WO2019101042A1 - 一种治疗代谢疾病的多结构域活性蛋白 - Google Patents

一种治疗代谢疾病的多结构域活性蛋白 Download PDF

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WO2019101042A1
WO2019101042A1 PCT/CN2018/116244 CN2018116244W WO2019101042A1 WO 2019101042 A1 WO2019101042 A1 WO 2019101042A1 CN 2018116244 W CN2018116244 W CN 2018116244W WO 2019101042 A1 WO2019101042 A1 WO 2019101042A1
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active protein
seq
glp
fgf21
multidomain
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PCT/CN2018/116244
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French (fr)
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黄岩山
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浙江道尔生物科技有限公司
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Publication of WO2019101042A1 publication Critical patent/WO2019101042A1/zh

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    • 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
    • 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
    • C07K14/50—Fibroblast growth factor [FGF]
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • 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
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • 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
    • C07K14/575—Hormones
    • C07K14/605—Glucagons
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • 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
    • C07K2319/00—Fusion polypeptide
    • 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 belongs to the field of biopharmaceuticals, and in particular relates to a multidomain active protein for treating metabolic diseases.
  • diabetes can be divided into two types: type 1 diabetes and type 2 diabetes.
  • Type 1 diabetes is mainly characterized by insufficient insulin secretion and the need to inject insulin daily; while type 2 diabetes is caused by the inability of the body to effectively use insulin.
  • type 2 diabetes patients account for the vast majority. It is estimated that approximately 80-90% of patients with type 2 diabetes are significantly obese (Center for disease control and prevention (CDC) National Diabetes Fact Sheet, 2014).
  • the protein drugs for type 2 diabetes are mainly GLP-1R (GLP-1 receptor) agonists, such as Dulaglutide (trade name: ), Albiglutide (trade name) ), liraglutide (Liraglutide, trade name and For the treatment of obesity and diabetes, respectively, Exenatide (trade name) ), lixisenatide (Lixisenatide, trade name ) and somaglutide (Semaglutide) and the like.
  • GLP-1R agonists have a significant hypoglycemic effect, and unlike insulin, the hypoglycemic effect of GLP-1R agonists is strictly blood glucose-dependent, does not easily cause hypoglycemia, and has the effect of reducing body weight.
  • duraglutide has a reduced weight of about 2.9 kilograms
  • Liraglutide (one dose per day, dose 3 mg) approved for weight loss loses about 8 kilograms.
  • the weight loss of these drugs is mainly controlled by appetite, and most of them do not exceed 10% of the average body weight.
  • incretin secretion is proliferated in patients undergoing surgical bariatric surgery (Obesity and Diabetes, New Surgical and Nonsurgical Approaches, Springer, 2015). Therefore, the current generation of diabetes drugs are mainly concentrated in the study of double-effect or multi-effect incretin receptor agonists, such as GLP-1R/GIPR and GLP-1R/GCGR double-acting agonists, even GLP- 1R/GIPR/GCGR agonist.
  • glucagon and GLP-1 are structurally related, but these two hormones play a diametrically opposite role in controlling glucose.
  • GLP-1 and its analogues are mainly used for glycemic control in diabetics, while glucagon is used for acute hypoglycemia.
  • GLP-1 and Glucagon seem to have positive addition or synergy.
  • Physiological effects such as Glucagon receptor (GCGR) and GLP-1 receptor (GLP-1R) dual agonists are more effective at weight loss than GLP-1R single agonists.
  • GCGR agitation may result in an increase in blood glucose levels, this risk can be appropriately offset by GLP-1R activation.
  • double-acting agonists of GLP-1R and GCGR are generally based on Oxyntomodulin or Glucagon, and are engineered to improve their short-acting and enzymatic defects (Oxyntomodulin analog or Glucagon analog).
  • Oxyntomodulin analog or Glucagon analog Most of these analogs mutate the second serine (Ser) to the non-natural amino acid Aib to resist the enzymatic hydrolysis of DPP-IV.
  • Ser serine
  • Glucagon and oxyntomodulin are similar to native GLP-1 and are highly susceptible to hydrolysis by DPP-IV protease in serum (Victor A. Gault et al., A novel GLP-1/glucagon hybrid).
  • FGF21 belongs to a family of polypeptide families that play an important role in a variety of physiological functions, which are widely expressed in development as well as in adult tissues. FGF21 is mainly expressed in pancreatic ⁇ -cell, liver, WAT, and skeletal muscle. It has recently been found to have low expression in thymus, vascular endothelium, kidney, and testicular tissues, and has obvious tissue specificity, and FGF15/19 and FGF23 belong to FGF. Family "endocrine" hormones.
  • FGF21 As an important metabolic regulator, FGF21 has been shown to improve a variety of metabolic abnormalities in pre-clinical models of type 2 diabetes (T2DM). FGF21 is a therapeutic drug for diabetic patients that improves insulin sensitivity, improves glycemic control, reduces body weight, lowers low-density lipoprotein cholesterol (LDL-C) and triglycerides, and increases high-density lipoprotein cholesterol levels (HDL-C). There are potential effects. In diabetic rats and monkeys, human FGF21 is able to reduce fasting serum glucose concentrations and reduce the concentration of fasting serum triglycerides, insulin and glucagon. Furthermore, in a rodent model of diet-induced obesity, administration of FGF21 resulted in a dose-dependent overall weight loss. Therefore, FGF21 has the potential to treat diseases such as diabetes, obesity, dyslipidemia and metabolic syndrome.
  • T2DM type 2 diabetes
  • FGF21 has a very short serum half-life: 30 minutes in mice and 2 hours in monkeys. Therefore, in order to maintain biological activity in vivo, daily injection or continuous infusion of the corresponding FGF21 protein is required.
  • circulating FGF21 levels are often elevated in obese, dyslipidemia, TDM2, and other insulin-related disease-related diseases. Studies have shown that increased FGF21 concentrations are associated with increased CVD risk and can lead to osteoporosis. Influencing reproduction (promoting metabolism and causing insufficient energy), etc. (Wei W, Dutchak PA, Wang X, Ding X, Wang X, Bookout AL, et al.
  • Fibroblast growth factor 21 promotes bone loss by potentiating the effects of peroxisome proliferator-activated receptor Gamma. Proc Natl Acad Sci USA. 2012; 109(8): 3143-8; Fibroblast growth factor 21 has no direct role in regulating fertility in female mice, Mol Metab, 5(8): 690-8, 2016).
  • the homology of the FGF family sequences and the widespread distribution of the FGFR1 receptor also raise concerns about the potential safety issues associated with the clinical use of large doses of FGF21 (Kharitonenkov A&DiMarchi R: Fibroblast growth factor 21 night watch:advances and uncertainties in the field.J Intern Med.2017 Mar;281(3):233-246.).
  • GLP-1 analogues and FGF21 are Fc-fused to form a double-acting active protein (YH25723, a Novel) Long-Acting GLP-1/FGF21 Dual Agonist Provides More Potent and Sustained Glycemic Control and Greater Weight Loss Compared with Single Agonists in Animal Models, The American Diabetes Association, 2016).
  • double-acting or even three-acting peptides currently modified based on Glucagon or oxyntomodulin generally require partial amino acid substitution of non-natural amino acids to improve stability and activity, and even modification by fatty acid or PEG, technically. It is extremely difficult to prepare a single molecule by fusion expression with FGF21 analog. There are currently no reports on the combination of a dual- or multi-effect agonist polypeptide with a FGF21 analog.
  • the multiplex active protein of the invention has significant weight-reducing effects, and can be clinically used for treating diabetes, weight loss, non-alcoholic fatty liver, hyperlipidemia and the like.
  • a multidomain active protein comprising a structure as shown in Formula I, wherein the structure of Formula I is: AL a -FL b -B, wherein A is GCGR/ GLP-1R double-acting agonist peptide, F is a long-acting protein unit, B is a natural FGF21 or FGF21 analog, L a is absent or is a linking chain, L b is absent or is a linking chain.
  • the multidomain active protein has at least GLP-1, GCG and FGF21 triple potency.
  • the A includes a structure as shown in Formula II, and the structure shown in Formula II is:
  • X 10 is selected from any of the V, L, or Y, or I;, E or K according to any of the selected S-X 12; X 13 is selected from any of the Q or of a Y; X 14 is selected from the Any one from L or M; the X 15 is selected from any one of D or E; the X 16 is selected from any one of S, E or G; and the X 17 is selected from any one of R, E or Q ; either the E or a, X 18 is selected from the R a; X 19 is selected from the any one of a or V;, any of the K or R X 20 is selected from a Q; X 21 is selected from the Any of D, L or E;
  • the X 23 is selected from any one of V or I; the X 24 is selected from any one of Q, A or E; the X 27 is selected from any one of M, K or V; and the X 28 is selected from N Or any one of K; said X 29 is selected from any one of G or T; said X 30 is G or a deletion; said X z is absent or selected from GPSSGAPPPS (SEQ ID NO. 3), PSSGAPPPS (SEQ ID NO. 4), any of SSGAPPPS (SEQ ID NO. 5), GPSSGAPPS (SEQ ID NO. 6), PSSGAPPS (SEQ ID NO. 7) or KRNRNN IA (SEQ ID NO. 8).
  • amino acid sequence of A is as shown in any one of SEQ ID NOS. 44 to 92.
  • the B is native FGF21 (SEQ ID NO. 136) or an FGF21 analog.
  • the B includes the following structure:
  • HPIPDSSPLLQFGGQVRQ X 19 YLYTDDAQQTE X 31 HLEI X 36 EDGTVG X 43 A X 45 DQSPESLLQL X 56 ALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRE X 98 LLEDGYNVYQSEAHGLPLH X 118 PGN X 122 SPHRDPAPRGP X 134 RFLPLPGLPPALPEPPGILAPQPPDVGSSDPL X 1 67 MV X 170 X 171 SQ X 174 RSPS X 179 X 180 X 181 , Wherein the N-terminal HPIPDSS may be deleted or partially deleted; X 19 is selected from R, Y, V, E or C; X 31 is selected from A or C; X 36 is selected from R or K; X 43 is selected from G or C; 45 is selected from A, K, E or V; X 56 is selected from K, R, V
  • the FGF21 analog is an active protein having the same or similar biological function as native FGF21 (SEQ ID NO. 136) and having a homology to native FGF21 (SEQ ID NO. 136) of 80% or more.
  • the FGF21 analog has a homology to native FGF21 (SEQ ID NO. 136) of 85% or more; more preferably, the FGF21 analog has 90 homology to native FGF21 (SEQ ID NO. 136). More preferably, the FGF21 analog has a homology to native FGF21 (SEQ ID NO. 136) of 95% or more.
  • the FGF21 analog may be selected from FGF21 similar to that described in patents or patent applications such as US20140213512, US8188040, US9493530, WO 2016114633, US 20150291677, US9422353, US 8541369, US7622445, US7576190, US20070142278, US9006400 or US20130252884. Or mutant.
  • the FGF21 analog is as set forth in SEQ ID NOs. 137-148.
  • the F is a F C moiety derived from a mammalian immunoglobulin.
  • the immunoglobulin is a polypeptide chain molecule containing a disulfide bond, generally having two light chains and two heavy chains.
  • the F C portion of the immunoglobulin used herein has the usual meaning of the terminology in the field of immunology. Specifically, the term refers to an antibody fragment obtained by removing two antigen-binding regions (Fab fragments) from an antibody.
  • the F C portion can include a hinge region and extend through the CH 2 and CH 3 domains to the C-terminus of the antibody.
  • the F C moiety can further comprise one or more glycosylation sites.
  • the human body has five human immunoglobulins with different effect characteristics and pharmacokinetic properties: IgG, IgA, IgM, IgD and IgE.
  • IgG is the highest immunoglobulin in serum.
  • IgG is also the longest serum half-life (about 23 days) in all immunoglobulins.
  • F may be selected from the intact F C portion of an immunoglobulin, a fragment of the F C portion of an immunoglobulin, or a mutant of the F C portion of an immunoglobulin.
  • the immunoglobulin F C moiety for use in the present invention is derived from the F C region of mammalian IgG1, IgG2 or IgG4 or a mutant thereof; preferably, it may be a F C region derived from human IgG1, IgG2 or IgG4 or a mutation thereof More preferably, it can be derived from the F C region of human IgG1 or IgG4 or a mutant thereof.
  • position 297 of the F C domain is replaced by glycine or alanine.
  • the above is in accordance with the EU index number of kabat (kabat, EA, etc., sequences of proteins of immunological interest, fifth edition, public health service, National Institutes of Health, Bethesda, MD (1991)).
  • the F C domain is derived from human IgG4 and is set forth in SEQ ID NO. In a preferred embodiment, the F C domain is derived from human IgG1 as set forth in SEQ ID NO.
  • the K at the end of the F C chain can be removed to facilitate the improvement of the homogeneity of the expression product.
  • amino acid sequence of F can be as set forth in any one of SEQ ID NO. 9-18.
  • the GCGR/GLP-1R double-acting agonist peptide A is fused with natural FGF21 or FGF21 analog B via a long-acting protein unit F to form at least GLP-1, GCG and FGF21
  • An active recombinant fusion protein. May be connected by the connecting link between A and F, such as L a, the connecting link may not be fused directly added.
  • F and B can be connected by a link chain, such as L b , or can be directly fused without adding a link chain.
  • L a and L b are a linking chain
  • the linking chain is a flexible polypeptide consisting of glycine (G), serine (S) and/or alanine (A) of a suitable length, thereby allowing adjacent proteins The domains are free to move relative to each other.
  • G glycine
  • S serine
  • A alanine
  • Preferred linker chains of the invention include units containing G, S and/or A, exemplified by (GS)n, (GGS)n, (GGSG)n, (GGGS)nA, (GGGGS)nA, ( GGGGA)nA, etc., n is an integer from 1 to 10, and in a preferred embodiment, the linker has an amino acid length of 5-26.
  • Exemplary linkers are each independently selected from the group consisting of SEQ ID NOS. 19-41.
  • amino acid sequence of the linker can be as shown in any one of SEQ ID NOS. 19 to 41.
  • amino acid sequence of the multidomain active protein is as shown in any one of SEQ ID NOS. 150 to 208.
  • the multidomain active protein provided by the present invention is an F C fusion protein which retains the conventional properties of F C , such as binding to FcRn to prolong the half-life in vivo, and high affinity with Protein A or G filler during separation and purification. The property of combining with high specificity for efficient purification purposes.
  • these multi-domain active proteins can effectively prevent the degradation of the N-terminus in addition to effectively resisting the degradation of the protein inside the serum.
  • N-terminal integrity is critical for determining its biological activity.
  • the natural half-life of Glucagon and GLP-1 in vivo is short, and in addition to the small molecular weight, it is more important because of the hydrolysis of DPP-IV in the receptor and the hydrolysis inside the polypeptide.
  • the native Glucagon when fused to F C , is still rapidly degraded by DPP-IV and inactivated; whereas the corresponding Glucagon analog is significantly resistant to DPP-IV attack.
  • an isolated polynucleotide is provided, the isolated polynucleotide encoding the aforementioned multidomain active protein.
  • a recombinant expression vector comprising the isolated polynucleotide described above is provided.
  • a host cell comprising the aforementioned recombinant expression vector or the above-described isolated polynucleotide integrated with exogenous in the genome is provided.
  • a method for producing the aforementioned multi-domain active protein comprising culturing the host cell under suitable conditions to express the multi-domain active protein, and then separating and purifying the multi-domain Active protein.
  • the use of the aforementioned multi-domain active protein for the preparation of a medicament for treating a diabetes-related disease is provided.
  • the multidomain active protein provided by the present invention can be used to treat metabolic syndrome.
  • Metabolic syndrome is usually characterized by clustering at least three of the following risk factors: (1) abdominal obesity (too much or less adipose tissue in the abdomen), (2) atherogenic dyslipidemia, dyslipidemia, including high Triglycerides, low HDL cholesterol and high LDL cholesterol, which enhance the accumulation of plaque in the arterial wall, (3) elevated blood pressure, (4) insulin resistance or glucose intolerance, (5) thrombotic state, such as blood Medium-high fibrin or plasminogen activator inhibitor-1, and (6) promotes an inflammatory state, such as elevated C-reactive protein in the blood.
  • risk factors can include aging, hormonal imbalances, and genetic factors.
  • the multidomain active proteins of the invention are also useful in the treatment of obesity.
  • the multi-domain active protein of the invention treats obesity by reducing appetite, reducing food intake, reducing fat levels in a patient, and increasing energy expenditure.
  • a method of treating a metabolic-related disease comprising administering the aforementioned multi-domain active protein to a subject.
  • the invention further provides a method of promoting weight loss or preventing weight gain comprising administering the multi-domain active protein in a subject.
  • composition comprising the aforementioned multi-domain active protein or a culture of the aforementioned host cell, and a pharmaceutically acceptable carrier.
  • a ninth aspect of the invention the use of the aforementioned multi-domain active protein for the preparation of a fusion protein is provided.
  • a fusion protein comprising the aforementioned multidomain active protein is provided in the structure.
  • composition for the treatment of metabolic and related diseases comprising a GCGR/GLP-1R double acting agonistic active protein and a long acting FGF21 analog.
  • the structure of the GCGR/GLP-1R double-acting agonistic active protein includes: AL a -F, and the structure of the A includes a structure represented by Formula II, and the structure shown in Formula II is:
  • X 10 is selected from any of the V, L, or Y, or I;, E or K according to any of the selected S-X 12; X 13 is selected from any of the Q or of a Y; X 14 is selected from the Any one from L or M; the X 15 is selected from any one of D or E; the X 16 is selected from any one of S, E or G; and the X 17 is selected from any one of R, E or Q ; either the E or a, X 18 is selected from the R a; X 19 is selected from the any one of a or V;, any of the K or R X 20 is selected from a Q; X 21 is selected from the Any of D, L or E;
  • the X 23 is selected from any one of V or I; the X 24 is selected from any one of Q, A or E; the X 27 is selected from any one of M, K or V; and the X 28 is selected from N Or any one of K; said X 29 is selected from any one of G or T; said X 30 is G or a deletion; said X z is absent or selected from GPSSGAPPPS (SEQ ID NO. 3), PSSGAPPPS (SEQ ID NO. 4), any of SSGAPPPS (SEQ ID NO. 5), GPSSGAPPS (SEQ ID NO. 6), PSSGAPPS (SEQ ID NO. 7) or KRNRNN IA (SEQ ID NO. 8).
  • the F protein is a long-acting unit, selected from mutant F F C F C portion of the complete portion of an immunoglobulin, fragment F C portion of an immunoglobulin or immunoglobulin. Further, the amino acid sequence of F is as shown in SEQ ID NO. 9-18.
  • L a is absent or is a linking chain, and when it is a linking chain, the linking strand is a flexible polypeptide of a suitable length consisting of glycine (G), serine (S) and/or alanine (A), thereby Adjacent protein domains are free to move relative to each other.
  • G glycine
  • S serine
  • A alanine
  • a longer link chain can be used when it is necessary to ensure that the two adjacent domains do not interfere with each other spatially.
  • the linker is exemplified by (GS)n, (GGS)n, (GGSG)n, (GGGS)nA, (GGGGS)nA, (GGGGA)nA, etc., and n is an integer of 1-10.
  • Exemplary linkers can each independently be selected from SEQ ID NOS. 19-41.
  • amino acid sequence of the GCGR/GLP-1R double-acting agonist active protein is SEQ ID NO. 96, SEQ ID NO. 98, SEQ ID NO. 100, SEQ ID NO. 102, SEQ ID NO. 104, SEQ Any of ID NO. 106, SEQ ID NO. 108, SEQ ID NO. 110, SEQ ID NO. 112, SEQ ID NO. 114-133.
  • the structure of the long-acting FGF21 analog includes: FL b -B, and the structure of the B includes:
  • HPIPDSSPLLQFGGQVRQ X 19 YLYTDDAQQTE X 31 HLEI X 36 EDGTVG X 43 A X 45 DQSPESLLQL X 56 ALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRE X 98 LLEDGYNVYQSEAHGLPLH X 118 PGN X 122 SPHRDPAPRGP X 134 RFLPLPGLPPALPEPPGILAPQPPDVGSSDPL X 1 67 MV X 170 X 171 SQ X 174 RSPS X 179 X 180 X 181 , Wherein the N-terminal HPIPDSS may be deleted or partially deleted; X 19 is selected from R, Y, V, E or C; X 31 is selected from A or C; X 36 is selected from R or K; X 43 is selected from G or C; 45 is selected from A, K, E or V; X 56 is selected from K, R, V
  • the FGF21 analog is an active protein having the same or similar biological function as native FGF21 (SEQ ID NO. 136) and having a homology to native FGF21 (SEQ ID NO. 136) of 80% or more.
  • the FGF21 analog has a homology to native FGF21 (SEQ ID NO. 136) of 85% or more; more preferably, the FGF21 analog has 90 homology to native FGF21 (SEQ ID NO. 136). More preferably, the FGF21 analog has a homology to native FGF21 (SEQ ID NO. 136) of 95% or more.
  • the FGF21 analog may be selected from FGF21 similar to that described in patents or patent applications such as US20140213512, US8188040, US9493530, WO 2016114633, US 20150291677, US9422353, US 8541369, US7622445, US7576190, US20070142278, US9006400 or US20130252884. Or mutant.
  • the FGF21 analog is shown in SEQ ID NOs. 137-148.
  • the F may be selected from the intact F C portion of an immunoglobulin, a fragment of the F C portion of an immunoglobulin, or a mutant of the F C portion of an immunoglobulin.
  • the amino acid sequence of F may be as shown in any one of SEQ ID NOS. 9 to 18.
  • L b is absent or is a linking chain, and when L b is a linking chain, the linking chain includes units containing G, S, and/or A, exemplified as (GS)n, (GGS)n, ( GGSG)n, (GGGS)nA, (GGGGS)nA, (GGGGA)nA, etc., n is an integer from 1 to 10, and in a preferred embodiment, the linker has an amino acid length of 5-26.
  • Exemplary linkers are each independently selected from the group consisting of SEQ ID NOS. 19-41.
  • amino acid sequence of the long-acting FGF21 analog is as shown in any one of SEQ ID NOs: 210-221.
  • a method of treating a metabolic-related disease comprising administering to a subject a composition comprising the aforementioned GCGR/GLP-1R double-acting agonist active protein and a long-acting FGF21 analog.
  • the invention further provides a method of promoting weight loss or preventing weight gain comprising administering a composition comprising a GCGR/GLP-1R double-acting agonist active protein and a long-acting FGF21 analog in a subject.
  • incretin-like hormone proteins such as GLP-1 analogs and Exendin-4 cause side effects such as nausea and vomiting, and are dose-related. Therefore, in the case of maintaining an ideal blood sugar level, minimizing the dose to be administered theoretically alleviates the patient's unpleasant side effects.
  • FGF21 has also been reported on the effects of osteoporosis and reproductive effects (Fibroblast growth factor-21concentration in serum and synovial fluid is associated with radiographic bone loss of backbone osteoarthritis. Scand J Clin Lab Invest.2015 Apr;75 (2 ): 121-5; Fibroblast growth factor 21 has no direct role in regulating fertility in female mice. Mol Metab. 5(8): 690-8, 2016).
  • the risk of drug side effects is directly proportional to the dose administered, and in the field of diabetes drugs, the safety of drugs is extremely high.
  • the inventors have found that the GCG/GLP-1/FGF21 three-way active protein and the above composition have a good effect of controlling blood sugar and body weight at a very low dose, and have little effect on the gastrointestinal tract, greatly reducing the dose of FGF21. , thus significantly reducing the risk of potential side effects.
  • the present invention has the following beneficial effects:
  • the multi-domain active protein of the present invention has a long half-life and supports a frequency of administration once a week;
  • the GLP-1R agonistic activity of the multidomain active protein of the present invention is up to 200 times or more;
  • the multidomain active protein of the present invention has good stability in vitro and in vivo, and has low immunogenicity
  • Figure 1 is a reduced electrophoresis pattern (10% SDS-PAGE) of some of the three-way active proteins obtained by purification.
  • Lanes 1-13 are C002L 13 F 4 L 10 W, C240L 12 F 8 L 12 M 1 , C495L 13 F 8 L, respectively.
  • M is a protein standard: 97.2, 66.4, 44.3, 29, 20.1, 14.3 KD.
  • Figure 2A A graph of the results of serum stability over time.
  • FIG. 2B Figure of the results of serum stability over time.
  • FIG. 2C Figure of the results of serum stability over time.
  • Fig. 3 is a graph showing the hypoglycemic effect of the active protein of Example 8 in normal ICR mice.
  • FIG. 4 Effect of active protein in Example 9 on body weight of DIO mice.
  • Figure 5 Effect of active protein in Example 9 on appetite of DIO mice; the intake of DIO mice in the PBS group was 100%, and the ordinate was the percentage of food intake of the other groups.
  • Figure 6 Effect of the active protein in Example 10 on the body weight of DIO mice.
  • Figure 7 Effect of active protein in Example 10 on appetite of DIO mice; the intake of DIO mice in the PBS group was 100%, and the ordinate was the percentage of food intake of the other groups.
  • Figure 8 is a graph showing the results of serum stability of active protein in Example 11 as a function of time.
  • Figure 9 is a graph showing the hypoglycemic effect of the active protein in Example 11 in normal ICR mice.
  • Figure 10 Effect of the active protein in Example 11 on the body weight of DIO mice.
  • Figure 11 Effect of active protein in Example 11 on appetite of DIO mice; the intake of DIO mice in the PBS group was 100%, and the ordinate was the percentage of food intake of the other groups.
  • Figure 12 Figure of hypoglycemic effect of three-way active protein in leptin receptor-deficient type 2 diabetes (dbdb) mice.
  • diabetes includes type 1 diabetes, type 2 diabetes, gestational diabetes, and other symptoms that cause hyperglycemia.
  • the term is used for metabolic disorders in which the pancreas does not produce enough insulin, or the cells of the body fail to respond appropriately to insulin, so the decrease in the efficiency of absorption of glucose by tissue cells results in the accumulation of glucose in the blood.
  • Type 1 diabetes also known as insulin-dependent diabetes and juvenile onset diabetes, is caused by beta cell destruction and usually leads to absolute insulin deficiency.
  • Type 2 diabetes also known as non-insulin-dependent diabetes and adult-onset diabetes, is generally associated with insulin resistance.
  • obese means an excess of adipose tissue, and when energy intake exceeds energy expenditure, excess calories are stored in fat, resulting in obesity.
  • BMI body mass index
  • Incretin is a gut hormone that regulates blood sugar by enhancing glucose-stimulated insulin secretion (also known as glucose-dependent insulin secretion, GSIS) (Drucker.D J, Nauck, MA, Lancet368 :1696-705, 2006). Incretin also slows the rate of nutrient absorption by directly delaying gastric emptying and directly reduces food absorption. At the same time, incretin also inhibits the secretion of glucagon by intestinal alpha cells. To date, there are two known incretins: glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP).
  • GLP-1 glucagon-like peptide-1
  • GIP glucose-dependent insulinotropic polypeptide
  • PreproGlucagon a 158 amino acid precursor polypeptide that is differentially processed in tissues to form a variety of structurally related proglucagon-derived peptides, including glucagon ( Glucagon), glucagon-like peptide-1 (GLP-1), glucagon-like peptide-2 (GLP-2), and Oxyntomodulin (OXM).
  • Glucagon glucagon
  • GLP-1 glucagon-like peptide-1
  • GLP-2 glucagon-like peptide-2
  • OXM Oxyntomodulin
  • GIP is a 42 amino acid peptide obtained by proteolytic processing of a 133 amino acid precursor (pre-pro-GIP) involved in various biological functions including glucose homeostasis, insulin secretion, gastric emptying and intestinal Growth and food intake regulation.
  • Glucagon-like peptide (GLP-1): the sequence is shown in SEQ ID NO: 1; is a 30 or 31 amino acid polypeptide incretin hormone secreted from intestinal L-cells, with GLP-1 (7-36) And GLP-1 (7-37) two active forms. GLP-1 is released into the circulation after a meal and exerts biological activity by activating the GLP-1 receptor. GLP-1 has many biological effects, including glucose-dependent insulin secretion, inhibition of glucagon production, delay of gastric emptying and appetite suppression (Tharakan G, Tan T, Bloom S. Emerging therapies in the treatment of 'diabesity ':beyond GLP-1.Trends Pharmacol Sci 2011; 32(1): 8-15.) et al.
  • Native GLP-1 limits its therapeutic potential due to its rapid degradation by dipeptidyl peptidase-4 (DPP-4), neutral endopeptidase (NEP), plasma kallikrein or plasmin. Since native GLP-1 has an ultra-short half-life of only about 2 minutes in the body, there has been a method of treating diabetes and obesity by utilizing chemical modification and/or formulation forms (Lorenz M, Evers A, Wagner M .Recent progress and future options in the development of GLP-1 receptor agonists for the treatment of diabesity.Bioorg Med Chem Lett 2013;23(14):4011-8;Tomlinson B,Hu M,Zhang Y,Chan P,Liu ZM .An overview of new GLP-1 receptor agonists for type 2 diabetes. Expert Opin Investig Drugs 2016;25(2):145-58).
  • DPP-4 dipeptidyl peptidase-4
  • NEP neutral endopeptidase
  • plasma kallikrein or plasmin Since
  • Oxyntomodulin is a 37 amino acid small peptide having the sequence set forth in SEQ ID NO: 2; it comprises the complete 29 amino acid sequence of glucagon Glucagon (SEQ ID NO: 42).
  • Glutathione is a dual agonist of GLP-1R and GCGR that is secreted together with GLP-1 by intestinal L-cells after a meal. Similar to glucagon Glucagon, oxyntomodulin produces significant weight loss in humans and rodents. The weight loss activity of oxyntomodulin has been compared to equimolar doses of selective GLP-1 agonists in obese mice.
  • oxyntomodulin has an antihyperglycemic effect compared to a selective GLP-1R agonist, which is capable of significantly reducing body weight and having lipid lowering activity (The Glucagon receptor is involved in mediating the body weight-lowering effects Of oxyntomodulin, Kosinski JR et al, Obesity (Silver Spring), 20): 1566-71, 2012).
  • a selective GLP-1R agonist which is capable of significantly reducing body weight and having lipid lowering activity.
  • Oxytocin is also shown to reduce human food intake and increase energy expenditure (Subcutaneous oxyntomodulin reduces body weight in overweight and obese subjects: a double-blind, randomized, controlled trial, Wynne K et al, Diabetes, 54: 2390- 5,2005; Oxyntomodulin increases energy expenditure in addition to decreasing energy intake in overweight and obese humans: a 12andomized controlled trial; Wynne K et al, Int J Obes (Lond), 30: 1729-36, 2006).
  • oxyntomodulin has a shorter half-life.
  • GLP-1R GLP-1 receptor
  • GCGR glucagon receptor
  • a mutation (the oxyntomodulin analog) was made in the defect of the solution, and most of the second serine Ser was mutated to ⁇ -aminoisobutyric acid (Aib) by introducing a non-natural amino acid to resist DPP-IV. Enzymatic hydrolysis.
  • the oxyntomodulin analogue showed initial hypoglycemic and lipid-lowering effects, its mechanism of action was still inaccurate.
  • the oxyntomodulin receptor has not been found and is currently only knocked out by GCGR or GLP-1R. Mouse or cell assays have demonstrated that oxyntomodulin binds to these two receptors.
  • Glucagon is a 29 amino acid peptide corresponding to amino acids 53-81 of proglucagon, as shown in SEQ ID NO: 42 (CGFanelli et al, Nutrition, Metabolism & Cardiovascular Diseases (2006). ) 16, S28-S34). Glucagon receptor activation has been shown to increase energy expenditure and reduce food intake in both rodents and humans (Habegger KM et al, the metabolic actions of Glucagon revisited, Nat. Rev. Endocrinol. 2010, 6, 689-697). And these effects are stable and sustained in rodents.
  • Glucagon has many physiological effects, such as by stimulating glycogenolysis and gluconeogenesis, increasing blood glucose levels under hypoglycemia, regulating hepatic ketone production, regulating bile acid metabolism, and satiety through the vagus nerve. In terms of treatment, glucagon has been used for acute hypoglycemia, and glucagon receptor activation reduces food intake and promotes lipolysis and weight loss in animals and humans.
  • receptor agonist can be defined as a polypeptide, protein or other small molecule that binds to a receptor and elicits a usual response to a natural ligand.
  • GLP-1 receptor (GLP-1R) agonist can be defined as a polypeptide, protein or other small molecule that binds to GLP-1R and is capable of eliciting a characteristic response similar or similar to native GLP-1.
  • GLP-1R agonists activate GLP-1R in whole or in part, which in turn causes a series of downstream signaling pathways in the cell to produce corresponding cellular activities: such as beta cells secreting insulin; typical GLP-1R agonists include native GLP-1 And mutants thereof, analogs such as exenatide, liraglutide and the like.
  • GLP-1 analogue As used herein, "GLP-1 analog” or “GLP-1 mutant” means a GLP-1R agonist and is versatile.
  • Glucagon receptor (GCGR) agonist a Glucagon receptor agonist, which can be defined as a polypeptide, protein or other small molecule that binds to GCGR and is capable of eliciting the same or similar characteristic response as native glucagon. molecule.
  • GCGR agonists activate GCGR in whole or in part, which in turn induces a series of downstream signaling pathways in cells that produce corresponding cellular activities such as hepatocyte glycogenolysis, gluconeogenesis, fatty acid oxidation, and ketogenic effects.
  • Glucagon analogs As used herein, “Glucagon analogs”, “GCG analogs”, “Glucagon mutants” and “GCG mutants” all mean Glucagon receptor agonists and are mutually versatile.
  • the GCGR/GLP-1R double-acting active peptide of the present invention includes a protein or polypeptide capable of simultaneously agonizing GLP-1R and GCGR.
  • Oxyntomodulin-based double-effect agonists Glucagon-Like Peptide 1/Glucagon Receptor Dual Agonism Reverses Obesity in Mice, Diabetes; 58(10): 2258-2266, 2009), or by Richard D. DiMarchi, etc., as reported by Alessandro Pocai et al.
  • Glucagon-based double-effect agonist US9018164B2
  • double-acting agonist or "bispecific active protein” or “double-acting active protein” are synonymous.
  • FGF21 Fibroblast growth factor 21, fibroblast growth factor 21
  • FGF15/19, FGF23 belong to the FGF family "endocrine" hormone.
  • FGF21 is an important hormone regulating glucose and lipid metabolism. Unlike the mechanism of insulin regulation, FGF21 promotes glucose uptake in adipocytes by upregulating the expression of GLUT1.
  • the binding of FGF21 to the receptor requires the transmembrane protein ⁇ -Klotho to assist in stimulating signaling by binding to the FGFR/ ⁇ -Klotho receptor complex, stimulating the biological effects of liver, adipose tissue and pancreas.
  • ⁇ -Klotho is selectively expressed in pancreas, liver, and fat, which also explains the specificity of FGF21 on these tissues (Kurosu H et al., Tissue-specific expression of beta Klotho and fibroblast growth factor (FGF) receptor isoforms determined metabolic activity of FGF19 and FGF21. J Biol Chem 282: 26687-26695, 2007; Kharitonenkov A, et al., (2008b) FGF-21/FGF-21 receptor interaction and activation is determined by beta Klotho. J Cell Physiol 215:1-7).
  • FGF21 can bind and activate three FGFR isoforms (1c, 2c and 3c), and other FGFR isoforms, such as FGFR1b, 2b and 3b, do not form a complex with ⁇ -Klotho, Therefore, it does not act as a FGF21 receptor.
  • FGFR1 plays a major role in regulating FGF21 activity in FGFR receptors that bind to FGF21.
  • the N and C termini of FGF21 are important for their biological activity, where the N-terminus binds to FGFR and the C-terminus binds to ⁇ -Klotho (Micanovic R, et al (2009) Different roles of N-and C-termini in the functional activity of FGF21 .J Cell Physiol 219:227-234).
  • the mouse FGF21 protein is composed of 210 amino acids, the amino terminal has a signal peptide consisting of 30 amino acids, the human FGF21 protein is composed of 209 amino acids, the amino terminus has a signal peptide consisting of 28 amino acids, and the human FGF21 protein has about 75 with mouse. % homology.
  • FGF21 is mainly expressed in pancreatic ⁇ cells, liver, WAT, and skeletal muscle, and has obvious tissue specificity. Human FGF21 is easily degraded by prolyl peptidase (FAP, a serine protease) in vivo, with a half-life of 30 min in mice and 2 h in monkeys.
  • FAP prolyl peptidase
  • a domain is a region of a biological macromolecule that has a specific structure and independent function, and particularly refers to such a region in a protein.
  • the domain In globular proteins, the domain has its own specific tertiary structure, and its function does not depend on the rest of the protein molecule, but different domains in the same protein can often be linked by short sequences without secondary structure. Different domains in the protein make up a multidomain.
  • the multidomain refers to a fusion protein comprising a GCG analog, a FGF21 and an analog thereof and a FC having GCGR agonistic activity, GLP-1R agonistic activity, and FGF21 activity.
  • Dimer The dimer referred to in the present invention is formed by the natural non-covalent and covalent interaction of the constant region (F C ) of immunoglobulin. If not otherwise indicated, the dimers formed by F C are homodimers as described for the dimers provided herein.
  • Three-way active protein In this context, "three-way agonistic active protein”, “three active agonistic active protein”, “trispecific dimeric active protein” and the like are synonymous and can be used interchangeably.
  • EC 50 concentration for 50% of maximal effect refers to the concentration required for a drug or substance to stimulate 50% of its corresponding biological response.
  • concentration for 50% of maximal effect refers to the concentration required for a drug or substance to stimulate 50% of its corresponding biological response.
  • the luciferase reporter assay is used for the GLP-1R and GCGR agonistic activity in vitro cell viability assays of the present invention. This method is based on the principle that GLP-1R and GCGR activate the downstream cAMP pathway after activation. The activity of FGF21 and its analogs was determined by co-transfection of FGF21R with ⁇ -klotho into the same CHO cell to detect changes in fluorescence caused by the signal.
  • the GLP-1R agonistic activity is increased by a staggering 200-fold or more (EC 50 of about 1.1 nM).
  • the GCG-1R agonistic activity ratio calculated according to the data disclosed in the corresponding patents and literatures by US9018164B2 and Joseph R. Chabenne et al., the GLP-1R agonistic activity ratio is only about 2 times before and after the increase of GPSSGAPPPS or the like (as in the article, natural Glucagon has a GLP-1R agonistic activity percentage of 0.7%, which increases to 1.6% after increasing the GPSSGAPPPS sequence. That is, the addition of the GPSSGAPPPS sequence at the C-terminus of the Glucagon polypeptide does not significantly increase the agonistic activity of its GLP-1R.
  • Natural Glucagon has multiple sensitive degradation sites, including the second DPP-IV degradation site and the 16-18 SRR site. Although it has been reported that F C can increase the chemical stability and serum stability of active proteins, the role of F C seems to be inconsistent for GLP-1 or Glucagon analogs that must be exposed at the N-terminus. After natural fusion of GLP-1 or Glucagon with F C , significant degradation under 37-degree serum conditions was still observed.
  • the present invention introduces a mutation resistant to protease hydrolysis on the basis of natural Glucagon to increase its stability. After the fusion of these mutants with F C , the stability was further improved.
  • DPP-IV-resistant mutations in the second position, such as L-type mutations to D-type amino acids (L-Ser mutation to D-Ser). ), or introduce the non-natural amino acid Aib et al (Matthias H. Etc. Unimolecular Polypharmacy for Treatment of Diabetes and Obesity, 24: 51–62, 2016).
  • the double-acting active protein conforming to Formula II and storing the native L-Ser in the second position exhibits very high serum stability and still has no significant degradation of DPP-IV at 24 hours.
  • IPGTT Peritoneal glucose tolerance test
  • mice administered multi-domain active protein exhibited extremely stable blood glucose fluctuations after glucose injection.
  • GCGR agonists have been reported to have potential weight loss effects.
  • natural Glucagon is easily degraded and has a very small molecular weight, the potential for medicine is extremely small.
  • Glucagon analogues are currently used primarily for acute hypoglycemia symptoms.
  • Clinical reports of long-acting GCG analogues for weight loss in diabetic patients are also emerging. It is well known that obesity is one of the causes of insulin resistance in diabetic patients, and weight loss is an important indicator for evaluating a hypoglycemic drug.
  • the multi-domain active protein of the present invention induced a significant decrease in body weight after administration in DIO mice.
  • Example 9 of the present invention the 30 nM/kg dose of the three-way active protein reduced weight by about 30%, while the appetite was substantially unchanged. Similarly, at the 10 nM/kg dose, the weight loss was also close to 15%.
  • the combination of the double-acting active protein and the long-acting FGF21 analog (AL a -F+FL b -B) in Example 10 also has a synergistic effect: the composition for co-administration can be reduced by 35 % or more body weight, however, an equal dose of the double-acting active protein can only reduce the body weight by no more than 13%, and the long-acting FGF21 analog has a weight loss of less than 10%.
  • the multiplex active proteins of the invention have potential pharmacokinetic properties that are suitable for administration once a week or more.
  • the dosage will depend on the frequency and mode of administration, the age, sex, weight and general condition of the subject being treated, the condition and severity of the treatment, any concomitant disease to be treated, and other factors apparent to those skilled in the art.
  • the multiplex active protein of the invention may be administered or applied in combination with one or more other therapeutically active compounds or substances, for example, other therapeutically active compounds may be selected including but not Limited to anti-diabetic drugs, anti-hyperlipidemic drugs, anti-obesity drugs, antihypertensive drugs and agents for treating complications arising from diabetes or diabetes.
  • Metabolic syndrome is associated with an increased risk of coronary heart disease and other conditions associated with vascular plaque accumulation, such as stroke and peripheral vascular disease, becoming atherosclerotic cardiovascular disease (ASCVD).
  • Patients with metabolic syndrome can progress from being in an early stage of insulin resistance to fully mature type 2 diabetes, and the risk of ASCVD is further increased.
  • the relationship between insulin resistance, metabolic syndrome, and vascular disease may involve one or more common pathogenesis, including insulin-stimulated vasodilation, resulting from increased oxidative stress.
  • Reduced availability of insulin resistance and abnormalities in adipogenic hormones such as adiponectin (Lteif, Mather, Can. J. Cardiol. 20 (Supp. B): 66B-76B, 2004)
  • the active proteins of the invention are also useful in the treatment of obesity.
  • the active proteins of the invention treat obesity by reducing appetite, reducing food intake, reducing fat levels in a patient's body, and increasing energy expenditure.
  • the active proteins of the invention are useful in the treatment of nonalcoholic fatty liver disease (NAFLD).
  • NAFLD refers to a broad spectrum of liver disease ranging from simple fatty liver (steatosis) to nonalcoholic steatosis hepatitis (NASH) to cirrhosis (reversible late scar formation of the liver). All stages of NAFLD have fat accumulation in liver cells. Simple fatty liver is abnormal accumulation of certain types of fat and triglyceride in liver cells, but no inflammation or scar formation. In NASH, fat accumulation is associated with varying degrees of liver inflammation (hepatitis) and scar formation (fibrosis). Inflammatory cells destroy liver cells (hepatocyte necrosis).
  • steatosis refers to fat infiltration
  • hepatitis refers to inflammation in the liver
  • necrosis refers to damaged liver cells.
  • NASH can eventually lead to liver scar formation (fibrosis) and then to irreversible late scar formation (cirrhosis), and cirrhosis caused by NASH is the last and most serious stage within the NAFLD spectrum.
  • the experimental methods, detection methods, and preparation methods disclosed in the present invention employ molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields conventional in the art. Conventional technology. These techniques are well described in the prior literature, see Sambrook et al.
  • MOLECULAR CLONING A LABORATORY MANUAL, Second edition, Cold Spring Harbor Laboratory Press, 1989 and Third edition, 2001; Ausubel et al, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, 1987 and periodic updates; the series METHODS IN ENZYMOLOGY, Academic Press, San Diego; Wolffe, CHROMATIN STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998; METHODS IN ENZYMOLOGY, Vol. 304, Chromatin (PM Wassarman and AP Wolffe, eds.), Academic Press, San Diego, 1999; and METHODS IN MOLECULAR BIOLOGY, Vol. 119, Chromatin Protocols (PBBecker, ed.) Humana Press, Totowa, 1999, and the like.
  • amino acid sequence of native GLP-1 is shown in SEQ ID NO. 1, specifically:
  • the amino acid sequence of the native Oxyntomodulin is shown in SEQ ID NO. 2, specifically:
  • the GCG analog of the present invention is designated A, and the A is a GCGR/GLP-1R double-acting agonist active peptide selected from all peptide chains capable of achieving GCGR and GLP-1R double-acting agonistic activity.
  • X 10 is selected from any of the V, L, or Y, or I;, E or K according to any of the selected S-X 12; X 13 is selected from any of the Q or of a Y; X 14 is selected from the Any one from L or M; the X 15 is selected from any one of D or E; the X 16 is selected from any one of S, E or G; and the X 17 is selected from any one of R, E or Q ; either the E or a, X 18 is selected from the R a; X 19 is selected from the any one of a or V;, any of the K or R X 20 is selected from a Q; X 21 is selected from the Any of D, L or E;
  • the X 23 is selected from any one of V or I; the X 24 is selected from any one of Q, A or E; the X 27 is selected from any one of M, K or V; and the X 28 is selected from N Or any one of K; wherein X 29 is selected from any one of G or T; said X 30 is G or a deletion; said X z is absent or selected from any one of SEQ ID NOS. 3 to 8.
  • the amino acid sequences of the exemplary GCG analogs may each independently be selected from the group consisting of SEQ ID NO. 42-92, and the corresponding polypeptide codes are C001, C002, C240, C241, C276, C225, C222, C163, C164, C271, C368, C495, C353, C352, C355, C382, C232, C227, C266, C137, C399, C398, C396, C392, C462, C228, C187, C363, C364, C209, C289, C611, C618, C623, C627, C654, C673, C563, C549, C555, C487, C488, C489, C503, C508, C711, C708, C743, C756, C788, C731.
  • a dimeric double-acting active protein AL a -F was obtained by fusion of a GCG analog with a linker L a and F.
  • A is the same as A in the first embodiment.
  • the F protein is a long-acting unit, selected from mutant F F C F C portion of the complete portion of an immunoglobulin, fragment F C portion of an immunoglobulin or immunoglobulin.
  • the amino acid sequence of the F is as shown in SEQ ID NO. 9-18, and the abbreviations corresponding to the sequences are respectively F1-F10.
  • La is a linking chain which is a flexible polypeptide of a suitable length consisting of glycine (G), serine (S) and/or alanine (A) such that adjacent protein domains are relative to each other Move freely.
  • G glycine
  • S serine
  • A alanine
  • the linker is exemplified by (GS)n, (GGS)n, (GGSG)n, (GGGS)nA, (GGGGS)nA, (GGGGA)nA, etc., and n is an integer of 1-10.
  • Exemplary linkers can each independently be selected from the group consisting of SEQ ID NO. 19-41, and the sequences corresponding to the sequences are L1-L23, respectively.
  • the amino acid sequences of the partial double-acting active protein A-La-F may each independently be selected from the group consisting of SEQ ID NO. 93-133, and the DNA sequence may be independently selected from the group consisting of SEQ ID NO. 222-262, the corresponding double-acting active protein A- la-F code-named C001L13F8, C002L13F8, CG283L13F8, C240L13F8, CG214L13F8, C382L13F8, CG267L13F8, C276L13F8, C308L13F8, C368L13F8, C224L13F8, C225L13F8, CG308L13F8, C495L13F8, C319L13F8, C364L13F8, C214L13F8, C232L13F8, C303L13F8, C392L13F8, CG303L13F8, C462L13F8, C240L12F8, C368L10F4, C364L10F4, C352
  • the preparation process is as follows:
  • the DNA sequence is designed based on the protein sequence and the amino acid codon table.
  • the polynucleotide DNA fragments corresponding to A, L a and F in the recombinant protein are respectively prepared, and each DNA fragment can be synthesized and spliced by conventional solid phase synthesis technology;
  • PCR splicing technology (including primer design, PCR introduction mutation and enzymatic cleavage, etc.) is a person skilled in the art. Well known techniques are well known. Those skilled in the art will appreciate that the PCR splicing process of this embodiment is not the only method, for example, the gene of interest can also be obtained by gene synthesis.
  • the target gene was cloned into the mammalian cell expression vector pTT5 (Yves Durocher) and transformed into E. coli Top10F'; after identification, the positive clone was inoculated into 500 ml LB medium, cultured overnight, and the cells were collected by centrifugation. Omega Endo-Free Plasmid Maxi Kit extracts the plasmid;
  • the three-way active protein of the present invention contains a multi-domain and has a three-way agonistic activity.
  • the structural formula of the three-way active protein is as shown in Formula I: AL a -FL b -B, wherein A is a GCGR/GLP-1R double acting agonistic active peptide, F is a long acting protein unit, and B is a natural FGF21 or FGF21 similar L a is a linking chain and L b is a linking chain.
  • the F may be selected from the intact F C portion of an immunoglobulin, a fragment of the F C portion of an immunoglobulin, or a mutant of the F C portion of an immunoglobulin, as set forth in SEQ ID NO. 9-18 .
  • the B is native FGF21 (SEQ ID NO. 136) or an FGF21 analog.
  • the structural formula of the B is:
  • HPIPDSSPLLQFGGQVRQ X 19 YLYTDDAQQTE X 31 HLEI X 36 EDGTVG X 43 A X 45 DQSPESLLQL X 56 ALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRE X 98 LLEDGYNVYQSEAHGLPLH X 118 PGN X 122 SPHRDPAPRGP X 134 RFLPLPGLPPALPEPPGILAPQPPDVGSSDPL X 1 67 MV X 170 X 171 SQ X 174 RSPS X 179 X 180 X 181 .
  • N-terminal HPIPDSS may be deleted or partially deleted;
  • X 19 is selected from R, Y, V, E or C;
  • X 31 is selected from A or C;
  • X 36 is selected from R or K;
  • X 43 is selected from G or C;
  • 45 is selected from A, K, E or V;
  • X 56 is selected from K, R, V or I;
  • X 98 is selected from L, R or D;
  • X 118 is selected from L or C;
  • X 122 is selected from K or R;
  • 134 is selected from A or C;
  • X 167 is selected from S, A or R;
  • X 170 is selected from G or E;
  • X 171 is selected from P or G;
  • X 174 is selected from G, A or L; and
  • X 179 is selected from Y, A.
  • Or F X 180 is selected from A or E;
  • X 181 is selected from S, K or a deletion.
  • the FGF21 analog is an active protein having the same or similar biological function as native FGF21 (SEQ ID NO. 136) and having a homology to native FGF21 (SEQ ID NO. 136) of 80% or more.
  • the FGF21 analog has a homology to native FGF21 (SEQ ID NO. 136) of 85% or more; more preferably, the FGF21 analog has 90 homology to native FGF21 (SEQ ID NO. 136). More preferably, the FGF21 analog has a homology to native FGF21 (SEQ ID NO. 136) of 95% or more.
  • the FGF21 analog is as set forth in SEQ ID NOs. 137-148.
  • L a is absent or is a linking chain
  • L b is absent or is a linking chain
  • the linking chain is the same as La in Example 2.
  • the FGF21 or FGF21 analog is fused at the C-terminus of F C by a ligation peptide chain to prepare an amino acid as shown in SEQ ID NO.
  • ID exemplary three-way active protein shown NO.263-322 respectively corresponding to the code C002L13F8L10W, C240L12F8L12M1, C240L9F7L13M2, C240L13F4L9M1, C240L9F2L13M3, C240L13F10L9M2, C225L10F10L14M2, C163L13F8L13M2, C271L9F4L8M2, C368L10F4L10M2, C495L13F8L13M1, C495L13F8L10M2, C495L9F10L9M1, C353L13F3L10M4, C352L13F4L9M3 , C382L9F3L9M2, C382L10F2L13M2, C382L13F10L9M1, C382L13F8L10M2, C382L12F7L9M2, C382L14F4L9M2, C232L9F3
  • F C code-named F 9
  • native FGF21 and FGF21 analog codenamed M 1 -M 12
  • the structural formula of the long-acting FGF21 analog is FL b -B .
  • the F may be selected from the intact F C portion of an immunoglobulin, a fragment of the F C portion of an immunoglobulin, or a mutant of the F C portion of an immunoglobulin.
  • the amino acid sequence of F may be as shown in any one of SEQ ID NOS. 9 to 18.
  • L b is absent or is a linking chain, and when L b is a linking chain, the linking chain is the same as La in Example 2.
  • n is an integer from 1 to 10
  • the linker chain has an amino acid length of 5-26.
  • the amino acid sequence of the linker can be as shown in any one of SEQ ID NOS. 19 to 41.
  • the B is native FGF21 (SEQ ID NO. 136) or an FGF21 analog.
  • the B is the same as B in the third embodiment.
  • the amino acid sequence of the long-acting FGF21 analog can be represented by SEQ ID NO. 209-SEQ ID NO. 221, respectively, and the codes are F9L10W, F9L10M1, F9L10M2, F9L10M3, F9L10M4, F9L10M5, F9L10M6, F9L10M7, F9L10M8, F9L10M9, F9L10M10, respectively.
  • the double-acting active protein obtained in Example 2 was assayed for in vitro activity, including GLP-1R agonistic activity assay and GCGR agonistic activity assay.
  • the luciferase reporter assay was used for GLP-1R agonistic activity assay (Jonathan W Day et al: Nat Chem Biol. 2009 Oct; 5(10): 749-57).
  • the human GLP-1R gene was cloned into the mammalian cell expression plasmid pCDNA3.1, and the recombinant expression plasmid pCDNA3.1-GLP-1R was constructed, and the full-length gene of luciferase was cloned into pCRE plasmid to obtain pCRE- Luc recombinant plasmid.
  • the pCDNA3.1-GLP-1R and pCRE-Luc plasmids were transfected into CHO cells at a ratio of 1:10, and the stably transfected expression strains were selected to obtain a recombinant CHO/GLP-1R stably transfected cell line.
  • the cells were cultured in a 9-cm cell culture dish in DMEM/F12 medium containing 10% FBS and 300 ⁇ g/ml G418. When the confluency was about 90%, the culture supernatant was discarded, and after 2 ml trypsin digestion for 3 min, Add 2 ml of DMEM/F12 medium containing 10% FBS and 300 ⁇ g/ml G418, transfer to a 15 ml centrifuge tube, centrifuge at 1000 rpm for 5 min, discard the supernatant, and add 2 ml of DMEM containing 10% FBS and 300 ⁇ g/ml G418. /F12 medium was resuspended and counted.
  • the cells were diluted with DMEM/F12 medium containing 10% FBS to 3 ⁇ 10 5 , 100 ⁇ l per well in a 96-well plate, ie 5 ⁇ 10 4 /well, and then affixed to DMEM/F12 containing 0.2% FBS. Base culture.
  • the purified recombinant protein (Table 1, Table 2) or natural Glucagon (Hangzhou Zhongpept Biochemical Co., Ltd., GLUC-004) and natural GLP-1 (Hangzhou Zhongpeptide Biochemistry) Co., Ltd., GLUC-016B) was diluted as a control with DMEM/F12 medium containing 0.1% FBS to a specified concentration, and added to the cell culture well, 100 ⁇ l/well, and stimulated for 6 hours. Detection was carried out according to the instructions of the Lucifersae reporter kit (Ray Biotech, Cat: 68-LuciR-S200).
  • luciferase reporter assay was also used for GCGR agonistic activity assays.
  • the GCGR gene was cloned into the mammalian cell expression plasmid pCDNA3.1, and the recombinant expression plasmid pCDNA3.1-GCGR was constructed.
  • the recombinant plasmid pKRE-Luc was co-transfected into HEK 293T cells and the stable cell line HEK 293T/GCGR was screened. Ibid.
  • the FGF21 activity assay was performed using a similar method in the literature and appropriately modified (Xu J et al, Polyethylene glycol modified FGF21 engineered to maximize potency and minimize vacuole formation, Bioconjug Chem.; 24(6): 915-25, 2013).
  • the puromycin resistance gene pac was amplified by PCR, cloned into pcDNA3.1(+), and the original G418 resistance gene was replaced.
  • the GAL4DBD-ELK1, IRES, and KLB ( ⁇ -klotho) genes were amplified by PCR and cloned into pcDNA-Puro plasmid in turn to construct plasmid pcDNA-GAL4DBD-ELK1-IRES-KLB-Puro for cell transfection screening. Plasmid using Omega Endo-Free Plasmid Midi Kit extracts spares.
  • the cell transfection process was as follows: Hek293T cells were plated in 6-well plates at 3 x 10 5 cells per well and cultured overnight.
  • Opti-MEM medium After washing the cells twice in Opti-MEM medium, 2 ml of Opti-MEM medium was added.
  • the cell transfection reagent was prepared in the following ratio: Lipofectamine 2000 (6 ⁇ l): pFR-Luc (4.6 ⁇ g): pcDNA-GAL4DBD-ELK1-IRES-KLB-Puro (1 ⁇ g). After standing for 20 min, slowly add to a 6-well plate and mix while mixing. After 6 hours of culture, DMEM + 10% FBS medium was changed, and the culture was continued at 37 ° C, 5% CO 2 . A stable cell line with a FGF21 activity response was obtained by screening.
  • the cells were trypsinized, and a cell suspension (1 x 105 cells/ml, DMEM + 5% FBS + 1 ⁇ g/ml puromycin) was prepared, and 96-well plates were plated at 100 ⁇ l per well, and cultured overnight. A gradient concentration of the sample to be tested was added for 6 hours, and fluorescence detection was performed using a Luciferase Reporter Assay Kit (68-LucifR-S200).
  • the protein number in the table is according to the following rules: peptide code + linker code + FC code, such as C240L 13 F 4 , indicating that the C240 polypeptide is fused to the IgG FC codenamed F 4 by a linker coded L 13 .
  • a The ratio of GLP-1R agonistic activity before and after insertion of a GPSSGAPPPS or similar sequence (also referred to as a Cex sequence, which is any of SEQ ID NOS. 3-8 in the present invention) between the GCG analog and the Fc chain.
  • the protein number in the table is as follows: peptide code + linker code + F C code + linker code + FGF21 mutant code, such as C209L 13 F 4 L 13 M 9 , indicating that the C209 polypeptide and the IgG F C codenamed F4 pass L is a linking chain fusion code 13, and code for further mutants M FGF21 9 L chain 13 is connected by fusion of the code.
  • GD Ser GS HD-Ser-QGTFTSDYSKYLDSQAAQDFVQWLMNGGPSSGAPPPS (SEQ ID NO. 134);
  • G Aib GS H-Aib-QGTFTSDYSKYLDSQAAQDFVQWLMNGGPSSGAPPPS (SEQ ID NO. 135);
  • HEK 293T/GCGR cells were passaged twice and then plated in 96-well plates to measure the activity of the samples. Residual activity: The activity value at 0 hours was 100%, and the value measured at the subsequent time point was obtained. Except for C002L 13 F 4 L 13 W, similar to the results in Table 4, there was no significant difference in serum stability of each of the three-way active proteins.
  • the relative activity of the exemplary three-way active protein as a function of time is shown in Figures 2A-C.
  • Example 9 Pharmacodynamic study of continuous administration of three-way active protein in induced obesity (DIO) mice
  • mice Seven-week-old male C57BL/6J male mice were fed with high fat diet (60% kcal from fat) for 16 weeks (23 weeks total) and tested at a body weight of approximately 55 g. Feeding conditions: 12h light/12h dark, free-feeding, single-cage feeding, mice were grouped according to body weight and body weight growth curve one day before administration (8/group), and treated subcutaneously the next day. According to Table 5, the active protein was administered at a dose of 10 nmol/kg body weight or 30 nmol/kg body weight once every 4 days; the negative control group was injected with physiological saline (PBS) at 5 ul/g body weight; the positive control group was injected with liraglutide.
  • PBS physiological saline
  • mice (30 nmol/kg body weight), administered once a day for 28 days, and the body weight and food intake of the mice were measured daily. The 5th day after the last administration was sacrificed. Eyes take blood. Plasma samples were stored at -80 °C. The average body weight change before and during sacrifice was calculated for each group of animals. The results of weight change are shown in Figure 4; the change in total food intake is shown in Figure 5.
  • This example differs from Example 9 in that the double-acting active protein is administered in combination with the long-acting FGF21 analog in a combination form.
  • Seven-week-old male C57BL/6J male mice were fed with high fat diet (60% kcal from fat) for 16 weeks (23 weeks total) and tested at a body weight of approximately 55 g.
  • Feeding conditions 12h light/12h dark, free-feeding, single-cage feeding, mice were grouped according to body weight and body weight growth curve one day before administration (8/group), and subcutaneously administered the next day.
  • the double-acting active protein and the long-acting FGF21 analog were mixed according to the dose shown in Table 6 before administration, and administered at a dose of 15 nmol/kg body weight or 30 nmol/kg body weight once every 4 days; the negative control group was injected at 5 ul/g body weight.
  • the three-way active protein of the present invention is the same as that of the third embodiment, and the fusion proteins C382L13F3L10M2 and C382L13F9L10M2 are obtained: the corresponding amino acids are represented by SEQ ID NO. 336 and SEQ ID NO. 338, respectively, and the corresponding nucleotides are respectively SEQ ID NO. 337 and SEQ ID NO. 339.
  • the preparation method used in this example is the same as that in Example 2.
  • the in vivo activity assay of the three-way active protein obtained in the present example including GLP-1R agonistic activity assay
  • Example 11 The triose active protein obtained in Example 11 was subjected to a serum stability test in the same manner as in Example 7. The relative activity of the three-way active protein changes with time as shown in FIG.
  • Example 11 The pharmacodynamic study of continuous administration of the three-way active protein obtained in Example 11 in induced obesity (DIO) mice was carried out in the same manner as in Example 9. The results of weight change are shown in Figure 10; the change in total food intake is shown in Figure 11.
  • the data in Figure 10 shows that the three-way active protein obtained in Example 11 was significantly different from liraglutide at 10 nmol/kg and 30 nmol/kg, and the weight loss was positively correlated with the dose at 30 nmol/kg. The weight loss effect was very significant; the data of Figure 11 shows that the three-way active protein obtained in Example 11 had no significant effect on feeding at doses of 10 nmol/kg and 30 nmol/kg.
  • mice Hypoglycemic assay in leptin receptor-deficient type 2 diabetes (db/db) mice.
  • the db/db mice were screened and balanced according to the three indicators of body weight, non-empty blood glucose and pre-medication OGTT reaction. Each group had 10, and the individuals who were too large or too small were excluded as much as possible. The non-fasting blood glucose was greater than 15 mM.
  • normal ICR mice were selected as the basic blood glucose control.
  • the active protein was injected subcutaneously at a dose of 20 nmol/kg body weight, once every 4 days, the first day was day 0, and the last time was day 24; the negative control group was saline (PBS) (5 ⁇ l / gram body weight), the positive control group liraglutide (10nmol / kg body weight) was administered once a day by subcutaneous injection for 26 days.
  • Random blood glucose values were measured at 9 am before the first injection and at 2d, 6d, 10d, 14d, 18d, 22d, and 26d. The results of random blood glucose changes are shown in Figure 12.
  • Figure 12 shows that the three-way active protein in Table 8 is significantly better than the positive control liraglutide in leptin receptor-deficient type 2 diabetes (dbdb) mice.
  • the blood glucose level of C382L 13 F 3 L 10 M 2 was even comparable to that of normal ICR mice (not shown).
  • nucleotide sequences corresponding to the active proteins described in the present specification are shown in Table 9.

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Abstract

本发明属于生物药物领域，具体涉及一种治疗代谢疾病的多结构域活性蛋白。所述多结构域活性蛋白，其结构式如式I所示：A-La-F-Lb-B，与现有技术相比，本发明的多结构域活性蛋白半衰期长、支持一周一次的给药频率；所述多结构域活性蛋白的GLP-1R激动活性最高提高到了200倍以上；多结构域活性蛋白体内外稳定性好，且免疫原性低；由于不需要引入非天然氨基酸，无需涉及化学合成及交联步骤，因此可通过重组方法制备，极大地简化了制备工序。

Description

一种治疗代谢疾病的多结构域活性蛋白 技术领域
本发明属于生物药物领域，具体涉及一种治疗代谢疾病的多结构域活性蛋白。
背景技术
糖尿病按病理特征可分为一型糖尿病和二型糖尿病两种。一型糖尿病主要表现为胰岛素分泌不足，需要每天注射胰岛素；而二型糖尿病则是由于人体无法有效利用胰岛素造成。其中二型糖尿病患者占绝大多数。据估计大约80-90％二型糖尿病患者明显肥胖(Center for disease control and prevention(CDC)National Diabetes Fact Sheet,2014)。
用于治疗二型糖尿病的常规化药如磺酰脲类，噻唑烷二酮类等降糖效果明显，但主要缺点是会导致体重增加(Kahn SE,Haffner SM,Heise MA,Herman WH,Holman RR,Jones NP,et al.Glycemic durability of rosiglitazone,metformin,or glyburide monotherapy.N Engl J Med 2006；355(23):2427-43.)。而用于二型糖尿病的蛋白类药物主要是GLP-1R(GLP-1受体)激动剂，如杜拉鲁肽(Dulaglutide，商品名：
Figure PCTCN2018116244-appb-000001
)、阿必鲁肽(Albiglutide，商品名
Figure PCTCN2018116244-appb-000002
)、利拉鲁肽(Liraglutide，商品名
Figure PCTCN2018116244-appb-000003
及
Figure PCTCN2018116244-appb-000004
分别用于治疗肥胖和糖尿病)、艾塞那肽(Exenatide，商品名
Figure PCTCN2018116244-appb-000005
)、利西拉肽(Lixisenatide，商品名
Figure PCTCN2018116244-appb-000006
)及索马鲁肽(Semaglutide)等。GLP-1R激动剂具有显著的降血糖效应，而且与胰岛素不同的是，GLP-1R激动剂的降糖作用是严格地血糖依赖性的，不容易造成低血糖，还有降体重的效果。例如，杜拉鲁肽的减重大约为2.9公斤，而被批准用于减肥的Liraglutide(一天一次，剂量3mg)减重约8公斤。这些药物的减重主要是通过食欲控制，而且大部分都不超过平均体重的10％。减肥手术(Bariatric surgery)虽然可以显著改善肥胖症和治疗糖尿病，然而其应用并不广泛，因为大部分的患者出于手术风险及长期后遗症的考虑，并不愿意接受这种手术(Obesity and Diabetes，New Surgical and Nonsurgical Approaches，Springer出版社，2015)。
据报道，经外科减肥手术的患者肠降血糖素(Incretin)分泌会激增(Obesity and Diabetes，New Surgical and Nonsurgical Approaches，Springer出版社，2015)。因此，目前新一代的糖尿病药物主要集中于双效或多效肠降血糖素(Incretin)受体激动剂的研究，如GLP-1R/GIPR 和GLP-1R/GCGR双效激动剂，甚至GLP-1R/GIPR/GCGR三效激动剂。
其中，胰高血糖素(Glucagon)和GLP-1(Glucagon-like peptide-1)的受体在结构上相关，但是这两种激素在控制葡萄糖中表现出截然相反的作用。临床上，GLP-1及其类似物主要用于糖尿病人的血糖控制，而胰高血糖素(Glucagon)则用于急性低血糖症。近年来，越来越多的研究证明，胰高血糖素(Glucagon)尽管存在升血糖的风险，但是却能有效减少体重；更重要的是，GLP-1与Glucagon似乎有正向的附加或协同生理作用，例如Glucagon受体(GCGR)与GLP-1受体(GLP-1R)双激动剂比GLP-1R单激动剂能更有效地减重。虽然GCGR激动可能会导致血糖水平增加，但是这个风险可以通过GLP-1R激动而适当地抵消。
目前GLP-1R和GCGR的双效激动剂普遍都是基于胃泌酸调节素(Oxyntomodulin)或Glucagon，并且为了改善其短效及酶解的缺陷而进行改造(Oxyntomodulin类似物或Glucagon类似物)。这些类似物大都将第二位的丝氨酸(Ser)突变成非天然氨基酸Aib来抵抗DPP-IV的酶解。这是因为天然的Glucagon与胃泌酸调节素都与天然GLP-1类似，极易遭受血清中DPP-IV蛋白酶的水解而导致失活(Victor A.Gault等，A novel GLP-1/glucagon hybrid peptide with triple-acting agonist activity at GIP,GLP-1 and glucagon receptors and therapeutic potential in high-fat fed mice，J Biol Chem.,288(49):35581-91.2013；Bhat VK等，A DPP-IV-resistant triple-acting agonist of GIP,GLP-1and glucagon receptors with potent glucose-lowering and insulinotropic actions in high-fat-fed mice，Diabetologia，56(6):1417-24.2013；John A.Pospisilik等；Metabolism of glucagon by dipeptidyl peptidase IV(CD26)，Regulatory Peptides 96:133–141,2001；Hinke SA等，Dipeptidyl peptidase IV(DPIV/CD26)degradation of glucagon.Characterization of glucagon degradation products and DPIV-resistant analogs，J Biol Chem 275:3827–3834,2000；Alessia Santoprete等，DPP-IV-resistant,long-acting oxyntomodulin derivatives,J.Pept.Sci.,17:270–280,2011)。
FGF21属于在发育以及成体组织中广泛表达的在多种生理功能中起重要作用的多肽家族成员。FGF21主要表达于胰腺β细胞、肝脏、WAT、骨骼肌，近来发现其在胸腺、血管内皮、肾脏、睾丸组织中也有较低表达，有明显的组织特异性，和FGF15/19、FGF23同属于FGF家族“内分泌”激素。
FGF21作为一种重要的代谢调节剂已经在II型糖尿病临床前模型(T2DM)实验中被证明能够改善多种代谢异常。FGF21作为糖尿病患者的治疗药物在提高胰岛素敏感性，改善血糖控制，减轻体重，降低低密度脂蛋白胆固醇(LDL-C)和甘油三酯，同时增加高密度脂蛋白胆固醇水平(HDL-C)这些方面有潜在作用。在糖尿病鼠和猴子中，人FGF21能 够降低空腹血清葡萄糖浓度，减少空腹血清甘油三酯，胰岛素和胰高血糖素的浓度。此外，在饮食诱导肥胖的啮齿动物模型中，FGF21给药导致剂量依赖的总体重减轻。因此，FGF21具有治疗糖尿病，肥胖，血脂异常和代谢综合征等疾病的潜力。
但是，FGF21具有非常短的血清半衰期：小鼠体内30分钟，猴子2小时。因此，为了维持体内生物活性需要每日注射或连续输注相应FGF21蛋白质。在人体研究中，在肥胖、血脂异常、TDM2及其他与胰岛素抵抗相关疾病的患者体内，循环FGF21水平往往升高，有研究表明FGF21浓度增加与CVD风险增加有关联，还会导致骨质疏松，影响生殖(促进新陈代谢进而导致能量不足)等(Wei W,Dutchak PA,Wang X,Ding X,Wang X,Bookout AL,et al.Fibroblast growth factor 21 promotes bone loss by potentiating the effects of peroxisome proliferator-activated receptor gamma.Proc Natl Acad Sci USA.2012；109(8):3143–8；Fibroblast growth factor 21 has no direct role in regulating fertility in female mice，Mol Metab，5(8):690-8，2016)。FGF家族序列的同源性以及FGFR1受体的广泛分布，也对临床大剂量使用FGF21所带来的潜在安全性问题带来关注(Kharitonenkov A&DiMarchi R:Fibroblast growth factor 21 night watch:advances and uncertainties in the field.J Intern Med.2017 Mar；281(3):233-246.)。
GCGR/GLP-1R双效激动剂及FGF21类似物都分别用于治疗糖尿病及减重，另外还有将GLP-1类似物与FGF21通过Fc融合制备成双效活性蛋白的报道(YH25723,a Novel Long-Acting GLP-1/FGF21 Dual Agonist Provides More Potent and Sustained Glycemic Control and Greater Weight Loss Compared with Single Agonists in Animal Models，The American Diabetes Association，2016)。如上所述，目前基于Glucagon或胃泌酸调节素改造而成的双效甚至三效多肽普遍需要将部分氨基酸取代非天然氨基酸来提高稳定性和活性，甚至还需要通过脂肪酸或PEG修饰，技术上极难与FGF21类似物融合表达制备成单一分子。目前也并无关于双效或者多效激动剂类多肽与FGF21类似物组合用药的报道。
发明内容
为了克服现有技术中所存在的问题，本发明的目的在于提供一种治疗代谢及相关疾病的多结构域活性蛋白及其制备与应用。本发明的多重活性蛋白具有显著的减重效果，临床可以用于治疗糖尿病、减肥、非酒精性脂肪肝、高血脂等相关疾病。
为了实现上述目的以及其他相关目的，本发明采用如下技术方案：
本发明的第一方面，提供一种多结构域活性蛋白，其结构中包括如式I所示的结构， 如式I所示结构为：A-L a-F-L b-B，其中，A为GCGR/GLP-1R双效激动活性肽，F为长效蛋白单元，B为天然FGF21或FGF21类似物，L a不存在或为连接链，L b不存在或为连接链。
所述多结构域活性蛋白具有至少GLP-1、GCG和FGF21三效活性。
进一步地，式I中，所述A包括如式II所示的结构，如式II所示结构为：
HSQGTFTSD-X 10-S-X 12-X 13-X 14-X 15-X 16-X 17-X 18-X 19-X 20-X 21-F-X 23-X 24-WL-X 27-X 28-X 29-X 30-X z。
其中，所述X 10选自V、L或Y之任一；所述X 12选自S、E或K之任一；所述X 13选自Y或Q之任一；所述X 14选自L或M之任一；所述X 15选自D或E之任一；所述X 16选自S、E或G之任一；所述X 17选自R、E或Q之任一；所述X 18选自R、E或A之任一；所述X 19选自A或V之任一；所述X 20选自Q、R或K之任一；所述X 21选自D、L或E之任一；
所述X 23选自V或I之任一；所述X 24选自Q、A或E之任一；所述X 27选自M、K或V之任一；所述X 28选自N或K之任一；所述X 29选自G或T之任一；所述X 30为G或缺失；所述X z不存在或选自GPSSGAPPPS(SEQ ID NO.3)、PSSGAPPPS(SEQ ID NO.4)、SSGAPPPS(SEQ ID NO.5)、GPSSGAPPS(SEQ ID NO.6)、PSSGAPPS(SEQ ID NO.7)或KRNRNN IA(SEQ ID NO.8)之任一。
进一步地，所述A的氨基酸序列如SEQ ID NO.44～92之任一所示。
式I中，所述的B为天然FGF21(SEQ ID NO.136)或者FGF21类似物。所述B包括如下结构：
HPIPDSSPLLQFGGQVRQ X 19YLYTDDAQQTE X 31HLEI X 36EDGTVG X 43A X 45DQSPESLLQL X 56ALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRE X 98LLEDGYNVYQSEAHGLPLH X 118PGN X 122SPHRDPAPRGP X 134RFLPLPGLPPALPEPPGILAPQPPDVGSSDPL X 1 67MV X 170 X 171SQ X 174RSPS X 179 X 180 X 181，其中，N端HPIPDSS可缺失或部分缺失；X 19选自R、Y、V、E或C；X 31选自A或C；X 36选自R或K；X 43选自G或C；X 45选自A、K、E或V；X 56选自K、R、V或I；X 98选自L、R或D；X 118选自L或C；X 122选自K或R；X 134选自A或C；X 167选自S、A或R；X 170选自G或E；X 171选自P或G；X 174选自G、A或L；X 179选自Y、A或F；X 180选自A或E；X 181选自S、K或缺失。
所述的FGF21类似物为与天然FGF21(SEQ ID NO.136)具有相同或相似生物学功能、且与天然FGF21(SEQ ID NO.136)同源性为80％以上的活性蛋白。优选地，所述FGF21 类似物与天然FGF21(SEQ ID NO.136)同源性为85％以上；更优选地，所述FGF21类似物与天然FGF21(SEQ ID NO.136)同源性为90％以上；更优选地，所述FGF21类似物与天然FGF21(SEQ ID NO.136)同源性为95％以上。示例性地，所述的FGF21类似物可选自如US20140213512、US8188040、US9493530、WO 2016114633、US 20150291677、US9422353、US 8541369、US7622445、US7576190、US20070142278、US9006400或US20130252884等专利或专利申请中所描述的FGF21类似物或突变体。
优选地，所述FGF21类似物如SEQ ID NO.137-148所示。
进一步地，所述F为源自哺乳动物免疫球蛋白的F C部分。所述免疫球蛋白是含有二硫键的多肽链分子，一般具有两条轻链和两条重链。此处用到的免疫球蛋白的F C部分具有免疫学领域术语的常用含义。具体的，该术语是指通过从抗体中去除两个抗原结合区(Fab片段)而得到的抗体片段。F C部分可以包括铰链区并且延伸通过CH 2和CH 3结构域到达抗体C末端。F C部分可以进一步包括一个或多个糖基化位点。人体有5种具有不同效应特征和药代动力学特性的人免疫球蛋白：IgG、IgA、IgM、IgD和IgE。IgG是血清中含量最高的免疫球蛋白。IgG在所有的免疫球蛋白中也是血清半衰期最长的(约23天)。
进一步地，F可选自免疫球蛋白的完整F C部分、免疫球蛋白的F C部分的片段或免疫球蛋白的F C部分的突变体。
用于本发明的免疫球蛋白F C部分来源于哺乳动物IgG1、IgG2或IgG4的F C区或其突变体；优选的，可以是来源自人的IgG1、IgG2或IgG4的F C区或其突变体；更优选的，可以来源自人IgG1或IgG4的F C区或其突变体。在一个优选的实施方案中，F C结构域的第297位被甘氨酸或丙氨酸替换。上述内容是依照kabat的EU索引编号(kabat,E.A.等，sequences of proteins of immunological interest，第五版，public health service,National Institutes of Health,Bethesda,MD(1991))。
在一个优选的实施方案中，F C结构域来自人IgG4，并如SEQ ID NO.16所示。在一个优选的实施方案中，所述F C结构域来自人IgG1，如SEQ ID NO.12所示。所述F C链末端的K可去除，便于提高表达产物的均一性。
在一些实施方式中，所述F的氨基酸序列可以如SEQ ID NO.9～18之任一所示。
本发明上述多结构域活性蛋白中，所述GCGR/GLP-1R双效激动活性肽A通过长效蛋白单元F与天然FGF21或FGF21类似物B融合，形成具有至少GLP-1、GCG和FGF21三效活性的重组融合蛋白。A与F之间可以通过连接链连接，如L a，也可以不加入连接链而直接融合。F与B之间可以通过连接链连接，如L b，也可以不加入连接链而直接融合。
当L a和L b为连接链时，所述连接链为一段合适长度的由甘氨酸(G)、丝氨酸(S)和/或丙氨酸(A)构成的柔性多肽，从而使相邻的蛋白质结构域可相对于彼此自由移动。当有必要保证所述两个相邻结构域空间上彼此互不干扰时，可以使用较长的连接链。
本发明优选的连接链包括含富含G、S和/或A的单元，示例性的如(GS)n，(GGS)n，(GGSG)n，(GGGS)nA，(GGGGS)nA、(GGGGA)nA、等，n为1-10的整数，在一个优选的实施方案中，所述的连接链的氨基酸长度为5-26。示例性的连接链各自独立的选自SEQ ID NO.19～41。
进一步地，所述连接链的氨基酸序列可如SEQ ID NO.19～41之任一所示。
进一步地，所述多结构域活性蛋白的氨基酸序列如SEQ ID NO.150～208之任一所示。
本发明提供的多结构域活性蛋白是一种F C融合蛋白，保留了F C的常规特性，如与FcRn结合而延长体内半衰期，以及在分离纯化过程中通过与Protein A或G类填料高亲和力和高特异性地结合而达到高效纯化目的的性质。另外，这些多结构域活性蛋白除了能有效地抵抗血清内的蛋白酶对蛋白内部的降解外，还能有效地防止N端的降解。对于Incretin类多肽如Glucagon或GLP-1而言，N端的完整性是决定其生物学活性的关键。天然Glucagon和GLP-1在体内的半衰期短，除了分子量小这个原因外，更重要的是由于受体内的DPP-IV酶水解及多肽内部的水解。在本发明的一个实施例中，天然Glucagon与F C融合后，依然会被DPP-IV快速地降解而失活；而对应的Glucagon类似物却能明显地抵抗DPP-IV的攻击。
本发明的第二方面，提供一种分离的多核苷酸，所述分离的多核苷酸编码前述多结构域活性蛋白。
本发明的第三方面，提供一种重组表达载体，包含前述分离的多核苷酸。
本发明的第四方面，提供一种宿主细胞，所述细胞含有前述重组表达载体或基因组中整合有外源的前述分离的多核苷酸。
本发明的第五方面，提供前述多结构域活性蛋白的制备方法，包括在合适的条件下培养前述宿主细胞，使之表达所述多结构域活性蛋白，而后分离及纯化获得所述多结构域活性蛋白。
本发明的第六方面，提供前述多结构域活性蛋白在制备治疗糖尿病代谢相关疾病的药物中的用途。
本发明提供的多结构域活性蛋白可以用于治疗代谢综合征。代谢综合征的特征通常在于簇集至少三种以上的以下风险因素：(1)腹部肥胖(腹部内或周围脂肪组织过多)，(2)致动脉粥样硬化血脂异常，血脂失调，包括高甘油三酯、低HDL胆固醇及高LDL胆固醇， 其增强动脉壁中斑块的积累，(3)血压升高，(4)胰岛素抗性或葡萄糖耐受不良，(5)血栓样状态，例如血液中高纤维蛋白或纤溶酶原活化因子抑制剂-1，以及(6)促发炎状态，例如血液中C反应性蛋白升高。其他风险因素可包括老化、激素失衡及遗传因素。
此外，本发明的多结构域活性蛋白还可用于治疗肥胖症。在一些方面中，本发明的多多结构域活性蛋白通过降低食欲、减少食物摄取、降低患者体内脂肪水平、提高能量消耗等机制来治疗肥胖症。
本发明的第七方面，提供一种治疗代谢相关疾病的方法，包括向对象施用前述多结构域活性蛋白。
本发明进一步提供一种促进体重减少或者防止体重增加的方法，包括在对象中施用所述的多结构域活性蛋白。
本发明的第八方面，提供一种组合物，含有前述多结构域活性蛋白或前述宿主细胞的培养物，以及药学上可接受的载体。
本发明的第九方面，提供前述多结构域活性蛋白在制备融合蛋白中的用途。
本发明的第十方面，提供一种融合蛋白，其结构中含有前述多结构域活性蛋白。
本发明的第十一方面，提供另外一种治疗代谢及相关疾病的组合物，包括GCGR/GLP-1R双效激动活性蛋白和长效FGF21类似物。
所述GCGR/GLP-1R双效激动活性蛋白的结构中包括：A-L a-F，所述A的结构中包括如式II所示结构，如式II所示结构为：
HSQGTFTSD-X 10-S-X 12-X 13-X 14-X 15-X 16-X 17-X 18-X 19-X 20-X 21-F-X 23-X 24-WL-X 27-X 28-X 29-X 30-X z。
其中，所述X 10选自V、L或Y之任一；所述X 12选自S、E或K之任一；所述X 13选自Y或Q之任一；所述X 14选自L或M之任一；所述X 15选自D或E之任一；所述X 16选自S、E或G之任一；所述X 17选自R、E或Q之任一；所述X 18选自R、E或A之任一；所述X 19选自A或V之任一；所述X 20选自Q、R或K之任一；所述X 21选自D、L或E之任一；
所述X 23选自V或I之任一；所述X 24选自Q、A或E之任一；所述X 27选自M、K或V之任一；所述X 28选自N或K之任一；所述X 29选自G或T之任一；所述X 30为G或缺失；所述X z不存在或选自GPSSGAPPPS(SEQ ID NO.3)、PSSGAPPPS(SEQ ID NO.4)、SSGAPPPS(SEQ ID NO.5)、GPSSGAPPS(SEQ ID NO.6)、PSSGAPPS(SEQ ID NO.7)或KRNRNN IA(SEQ ID NO.8)之任一。
所述F为长效蛋白单元，F可选自免疫球蛋白的完整F C部分、免疫球蛋白的F C部分的片段或免疫球蛋白的F C部分的突变体。进一步地，所述F的氨基酸序列如SEQ ID NO.9-18所示。
L a不存在或为连接链，当为连接链时，所述连接链为一段合适长度的由甘氨酸(G)、丝氨酸(S)和/或丙氨酸(A)构成的柔性多肽，从而使相邻的蛋白质结构域可相对于彼此自由移动。当有必要保证所述两个相邻结构域空间上彼此互不干扰时，可以使用较长的连接链。所述连接链示例性的如(GS)n，(GGS)n，(GGSG)n，(GGGS)nA，(GGGGS)nA、(GGGGA)nA等，n为1-10的整数。示例性的连接链可各自独立的选自SEQ ID NO.19～41。
进一步地，所述GCGR/GLP-1R双效激动活性蛋白的氨基酸序列如SEQ ID NO.96、SEQ ID NO.98、SEQ ID NO.100、SEQ ID NO.102、SEQ ID NO.104、SEQ ID NO.106、SEQ ID NO.108、SEQ ID NO.110、SEQ ID NO.112、SEQ ID NO.114-133之任一所示。
所述长效FGF21类似物的结构中包括：F-L b-B，所述B的结构中包括：
HPIPDSSPLLQFGGQVRQ X 19YLYTDDAQQTE X 31HLEI X 36EDGTVG X 43A X 45DQSPESLLQL X 56ALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRE X 98LLEDGYNVYQSEAHGLPLH X 118PGN X 122SPHRDPAPRGP X 134RFLPLPGLPPALPEPPGILAPQPPDVGSSDPL X 1 67MV X 170 X 171SQ X 174RSPS X 179 X 180 X 181，其中，N端HPIPDSS可缺失或部分缺失；X 19选自R、Y、V、E或C；X 31选自A或C；X 36选自R或K；X 43选自G或C；X 45选自A、K、E或V；X 56选自K、R、V或I；X 98选自L、R或D；X 118选自L或C；X 122选自K或R；X 134选自A或C；X 167选自S、A或R；X 170选自G或E；X 171选自P或G；X 174选自G、A或L；X 179选自Y、A或F；X 180选自A或E；X 181选自S、K或缺失。
所述的FGF21类似物为与天然FGF21(SEQ ID NO.136)具有相同或相似生物学功能、且与天然FGF21(SEQ ID NO.136)同源性为80％以上的活性蛋白。优选地，所述FGF21类似物与天然FGF21(SEQ ID NO.136)同源性为85％以上；更优选地，所述FGF21类似物与天然FGF21(SEQ ID NO.136)同源性为90％以上；更优选地，所述FGF21类似物与天然FGF21(SEQ ID NO.136)同源性为95％以上。示例性地，所述的FGF21类似物可选自如US20140213512、US8188040、US9493530、WO 2016114633、US 20150291677、US9422353、US 8541369、US7622445、US7576190、US20070142278、US9006400或US20130252884等专利或专利申请中所描述的FGF21类似物或突变体。所述FGF21类似物如 SEQ ID NO.137-148所示。
所述F可选自免疫球蛋白的完整F C部分、免疫球蛋白的F C部分的片段或免疫球蛋白的F C部分的突变体。所述F的氨基酸序列可以如SEQ ID NO.9～18之任一所示。L b不存在或为连接链，当L b为连接链时，所述连接链包括含富含G、S和/或A的单元，示例性的如(GS)n，(GGS)n，(GGSG)n，(GGGS)nA，(GGGGS)nA、(GGGGA)nA、等，n为1-10的整数，在一个优选的实施方案中，所述的连接链的氨基酸长度为5-26。示例性的连接链各自独立的选自SEQ ID NO.19～41。
进一步地，所述长效FGF21类似物的氨基酸序列如SEQ ID NO:210-221之任一所示。
本发明的第十二方面，提供前述组合物在制备治疗代谢相关疾病药物中的用途。
本发明的第十三方面，提供另外一种治疗代谢相关疾病的方法，包括向对象施用前述包括GCGR/GLP-1R双效激动活性蛋白和长效FGF21类似物的组合物。
本发明进一步提供一种促进体重减少或者防止体重增加的方法，包括在对象中施用前述包括GCGR/GLP-1R双效激动活性蛋白和长效FGF21类似物的组合物。
众所周知，肠降血糖素(Incretin)类激素蛋白如GLP-1类似物、Exendin-4等会让患者产生恶心、呕吐等副作用，而且是剂量相关的。因此在能维持理想的血糖水平的情况下，尽量减少给药剂量理论上会缓解患者的不适副作用。另外，FGF21对于骨质疏松及生殖影响方面的报道也时有报道(Fibroblast growth factor-21concentration in serum and synovial fluid is associated with radiographic bone loss of knee osteoarthritis.Scand J Clin Lab Invest.2015 Apr；75(2):121-5；Fibroblast growth factor 21 has no direct role in regulating fertility in female mice.Mol Metab.5(8):690-8，2016)。理论上，药物副作用产生的风险与给药剂量成正比，在糖尿病药物领域，对药物的安全性极高。发明人发现GCG/GLP-1/FGF21三效活性蛋白以及上述组合物在很低剂量下就有很好的控制血糖和体重的效果，并且对胃肠道影响小，大大地减少了FGF21的剂量，因而显著降低起潜在副作用风险。
与现有技术相比，本发明具有如下有益效果：
(1)本发明的多结构域活性蛋白半衰期长、支持一周一次的给药频率；
(2)本发明的多结构域活性蛋白的GLP-1R激动活性最高提高到了200倍以上；
(3)本发明的多结构域活性蛋白体内外稳定性好，且免疫原性低；
(4)由于不需要引入非天然氨基酸，无需涉及化学合成及交联步骤，因此可通过重组 方法制备，极大地简化了制备工序。
附图说明
图1为纯化获得的部分三效活性蛋白还原电泳图(10％SDS-PAGE)，泳道1-13分别为C002L 13F 4L 10W、C240L 12F 8L 12M 1、C495L 13F 8L 10M 1、C266L 13F 7L 13M 4、C462L 9F 2L 9M 10、C611L 11F 4L 11M 11、C563L 14F 8L 9M 3、C382L 13F 8L 10M 2、C623L 9F 3L 10M 1、C731L 5F 2L 9M 9、C353L 13F 3L 10M 4、C227L 12F 5L 14M 4、C137L 10F 8L 9M 5；M为蛋白标准品：97.2、66.4、44.3、29、20.1、14.3KD。
图2A：血清稳定性随时间变化的结果图。
图2B：血清稳定性随时间变化的结果图。
图2C：血清稳定性随时间变化的结果图。
图3：实施例8中的活性蛋白在正常ICR小鼠体内的降糖效果图。
图4：实施例9中的活性蛋白对DIO小鼠体重的影响作用。
图5：实施例9中的活性蛋白对DIO小鼠食欲的影响作用；以PBS组DIO鼠的摄食量为100％，纵坐标为其他组的摄食量与之相比的百分比。
图6：实施例10中的活性蛋白对DIO小鼠体重的影响作用。
图7：实施例10中的活性蛋白对DIO小鼠食欲的影响作用；以PBS组DIO鼠的摄食量为100％，纵坐标为其他组的摄食量与之相比的百分比。
图8：实施例11中的活性蛋白血清稳定性随时间变化的结果图。
图9：实施例11中的活性蛋白在正常ICR小鼠体内的降糖效果图。
图10：实施例11中的活性蛋白对DIO小鼠体重的影响作用。
图11：实施例11中的活性蛋白对DIO小鼠食欲的影响作用；以PBS组DIO鼠的摄食量为100％，纵坐标为其他组的摄食量与之相比的百分比。
图12：三效活性蛋白在瘦素受体缺陷二型糖尿病(dbdb)小鼠中降糖效果图。
具体实施方式
术语解释：
术语“糖尿病”包括一型糖尿病、二型糖尿病、妊娠糖尿病以及引起高血糖症的其他症状。该术语用于代谢紊乱，其中胰腺产生不了足够的胰岛素，或身体的细胞未能适当响应 胰岛素，因此组织细胞吸收葡萄糖效率下降导致葡萄糖在血液中积累。
一型糖尿病也称为胰岛素依赖性糖尿病和幼年发病型糖尿病，通过β细胞破坏引起，通常导致绝对胰岛素缺乏。
二型糖尿病也称为非胰岛素依赖性糖尿病和成年发病型糖尿病，普遍与胰岛素抗性相关。
术语“肥胖”意指脂肪组织的过量，当能量摄取超过能量消耗时，过量卡路里贮存于脂肪中，则导致肥胖。在本文中体重指数(BMI＝体重(千克)除以身高(米)的平方)超过25的个体视为肥胖。
肠降血糖素(Incretin)：肠降血糖素是通过增强葡萄糖刺激的胰岛素分泌(亦称为葡萄糖依赖的胰岛素分泌，GSIS)来调控血糖的胃肠激素(Drucker.D J,Nauck,MA,Lancet368:1696-705,2006)。肠降血糖素还可通过延缓胃排空的方式减慢营养吸收的速度并直接地减少食物吸收。同时，肠降血糖素还抑制肠道α细胞分泌胰高血糖素(Glucagon)。迄今为止有两种已知的肠降血糖素：胰高血糖素样肽-1(GLP-1)和葡萄糖依赖性促胰岛素多肽(GIP)。
前胰高血糖素原(preproGlucagon)：是158个氨基酸组成的前体多肽，其在组织中被差异性加工而形成多种结构上相关的胰高血糖素原衍生肽，包括胰高血糖素(Glucagon)、胰高血糖素样肽-1(GLP-1)、胰高血糖素样肽-2(GLP-2)和胃泌酸调节素(Oxyntomodulin，OXM)。
GIP：是由133个氨基酸的前体(pre-pro-GIP)通过蛋白水解加工得到的42个氨基酸的肽，这些分子参与多种生物功能，包括葡萄糖体内平衡、胰岛素分泌、胃排空和肠生长以及食物摄取调节。
胰高血糖素样肽(GLP-1)：序列如SEQ ID NO：1所示；是从肠L-细胞分泌的30或31个氨基酸的多肽肠促胰岛素激素，有GLP-1(7-36)和GLP-1(7-37)两种活性形式。GLP-1在进餐后释放到循环中，并通过激活GLP-1受体发挥生物活性。GLP-1具有许多生物学作用，包括葡萄糖依赖性的促胰岛素分泌，抑制胰高血糖素生成，延缓胃排空和抑制食欲(Tharakan G,Tan T,Bloom S.Emerging therapies in the treatment of‘diabesity’:beyond GLP-1.Trends Pharmacol Sci 2011；32(1):8-15.)等。天然GLP-1由于能够被二肽基肽酶-4(DPP-4)，中性肽链内切酶(NEP)，血浆激肽释放酶或纤溶酶等快速降解因而限制了其治疗潜力。由于天然GLP-1在体内仅有大约2分钟的超短半衰期，因此，出现了通过利用化学修饰和/或制剂形式来改善功效以治疗糖尿病和肥胖症的方法(Lorenz M,Evers A, Wagner M.Recent progress and future options in the development of GLP-1 receptor agonists for the treatment of diabesity.Bioorg Med Chem Lett 2013；23(14):4011-8；Tomlinson B,Hu M,Zhang Y,Chan P,Liu ZM.An overview of new GLP-1 receptor agonists for type 2 diabetes.Expert Opin Investig Drugs 2016；25(2):145-58)。
胃泌酸调节素(Oxyntomodulin)是37个氨基酸的小肽，序列如SEQ ID NO：2所示；其包含胰高血糖素Glucagon(SEQ ID NO：42)完整的29个氨基酸序列。胃泌酸调节素是GLP-1R和GCGR的双重激动剂，在进餐后通过肠L-细胞与GLP-1一起分泌。与胰高血糖素Glucagon类似，胃泌酸调节素在人和啮齿动物中产生显著的体重减轻。胃泌酸调节素的减肥活性已在肥胖小鼠中与等摩尔剂量的选择性GLP-1激动剂进行比较。已经发现，与选择性的GLP-1R激动剂相比，胃泌酸调节素具有抗高血糖作用，能够显著的减轻体重和具有降脂活性(The Glucagon receptor is involved in mediating the body weight-lowering effects of oxyntomodulin，Kosinski JR等，Obesity(Silver Spring)，20):1566-71，2012)。在超重和肥胖患者中，皮下施用天然胃泌酸调节素在四周内减少体重1.7公斤。胃泌酸调节素也被证明可以减少人类的食物摄取和增加能量消耗(Subcutaneous oxyntomodulin reduces body weight in overweight and obese subjects:a double-blind,randomized,controlled trial，Wynne K等，Diabetes，54:2390-5，2005；Oxyntomodulin increases energy expenditure in addition to decreasing energy intake in overweight and obese humans:a 12andomized controlled trial；Wynne K等，Int J Obes(Lond)，30:1729-36，2006)。但是同样由于分子量偏小及DPP-IV的降解，胃泌酸调节素具有较短的半衰期。目前GLP-1受体(GLP-1R)和胰高血糖素受体(GCGR)的双效激动剂普遍都是基于胃泌酸调节素的，并且为了改善胃泌酸调节素的短效及酶解的缺陷而做了突变(胃泌酸调节素类似物)，且大都采用第二位丝氨酸Ser突变为α-氨基异丁酸(Aib)的方法，通过引入非天然氨基酸来抵抗DPP-IV的酶解。胃泌酸调节素类似物虽然表现出了初步的降糖及减脂效果，但是其作用机制仍不确切，胃泌酸调节素受体一直没有发现，目前仅仅是通过GCGR或GLP-1R敲除的小鼠或者细胞试验验证胃泌酸调节素能与这2种受体结合而起作用。
胰高血糖素(Glucagon)是29个氨基酸的肽，其对应于前胰高血糖素原的53-81位氨基酸，序列如SEQ ID NO：42所示(C.G.Fanelli等，Nutrition,Metabolism&Cardiovascular Diseases(2006)16,S28-S34)。胰高血糖素受体活化已显示在啮齿类动物和人两者中增加能量消耗且减少食物摄入(Habegger K.M.等人，the metabolic actions of Glucagon revisited,Nat.Rev.Endocrinol.2010,6,689-697)并且这些效应在啮齿类动物中是稳定和持续的。胰高血糖 素具有许多生理效应，例如通过刺激糖原分解和糖异生，增加低血糖状况下的血糖水平，调节肝酮生成，调节胆汁酸代谢和通过迷走神经的饱腹效应。在治疗上，胰高血糖素已经用于急性低血糖症，胰高血糖素受体激活减少食物摄取并促进动物和人的脂肪分解和体重减轻。
术语“受体激动剂”可以定义为与受体结合且引发天然配体的通常应答的多肽、蛋白或其他小分子。
“GLP-1受体(GLP-1R)激动剂”可以定义为与GLP-1R结合且能够引发与天然GLP-1相同或类似的特征性反应的多肽、蛋白或其他小分子。GLP-1R激动剂通过完全或部分激活GLP-1R，继而引起一系列细胞内的下游信号通路反应，产生相应的细胞活性：如β细胞分泌胰岛素；典型的GLP-1R激动剂包括天然GLP-1及其突变体、类似物，如埃塞那肽、利拉鲁肽(Liraglutide)等。
GLP-1类似物：在本文中，“GLP-1类似物”或“GLP-1突变体”皆意为GLP-1R激动剂，可互相通用。
胰高血糖素受体(GCGR)激动剂:即Glucagon受体激动剂，可以定义为与GCGR结合且能够引发与天然胰高血糖素(Glucagon)相同或类似特征性反应的多肽、蛋白或其他小分子。GCGR激动剂通过完全或部分激活GCGR，继而引起一系列细胞内的下游信号通路反应，产生相应的细胞活性：如肝细胞糖原分解、糖质新生、脂肪酸氧化及生酮作用等。
Glucagon类似物：在本文中，“Glucagon类似物”、“GCG类似物”、“Glucagon突变体”和“GCG突变体”皆意为Glucagon受体激动剂，可互相通用。
GCGR/GLP-1R双效活性肽：本发明的GCGR/GLP-1R双效活性肽包括能同时激动GLP-1R和GCGR的蛋白或多肽。如Alessandro Pocai等报道的基于Oxyntomodulin的双效激动剂(Glucagon-Like Peptide 1/Glucagon Receptor Dual Agonism Reverses Obesity in Mice，Diabetes；58(10):2258-2266，2009)，或者Richard D.DiMarchi等报道的基于Glucagon的双效激动剂(US9018164B2)。在本文中，“双效激动剂“或“双特异性活性蛋白”或“双效活性蛋白”皆同义。
FGF21(Fibroblast growth factor 21，成纤维细胞生长因子21)以及FGF15/19、FGF23，同属于FGF家族“内分泌”激素。FGF21是调节葡萄糖和脂质代谢的重要激素。与胰岛素调节的机制不同，FGF21通过上调节GLUT1的表达促进脂肪细胞中的葡萄糖摄取。FGF21与受体结合需跨膜蛋白β-Klotho辅助，通过结合FGFR/β-Klotho受体复合物从而刺激信号传导，激发肝、脂肪组织和胰腺的生物学作用。β-Klotho在胰腺、肝脏、脂肪中选择性表 达，这也解释了FGF21对这些组织作用的特异性(Kurosu H等，Tissue-specific expression of betaKlotho and fibroblast growth factor(FGF)receptor isoforms determines metabolic activity of FGF19 and FGF21.J Biol Chem 282:26687-26695，2007；Kharitonenkov A等，(2008b)FGF-21/FGF-21receptor interaction and activation is determined by betaKlotho.J Cell Physiol 215:1-7)。在辅助受体β-Klotho存在下，FGF21可以结合并激活3种FGFR亚型(1c，2c和3c)，其他FGFR亚型，例如FGFR1b，2b和3b，由于不与β-Klotho形成复合物，因此不作为FGF21受体。证据表明，在结合FGF21的FGFR受体中，FGFR1在调节FGF21活性中占主要地位。FGF21的N和C末端对于其生物活性非常重要的，其中N端结合FGFR，C端结合β-Klotho(Micanovic R,et al(2009)Different roles of N-and C-termini in the functional activity of FGF21.J Cell Physiol 219:227-234)。小鼠FGF21蛋白由210个氨基酸组成，氨基端有30个氨基酸组成的信号肽，人FGF21蛋白由209个氨基酸组成，氨基端有28个氨基酸组成的信号肽，人FGF21蛋白与小鼠具有约75％的同源性。FGF21主要表达于胰腺β细胞、肝脏、WAT、骨骼肌，有明显的组织特异性。人FGF21在体内容易被脯氨酰肽酶(FAP，一种丝氨酸蛋白酶)降解，在小鼠体内的半衰期为30min，猴子体内2h。
多结构域：结构域是生物大分子中具有特异结构和独立功能的区域，特别指蛋白质中这样的区域。在球形蛋白中，结构域具有自己特定的三级结构,其功能不依赖于蛋白质分子中的其余部分，但是同一种蛋白质中不同结构域间常可通过不具二级结构的短序列连接起来。蛋白质中不同的结构域组成了多结构域。在本发明中，多结构域指包含了GCG类似物、FGF21及其类似物与FC形成的具有GCGR激动活性、GLP-1R激动活性及FGF21活性的融合蛋白。
二聚体：本发明所指的二聚体是通过免疫球蛋白的恒定区(F C)天然的非共价及共价作用而形成。如果没有其他特别指出，F C形成的二聚体皆为同二聚体，如本发明提供的二聚体所述。本发明中式I所述的活性蛋白，由于具有F C，因而都会形成二聚体。
三效活性蛋白：在本文中，“三效激动活性蛋白”、“三活性激动活性蛋白”、“三特异性二聚体活性蛋白”等皆同义，可互换使用。
EC 50(concentration for 50％of maximal effect)是指某一药物或者物质在刺激其相应的生物学反应的50％时所需的浓度。EC 50值越低，表明该药物或物质的刺激或激动能力越强，例如，更直观地可表现为引起的细胞内信号越强，从而诱导某激素产生的能力越佳。
细胞生物学活性
本发明的GLP-1R和GCGR激动活性体外细胞活性测定都采用荧光素酶报告基因检测法。此法基于GLP-1R和GCGR激动后能激活下游cAMP途径的原理。FGF21及其类似物的活性测定则是通过将FGF21R与β-klotho共转染同一个CHO细胞，检测信号引起的荧光变化而获得。
Joseph R.Chabenne等和Richard D.DiMarchi等曾报道，在Glucagon的C末端增加一段Exendin-4的C末端小肽cex(GPSSGAPPPS)能使GLP-1R的激动活性提高2倍左右(Optimization of the Native Glucagon Sequence for Medicinal Purposes，J Diabetes Sci Technol.4(6):1322–1331，2010及专利US9018164 B2)，但是GCGR激动活性与GLP-1R激动活性的比例仅达到35：1左右。另外，Evers A等报道(Evers A等，Design of Novel Exendin-Based Dual Glucagon-like Peptide 1(GLP-1)/Glucagon Receptor Agonists，J Med Chem.；60(10):4293-4303.2017)在GCG类似物的C末端加上cex序列后，GLP-1R激动活性反而下降了原来的3倍左右，GCG的活性下降了14倍左右(文章中表2，肽7和8)。
在本发明的一个实施例中，将含GPSSGAPPPS或类似序列的GCG类似物进一步融合至F C链时，GLP-1R激动活性提高了惊人的200倍以上(EC 50约1.1nM)。而根据US9018164B2及Joseph R.Chabenne等在相应专利及文献中披露的数据计算获得的GCG类似物，其GLP-1R激动活性比例在增加GPSSGAPPPS或类似序列前后变化仅在2倍左右(如文章中天然Glucagon的GLP-1R激动活性百分比为0.7％，增加了GPSSGAPPPS序列后提高至1.6％)。就是说，在Glucagon多肽的C末端增加GPSSGAPPPS序列并不会显著提高其GLP-1R的激动活性。
多结构域活性蛋白的稳定性
天然Glucagon有多处敏感的降解位点，包括第二位的DPP-IV降解位点，以及16-18位的SRR位点。尽管有报道认为F C能提高活性蛋白的化学稳定性及血清稳定性，然而对于N端必须暴露的GLP-1或Glucagon类似物，F C的作用似乎是无法一概而论的。天然的GLP-1或Glucagon与F C融合后，仍能观察到其在37度血清条件下的明显降解。本发明在天然Glucagon的基础上引入抵抗蛋白酶水解的突变，以增加其稳定性。这些突变体与F C融合后，稳定性进一步获得提高。
目前几乎所有基于Oxyntomodulin和Glucagon设计开发的GCGR/GLP-1R双效激动剂都在第二位上引入抵抗DPP-IV的突变，如L型突变为D型氨基酸(L-Ser突变为D-Ser)，或引入非天然氨基酸Aib等(Matthias H.
Figure PCTCN2018116244-appb-000007
等，Unimolecular Polypharmacy for Treatment of Diabetes and Obesity，24：51–62,2016)。然而，在本发明的实施例中，符合式II的且第二位保存天然L-Ser的双效活性蛋白呈现出非常高的血清稳定性，在24小时仍并没有显著的 DPP-IV降解的迹象，而没有融合F C的对应多肽则快速地被DPP-IV水解(表4)。本发明人制备了天然Glucagon与F C融合的活性蛋白C001L 13F 8(SEQ ID NO.93)以及Joseph R.Chabenne等报道的Glucagon-cex与F C融合的活性蛋白C002L 13F 8(SEQ ID NO.94)作为对照验证是否F C融合提高了稳定性。然而，C001L 13F 8(SEQ ID NO.93)和C002L 13F 8(SEQ ID NO.94)均无呈现出明显的抵抗DPP-IV的迹象。虽然有报道指出结合在血清白蛋白(如HSA)上可能会有助于提高蛋白的稳定性(如利拉鲁肽)，然而如果第二位不进行突变，根本无法使半衰期持续12小时以上，即不可能支持一周一次的给药频率。而本发明提供的GCG类似物药代及药效试验显示足以支持一周一次的给药频率，而非普遍报道的一天一次(如结合白蛋白的利拉鲁肽)。天然氨基酸的保留进一步减低免疫原性的风险，避免了化学交联还使制备过程更简易方便。
腹腔糖耐量测试(IPGTT)
在其中一个实施例中，进行了IPGTT实验。施用了多结构域活性蛋白的小鼠在注射葡萄糖后，呈现出极平稳的血糖波动。
DIO小鼠的减重及食欲控制、药代实验
GCGR激动剂具有潜在的减重效果已有多篇报道。然而天然Glucagon由于易被降解且分子量极小，因此成药的潜力极小。目前Glucagon类似物主要用于急性的低血糖症状。长效GCG类似物用于糖尿病患者减重的临床报道也不断涌现。众所周知，肥胖是导致糖尿病患者胰岛素抵抗的原因之一，而体重减轻量是评价一个降糖药物的重要指标。此外，本发明的多结构域活性蛋白在DIO小鼠施用后，诱导了体重的显著降低。GCGR/GLP-1R双效激动剂、FGF21及其类似物在降低血糖及脂质代谢中的作用已广为人知。Stanislaus S等报道(Stanislaus S等，A novel Fc FGF21with improved resistance to proteolysis,increased affinity towardsβ-Klotho and enhanced efficacy in mice and cynomolgus monkeys Endocrinology.2017May 1；158(5):1314-1327.)F C-FGF21类似物在3mg/kg(约30nM/kg)的剂量下，在连续4周给药后，体重减轻为15％以上。在本发明的实施例9中，30nM/kg剂量的三效活性蛋白减重达到约30％，而食欲则基本无明显变化。同样地，10nM/kg剂量时，减重也接近15％。另外，实施例10中的双效活性蛋白与长效FGF21类似物的组合物(A-L a-F+F-L b-B)，也共同起到了协同作用的效果：联合给药的组合物可以减少35％或以上体重，然而等剂量的双效活性蛋白只能减轻不超过13％的体重，长效FGF21类似物对体重的减轻则在10％以下。
临床应用展望
在临床上，本发明的多重活性蛋白具有潜在的适合每周或以上施用一次的药物代谢动力学特性。剂量取决于施用频率和模式，受治疗的受试者的年龄、性别、重量和一般状况，治疗的状况和严重性，待治疗的任何伴随性疾病以及对于本领域技术人员显而易见的其他因素。同时，根据受治疗者的情况和其他病理状况，本发明的多重活性蛋白可以与一种或多种其他的治疗活性化合物或物质组合施用或应用，例如可以选择的其他的治疗活性化合物包括但不限于抗糖尿病药、抗高血脂药、抗肥胖药，抗高血压药和用于治疗起因于糖尿病或与糖尿病有关的并发症的试剂。
代谢综合征与冠心病和血管斑块积累相关的其他病症风险增加相关，例如中风和外周血管疾病，成为粥样动脉硬化心血管病(ASCVD)。患有代谢综合征的患者可自处于早期的胰岛素抗性状态发展成完全成熟的二型糖尿病，并且ASCVD的风险进一步增加。不旨在受限于任何特定理论，胰岛素抗性、代谢综合征及血管疾病之间的关系可以涉及一种或多种共同发病机制，包括胰岛素刺激的血管舒张障碍、由氧化应力增强所致的胰岛素抗性相关性可用性降低，以及脂肪细胞衍生激素(诸如脂联素)异常(Lteif,Mather,Can.J.Cardiol.20(增刊B)：66B-76B，2004)
本发明的活性蛋白还可用于治疗肥胖症。在一些方面中，本发明的活性蛋白通过降低食欲、减少食物摄取、降低患者体内脂肪水平、提高能量消耗等机制来治疗肥胖症。
在一些潜在的实施方案中，本发明的活性蛋白可用于治疗非酒精性脂肪肝病(NAFLD)。NAFLD是指广谱肝脏疾病，范围自单纯脂肪肝(脂肪变性)至非酒精性脂肪变性肝炎(NASH)至肝硬化(肝脏的不可逆晚期瘢痕形成)。NAFLD的所有病期均具有在肝脏细胞中的脂肪累积。单纯脂肪肝为某类型的脂肪、甘油三酯在肝脏细胞中异常积累，但无发炎或瘢痕形成。在NASH中，脂肪累积与不同程度的肝脏发炎(肝炎)和瘢痕形成(纤维化)相关。发炎性细胞可破坏肝脏细胞(肝细胞坏死)。在术语“脂肪变性肝炎”和“脂肪变性坏死”中，脂肪变性是指脂肪浸润，肝炎是指肝脏中的炎症，并且坏死是指经破坏的肝脏细胞。NASH可最终导致肝脏瘢痕形成(纤维化)且接着导致不可逆晚期瘢痕形成(肝硬化)，由NASH导致的肝硬化为NAFLD谱内的最后且最严重的病期。
在进一步描述本发明具体实施方式之前，应理解，本发明的保护范围不局限于下述特定的具体实施方案；还应当理解，本发明实施例中使用的术语是为了描述特定的具体实施方案，而不是为了限制本发明的保护范围。下列实施例中未注明具体条件的试验方法，通常按照常规条件，或者按照各制造商所建议的条件。
当实施例给出数值范围时，应理解，除非本发明另有说明，每个数值范围的两个端点以及两个端点之间任何一个数值均可选用。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。
除非另外说明，本发明中所公开的实验方法、检测方法、制备方法均采用本技术领域常规的分子生物学、生物化学、染色质结构和分析、分析化学、细胞培养、重组DNA技术及相关领域的常规技术。这些技术在现有文献中已有完善说明，具体可参见Sambrook等MOLECULAR CLONING：A LABORATORY MANUAL，Second edition，Cold Spring Harbor Laboratory Press，1989 and Third edition，2001；Ausubel等，CURRENT PROTOCOLS IN MOLECULAR BIOLOGY，John Wiley&Sons，New York，1987and periodic updates；the series METHODS IN ENZYMOLOGY，Academic Press，San Diego；Wolffe，CHROMATIN STRUCTURE AND FUNCTION，Third edition，Academic Press，San Diego，1998；METHODS IN ENZYMOLOGY，Vol.304，Chromatin(P.M.Wassarman and A.P.Wolffe，eds.)，Academic Press，San Diego，1999；和METHODS IN MOLECULAR BIOLOGY，Vol.119，Chromatin Protocols(P.B.Becker，ed.)Humana Press，Totowa，1999等。
实施例1 GCG类似物的筛选(胰高血糖类似物的筛选)
天然GLP-1的氨基酸序列如SEQ ID NO.1所示，具体为：
HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG。
天然Oxyntomodulin的氨基酸序列如SEQ ID NO.2所示，具体为：
HSQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNN IA。
本发明的GCG类似物记为A，所述A为GCGR/GLP-1R双效激动活性肽，选自所有能实现GCGR及GLP-1R双效激动活性的肽链。
所述A的结构式如式II所示：
HSQGTFTSD-X 10-S-X 12-X 13-X 14-X 15-X 16-X 17-X 18-X 19-X 20-X 21-F-X 23-X 24-WL-X 27-X 28-X 29-X 30-X z。
其中，所述X 10选自V、L或Y之任一；所述X 12选自S、E或K之任一；所述X 13选自Y或Q之任一；所述X 14选自L或M之任一；所述X 15选自D或E之任一；所述X 16选 自S、E或G之任一；所述X 17选自R、E或Q之任一；所述X 18选自R、E或A之任一；所述X 19选自A或V之任一；所述X 20选自Q、R或K之任一；所述X 21选自D、L或E之任一；
所述X 23选自V或I之任一；所述X 24选自Q、A或E之任一；所述X 27选自M、K或V之任一；所述X 28选自N或K之任一；所述X 29选自G或T之任一；所述X 30为G或缺失；所述X z不存在或选自SEQ ID NO.3～8之任一。
示例性的GCG类似物的氨基酸序列可各自独立的选自SEQ ID NO.42-92，所对应的多肽代号分别为C001、C002、C240、C241、C276、C225、C222、C163、C164、C271、C368、C495、C353、C352、C355、C382、C232、C227、C266、C137、C399、C398、C396、C392、C462、C228、C187、C363、C364、C209、C289、C611、C618、C623、C627、C654、C673、C563、C549、C555、C487、C488、C489、C503、C508、C711、C708、C743、C756、C788、C731。
实施例2双效活性蛋白A-L a-F的制备
本实施例中，由GCG类似物与连接链L a及F融合而获得二聚体双效活性蛋白A-L a-F。其中，A同实施例1中的A。
所述F为长效蛋白单元，F可选自免疫球蛋白的完整F C部分、免疫球蛋白的F C部分的片段或免疫球蛋白的F C部分的突变体。所述F的氨基酸序列如SEQ ID NO.9-18所示，序列对应的简称分别为F1-F10。
La为连接链，所述连接链为一段合适长度的由甘氨酸(G)、丝氨酸(S)和/或丙氨酸(A)构成的柔性多肽，从而使相邻的蛋白质结构域可相对于彼此自由移动。当有必要保证所述两个相邻结构域空间上彼此互不干扰时，可以使用较长的连接链。所述连接链示例性的如(GS)n，(GGS)n，(GGSG)n，(GGGS)nA，(GGGGS)nA、(GGGGA)nA等，n为1-10的整数。示例性的连接链可各自独立的选自SEQ ID NO.19-41，序列对应的代号分别为L1-L23。
部分双效活性蛋白A-La-F的氨基酸序列可各自独立的选自SEQ ID NO.93-133，DNA序列可独自的选自SEQ ID NO.222-262，对应的双效活性蛋白A-La-F代号分别为C001L13F8、C002L13F8、CG283L13F8、C240L13F8、CG214L13F8、C382L13F8、CG267L13F8、C276L13F8、C308L13F8、C368L13F8、C224L13F8、C225L13F8、 CG308L13F8、C495L13F8、C319L13F8、C364L13F8、C214L13F8、C232L13F8、C303L13F8、C392L13F8、CG303L13F8、C462L13F8、C240L12F8、C368L10F4、C364L10F4、C352L13F4、C225L10F10、C228L13F4、C187L9F4、C618L12F4、C623L9F3、C228L13F4、C498L13F4、C503L15F2、C508L7F4、C756L20F4、C788L1F5、C289L12F4、C611L11F4、C209L13F8、C627L12F10。
在知晓了A-L a-F的氨基酸序列的基础上，本领域技术人员能够采用现有技术对其进行制备：由于带有F C序列，因此可通过高亲和力和高特异性的Protein A树脂层析进行蛋白纯化，在这里仅示例性地给出一种可行的制备方法。
制备过程如下：
(1)根据蛋白序列及氨基酸密码子表，设计其DNA序列。分别制备重组蛋白中A、L a、F对应的多核苷酸DNA片段，各DNA片段均可由常规的固相合成技术合成拼接；
(2)设计引物进行巢式PCR扩增，拼接A、L a、F分别对应的DNA片段，获得目的基因，PCR拼接技术(包括引物设计、PCR引入突变及酶切等)为本领域技术人员所熟知的公知技术。本领域技术人员应当知晓，本实施例的PCR拼接过程并非是唯一的方法，例如，通过基因合成也可以获得目的基因。成功获得目的基因后，将目的基因克隆至哺乳动物细胞表达载体pTT5(Yves Durocher)，转化大肠杆菌Top10F’；阳性克隆鉴定后，接种于500ml LB培养基中，过夜培养，离心收集菌体，使用Omega
Figure PCTCN2018116244-appb-000008
Endo-Free Plasmid Maxi Kit提取质粒；
(3)转染Hek293F细胞及细胞表达：取1.0mg质粒，使用Freestyle 293表达培养基(Thermofisher)稀释至25ml；取3.0mg PEI(线性，25KD)，使用Freestyle 293表达培养基稀释至25ml，加入到质粒溶液中，混匀，室温孵育30分钟；与此同时，取对数期生长的Hek293F细胞(活率>95％)，计数；1100RPM，离心10分钟，弃上清；使用450ml Freestyle 293表达培养基重悬细胞；PEI质粒混合液孵育结束后，加入到细胞悬液中，37℃，5％CO 2，140RPM震荡培养；7小时后，使用1000ml 293 SFM II培养基(Thermofisher)置换Freestyle 293表达培养基，继续培养7天；
(4)重组蛋白的纯化：将细胞培养液以8000rpm，高速离心10min，得到上清上样到预先用平衡液(20mM PB，0.5M NaCl，pH7)平衡好的Protein A柱(博格隆(上海)生物技术有限公司)，100％洗脱(洗脱液为0.1M Gly-HCl，pH3.0)；收集管中预先加入中和液为(1M Tris-HCl，pH8.0)，收集洗脱样品；最后加中和液到洗脱样品体积的1/10，并采用常 规Bradford法测定蛋白浓度；
(5)重组蛋白的理化性质鉴定：对纯化获得的重组蛋白进行SDS-PAGE电泳或氨基酸序列验证，均与预期一致。
实施例3三效活性蛋白的制备
本发明的三效活性蛋白，含有多结构域，具有三效激动活性。所述三效活性蛋白的结构式如式I所示：A-L a-F-L b-B，其中A为GCGR/GLP-1R双效激动活性肽，F为长效蛋白单元，B为天然FGF21或FGF21类似物，L a为连接链，L b为连接链。
式I中，所述A同实施例1中的A。
式I中，所述F可选自免疫球蛋白的完整F C部分、免疫球蛋白的F C部分的片段或免疫球蛋白的F C部分的突变体，如SEQ ID NO.9-18所示。
式I中，所述的B为天然FGF21(SEQ ID NO.136)或者FGF21类似物。所述B的结构式为：
HPIPDSSPLLQFGGQVRQ X 19YLYTDDAQQTE X 31HLEI X 36EDGTVG X 43A X 45DQSPESLLQL X 56ALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRE X 98LLEDGYNVYQSEAHGLPLH X 118PGN X 122SPHRDPAPRGP X 134RFLPLPGLPPALPEPPGILAPQPPDVGSSDPL X 1 67MV X 170X 171SQ X 174RSPS X 179 X 180 X 181。
其中，N端HPIPDSS可缺失或部分缺失；X 19选自R、Y、V、E或C；X 31选自A或C；X 36选自R或K；X 43选自G或C；X 45选自A、K、E或V；X 56选自K、R、V或I；X 98选自L、R或D；X 118选自L或C；X 122选自K或R；X 134选自A或C；X 167选自S、A或R；X 170选自G或E；X 171选自P或G；X 174选自G、A或L；X 179选自Y、A或F；X 180选自A或E；X 181选自S、K或缺失。
所述的FGF21类似物为与天然FGF21(SEQ ID NO.136)具有相同或相似生物学功能、且与天然FGF21(SEQ ID NO.136)同源性为80％以上的活性蛋白。优选地，所述FGF21类似物与天然FGF21(SEQ ID NO.136)同源性为85％以上；更优选地，所述FGF21类似物与天然FGF21(SEQ ID NO.136)同源性为90％以上；更优选地，所述FGF21类似物与天然FGF21(SEQ ID NO.136)同源性为95％以上。优选地，所述FGF21类似物如SEQ ID NO.137-148所示。
L a不存在或为连接链，L b不存在或为连接链。当L a和L b为连接链时，所述连接链同 实施例2中的La。
在GCGR/GLP-1R双效活性蛋白的基础上，在F C的C末端通过连接肽链融合FGF21或FGF21类似物，制备成氨基酸如SEQ ID NO.149-208所示、核苷酸如SEQ ID NO.263-322所示的示例性三效活性蛋白，对应的代号分别为C002L13F8L10W、C240L12F8L12M1、C240L9F7L13M2、C240L13F4L9M1、C240L9F2L13M3、C240L13F10L9M2、C225L10F10L14M2、C163L13F8L13M2、C271L9F4L8M2、C368L10F4L10M2、C495L13F8L13M1、C495L13F8L10M2、C495L9F10L9M1、C353L13F3L10M4、C352L13F4L9M3、C382L9F3L9M2、C382L10F2L13M2、C382L13F10L9M1、C382L13F8L10M2、C382L12F7L9M2、C382L14F4L9M2、C232L9F3L10M3、C227L12F5L14M4、C266L13F7L13M4、C137L10F8L9M5、C399L12F4L19M8、C392L11F7L10M5、C462L9F2L9M10、C462L10F5L10M3、C462L11F8L13M1、C462L13F4L10M2、C462L13F8L10M2、C462L13F10L9M4、C228L13F4L13M12、C187L9F4L12M7、C364L10F4L12M8、C209L13F8L13M9、C289L12F4L8M10、C611L11F4L11M11、C618L13F7L13M1、C618L12F4L12M2、C623L9F3L10M1、C623L9F8L9M2、C627L12F10L9M6、C654L13F9L6M3、C673L8F3L8M8、C563L14F8L9M3、C549L12F4L9M4、C555L10F6L12M1、C487L13F7L13M4、C488L9F9L9M2、C498L13F4L13M4、C503L15F2L9M5、C508L7F4L9M3、C711L13F5L10M4、C708L10F4L14M7、C743L18F7L8M10、C756L20F4L10M8、C788L1F5L5M5、C731L5F2L9M9。
在知晓了A-L a-F-L b-B的氨基酸序列的基础上，本领域技术人员能够采用现有技术对其进行制备。由于带有F C序列，因此可通过高亲和力和高特异性的Protein A树脂层析进行蛋白纯化。具体方法可参照实施例2中的制备方法。对纯化获得的重组蛋白进行SDS-PAGE电泳或氨基酸序列验证，均与预期一致。其中，图1为部分纯化样品的SDS-PAGE电泳图。
实施例4融合F C的FGF21类似物的制备
本实施例将代号为F 9的F C与天然FGF21及代号为M 1-M 12的FGF21类似物融合，分别获得长效FGF21类似物，所述长效FGF21类似物的结构式为F-L b-B。所述F可选自免疫球蛋白的完整F C部分、免疫球蛋白的F C部分的片段或免疫球蛋白的F C部分的突变体。所述F的氨基酸序列可以如SEQ ID NO.9～18之任一所示。L b不存在或为连接链，当L b为连接链时，所述连接链同实施例2中的La。
包括含富含G、S和/或A的单元，示例性的如(GS)n，(GGS)n，(GGSG)n，(GGGS)nA， (GGGGS)nA、(GGGGA)nA、等，n为1-10的整数，在一个优选的实施方案中，所述的连接链的氨基酸长度为5-26。进一步地，所述连接链的氨基酸序列可如SEQ ID NO.19～41之任一所示。所述B为为天然FGF21(SEQ ID NO.136)或者FGF21类似物。所述B同实施例3中的B。
所述长效FGF21类似物氨基酸序列可分别如SEQ ID NO.209-SEQ ID NO.221所示，代号分别为F9L10W、F9L10M1、F9L10M2、F9L10M3、F9L10M4、F9L10M5、F9L10M6、F9L10M7、F9L10M8、F9L10M9、F9L10M10、F9L10M11、F9L10M12。根据蛋白质序列合成DNA并亚克隆至重组表达载体为本技术领域的常规方法，同实施例2，转染Hek293F细胞及细胞表达。同样，由于带有F C序列，分离纯化的过程也可参照实施例2。
实施例5体外细胞活性检测
将实施例2中获得的双效活性蛋白进行体外活性测定，包括GLP-1R激动活性检测和GCGR激动活性检测。
GLP-1R激动活性检测：
GLP-1R激动活性检测采用荧光素酶报告基因检测法(Jonathan W Day等：Nat Chem Biol.2009Oct；5(10):749-57)。将人源GLP-1R基因克隆至哺乳动物细胞表达质粒pCDNA3.1中，构建成重组表达质粒pCDNA3.1-GLP-1R，同时荧光素酶(luciferase)全长基因克隆至pCRE质粒上得到pCRE-Luc重组质粒。pCDNA3.1-GLP-1R和pCRE-Luc质粒按摩尔比1:10的比例转染CHO细胞，筛选稳转表达株，获得重组CHO/GLP-1R稳转细胞株。
在9-cm细胞培养皿中用含10％FBS和300μg/ml G418的DMEM/F12培养基培养细胞，等汇合度至90％左右时，弃去培养上清，加入2ml胰酶消化3min后，加入2ml含10％FBS和300μg/ml G418的DMEM/F12培养基中和，转移至15ml离心管中，1000rpm离心5min后，弃去上清，加入2ml含10％FBS和300μg/ml G418的DMEM/F12培养基重悬，计数。用含10％FBS的DMEM/F12培养基稀释细胞至3×10 5，96孔板中每孔铺100μl，即5×10 4/孔，贴壁后换成含0.2％FBS的DMEM/F12培养基培养。铺在96孔板的细胞弃去上清后，将纯化的重组蛋白(表1、表2)或天然Glucagon(杭州中肽生化有限公司，GLUC-004)与天然GLP-1(杭州中肽生化有限公司,GLUC-016B)作为对照，用含0.1％FBS的DMEM/F12培养基稀释至一系列指定浓度，加入到细胞培养孔中，100μl/孔，刺激6h后检测。根据萤光素酶报告检测试剂盒(Lucifersae reporter kit，Ray Biotech,Cat:68-LuciR-S200)说明书进行检测。
GCGR激动活性检测方法：
GCGR激动活性检测同样也采用类似的荧光素酶报告基因检测法。将GCGR基因克隆至哺乳动物细胞表达质粒pCDNA3.1中，构建成重组表达质粒pCDNA3.1-GCGR，与pCRE-Luc重组质粒共转染HEK 293T细胞并筛选稳转细胞株HEK 293T/GCGR的构建同上。
FGF21活性检测方法：
FGF21活性测定采用文献类似方法并做适当改进(Xu J等，Polyethylene glycol modified FGF21 engineered to maximize potency and minimize vacuole formation，Bioconjug Chem.；24(6):915-25，2013)。PCR扩增嘌呤霉素抗性基因pac，克隆到pcDNA3.1(+)，替换原有的G418抗性基因。PCR扩增GAL4DBD-ELK1、IRES、KLB(β-klotho)基因，依次克隆到pcDNA-Puro质粒上，构建质粒pcDNA-GAL4DBD-ELK1-IRES-KLB-Puro用于细胞转染筛选。质粒采用Omega
Figure PCTCN2018116244-appb-000009
Endo-Free Plasmid Midi Kit提取备用。细胞转染过程如下：Hek293T细胞铺6孔板，每孔3 x 10 5个细胞，培养过夜。
Opti-MEM培养基洗涤细胞两次后，加入2ml Opti-MEM培养基。按照如下比例配制细胞转染试剂：Lipofectamine 2000(6μl)：pFR-Luc(4.6μg)：pcDNA-GAL4DBD-ELK1-IRES-KLB-Puro(1μg)。静置20min后，缓慢加入到6孔板中，边加边混匀。培养6h后换DMEM+10％FBS培养基，37℃，5％CO 2继续培养。筛选获得具有FGF21活性响应的稳转细胞株。细胞长满培养皿后，胰酶消化，制备细胞悬液(1 x 105细胞/ml,DMEM+5％FBS+1μg/ml puromycin)，铺96孔板，每孔100μl，过夜培养。添加梯度浓度的待测样品，作用6h，使用Luciferase Reporter Assay Kit(68-LucifR-S200)进行荧光检测。
部分双效活性蛋白的活性测定结果如表1、表2所示：
表1
Figure PCTCN2018116244-appb-000010
Figure PCTCN2018116244-appb-000011
说明：表中蛋白编号按照如下规则：多肽代号+连接链代号+FC代号，如C240L 13F 4，表示C240多肽与代号为F 4的IgG FC通过代号为L 13的连接链融合。
a.为GCG类似物与Fc链之间***GPSSGAPPPS或类似序列(又称为Cex序列，在本发明中为SEQ ID NO.3-8之任一)前后的GLP-1R激动活性比例。
b.为根据US9018164 B2表2中披露的天然Glucagon与Glucagon Cex的GLP-1R激动活性数据计算获得的比例。
c.为根据Joseph R.Chabenne等(Joseph R.Chabenne等，Optimization of the Native Glucagon Sequence for Medicinal Purposes，J Diabetes Sci Technol.4(6):1322–1331，2010)文章中表1披露的天然Glucagon与Glucagon Cex的GLP-1R激动活性数据计算获得的比例。
如表1-表2所示，当把含CEX延伸肽的序列通过(GGGGS) 3A(SEQ ID NO.31)与F(SEQ ID NO.16)融合制备成二聚体后，对GLP-1R的激动活性提高了200倍以上，而对GCGR的激动活性则无明显差异。
表2
Figure PCTCN2018116244-appb-000012
Figure PCTCN2018116244-appb-000013
三效活性蛋白的活性检测
实施例3制备的三效活性蛋白活性结果如表3所示：
表3
Figure PCTCN2018116244-appb-000014
Figure PCTCN2018116244-appb-000015
Figure PCTCN2018116244-appb-000016
表中蛋白编号按照如下规则：多肽代号+连接链代号+F C代号+连接链代号+FGF21突变体代号，如C209L 13F 4L 13M 9，表示C209多肽与代号为F4的IgG F C通过代号为L 13的连接链融合，再进一步与代号为M 9的FGF21突变体通过代号为L 13的连接链融合。
实施例6三效活性蛋白的DPP-IV耐酶稳定性
将纯化的三效活性蛋白5uM溶解于10mM的HEPES缓冲液(含0.05mg/ml BSA中，加入终浓度为10nM的重组DPP-IV酶，于37℃温浴24小时后，检测体外GCGR细胞活性检测。活性保留率＝(DPP-IV酶处理后活性/处理前活性)×100％。
本实施例中采用了第二位引入非天然氨基酸Aib或D-Ser的GCG类似物作为对照：
GD SerGS：H-D-Ser-QGTFTSDYSKYLDSQAAQDFVQWLMNGGPSSGAPPPS(SEQ ID NO.134)；
G AibGS：H-Aib-QGTFTSDYSKYLDSQAAQDFVQWLMNGGPSSGAPPPS(SEQ ID NO.135)；
C364(SEQ ID NO.70)、C382(SEQ ID NO.57)、C495(SEQ ID NO.53)、C462(SEQ ID NO.66)、C225(SEQ ID NO.47)及C209(SEQ ID NO.71)作为本实施例中稳定性实验的对照。
结果如表4所示：
表4
Figure PCTCN2018116244-appb-000017
Figure PCTCN2018116244-appb-000018
实施例7三效活性蛋白的血清稳定性试验
体外细胞检测法：
(1)取三效活性蛋白，先超滤浓缩，再用20mM PB pH7.4稀释到1.6mg/ml，除菌过滤后，血清(FBS，GEMINI 900-108，A97E00G)稀释10倍，混匀，分装到无菌离心管中；
(2)另外取Glucagon(SEQ ID NO：42，杭州中肽生化有限公司，GLUC-004)，稀释到0.2mg/ml，除菌过滤后，血清稀释10倍，混匀，分装到无菌离心管中；
(3)上述样品1-2管于-20度冻存作为对照，另几管置37℃培养箱，于不同时间点取样检测GCGR激动活性；
(4)HEK 293T/GCGR细胞传代两次后铺96孔板，检测样品活性。残留活性：以0小时的活性值为100％，后续时间点测得的值与之相比而获得。除了C002L 13F 4L 13W外，同表4中的结果相似，各三效活性蛋白的血清稳定性无显著差异。示例性的三效活性蛋白相对活性随着时间的变化如图2A-C所示。
实施例8正常ICR小鼠的葡萄糖耐量试验(IPGTT)
正常ICR小鼠分组，每组8只。禁食过夜，尾部采血(记为t＝0分钟血糖样)，皮下注射三效活性蛋白(40nmol/kg，醋酸盐缓冲液)、组合物(联合给药组)或生理盐水PBS。联合给药组在给药前预先混合(各40nmol/kg，醋酸盐缓冲液)。15分钟后腹腔注射葡萄糖(2克/千克体重)，并在t＝30分钟、t＝60分钟、t＝120分钟和t＝240分钟测量血液葡萄糖水平。在实验期间动物仍禁食以防止食物摄取的干扰。结果如图3所示。
实施例9三效活性蛋白连续给药在诱导肥胖(DIO)小鼠中的药效研究
7周龄雄性C57BL/6J雄性小鼠给予高脂饲料(60％kcal from fat)继续饲养16周(共23周)，到体重约为55g时进行试验。饲养条件：12h光照/12h黑暗，自由采食，单笼饲养，给药前一天根据体重和体重生长曲线对小鼠进行分组(8只/组)，第二天皮下给药处理。根据表5所示，活性蛋白按照10nmol/千克体重或30nmol/千克体重的剂量给药，4天一次；阴性对照组按5ul/克体重注射生理盐水(PBS)；阳性对照组注射利拉鲁肽(30nmol/千克体重)，每天一次给药，连续给药28天，每天测量小鼠体重及进食量。最后一次给药后的第5天处死。眼眶取血。将血浆标本储存在-80℃。计算每组动物给药前及处死时的平均体重变化。体重变化结果如图4所示；总摄食量的变化如图5所示。
表5
样品 SEQ ID NO. 剂量(nM)
C002L 13F 8L 10W 149 10
C495L 13F 8L 10M 2 160 10
C382L 13F 8L 10M 2 167 10
C462L 13F 8L 10M 2 180 10
C495L 13F 8L 10M 2 160 30
C382L 13F 8L 10M 2 167 30
C462L 13F 8L 10M 2 180 30
实施例10联合给药在诱导肥胖(DIO)小鼠中的药效研究
本实施例与实施例9的不同之处在于双效活性蛋白与长效FGF21类似物采用复方的形式联合给药。7周龄雄性C57BL/6J雄性小鼠给予高脂饲料(60％kcal from fat)继续饲养16周(共23周)，到体重约为55g时进行试验。饲养条件：12h光照/12h黑暗，自由采食，单笼饲养，给药前一天根据体重和体重生长曲线对小鼠进行分组(8只/组)，第二天皮下 给药处理。双效活性蛋白与长效FGF21类似物在给药前根据表6所示剂量混合，按照15nmol/千克体重或30nmol/千克体重的剂量给药，4天一次；阴性对照组按5ul/克体重注射生理盐水(PBS)；阳性对照组注射利拉鲁肽(30nmol/千克体重)，每天一次给药，连续给药28天，每天测量小鼠体重及进食量。最后一次给药后的第5天处死。眼眶取血。将血浆标本储存在-80℃。计算每组动物给药前及处死时的平均体重变化。体重变化结果如图6所示；总摄食量的变化如图7所示。
表6
样品 SEQ ID NO. 剂量(nM)
C495 L 13F 8+F 9L 10M 1 106、210 各30
C495 L 13F 8+F 9L 10M 2 106、211 各30
C382 L 13F 8+F 9L 10M 2 98、211 各30
C382 L 13F 8+F 9L 10M 3 98、212 各30
C462 L 13F 8+F 9L 10M 1 114、210 各30
C462 L 13F 8+F 9L 10M 2 114、211 各30
C462 L 13F 8+F 9L 10M 3 114、212 各30
C462 L 13F 8 114 30
C382 L 13F 8 98 30
C495 L 13F 8 106 30
F 9L 10M 1 210 30
F 9L 10M 2 211 30
F 9L 10M 3 212 30
实施例11三效活性蛋白的构建及活性测定
本发明的三效活性蛋白，所述结构同实施例3，构建获得融合蛋白C382L13F3L10M2和C382L13F9L10M2：对应的氨基酸分别为SEQ ID NO.336和SEQ ID NO.338所示，对应的核苷酸分别为SEQ ID NO.337和SEQ ID NO.339所示。
本实施例中所使用的制备方法同实施例2。
将本实施例中获得的三效活性蛋白进行体外活性测定，包括GLP-1R激动活性检
测、GCGR激动活性检测、FGF21活性检测。检测方法同实施例5。
检测结果如表7所示。
表7
Figure PCTCN2018116244-appb-000019
Figure PCTCN2018116244-appb-000020
实施例12三效活性蛋白的血清稳定性试验
将实施例11中获得的三效活性蛋白进行血清稳定性试验，检测方法同实施例7。所述三效活性蛋白相对活性随着时间的变化如图8所示。
图8中的数据显示出所述三效活性蛋白在血清中孵育7d还保留一定地活性，相对于天然GLP1或Glucagon小肽的稳定性大大提高。
实施例13正常ICR小鼠的葡萄糖耐量试验(IPGTT)
正常ICR小鼠分组，每组8只。禁食过夜，尾部采血(记为t＝0分钟血糖样)，皮下注射实施例11中获得的三效活性蛋白和C002L13F8L10W(40nmol/kg，醋酸盐缓冲液)、或生理盐水PBS。15分钟后腹腔注射葡萄糖(2克/千克体重)，并在t＝30分钟、t＝60分钟、t＝120分钟和t＝240分钟测量血液葡萄糖水平。在实验期间动物仍禁食以防止食物摄取的干扰。结果如图9所示。
图9中的数据显示出相比C002L13F8L10W，C382L13F3L10M2和C382L13F9L10M2有显著的降糖效果。
实施例14三效活性蛋白连续给药在诱导肥胖(DIO)小鼠中的药效研究
将实施例11中获得的三效活性蛋白进行连续给药在诱导肥胖(DIO)小鼠中的药效研究，方法同实施例9。体重变化结果如图10所示；总摄食量的变化如图11所示。
图10数据显示实施例11中获得的三效活性蛋白在10nmol/kg和30nmol/kg剂量下都与利拉鲁肽有显著性差异，且其减重与剂量呈正相关，在30nmol/kg剂量下的减重效果非常明显；图11数据显示实施例11中获得的三效活性蛋白在10nmol/kg和30nmol/kg剂量下对摄食并没有明显的影响。
实施例15三效活性蛋白连续给药在db/db小鼠给药后随机血糖检测
瘦素受体缺陷二型糖尿病(db/db)小鼠中的降糖实验。db/db小鼠主要按照体重、非空 腹血糖，药前OGTT反应三个指标进行筛选并均衡分组，每组10只，尽量排除过大或者过小的个体，非空腹血糖要大于15mM。另外选取正常ICR鼠作为基础血糖对照。根据表8所示，活性蛋白按照20nmol/千克体重的剂量皮下注射，每4天给药一次，第一次为第0天，最后一次为第24天；阴性对照组生理盐水(PBS)(5μl/克体重)、阳性对照组利拉鲁肽(10nmol/千克体重)皮下注射方式每天给药一次，连续给药26天。在第一次注射前及第2d、6d、10d、14d、18d、22d、26d早上9点测定随机血糖值。随机血糖变化结果如图12所示。
图12显示表8中的三效活性蛋白在瘦素受体缺陷二型糖尿病(dbdb)小鼠中降糖效果明显优于阳性对照利拉鲁肽。其中C382L 13F 3L 10M 2的血糖水平甚至与正常ICR鼠对照相当(图中未显示)。
表8
样品 SEQ ID NO. 剂量(nM)
C462L 13F 4L 10M 2 179 20
C382L 13F 3L 10M 2 336 20
C382L 13F 9L 10M 2 338 20
C495L 13F 3L 10M 2 340 20
本说明书中所述及的活性蛋白所对应的核苷酸序列如表9中所示。
表9
Figure PCTCN2018116244-appb-000021
Figure PCTCN2018116244-appb-000022
Figure PCTCN2018116244-appb-000023
以上所述，仅为本发明的较佳实施例，并非对本发明任何形式上和实质上的限制，应当指出，对于本技术领域的普通技术人员，在不脱离本发明方法的前提下，还将可以做出若干改进和补充(包括本发明所述示例性的GCG类似物与各种Fc、各种FGF21的融合)，这些改进和补充也应视为本发明的保护范围。凡熟悉本专业的技术人员，在不脱离本发明的精神和范围的情况下，当可利用以上所揭示的技术内容而做出的些许更动、修饰与演变的等同变化，均为本发明的等效实施例；同时，凡依据本发明的实质技术对上述实施例所作的任何等同变化的更动、修饰与演变，均仍属于本发明的技术方案的范围内。

Claims (14)

  1. 一种多结构域活性蛋白，其结构中包括如式I所示的结构，如式I所示结构为：
    A-L a-F-L b-B，其中，A为GCGR/GLP-1R双效激动活性肽，F为长效蛋白单元，B为天然FGF21或FGF21类似物，L a不存在或为连接链，L b不存在或为连接链。
  2. 根据权利要求1所述的多结构域活性蛋白，其特征在于，所述A包括如式II所示的结构，如式II所示结构为：
    HSQGTFTSD-X 10-S-X 12-X 13-X 14-X 15-X 16-X 17-X 18-X 19-X 20-X 21-F-X 23-X 24-WL-X 27-X 28-X 29-X 30-X z，所述X 10选自V、L或Y之任一；所述X 12选自S、E或K之任一；所述X 13选自Y或Q之任一；所述X 14选自L或M之任一；所述X 15选自D或E之任一；所述X 16选自S、E或G之任一；所述X 17选自R、E或Q之任一；所述X 18选自R、E或A之任一；所述X 19选自A或V之任一；所述X 20选自Q、R或K之任一；所述X 21选自D、L或E之任一；所述X 23选自V或I之任一；所述X 24选自Q、A或E之任一；所述X 27选自M、K或V之任一；所述X 28选自N或K之任一；所述X 29选自G或T之任一；所述X 30为G或缺失；所述X z不存在或选自GPSSGAPPPS、PSSGAPPPS、SSGAPPPS、GPSSGAPPS、PSSGAPPS或KRNRNNIA之任一。
  3. 根据权利要求1所述的多结构域活性蛋白，其特征在于，所述B包括如下结构：
    HPIPDSSPLLQFGGQVRQ X 19YLYTDDAQQTE X 31HLEI X 36EDGTVG X 43A X 45DQSPESLLQL X 56ALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRE X 98LLEDGYNVYQSEAHGLPLH X 118PGN X 122SPHRDPAPRGP X 134RFLPLPGLPPALPEPPGILAPQPPDVGSSDPL X 167MV X 170 X 171SQ X 174RSPS X 179 X 180 X 181，其中，N端HPIPDSS可缺失或部分缺失；X 19选自R、Y、V、E或C；X 31选自A或C；X 36选自R或K；X 43选自G或C；X 45选自A、K、E或V；X 56选自K、R、V或I；X 98选自L、R或D；X 118选自L或C；X 122选自K或R；X 134选自A或C；X 167选自S、A或R；X 170选自G或E；X 171选自P或G；X 174选自G、A或L；X 179选自Y、A或F；X 180选自A或E；X 181选自S、K或缺失。
  4. 根据权利要求1所述的多结构域活性蛋白，其特征在于，还包括以下特征的任一项或多项：(1)所述F为源自哺乳动物免疫球蛋白的F C部分；(2)当L a为连接链时，所述L a为富含G、S和/或A的连接链；(3)当L b为连接链时，所述L b为富含G、S和/或A的连接链。
  5. 根据权利要求1所述的多结构域活性蛋白，其特征在于，还包括以下特征的任一项或多项：(1)所述A的氨基酸序列如SEQ ID NO.44～92之任一所示；(2)所述B的氨 基酸序列如SEQ ID NO.136-148之任一所示；(3)所述F的氨基酸序列可以如SEQ ID NO.9～18之任一所示；(4)当L a为连接链时，所述L a的氨基酸序列可如SEQ ID NO.19～41之任一所示；(5)当L b为连接链时，所述L b的氨基酸序列可如SEQ ID NO.19～41之任一所示。
  6. 一种分离的多核苷酸，所述分离的多核苷酸编码如权利要求1-5之任一项所述多结构域活性蛋白。
  7. 一种重组表达载体，包含如权利要求6所述分离的多核苷酸。
  8. 一种宿主细胞，所述细胞含有如权利要求7所述重组表达载体或基因组中整合有外源的如权利要求6所述分离的多核苷酸。
  9. 如权利要求1-5之任一项所述多结构域活性蛋白的制备方法，其特征在于，在合适的条件下培养如权利要求8所述宿主细胞，使之表达所述多结构域活性蛋白，而后分离及纯化获得所述多结构域活性蛋白。
  10. 如权利要求1-5之任一项所述多结构域活性蛋白在制备治疗代谢相关疾病的药物中的用途。
  11. 一种组合物，含有如权利要求1-5之任一项所述多结构域活性蛋白或如权利要求8所述宿主细胞的培养物，以及药学上可接受的载体。
  12. 如权利要求1-5之任一项所述多结构域活性蛋白在制备融合蛋白中的用途。
  13. 一种治疗代谢及相关疾病的组合物，包括GCGR/GLP-1R双效激动活性蛋白和长效FGF21类似物，所述GCGR/GLP-1R双效激动活性蛋白的结构中包括：A-L a-F，所述长效FGF21类似物的结构中包括：F-L b-B。
  14. 一种促进体重减少或者防止体重增加的方法，包括在对象中施用如权利要求1-5之任一项所述多结构域活性蛋白或如权利要求11所述组合物或如权利要求13所述治疗代谢及相关疾病的组合物。
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