1为4，m为10，n2为1，q2为4。
在本发明的一些实施方式中，所述的肽接头包含(G 4S) 5(SEQ ID NO:123)；在本发明的一些实施方式中，所述的肽接头包含(G 4S) 8(SEQ ID NO:124)；在本发明的一些实施方式中，所述的肽接头包含G 4(AP) 10G 4(SEQ ID NO:125)；在本发明的另一些实施方式中，所述的肽接头包含(G 4S) 2(AP) 10(G 4S) 2(SEQ ID NO:126)；在本发明的又一些实施方式中，所述的肽接头包含(G 4S) 2(EP) 10(G 4S) 2(SEQ ID NO:127)。发明人经过大量研究发现，采用刚性肽接头，或者刚性/柔性杂合肽接头，能显著提高本发明融合蛋白的表达量。
在一些实施方式中，所述融合蛋白包括选自下组之一的氨基酸序列：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。
表3 融合蛋白
Figure PCTCN2022114143-appb-000007
Figure PCTCN2022114143-appb-000008
注：表3中，Fc变体的突变注释中表示氨基酸位置的数字编号依照EU编号；GDF15活性结构域中表示氨基酸位置的数字编号方式为：以SEQ ID NO：46的N末端第一个氨基酸为第一位，依次向C末端方向进行编号。
在另一实施方式中，所述融合蛋白包含与下组序列之一具有至少85％、90％、 95％或99％序列同一性的氨基酸序列：SEQ ID NO：67-78，SEQ ID NO：80-102，SEQ ID NO：105-118。在另一实施方式中，所述融合蛋白包含下组之一的氨基酸序列，或者与下组之一具有至少95％序列同一性的氨基酸序列：SEQ ID NO：67-78，SEQ ID NO：80-102，SEQ ID NO：105-118；优选地，所述融合蛋白包含下组之一的氨基酸序列：SEQ ID NO：67-78，SEQ ID NO：80-102，SEQ ID NO：105-118。
本发明的第二方面，提供一种同源二聚体融合蛋白用于治疗肥胖相关疾病的方法，其由本发明第一方面所述的融合蛋白所构成。
本发明的第三方面，提供一种生物材料用于治疗肥胖相关疾病的方法，所述生物材料为(i)、(ii)、(iii)中的任一种：
(i)核酸，所述核酸包括编码本发明所述的融合蛋白的核苷酸序列；
(ii)载体，所述载体包含(i)中的核酸；
(iii)宿主细胞，所述宿主细胞含有(i)中的核酸和/或(ii)中的载体。
本发明的第四方面，提供一种药物组合物用于治疗肥胖相关疾病的方法，所述药物组合物包括本发明所述的同源二聚体融合蛋白作为活性成分，所述同源二聚体融合蛋白以治疗有效量存在于所述药物组合物中。
进一步，所述药物组合物还包括药学上可接受的载体。
与现有技术相比，本发明具有以下进步：相对于未突变IgG Fc与GDF15活性结构域的融合蛋白，通过将本发明提供的Fc变体与任意保留生物学活性的GDF15结构域(成熟GDF15、截短的GDF15、或其变体)融合，使得Fc-GDF15融合蛋白具有显著提高的理化性质和重组表达量，具有与天然GDF15分子相当或更高的体外活性，并且具有显著更长的体内循环半衰期，在治疗肥胖时，可以支持两周一次甚至一个月一次的给药频率。此外，本发明Fc-GDF15融合蛋白相比现有技术中的异源二聚体形成的融合蛋白，制备工艺更简单，成本更低，患者的依从性更好。
附图说明
通过阅读下文优选实施方式的详细描述，各种其他的优点和益处对于本领域普通技术人员将变得清楚明了。附图仅用于示出优选实施方式的目的，而并不认为是对本发明的限制。在附图中：
图1：Fc-GDF15融合蛋白所形成的同源二聚体的示意图；
图2：Fc-GDF15融合蛋白单次给药治疗对正常小鼠体重的影响(监测14天)；
图3：Fc-GDF15融合蛋白单次给药治疗对正常小鼠食物摄入量的影响(监测14天)；
图4：Fc-GDF15融合蛋白单次给药治疗对正常小鼠体重的影响(监测30天)；
图5：Fc-GDF15融合蛋白单次给药治疗对正常小鼠食物摄入量的影响(监测30天)；
图6：Fc-GDF15融合蛋白单次给药治疗对正常小鼠体重的影响(监测56天)；
图7：Fc-GDF15融合蛋白单次给药治疗对正常小鼠食物摄入量的影响(监测56天)；
图8：Fc-GDF15融合蛋白重复给药对饮食诱导肥胖小鼠体重的影响(监测42天)；
图9：接受Fc-GDF15融合蛋白重复给药的饮食诱导肥胖小鼠的耐糖量测试结果；
图10：不同剂量下M39构建体重复给药对饮食诱导肥胖小鼠体重的影响(监测49天)；
图11：接受不同剂量M39构建体重复给药后饮食诱导肥胖小鼠空腹血糖水平(第46天)(***-p<0.001相对于溶媒；统计分析方法：one-way ANOVA,followed by Dunnett’s)；
图12：a.接受不同剂量M39构建体重复给药后饮食诱导肥胖小鼠糖耐量测试结果(第46天)；b.糖耐量测试曲线下面积统计(**-p<0.01,***-p<0.001相对于溶媒；统计分析方法：one-way ANOVA,followed by Dunnett’s)；
图13：接受不同剂量M39构建体重复给药后饮食诱导肥胖小鼠脂肪指数水平(**-p<0.01,***-p<0.001,#-p<0.05,##-p<0.01,###-p<0.001相对于溶媒；统计分析方法：one-way ANOVA,followed by Dunnett’s)；
图14：接受不同剂量M39构建体重复给药后饮食诱导肥胖小鼠谷丙转氨 酶水平(***-p<0.001相对于溶媒；统计分析方法：one-way ANOVA,followed by Dunnett’s)；
图15：接受不同剂量M39构建体重复给药后饮食诱导肥胖小鼠谷草转氨酶水平(***-p<0.001相对于溶媒；统计分析方法：one-way ANOVA,followed by Dunnett’s)；
图16：不同剂量下M6和M39构建体重复给药对ob/ob肥胖小鼠体重的影响(监测52天)。a.体重变化相对于基线；b.体重变化相对于溶媒对照；
图17不同剂量下M6和M39构建体重复给药对ob/ob肥胖小鼠摄食量的影响(监测52天)；
图18：接受不同剂量M6和M39构建体重复给药后ob/ob肥胖小鼠肝脏指数水平(***-p<0.001相对于溶媒，###-p<0.001相对于司美格鲁肽；统计分析方法：one-way ANOVA,followed by Dunnett’s)；
图19：接受不同剂量M6和M39构建体重复给药后ob/ob肥胖小鼠谷丙转氨酶水平(***-p<0.001相对于溶媒，##-p<0.01相对于司美格鲁肽；统计分析方法：one-way ANOVA,followed by Dunnett’s)；
图20：接受不同剂量M6和M39构建体重复给药后ob/ob肥胖小鼠谷草转氨酶水平(*-p<0.05,**-p<0.01,***-p<0.001相对于溶媒，#-p<0.05相对于司美格鲁肽；统计分析方法：one-way ANOVA,followed by Dunnett’s)；
图21：接受不同剂量M6和M39构建体重复给药后ob/ob肥胖小鼠肝病理改善效果(***-p<0.001相对于溶媒，###-p<0.001相对于司美格鲁肽；统计分析方法：one-way ANOVA,followed by Dunnett’s)。
具体实施方式
下面将参照附图更详细地描述本公开的示例性实施方式。虽然附图中显示了本公开的示例性实施方式，然而应当理解，可以以各种形式实现本公开而不应被这里阐述的实施方式所限制。相反，提供这些实施方式是为了能够更透彻地理解本公开，并且能够将本公开的范围完整的传达给本领域的技术人员。
除非另外定义，本发明中使用的所有技术和科学术语与本技术领域技术人员通常理解的意义相同。除实施例中使用的具体方法、设备、材料外，根据本技术领域的技术人员对现有技术的掌握及本发明的记载，还可以使用与本发明 实施例中所述的方法、设备、材料相似或等同的现有技术的任何方法、设备和材料来实现本发明。
本文中，术语“天然”、“野生型”或“WT”指不包含任何遗传工程突变的、天然存在或可从环境分离的蛋白质或多肽。
本文中，氨基酸“取代”或“置换”指将多肽中一个氨基酸用另一种氨基酸替换。
本文中，氨基酸的取代由第一个字母后跟数字后跟第二个字母来表示。第一个字母表示野生型蛋白质中的氨基酸；数字是指其中被置换的氨基酸位置；第二个字母表示用于替代野生型氨基酸的氨基酸。
本文中，蛋白质或多肽的氨基末端的缺失由ΔN后跟数字来表示，其中数字表示氨基末端缺失的氨基酸的数目。
本文中，“IgG”或“IgG抗体”指具有天然存在免疫球蛋白G分子的结构的抗体。IgG抗体包括例如IgG1、IgG2、IgG3和IgG4。IgG1 Fc、IgG2 Fc、IgG3 Fc、IgG4 Fc分别表示IgG1、IgG2、IgG3和IgG4的Fc或Fc区。本文中融合蛋白中的IgG Fc可以为IgG1 Fc，IgG2 Fc，IgG3 Fc，IgG4 Fc，优选是IgG1 Fc，IgG4 Fc。
本文中，“肽接头”指将两个蛋白质连接在一起的单个氨基酸或者多肽序列。肽接头的长度可以为例如约1-40个氨基酸，包含例如重复的丙氨酸，甘氨酸和丝氨酸。常见的肽接头包括柔性肽接头，例如甘氨酸和丝氨酸的组合，如(GGGGS) n，通过肽接头中重复单元的数量可以调节其连接的蛋白之间的距离；仅由甘氨酸组成的肽接头，例如G n，常见的包括G 6、G 8等；刚性肽接头，例如具有(EAAAK) n序列的α-螺旋形肽接头；具有富含脯氨酸的肽接头(XP) n，其中X可以指定任何氨基酸，常见的包括丙氨酸、赖氨酸或谷氨酸，(XP) n序列没有螺旋结构，肽接头中存在的脯氨酸可以增加骨架硬度，并且有效分离结构域。富含脯氨酸序列的结构在机体中广泛的存在，例如骨骼肌蛋白的N端就存在(AP) 7的结构。肽接头的选择本领域技术人员可根据通常的实验进行确定，例如仅使用柔性肽接头、仅使用刚性肽接头、使用柔性肽接头与刚性肽接头的组合等。
本文中，“Fc”或“Fc区”用于定义抗体重链中至少含有恒定区的一部分 的C端区域。Fc区可以是天然序列Fc区或变体Fc区。本领域技术人员知晓，Fc区一般包含两个恒定结构域：CH2和CH3，而Fc区的边界可以变化。在本文中，Fc区的C末端即为抗体重链的C末端；而Fc区的N末端可以变化，在一些实施方式中，Fc区的N末端可以从例如第231位(EU编号)开始，或第233位(EU编号)开始，或第236位(EU编号)开始，或从第237位(EU编号)开始；在另一些实施方式中，本发明的Fc区的N末端不包含铰链区；在又一些实施方式中，本发明的Fc区的N末端包含铰链区。本文中，Fc区中氨基酸残基的编号方式依照EU编号系统，也称为EU索引，如记载于Edelman et al.,The covalent structure of an entireγG immunoglobulin molecule.Proc.Natl.Acad.Sci.,USA,1969。
GDF15需要形成二聚体以行使活性，在本发明研究过程中发现，尽管IgG Fc能够自然形成二聚体，然而IgG Fc二聚体中两个单体Fc的羧基端的空间距离与GDF15单体氨基端的空间距离存在明显差异；即使使用柔性或刚性的肽接头，经过IgG Fc的C末端与GDF15的N末端形成的融合蛋白在组装时仍然存在不稳定性，表达产量低等问题。本发明提供的Fc变体，通过在Fc的特定位点如439位或356位引入突变，显著改善了Fc-GDF15分子的稳定性，实现Fc-GDF15融合蛋白的高效组装，提高了融合蛋白的表达产量，制备工艺简单，且不破坏同源二聚体的形成。此外，本发明的Fc变体可适用于所有保留生物学活性的GDF15蛋白或其变体，无论是成熟GDF15蛋白，或其截短的GDF15，或其变体，均可以与本发明的Fc变体形成融合蛋白而发挥作用。
本文中，“二聚体”指蛋白二聚体，其由两个蛋白质分子组合而成，是一种蛋白质的四级结构。组成二聚体的两个蛋白质分子可称为该二聚体的单体分子或单体蛋白质。二聚体可包括“同源二聚体”和“异源二聚体”两种情况，其中，同源二聚体由两个相同的单体分子组合而成；异源二聚体由两种不同的单体分子组合而成。通常认为，同源二聚体相对于异源二聚体，具有更简单的制备方法，因为只需合成一种肽。在本发明中，本发明提供的Fc变体具有形成同源二聚体的能力；本发明提供的Fc变体与GDF15活性结构域的融合蛋白也具有形成同源二聚体的能力，参照图1，在由该融合蛋白形成的同源二聚体中，两个单体Fc变体之间、两个单体GDF15活性结构域之间分别都存在着同源二 聚作用。
本文中，“生长分化因子15”，“GDF15”或“GDF15活性结构域”在有些实施例中是天然的成熟人GDF15或其结构域，在某些实施例中指的是GDF15或其结构域的突变体。成熟GDF15由总共308个氨基酸(UniProt Q99988)中除信号肽和前肽外的第197位(A)至第308位(I)氨基酸组成(GDF15(197-308)(SEQ ID NO:46))，或与所述氨基酸序列在维持GDF15的独特活性和结构的范围内具有至少85％、90％、95％、99％的序列同一性的多肽。所述的突变体包含截短的，和/或者一个、多个氨基酸突变。在有些实施例中，截短的GDF15可以是N端缺失变体，比如N末端处截短1-14个氨基酸的序列，或与截短氨基酸序列至少85％、90％、95％、99％的序列同一性的多肽；在有些实施例中，所述的GDF15变体，是指相比于成熟GDF15或截短的GDF15存在至少1个氨基酸位点的置换、插入或缺失，只要其保持GDF15蛋白的生物活性中的至少一者，如其对食物摄取量、血糖水平、胰岛素抗性和体重等的影响，都在本发明保护范围内。
所述的GDF15变体，也可通过在GDF15多肽的特定位置上引入一个或多个保守或非保守氨基酸取代和使用天然存在或非天然存在的氨基酸或者通过缺失特定残基或残基的区段而产生。
“保守氨基酸取代”可涉及天然氨基酸残基(即，在野生型GDF15多肽序列的给定位置上发现的残基)被非天然残基(即，不是在野生型GDF15多肽序列的给定位置上发现的残基)取代，以使对所述位置处的氨基酸残基的极性或电荷影响很小或没有影响。保守氨基酸取代还涵盖通常通过化学肽合成而不是通过在生物系统中的合成并入的非天然存在的氨基酸残基(如本文所定义)。这些包括肽模拟物以及氨基酸部分的其它反向或倒转形式。
天然存在的残基可基于共同侧链特性分成以下种类：
(1)疏水性：Met、Ala、Val、Leu、Ile；
(2)中性亲水性：Cys、Ser、Thr、Asn、Gln；
(3)酸性：Asp、Glu；
(4)碱性：His、Lys、Arg；
(5)影响链取向的残基：Gly、Pro；以及
(6)芳族：Trp、Tyr、Phe。
某些保守氨基酸取代的示例见下表：
氨基酸 保守氨基酸取代
Ala D-Ala，Gly，Aib，β-Ala，L-Cys，D-Cys
Arg D-Arg，Lys，Orn，D-Orn
Asn D-Asn，Asp，D-Asp，Glu，D-Glu，Gln，D-Gln
Asp D-Asp，D-Asn，Asn，Glu，D-Glu，Gln，D-Gln
Cys D-Cys，S-Me-Cys，Met，D-Met，Thr，D-Thr，L-Ser，D-Ser
Gln D-Gln，Asn，D-Asn，Glu，D-Glu，Asp，D-Asp
Glu D-Glu，Asp，D-Asp，Asn，D-Asn，Gln，D-Gln
Gly Ala，D-Ala，Pro，D-Pro，Aib，β-Ala
Ile D-Ile，Val，D-Val，Leu，D-Leu，Met，D-Met
Leu D-Leu，Val，D-Val，Met，D-Met，Ile，D-Ile
Lys D-Lys，Arg，D-Arg，Orn，D-Orn
Met D-Met，S-Me-Cys，Ile，D-Ile，Leu，D-Leu，Val，D-Val
Phe D-Phe，Tyr，D-Tyr，His，D-His，Trp，D-Trp
Pro D-Pro
Ser D-Ser,Thr,D-Thr,allo-Thr,L-Cys,D-Cys
Thr D-Thr，Ser，D-Ser，allo-Thr，Met，D-Met，Val，D-Val
Tyr D-Tyr，Phe，D-Phe，His，D-His，Tip，D-Trp
Val D-Val，Leu，D-Leu，Ile，D-Ile，Met，D-Met
保守取代可涉及这些种类之一的成员与相同种类的另一成员的交换。非保守取代可涉及这些种类之一的成员与另一种类的成员的交换。
本文中，“同一性”或“同源性”，通常是指两个肽或蛋白质之间或两个核酸分子之间的序列相似性。“同一性”或“同源性”百分比，指所比较的分子中的氨基酸或核苷酸之间相同残基的百分比，并且基于正在比较的分子中最小分子的大小来计算。
本发明中的Fc变体与天然的成熟人GDF15或其变体所形成的融合蛋白， 均可以达到本发明的目的。本文中，“载体”指能够增殖与其连接的另一种核酸的核酸分子，包括作为自身复制型核酸结构的载体及整合入接受其导入的宿主细胞的基因组中的载体。某些载体能够指导与其可操作连接的核酸的表达，此类载体在本文中称为“表达载体”。
本文中，“宿主细胞”指已引入外源核酸的细胞，包括这类细胞的后代。宿主细胞包括初始转化的细胞和自其衍生的后代(不考虑传代数)。后代在核酸内含物上可能与亲本细胞不完全相同，但可以含有突变。本文中包括具有如原始转化细胞中筛选或选择的相同的功能或生物学活性的突变体后代。宿主细胞是能用于生成本发明的融合蛋白的任意类型的细胞系统。宿主细胞包括哺乳动物培养细胞如CHO细胞、BHK细胞、NS0细胞、SP2/0细胞、YO骨髓瘤细胞、P3X63小鼠骨髓瘤细胞、PER细胞、PER.C6细胞或杂交瘤细胞、酵母细胞、细菌细胞如大肠杆菌，昆虫细胞和植物细胞等，而且还包括在转基因动物、转基因植物或培养的植物或动物组织中包含的细胞。
本文中，“药物组合物”指其形式使得容许其中含有的活性成分的生物学活性有效，且不含对会接受药物组合物施用的受试者有不可接受的毒性的别的成分的制剂。
本文中，“治疗有效量”指有效实现期望的治疗或预防结果的量，期望的治疗或预防结果包括例如消除、降低、延迟、最小化或预防疾病的不良作用。
本文中，“药学上可接受的载体”指药物组合物中活性成分以外的对受试者无毒的成分。药学上可接受的载体包括但不限于缓冲剂、赋形剂、稳定剂或防腐剂。
本文中，“治疗”指试图改变治疗个体中疾病的自然进程，并且可以是为了预防或在临床病理学的过程期间实施的临床干预。治疗的期望效果包括但不限于预防疾病的发生或复发、缓解症状、降低疾病的任何直接或间接病理学后果、减缓疾病进展速率、改善或消除疾病状态、及消退或改善的预后。
本文中，“个体”或“受试者”是哺乳动物。哺乳动物包括但不限于灵长类(例如人和非人灵长类如猴)或其他哺乳动物(例如牛、羊、猫、犬、马、兔和啮齿动物如小鼠和大鼠)。特别地，所述个体或受试者是人。
在本文中，使用天然氨基酸的常规单字母或三字母代码：
Figure PCTCN2022114143-appb-000009
实施例1 Fc变体设计
本实施例测试了引入不同突变对Fc结构性质的影响，部分突变体的测试结果如表4所示。试验步骤包括：
采用分子克隆方法将Fc核酸分子序列插入到哺乳动物细胞表达载体中，采用ExpiCHO Fectamine TM CHO Transfection Kit(ThermoFisher Scientific)，在CHO-S细胞中进行瞬时转染表达；经过Protein A层析柱纯化后，获得目标蛋白，采用尺寸排阻高效液相色谱(SEC)对目标蛋白进行分析。
SEC分析的主要原理是基于待分析物(如蛋白质)的分子量大小和三维结构，一般分子量大的分析物会更加快速地通过色谱柱，因此具有较短的色谱柱保留时间，反之，分子量更小的分析物则具有更长的保留时间，因此SEC可用来分析蛋白分子的聚体情况。
天然免疫球蛋白Fc片段会自然形成同源二聚体结构，而为了提高融合蛋白稳定性，需要对Fc进行突变，但突变后可能会破坏Fc同源二聚体的结构。而发明人惊奇地发现，在第439位引入突变后的Fc分子(K439D或K439E)与未引入突变的Fc分子在SEC分析中保留时间一致，表明该引入的突变不会破坏Fc二聚体形成。作为对照，本实施例还测试了氨基酸序列为SEQ ID NO：24的Fc分子(包含F405Q/Y407E突变)，根据此前专利文献WO2019195091报道，F405Q/Y407E突变可破坏Fc二聚体而形成单体Fc分子，本实验中发现该突变具有明显延长的保留时间，表明该Fc分子确实以单体形式存在。
表4 Fc变体二聚体形成特性
序列 Fc变体 保留时间 聚体状态
SEQ ID NO：23 IgG4 Fc(AA) 13.341min 二聚体
SEQ ID NO：3 IgG4 Fc(AA,K439E) 13.156min 二聚体
SEQ ID NO：7 IgG4 Fc(AA,K439D) 13.093min 二聚体
SEQ ID NO：24 IgG4 Fc(AA,F405Q/Y407E) 15.076min 单体
实施例2 Fc-GDF15构建体的制备
构建表达载体：将表3所示的Fc-GDF15分子分别插入至哺乳动物细胞载体pXC17.4的多克隆酶切位点，构建得到分别表达表3所示的Fc-GDF15的表达载体；采用去内毒素的质粒提取试剂盒(OMEGA)提取Fc-GDF15的表达载体，用于细胞转染。
细胞转染与培养：将CHO-S细胞复苏后传代培养，至密度约为6×10 6细胞/mL时收集细胞，进行细胞转染。细胞转染采用ExpiCHO Fectamine TM CHO Transfection Kit(ThermoFisher Scientific)，其中Fc-GDF15的表达载体的终浓度为1μg/ml。转染约20h后加入ExpiCHO Fectamine CHO Enhancer以及ExpiCHO Feed维持转染细胞的生长。待细胞活率降至约80％时收获细胞培养液。
蛋白纯化：将细胞培养液离心后收集上层清液，并用0.22μm滤膜过滤以获得细胞培养液上清。采用两步层析法对所述细胞培养液上清进行纯化：第一层析：将细胞培养液上清应用至经磷酸缓冲盐溶液(PBS)平衡的AT Protein A Diamond层析柱(博格隆公司)，待目标蛋白结合后，用平衡液冲洗3CV(柱体积，column volume)以上，以除去未结合杂质，用100mM乙酸-乙酸钠(pH 3.0)缓冲液洗脱并收集目标蛋白，然后迅速用1.0M Tris-HCl pH 8.0溶液调节洗脱收集样品pH至7.2-7.6，对已调节pH样品用纯化水进行稀释至其电导值低于5ms/cm，得到第一样品；第二步层析：使用Bestarose Q HP层析柱对经第一层层析获得的第一样品进一步精纯，用20mM Tris-HCl缓冲液平衡层析柱，将第一样品上样，20mM Tris-HCl缓冲液冲洗3CV，20mM PB缓冲液冲洗3CV以上以置换缓冲体系，最终用PBS洗脱并收集洗脱组分，获得目标蛋白样品。目标蛋白样品的性质和纯度通过十二烷基硫酸钠聚丙烯酰胺凝胶电泳 (SDS-PAGE)和质谱分析进行评价。目标蛋白样品通过微量核酸蛋白测定仪(NanoDrop 2000/2000c Spectrophotometer)进行定量。
实施例3 Fc变体对Fc-GDF15融合蛋白表达量影响
采用实施例2中描述的方法表达得到了表3所记载的Fc-GDF15融合蛋白，比较应用不同Fc变体对Fc-GDF15融合蛋白表达产量的影响。测试的结果在表5中示出。
根据测试结果，发现未对Fc的K439(EU编号)或E356(EU编号)进行突变的Fc-GDF15融合蛋白，如M1(单体具有SEQ ID NO：119序列的同源二聚体)、M14(单体具有SEQ ID NO：79序列的同源二聚体)表达量较低，而对Fc K439和/或E356进行突变的Fc-GDF15融合蛋白，如M2(单体具有SEQ ID NO：67序列的同源二聚体)、M3(单体具有SEQ ID NO：68序列的同源二聚体)、M4(单体具有SEQ ID NO：69序列的同源二聚体)、M5(单体具有SEQ ID NO：70序列的同源二聚体)、M6(单体具有SEQ ID NO：71序列的同源二聚体)、M7(单体具有SEQ ID NO：72序列的同源二聚体)、M8(单体具有SEQ ID NO：73序列的同源二聚体)、M9(单体具有SEQ ID NO：74序列的同源二聚体)、M10(单体具有SEQ ID NO：75序列的同源二聚体)、M11(单体具有SEQ ID NO：76序列的同源二聚体)、M15(单体具有SEQ ID NO：80序列的同源二聚体)、M39(单体具有SEQ ID NO：105序列的同源二聚体)、M50(单体具有SEQ ID NO：116序列的同源二聚体)，具有提高的融合蛋白表达产量，表明在Fc第356位(EU编号)或第439位(EU编号)氨基酸进行的突变更有利于Fc-GDF15融合蛋白的组装表达。
表5 包含不同Fc变体的Fc-GDF15融合蛋白表达产量
Figure PCTCN2022114143-appb-000010
Figure PCTCN2022114143-appb-000011
实施例4 Fc-GDF15融合蛋白的理化性质
蛋白分子的理化性质比如聚集和降解倾向会影响分子的成药性，因此理想的药物分子需要具有稳定单一的组分，即有更少的聚集和降解发生。
为了测试Fc-GDF15融合蛋白的理化性质，采用实施例2中描述的方法表达纯化出Fc-GDF15融合蛋白，然后利用尺寸排阻色谱高效液相色谱(SEC)对融合蛋白进行分析。具体地，SEC分析采用Waters Xbridge BEH 200A，3.5μm(7.8×300mm)色谱柱，流动相为(150mM磷酸盐缓冲液：乙腈＝9:1，pH 6.5)，流速为0.5ml/min。检测结果中，高分子量(HMW)组分占比代表聚集的比例，低分子量(LMW)代表降解的比例。检测结果如表6所示。
表6 不同Fc-GDF15融合蛋白理化性质比较
Figure PCTCN2022114143-appb-000012
Figure PCTCN2022114143-appb-000013
Figure PCTCN2022114143-appb-000014
表6结果表明，未对Fc的K439(EU编号)或E356(EU编号)进行突变的Fc-GDF15融合蛋白，如M1和M14分别具有50.2％和67.6％的高分子(HMW)聚集；而对Fc K439(EU编号)和/或E356(EU编号)进行突变的Fc-GDF15融合蛋白，均具有显著降低的高分子聚集比例，并且绝大部分融合蛋白具有低于5％的高分子聚集，说明Fc K439(EU编号)或E356(EU编号)的突变能带来更好的Fc-GDF15融合蛋白成药性。
实施例5 Fc-GDF15融合蛋白的体外活性
已有研究表明，GDF15在体内发挥功能需要通过其特异性受体GFRAL和辅助受体RET进行信号转导。GDF15与细胞表面受体结合后可激活下游信号通路，其中一条通路为ERK1/2磷酸化。因此可通过检测细胞内ERK1/2磷酸化水平来评价GDF15的体外活性。
将人GFRAL(UniProtKB-Q6UXV0)和人RET(UniProtKB-P07949)的表达基因通过IRES元件连接并置于CMV启动子下游后转染HEK293T细胞(ATCC)，加入嘌呤霉素(Gibco)筛选得到稳定表达两个受体的阳性细胞，简称为受体表达细胞，将其用于磷酸化水平检测试验。细胞内ERK1/2磷酸化水平采用Advanced phospho-ERK1/2(Thr202/Tyr204)试剂盒(cisbio)基于HTRF(Homogeneous Time-Resolved Fluorescence，均相时间分辨荧光)技术进行检测，按照试剂盒手册进行操作。
具体地，将受体表达细胞按16000细胞/孔的密度铺384孔板，饥饿培养后用待测Fc-GDF15融合蛋白或天然GDF15(ACROBiosystems,GD5-H5149)刺激细胞，细胞裂解后加入试剂盒中经标记的抗体，室温孵育过夜后用多功能酶标仪(SpectraMax i3X，Molecular devices)以337nm为激发波长，检测665nm、620nm荧光值。响应值通过在665nm及620nm处的荧光值的比值Ratio计算得到，公式如下：
Figure PCTCN2022114143-appb-000015
其中：A665为检测在665nm波长下的荧光值；A620为检测在620nm波长 下的荧光值。响应值强度即对应细胞内ERK1/2磷酸化强度。
采用GraphPad Prism通过四参数拟合剂量-响应曲线，测定Fc-GDF15融合蛋白体外活性的EC 50值，然后和天然成熟GDF15的活性EC 50值比较，计算得到相对活性比值(天然GDF15 EC 50：Fc-GDF15构建体EC 50)，结果如表7所示。
表7 Fc-GDF15融合蛋白的体外活性
Figure PCTCN2022114143-appb-000016
Figure PCTCN2022114143-appb-000017
结果表明，除M35具有显著降低的体外活性之外，其余Fc-GDF15融合蛋白均具有与天然GDF15相当或优于天然GDF15的体外活性。
实施例6 Fc-GDF15融合蛋白的热稳定性(DSC)
本实施例采用差示扫描热分析法(DSC)测试了不同Fc-GDF15融合蛋白的热稳定性。DSC图谱上的吸热转变曲线代表了蛋白分子变性解折叠过程，可以通过热转变中点(Tm)值来表征蛋白热稳定性。具体地，样品以120℃/小时的加热速率从30℃加热到110℃，测定不同Fc-GDF15融合蛋白的Tm值，结果如表8所示。实验结果表明，Fc-GDF15融合蛋白具有比较好的热稳定性，适合进行药物开发。
表8 Fc-GDF15融合蛋白的Tm值
构建体 Tm(℃)
M3 63.94
M4 63.93
M5 62.74
M6 65.69
M15 65.23
M39 65.71
实施例7 单次施与Fc-GDF15融合蛋白在正常小鼠体内的药效
GDF15能够抑制食物摄入，降低体重，具有治疗肥胖等相关代谢疾病的潜力。但天然GDF15分子在人体内只有约3小时的半衰期，不适合直接应用于药物治疗，本发明提供的Fc-GDF15融合蛋白可延长药物分子半衰期。本实施例测试了单次施与不同Fc-GDF15融合蛋白对正常小鼠食物摄入量和体重的影响，以评估不同Fc-GDF15融合蛋白的体内活性与药效维持时间。
具体地，在第0天对8-9周龄C57BL/6(湖南斯莱克景达公司)小鼠称重并单次皮下给予待测试Fc-GDF15融合蛋白或溶媒(PBS)，并在给药后连续监测动物摄食量和体重的变化。其中体重变化百分比计算方式为：体重变化(％)＝(实测体重-初始体重)/初始体重×100％
正常小鼠单次给药治疗14天的摄食量和体重结果显示于表9和图2、图3中。
表9 单次给药治疗对正常小鼠体重和食物摄入量影响(监测14天)
Figure PCTCN2022114143-appb-000018
Figure PCTCN2022114143-appb-000019
注：数值代表每组7只动物数据的平均值±SEM；***-p<0.001，相对于溶媒；#-p<0.05，相对于MonoFc-GDF15；##-p<0.01，相对于MonoFc-GDF15，统计分析方法：T检验。
结果表明，正常小鼠以3nmol/kg剂量单次施用Fc-GDF15融合蛋白，在第8天和第14天相对于溶媒能抑制食物摄入并显著降低体重。其中，包含单体Fc的GDF15融合蛋白monoFc-GDF15(专利WO2019195091中公开的“Compound 2”，单体具有SEQ ID NO：104序列；Fc具有SEQ ID NO：18序列，其中F405Q，Y407E)在第8天左右药效达到最大值；对Fc K439进行突变的Fc-GDF15融合蛋白，如M3(单体具有SEQ ID NO：68序列的同源二聚体)和M15(单体具有SEQ ID NO：80序列的同源二聚体)；以及进一步包含GDF15不同氨基酸位点突变的Fc-GDF15融合蛋白，如M20(单体具有SEQ ID NO：85序列的同源二聚体)、M23(单体具有SEQ ID NO：88序列的同源二聚体)、M24(单体具有SEQ ID NO：89序列的同源二聚体)和M35(单体具有SEQ ID NO：100序列的同源二聚体)在给药第14天时小鼠体重仍有下降趋势，并且体重降低显著大于monoFc-GDF15融合蛋白的效果。
正常小鼠单次给药治疗30天的摄食量和体重结果显示于表10和图4、图5中。
表10 单次给药治疗对正常小鼠体重和食物摄入量影响(监测30天)
Figure PCTCN2022114143-appb-000020
Figure PCTCN2022114143-appb-000021
注：数值代表每组7-8只动物数据的平均值±SEM；**-p<0.01，***-p<0.001，相对于溶媒；##-p<0.01，###-p<0.001，相对于MonoFc-GDF15，统计分析方法：T检验。
结果表明，对正常小鼠以1nmol/kg剂量单次施用Fc-GDF15融合蛋白，能够显著降低小鼠体重。其中，包含单体Fc的GDF15融合蛋白MonoFc-GDF15降体重效果较弱，并且在14天时小鼠已经恢复到初始体重。而包含Fc K439位不同氨基酸突变的Fc-GDF15构建体，如M3(单体具有SEQ ID NO：68序列的同源二聚体)、M6(单体具有SEQ ID NO：71序列的同源二聚体)、M7(单体具有SEQ ID NO：72序列的同源二聚体)、M16(单体具有SEQ ID NO：81序列的同源二聚体)和M51(单体具有SEQ ID NO：117序列的同源二聚体)，具有更加显著的降体重效果，并且能够在第21-28天内持续降低小鼠体重，具有显著延长的药效维持时间。
正常小鼠单次给药治疗56天的摄食量和体重结果显示于表11和图6、图7中。
表11 单次给药治疗对正常小鼠体重和食物摄入量影响(监测56天)
Figure PCTCN2022114143-appb-000022
Figure PCTCN2022114143-appb-000023
注：数值代表每组8只动物数据的平均值±SEM；***-p<0.001，相对于溶媒；统计分析方法：T检验。
结果表明，正常小鼠以1nmol/kg剂量单次施用M9(单体具有SEQ ID NO：74序列的同源二聚体)、M18(单体具有SEQ ID NO：83序列的同源二聚体)、M22(单体具有SEQ ID NO：87序列的同源二聚体)或M35(单体具有SEQ ID NO：100序列的同源二聚体)，能抑制小鼠食物摄入并显著降低体重。并且，小鼠体重持续降低直到第21天之后才趋于平稳和回升，在第56天基本恢复至初始体重，表明对Fc K439或E356进行突变的Fc-GDF15融合蛋白，如M9和M18以及进一步包含GDF15不同氨基酸位点突变的融合蛋白如M22和M35均具有超长的药效维持时间，单次给药治疗后药效维持可达1个月左右。同时发明人惊奇地发现，虽然构建体M35具有显著降低的体外活性，其仍然保持了与其他构建体类似的体内药效。
实施例8 重复施与Fc-GDF15融合蛋白在饮食诱导肥胖(DIO)小鼠体内药效
本实施例测试了Fc-GDF15融合蛋白重复给药在DIO小鼠中对食欲抑制、降低体重，以及改善各项代谢指标的效果。C57BL/6小鼠经过高脂饮食诱导四个月后，根据体重进行随机分组，随后分别皮下给予溶媒(PBS，两周一次)、不同Fc-GDF15融合蛋白(1nmol/kg，每两周一次)或司美格鲁肽(3nmol/kg，每天一次)，试验持续49天。试验期间持续监测小鼠体重和食物摄入量变化，在初次给药后第45天时对小鼠进行糖耐量测试，并且在第49天对小鼠进行解剖，分析了脂肪含量以及各项血清生化指标。
小鼠体重和食物摄入量结果显示于表12中，体重变化曲线显示于图8中；糖耐量曲线显示于图9中，糖耐曲线下面积、脂肪指数、血清总胆固醇、甘油三脂、谷丙转氨酶、谷草转氨酶和低密度脂蛋白参数显示于表13中。
表12 Fc-GDF15构建体重复给药对DIO小鼠体重和食物摄入量的影响(第42天)
Figure PCTCN2022114143-appb-000024
注：数值代表每组9只动物数据的平均值±SEM。
表13 Fc-GDF15构建体重复给药对DIO小鼠糖耐量、脂肪指数、血清总胆固醇(TC)、甘油三脂(TG)、谷丙转氨酶(ALT)、谷草转氨酶(AST)和低密度脂蛋白(LDL)的影响
Figure PCTCN2022114143-appb-000025
Figure PCTCN2022114143-appb-000026
注：数值代表每组7-9只动物数据的平均值±SEM；*-p<0.05，**-p<0.01，***-p<0.001，ns-无显著性差异，相对于溶媒；统计分析方法：one-way ANOVA,followed by Dunnett’s。
结果表明，以1nmol/kg剂量每两周一次给予Fc-GDF15融合蛋白M4(单体具有SEQ ID NO：69序列的同源二聚体)、M5(单体具有SEQ ID NO：70序列的同源二聚体)或M9(单体具有SEQ ID NO：74序列的同源二聚体)、治疗DIO小鼠能够抑制食物摄入并且显著降低小鼠体重，体重减轻效果与每天给予3nmol/kg司美格鲁肽效果相当，均能使小鼠体重达到正常水平；M4和M5还能显著改善DIO小鼠糖耐量水平；M4、M5和M9能显著降低DIO小鼠的脂肪含量，同时还能改善血脂如显著降低总胆固醇、甘油三脂和低密度脂蛋白；M4、M5和M9也能改善肝功能如显著降低ALT和AST水平。表明本发明的Fc-GDF15构建体可以应用于肥胖、糖尿病、高血脂或脂肪肝/脂肪肝炎的疾病的治疗。
在另一次实验中测试了不同剂量的构建体M39(单体具有SEQ ID NO：105序列的同源二聚体)重复给药在DIO小鼠中对食欲抑制、降低体重，以及改善各项代谢指标的效果。DIO小鼠随机分组(每组10-12只小鼠)，分别皮下给予溶媒(PBS，每周一次)、不同剂量的M39(0.03nmol/kg,0.1nmol/kg和1nmol/kg，每周一次；3nmol/kg，每两周一次)或司美格鲁肽(3nmol/kg，每天一次)。试验期间持续监测小鼠体重和食物摄入量变化，在初次给药后第46天时对小鼠进行空腹血糖及糖耐量测定，并且在第49天对小鼠进行解剖，分析了脂肪含量、各项血清生化指标。
小鼠在治疗末期体重变化显示于表14中，体重变化曲线、治疗46天后空腹血糖水平和IPGTT及曲线下面积、脂肪指数、转氨酶水平分别显示于图10 到15中。
表14 不同剂量M39重复给药对DIO小鼠降体重效果(第48天)
组别 体重变化(相对于基线％)
溶媒 1.2±1.1
司美格鲁肽QD(3nmol/kg) -24.7±2.0 ***
M39 QW(0.03nmol/kg) -5.4±1.6
M39 QW(0.1nmol/kg) -19.4±2.2 ***
M39 QW(1nmol/kg) -33.6±1.8 ***#
M39 Q2W(3nmol/kg) -30.1±3.2 ***
注：数值代表每组10-12只动物数据的平均值±SEM；***-p<0.001相对于溶媒，#-p<0.05相对于司美格鲁肽；统计分析方法：one-way ANOVA,followed by Dunnett’s。
以上结果表明，每周一次或每两周一次皮下注射M39能够剂量依赖性降低DIO小鼠体重，并且在每周一次1nmol/kg剂量下减重效果显著强于每天一次注射3nmol/kg司美格鲁肽。同时M39还能显著降低小鼠脂肪含量，改善小鼠空腹血糖水平、糖耐量，以及肝功能指标。
实施例9 Fc-GDF15融合蛋白在ob/ob肥胖小鼠模型体内药效
本实施例测试了构建体M6和M39在ob/ob肥胖小鼠模型中对食欲抑制、降低体重，以及改善各项代谢指标的效果。6-7周龄ob/ob小鼠(江苏集萃药康生物科技有限公司)经过适应性饲养后根据体重进行随机分组，分别皮下给予溶媒(PBS)、不同剂量的M6或M39(0.1nmol/kg，第0天和第24天各给药一次；1nmol/kg、10nmol/kg，第0天和第36天各给药一次)或司美格鲁肽(3nmol/kg，每天一次)。试验期间持续监测小鼠体重和食物摄入量变化，在第52天对小鼠进行解剖，分析了各项血清生化指标和肝病理情况。
小鼠在治疗末期体重变化显示于表15中，体重变化曲线、累计摄食量、肝体比、转氨酶水平以及肝病理结果分别显示于图16到21中。
表15 不同剂量M6和M39改善ob/ob小鼠体重效果(第52天)
组别 体重变化(相对于基线％)
溶媒 47.9±4.5
司美格鲁肽(3nmol/kg) 30.6±4.5 ***
M6(0.1nmol/kg) 25.9±1.7 ***
M6(1nmol/kg) 0.1±2.0 ***###
M6(10nmol/kg) -1.9±2.2 ***###
M39(0.1nmol/kg) 27.2±2.8 ***
M39(1nmol/kg) 2.7±2.1 ***###
M39(10nmol/kg) -0.7±1.7 ***###
注：数值代表每组8只动物数据的平均值±SEM；***-p<0.001相对于溶媒，###-p<0.001相对于司美格鲁肽；统计分析方法：one-way ANOVA,followed by Dunnett’s。
以上结果表明，本发明设计的Fc-GDF15构建体具有超长的药效维持时间，M6和M39低剂量下在第0天和第24天、以及中高剂量下在第0天和第36天分别给药一次，能够显著降低ob/ob小鼠摄食量，对体重的改善显著强于每天给药的司美格鲁肽，同时还能显著改善ob/ob小鼠的肝功能以及肝脂肪变性，效果也显著好于司美格鲁肽。
实施例10 Fc-GDF15融合蛋白在小鼠体内药代动力学
本实施例测试了构建体M38(单体具有SEQ ID NO：103序列的同源二聚体)、M3(单体具有SEQ ID NO：68序列的同源二聚体)、M15(单体具有SEQ ID NO：80序列的同源二聚体)在小鼠体内的药代动力学表现。将上述融合蛋白用10mM的PBS稀释至0.05mg/ml，然后以0.25mg/kg的剂量分别皮下注射C57BL/6小鼠，每种融合蛋白注射3只小鼠。每只动物给药前、给药后2h、7h、24h、48h、96h、168h、356h、504h、672h分别采血，采用夹心ELISA方法测定血浆Fc-GDF15浓度。具体地，采用鼠抗人GDF15单克隆抗体(义翘神州公司)包板，然后用鼠抗人IgG4 Fc-HRP检测目标蛋白，并进行药代数据计算，结果如表16所示。
表16 Fc-GDF15融合蛋白在小鼠体内药代参数
Figure PCTCN2022114143-appb-000027
Figure PCTCN2022114143-appb-000028
注：AUC INF_obs表示从给药时间开始到理论外推无穷远的时间的曲线下面积；C max表示检测到的最高血浆浓度；T 1/2表示半衰期；T max表示达到最高血浆浓度的时间。
结果表明，包含单体Fc和GDF15的融合蛋白M38在小鼠体内的半衰期为58h；惊奇地发现，具有Fc变体的Fc-GDF15融合蛋白M15和M3在小鼠体内的半衰期则达到了244和306h，远长于常规Fc融合蛋白在小鼠体内的半衰期。
实施例11 Fc-GDF15融合蛋白在食蟹猴体内药代动力学
本实施例测试了构建体M39在食蟹猴体内的药代动力学表现。对食蟹猴分别以1mg/kg和2mg/kg剂量皮下注射给予M39，每个剂量雌雄各三只动物。每只动物给药前、给药后0.5h、2h、6h、48h、168h、356h、504h、672h分别采血，采用夹心ELISA方法测定血浆Fc-GDF15浓度。具体地，采用鼠抗人GDF15单克隆抗体(义翘神州公司)包板，然后用鼠抗人IgG4 Fc-HRP检测目标蛋白，并进行药代数据计算，结果如表17所示。
表17 构建体M39在食蟹猴体内药代参数
Figure PCTCN2022114143-appb-000029
结果表明，发明人惊奇地发现，构建体M39在食蟹猴中也体现出了超长半衰期，达到200-300小时，远长于常规非抗体类Fc融合蛋白在食蟹猴中约100h左右的半衰期。
以上所述，仅为本发明较佳的具体实施方式，但本发明的保护范围并不局限于此，任何熟悉本技术领域的技术人员在本发明揭露的技术范围内，可轻易想到的变化或替换，都应涵盖在本发明的保护范围之内。因此，本发明的保护范围应以所述权利要求的保护范围为准。

Claims (40)

  1. 用于治疗肥胖相关疾病的Fc-GDF15融合蛋白。
  2. 如权利要求1所述的融合蛋白，其特征在于，所述融合蛋白包括GDF15活性结构域和Fc变体；所述Fc变体的C末端直接或者通过肽接头连接至所述GDF15活性结构域的N末端；所述Fc变体依照EU编号在IgG Fc的第356位和/或第439位处具有氨基酸取代。
  3. 如权利要求2所述的融合蛋白，其特征在于，所述Fc变体具有形成同源二聚体的能力。
  4. 如权利要求2-3任一项所述的融合蛋白，其特征在于，所述Fc变体依照EU编号将IgG Fc的第356位的氨基酸取代为：除天冬氨酸(D)、谷氨酸(E)和半胱氨酸(C)以外的氨基酸；和/或所述Fc变体依照EU编号将IgG Fc的第439位的氨基酸取代为：除精氨酸(R)、组氨酸(H)、赖氨酸(K)和半胱氨酸(C)以外的氨基酸；
    优选地，所述Fc变体依照EU编号将IgG Fc的第356位的氨基酸取代为：甘氨酸(G)、丝氨酸(S)、丙氨酸(A)、苏氨酸(T)、缬氨酸(V)、天冬酰胺(N)、亮氨酸(L)、异亮氨酸(I)、谷氨酰胺(Q)、酪氨酸(Y)、苯丙氨酸(F)、组氨酸(H)、脯氨酸(P)、甲硫氨酸(M)、赖氨酸(K)、精氨酸(R)中的一种，和/或所述Fc变体依照EU编号将IgG Fc的第439位的氨基酸取代为：甘氨酸(G)、丝氨酸(S)、丙氨酸(A)、苏氨酸(T)、缬氨酸(V)、天冬氨酸(D)、天冬酰胺(N)、亮氨酸(L)、异亮氨酸(I)、谷氨酸(E)、谷氨酰胺(Q)、酪氨酸(Y)、苯丙氨酸(F)、脯氨酸(P)、甲硫氨酸(M)中的一种；
    更优选地，所述Fc变体含有以下突变：E356R、E356Q、E356A、E356N中的一种，和/或K439D、K439E、K439Q、K439A、K439N中的一种。
  5. 如权利要求2-3任一项所述的融合蛋白，其特征在于，所述Fc变体含有以下突变中的一种：K439D、K439E、K439Q、K439A、K439N；
    优选地，所述Fc变体含有以下突变中的一种：K439D、K439E、K439Q。
  6. 如权利要求2-3任一项所述的融合蛋白，其特征在于，所述Fc变体含有以下突变中的一种：E356R、E356Q、E356A、E356N；
    优选地，所述Fc变体含有E356R突变。
  7. 如权利要求1-6任一项所述的融合蛋白，其特征在于，所述Fc变体还含有以下突变：依照EU编号将IgG Fc的第234位和第235位的氨基酸取代为丙氨酸(AA)，和/或第447位的氨基酸删除。
  8. 如权利要求1-7任一项所述的融合蛋白，其特征在于，所述Fc变体包含下组之一的氨基酸序列，或者与下组之一具有至少85％、90％、95％或99％序列同一性的氨基酸序列：SEQ ID NO：1-22，SEQ ID NO：27，SEQ ID NO：29-45。
  9. 如权利要求2-3任一项所述的融合蛋白，其特征在于，所述GDF15活性结构域为全长成熟GDF15蛋白、N端截短的GDF15蛋白、或保留GDF15生物学活性的任意变体。
  10. 如权利要求9所述的融合蛋白，其特征在于，所述GDF15活性结构域包含选自下组之一的氨基酸序列或与下组之一的氨基酸序列具有至少85％、90％、95％或99％序列同一性的氨基酸序列：
    SEQ ID NO：46；
    在SEQ ID NO：46的N末端有1-14个氨基酸截短，和/或在SEQ ID NO：46中含有1-3个氨基酸取代。
  11. 如权利要求10所述的融合蛋白，其特征在于，所述GDF15活性结构域包含选自下组之一的氨基酸序列：
    SEQ ID NO：46；
    在SEQ ID NO：46的N末端有1-14个氨基酸截短，和/或在SEQ ID NO：46中含有1-3个氨基酸取代。
  12. 如权利要求10-11任一项所述的融合蛋白，其特征在于，所述SEQ ID NO：46中的氨基酸取代的位置选自下组中的一个、两个、或三个：第5位、第6位、第21位、第26位、第30位、第47位、第54位、第55位、第57位、第67位、第69位、第81位、第94位、第107位；
    优选地，所述SEQ ID NO：46中的氨基酸取代选自下组中的一个、不同位置的任两个、或者不同位置的任三个：D5E、H6D、H6E、R21Q、R21H、D26E、A30S、A47D、A54S、A55E、M57T、R67Q、K69R、A81S、T94E、K107Q。
  13. 如权利要求10-11任一项所述的融合蛋白，其特征在于，所述SEQ ID  NO：46的N末端有3个、4个或14个氨基酸截短。
  14. 如权利要求9-13任一项所述的融合蛋白，其特征在于，所述GDF15活性结构域包含下组之一的氨基酸序列，或者与下组之一具有至少85％、90％、95％或99％序列同一性的氨基酸序列：SEQ ID NO：46-66。
  15. 如权利要求14所述的融合蛋白，其特征在于，所述GDF15活性结构域包含下组之一的氨基酸序列，或者与下组之一具有至少95％同一性的氨基酸序列：SEQ ID NO：46-66；
    优选地，所述GDF15活性结构域包含下组之一的氨基酸序列：SEQ ID NO：46-66。
  16. 如权利要求1-15任一项所述的融合蛋白，其特征在于，所述融合蛋白包含下组之一的氨基酸序列，或者与下组之一具有至少85％、90％、95％或99％序列同一性的氨基酸序列：SEQ ID NO：67-78，SEQ ID NO：80-102，SEQ ID NO：105-118。
  17. 如权利要求16所述的融合蛋白，其特征在于，所述融合蛋白包含下组之一的氨基酸序列，或者与下组之一具有至少95％序列同一性的氨基酸序列：SEQ ID NO：67-78，SEQ ID NO：80-102，SEQ ID NO：105-118；
    优选地，所述融合蛋白包含下组之一的氨基酸序列：SEQ ID NO：67-78，SEQ ID NO：80-102，SEQ ID NO：105-118。
  18. 用于治疗肥胖相关的疾病的同源二聚体融合蛋白，其由权利要求1-17任一项所述的融合蛋白所构成。
  19. 用于治疗肥胖相关的疾病的生物材料，其特征在于，所述生物材料为以下(i)、(ii)、(iii)中的任一种：
    (i)核酸，所述核酸包括编码权利要求1-16任一项所述的融合蛋白的核苷酸序列；
    (ii)载体，所述载体包含(i)中所述的核酸；
    (iii)宿主细胞，所述宿主细胞含有(i)中所述的核酸和/或(ii)中所述的载体。
  20. 用于治疗肥胖相关的疾病的药物组合物，其特征在于，所述药物组合物包括权利要求17所述的同源二聚体融合蛋白作为活性成分，所述同源二聚体融 合蛋白以治疗有效量存在于所述药物组合物中；
    优选地，所述药物组合物还包括药学上可接受的载体。
  21. 一种治疗与肥胖相关的疾病的方法，其特征在于，所述的方法包括给予受试者治疗有效量的Fc-GDF15融合蛋白。
  22. 如权利要求21所述的方法，其特征在于，所述融合蛋白包括GDF15活性结构域和Fc变体；所述Fc变体的C末端直接或者通过肽接头连接至所述GDF15活性结构域的N末端；所述Fc变体依照EU编号在IgG Fc的第356位和/或第439位处具有氨基酸取代。
  23. 如权利要求22所述的方法，其特征在于，所述Fc变体具有形成同源二聚体的能力。
  24. 如权利要求22所述的方法，其特征在于，所述Fc变体依照EU编号将IgG Fc的第356位的氨基酸取代为：除天冬氨酸(D)、谷氨酸(E)和半胱氨酸(C)以外的氨基酸；和/或所述Fc变体依照EU编号将IgG Fc的第439位的氨基酸取代为：除精氨酸(R)、组氨酸(H)、赖氨酸(K)和半胱氨酸(C)以外的氨基酸；
    优选地，所述Fc变体依照EU编号将IgG Fc的第356位的氨基酸取代为：甘氨酸(G)、丝氨酸(S)、丙氨酸(A)、苏氨酸(T)、缬氨酸(V)、天冬酰胺(N)、亮氨酸(L)、异亮氨酸(I)、谷氨酰胺(Q)、酪氨酸(Y)、苯丙氨酸(F)、组氨酸(H)、脯氨酸(P)、甲硫氨酸(M)、赖氨酸(K)、精氨酸(R)中的一种，和/或所述Fc变体依照EU编号将IgG Fc的第439位的氨基酸取代为：甘氨酸(G)、丝氨酸(S)、丙氨酸(A)、苏氨酸(T)、缬氨酸(V)、天冬氨酸(D)、天冬酰胺(N)、亮氨酸(L)、异亮氨酸(I)、谷氨酸(E)、谷氨酰胺(Q)、酪氨酸(Y)、苯丙氨酸(F)、脯氨酸(P)、甲硫氨酸(M)中的一种；
    更优选地，所述Fc变体含有以下突变：E356R、E356Q、E356A、E356N中的一种，和/或K439D、K439E、K439Q、K439A、K439N中的一种。
  25. 如权利要求24所述的方法，其特征在于，所述Fc变体含有以下突变中的一种：K439D、K439E、K439Q、K439A、K439N；
    优选地，所述Fc变体含有以下突变中的一种：K439D、K439E、K439Q。
  26. 如权利要求24所述的方法，其特征在于，所述Fc变体含有以下突变中的一种：E356R、E356Q、E356A、E356N；
    优选地，所述Fc变体含有E356R突变。
  27. 如权利要求21-26任一项所述的方法，其特征在于，所述Fc变体还含有以下突变：依照EU编号将IgG Fc的第234位和第235位的氨基酸取代为丙氨酸(AA)，和/或第447位的氨基酸删除。
  28. 如权利要求27所述的方法，其特征在于，所述Fc变体包含下组之一的氨基酸序列，或者与下组之一具有至少85％、90％、95％或99％序列同一性的氨基酸序列：SEQ ID NO：1-22，SEQ ID NO：27，SEQ ID NO：29-45。
  29. 如权利要求22所述的方法，其特征在于，所述GDF15活性结构域为全长成熟GDF15蛋白、N端截短的GDF15蛋白、或保留GDF15生物学活性的任意变体。
  30. 如权利要求29所述的方法，其特征在于，所述GDF15活性结构域包含选自下组之一的氨基酸序列或与下组之一的氨基酸序列具有至少85％、90％、95％或99％序列同一性的氨基酸序列：
    SEQ ID NO：46；
    在SEQ ID NO：46的N末端有1-14个氨基酸截短，和/或在SEQ ID NO：46中含有1-3个氨基酸取代。
  31. 如权利要求30所述的方法，其特征在于，所述GDF15活性结构域包含选自下组之一的氨基酸序列：
    SEQ ID NO：46；
    在SEQ ID NO：46的N末端有1-14个氨基酸截短，和/或在SEQ ID NO：46中含有1-3个氨基酸取代。
  32. 如权利要求30所述的方法，其特征在于，所述SEQ ID NO：46中的氨基酸取代的位置选自下组中的一个、两个、或三个：第5位、第6位、第21位、第26位、第30位、第47位、第54位、第55位、第57位、第67位、第69位、第81位、第94位、第107位；
    优选地，所述SEQ ID NO：46中的氨基酸取代选自下组中的一个、不同位置的任两个、或者不同位置的任三个：D5E、H6D、H6E、R21Q、R21H、D26E、 A30S、A47D、A54S、A55E、M57T、R67Q、K69R、A81S、T94E、K107Q。
  33. 如权利要求30所述的方法，其特征在于，所述SEQ ID NO：46的N末端有3个、4个或14个氨基酸截短。
  34. 如权利要求30-33任一项所述的方法，其特征在于，所述GDF15活性结构域包含下组之一的氨基酸序列，或者与下组之一具有至少85％、90％、95％或99％序列同一性的氨基酸序列：SEQ ID NO：46-66。
  35. 如权利要求34所述的方法，其特征在于，所述GDF15活性结构域包含下组之一的氨基酸序列，或者与下组之一具有至少95％同一性的氨基酸序列：SEQ ID NO：46-66；
    优选地，所述GDF15活性结构域包含下组之一的氨基酸序列：SEQ ID NO：46-66。
  36. 如权利要求21-35任一项所述的方法，其特征在于，所述融合蛋白包含下组之一的氨基酸序列，或者与下组之一具有至少85％、90％、95％或99％序列同一性的氨基酸序列：SEQ ID NO：67-78，SEQ ID NO：80-102，SEQ ID NO：105-118。
  37. 如权利要求36所述的方法，其特征在于，所述融合蛋白包含下组之一的氨基酸序列，或者与下组之一具有至少95％序列同一性的氨基酸序列：SEQ ID NO：67-78，SEQ ID NO：80-102，SEQ ID NO：105-118；
    优选地，所述融合蛋白包含下组之一的氨基酸序列：SEQ ID NO：67-78，SEQ ID NO：80-102，SEQ ID NO：105-118。
  38. 一种使用同源二聚体融合蛋白治疗与肥胖相关的疾病的方法，其特征在于，所述的同源二聚体融合蛋白由权利要求21-37任一项所述的融合蛋白所构成。
  39. 一种使用生物材料治疗与肥胖相关的疾病的方法，其特征在于，所述生物材料为以下(i)、(ii)、(iii)中的任一种：
    (i)核酸，所述核酸包括编码权利要求1-17任一项所述的融合蛋白的核苷酸序列；
    (ii)载体，所述载体包含(i)中所述的核酸；
    (iii)宿主细胞，所述宿主细胞含有(i)中所述的核酸和/或(ii)中所述 的载体。
  40. 一种使用药物组合物治疗与肥胖相关的疾病的方法，其特征在于，所述药物组合物包括权利要求38所述的同源二聚体融合蛋白作为活性成分，所述同源二聚体融合蛋白以治疗有效量存在于所述药物组合物中；
    所述药物组合物还包括药学上可接受的载体。
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