WO2019101036A1 - Multiple-active protein for treating metabolic diseases - Google Patents

Multiple-active protein for treating metabolic diseases Download PDF

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WO2019101036A1
WO2019101036A1 PCT/CN2018/116171 CN2018116171W WO2019101036A1 WO 2019101036 A1 WO2019101036 A1 WO 2019101036A1 CN 2018116171 W CN2018116171 W CN 2018116171W WO 2019101036 A1 WO2019101036 A1 WO 2019101036A1
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seq
active protein
glp
amino acid
glucagon
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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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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/26—Glucagons
    • 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
    • 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
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/50—Fibroblast growth factor [FGF]
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or 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
    • 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 multiplex 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 may be listed soon, such as Semaglutide.
  • 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).
  • Glucagon analogues of the second cross-linked fatty acid that retains natural Ser
  • This analog consists of 30 amino acids and is mutated by 9 amino acids compared to native Glucagon.
  • the second place of MEDI0382 also retains the natural Ser amino acid, it can only support the frequency of dosing once a day.
  • oxyntomodulin analogue showed initial hypoglycemic and lipid-lowering effects, its mechanism of action is still inaccurate: the oxyntomodulin receptor has not been found, and is currently only knocked out by GCGR or GLP-1R. Mouse or cell assays to verify that oxyntomodulin binds to these two receptors.
  • oxyntomodulin can agonize GLP-1R and GCGR, its activity is quite low (about one-tenth and one-hundredth of that of natural GLP-1 and Glucagon, respectively), and the stomach used in general research.
  • the agonistic activity of the oxyntomodulin analogues in GLP-1R and GCGR is generally designed to be about 1:1.
  • the GLP-1R agonist hypoglycemic agent Taspoglutide (introduced with the non-natural amino acid Aib) developed by Roche and Epson, the antibody production rate reached 49%, and finally stopped all phase III clinical studies (Julio Rosenstock et al, The Fate of Taspoglutide, a Weekly GLP-1 Receptor Agonist, Versus wice-Daily, Exenatide for Type 2, Diabetes Care, 36: 498-504, 2013).
  • Another issue relates to the half-life problem of GLP-1R/GCGR double-acting agonists.
  • Fatty acid cross-linking can minimize the loss of activity.
  • most of the half-life can only be maintained for about 12 hours, so it can only be administered in a daily manner.
  • PEG cross-linking is more effective in prolonging the half-life than fatty acid, but the activity damage is caused. very serious.
  • fusion Glucagon analogs or GLP-1R/GCGR bispecific agonists that are effective against DPP-IV degradation have not been reported.
  • HM12525A is prepared by cross-linking human F C on the basis of GLP-1R/GCGR bispecific small peptide.
  • Kang et al The ultra-long acting LAPSGLP/GCG dual agonist, HM12525A, demonstrated safety and prolonged pharmacokinetics in healthy volunteers: a phase 1 first-in-human study, 51st European Association for the Study of Diabetes (EASD), Sweden; September 14-18, 2015).
  • 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 first aspect of the invention provides a multiplex active protein, the structure of the multiplexed live protein comprising
  • A is a GCGR/GLP-1R double-acting agonist
  • F is a long-acting protein unit
  • L is a linking chain connecting the A and F.
  • the GCGR/GLP-1R double-acting agonist is selected from an analog of native Glucagon (SEQ ID NO. 44) or other polypeptide or protein having GCGR/GLP-1R double-acting agonistic activity.
  • the structure of the A includes a structure as shown in Formula II, and the structure shown in Formula II is:
  • HSQGTFTSDYSKYLD X 16 X 17 X 18 AQDFVQWLMN X 29 X z (SEQ ID NO. 141) Formula II, wherein X 16 is selected from any one of amino acids other than Y, N, W, and H; X 17 is selected from P Any one of amino acids other than L, T, F and H; X 18 except for one of amino acids other than P, F, H and W; and X 17 and X 18 may not be R at the same time, X 29 is T or Deletion, X z is selected from GGPSSGAPPPS (SEQ ID NO. 3), GPSSGAPPPS (SEQ ID NO. 4), PSSGAPPPS (SEQ ID NO.
  • SSGAPPPS SEQ ID NO. 6
  • GGPSSGAPPS SEQ ID NO. 7
  • Any of GPSSGAPPS SEQ ID NO. 8
  • PSSGAPPS SEQ ID NO. 9
  • SSGAPPS SEQ ID NO. 10
  • the A is resistant to proteolytic hydrolysis in the body.
  • amino acid sequence of A may be as shown in any one of SEQ ID NO. 46, SEQ ID NO. 55, SEQ ID NO. 59, SEQ ID NO. 68, SEQ ID NO.
  • a in the multiplexed protein ALF structure is a GCG analog
  • the multiplexed active protein of the ALF structure is highly resistant to DPP-IV when compared to a corresponding small peptide that does not contain the LF moiety. The ability to hydrolyze.
  • the second Ser a small peptide that does not contain the LF moiety, is highly susceptible to DPP-IV attack and is inactivated.
  • the stability of the multiplexed active protein of the ALF structure in serum is comparable to that of the second analog introducing the unnatural amino acid Aib or D-Ser.
  • DPP-IV enzyme resistance of GCG analogs by fusing F C.
  • the second position of GLP-1 is A, and even after fusion with F C , GLP-1 is easily degraded by DPP-IV to hydrolyze the first two amino acids HA. Therefore, long-acting GLP-1 analogues that are currently on the market and once a week in clinical doses must mutate the second amino acid to glycine Gly (such as duraglutide and albendide) or Aib (such as Somaru).
  • the inventors have surprisingly found that the preferred GCG analogs provided by the present invention, upon fusion with F C , achieve extremely high DPP-IV resistance, and pharmacodynamic experiments have shown that stability can support a once-a-week dosing frequency. This effect has been achieved through integration, which is currently not seen. Retaining the second Ser, on the one hand, reduces the risk of immunogenicity, on the other hand, it retains the agonistic activity of GCGR to the greatest extent, and can effectively achieve the weight loss effect.
  • MEDI0382 Compared to the native Glucagon, 9 mutations were introduced. At the same time, although the second place of MEDI0382 also retains the natural Ser amino acid, it can only support the frequency of dosing once a day.
  • the GCG analogue in the present invention retains the N-terminal second natural S amino acid on the one hand, and only undergoes mutations of not more than 3 amino acids, and is fused to a long-acting unit (such as F C ) to support once a week. Dosing frequency.
  • F C long-acting unit
  • the C-terminal T is further deleted.
  • the T deletion at the C-terminus has no effect on the stability of the Glucagon analog. Therefore, the greatest advantage of the present invention is that the use of fewer site mutations achieves the optimal receptor agonistic activity and stability, and does not introduce unnatural amino acids when mutated, while reducing potential immunogenicity. It is convenient to directly prepare products using recombinant techniques.
  • Example 4 The study found that the active protein (C002G12S3A1F4, SEQ ID NO. 76) obtained by the fusion of the Glucagon-cex sequence reported by Joseph R. Chabenne et al. with F C had no significant DPP-IV resistance (Example 4). In the IPCR mouse IPGTT test and the DIO mouse weight loss test (Examples 8 and 9), C002G12S3A1F4 also showed no significant hypoglycemic and sustained weight loss effects. Meanwhile, in Example 4 it can also be seen, although the same position is mutated 16-18 F C GCG fused analogs, different dimers bis difference in stability has a great effect active protein.
  • the spatial conformation of the protein is extremely complex, so that the mutation in the formula II not only improves the stability of the interior of the peptide chain, but is more likely to change the interaction conformation between the GCG analog and the F C chain, further enhancing the fusion protein N. Stability of the end. More importantly, the ALF structural proteins provided by the present invention have significantly enhanced GLP-1R agonistic activity compared to existing GCG and mutants.
  • the inventors have unexpectedly discovered that after fusion of a GCG analog with an Fc, the effect on the activity of GLP-1R and GCGR is completely different.
  • the C-terminus of the GCG analog is additionally added with cex or a similar sequence (SEQ ID NO. 3-10) and further fused to the F C chain, the structure containing the cex sequence is GLP compared to the fusion of the GCG analog directly with the F C chain.
  • the retention rate of -1R agonistic activity was significantly increased, and the highest was increased by more than 200 times, but the retention rate of GCG activity was basically unchanged or even decreased slightly.
  • the present invention provides a multiplex active protein having GCGR/GLP-1R double-acting agonistic activity.
  • double-acting agonists of GLP-1R and GCGR are generally engineered based on the native Oxyntomodulin (SEQ ID NO. 2) or Glucagon (SEQ ID NO. 44) sequences.
  • the agonistic activity of analogs used in general research in GLP-1R and GCGR is generally designed to be about 1:1. Most studies consider that the activity of 1:1 is the best for hypoglycemic and lipid-lowering effects (Peptide-based GLP-1/).
  • Glucagon co-agonists a double-edged sword to combat diabesity, Hitesh Soni, 95:5–9, 2016).
  • Oxyntomodulin (SEQ ID NO. 2) has a relatively low agonistic activity for both receptors, whereas Glucagon (SEQ ID NO. 44), after sequence mutation, has an increased agonistic activity against GLP-1R, but After PEG or fatty acids, the activity will inevitably decrease, especially in the case of PEG modification.
  • the double-acting active protein of the present invention retains most of the intact GLP-1R and GCGR agonistic activity. An increase in activity predicts a reduction in dose, and a lower dose results in a smoother glycemic control, which improves the ease of administration and reduces the risk of potential immunogenicity.
  • GLP-1 analogs and their fusion proteins are dose dependent, and lowering the dose can reduce gastrointestinal side effects.
  • Glucagon can increase the metabolic rate, increase fat consumption, play a more significant weight loss effect, and also reduce the risk of hypoglycemia, suitable for combination with other hypoglycemic agents, such as insulin.
  • GLP-1R and GCGR agonism and downstream signaling and their physiological effects are extremely complex and have not been fully understood to date. It is currently agreed that roughly: glucose enters the pancreatic beta cells through GLUT2 and undergoes glycolysis and produces pyruvate. Pyruvate enters the mitochondria for oxidative metabolism and produces ATP. An increase in intracellular ATP will turn off K ATP (ATP-sensitive potassium) channels, depolarizing the membrane, opening calcium channels, and increasing the influx of extracellular calcium ions. This series of changes Lead to the exocrine of insulin.
  • GLP-1 increases insulin exocrine through a series of mechanisms: GLP-1R binds to G ⁇ s, activates adenylate cyclase, adenylate cyclase converts ATP to cAMP, and mobilizes downstream PKA and Epac signaling factors. This leads to a series of cellular responses, including the closure of the K ATP channel, which promotes the fusion of insulin-secreting granules with the membrane (Chris de Graaf et al, Glucagon-Like Peptide-1 and Its Class BG Protein–Coupled Receptors: A Long March to Therapeutic Successes, Pharmacological Reviews, 68 (4) 954-1013, 2016).
  • GCGR is similar to GLP-1R, and intracellular cAMP is upregulated after binding to Glucagon. Both GLP-1R and GCGR belong to the GPCR family and have seven transmembrane regions. Upon binding to the respective ligand, the C-terminal phosphorylation of the receptor, ⁇ -arrestin is enriched and binds to the receptor, ultimately leading to endocytosis of the receptor (Jorgensen, R et al, Oxyntomodulin differentially affects Glucagon-like peptide-1 receptor-arrestin recruitment and signaling through G ⁇ , J. Pharmacol. Exp. Ther. 322, 148-154, 2007).
  • GCGR and GLP-1R have different endocytic efficiencies, and receptor endocytosis affects downstream signal transduction (Functional Consequences of Glucagon-like Peptide-1 Receptor Cross-talk and Trafficking, J Biol Chem, Sarah Noerklit Roed et al. 290(2): 1233–1243, 2015) may ultimately affect the physiological function of tissue cells.
  • Kuna, RS et al. GLP-1R endocytosis will reduce insulin release from pancreatic cells (Glucagon-like peptide-1)-mediated endosomal cAMP generation promotes glucose-stimulated insulin secretion in pancreatic-cells, Kuna, RS, etc. .J.
  • GLP-1R and GCGR double-acting agonists are constructed on the basis of F C bivalent protein, it is possible to simultaneously bind two receptors of GLP-1R and GCGR, respectively, resulting in heterodimerization and cross-linking of the two receptors. It affects the endocytosis of the respective receptors, and the intracellular signal transmission affects its normal physiological functions.
  • the linker is a flexible polypeptide of a suitable length consisting of glycine (G), serine (S) and/or alanine (A), and preferably obtains a suitable sequence and length reduction
  • 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. 21-43.
  • amino acid sequence of the linker L in the formula I can be as shown in any one of SEQ ID NOS. 21 to 43.
  • 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 IgG1 and is set forth in SEQ ID NO. In a preferred embodiment, the F C domain is derived from human IgG4, as set forth in SEQ ID NO.
  • the K at the end of the F C chain can be removed to facilitate the uniformity of the expression product.
  • the amino acid sequence of F may be as shown in any one of SEQ ID NOS. 11-20.
  • the multiplex active protein provided by the present invention is an F C fusion protein that retains the conventional properties of F C , such as binding to FcRn to prolong the half-life in vivo.
  • these multiple active proteins can effectively prevent the degradation of the N-terminus in addition to the degradation of the protein inside the serum by the protease in 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 due to the hydrolysis of DPP-IV in the receptor.
  • 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.
  • liraglutide and MEDI0382 described above both retain the second natural amino acid, the dosing cycle can only be supported once a day. While the multiplex active protein of the present invention retains the natural amino acid in the second position, the significant increase in stability is sufficient to support the frequency of administration once a week.
  • the second aspect of the present invention provides a multiplex active protein having a three-way agonistic activity, wherein the structural formula includes a structure represented by Formula III, and the structure represented by Formula III is: AL 1 -FL 2 -B, wherein A is a GCGR/GLP-1R double-acting agonist, F is a long-acting protein unit, B is native FGF21 (SEQ ID NO. 143) or FGF21 analog, L 1 is a linking chain, and the sequence is selected from SEQ ID NO. 21- Any of 43; L 2 is absent or selected from any one of SEQ ID NOS. 21-43.
  • the structure of A includes a structure as shown in Formula II, and the structure shown in Formula II is:
  • HSQGTFTSDYSKYLD X 16 X 17 X 18 AQDFVQWLMN X 29 X z (SEQ ID NO. 141) Formula II, wherein X 16 is selected from any one of amino acids other than Y, N, W, and H; X 17 is selected from P Any one of amino acids other than L, T, F and H; X 18 is selected from any one of amino acids other than P, F, H and W; further X 17 and X 18 cannot be R at the same time, X 29 is T or Deletion, X z is selected from GGPSSGAPPPS (SEQ ID NO. 3), GPSSGAPPPS (SEQ ID NO. 4), PSSGAPPPS (SEQ ID NO.
  • SSGAPPPS SEQ ID NO. 6
  • GGPSSGAPPS SEQ ID NO. 7
  • Any of GPSSGAPPS SEQ ID NO. 8
  • PSSGAPPS SEQ ID NO. 9
  • SSGAPPS SEQ ID NO. 10
  • amino acid sequence of A may be as shown in any one of SEQ ID NO. 46, SEQ ID NO. 54, SEQ ID NO. 55, SEQ ID NO.
  • the FGF21 analogs of Formula III may be selected from FGF21 as described in US Patent No. 20140213512, US Pat. No. 8,818,040, US Pat. No. 9,949,530, WO 2016114633, US 20150291677, US Pat. No. 9 422 353, US Pat. No. 8 541 369, US Pat. No. 7,622,445, US Pat. No. 7,576,190, US Pat. Analog or mutant. Further, the amino acid sequence of the FGF21 analog is as shown in SEQ ID NO. 144, SEQ ID NO. 145 or SEQ ID NO.
  • the tri-active protein group has a more significant weight-reducing effect than the co-administered group of the same dose of the double-acting active protein + FGF21 analog. But the impact on appetite has little effect. It was demonstrated that the side effects of the triactive protein may be lower and the safety is improved (Example 12).
  • the B of Formula III may also be a native leptin (SEQ ID NO. 155) and analogs thereof, which may be selected from the variants, derivatives or derivatives described in US Pat. No. 7,307,142, US Pat. analog;
  • B described in Formula III may also be Amylin and its analogs.
  • an isolated polynucleotide is provided, the isolated polynucleotide encoding the aforementioned multiplex 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 an exogenous source in the genome is provided.
  • a method for producing the aforementioned multiplex active protein which comprises culturing the aforementioned host cell under suitable conditions to express the multiplex active protein, and then isolating and purifying to obtain the multiplex active protein.
  • the use of the aforementioned multiplex active protein for the preparation of a medicament for treating a diabetes-related disease is provided.
  • 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 multiplex active proteins of the invention can also be used to treat obesity.
  • the multiplex active proteins of the invention treat 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 multiplex active protein to a subject.
  • the invention further provides a method of promoting weight loss or preventing weight gain comprising administering the multiplex active protein in a subject.
  • composition comprising the aforementioned multiplex active protein or a culture of the aforementioned host cell, and a pharmaceutically acceptable carrier.
  • the use of the aforementioned multiplex active protein for the preparation of a fusion protein is provided.
  • a multidomain protein comprising the aforementioned multiplex active protein is contained in the structure of the multidomain protein.
  • the present invention has the following beneficial effects as compared with the prior art:
  • the multiplex active protein of the present invention has a long half-life and supports a frequency of administration once a week;
  • the agonistic activity of GLP-1R of the multiplex active protein of the present invention is up to 200 times or more;
  • the multiplex active protein of the present invention has good stability in vitro and in vivo, and has low immunogenicity.
  • Figure 1 is a partial electrophoresis map of purified recombinant protein (10% SDS-PAGE). Lanes 1-6 are C240G12S3A1F4, C368G12S3A1F4, C225G12S3A1F4, C495G12S3A1F4, C382G12S3A1F4 and C462G12S3A1F4 non-reduced samples; 7-12 are C240G12S3A1F4 and C368G12S3A1F4, respectively.
  • M is a protein standard: 97.2, 66.4, 44.3, 29, 20.1, 14.3 KD.
  • Figure 2A is a graph showing the results of GCGR measurement.
  • Fig. 2B is a graph showing the results of GLP-1R measurement.
  • Figure 3A A graph of the results of serum stability over time.
  • FIG. 3B Figure of the results of serum stability over time.
  • FIG. 3C Figure of the results of serum stability over time.
  • Figure 3D Figure of the results of serum stability over time.
  • Figure 4 Schematic diagram of the stimulation of islet cells by dimeric recombinant proteins obtained by fusion of GCG analogs with different length linkers and F (SEQ ID NO. 12) in Examples 6 and 7.
  • Fig. 5 is a graph showing the hypoglycemic effect of the dimeric recombinant protein of Example 8 in normal ICR mice.
  • Figure 6 Effect of the dimeric recombinant protein of Example 9 on the body weight of DIO mice.
  • FIG. 7 Electropherogram (10% SDS-PAGE) of the fusion protein obtained in the purification of Example 11, lanes 1-6 are non-reduced samples of C382F4FGF1, C382F4FGF2, C382F4FGF3, C495F4FGF1, C495F4FGF2 and C495F4FGF3, respectively; Samples of C382F4FGF1, C382F4FGF2, C382F4FGF3, C495F4FGF1, C495F4FGF2 and C495F4FGF3 reduced; M is a self-made protein standard: 140, 97.2, 66.4, 44.3, 29, 20.1, 14.3 KD.
  • Figure 8 Effect of the triactive protein in Example 12 on the body weight of DIO mice.
  • Figure 9 Effect of the triactive protein in Example 12 on the food intake of DIO mice.
  • the food intake of the DIO rats in the PBS group was 100%, and the ordinate was the percentage of the food intake of the other groups.
  • Figure 10 Effect of the tri-active protein in Example 14 on the body weight of DIO mice.
  • Figure 11 Effect of the tri-active protein in Example 14 on the food intake of DIO mice.
  • the food intake of the DIO rats in the PBS group was 100%, and the ordinate was the percentage of the food intake of the other groups.
  • 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.
  • 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, Lancet 368: 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.
  • GLP-1 Glucagon-like peptide: 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 improving the efficacy by using chemical modification and/or formulation to treat diabetes and obesity (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
  • 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: 44).
  • 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 consumption in addition to decreasing energy intake in overweight and obese humans: a andomized 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: 44 (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-1R analog As used herein, "GLP-1 analog” or “GLP-1 mutant” means a GLP-1R agonist and is mutually 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 agonist 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 (FGF), also known as heparin binding growth factor, is a type of polypeptide substance secreted mainly by the pituitary and hypothalamus. FGF has various functions, such as promoting fibroblast mitosis, mesoderm cell growth, stimulating blood vessel formation and the like. While FGF21 is an important member of the FGF family, the hormone is currently being developed as a diet drug and a drug for diabetes, and the drug has entered clinical trials. FGF21 exerts a physiological action through the FGF21 receptor and its co-receptor ⁇ -klotho.
  • Leptin Produced mainly by white adipose tissue. Its precursor consists of 167 amino acid residues and contains a N-terminal 21 amino acid signal peptide. The signal peptide of the precursor is cleaved off in the blood to become a 146 amino acid leptin mature peptide (leptin). Leptin has a wide range of biological effects, such as acting on the metabolic regulation center of the hypothalamus, exerting appetite suppression, reducing energy intake, increasing energy expenditure, and inhibiting fat synthesis.
  • 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.
  • Dimer double-acting active protein In this context, “double-acting active protein”, “dimer double-acting activity”, and “double-acting agonistic active protein” are synonymous and can be used interchangeably. Means a fusion protein having both GCGR and GLP-1R agonistic activity, which has a F C moiety and thus has two peptide chains, which are formed by non-covalent and covalent interactions between the two peptide chains. The structure of the dimer.
  • Dimeric tri-acting active protein In this context, "three-way active protein”, “three-way agonistic active protein”, “three active agonistic active protein”, “dimeric three-way active protein” are synonymous, and can Change to use. Means a fusion protein having both GCGR, GLP-1R agonistic activity and FGF21 activity (or leptin activity), which has a F C moiety and therefore has two peptide chains, which are passed between the two peptide chains. Covalent and covalent interactions form the structure of the dimer.
  • IC 50 half maximal inhibitory concentration refers to the semi-inhibitory concentration of the antagonist being measured. It indicates the concentration required for a drug or substance (inhibitor) to inhibit its corresponding 50% biological response (or some of the substances involved in the reaction, such as enzymes, cell receptors or microorganisms) .
  • 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.
  • LDL Low-density lipoprotein
  • a type of plasma lipoprotein is the main carrier of cholesterol in the blood, which tends to deposit cholesterol on the arterial wall.
  • White blood cells try to digest low-density lipoproteins, but in the process they turn them into toxins. More and more white blood cells are attracted to the changing areas, causing the arterial wall to become inflamed. Over time, as the process continues, these plaque deposits can accumulate on the arterial wall, making the channel very narrow and lacking toughness. If too many plaques accumulate, the artery can be completely blocked.
  • LDL-C LDL and cholesterol
  • High-density protein Helps clear LDL on the arteries, acts as a scavenger, clears LDL from the artery and returns to the liver.
  • Triglyceride Another type of fat used to store excess energy in the diet.
  • High levels of triglycerides in the blood are associated with atherosclerosis.
  • High triglycerides can be caused by overweight and obesity, lack of exercise, smoking, excessive alcohol consumption, and high carbohydrate intake (more than 60% of total calories).
  • Sometimes basic or genetic diseases are the cause of high triglycerides.
  • People with high triglycerides typically have high total cholesterol levels, including high LDL cholesterol and low HDL cholesterol, and many people with heart disease or diabetes also have high triglyceride levels.
  • 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 (EC50 of about 1.1 nM).
  • the GLG-1R agonistic activity ratio calculated according to the data disclosed in the corresponding patents and literatures by US9018164 B2 and Joseph R. Chabenne et al., the GLP-1R agonistic activity ratio changes only before and after increasing the Cendrin extended peptide cex sequence of Exendin-4. The ratio is about 0.7% of the GLP-1R agonistic activity of the natural Glucagon in the article, and increases to 1.6% after the GPSSGAPPPS sequence is increased. 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 against the hydrolysis of the 16-18th protease on the basis of the natural Glucagon to increase its stability. After the fusion of these mutants with F C , the stability was further improved. In the examples of the present invention, about 50% of the GCGR agonistic activity was still detected at 72 hours. At this time, the dimer (SEQ ID NO. 76) formed by fusion of the native Glucagon containing the cex sequence with F C has almost no activity detected.
  • GCGR/GLP-1R double-acting agonists designed and developed based on Oxyntomodulin and Glucagon have introduced 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 second dimeric active protein preserving natural L-Ser exhibits very high serum stability, and there is no significant sign of DPP-IV degradation at 24 hours, but no The corresponding polypeptide fused to F C was rapidly hydrolyzed by DPP-IV (Table 5).
  • the present inventors prepared the active protein C001G12S3A1F4 (SEQ ID NO. 75) fused with native Glucagon and F C and the active protein C002G12S3A1F4 (SEQ ID NO. 76) fused by Glucagon-cex and F C as reported by Joseph R. Chabenne et al. Verify that F C fusion improves stability.
  • C001G12S3A1F4 SEQ ID NO. 75
  • C002G12S3A1F4 SEQ ID NO. 76
  • binding to serum albumin may help to improve protein stability (such as liraglutide)
  • protein stability such as liraglutide
  • the second is not mutated, the half-life cannot be sustained for more than 12 hours. That is, it is impossible to support the frequency of administration once a week.
  • the GCG analog pharmacokinetic and pharmacodynamic tests provided by the present invention have been shown to be sufficient to support the frequency of dosing once a week, rather than once a day (eg, albumin-binding liraglutide).
  • the retention of natural amino acids further reduces the risk of immunogenicity, avoids chemical cross-linking and makes the preparation process easier and more convenient.
  • GSIS Glucose-stimulated insulin secretion assay
  • GLP-1 or its analog acts on islet ⁇ cells by agonizing GLP-1R, promoting transcription of insulin genes, synthesis and secretion of insulin. Clinically, GLP-1 analogs are often used in combination with insulin. Although the detailed mechanism of GLP-1R agonism has not been fully revealed, the cAMP signal production after receptor activation and rapid endocytosis of receptors are relatively clear facts.
  • a portion of the GCGR/GLP-1R agonist induces a significant decrease in insulin secretion.
  • Studies have shown that a variety of human gland receptors are present on the surface of a variety of human cells (Dominik Schelshorn et al, Lateral Allosterism in the Glucagon Receptor Family: Glucagon-Like Peptide 1 Induces G-Protein-Coupled Receptor Heteromer Formation, Molecular Pharmacology , 81 (3) 309-318, 2012).
  • the GCGR/GLP-1R double-acting agonist binds to both GLP-1R and GCGR receptors, it may not only reduce its original physiological effects, but some potential unknown effects are even more difficult to predict. In addition to the reduction in insulin secretion, there may be unpredictable side effects. The safety of diabetes medication is extremely high, and therefore, the inventors believe that a dimer which does not cause cross-linking of the receptor should be superior.
  • IPGTT Peritoneal glucose tolerance test
  • mice administered the bispecific 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 multiplex active protein of the present invention induced a significant decrease in body weight after administration in DIO mice.
  • bispecific active proteins of the invention have improved pharmacokinetic properties, i.e., they have an extended half-life in vivo.
  • various bispecific active proteins are administered subcutaneously in rats, and serum concentrations are determined at different time points to assess their pharmacokinetic properties.
  • 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 ( PMWassarman 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.
  • the GCG analog of the present invention is referred to as A, and the structural formula of A is as shown in Formula II (SEQ ID NO. 141):
  • X 16 is selected from any one of amino acids other than Y, N, W, and H
  • X 17 is selected from any one of amino acids other than P, L, T, F, and H
  • X 18 is selected from P, Any one of amino acids other than F, H and W; further, X 17 and X 18 may not be R at the same time
  • X 29 is T or a deletion
  • X z is selected from any one of SEQ ID NOS. 3 to 10.
  • amino acid sequences of the exemplary GCG analogs may each independently be selected from the group consisting of SEQ ID NO. 44-74, and the corresponding polypeptide codes are C001, C002, C240, C320, C276, C225, C302, C163, C350, C271, C368, C495, C353, C352, C307, C382, C232, C227, C266, C137, C399, C395, C394, C392, C462, C228, C187, C334, C364, C209, C289.
  • HSQGTFTSDYSKYLD X 16 X 17 X 18 AQDFVQWLMN X 29 X z (SEQ ID NO. 141), wherein X 16 is selected from the group consisting of Y, N, W, and H. any amino acid of a; X 17 is selected from any one of amino acid except P, L, T, F and H a; X 18 is selected from any of the amino acid except P, F, H and W a; and additional X 17 X 18 cannot be R at the same time, X 29 is T or a deletion, and X z is selected from GGPSSGAPPPS (SEQ ID NO.
  • GPSSGAPPPS SEQ ID NO. 4
  • PSSGAPPPS PSSGAPPPS
  • SEQ ID NO. 5 PSSGAPPPS
  • SEQ ID NO) .6 GGPSSGAPPS
  • GPSSGAPPS SEQ ID NO. 8
  • PSSGAPPS PSSGAPPS
  • SEQ ID NO. 9 SSGAPPS (SEQ ID NO. 10).
  • F is a long-acting protein unit, and 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, such as SEQ ID NO. 11-20.
  • F may be the complete FC portion of native IgG1, the amino acid sequence is set forth in SEQ ID NO. 11, F may be a mutant of the FC portion of native IgG1, the amino acid sequence is set forth in SEQ ID NO. 12, and F may be natural.
  • the complete FC portion of IgG2 the amino acid sequence is set forth in SEQ ID NO. 13
  • F may be the complete FC portion of native IgG4
  • the amino acid sequence is set forth in SEQ ID NO. 14
  • F may be a mutant of the FC portion of native IgG4
  • the amino acid sequence is set forth in SEQ ID NO. 15-18, and F may be a mutant of the FC portion of native IgG1, and the amino acid sequence may be as shown in SEQ ID NO. 19-20.
  • L is a linking chain which is a flexible polypeptide of glycine (G), serine (S) and/or alanine (A) of suitable length such that adjacent protein domains They are free to move relative to each other.
  • G glycine
  • S serine
  • A alanine
  • the linker is exemplified by (GS)n, (GGS)n, (GGSG)n, (GGGS)nA, (GGGGS)nA, (GGGGA)nA, etc., n is an integer of 1-10, in a preferred In an 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. 21-43.
  • Example 1 the amino acid sequence of the dimeric double-acting active protein (the structural formula is as shown in Formula I) obtained by fusion of the GCG analog with the linker and F is selected from the group consisting of SEQ ID NO. 75-105 and SEQ ID NO. 142, the corresponding nucleotide sequences are SEQ ID NO. 161-191 and SEQ ID NO.
  • the corresponding double-acting active protein ALF codes are C001G12S3A1F4, C002G12S3A1F4, C240G12S3A1F4, C320G12S3A1F4, C276G12S3A1F4, C368G12S3A1F4, C225G12S3A1F4, C302G12S3A1F4, respectively.
  • 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 and F in the recombinant protein are respectively prepared, and each DNA fragment can be synthesized and spliced by conventional solid phase synthesis technology;
  • 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 to detect GLP-1R agonistic activity.
  • 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 the pCRE-EGFP plasmid.
  • the EGFP gene was replaced to obtain a 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) or natural Glucagon (Hangzhou Zhongpept Biochemical Co., Ltd., GLUC-004) and natural GLP-1 (Hangzhou Zhongpept Biochemical 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).
  • the luciferase reporter assay is 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, and the transfected HEK 293T cells and the stably transfected cell line HEK 293T/GCGR were constructed as above.
  • Figure 2 is a graph showing the results of the measured EC50 of the partially dimeric double-acting active protein.
  • CG283G12S3A1F4 (SEQ ID NO. 106), CG214G12S3A1F4 (SEQ ID NO. 107), CG267G12S3A1F4 (SEQ ID NO. 108), C308G12S3A1F4 (SEQ ID NO. 109), C224G12S3A1F4 (SEQ ID NO. 110), CG308G12S3A1F4 (SEQ ID NO. 111), C319G12S3A1F4 (SEQ ID NO. 112), C214G12S3A1F4 (SEQ ID NO.
  • C303G12S3A1F4 (SEQ ID NO. 114)
  • CG303G12S3A1F4 (SEQ ID NO. 115) was assayed for in vitro activity, including GLP-1R agonistic activity assay and GCGR agonistic activity assay.
  • GLP-1R As shown in Tables 1 and 2, when the sequence containing the extended peptide was prepared by fusion of (GGGGS) 3 A (SEQ ID NO. 33) and F (SEQ ID NO. 16) to form a dimer, GLP-1R was used. The agonistic activity was increased by more than 200 times, while the agonistic activity of GCGR was not significantly different.
  • GDSerGS H-D-Ser-QGTFTSDYSKYLDSQAAQDFVQWLMNGGPSSGAPPPS (SEQ ID NO. 116);
  • GAibGS H-Aib-QGTFTSDYSKYLDSQAAQDFVQWLMNGGPSSGAPPPS (SEQ ID NO. 117);
  • Residual activity The activity value at 0 hours was 100%, and the value measured at the subsequent time point was obtained.
  • the GCG analog of Example 1 was fused to the N-terminus of human IgG1 F C (SEQ ID NO. 12) by a ligation chain of different length. That is, the dimeric double-acting active proteins obtained by fusion of the GCG analogs with the different length linkages and F (SEQ ID NO. 12) are C382G4A1F1, C382G4S1F1, C382G6S2F1, C382G4S4F1, C382G8S2F1, C382G8A2F1, C382G9S3F1, C382G9S2A1F1, respectively.
  • the preparation method used in this example is the same as that in Example 2.
  • the dimeric double-acting active protein obtained in the present example was assayed for in vitro activity, including GLP-1R agonistic activity assay and GCGR agonistic activity assay.
  • the detection method is the same as in the third embodiment.
  • Rat BRIN-BD11 cells were used to determine insulin release induced by active protein stimulation, but with minor modifications, ie in a 24-well plate (Orange Scientific, Brainel) 1.0 ⁇ 10 6 cells were added to each well in 'Alleud, Belgium, cultured at 37 ° C overnight, and the supernatant was centrifuged, and 1.0 ml of KRB (115 mM NaCl, 4.7 mM KCl, 1.28 mM CaCl 2 , 1.2 mM MgSO 4 ) was added to each well.
  • KRB 115 mM NaCl, 4.7 mM KCl, 1.28 mM CaCl 2 , 1.2 mM MgSO 4
  • 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.
  • mice Male Sprague-Dawley rats of about 6 weeks were grouped into groups of 8 rats. Administration according to the protocol of Table 5 or physiological saline. At the end of the administration, all rats began to drink freely; the time point at which the administration was completed was set to 0, and was used as a time reference for subsequent experiments. The pharmacokinetic detection of dimeric double-acting active protein was carried out by sandwich ELISA.
  • Ie mouse anti-human IgG4FC mAb (9002-01, I2013-NG 56, 0.25mg/ml); coated plate (96-well plate, corning, 42592), 200ng / well, 4 ° C overnight; PBST wash plate 4 times 5% milk powder solution was blocked, room temperature was 1 h; PBST was washed 4 times; rat serum was diluted with PBST, then incubated at 37 ° C for 2 h; PBST was washed 6 times; and rabbit anti-Glucagon antibody was diluted with 1% BSA solution (home-made: The natural Glucagon polypeptide (SEQ ID NO.
  • the FGF21 analog is further fused to construct a fusion protein (a tri-fold active protein fused to the FGF21 analog): native FGF21 (SEQ ID NO. 143), FGF21 analog 1 ( SEQ ID NO. 144), FGF21 Analog 2 (SEQ ID NO. 145), FGF21 Analog 3 (SEQ ID NO. 146), C382F4FGF (SEQ ID NO. 147), C382F4FGF1 (SEQ ID NO. 148), C382F4FGF2 (SEQ ID NO. 149), C382F4FGF3 (SEQ ID NO. 150), C495F4FGF (SEQ ID NO. 151), C495F4FGF1 (SEQ ID NO. 152), C495F4FGF2 (SEQ ID NO. 153), C495 F4FGF3 (SEQ ID NO.154).
  • native FGF21 SEQ ID NO. 143
  • FGF21 analog 1 SEQ ID NO. 144
  • the preparation method of the fusion protein belongs to the prior art: since the F C sequence is carried out, protein purification can be carried out by high affinity and high specificity Protein A resin chromatography, and the method in Example 2 can be referred to.
  • the electropherogram of the purified fusion protein is shown in Fig. 7.
  • the preparation of natural FGF21 and FGF21 analogs is described in Xu J et al. (Xu J et al., Polyethylene glycol modified FGF21 engineered to maximize potency and minimize vacuole formation, Bioconjug Chem.; 24(6): 915-25, 2013) and is as follows Modification: The expression vector was PET30 and the host strain was BL21 (DE3) (Merck China).
  • the inclusion bodies were washed four times with washing solution (50 mM Tris, 150 mM NaCl, 2 M urea, pH 8.0) and weighed; 1 ml of denaturant (50 mM Tris, 150 mM NaCl) was added per 0.1 g of inclusion body according to (1:10 mass to volume ratio). , 8M urea, 10mM DTT, pH 8.5), gently mix and dissolve for more than 5h at room temperature shaker; dilute renaturation according to the ratio of 1:100-200.
  • the denaturant was slowly added dropwise to the reconstituted solution, and stirring was continued during the process; after the addition was completed, the protein-containing reconstituted solution was placed at 4 ° C for 24 h; after completion, it was filtered with a 0.45 um filter for chromatographic purification. .
  • 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.
  • 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 subcutaneous administration was performed the next day (Table 8). .
  • the double-acting active protein was administered at a dose of 30 nmol/kg body weight or 90 nmol/kg body weight once every 4 days; the FGF21 analog was administered twice daily at 30 nmol/kg body weight; and the negative control group was injected with physiological saline at 5 ul/g body weight ( PBS); The positive control group was injected with liraglutide (30 nmol/kg body weight) once a day for 28 days, and the body weight and food intake of the mice were measured every day. 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 8; the change in total food intake is shown in Figure 9.
  • native leptin SEQ ID NO. 155
  • fusion protein trimeric protein of fusion leptin
  • 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.
  • the negative control group was injected with normal saline (PBS) at 5ul/g body weight; the double-acting active protein was administered once every 4 days, and other days were given PBS sham injection for 28 days, leptin was 30nmol / kg body weight was administered twice daily, and the body weight and food intake of the mice were measured daily.
  • PBS normal saline
  • 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 10; the change in total food intake is shown in Figure 11.
  • nucleotide sequences corresponding to the active proteins described in the present specification are shown in Table 10.

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Abstract

The present invention falls within the field of biological drugs, and in particular relates to a multiple-active protein for treating metabolic diseases. The multiple-active protein of the present invention has the structural formula: A-L-F or A-L1-F-L2-B. Compared to the the prior art, the multiple-active protein of the present invention has the following beneficial effects: the multiple-active protein has a long half-life, and supports a dosage frequency of once a week; the GLP-1R agonistic activity of the multiple-active protein increases up to at least 200 times, with the ratio of GCG to GLP-1 reaching approximately 1 : 1; and the multiple-active protein has a good stability and a low immunogenicity.

Description

一种治疗代谢疾病的多重活性蛋白Multiple active protein for treating metabolic diseases 技术领域Technical field
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重活性蛋白。The invention belongs to the field of biopharmaceuticals, and in particular relates to a multiplex active protein for treating metabolic diseases.
背景技术Background technique
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)。According to the pathological features, 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. Among them, 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).
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 PCTCN2018116171-appb-000001
)、阿必鲁肽(Albiglutide，商品名
Figure PCTCN2018116171-appb-000002
)、利拉鲁肽(Liraglutide，商品名
Figure PCTCN2018116171-appb-000003
及
Figure PCTCN2018116171-appb-000004
分别用于治疗肥胖和糖尿病)、艾塞那肽(Exenatide，商品名
Figure PCTCN2018116171-appb-000005
)、利西拉肽(Lixisenatide，商品名
Figure PCTCN2018116171-appb-000006
)及可能即将上市的索马鲁肽(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)。 Conventional chemicals for the treatment of type 2 diabetes, such as sulfonylureas and thiazolidinediones, have significant hypoglycemic effects, but the main disadvantage is that they lead to weight gain (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.). The protein drugs for type 2 diabetes are mainly GLP-1R (GLP-1 receptor) agonists, such as Dulaglutide (trade name:
Figure PCTCN2018116171-appb-000001
), Albiglutide (trade name)
Figure PCTCN2018116171-appb-000002
), liraglutide (Liraglutide, trade name
Figure PCTCN2018116171-appb-000003
and
Figure PCTCN2018116171-appb-000004
For the treatment of obesity and diabetes, respectively, Exenatide (trade name)
Figure PCTCN2018116171-appb-000005
), lixisenatide (Lixisenatide, trade name
Figure PCTCN2018116171-appb-000006
) and may be listed soon, such as Semaglutide. 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. For example, duraglutide has a reduced weight of about 2.9 kilograms, while 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. Although bariatric surgery can significantly improve obesity and treat diabetes, its application is not widespread, as most patients are reluctant to undergo such surgery because of surgical risks and long-term sequelae (Obesity and Diabetes, New Surgical and Nonsurgical Approaches, Springer Press, 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。It has been reported that 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.
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。Among them, the receptors of glucagon and GLP-1 (Glucagon-like peptide-1) are structurally related, but these two hormones play a diametrically opposite role in controlling glucose. Clinically, GLP-1 and its analogues are mainly used for glycemic control in diabetics, while glucagon is used for acute hypoglycemia. In recent years, more and more studies have shown that glucagon can effectively reduce body weight despite the risk of raising blood sugar; more importantly, 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. Although GCGR agitation may result in an increase in blood glucose levels, this risk can be appropriately offset by GLP-1R activation.
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-1 and 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 Peptides96:133–141,2001；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)。突变产生的免疫原性风险极高。也有少数报道第二位保持天然Ser的交联脂肪酸的Glucagon类似物(Henderson SJ等，Robust anti-obesity and metabolic effects of a dual GLP-1/Glucagon receptor peptide agonist in rodents and non-human primates,Diabetes Obes Metab，2016)。该类似物(MEDI0382)包括30个氨基酸，与天然Glucagon相比，突变了9个氨基酸。同时，虽然MEDI0382的第二位也保留了天然的Ser氨基酸，但是仅能支持一天一次的给药频率。Currently, 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). Most of these analogs mutate the second serine (Ser) to the non-natural amino acid Aib to resist the enzymatic hydrolysis of DPP-IV. This is because both natural 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). 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 et al, A DPP-IV-resistant Triple-acting agonist of GIP, GLP-1 and glucagon receptors with potent glucose-lowering and insulinotropic actions in high-fat-fed mice, Diabetologia, 56(6): 1417-24.2013; John A. Pospisilik et al; Metabolism of glucagon by Dipeptidyl peptidase IV (CD26), Regulatory Peptides 96: 133–141, 2001; Hinke SA et al, 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 et al, DPP-IV-resistant, long-acting oxyntomodulin de Rivers, J.Pept.Sci., 17:270–280, 2011). Mutations produce an extremely high risk of immunogenicity. There are also a few reports of Glucagon analogues of the second cross-linked fatty acid that retains natural Ser (Henderson SJ et al, Robust anti-obesity and metabolic effects of a dual GLP-1/Glucagon receptor peptide agonist in rodents and non-human primates, Diabetes Obes Metab, 2016). This analog (MEDI0382) consists of 30 amino acids and is mutated by 9 amino acids compared to native Glucagon. At the same time, although the second place of MEDI0382 also retains the natural Ser amino acid, it can only support the frequency of dosing once a day.
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来验证胃泌酸调节素能与这2种受体结合而起作用。另外，胃泌酸调节素虽然能激动 GLP-1R及GCGR，但是活性相当的低(大约分别是天然GLP-1和Glucagon的十分之一和百分之一左右)，而一般研究所用的胃泌酸调节素类似物在GLP-1R及GCGR的激动活性一般是设计成1:1左右，多数研究认为活性1:1左右时的降糖效果及减脂效果最好(Peptide-based GLP-1/Glucagon co-agonists:a double-edged sword to combat diabesity，Hitesh Soni，95:5–9，2016)。另外也有在基于Glucagon序列基础上进行改造的方法，同样的也大多是采用引入非天然氨基酸的形式。由于降糖药物的安全性要求极高，这类受体激动剂开发的难点之一为需要综合考虑到免疫原性的影响，一般情况下与人源序列具有越高的同源性，那么在人体中免疫原性风险就相对的越低。罗氏与益普生合作研发的GLP-1R激动剂降糖药Taspoglutide(引入了非天然氨基酸Aib)，抗体生成率达到了49％，最终停止了所有Ⅲ期临床研究(Julio Rosenstock等，The Fate of Taspoglutide,a Weekly GLP-1 Receptor Agonist,Versus wice-Daily，Exenatide for Type 2,Diabetes Care,36:498-504,2013)。Although the oxyntomodulin analogue showed initial hypoglycemic and lipid-lowering effects, its mechanism of action is still inaccurate: the oxyntomodulin receptor has not been found, and is currently only knocked out by GCGR or GLP-1R. Mouse or cell assays to verify that oxyntomodulin binds to these two receptors. In addition, although oxyntomodulin can agonize GLP-1R and GCGR, its activity is quite low (about one-tenth and one-hundredth of that of natural GLP-1 and Glucagon, respectively), and the stomach used in general research. The agonistic activity of the oxyntomodulin analogues in GLP-1R and GCGR is generally designed to be about 1:1. Most studies consider that the hypoglycemic effect and the fat-reducing effect are best when the activity is about 1:1 (Peptide-based GLP-1). /Glucagon co-agonists: a double-edged sword to combat diabesity, Hitesh Soni, 95:5–9, 2016). There are also methods based on the Glucagon sequence, and most of them are also introduced in the form of unnatural amino acids. Because of the high safety requirements of hypoglycemic drugs, one of the difficulties in the development of such receptor agonists is the need to take into account the effects of immunogenicity, and generally have higher homology with human sequences, then The risk of immunogenicity in the human body is relatively low. The GLP-1R agonist hypoglycemic agent Taspoglutide (introduced with the non-natural amino acid Aib) developed by Roche and Epson, the antibody production rate reached 49%, and finally stopped all phase III clinical studies (Julio Rosenstock et al, The Fate of Taspoglutide, a Weekly GLP-1 Receptor Agonist, Versus wice-Daily, Exenatide for Type 2, Diabetes Care, 36: 498-504, 2013).
另一个问题则涉及GLP-1R/GCGR双效激动剂的半衰期问题。目前大多研究都采用脂肪酸或PEG交联的方式。脂肪酸交联可以最大程度地减少活性损失，然而半衰期大多只能维持12小时左右，因此只能采取每天给药的方式；PEG交联相对于脂肪酸虽然更有效地延长半衰期，但是导致的活性损伤问题非常严重。更重要的是，目前还未有报道能有效抵御DPP-IV降解的融合Glucagon类似物或者GLP-1R/GCGR双特异性激动剂。根据现有技术，将Glucagon类似物与F C或人血清白蛋白等长效单元融合表达并在第二位引入非天然氨基酸Aib是极难实现的。而如果模仿天然GLP-1，将天然Glucagon的第二位Ser突变为其他天然氨基酸，则会导致GCGR激动活性明显下降(Alessia Santoprete等，DPP-IV-resistant,long-acting oxyntomodulin derivatives,J.Pept.Sci.,17:270–280,2011)。常用的方法是先化学合成带非天然氨基酸的小肽，再用F C、PEG或脂肪酸等进行交联。如HM12525A就是在GLP-1R/GCGR双特异性小肽的基础上交联了人源F C制备而成。(Jahoon Kang等，The ultra-long acting LAPSGLP/GCG dual agonist,HM12525A,demonstrated safety and prolonged pharmacokinetics in healthy volunteers:a phase 1 first-in-human study，51st European Association for the Study of Diabetes(EASD),Stockholm,Sweden；September 14-18,2015)。 Another issue relates to the half-life problem of GLP-1R/GCGR double-acting agonists. At present, most studies use fatty acid or PEG cross-linking. Fatty acid cross-linking can minimize the loss of activity. However, most of the half-life can only be maintained for about 12 hours, so it can only be administered in a daily manner. PEG cross-linking is more effective in prolonging the half-life than fatty acid, but the activity damage is caused. very serious. More importantly, fusion Glucagon analogs or GLP-1R/GCGR bispecific agonists that are effective against DPP-IV degradation have not been reported. According to the prior art, it is extremely difficult to fuse a Glucagon analog with a long acting unit such as F C or human serum albumin and introduce a non-natural amino acid Aib in the second position. However, if the natural GLP-1 is mimicked and the second Ser of the natural Glucagon is mutated to other natural amino acids, the GCGR agonistic activity is significantly reduced (Alessia Santoprete et al., DPP-IV-resistant, long-acting oxyntomodulin derivatives, J.Pept). .Sci., 17:270–280, 2011). A common method is to chemically synthesize small peptides with unnatural amino acids, and then crosslink them with F C , PEG or fatty acids. For example, HM12525A is prepared by cross-linking human F C on the basis of GLP-1R/GCGR bispecific small peptide. (Jahoon Kang et al, The ultra-long acting LAPSGLP/GCG dual agonist, HM12525A, demonstrated safety and prolonged pharmacokinetics in healthy volunteers: a phase 1 first-in-human study, 51st European Association for the Study of Diabetes (EASD), Stockholm , Sweden; September 14-18, 2015).
发明内容Summary of the invention
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。In order to overcome the problems in the prior art, it is an object of the present invention to provide a multidomain active protein for the treatment of metabolic and related diseases, and the preparation and use thereof. 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.
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：In order to achieve the above and other related objects, the present invention adopts the following technical solutions:
本发明的第一方面提供一种多重活性蛋白，所述多重活蛋白的结构中包括如式I所A first aspect of the invention provides a multiplex active protein, the structure of the multiplexed live protein comprising
示结构，如如式I所示结构为：A-L-F式IThe structure is as shown in formula I: A-L-F I
式I中，A为GCGR/GLP-1R双效激动剂，F为长效蛋白单元，L为连接所述A与F的连接链。In Formula I, A is a GCGR/GLP-1R double-acting agonist, F is a long-acting protein unit, and L is a linking chain connecting the A and F.
进一步地，所述GCGR/GLP-1R双效激动剂选自天然Glucagon(SEQ ID NO.44)的类似物或者其他具有GCGR/GLP-1R双效激动活性的多肽或蛋白。在本发明的实施例中，所述A的结构中包括如式II所示结构，如式II所示结构为：Further, the GCGR/GLP-1R double-acting agonist is selected from an analog of native Glucagon (SEQ ID NO. 44) or other polypeptide or protein having GCGR/GLP-1R double-acting agonistic activity. In an embodiment of the present invention, the structure of the A includes a structure as shown in Formula II, and the structure shown in Formula II is:
HSQGTFTSDYSKYLD X 16X 17X 18 AQDFVQWLMN X 29 X z(SEQ ID NO.141)式II，其中，X 16选自除Y、N、W、和H外的氨基酸之任一；X 17选自除P、L、T、F和H外的氨基酸之任一；X 18除选自P、F、H和W外的氨基酸之任一；另外X 17与X 18不能同时为R，X 29为T或缺失，X z选自GGPSSGAPPPS(SEQ ID NO.3)、GPSSGAPPPS(SEQ ID NO.4)、PSSGAPPPS(SEQ ID NO.5)、SSGAPPPS(SEQ ID NO.6)、GGPSSGAPPS(SEQ ID NO.7)、GPSSGAPPS(SEQ ID NO.8)、PSSGAPPS(SEQ ID NO.9)或SSGAPPS(SEQ ID NO.10)之任一。 HSQGTFTSDYSKYLD X 16 X 17 X 18 AQDFVQWLMN X 29 X z (SEQ ID NO. 141) Formula II, wherein X 16 is selected from any one of amino acids other than Y, N, W, and H; X 17 is selected from P Any one of amino acids other than L, T, F and H; X 18 except for one of amino acids other than P, F, H and W; and X 17 and X 18 may not be R at the same time, X 29 is T or Deletion, X z is selected from GGPSSGAPPPS (SEQ ID NO. 3), GPSSGAPPPS (SEQ ID NO. 4), PSSGAPPPS (SEQ ID NO. 5), SSGAPPPS (SEQ ID NO. 6), GGPSSGAPPS (SEQ ID NO. 7) Any of GPSSGAPPS (SEQ ID NO. 8), PSSGAPPS (SEQ ID NO. 9) or SSGAPPS (SEQ ID NO. 10).
所述A能抵御体内蛋白酶水解。The A is resistant to proteolytic hydrolysis in the body.
所述A的氨基酸序列可以如SEQ ID NO.46、SEQ ID NO.55、SEQ ID NO.59、SEQ ID NO.68、SEQ ID NO.74之任一所示。The amino acid sequence of A may be as shown in any one of SEQ ID NO. 46, SEQ ID NO. 55, SEQ ID NO. 59, SEQ ID NO. 68, SEQ ID NO.
目前，无论是基于Glucagon还是胃泌酸调节素的杂合肽，大多在第二位上进行了改造，将Ser突变为非天然的氨基酸Aib或者D型氨基酸(D-Ser)(详见综述文献Peptide-based GLP-1/Glucagon co-agonists:a double-edged sword to combat diabesity，Hitesh Soni，95:5–9，2016)以抵御血清DPP-IV酶的水解。与天然GLP-1(SEQ ID NO:1)类似，天然的Glucagon(SEQ ID NO:44)极易遭受血清中DPP-IV的水解而导致失活(Victor A.Gault等，See comment in PubMed Commons below 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-1 and 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)。然而本发明人发现，本发明筛选获得的GCG类似物即使保留了天然第二位的Ser，在与F C融合后，稳定性也足以支持一周一次的给药频率，降低了免疫原性的潜在风险。而在16、17和18位上的改造，除了减弱内肽酶对GCG类似物的降解外，也能较好地保持GCGR激动活性。在本发明的其中一个实施例中，多重活性蛋白A-L-F结构中的A为GCG类似物，当与不含L-F部分的对应小肽相比，A-L-F结构的多重活性蛋白具有极高的抵御DPP-IV水解的能力。相反地，如报道所述，不含L-F部分的小肽第二位Ser极易受到DPP-IV攻击而失活。而且，A-L-F结构的多重活性蛋白在血清中的稳定性与第二位引入非天然氨基酸Aib或D-Ser的类似物相当。目前并无现有技术揭示可通过融合F C来增加GCG类似物的DPP-IV酶抵抗能力。众所周知，GLP-1的第二位为A，即便与F C融合后，GLP-1仍极易被DPP-IV降解水解前两个氨基酸HA。因此，目前上市及临床中一周一次给药频率的长效GLP-1类似物必须将第二位氨基酸突变成甘氨酸Gly(如杜拉鲁肽和阿必鲁肽)或Aib(如索马鲁肽)才能保持N端的稳定性。同样地，对于Glucagon类似物多肽，如上所述有众多的报道表明，第二位氨基酸为天然Ser的GCG类似物极易受DPP-IV攻击而失活，同样必须通过将第二位突变为非天然氨基酸才能避免降解，同时保留GCGR激动活性。根据现有技术，将GCG类似物与F C融合并进行第二位氨基酸的修饰，是无法实现的。而如果模仿天然GLP-1，将天然Glucagon的第二位氨基酸Ser突变为其他天然氨基酸，很可能会导致GCGR激动活性明显下降(Alessia Santoprete等，DPP-IV-resistant,long-acting oxyntomodulin derivatives,J.Pept.Sci.,17:270–280,2011)。唯一的既能实现一周一次给药周期(长半衰期)又能保留高活性的方法，只有将第二位的Ser替换为非天然氨基酸，同时用PEG或者F C等大分子进行交联反应(Jahoon Kang等，The ultra-long acting  LAPSGLP/GCG dual agonist,HM12525A,demonstrated safety and prolonged pharmacokinetics in healthy volunteers:a phase 1 first-in-human study，51 st European Association for the Study of Diabetes(EASD),Stockholm,Sweden；September 14-18,2015)。而本发明人惊奇地发现，本发明提供的优选GCG类似物与F C融合后，获得了极高的DPP-IV抵抗能力，而且药效学实验表明稳定性能支持一周一次的给药频率。通过融合的方式，达到这种效果，是目前未有所见的。保留第二位的Ser，一方面降低了免疫原性风险，另一方面则是最大程度地保留了GCGR的激动活性，能有效地达到减重效果。 At present, most of the hybrid peptides based on Glucagon or oxyntomodulin have been modified in the second position, and the Ser is mutated to the non-natural amino acid Aib or D-amino acid (D-Ser) (see review literature for details). Peptide-based GLP-1/Glucagon co-agonists: a double-edged sword to combat diabesity, Hitesh Soni, 95: 5–9, 2016) to resist hydrolysis of the serum DPP-IV enzyme. Similar to native GLP-1 (SEQ ID NO: 1), native Glucagon (SEQ ID NO: 44) is highly susceptible to hydrolysis by DPP-IV in serum resulting in inactivation (Victor A. Gault et al., See comment in PubMed Commons) Below 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 et al, A DPP-IV-resistant triple-acting agonist of GIP, GLP-1 and glucagon receptors with potent glucose-lowering and insulinotropic actions in high-fat-fed mice, Diabetologia, 56(6): 1417-24.2013; John A. Pospisilik et al; Metabolism of glucagon by dipeptidyl peptidase IV (CD26), Regulatory Peptides 96: 133–141, 2001; Hinke SA et al, Dipeptidyl peptidase IV (DPIV/CD26) degradation of glucagon. Characterization of glucagon degradation products and DPIV- Resistant analogs, J Biol Chem 275: 3827–3834, 2000). However, the inventors have found that the GCG analog obtained by the screening of the present invention retains the natural second-position Ser, and after fusion with F C , the stability is sufficient to support the frequency of administration once a week, and the potential for immunogenicity is lowered. risk. Modifications at positions 16, 17 and 18, in addition to attenuating the degradation of the GCG analog by endopeptidases, also maintained GCGR agonistic activity. In one embodiment of the invention, A in the multiplexed protein ALF structure is a GCG analog, and the multiplexed active protein of the ALF structure is highly resistant to DPP-IV when compared to a corresponding small peptide that does not contain the LF moiety. The ability to hydrolyze. Conversely, as reported, the second Ser, a small peptide that does not contain the LF moiety, is highly susceptible to DPP-IV attack and is inactivated. Moreover, the stability of the multiplexed active protein of the ALF structure in serum is comparable to that of the second analog introducing the unnatural amino acid Aib or D-Ser. There is currently no prior art disclosure to increase the DPP-IV enzyme resistance of GCG analogs by fusing F C. It is well known that the second position of GLP-1 is A, and even after fusion with F C , GLP-1 is easily degraded by DPP-IV to hydrolyze the first two amino acids HA. Therefore, long-acting GLP-1 analogues that are currently on the market and once a week in clinical doses must mutate the second amino acid to glycine Gly (such as duraglutide and albendide) or Aib (such as Somaru). Peptide) to maintain the stability of the N-terminus. Similarly, for Glucagon analog polypeptides, there are numerous reports as described above that the GCG analog whose second amino acid is native Ser is highly susceptible to DPP-IV challenge and is also inactivated by mutating the second position to non- Natural amino acids can avoid degradation while retaining GCGR agonistic activity. According to the prior art, fusion of a GCG analog to F C and modification of the second amino acid is not possible. However, if the natural GLP-1 is mimicked, the second amino acid Ser of the natural Glucagon is mutated to other natural amino acids, which may result in a significant decrease in GCGR agonistic activity (Alessia Santoprete et al., DPP-IV-resistant, long-acting oxyntomodulin derivatives, J). .Pept.Sci., 17:270–280, 2011). The only way to achieve a once-a-week dosing cycle (long half-life) while retaining high activity is to replace the second Ser with an unnatural amino acid and crosslink with a macromolecule such as PEG or F C (Jahoon) Kang et al, The ultra-long acting LAPS GLP/GCG dual agonist, HM12525A, demonstrated safety and prolonged pharmacokinetics in healthy volunteers: a phase 1 first-in-human study, 51 st European Association for the Study of Diabetes (EASD), Stockholm , Sweden; September 14-18, 2015). The inventors have surprisingly found that the preferred GCG analogs provided by the present invention, upon fusion with F C , achieve extremely high DPP-IV resistance, and pharmacodynamic experiments have shown that stability can support a once-a-week dosing frequency. This effect has been achieved through integration, which is currently not seen. Retaining the second Ser, on the one hand, reduces the risk of immunogenicity, on the other hand, it retains the agonistic activity of GCGR to the greatest extent, and can effectively achieve the weight loss effect.
Ritzel U等报道了一种第二位从Ala突变为Ser的GLP-1类似物多肽(Ritzel U等，A synthetic glucagon-like peptide-1 analog with improved plasma stability，J Endocrinol.， 159(1):93-102，1998)具有抵抗DPP-IV效果。基于此，Picha KM等(Picha KM等，Protein engineering strategies for sustained glucagon-like peptide-1 receptor-dependent control of glucose homeostasis，Diabetes，57(7):1926-34，2008)亦报道了一种N端两位氨基酸为HS的GLP-1与抗体F C融合的活性蛋白(CNTO736)。然而，如上所述，众多报道表明Glucagon类似物的N端保留天然HS序列时，是非常不稳定的，即便是交联了脂肪酸，半衰期都很难持续到12小时以上；而且发明人也发现，将具有HS序列的天然Glucagon直接与F C融合(SEQ ID NO.75)，或将Joseph R.Chabenne等报道的Glucagon-cex序列(Joseph R.Chabenne等，Optimization of the Native Glucagon Sequence for Medicinal Purposes，J Diabetes Sci Technol.4(6):1322–1331，2010)与F C融合(SEQ ID NO.76)，仍不具备显著的抵抗DPP-IV酶的性质。这说明虽然GLP-1与Glucagon同属于肠降血糖素(Incretin)家族成员，然而不同的序列本身具有较大的构象差异，因而对于蛋白酶的抵抗能力也各有不同。 Ritzel U et al. reported a second GLP-1 analogue polypeptide mutated from Ala to Ser (Ritzel U et al, A synthetic glucagon-like peptide1) with improved plasma stability, J Endocrinol., 159(1): 93-102, 1998) has a DPP-IV resistance. Based on this, Picha KM et al. (Picha KM et al., Protein engineering strategies for sustained glucagon-like peptide-1 receptor-dependent control of glucose homeostasis, Diabetes, 57(7): 1926-34, 2008) also reported an N-terminus. The two amino acids are the active protein (CNTO736) in which GLP-1 of HS is fused to antibody F C. However, as noted above, numerous reports indicate that the N-terminus of the Glucagon analog is very unstable when it retains the native HS sequence, and even if the fatty acid is cross-linked, the half-life is difficult to sustain for more than 12 hours; and the inventors have also discovered that The native Glucagon with the HS sequence is directly fused to F C (SEQ ID NO. 75), or the Glucagon-cex sequence reported by Joseph R. Chabenne et al. (Joseph R. Chabenne et al., Optimization of the Native Glucagon Sequence for Medicinal Purposes, J Diabetes Sci Technol. 4(6): 1322–1331, 2010) is fused to F C (SEQ ID NO. 76) and still does not possess significant resistance to DPP-IV enzymes. This suggests that although GLP-1 and Glucagon belong to the Incretin family, the different sequences themselves have large conformational differences and thus have different resistance to proteases.
另外，有报道表明Glucagon的16-18位也是易遭受降解的位点，因此大多数的Glucagon类似物都在此位点进行改造或修饰(PEG或脂肪酸等)，而为了平衡GCGR和GLP-1R激动活性，还需要在另外的位点引入更多突变，从而获得对两个受体都有高活性的多肽。比如目前处于临床研究中的MEDI0382(Henderson SJ等，Robust anti-obesity and metabolic effects of a dual GLP-1/Glucagon receptor peptide agonist in rodents and non-human primates,Diabetes Obes Metab，2016)，包括30个氨基酸。与天然Glucagon相比，引入了9个突变。同时，虽然MEDI0382的第二位也保留了天然的Ser氨基酸，但是仅能支持一天一次的给药频率。在本发明中的GCG类似物一方面保留了N端第二位的天然S氨基酸，仅进行了不超过3个氨基酸的突变，再与长效单元(如F C)融合后即足以支持一周一次的给药频率。在本发明的特定实施例中，为了降低GCGR的激动活性以平衡GCGR与GLP-1R的活性比例，还进一步把C末端的T删除。C末端的T删除对于Glucagon类似物的稳定性并无影响。因此，本发明的最大优势是采用了较少的位点突变达到了最理想的受体激动活性及稳定性的目的，并且突变时不引入非天然氨基酸，在降低了潜在的免疫原性的同时便于利用重组技术直接制备产物。 In addition, it has been reported that the 16-18 position of Glucagon is also a site susceptible to degradation, so most Glucagon analogs are modified or modified at this site (PEG or fatty acids, etc.), in order to balance GCGR and GLP-1R. For agonistic activity, it is also necessary to introduce more mutations at another site to obtain a polypeptide having high activity for both receptors. For example, MEDI0382 (Henderson SJ et al, Robust anti-obesity and metabolic effects of a dual GLP-1/Glucagon antibody peptide agonist in rodents and non-human primates, Diabetes Obes Metab, 2016) currently in clinical research, including 30 amino acids . Compared to the native Glucagon, 9 mutations were introduced. At the same time, although the second place of MEDI0382 also retains the natural Ser amino acid, it can only support the frequency of dosing once a day. The GCG analogue in the present invention retains the N-terminal second natural S amino acid on the one hand, and only undergoes mutations of not more than 3 amino acids, and is fused to a long-acting unit (such as F C ) to support once a week. Dosing frequency. In a particular embodiment of the invention, in order to reduce the agonistic activity of GCGR to balance the activity ratio of GCGR to GLP-1R, the C-terminal T is further deleted. The T deletion at the C-terminus has no effect on the stability of the Glucagon analog. Therefore, the greatest advantage of the present invention is that the use of fewer site mutations achieves the optimal receptor agonistic activity and stability, and does not introduce unnatural amino acids when mutated, while reducing potential immunogenicity. It is convenient to directly prepare products using recombinant techniques.
研究发现，将Joseph R.Chabenne等报道的Glucagon-cex序列与F C融合的制备获得的活性蛋白(C002G12S3A1F4，SEQ ID NO.76)并无显著的DPP-IV抵御能力(实施例4)。在ICR鼠的IPGTT试验及DIO鼠减肥试验(实施例8和9)中显示，C002G12S3A1F4也无明显的降血糖及持续的减重效果。同时，实施例4中还可见，虽然同样是16-18位突变的F C融合的GCG类似物，不同二聚体双效活性蛋白的稳定性差异却极大。蛋白的空间构象极 其复杂，因此符合式II中的突变不但提高了肽链内部的稳定性，更有可能是改变了GCG类似物与F C链间的相互作用构象，进一步地提高了融合蛋白N端的稳定性。更重要的是，本发明提供的A-L-F结构蛋白与现有的GCG及突变体相比，具有显著提升的GLP-1R激动活性。虽然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(SEQ ID NO.4,GPSSGAPPPS)后，使得其GLP-1R激动活性从0.7％增至1.6％，提高了约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US9018164B2)，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)。 The study found that the active protein (C002G12S3A1F4, SEQ ID NO. 76) obtained by the fusion of the Glucagon-cex sequence reported by Joseph R. Chabenne et al. with F C had no significant DPP-IV resistance (Example 4). In the IPCR mouse IPGTT test and the DIO mouse weight loss test (Examples 8 and 9), C002G12S3A1F4 also showed no significant hypoglycemic and sustained weight loss effects. Meanwhile, in Example 4 it can also be seen, although the same position is mutated 16-18 F C GCG fused analogs, different dimers bis difference in stability has a great effect active protein. The spatial conformation of the protein is extremely complex, so that the mutation in the formula II not only improves the stability of the interior of the peptide chain, but is more likely to change the interaction conformation between the GCG analog and the F C chain, further enhancing the fusion protein N. Stability of the end. More importantly, the ALF structural proteins provided by the present invention have significantly enhanced GLP-1R agonistic activity compared to existing GCG and mutants. Although Joseph R. Chabenne et al. and Richard D. DiMarchi et al. reported that a C-terminal small peptide cex (SEQ ID NO. 4, GPSSGAPPPS) of Exendin-4 was added to the C-terminus of Glucagon, its GLP-1R agonistic activity was 0.7% increased to 1.6%, an increase of about 2 times (Optimization of the Native Glucagon Sequence for Medicinal Purposes, J Diabetes Sci Technol. 4 (6): 1322–1331, 2010 and patent US9018164B2), but GCGR agonistic activity and GLP The ratio of -1R agonistic activity is only about 35:1. In addition, Evers A et al. (Evers A et al., Design of Novel Exendin-Based Dual Glucagon-like Peptide 1 (GLP-1)/Glucagon Receptor Agonists, J Med Chem.; 60(10): 4293-4303.2017) is similar in GCG When the C-terminus of the substance was added with the cex sequence, the agonistic activity of GLP-1R decreased by about 3 times, and the activity of GCG decreased by about 14 times (Table 2, peptides 7 and 8 in the article).
就是说，仅简单在天然Glucagon的C末端增加Exendin-4的C末端肽cex序列(如GPSSGAPPPS)并不会显著提高甚至会进一步减弱其GLP-1R的激动活性。另一方面，Glucagon、GLP-1这类小肽与Fc，白蛋白这类载体融合蛋白进行融合表达的时候，由于空间位阻效应，活性往往是显著下降的(YAN-SHAN HUANG等,Preparation and characterization of a novel exendin-4 human serum albumin fusion protein expressed in Pichia pastoris，J.Pept.Sci.2008；14:588–595)，并且这种活性变化是不可预测的。本发明人意外地发现，将GCG类似物与Fc融合以后，对GLP-1R与GCGR的活性影响完全是不一样的。GCG类似物的C末端额外加入cex或类似序列(SEQ ID NO.3-10)再进一步融合至F C链时，与GCG类似物直接与F C链融合相比，含有cex序列的结构其GLP-1R激动活性保留率明显提高，最高提高了200倍以上，但是GCG活性保留率却是基本不变，甚至略有下降的。 That is, simply increasing the C-terminal peptide cex sequence of Exendin-4 (such as GPSSGAPPPS) at the C-terminus of native Glucagon does not significantly increase or even further attenuate the agonistic activity of its GLP-1R. On the other hand, when small peptides such as Glucagon and GLP-1 are expressed in fusion with Fc and albumin, the activity is often significantly decreased due to steric hindrance (YAN-SHAN HUANG et al., Preparation and Characterization of a novel exendin-4 human serum albumin fusion protein expressed in Pichia pastoris, J. Pept. Sci. 2008; 14: 588-595), and this change in activity is unpredictable. The inventors have unexpectedly discovered that after fusion of a GCG analog with an Fc, the effect on the activity of GLP-1R and GCGR is completely different. When the C-terminus of the GCG analog is additionally added with cex or a similar sequence (SEQ ID NO. 3-10) and further fused to the F C chain, the structure containing the cex sequence is GLP compared to the fusion of the GCG analog directly with the F C chain. The retention rate of -1R agonistic activity was significantly increased, and the highest was increased by more than 200 times, but the retention rate of GCG activity was basically unchanged or even decreased slightly.
另外，对于本领域技术人员，尤其是重组蛋白药的科研技术人员，一个公知的概念是：在蛋白序列上任意一个位点引入突变，都是无法准确预料其结果的，尤其是对于GLP-1、Exendin-4或Glucagon这类氨基酸数量只有30-40个的小肽而言，单个位点突变或者若干个位点同时突变的效果更是难以预测。比如Joseph Chabenne等报道(Joseph Chabenne等，A Glucagon analog chemically stabilized for immediate treatment of life-threatening hypoglycemia，Molecular Metabolism,3：293-300，2014)对Glucagon(SEQ ID NO.44)进行了丙氨酸扫描(Ala scan)，Glucagon的各个位点独立地被丙氨酸取代后，相对残留活性保留跨度从0.2％-100％，并认为Glucagon的第1、2、3、4、6-12、14、15、22、23、25-27、29位突变 都会使GCGR激动活性大幅减弱(文章中的表4)。然而我们也可以在其他的报道中看到在上述这些位点中取若干个位点同时进行突变，用其他的氨基酸取代时，活性的变化并不总是与丙氨酸扫描的结果一致。如Jonathan W Day等(Jonathan W Day等，A new Glucagon and GLP-1 co-agonist eliminates obesity in rodents，Nature Chemical Biology，5：749-757，2009)报道的杂合肽(文中的Chimera 2)，将23位的Val突变为Ile，会使GCGR的激动活性略微提升。但是丙氨酸扫描结果显示，在23位用丙氨酸取代，将导致GCGR激动活性基本完全丧失(降低至仅剩1.1％)。In addition, a well-known concept for those skilled in the art, especially those of recombinant protein drugs, is that the introduction of a mutation at any one site in a protein sequence cannot accurately predict the result, especially for GLP-1. For small peptides with only 30-40 amino acids, such as Exendin-4 or Glucagon, the effect of single site mutation or simultaneous mutation of several sites is more difficult to predict. For example, Joseph Chabenne et al. (Joseph Chabenne et al, A Glucagon analog chemically stabilized for immediate treatment of life-threatening hypoglycemia, Molecular Metabolism, 3: 293-300, 2014) performed alanine scanning on Glucagon (SEQ ID NO. 44). (Ala scan), after each site of Glucagon is independently replaced by alanine, the relative residual activity retention span is from 0.2% to 100%, and that Glucagon's 1, 2, 3, 4, 6-12, 14, Mutations 15, 22, 23, 25-27, and 29 all significantly attenuated GCGR agonistic activity (Table 4 in the article). However, we can also see in other reports that several sites in these sites are simultaneously mutated. When substituted with other amino acids, the change in activity is not always consistent with the results of alanine scanning. Hybrid peptides (Chimera 2 in the text) as reported by Jonathan W Day et al. (Jonathan W Day et al, A new Glucagon and GLP-1 co-agonist eliminates obesity in rodents, Nature Chemical Biology, 5: 749-757, 2009), Mutation of the 23-position Val to Ile resulted in a slight increase in the agonistic activity of GCGR. However, the alanine scan showed that substitution with alanine at position 23 resulted in a complete loss of GCGR agonistic activity (reduced to only 1.1%).
最重要的是，本发明提供一种具有GCGR/GLP-1R双效激动活性的多重活性蛋白。目前GLP-1R和GCGR的双效激动剂普遍都是基于天然Oxyntomodulin(SEQ ID NO.2)或Glucagon(SEQ ID NO.44)序列而改造的。而一般研究所用的类似物在GLP-1R及GCGR的激动活性一般是设计成1:1左右，多数研究认为活性1:1的降糖效果及减脂效果最好(Peptide-based GLP-1/Glucagon co-agonists:a double-edged sword to combat diabesity，Hitesh Soni，95:5–9，2016)。Most importantly, the present invention provides a multiplex active protein having GCGR/GLP-1R double-acting agonistic activity. Currently, double-acting agonists of GLP-1R and GCGR are generally engineered based on the native Oxyntomodulin (SEQ ID NO. 2) or Glucagon (SEQ ID NO. 44) sequences. The agonistic activity of analogs used in general research in GLP-1R and GCGR is generally designed to be about 1:1. Most studies consider that the activity of 1:1 is the best for hypoglycemic and lipid-lowering effects (Peptide-based GLP-1/). Glucagon co-agonists: a double-edged sword to combat diabesity, Hitesh Soni, 95:5–9, 2016).
Oxyntomodulin(SEQ ID NO.2)对于两个受体的激动活性本身就比较低，而Glucagon(SEQ ID NO.44)经序列突变后，虽然对GLP-1R的激动活性有所提高，但是在加上PEG或者脂肪酸后，活性仍会不可避免地下降，尤其是PEG修饰的情况。然而本发明的双效活性蛋白保持了大部分完整的GLP-1R和GCGR激动活性。活性的提高预示着剂量的减少，更低剂量即可达到更平稳的血糖控制，从而很好的提高了用药便利性，并减少了潜在的免疫原性的风险。众所周知，GLP-1类似物及其融合蛋白的副作用包括头晕、恶心等，是剂量依赖的，降低剂量可以减少胃肠道副作用。同时Glucagon可以提高代谢速率，增加脂肪消耗，起到更显著的减重效果，同时也能减少低血糖的风险，适合于与其他降糖药，如胰岛素等联合用药。Oxyntomodulin (SEQ ID NO. 2) has a relatively low agonistic activity for both receptors, whereas Glucagon (SEQ ID NO. 44), after sequence mutation, has an increased agonistic activity against GLP-1R, but After PEG or fatty acids, the activity will inevitably decrease, especially in the case of PEG modification. However, the double-acting active protein of the present invention retains most of the intact GLP-1R and GCGR agonistic activity. An increase in activity predicts a reduction in dose, and a lower dose results in a smoother glycemic control, which improves the ease of administration and reduces the risk of potential immunogenicity. It is well known that the side effects of GLP-1 analogs and their fusion proteins, including dizziness, nausea, etc., are dose dependent, and lowering the dose can reduce gastrointestinal side effects. At the same time, Glucagon can increase the metabolic rate, increase fat consumption, play a more significant weight loss effect, and also reduce the risk of hypoglycemia, suitable for combination with other hypoglycemic agents, such as insulin.
GLP-1R和GCGR激动和下游信号传递与其生理效应是极其复杂的，至今尚未完全清楚。目前一致认为大致是：葡萄糖通过GLUT2进入胰腺β细胞后发生糖酵解并产生丙酮酸。丙酮酸进入线粒体进行氧化代谢及产生ATP。细胞内的ATP增加将会关闭K ATP(对ATP敏感的钾离子，ATP-sensitive potassium)通道，使得胞膜去极化，打开钙离子通道，增加胞外钙离子的流入，这一系列的变化导致了胰岛素的外分泌。GLP-1通过一系列的机制增加胰岛素的外分泌：GLP-1R与Gαs结合，激活腺苷酸环化酶，腺苷酸环化酶将ATP转化为cAMP，并调动下游的PKA和Epac信号因子。这会导致一系列的细胞反应，包括关闭K ATP通道，促进胰岛素分泌颗粒与胞膜的融合(Chris de Graaf等，Glucagon-Like Peptide-1 and Its Class  B G Protein–Coupled Receptors:A Long March to Therapeutic Successes，Pharmacological Reviews,68(4)954-1013，2016)。GCGR与GLP-1R类似，在与Glucagon结合后，胞内出现cAMP上调。GLP-1R及GCGR都属于GPCR家族，具有7个跨膜区域。在与各自的配体结合后，受体的C末端磷酸化，β-抑制蛋白(β-arrestin)富集并与受体结合，最终导致受体的内吞(Jorgensen,R等，Oxyntomodulin differentially affects Glucagon-like peptide-1 receptor-arrestin recruitment and signaling through Gα，J.Pharmacol.Exp.Ther.322,148–154，2007)。 GLP-1R and GCGR agonism and downstream signaling and their physiological effects are extremely complex and have not been fully understood to date. It is currently agreed that roughly: glucose enters the pancreatic beta cells through GLUT2 and undergoes glycolysis and produces pyruvate. Pyruvate enters the mitochondria for oxidative metabolism and produces ATP. An increase in intracellular ATP will turn off K ATP (ATP-sensitive potassium) channels, depolarizing the membrane, opening calcium channels, and increasing the influx of extracellular calcium ions. This series of changes Lead to the exocrine of insulin. GLP-1 increases insulin exocrine through a series of mechanisms: GLP-1R binds to Gαs, activates adenylate cyclase, adenylate cyclase converts ATP to cAMP, and mobilizes downstream PKA and Epac signaling factors. This leads to a series of cellular responses, including the closure of the K ATP channel, which promotes the fusion of insulin-secreting granules with the membrane (Chris de Graaf et al, Glucagon-Like Peptide-1 and Its Class BG Protein–Coupled Receptors: A Long March to Therapeutic Successes, Pharmacological Reviews, 68 (4) 954-1013, 2016). GCGR is similar to GLP-1R, and intracellular cAMP is upregulated after binding to Glucagon. Both GLP-1R and GCGR belong to the GPCR family and have seven transmembrane regions. Upon binding to the respective ligand, the C-terminal phosphorylation of the receptor, β-arrestin is enriched and binds to the receptor, ultimately leading to endocytosis of the receptor (Jorgensen, R et al, Oxyntomodulin differentially affects Glucagon-like peptide-1 receptor-arrestin recruitment and signaling through Gα, J. Pharmacol. Exp. Ther. 322, 148-154, 2007).
有研究表示，GCGR跟GLP-1R的内吞效率不一样，而受体的内吞又影响着下游信号转导(Functional Consequences of Glucagon-like Peptide-1 Receptor Cross-talk and Trafficking，J Biol Chem，Sarah Noerklit Roed等290(2):1233–1243，2015)，最终可能影响组织细胞的生理功能。如Kuna,R.S.等表示，GLP-1R内吞受阻会减少胰腺细胞的胰岛素释放(Glucagon-like peptide-1 receptor-mediated endosomal cAMP generation promotes glucose-stimulated insulin secretion in pancreatic-cells，Kuna,R.S.等，Am.J.Physiol.Endocrinol Metab，305,E161–E170，2013)。另外，两型受体的内吞效率也存在差异，如GCGR的内吞效率比GLP-1R低。为了避免GLP-1R及GCGR双效激动剂同时结合于GLP-1R及GCGR，现有的GLP-1R及GCGR的双效激动剂，绝大多数是基于一条肽链结构的。但是，如果在F C二价蛋白基础上构建GLP-1R和GCGR双效激动剂，有可能会同时分别结合GLP-1R及GCGR两个受体，造成两个受体异二聚化交联而影响了各自受体的内吞，胞内信号传递从而影响其正常的生理功能。目前已经不少提及GCGR与GLP-1R在同一组织细胞表面共表达的报道(Dominik Schelshorn等，Lateral Allosterism in the Glucagon Receptor Family:Glucagon-Like Peptide 1 Induces G-Protein-Coupled Receptor Heteromer Formation，Molecular Pharmacology，81(3)309-318，2012)。在本发明研究过程中也发现，不同结构的GCGR/GLP-1R双效激动活性蛋白在大鼠BRIN-BD11细胞诱生胰岛素的能力是完全不一样的。多种因素会影响其异二聚化产生的效应。因此，如何获得正常生理效应的GLP-1R及GCGR双效激动剂是一件极其困难的工作。如GLP-1R/GCGR受体激动剂与F C之间的连接肽长度和结构不仅会影响蛋白的活性，也与GLP-1R及GCGR交联有关。在一个优选的实施方案中，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，并优选获得合适的序列和长度减少潜在的受体异二聚化，使得二价的双效激动剂仍具有最好的GLP-1R和GCGR激动活性。 Studies have shown that GCGR and GLP-1R have different endocytic efficiencies, and receptor endocytosis affects downstream signal transduction (Functional Consequences of Glucagon-like Peptide-1 Receptor Cross-talk and Trafficking, J Biol Chem, Sarah Noerklit Roed et al. 290(2): 1233–1243, 2015) may ultimately affect the physiological function of tissue cells. For example, Kuna, RS et al., GLP-1R endocytosis will reduce insulin release from pancreatic cells (Glucagon-like peptide-1)-mediated endosomal cAMP generation promotes glucose-stimulated insulin secretion in pancreatic-cells, Kuna, RS, etc. .J. Physiol. Endocrinol Metab, 305, E161–E170, 2013). In addition, there are also differences in the endocytosis efficiency of the two types of receptors, such as GCGR, which has lower endocytic efficiency than GLP-1R. In order to avoid simultaneous binding of GLP-1R and GCGR double-acting agonists to GLP-1R and GCGR, the majority of the dual-effect agonists of GLP-1R and GCGR are based on a single peptide chain structure. However, if GLP-1R and GCGR double-acting agonists are constructed on the basis of F C bivalent protein, it is possible to simultaneously bind two receptors of GLP-1R and GCGR, respectively, resulting in heterodimerization and cross-linking of the two receptors. It affects the endocytosis of the respective receptors, and the intracellular signal transmission affects its normal physiological functions. There have been many reports of co-expression of GCGR and GLP-1R on the same tissue surface (Dominik Schelshorn et al, Lateral Allosterism in the Glucagon Receptor Family: Glucagon-Like Peptide 1 Induces G-Protein-Coupled Receptor Heteromer Formation, Molecular Pharmacology , 81 (3) 309-318, 2012). It has also been found during the course of the present invention that the ability of different structures of GCGR/GLP-1R double acting agonistic active protein to induce insulin in rat BRIN-BD11 cells is completely different. A variety of factors can affect the effects of heterodimerization. Therefore, how to obtain normal physiological effects of GLP-1R and GCGR double-acting agonists is an extremely difficult task. For example, the length and structure of the linker between the GLP-1R/GCGR receptor agonist and F C not only affects the activity of the protein, but also is related to the cross-linking of GLP-1R and GCGR. In a preferred embodiment, the linker is a flexible polypeptide of a suitable length consisting of glycine (G), serine (S) and/or alanine (A), and preferably obtains a suitable sequence and length reduction The potential receptor heterodimerization allows the bivalent double-acting agonist to still have the best GLP-1R and GCGR agonistic activity.
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.21～43。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. 21-43.
进一步地，所述式I中的连接链L的氨基酸序列可如SEQ ID NO.21～43之任一所示。Further, the amino acid sequence of the linker L in the formula I can be as shown in any one of SEQ ID NOS. 21 to 43.
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)。 Further, 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.
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。 Further, 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.
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))。 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. In a preferred embodiment, 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)).
在一个优选的实施方案中，F C结构域来自人IgG1，并如SEQ ID NO.12所示。在一个优选的实施方案中，所述F C结构域来自人IgG4，如SEQ ID NO.16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。 In a preferred embodiment, the F C domain is derived from human IgG1 and is set forth in SEQ ID NO. In a preferred embodiment, the F C domain is derived from human IgG4, as set forth in SEQ ID NO. The K at the end of the F C chain can be removed to facilitate the uniformity of the expression product.
所述F的氨基酸序列可以如SEQ ID NO.11～20之任一所示。The amino acid sequence of F may be as shown in any one of SEQ ID NOS. 11-20.
本发明提供的多重活性蛋白是一种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。另外，这些多重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。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。利拉鲁肽与上述的MEDI0382虽然都保持了第二位 的天然氨基酸，但是给药周期只能支持一天一次。而本发明的多重活性蛋白即使保留了第二位的天然氨基酸，稳定性的显著提高却足以支持一周一次的给药频率。 The multiplex active protein provided by the present invention is an F C fusion protein that retains the conventional properties of F C , such as binding to FcRn to prolong the half-life in vivo. In addition, these multiple active proteins can effectively prevent the degradation of the N-terminus in addition to the degradation of the protein inside the serum by the protease in the serum. For Incretin-like polypeptides such as Glucagon or GLP-1, 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 due to the hydrolysis of DPP-IV in the receptor. In one embodiment of the invention, 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. Although liraglutide and MEDI0382 described above both retain the second natural amino acid, the dosing cycle can only be supported once a day. While the multiplex active protein of the present invention retains the natural amino acid in the second position, the significant increase in stability is sufficient to support the frequency of administration once a week.
进一步地，本发明第二方面提供一种多重活性蛋白，具有三效激动活性，其结构式中包括如式III所示结构，如式III所示结构为：A-L 1-F-L 2-B，其中，A为GCGR/GLP-1R双效激动剂，F为长效蛋白单元，B为天然FGF21(SEQ ID NO.143)或FGF21类似物，L 1为连接链，序列选自SEQ ID NO.21-43之任一；L 2不存在或选自SEQ ID NO.21-43之任一。 Further, the second aspect of the present invention provides a multiplex active protein having a three-way agonistic activity, wherein the structural formula includes a structure represented by Formula III, and the structure represented by Formula III is: AL 1 -FL 2 -B, wherein A is a GCGR/GLP-1R double-acting agonist, F is a long-acting protein unit, B is native FGF21 (SEQ ID NO. 143) or FGF21 analog, L 1 is a linking chain, and the sequence is selected from SEQ ID NO. 21- Any of 43; L 2 is absent or selected from any one of SEQ ID NOS. 21-43.
进一步地，A的结构中包括如式II所示结构，如式II所示结构为：Further, the structure of A includes a structure as shown in Formula II, and the structure shown in Formula II is:
HSQGTFTSDYSKYLD X 16X 17X 18 AQDFVQWLMN X 29 X z(SEQ ID NO.141)式II，其中，X 16选自除Y、N、W、和H外的氨基酸之任一；X 17选自除P、L、T、F和H外的氨基酸之任一；X 18选自除P、F、H和W外的氨基酸之任一；另外X 17与X 18不能同时为R，X 29为T或缺失，X z选自GGPSSGAPPPS(SEQ ID NO.3)、GPSSGAPPPS(SEQ ID NO.4)、PSSGAPPPS(SEQ ID NO.5)、SSGAPPPS(SEQ ID NO.6)、GGPSSGAPPS(SEQ ID NO.7)、GPSSGAPPS(SEQ ID NO.8)、PSSGAPPS(SEQ ID NO.9)或SSGAPPS(SEQ ID NO.10)之任一。 HSQGTFTSDYSKYLD X 16 X 17 X 18 AQDFVQWLMN X 29 X z (SEQ ID NO. 141) Formula II, wherein X 16 is selected from any one of amino acids other than Y, N, W, and H; X 17 is selected from P Any one of amino acids other than L, T, F and H; X 18 is selected from any one of amino acids other than P, F, H and W; further X 17 and X 18 cannot be R at the same time, X 29 is T or Deletion, X z is selected from GGPSSGAPPPS (SEQ ID NO. 3), GPSSGAPPPS (SEQ ID NO. 4), PSSGAPPPS (SEQ ID NO. 5), SSGAPPPS (SEQ ID NO. 6), GGPSSGAPPS (SEQ ID NO. 7) Any of GPSSGAPPS (SEQ ID NO. 8), PSSGAPPS (SEQ ID NO. 9) or SSGAPPS (SEQ ID NO. 10).
所述A的氨基酸序列可以如SEQ ID NO.46、SEQ ID NO.54、SEQ ID NO.55、SEQ ID NO.68之任一所示。The amino acid sequence of A may be as shown in any one of SEQ ID NO. 46, SEQ ID NO. 54, SEQ ID NO. 55, SEQ ID NO.
式III所述的FGF21类似物可选自如US20140213512、US8188040、US9493530、WO 2016114633、US 20150291677、US 9422353、US 8541369、US7622445、US7576190、US20070142278、US 9006400或US 20130252884等专利或专利申请中所描述的FGF21类似物或突变体。进一步地，所述FGF21类似物的氨基酸序列如SEQ ID NO.144、SEQ ID NO.145或SEQ ID NO.146所示。在本发明的另一个动物体内药效实施例中，三活性蛋白组与相同剂量的双效活性蛋白+FGF21类似物联合给药组相比具有更显著的减重效果。但是对于食欲的影响却并无太大影响。证明三活性蛋白的副作用可能更低，安全性提高(实施例12)。The FGF21 analogs of Formula III may be selected from FGF21 as described in US Patent No. 20140213512, US Pat. No. 8,818,040, US Pat. No. 9,949,530, WO 2016114633, US 20150291677, US Pat. No. 9 422 353, US Pat. No. 8 541 369, US Pat. No. 7,622,445, US Pat. No. 7,576,190, US Pat. Analog or mutant. Further, the amino acid sequence of the FGF21 analog is as shown in SEQ ID NO. 144, SEQ ID NO. 145 or SEQ ID NO. In another in vivo pharmacodynamic embodiment of the invention, the tri-active protein group has a more significant weight-reducing effect than the co-administered group of the same dose of the double-acting active protein + FGF21 analog. But the impact on appetite has little effect. It was demonstrated that the side effects of the triactive protein may be lower and the safety is improved (Example 12).
式III所述的B还可以是天然瘦素(SEQ ID NO.155)及其类似物，可选自US 7307142、US 7423113或US 20140018290等专利或专利申请中所描述的变体，衍生物或类似物；The B of Formula III may also be a native leptin (SEQ ID NO. 155) and analogs thereof, which may be selected from the variants, derivatives or derivatives described in US Pat. No. 7,307,142, US Pat. analog;
式III所述的B还可以是Amylin及其类似物。B described in Formula III may also be Amylin and its analogs.
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重活性蛋白。In a second aspect of the invention, an isolated polynucleotide is provided, the isolated polynucleotide encoding the aforementioned multiplex active protein.
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。In a third aspect of the invention, a recombinant expression vector comprising the isolated polynucleotide described above is provided.
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。According to a fourth aspect of the present invention, a host cell comprising the aforementioned recombinant expression vector or the above-described isolated polynucleotide integrated with an exogenous source in the genome is provided.
本发明的第五方面，提供前述多重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，使之表达所述多重活性蛋白，而后分离及纯化获得所述多重活性蛋白。According to a fifth aspect of the present invention, there is provided a method for producing the aforementioned multiplex active protein, which comprises culturing the aforementioned host cell under suitable conditions to express the multiplex active protein, and then isolating and purifying to obtain the multiplex active protein.
本发明的第六方面，提供前述多重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。According to a sixth aspect of the present invention, the use of the aforementioned multiplex active protein for the preparation of a medicament for treating a diabetes-related disease is provided.
本发明提供的多重活性蛋白可以用于治疗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。The multiplex active proteins provided by the present invention can be used for the treatment of 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. Other risk factors can include aging, hormonal imbalances, and genetic factors.
此外，本发明的多重活性蛋白还可用于治疗肥胖症。在一些方面中，本发明的多重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。Furthermore, the multiplex active proteins of the invention can also be used to treat obesity. In some aspects, the multiplex active proteins of the invention treat obesity by reducing appetite, reducing food intake, reducing fat levels in a patient, and increasing energy expenditure.
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重活性蛋白。In a seventh aspect of the invention, a method of treating a metabolic-related disease comprising administering the aforementioned multiplex active protein to a subject.
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重活性蛋白。The invention further provides a method of promoting weight loss or preventing weight gain comprising administering the multiplex active protein in a subject.
本发明的第八方面，提供一种组合物，含有前述多重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。In an eighth aspect of the invention, there is provided a composition comprising the aforementioned multiplex active protein or a culture of the aforementioned host cell, and a pharmaceutically acceptable carrier.
本发明的第九方面，提供前述多重活性蛋白在制备融合蛋白中的用途。In a ninth aspect of the invention, the use of the aforementioned multiplex active protein for the preparation of a fusion protein is provided.
本发明的第十方面，提供一种多结构域蛋白，所述多结构域蛋白的结构中含有前述多重活性蛋白。In a tenth aspect of the invention, a multidomain protein comprising the aforementioned multiplex active protein is contained in the structure of the multidomain protein.
综上所述，与现有技术相比，本发明具有如下有益效果：In summary, the present invention has the following beneficial effects as compared with the prior art:
(1)本发明的多重活性蛋白半衰期长、支持一周一次的给药频率；(1) The multiplex active protein of the present invention has a long half-life and supports a frequency of administration once a week;
(2)本发明的多重活性蛋白的GLP-1R激动活性最高提高到了200倍以上；(2) The agonistic activity of GLP-1R of the multiplex active protein of the present invention is up to 200 times or more;
(3)本发明的多重活性蛋白体内外稳定性好，且免疫原性低。(3) The multiplex active protein of the present invention has good stability in vitro and in vivo, and has low immunogenicity.
(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。(4) Since it is not necessary to introduce an unnatural amino acid, it does not need to involve a chemical synthesis and crosslinking step, and thus can be prepared by a recombinant method, which greatly simplifies the preparation process.
附图说明DRAWINGS
图1为纯化获得的部分重组蛋白电泳图(10％SDS-PAGE)，泳道1-6分别为C240G12S3A1F4、C368G12S3A1F4、C225G12S3A1F4、C495G12S3A1F4、C382G12S3A1F4及C462G12S3A1F4非还原处理的样品；7-12分别为C240G12S3A1F4、C368G12S3A1F4、C225G12S3A1F4、C495G12S3A1F4、C382G12S3A1F4及C462G12S3A1F4还原处理的样品；M为蛋白标准品：97.2、66.4、44.3、29、20.1、14.3KD。Figure 1 is a partial electrophoresis map of purified recombinant protein (10% SDS-PAGE). Lanes 1-6 are C240G12S3A1F4, C368G12S3A1F4, C225G12S3A1F4, C495G12S3A1F4, C382G12S3A1F4 and C462G12S3A1F4 non-reduced samples; 7-12 are C240G12S3A1F4 and C368G12S3A1F4, respectively. , C225G12S3A1F4, C495G12S3A1F4, C382G12S3A1F4 and C462G12S3A1F4 reduced treated samples; M is a protein standard: 97.2, 66.4, 44.3, 29, 20.1, 14.3 KD.
图2A为GCGR测活结果图。Figure 2A is a graph showing the results of GCGR measurement.
图2B为GLP-1R测活结果图。Fig. 2B is a graph showing the results of GLP-1R measurement.
图3A：血清稳定性随时间变化的结果图。Figure 3A: A graph of the results of serum stability over time.
图3B：血清稳定性随时间变化的结果图。Figure 3B: Figure of the results of serum stability over time.
图3C：血清稳定性随时间变化的结果图。Figure 3C: Figure of the results of serum stability over time.
图3D：血清稳定性随时间变化的结果图。Figure 3D: Figure of the results of serum stability over time.
图4：实施例6和7中由GCG类似物与不同长度连接链及F(SEQ ID NO.12)融合而获得的二聚体重组蛋白对胰岛细胞的刺激作用图。Figure 4: Schematic diagram of the stimulation of islet cells by dimeric recombinant proteins obtained by fusion of GCG analogs with different length linkers and F (SEQ ID NO. 12) in Examples 6 and 7.
图5：实施例8中的二聚体重组蛋白在正常ICR小鼠体内的降糖效果图。Fig. 5 is a graph showing the hypoglycemic effect of the dimeric recombinant protein of Example 8 in normal ICR mice.
图6：实施例9中的二聚体重组蛋白对DIO小鼠体重的影响作用。Figure 6: Effect of the dimeric recombinant protein of Example 9 on the body weight of DIO mice.
图7：实施例11纯化获得的融合蛋白的电泳图(10％SDS-PAGE)，泳道1-6分别为C382F4FGF1、C382F4FGF2、C382F4FGF3、C495F4FGF1、C495F4FGF2及C495F4FGF3非还原处理的样品；7-12分别为C382F4FGF1、C382F4FGF2、C382F4FGF3、C495F4FGF1、C495F4FGF2及C495F4FGF3还原处理的样品；M为自制蛋白标准品：140、97.2、66.4、44.3、29、20.1、14.3KD。Figure 7: Electropherogram (10% SDS-PAGE) of the fusion protein obtained in the purification of Example 11, lanes 1-6 are non-reduced samples of C382F4FGF1, C382F4FGF2, C382F4FGF3, C495F4FGF1, C495F4FGF2 and C495F4FGF3, respectively; Samples of C382F4FGF1, C382F4FGF2, C382F4FGF3, C495F4FGF1, C495F4FGF2 and C495F4FGF3 reduced; M is a self-made protein standard: 140, 97.2, 66.4, 44.3, 29, 20.1, 14.3 KD.
图8：实施例12中的三活性蛋白的对DIO小鼠体重的影响作用。Figure 8: Effect of the triactive protein in Example 12 on the body weight of DIO mice.
图9：实施例12中的三活性蛋白对DIO小鼠摄食量的影响作用。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。Figure 9: Effect of the triactive protein in Example 12 on the food intake of DIO mice. The food intake of the DIO rats in the PBS group was 100%, and the ordinate was the percentage of the food intake of the other groups.
图10：实施例14中的三活性蛋白对DIO小鼠体重的影响作用。Figure 10: Effect of the tri-active protein in Example 14 on the body weight of DIO mice.
图11：实施例14中的三活性蛋白对DIO小鼠摄食量的影响作用。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。Figure 11: Effect of the tri-active protein in Example 14 on the food intake of DIO mice. The food intake of the DIO rats in the PBS group was 100%, and the ordinate was the percentage of the food intake of the other groups.
具体实施方式Detailed ways
术语解释：Explanation of terms:
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。The term "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.
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。 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.
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。 Type 2 diabetes, also known as non-insulin-dependent diabetes and adult-onset diabetes, is generally associated with insulin resistance.
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，过量卡里路贮存于脂肪中，则导致肥胖。在本文中，肥胖最佳地被视为任何程度的危害健康的过量脂肪组织的形成。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。The term "obesity" means an excess of adipose tissue, and when energy intake exceeds energy expenditure, excess calories are stored in fat, resulting in obesity. In this context, obesity is best viewed as the formation of any excess of adipose tissue that is hazardous to health. Individuals with a body mass index (BMI = body weight (kg) divided by height (m) square) of more than 25 are considered obese herein.
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,Lancet 368:1696-705,2006)。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)。Incretin: 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, Lancet 368: 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).
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)。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).
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。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.
胰高血糖素样肽(GLP-1)：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)。Glucagon-like peptide (GLP-1): 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 improving the efficacy by using chemical modification and/or formulation to treat diabetes and obesity (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：44)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着的体重减轻。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  andomized 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。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: 44). 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. It has been found that 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). In overweight and obese patients, subcutaneous administration of natural oxyntomodulin reduced body weight by 1.7 kg in four weeks. 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 consumption in addition to decreasing energy intake in overweight and obese humans: a andomized controlled trial; Wynne K et al, Int J Obes (Lond), 30: 1729-36, 2006). However, due to the small molecular weight and degradation of DPP-IV, oxyntomodulin has a shorter half-life. Currently, double-acting agonists of GLP-1 receptor (GLP-1R) and glucagon receptor (GCGR) are generally based on oxyntomodulin, and in order to improve the short-acting and enzymes of oxyntomodulin A mutation (the oxyntomodulin analog) was made in the defect of the solution, and most of the second serine Ser was mutated to α-aminoisobutyric acid (Aib) by introducing a non-natural amino acid to resist DPP-IV. Enzymatic hydrolysis. Although 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.
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：44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。Glucagon is a 29 amino acid peptide corresponding to amino acids 53-81 of proglucagon, as shown in SEQ ID NO: 44 (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.
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。The term "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。A "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-1R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。GLP-1R analog: As used herein, "GLP-1 analog" or "GLP-1 mutant" means a GLP-1R agonist and is mutually versatile.
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 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。Glucagon Analogs: As used herein, "Glucagon analogs", "GCG analogs", "Glucagon mutants" and "GCG mutants" all mean Glucagon receptor agonists and are mutually versatile.
GCGR/GLP-1R双效激动剂：本发明的GCGR/GLP-1R双效激动剂包括能同时激动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。GCGR/GLP-1R double-acting agonist: The GCGR/GLP-1R double-acting agonist 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). As used herein, "double-acting agonist" or "bispecific active protein" or "double-acting active protein" are synonymous.
FGF21：成纤维细胞生长因子(fibroblast growth factor，FGF)又叫肝素结合生长因子(heparin binding growth factor)，是主要由垂体和下丘脑分泌的一类多肽物质。FGF具有多种功效，如促进成纤维细胞有丝分裂、中胚层细胞的生长，刺激血管形成等。而FGF21是FGF家族中的重要一员，目前该激素被当作药物开发成减肥药以及治疗糖尿病药物，并且该药物已经进入了临床试验阶段。FGF21通过FGF21受体及其共受体β-klotho发挥生理作用。FGF21: fibroblast growth factor (FGF), also known as heparin binding growth factor, is a type of polypeptide substance secreted mainly by the pituitary and hypothalamus. FGF has various functions, such as promoting fibroblast mitosis, mesoderm cell growth, stimulating blood vessel formation and the like. While FGF21 is an important member of the FGF family, the hormone is currently being developed as a diet drug and a drug for diabetes, and the drug has entered clinical trials. FGF21 exerts a physiological action through the FGF21 receptor and its co-receptor β-klotho.
瘦素(leptin)：主要由白色脂肪组织产生。其前体由167个氨基酸残基组成，包含N端21个氨基酸的信号肽，前体的信号肽在血液中被切掉而成为146氨基酸的瘦素成熟肽(leptin)。瘦素具有广泛的生物学效应，如作用于下丘脑的代谢调节中枢、发挥抑制食欲、减少能量摄取、增加能量消耗及抑制脂肪合成等的作用。Leptin: Produced mainly by white adipose tissue. Its precursor consists of 167 amino acid residues and contains a N-terminal 21 amino acid signal peptide. The signal peptide of the precursor is cleaved off in the blood to become a 146 amino acid leptin mature peptide (leptin). Leptin has a wide range of biological effects, such as acting on the metabolic regulation center of the hypothalamus, exerting appetite suppression, reducing energy intake, increasing energy expenditure, and inhibiting fat synthesis.
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。 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.
二聚体双效活性蛋白：在本文中，“双效活性蛋白”、“二聚体双效活性”、“双效激动活性蛋白”皆同义，可互换使用。意指同时具有GCGR和GLP-1R激动活性的融合蛋白，该种融合蛋白由于具有F C部分，因此具有两条肽链，通过两条肽链之间的非共价和共价作用而形成了二聚体的结构。 Dimer double-acting active protein: In this context, "double-acting active protein", "dimer double-acting activity", and "double-acting agonistic active protein" are synonymous and can be used interchangeably. Means a fusion protein having both GCGR and GLP-1R agonistic activity, which has a F C moiety and thus has two peptide chains, which are formed by non-covalent and covalent interactions between the two peptide chains. The structure of the dimer.
二聚体三效活性蛋白：在本文中，“三效活性蛋白”、“三效激动活性蛋白”、“三活性激动活性蛋白”、“二聚体三效活性蛋白”皆同义，可互换使用。意指同时具有GCGR、GLP-1R激动活性及FGF21活性(或瘦素活性)的融合蛋白，该种融合蛋白由于具有F C部分，因此具有两条肽链，通过两条肽链之间的非共价和共价作用而形成了二聚体的结构。 Dimeric tri-acting active protein: In this context, "three-way active protein", "three-way agonistic active protein", "three active agonistic active protein", "dimeric three-way active protein" are synonymous, and can Change to use. Means a fusion protein having both GCGR, GLP-1R agonistic activity and FGF21 activity (or leptin activity), which has a F C moiety and therefore has two peptide chains, which are passed between the two peptide chains. Covalent and covalent interactions form the structure of the dimer.
IC 50(half maximal inhibitory concentration)是指被测量的拮抗剂的半抑制浓度。它能指示某一药物或者物质(抑制剂)在抑制其相应的50％生物学反应(或者是包含在此反应中的某些物质，比如酶，细胞受体或是微生物)时所需的浓度。IC50值越低，表明该药物或物质的抑制能力越强，例如，更直观地表现为与受体的结合亲和力越佳。是物质在抑制特定生物或生化功能中的有效性的量度标准。 IC 50 (half maximal inhibitory concentration) refers to the semi-inhibitory concentration of the antagonist being measured. It indicates the concentration required for a drug or substance (inhibitor) to inhibit its corresponding 50% biological response (or some of the substances involved in the reaction, such as enzymes, cell receptors or microorganisms) . The lower the IC50 value, the stronger the inhibitory ability of the drug or substance, for example, the more intuitively, the better the binding affinity to the receptor. It is a measure of the effectiveness of a substance in inhibiting a particular biological or biochemical function.
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。EC50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。 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. The lower the EC50 value, the stronger the stimulating or stimulating ability of the drug or substance, for example, the more intuitively, the stronger the intracellular signal that is caused, the better the ability to induce the production of a hormone.
低密度脂蛋白(LDL)：属于血浆脂蛋白的一种，是血液中胆固醇的主要载体,其倾向于将胆固醇沉积在动脉壁上。白细胞试图消化低密度脂蛋白，但在这个过程却将它们变成毒素。越来越多的白细胞被吸引到发生变化的区域，致使动脉壁可能发炎。随着时间的推移，随着该过程继续，这些斑块沉积物可以积聚在动脉壁上，使得通道变得非常窄和缺乏韧性。如果太多的斑块累积，则动脉可以完全阻塞。当LDL与胆固醇形成的复合物(LDL-C)在动脉壁上产生太多斑块时，血液将不能自由流过动脉。斑块随时可能会在动脉中突然塌陷，导致血管堵塞，最终引发心脏病。Low-density lipoprotein (LDL), a type of plasma lipoprotein, is the main carrier of cholesterol in the blood, which tends to deposit cholesterol on the arterial wall. White blood cells try to digest low-density lipoproteins, but in the process they turn them into toxins. More and more white blood cells are attracted to the changing areas, causing the arterial wall to become inflamed. Over time, as the process continues, these plaque deposits can accumulate on the arterial wall, making the channel very narrow and lacking toughness. If too many plaques accumulate, the artery can be completely blocked. When the complex of LDL and cholesterol (LDL-C) produces too many plaques on the arterial wall, blood will not flow freely through the artery. Plaques can suddenly collapse in the arteries at any time, causing blood vessels to clog and eventually lead to heart disease.
高密度蛋白(HDL)：有助于清除动脉上的LDL，起到清道夫的作用，将LDL从动脉上清走并回到肝脏。High-density protein (HDL): Helps clear LDL on the arteries, acts as a scavenger, clears LDL from the artery and returns to the liver.
甘油三酯(TG)：是另一种类型的脂肪，用于储存饮食过多的能量。血液中高水平的甘油三酯与动脉粥样硬化有关。高甘油三酯可以由超重和肥胖，身体缺乏运动，吸烟，过量的酒精消耗和高碳水化合物(超过总卡路里的60％)摄入引起。有时基础疾病或遗传疾病是高甘油三酯的原因。具有高甘油三酯的人通常具有高的总胆固醇水平，包括高LDL胆固醇和低HDL胆固醇，许多患有心脏病或糖尿病的人也具有高甘油三酯水平。Triglyceride (TG): Another type of fat used to store excess energy in the diet. High levels of triglycerides in the blood are associated with atherosclerosis. High triglycerides can be caused by overweight and obesity, lack of exercise, smoking, excessive alcohol consumption, and high carbohydrate intake (more than 60% of total calories). Sometimes basic or genetic diseases are the cause of high triglycerides. People with high triglycerides typically have high total cholesterol levels, including high LDL cholesterol and low HDL cholesterol, and many people with heart disease or diabetes also have high triglyceride levels.
细胞生物学活性Cellular biological activity
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。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 β-klotho into the same CHO cell to detect changes in fluorescence caused by the signal.
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US9018164B2)。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3倍左右，GCG的活性下降了14倍左右(文章中表2，肽7和8)。Joseph R. Chabenne et al. and Richard D. DiMarchi et al. reported that adding a C-terminal small peptide cex (GPSSGAPPPS) of Exendin-4 at the C-terminus of Glucagon increased the agonistic activity of GLP-1R by about 2 fold (Optimization of the Native). Glucagon Sequence for Medicinal Purposes, J Diabetes Sci Technol. 4(6): 1322 - 1331, 2010 and patent US9018164B2). In addition, Evers A et al. (Evers A et al., Design of Novel Exendin-Based Dual Glucagon-like Peptide 1 (GLP-1)/Glucagon Receptor Agonists, J Med Chem.; 60(10): 4293-4303.2017) is similar in GCG When the C-terminus of the compound was added with the cex sequence, the agonistic activity of GLP-1R decreased by about 3 times, and the activity of GCG decreased by about 14 times (Table 2, peptides 7 and 8 in the article).
在本发明的一个实施例中，含Exendin-4的C末端延伸肽的GCG类似物进一步融合至F C链时，GLP-1R激动活性提高到了惊人的200倍以上(EC50约1.1nM)。而根据US9018164 B2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Exendin-4的C末端延伸肽cex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。 In one embodiment of the invention, when the GCG analog comprising the C-terminal extension peptide of Exendin-4 is further fused to the F C chain, the GLP-1R agonistic activity is increased by a staggering 200-fold or more (EC50 of about 1.1 nM). The GLG-1R agonistic activity ratio calculated according to the data disclosed in the corresponding patents and literatures by US9018164 B2 and Joseph R. Chabenne et al., the GLP-1R agonistic activity ratio changes only before and after increasing the Cendrin extended peptide cex sequence of Exendin-4. The ratio is about 0.7% of the GLP-1R agonistic activity of the natural Glucagon in the article, and increases to 1.6% after the GPSSGAPPPS sequence is increased. 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.
多重活性蛋白的稳定性Multiple active protein stability
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16-18位蛋白酶水解的突变，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。在本发明的实施例中，在72小时仍然能检测到50％左右的GCGR激动活性。而此时含cex序列的天然Glucagon与F C融合形成的二聚体(SEQ ID NO.76)已几乎检测不到任何的活性。 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 against the hydrolysis of the 16-18th protease on the basis of the natural Glucagon to increase its stability. After the fusion of these mutants with F C , the stability was further improved. In the examples of the present invention, about 50% of the GCGR agonistic activity was still detected at 72 hours. At this time, the dimer (SEQ ID NO. 76) formed by fusion of the native Glucagon containing the cex sequence with F C has almost no activity detected.
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 PCTCN2018116171-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，第二位保存天然L-Ser的二聚体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5)。本发明人制备了天然Glucagon与F C融合的活性蛋白C001G12S3A1F4(SEQ ID NO.75)以及Joseph R.Chabenne等报道的Glucagon-cex与F C融合的活性蛋白C002G12S3A1F4(SEQ ID NO.76)作为对照验证是否F C融合提高了稳定性。然而，C001G12S3A1F4(SEQ ID NO.75)和C002G12S3A1F4(SEQ ID NO.76)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。 At present, almost all GCGR/GLP-1R double-acting agonists designed and developed based on Oxyntomodulin and Glucagon have introduced 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.
Figure PCTCN2018116171-appb-000007
Etc. Unimolecular Polypharmacy for Treatment of Diabetes and Obesity, 24: 51–62, 2016). However, in the examples of the present invention, the second dimeric active protein preserving natural L-Ser exhibits very high serum stability, and there is no significant sign of DPP-IV degradation at 24 hours, but no The corresponding polypeptide fused to F C was rapidly hydrolyzed by DPP-IV (Table 5). The present inventors prepared the active protein C001G12S3A1F4 (SEQ ID NO. 75) fused with native Glucagon and F C and the active protein C002G12S3A1F4 (SEQ ID NO. 76) fused by Glucagon-cex and F C as reported by Joseph R. Chabenne et al. Verify that F C fusion improves stability. However, none of C001G12S3A1F4 (SEQ ID NO. 75) and C002G12S3A1F4 (SEQ ID NO. 76) showed significant signs of resistance to DPP-IV. Although it has been reported that binding to serum albumin (such as HSA) may help to improve protein stability (such as liraglutide), if the second is not mutated, the half-life cannot be sustained for more than 12 hours. That is, it is impossible to support the frequency of administration once a week. While the GCG analog pharmacokinetic and pharmacodynamic tests provided by the present invention have been shown to be sufficient to support the frequency of dosing once a week, rather than once a day (eg, albumin-binding liraglutide). The retention of natural amino acids further reduces the risk of immunogenicity, avoids chemical cross-linking and makes the preparation process easier and more convenient.
葡萄糖刺激的胰岛素分泌实验(GSIS)Glucose-stimulated insulin secretion assay (GSIS)
众所周知，GLP-1或其类似物通过激动GLP-1R，作用于胰岛β细胞，促进胰岛素基因的转录、胰岛素的合成和分泌。临床上，GLP-1类似物常与胰岛素联合使用。虽然GLP-1R激动的详细机制仍未完全揭开，然而受体激动后的cAMP信号产生及受体迅速内吞却是比 较明确的事实。有多个研究表明，cAMP信号与GLP-1R受体内吞属于不同的信号通路，但是都会影响着胰岛素的分泌(Agonist-induced internalization of the Glucagon-like peptide-1 receptor is mediated by the Gaq pathway，Aiysha Thompson,等，Biochemical Pharmacology，93:72–84，2015；Molecular Characterisation of Small Molecule Agonists Effect on the Human Glucagon Like Peptide-1Receptor Internalisation，Aiysha Thompson等，PLOS ONE)。因此，cAMP信号与受体内吞的减弱，理论上都会破坏胰岛素的分泌从而影响GLP-1类似物的生理效能。GLP-1R与GCGR交联形成异二聚体后，内吞明显减弱，而影响胰岛素的分泌。It is well known that GLP-1 or its analog acts on islet β cells by agonizing GLP-1R, promoting transcription of insulin genes, synthesis and secretion of insulin. Clinically, GLP-1 analogs are often used in combination with insulin. Although the detailed mechanism of GLP-1R agonism has not been fully revealed, the cAMP signal production after receptor activation and rapid endocytosis of receptors are relatively clear facts. A number of studies have shown that cAMP signaling and GLP-1R receptor endocytosis belong to different signaling pathways, but they all affect insulin secretion (Agonist-induced internalization of the Glucagon-like peptide-1 peptide is mediated by the Gaq pathway, Aiysha Thompson, et al, Biochemical Pharmacology, 93: 72-84, 2015; Molecular Characterisation of Small Molecule Agonists Effect on the Human Glucagon Like Peptide-1 Receptor Internalisation, Aiysha Thompson et al, PLOS ONE). Therefore, the attenuation of cAMP signaling and receptor endocytosis theoretically destroys the secretion of insulin and affects the physiological efficacy of GLP-1 analogues. After GLP-1R cross-links with GCGR to form a heterodimer, endocytosis is significantly attenuated, which affects insulin secretion.
在本发明的一个实施例中，部分GCGR/GLP-1R激动剂诱导的胰岛素分泌明显减少。已有研究显示多种人体细胞表面存在同时表达多种肠道激素受体的现象(Dominik Schelshorn等，Lateral Allosterism in the Glucagon Receptor Family:Glucagon-Like Peptide 1Induces G-Protein-Coupled Receptor Heteromer Formation，Molecular Pharmacology，81(3)309-318，2012)。因此，若GCGR/GLP-1R双效激动剂同时结合于GLP-1R和GCGR受体，不仅可能降低了其原有的生理效应，一些潜在未知作用更是很难预测的。除了胰岛素分泌的减少，还有可能会产生难以预料的毒副作用。糖尿病用药的安全性要求极高，因此，本发明人认为不会使受体产生交联的二聚体应该是更为优越的。In one embodiment of the invention, a portion of the GCGR/GLP-1R agonist induces a significant decrease in insulin secretion. Studies have shown that a variety of human gland receptors are present on the surface of a variety of human cells (Dominik Schelshorn et al, Lateral Allosterism in the Glucagon Receptor Family: Glucagon-Like Peptide 1 Induces G-Protein-Coupled Receptor Heteromer Formation, Molecular Pharmacology , 81 (3) 309-318, 2012). Therefore, if the GCGR/GLP-1R double-acting agonist binds to both GLP-1R and GCGR receptors, it may not only reduce its original physiological effects, but some potential unknown effects are even more difficult to predict. In addition to the reduction in insulin secretion, there may be unpredictable side effects. The safety of diabetes medication is extremely high, and therefore, the inventors believe that a dimer which does not cause cross-linking of the receptor should be superior.
腹腔糖耐量测试(IPGTT)Peritoneal glucose tolerance test (IPGTT)
在其中一个实施例中，进行了IPGTT实验。施用了双特异性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。In one of the examples, an IPGTT experiment was performed. Mice administered the bispecific active protein exhibited extremely stable blood glucose fluctuations after glucose injection.
DIO小鼠的减重实验Weight loss experiment in DIO mice
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，本发明的多重活性蛋白在DIO小鼠施用后，诱导了体重的显著降低。大鼠药代动力学研究本发明的双特异性活性蛋白具有改善的药物代谢动力学特性，即，它们在体内具有延长的半衰期。在一个实施例中，在大鼠皮下施用各种双特异性活性蛋白，并在不同时间点取血清测定血药浓度，以评价其药物代谢动力学特性。GCGR agonists have been reported to have potential weight loss effects. However, since 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. Furthermore, the multiplex active protein of the present invention induced a significant decrease in body weight after administration in DIO mice. Rat Pharmacokinetic Studies The bispecific active proteins of the invention have improved pharmacokinetic properties, i.e., they have an extended half-life in vivo. In one embodiment, various bispecific active proteins are administered subcutaneously in rats, and serum concentrations are determined at different time points to assess their pharmacokinetic properties.
临床应用展望Prospect of clinical application
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。Clinically, 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. In the meantime, depending on the condition of the subject and other pathological conditions, 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.
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)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. Without intending to be bound by any particular theory, 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)
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。The active proteins of the invention are also useful in the treatment of obesity. In some aspects, 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.
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。In some potential embodiments, 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). In the terms "steatogenic hepatitis" and "fatty degeneration necrosis", steatosis refers to fat infiltration, hepatitis refers to inflammation in the liver, and 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.
具体实施方式Detailed ways
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。Before the present invention is further described, it is to be understood that the scope of the present invention is not limited to the specific embodiments described below; It is not intended to limit the scope of the invention. The test methods which do not specify the specific conditions in the following examples are usually carried out according to conventional conditions or according to the conditions recommended by each manufacturer.
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。When the numerical values are given by the examples, it is to be understood that the two endpoints of each numerical range and any one of the two. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning meaning In addition to the specific methods, devices, and materials used in the embodiments, the methods, devices, and materials described in the embodiments of the present invention may also be used according to the prior art and the description of the present invention by those skilled in the art. Any method, apparatus, and material of the prior art, similar or equivalent, is used to practice the invention.
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，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(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。Unless otherwise stated, 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 ( PMWassarman 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.
实施例1 GCG类似物的筛选(胰高血糖类似物的筛选)Example 1 Screening of GCG Analogs (Screening of Pancreatic Hyperglycemia Analogs)
本发明的GCG类似物记为A，A的结构式如式II(SEQ ID NO.141)所示：The GCG analog of the present invention is referred to as A, and the structural formula of A is as shown in Formula II (SEQ ID NO. 141):
HSQGTFTSDYSKYLD X 16X 17X 18 AQDFVQWLMN X 29 X z(SEQ ID NO.141)。其中，X 16选自除Y、N、W、和H外的氨基酸之任一；X 17选自除P、L、T、F和H外的氨基酸之任一；X 18选自除P、F、H和W外的氨基酸之任一；另外X 17与X 18不能同时为R，X 29为T或缺失，X z选自SEQ ID NO.3～10之任一。 HSQGTFTSDYSKYLD X 16 X 17 X 18 AQDFVQWLMN X 29 X z (SEQ ID NO. 141). Wherein X 16 is selected from any one of amino acids other than Y, N, W, and H; X 17 is selected from any one of amino acids other than P, L, T, F, and H; X 18 is selected from P, Any one of amino acids other than F, H and W; further, X 17 and X 18 may not be R at the same time, X 29 is T or a deletion, and X z is selected from any one of SEQ ID NOS. 3 to 10.
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.44-74，所对应的多肽代号分别为C001、C002、C240、C320、C276、C225、C302、C163、C350、C271、C368、C495、C353、C352、C307、C382、C232、C227、C266、C137、C399、C395、C394、C392、C462、C228、C187、C334、C364、C209、C289。The amino acid sequences of the exemplary GCG analogs may each independently be selected from the group consisting of SEQ ID NO. 44-74, and the corresponding polypeptide codes are C001, C002, C240, C320, C276, C225, C302, C163, C350, C271, C368, C495, C353, C352, C307, C382, C232, C227, C266, C137, C399, C395, C394, C392, C462, C228, C187, C334, C364, C209, C289.
进一步地，本发明的重组蛋白的结构式如式I所示：A-L-F式I，Further, the structural formula of the recombinant protein of the present invention is as shown in Formula I: A-L-F Formula I,
式I中，A的结构式如上述式II所示： HSQGTFTSDYSKYLD X 16X 17X 18 AQDFVQWLMN X 29 X z(SEQ ID NO.141)，其中，X 16选自除Y、N、W、和H外的氨基酸之任一；X 17选自除P、L、T、F和H外的氨基酸之任一；X 18选自除P、F、H和W外的氨基酸之任一；另外X 17与X 18不能同时为R，X 29为T或缺失，X z选自GGPSSGAPPPS(SEQ ID NO.3)、GPSSGAPPPS(SEQ ID NO.4)、PSSGAPPPS(SEQ ID NO.5)、SSGAPPPS(SEQ ID NO.6)、GGPSSGAPPS(SEQ ID NO.7)、GPSSGAPPS(SEQ ID NO.8)、PSSGAPPS(SEQ ID NO.9)或SSGAPPS(SEQ ID NO.10)之任一。 In Formula I, the structural formula of A is as shown in the above formula II: HSQGTFTSDYSKYLD X 16 X 17 X 18 AQDFVQWLMN X 29 X z (SEQ ID NO. 141), wherein X 16 is selected from the group consisting of Y, N, W, and H. any amino acid of a; X 17 is selected from any one of amino acid except P, L, T, F and H a; X 18 is selected from any of the amino acid except P, F, H and W a; and additional X 17 X 18 cannot be R at the same time, X 29 is T or a deletion, and X z is selected from GGPSSGAPPPS (SEQ ID NO. 3), GPSSGAPPPS (SEQ ID NO. 4), PSSGAPPPS (SEQ ID NO. 5), SSGAPPPS (SEQ ID NO) .6), GGPSSGAPPS (SEQ ID NO. 7), GPSSGAPPS (SEQ ID NO. 8), PSSGAPPS (SEQ ID NO. 9) or SSGAPPS (SEQ ID NO. 10).
式I中，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，如SEQ ID NO.11-20所示。 In Formula I, F is a long-acting protein unit, and 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, such as SEQ ID NO. 11-20.
例如，F可以是天然IgG1的完整FC部分，氨基酸序列如SEQ ID NO.11所示，F可以是天然IgG1的FC部分的突变体，氨基酸序列如SEQ ID NO.12所示，F可以是天然IgG2的完整FC部分，氨基酸序列如SEQ ID NO.13所示，F可以是天然IgG4的完整FC部分，氨基酸序列如SEQ ID NO.14所示，F可以是天然IgG4的FC部分的突变体，氨基酸序列如SEQ ID NO.15-18所示，F可以是天然IgG1的FC部分的突变体，氨基酸序列可以如SEQ ID NO.19-20所示。For example, F may be the complete FC portion of native IgG1, the amino acid sequence is set forth in SEQ ID NO. 11, F may be a mutant of the FC portion of native IgG1, the amino acid sequence is set forth in SEQ ID NO. 12, and F may be natural. The complete FC portion of IgG2, the amino acid sequence is set forth in SEQ ID NO. 13, F may be the complete FC portion of native IgG4, the amino acid sequence is set forth in SEQ ID NO. 14, and F may be a mutant of the FC portion of native IgG4, The amino acid sequence is set forth in SEQ ID NO. 15-18, and F may be a mutant of the FC portion of native IgG1, and the amino acid sequence may be as shown in SEQ ID NO. 19-20.
式I中，L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，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.21-43。In formula I, L is a linking chain which is a flexible polypeptide of glycine (G), serine (S) and/or alanine (A) of suitable length such that adjacent protein domains They are free to move relative to each other. 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., n is an integer of 1-10, in a preferred In an 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. 21-43.
实施例2 二聚体双效活性蛋白的制备Example 2 Preparation of Dimer Double-acting Active Protein
实施例1中，由GCG类似物与连接链及F融合而获得的二聚体双效活性蛋白(结构式如式I所示)的氨基酸序列选自SEQ ID NO.75-105和SEQ ID NO.142，对应的核苷酸序列分别为SEQ ID NO.161-191、SEQ ID NO.225，对应的双效活性蛋白A-L-F代号分别为C001G12S3A1F4、C002G12S3A1F4、C240G12S3A1F4、C320G12S3A1F4、C276G12S3A1F4、C368G12S3A1F4、C225G12S3A1F4、C302G12S3A1F4、C163G12S3A1F4、C350G12S3A1F4、C271G12S3A1F4、C232G12S3A1F4、 C495G12S3A1F4、C307G12S3A1F4、C382G12S3A1F4、C227G12S3A1F4、C266G12S3A1F4、C137G12S3A1F4、C399G12S3A1F4、C395G12S3A1F4、C394G12S3A1F4、C392G12S3A1F4、C353G12S3A1F4、C352G12S3A1F4、C228G12S3A1F4、C462G12S3A1F4、C187G12S3A1F4、C334G12S3A1F4、C364G12S3A1F4、C209G12S3A1F4、C289G12S3A1F4、C0382G12S3A1F4。In Example 1, the amino acid sequence of the dimeric double-acting active protein (the structural formula is as shown in Formula I) obtained by fusion of the GCG analog with the linker and F is selected from the group consisting of SEQ ID NO. 75-105 and SEQ ID NO. 142, the corresponding nucleotide sequences are SEQ ID NO. 161-191 and SEQ ID NO. 225, respectively, and the corresponding double-acting active protein ALF codes are C001G12S3A1F4, C002G12S3A1F4, C240G12S3A1F4, C320G12S3A1F4, C276G12S3A1F4, C368G12S3A1F4, C225G12S3A1F4, C302G12S3A1F4, respectively. C163G12S3A1F4, C350G12S3A1F4, C271G12S3A1F4, C232G12S3A1F4, C495G12S3A1F4, C307G12S3A1F4, C382G12S3A1F4, C227G12S3A1F4, C266G12S3A1F4, C137G12S3A1F4, C399G12S3A1F4, C395G12S3A1F4, C394G12S3A1F4, C392G12S3A1F4, C353G12S3A1F4, C352G12S3A1F4, C228G12S3A1F4, C462G12S3A1F4, C187G12S3A1F4, C334G12S3A1F4, C364G12S3A1F4, C209G12S3A1F4, C289G12S3A1F4, C0382G12S3A1F4.
在知晓了二聚体双效活性蛋白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。 Based on the knowledge of the amino acid sequence of the dimeric double-acting active protein, those skilled in the art can prepare it by the prior art: due to the F C sequence, it can pass the high affinity and high specificity of Protein A. Resin chromatography is used for protein purification, and only one possible preparation method is exemplified herein.
制备过程如下：The preparation process is as follows:
(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、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；(1) The DNA sequence is designed based on the protein sequence and the amino acid codon table. The polynucleotide DNA fragments corresponding to A, L and F in the recombinant protein are respectively prepared, and each DNA fragment can be synthesized and spliced by conventional solid phase synthesis technology;
(2)设计引物进行巢式PCR扩增，拼接A、L、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 PCTCN2018116171-appb-000008
Endo-Free Plasmid Maxi Kit提取质粒。 (2) Design primers for nested PCR amplification, splicing the DNA fragments corresponding to A, L, and F respectively to obtain the target gene, and PCR splicing technology (including primer design, PCR introduction mutation and enzyme digestion, 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. After successfully obtaining the target gene, 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
Figure PCTCN2018116171-appb-000008
Endo-Free Plasmid Maxi Kit extracts the plasmid.
(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。 (3) Transfection of Hek293F cells and cell expression: 1.0 mg of plasmid was diluted to 25 ml with Freestyle 293 expression medium (Thermofisher); 3.0 mg of PEI (linear, 25 KD) was taken and diluted to 25 ml with Freestyle 293 expression medium. Into the plasmid solution, mix and incubate for 30 minutes at room temperature; at the same time, take log phase growth of Hek293F cells (live rate >95%), count; 1100 RPM, centrifuge for 10 minutes, discard the supernatant; use 450ml Freestyle 293 expression The medium was resuspended in the medium; after the incubation of the PEI plasmid mixture, it was added to the cell suspension, and cultured at 37 ° C, 5% CO 2 , 140 RPM; after 7 hours, the Freestyle 293 was replaced with 1000 ml of 293 SFM II medium (Thermofisher). The expression medium was continued and cultured for 7 days.
(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)，再次平衡后，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。(4) Purification of recombinant protein: The cell culture medium was centrifuged at 8000 rpm for 10 min at high speed, and the supernatant was applied to a Protein A column (Boglon () which was previously equilibrated with an equilibration solution (20 mM PB, 0.5 M NaCl, pH 7). Shanghai) Biotechnology Co., Ltd.), after re-equilibration, 100% elution (eluent is 0.1M Gly-HCl, pH 3.0); the neutralizing solution is added to the collection tube (1M Tris-HCl, pH 8.0) The eluted sample was collected; finally, the neutralizing solution was added to 1/10 of the eluted sample volume, and the protein concentration was determined by the conventional Bradford method.
(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，图1为示例性的纯化样品的SDS-PAGE电泳图。(5) Identification of physicochemical properties of recombinant protein: SDS-PAGE electrophoresis was performed on the purified recombinant protein, and Figure 1 is an SDS-PAGE electrophoresis pattern of an exemplary purified sample.
实施例3 体外细胞活性检测Example 3 In vitro cell viability assay
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。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.
GLP-1R激动活性检测：GLP-1R agonistic activity assay:
GLP-1R激动活性检测采用荧光素酶报告基因检测法。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-EGFP质粒上，替换EGFP基因，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。The luciferase reporter assay was used to detect GLP-1R agonistic activity. 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 the pCRE-EGFP plasmid. The EGFP gene was replaced to obtain a 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.
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)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。 The cells were cultured in a 9-cm cell culture dish in DMEM/F12 medium containing 10% FBS and 300 μg/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 μg/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 μg/ml G418. /F12 medium was resuspended and counted. The cells were diluted with DMEM/F12 medium containing 10% FBS to 3×10 5 , 100 μl per well in a 96-well plate, ie 5×10 4 /well, and then affixed to DMEM/F12 containing 0.2% FBS. Base culture. After disposing the supernatant in the 96-well plate, the purified recombinant protein (Table 1) or natural Glucagon (Hangzhou Zhongpept Biochemical Co., Ltd., GLUC-004) and natural GLP-1 (Hangzhou Zhongpept Biochemical 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 μl/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).
GCGR激动活性检测方法：GCGR agonistic activity detection method:
GCGR激动活性检测同样也采用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，转染HEK 293T细胞及稳转细胞株HEK 293T/GCGR的构建同上。图2为部分二聚体双效活性蛋白的测活EC50结果图。The luciferase reporter assay is 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, and the transfected HEK 293T cells and the stably transfected cell line HEK 293T/GCGR were constructed as above. Figure 2 is a graph showing the results of the measured EC50 of the partially dimeric double-acting active protein.
结果如表1所示：The results are shown in Table 1:
表1Table 1
Figure PCTCN2018116171-appb-000009
Figure PCTCN2018116171-appb-000009
此外，还对其他不带有Exendin-4C末端序列的二聚体活性蛋白：CG283G12S3A1F4(SEQ ID NO.106)、CG214G12S3A1F4(SEQ ID NO.107)、CG267G12S3A1F4(SEQ ID NO.108)、C308G12S3A1F4(SEQ ID NO.109)、C224G12S3A1F4(SEQ ID NO.110)、CG308G12S3A1F4(SEQ ID NO.111)、C319G12S3A1F4(SEQ ID NO.112)、C214G12S3A1F4(SEQ ID NO.113)、C303G12S3A1F4(SEQ ID NO.114)、CG303G12S3A1F4(SEQ ID NO.115)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。In addition, other dimeric active proteins without the Exendin-4 C-terminal sequence: CG283G12S3A1F4 (SEQ ID NO. 106), CG214G12S3A1F4 (SEQ ID NO. 107), CG267G12S3A1F4 (SEQ ID NO. 108), C308G12S3A1F4 (SEQ ID NO. 109), C224G12S3A1F4 (SEQ ID NO. 110), CG308G12S3A1F4 (SEQ ID NO. 111), C319G12S3A1F4 (SEQ ID NO. 112), C214G12S3A1F4 (SEQ ID NO. 113), C303G12S3A1F4 (SEQ ID NO. 114) CG303G12S3A1F4 (SEQ ID NO. 115) was assayed for in vitro activity, including GLP-1R agonistic activity assay and GCGR agonistic activity assay.
测定结果与本发明的重组蛋白进行比较，结果如表2所示：The results of the assay were compared with the recombinant protein of the present invention, and the results are shown in Table 2:
表2Table 2
Figure PCTCN2018116171-appb-000010
Figure PCTCN2018116171-appb-000010
说明：a.为加上Exendin-4的C末端延伸肽Cex(在本发明中为SEQ ID NO.3-10之任一)前后的GLP-1R激动活性比例。Description: a. The ratio of GLP-1R agonistic activity before and after the addition of the C-terminal extension peptide Cex (any of SEQ ID NOS. 3-10 in the present invention) of Exendin-4.
b.为根据US9018164B2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。b. Calculated for the GLP-1R agonistic activity data of native Glucagon and Glucagon Cex as disclosed in Table 2 of US9018164B2.
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。c. Natural Glucagon disclosed in Table 1 of the article by Joseph R. Chabenne et al. (Joseph R. Chabenne et al., Optimization of the Native Glucagon Sequence for Medicinal Purposes, J Diabetes Sci Technol. 4(6): 1322–1331, 2010). The ratio obtained from the GLP-1R agonistic activity data of Glucagon Cex was calculated.
如表1和表2所示，当把含延伸肽的序列通过(GGGGS) 3A(SEQ ID NO.33)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。 As shown in Tables 1 and 2, when the sequence containing the extended peptide was prepared by fusion of (GGGGS) 3 A (SEQ ID NO. 33) and F (SEQ ID NO. 16) to form a dimer, GLP-1R was used. The agonistic activity was increased by more than 200 times, while the agonistic activity of GCGR was not significantly different.
实施例4 DPP-IV耐酶稳定性Example 4 DPP-IV resistance to enzyme stability
将纯化的二聚体双效活性蛋白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％。The purified dimeric double-acting active protein 5uM was dissolved in 10 mM HEPES buffer (containing 0.05 mg/ml BSA, and the final concentration of 10 nM recombinant DPP-IV enzyme was added, and the GCGR was detected in vitro after being incubated at 37 ° C for 24 hours. Cell viability assay. Activity retention rate = (activity after DPP-IV enzyme treatment / activity before treatment) x 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：In this example, a second GCG analog introduced with the unnatural amino acid Aib or D-Ser was used as a control:
GDSerGS：H-D-Ser-QGTFTSDYSKYLDSQAAQDFVQWLMNGGPSSGAPPPS(SEQ ID NO.116)；GDSerGS: H-D-Ser-QGTFTSDYSKYLDSQAAQDFVQWLMNGGPSSGAPPPS (SEQ ID NO. 116);
GAibGS：H-Aib-QGTFTSDYSKYLDSQAAQDFVQWLMNGGPSSGAPPPS(SEQ ID NO.117)；GAibGS: H-Aib-QGTFTSDYSKYLDSQAAQDFVQWLMNGGPSSGAPPPS (SEQ ID NO. 117);
C364(SEQ ID NO.72)、C382(SEQ ID NO.59)、C495(SEQ ID NO.55)、C462(SEQ ID NO.68)、C225(SEQ ID NO.49)及C209(SEQ ID NO.73)作为本实施例中稳定性实验的对照。C364 (SEQ ID NO. 72), C382 (SEQ ID NO. 59), C495 (SEQ ID NO. 55), C462 (SEQ ID NO. 68), C225 (SEQ ID NO. 49), and C209 (SEQ ID NO) .73) As a control for the stability experiment in this example.
结果如表3所示：The results are shown in Table 3:
表3table 3
Figure PCTCN2018116171-appb-000011
Figure PCTCN2018116171-appb-000011
Figure PCTCN2018116171-appb-000012
Figure PCTCN2018116171-appb-000012
实施例5 血清稳定性试验Example 5 Serum stability test
体外细胞检测法：In vitro cell assay:
(1)取二聚体双效活性蛋白，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；(1) Take the dimeric double-acting active protein, concentrate it by ultrafiltration, dilute to 1.6 mg/ml with 20 mM PB pH 7.4, sterilize and filter, and serum (FBS, GEMINI 900-108, A97E00G) diluted 10 times. , mix and dispense into a sterile centrifuge tube;
(2)另外取Glucagon(SEQ ID NO：44，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；(2) Take Glucagon (SEQ ID NO: 44, Hangzhou Zhongpept Biochemical Co., Ltd., GLUC-004), dilute to 0.2mg/ml, sterilize and filter, serum diluted 10 times, mix, and dispense into sterile In a centrifuge tube;
(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；(3) The above sample 1-2 tubes were frozen at -20 degrees as a control, and the other tubes were placed at 37 ° C incubator, and the GCGR agonistic activity was sampled at different time points;
(4)HEK 293T/GCGR细胞传代两次后铺96孔板，检测样品活性。相对活性随着时间的变化如图3A-D所示。从图3结果可见C240G12S3A1F4、C276G12S3A1F4、C368G12S3A1F4、C225G12S3A1F4、C163G12S3A1F4、C232G12S3A1F4、C495G12S3A1F4、C382G12S3A1F4、C271G12S3A1F4、C227G12S3A1F4、C266G12S3A1F4、C399G12S3A1F4、C392G12S3A1F4、C353G12S3A1F4、C137G12S3A1F4、C289G12S3A1F4、C209G12S3A1F4、C352G12S3A1F4、C228G12S3A1F4、C462G12S3A1F4、C187G12S3A1F4、C364G12S3A1F4在血清中的稳定性相对其他二聚体明显较高。(4) HEK 293T/GCGR cells were passaged twice and then plated in 96-well plates to measure the activity of the samples. The relative activity changes over time as shown in Figures 3A-D. It is seen from the results shown in figure C240G12S3A1F4, C276G12S3A1F4, C368G12S3A1F4, C225G12S3A1F4, C163G12S3A1F4, C232G12S3A1F4, C495G12S3A1F4, C382G12S3A1F4, C271G12S3A1F4, C227G12S3A1F4, C266G12S3A1F4, C399G12S3A1F4, C392G12S3A1F4, C353G12S3A1F4, C137G12S3A1F4, C289G12S3A1F4, C209G12S3A1F4, C352G12S3A1F4, C228G12S3A1F4, C462G12S3A1F4, C187G12S3A1F4, C364G12S3A1F4 serum The stability in the medium is significantly higher than in other dimers.
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。Residual activity: The activity value at 0 hours was 100%, and the value measured at the subsequent time point was obtained.
实施例6 不同连接链长度的二聚体双效活性蛋白的活性测定Example 6 Activity Determination of Dimeric Double-acting Active Proteins with Different Link Lengths
将实施例1中的GCG类似物通过不同长度的连接链融合于人IgG1F C(SEQ ID NO.12)的N端。亦即，由GCG类似物与不同长度连接链及F(SEQ ID NO.12)融合而获得的二聚体双效活性蛋白分别为C382G4A1F1、C382G4S1F1、C382G6S2F1、C382G4S4F1、C382G8S2F1、C382G8A2F1、C382G9S3F1、C382G9S2A1F1、C382G12S3F1、C382G12A3F1、C382G12A1S3F1、C382G12S3A1F1、C382G12A4F1、C382G12S1A3F1、C382G13S4F1、C382G16A4F1、C382G16S4F1、C382G17S5F1、C382G20S5F1、C382G20S5A1F1、C382G24S6F1、C382G28S7F1、C382G32S8F1，对应的氨基酸序列SEQ ID NO.118-140，对应的核苷酸序列SEQ ID NO.202-224。 The GCG analog of Example 1 was fused to the N-terminus of human IgG1 F C (SEQ ID NO. 12) by a ligation chain of different length. That is, the dimeric double-acting active proteins obtained by fusion of the GCG analogs with the different length linkages and F (SEQ ID NO. 12) are C382G4A1F1, C382G4S1F1, C382G6S2F1, C382G4S4F1, C382G8S2F1, C382G8A2F1, C382G9S3F1, C382G9S2A1F1, respectively. C382G12S3F1, C382G12A3F1, C382G12A1S3F1, C382G12S3A1F1, C382G12A4F1, C382G12S1A3F1, C382G13S4F1, C382G16A4F1, C382G16S4F1, C382G17S5F1, C382G20S5F1, C382G20S5A1F1, C382G24S6F1, C382G28S7F1, C382G32S8F1, corresponding amino acid sequence SEQ ID NO. 118-140, corresponding nucleotide sequence SEQ ID NO.202-224.
在知晓了二聚体双效活性蛋白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。Based on the knowledge of the amino acid sequence of the dimeric double-acting active protein, those skilled in the art can prepare it using the prior art.
本实施例中所使用的制备方法同实施例2。The preparation method used in this example is the same as that in Example 2.
将本实施例中获得的二聚体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。检测方法同实施例3。The dimeric double-acting active protein obtained in the present example was assayed for in vitro activity, including GLP-1R agonistic activity assay and GCGR agonistic activity assay. The detection method is the same as in the third embodiment.
检测结果如表4所示。The test results are shown in Table 4.
表4Table 4
Figure PCTCN2018116171-appb-000013
Figure PCTCN2018116171-appb-000013
实施例7 葡萄糖刺激的胰岛素分泌实验(GSIS)Example 7 Glucose-stimulated insulin secretion assay (GSIS)
本实施例参照Aisling M.Lynch等人的方法(A novel DPP IV-resistant C-terminally extended Glucagon analogue exhibits weight-lowering and diabetes-protective effects in high-fat-fed mice mediated through Glucagon and GLP-1 receptor activation，Aisling M.Lynch等，Diabetologia，57:1927–1936，2014)采用大鼠BRIN-BD11细胞用于测定活性蛋白刺激引起的胰岛素释放，但稍作修改，即在24孔板(Orange Scientific,Brainel’Alleud,Belgium)中每孔加入1.0×10 6个细胞，37℃培养过夜后离心去上清，再每孔加入1.0ml KRB(115mM NaCl、4.7mM KCl、1.28mM CaCl 2、1.2mM MgSO 4、1.2mM KH 2PO 4、25mM HEPES、10mM NaHCO 3，NaOH调节pH至7.4)、0.1％(wt/vol.)BSA和1.1mM葡萄糖。细胞置于37℃培养40分钟后，离心去上清并替换成1.0ml新鲜的KRB溶液与梯度浓度的表4中的活性蛋白。37℃培养20分钟后，离心去除缓冲液并于-20℃储存过夜，再作免疫放射检测胰岛素含量。结果 如图4所示。 In this embodiment, the method of Aisling M. Lynch et al. (A novel DPP IV-resistant C-terminally extended Glucagon analogue exhibits weight-lowering and diabetes-protective effects in high-fat-fed mice mediated through Glucagon and GLP-1 receptor activation , Aisling M. Lynch et al, Diabetologia, 57: 1927–1936, 2014) Rat BRIN-BD11 cells were used to determine insulin release induced by active protein stimulation, but with minor modifications, ie in a 24-well plate (Orange Scientific, Brainel) 1.0×10 6 cells were added to each well in 'Alleud, Belgium, cultured at 37 ° C overnight, and the supernatant was centrifuged, and 1.0 ml of KRB (115 mM NaCl, 4.7 mM KCl, 1.28 mM CaCl 2 , 1.2 mM MgSO 4 ) was added to each well. 1.2 mM KH 2 PO 4 , 25 mM HEPES, 10 mM NaHCO 3 , NaOH adjusted to pH 7.4), 0.1% (wt/vol.) BSA and 1.1 mM glucose. After the cells were incubated at 37 ° C for 40 minutes, the supernatant was centrifuged and replaced with 1.0 ml of fresh KRB solution and a gradient concentration of the active protein in Table 4. After incubation at 37 ° C for 20 minutes, the buffer was removed by centrifugation and stored at -20 ° C overnight, and then subjected to immunoradiometric detection of insulin content. The result is shown in Figure 4.
实施例8 正常ICR小鼠的葡萄糖耐量试验(IPGTT)Example 8 Glucose Tolerance Test (IPGTT) in Normal ICR Mice
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)，皮下注射二聚体双效活性蛋白(40nmol/kg，醋酸盐缓冲液)或生理盐水PBS，其中C002G12S3A1F4给药剂量为250nmol/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5所示。Normal ICR mice were grouped into groups of 8 animals. Fasting overnight, tail blood sampling (denoted as t = 0 minute blood glucose), subcutaneous injection of dimeric double-acting active protein (40nmol / kg, acetate buffer) or saline PBS, of which C002G12S3A1F4 dose was 250nmol / Kg (acetate buffer). After 15 minutes, glucose (2 g/kg body weight) was injected intraperitoneally, and blood glucose levels were measured at t = 30 minutes, t = 60 minutes, t = 120 minutes, and t = 240 minutes. Animals were still fasted during the experiment to prevent interference with food intake. The result is shown in Figure 5.
实施例9 连续给药在诱导肥胖(DIO)小鼠中的药效研究Example 9 Study on the efficacy of continuous administration in induced obesity (DIO) mice
本实施例的目的是研究不同的二聚体双效活性蛋白对DIO小鼠体重的影响作用。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。按照50nmol/千克体重的剂量给药，4天一次，其他日期予以PBS假注射，连续给药28天；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(40nmol/千克体重)，每天一次给药，每天测量小鼠体重。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。结果如图6所示。The purpose of this example was to investigate the effect of different dimeric double-acting active proteins on the body weight of DIO 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 the dose of 50nmol/kg body weight, once every 4 days, other days were given PBS sham injection for 28 days; the negative control group was injected with normal saline (PBS) at 5 ul/g body weight; the positive control group was injected with liraglutide. (40 nmol/kg body weight), once daily, the body weight of the mice was 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 result is shown in Figure 6.
实施例10 大鼠的药物代谢动力学参数(PK)测定Example 10 Determination of pharmacokinetic parameters (PK) in rats
将6周左右的雄性SD大鼠分组，每组8只。根据表5的方案给药或生理盐水。给药结束后所有大鼠开始自由饮水进食；给药结束的时间点设置为0，并作为后续实验的时间参照。二聚体双效活性蛋白的药代检测采用夹心ELISA法。即鼠抗人IgG4FC单抗(9002-01，I2013-NG 56，0.25mg/ml)；包板(96孔板，corning，42592)，200ng/孔，4℃过夜；PBST洗板4次后用5％奶粉溶液封闭，室温1h；再PBST洗板4次；用PBST稀释大鼠血清后，37℃孵育2h；PBST洗板6次；再用1％BSA溶液稀释兔抗Glucagon抗体(自制：用天然Glucagon多肽(SEQ ID NO.44)免疫家兔，通过Protein G纯化得到抗Glucagon抗体(杭州华安生物技术有限公司)，生物素标记，200×，0.2mg/ml)，1:2000，37℃孵育2h；PBST洗板6次；1％BSA溶液稀释strep-HRP，1:5万，37℃孵育1.5h；PBST洗板6次；最后TMB 显色，OD450读数。Male Sprague-Dawley rats of about 6 weeks were grouped into groups of 8 rats. Administration according to the protocol of Table 5 or physiological saline. At the end of the administration, all rats began to drink freely; the time point at which the administration was completed was set to 0, and was used as a time reference for subsequent experiments. The pharmacokinetic detection of dimeric double-acting active protein was carried out by sandwich ELISA. Ie mouse anti-human IgG4FC mAb (9002-01, I2013-NG 56, 0.25mg/ml); coated plate (96-well plate, corning, 42592), 200ng / well, 4 ° C overnight; PBST wash plate 4 times 5% milk powder solution was blocked, room temperature was 1 h; PBST was washed 4 times; rat serum was diluted with PBST, then incubated at 37 ° C for 2 h; PBST was washed 6 times; and rabbit anti-Glucagon antibody was diluted with 1% BSA solution (home-made: The natural Glucagon polypeptide (SEQ ID NO. 44) was immunized to rabbits and purified by Protein G to obtain anti-Glucagon antibody (Hangzhou Huaan Biotechnology Co., Ltd.), biotin label, 200×, 0.2 mg/ml), 1:2000, 37 °C. Incubate for 2 h; wash the plate 6 times with PBST; dilute strep-HRP with 1% BSA solution, 1:50,000, incubate for 1.5 h at 37 °C; wash plate 6 times with PBST; finally TMB color development, OD450 reading.
表5table 5
Figure PCTCN2018116171-appb-000014
Figure PCTCN2018116171-appb-000014
结果如表6所示：The results are shown in Table 6:
表6Table 6
二聚体双效活性蛋白Dimeric double-acting active protein T 1/2(小时) T 1/2 (hours) T max(小时) T max (hours)
C240G12S3A1F4(SEQ ID NO.77)C240G12S3A1F4 (SEQ ID NO. 77) 41.241.2 24twenty four
C382G12S3A1F4(SEQ ID NO.89)C382G12S3A1F4 (SEQ ID NO. 89) 42.542.5 24twenty four
C495G12S3A1F4(SEQ ID NO.87)C495G12S3A1F4 (SEQ ID NO. 87) 43.743.7 24twenty four
C364G12S3A1F4(SEQ ID NO.103)C364G12S3A1F4 (SEQ ID NO. 103) 42.642.6 24twenty four
C462G12S3A1F4(SEQ ID NO.100)C462G12S3A1F4 (SEQ ID NO. 100) 39.539.5 24twenty four
C227G12S3A1F4(SEQ ID NO.90)C227G12S3A1F4 (SEQ ID NO. 90) 42.242.2 24twenty four
C368G12S3A1F4(SEQ ID NO.80)C368G12S3A1F4 (SEQ ID NO. 80) 37.837.8 24twenty four
C266G12S3A1F4(SEQ ID NO.91)C266G12S3A1F4 (SEQ ID NO. 91) 38.438.4 24twenty four
C002G12S3A1F4(SEQ ID NO.76)C002G12S3A1F4 (SEQ ID NO. 76) 8.58.5 66
实施例11 融合FGF21类似物的二聚体三效活性蛋白的构建及活性测定Example 11 Construction and Activity Determination of Dimeric Triple-acting Active Protein Fusion FGF21 Analogue
在前述二聚体双效活性蛋白的C末端，进一步融合FGF21类似物，构建获得融合蛋白(融合FGF21类似物的三效活性蛋白)：天然FGF21(SEQ ID NO.143)、FGF21类似物1(SEQ ID NO.144)、FGF21类似物2(SEQ ID NO.145)、FGF21类似物3(SEQ ID NO.146)、C382F4FGF(SEQ ID NO.147)、C382F4FGF1(SEQ ID NO.148)、C382F4FGF2(SEQ ID NO.149)所示，C382F4FGF3(SEQ ID NO.150)、C495F4FGF(SEQ ID NO.151)、C495F4FGF1(SEQ ID NO.152)、C495F4FGF2(SEQ ID NO.153)、C495 F4FGF3(SEQ ID NO.154)。At the C-terminus of the aforementioned dimeric double-acting active protein, the FGF21 analog is further fused to construct a fusion protein (a tri-fold active protein fused to the FGF21 analog): native FGF21 (SEQ ID NO. 143), FGF21 analog 1 ( SEQ ID NO. 144), FGF21 Analog 2 (SEQ ID NO. 145), FGF21 Analog 3 (SEQ ID NO. 146), C382F4FGF (SEQ ID NO. 147), C382F4FGF1 (SEQ ID NO. 148), C382F4FGF2 (SEQ ID NO. 149), C382F4FGF3 (SEQ ID NO. 150), C495F4FGF (SEQ ID NO. 151), C495F4FGF1 (SEQ ID NO. 152), C495F4FGF2 (SEQ ID NO. 153), C495 F4FGF3 (SEQ ID NO.154).
所述融合蛋白的制备方法属于现有技术：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，可参照实施例2中的方法。纯化获得的融合蛋白的电泳图如图7所示。天然FGF21、FGF21类似物的制备参考Xu J等的报道(Xu J等，Polyethylene glycol modified FGF21 engineered to maximize potency and minimize vacuole formation，Bioconjug Chem.；24(6):915-25，2013)并作如下修改：表达载体为PET30，宿主菌为BL21(DE3)(默克中国)。包涵体用洗涤液(50mM Tris,150mM NaCl,2M尿素，pH 8.0)洗涤四次，称重；按照(1:10的质量体积比)每0.1g包涵体加入1ml变性液(50mM Tris,150mM NaCl,8M urea,10mM DTT，pH 8.5)，室温摇床轻轻混合溶解5h以上；按照1:100-200的比例稀释复性。将变性液缓慢滴加到复性液中，过程中不断搅拌；滴加完成之后，将含有蛋白的复性液放于4℃，24h；结束后用0.45um滤膜抽滤用于层析纯化。 The preparation method of the fusion protein belongs to the prior art: since the F C sequence is carried out, protein purification can be carried out by high affinity and high specificity Protein A resin chromatography, and the method in Example 2 can be referred to. The electropherogram of the purified fusion protein is shown in Fig. 7. The preparation of natural FGF21 and FGF21 analogs is described in Xu J et al. (Xu J et al., Polyethylene glycol modified FGF21 engineered to maximize potency and minimize vacuole formation, Bioconjug Chem.; 24(6): 915-25, 2013) and is as follows Modification: The expression vector was PET30 and the host strain was BL21 (DE3) (Merck China). The inclusion bodies were washed four times with washing solution (50 mM Tris, 150 mM NaCl, 2 M urea, pH 8.0) and weighed; 1 ml of denaturant (50 mM Tris, 150 mM NaCl) was added per 0.1 g of inclusion body according to (1:10 mass to volume ratio). , 8M urea, 10mM DTT, pH 8.5), gently mix and dissolve for more than 5h at room temperature shaker; dilute renaturation according to the ratio of 1:100-200. The denaturant was slowly added dropwise to the reconstituted solution, and stirring was continued during the process; after the addition was completed, the protein-containing reconstituted solution was placed at 4 ° C for 24 h; after completion, it was filtered with a 0.45 um filter for chromatographic purification. .
测活细胞的构建：Measuring the construction of living cells:
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 PCTCN2018116171-appb-000015
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3x 10 5个细胞，培养过夜。 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 (β-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
Figure PCTCN2018116171-appb-000015
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。 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 μl): pFR-Luc (4.6 μg): pcDNA-GAL4DBD-ELK1-IRES-KLB-Puro (1 μg). 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.
细胞测活：Cell measurement:
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。结果如表7所示：After the cells were overgrown, the cells were trypsinized, and a cell suspension (1 x 105 cells/ml, DMEM + 5% FBS + 1 μg/ml puromycin) was prepared, and 96-well plates were plated at 100 μl 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 results are shown in Table 7:
表7Table 7
Figure PCTCN2018116171-appb-000016
Figure PCTCN2018116171-appb-000016
Figure PCTCN2018116171-appb-000017
Figure PCTCN2018116171-appb-000017
实施例12 融合FGF21及其类似物的三活性蛋白在DIO小鼠中的药效研究Example 12 Study on the pharmacodynamics of tri-active protein fused to FGF21 and its analogs in DIO mice
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(表8)。双效活性蛋白按照30nmol/千克体重或90nmol/千克体重的剂量给药，4天一次；FGF21类似物按30nmol/千克体重，每天两次给药；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8所示；总摄食量的变化如图9所示。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 subcutaneous administration was performed the next day (Table 8). . The double-acting active protein was administered at a dose of 30 nmol/kg body weight or 90 nmol/kg body weight once every 4 days; the FGF21 analog was administered twice daily at 30 nmol/kg body weight; and the negative control group was injected with physiological saline at 5 ul/g body weight ( PBS); The positive control group was injected with liraglutide (30 nmol/kg body weight) once a day for 28 days, and the body weight and food intake of the mice were measured every day. 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 8; the change in total food intake is shown in Figure 9.
表8Table 8
Figure PCTCN2018116171-appb-000018
Figure PCTCN2018116171-appb-000018
实施例13 融合瘦素的三活性蛋白的构建及活性测定Example 13 Construction and Activity Determination of Tri-active Proteins Fusion with Leptin
在前述二聚体双效活性蛋白的C末端，可进一步融合天然瘦素(SEQ ID NO.155)及其类 似物，构建获得表9中的融合蛋白(融合瘦素的三活性蛋白)。At the C-terminus of the aforementioned dimeric double-acting active protein, native leptin (SEQ ID NO. 155) and its analogs can be further fused to construct a fusion protein (trimeric protein of fusion leptin) obtained in Table 9.
表9Table 9
Figure PCTCN2018116171-appb-000019
Figure PCTCN2018116171-appb-000019
实施例14 融合瘦素的三活性蛋白在DIO小鼠中的药效研究Example 14 Study on the efficacy of trimeric active protein with leptin in DIO mice
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。按照30nmol/千克体重的剂量给药，阴性对照组按5ul/克体重注射生理盐水(PBS)；双效活性蛋白4天一次，其他日期予以PBS假注射，连续给药28天，瘦素按30nmol/千克体重每天两次给药，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10所示；总摄食量的变化如图11所示。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 the dose of 30nmol/kg body weight, the negative control group was injected with normal saline (PBS) at 5ul/g body weight; the double-acting active protein was administered once every 4 days, and other days were given PBS sham injection for 28 days, leptin was 30nmol / kg body weight was administered twice daily, 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 10; the change in total food intake is shown in Figure 11.
本说明书中所述及的活性蛋白所对应的核苷酸序列如表10中所示。The nucleotide sequences corresponding to the active proteins described in the present specification are shown in Table 10.
表10Table 10
序号Serial number 活性蛋白Active protein 对应的核苷酸序列Corresponding nucleotide sequence 序号Serial number 活性蛋白Active protein 对应的核苷酸序列 Corresponding nucleotide sequence
11 C001G12S3A1F4C001G12S3A1F4 SEQ ID NO.161SEQ ID NO.161 4040 C303G12S3A1F4C303G12S3A1F4 SEQ ID NO.200SEQ ID NO.200
22 C002G12S3A1F4C002G12S3A1F4 SEQ ID NO.162SEQ ID NO.162 4141 CG303G12S3A1F4CG303G12S3A1F4 SEQ ID NO.201SEQ ID NO.201
33 C240G12S3A1F4C240G12S3A1F4 SEQ ID NO.163SEQ ID NO.163 4242 C382G4A1F1C382G4A1F1 SEQ ID NO.202SEQ ID NO.202
44 C320G12S3A1F4C320G12S3A1F4 SEQ ID NO.164SEQ ID NO.164 4343 C382G4S1F1C382G4S1F1 SEQ ID NO.203SEQ ID NO.203
55 C276G12S3A1F4C276G12S3A1F4 SEQ ID NO.165SEQ ID NO.165 4444 C382G6S2F1C382G6S2F1 SEQ ID NO.204SEQ ID NO.204
66 C368G12S3A1F4C368G12S3A1F4 SEQ ID NO.166SEQ ID NO.166 4545 C382G4S4F1C382G4S4F1 SEQ ID NO.205SEQ ID NO.205
77 C225G12S3A1F4C225G12S3A1F4 SEQ ID NO.167SEQ ID NO.167 4646 C382G8S2F1C382G8S2F1 SEQ ID NO.206SEQ ID NO.206
88 C302G12S3A1F4C302G12S3A1F4 SEQ ID NO.168SEQ ID NO.168 4747 C382G8A2F1C382G8A2F1 SEQ ID NO.207SEQ ID NO.207
99 C163G12S3A1F4C163G12S3A1F4 SEQ ID NO.169SEQ ID NO.169 4848 C382G9S3F1C382G9S3F1 SEQ ID NO.208SEQ ID NO.208
1010 C350G12S3A1F4C350G12S3A1F4 SEQ ID NO.170SEQ ID NO.170 4949 C382G9S2A1F1C382G9S2A1F1 SEQ ID NO.209SEQ ID NO.209
1111 C271G12S3A1F4C271G12S3A1F4 SEQ ID NO.171SEQ ID NO.171 5050 C382G12S3F1C382G12S3F1 SEQ ID NO.210SEQ ID NO.210
1212 C232G12S3A1F4C232G12S3A1F4 SEQ ID NO.172SEQ ID NO.172 5151 C382G12A3F1C382G12A3F1 SEQ ID NO.211SEQ ID NO.211
1313 C495G12S3A1F4C495G12S3A1F4 SEQ ID NO.173SEQ ID NO.173 5252 C382G12A1S3F1C382G12A1S3F1 SEQ ID NO.212SEQ ID NO.212
1414 C307G12S3A1F4C307G12S3A1F4 SEQ ID NO.174SEQ ID NO.174 5353 C382G12S3A1F1C382G12S3A1F1 SEQ ID NO.213SEQ ID NO.213
1515 C382G12S3A1F4C382G12S3A1F4 SEQ ID NO.175SEQ ID NO.175 5454 C382G12A4F1C382G12A4F1 SEQ ID NO.214SEQ ID NO.214
1616 C227G12S3A1F4C227G12S3A1F4 SEQ ID NO.176SEQ ID NO.176 5555 C382G12S1A3F1C382G12S1A3F1 SEQ ID NO.215SEQ ID NO.215
1717 C266G12S3A1F4C266G12S3A1F4 SEQ ID NO.177SEQ ID NO.177 5656 C382G13S4F1C382G13S4F1 SEQ ID NO.216SEQ ID NO.216
1818 C137G12S3A1F4C137G12S3A1F4 SEQ ID NO.178SEQ ID NO.178 5757 C382G16A4F1C382G16A4F1 SEQ ID NO.217SEQ ID NO.217
1919 C399G12S3A1F4C399G12S3A1F4 SEQ ID NO.179SEQ ID NO.179 5858 C382G16S4F1C382G16S4F1 SEQ ID NO.218SEQ ID NO.218
2020 C395G12S3A1F4C395G12S3A1F4 SEQ ID NO.180SEQ ID NO.180 5959 C382G17S5F1C382G17S5F1 SEQ ID NO.219SEQ ID NO.219
21twenty one C394G12S3A1F4C394G12S3A1F4 SEQ ID NO.181SEQ ID NO.181 6060 C382G20S5F1C382G20S5F1 SEQ ID NO.220SEQ ID NO.220
22twenty two C392G12S3A1F4C392G12S3A1F4 SEQ ID NO.182SEQ ID NO.182 6161 C382G20S5A1F1C382G20S5A1F1 SEQ ID NO.221SEQ ID NO.221
23twenty three C353G12S3A1F4C353G12S3A1F4 SEQ ID NO.183SEQ ID NO.183 6262 C382G24S6F1C382G24S6F1 SEQ ID NO.222SEQ ID NO.222
24twenty four C352G12S3A1F4C352G12S3A1F4 SEQ ID NO.184SEQ ID NO.184 6363 C382G28S7F1C382G28S7F1 SEQ ID NO.223SEQ ID NO.223
2525 C228G12S3A1F4C228G12S3A1F4 SEQ ID NO.185SEQ ID NO.185 6464 C382G32S8F1C382G32S8F1 SEQ ID NO.224SEQ ID NO.224
2626 C462G12S3A1F4C462G12S3A1F4 SEQ ID NO.186SEQ ID NO.186 6565 C0382G12S3A1F4C0382G12S3A1F4 SEQ ID NO.225SEQ ID NO.225
2727 C187G12S3A1F4C187G12S3A1F4 SEQ ID NO.187SEQ ID NO.187 6666 C382F4FGFC382F4FGF SEQ ID NO.226SEQ ID NO.226
2828 C334G12S3A1F4C334G12S3A1F4 SEQ ID NO.188SEQ ID NO.188 6767 C382F4FGF1C382F4FGF1 SEQ ID NO.227SEQ ID NO.227
2929 C364G12S3A1F4C364G12S3A1F4 SEQ ID NO.189SEQ ID NO.189 6868 C382F4FGF2C382F4FGF2 SEQ ID NO.228SEQ ID NO.228
3030 C209G12S3A1F4C209G12S3A1F4 SEQ ID NO.190SEQ ID NO.190 6969 C382F4FGF3C382F4FGF3 SEQ ID NO.229SEQ ID NO.229
3131 C289G12S3A1F4C289G12S3A1F4 SEQ ID NO.191SEQ ID NO.191 7070 C495F4FGFC495F4FGF SEQ ID NO.230SEQ ID NO.230
3232 CG283G12S3A1F4CG283G12S3A1F4 SEQ ID NO.192SEQ ID NO.192 7171 C495F4FGF1C495F4FGF1 SEQ ID NO.231SEQ ID NO.231
3333 CG214G12S3A1F4CG214G12S3A1F4 SEQ ID NO.193SEQ ID NO.193 7272 C495F4FGF2C495F4FGF2 SEQ ID NO.232SEQ ID NO.232
3434 CG267G12S3A1F4CG267G12S3A1F4 SEQ ID NO.194SEQ ID NO.194 7373 C495F4FGF3C495F4FGF3 SEQ ID NO.233SEQ ID NO.233
3535 C308G12S3A1F4C308G12S3A1F4 SEQ ID NO.195SEQ ID NO.195 7474 C382F4LepC382F4Lep SEQ ID NO.234SEQ ID NO.234
3636 C224G12S3A1F4C224G12S3A1F4 SEQ ID NO.196SEQ ID NO.196 7575 C495F4LepC495F4Lep SEQ ID NO.235SEQ ID NO.235
3737 CG308G12S3A1F4CG308G12S3A1F4 SEQ ID NO.197SEQ ID NO.197 7676 C462F4LepC462F4Lep SEQ ID NO.236SEQ ID NO.236
3838 C319G12S3A1F4C319G12S3A1F4 SEQ ID NO.198SEQ ID NO.198 7777 C364F4LepC364F4Lep SEQ ID NO.237SEQ ID NO.237
3939 C214G12S3A1F4C214G12S3A1F4 SEQ ID NO.199SEQ ID NO.199 7878 C289F4LepC289F4Lep SEQ ID NO.238SEQ ID NO.238
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，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。The above is only a preferred embodiment of the present invention, and is not intended to limit the scope of the present invention. It should be noted that those skilled in the art will also A number of improvements and additions may be made which are also considered to be within the scope of the invention. All of the equivalents of the changes, modifications, and evolutions that can be made by the above-disclosed technical content are all those skilled in the art without departing from the spirit and scope of the present invention. Equivalent embodiments; at the same time, any changes, modifications and evolutions of any equivalent changes made to the above-described embodiments in accordance with the essential techniques of the present invention are still within the scope of the technical solutions of the present invention.

Claims (19)

  1. 一种多重活性蛋白，所述多重活蛋白的结构中包括如式I所示结构，如式I所示结构为：A-L-F，式I中，A为GCGR/GLP-1R双效激动剂，F为长效蛋白单元，L为连接所述A与F的连接链。A multiplex active protein comprising a structure as shown in Formula I, wherein the structure of Formula I is: ALF, in Formula I, A is a GCGR/GLP-1R double-acting agonist, and F is Long acting protein unit, L is the linking chain connecting the A and F.
  2. 根据权利要求1所述的多重活性蛋白，其特征在于，所述A的结构中包括如式II所示结构，如式II所示结构为：HSQGTFTSDYSKYLD X 16X 17X 18 AQDFVQWLMN X 29 X z式II，其中，其中，X 16选自除Y、N、W、和H外的氨基酸之任一；X 17选自除P、L、T、F和H外的氨基酸之任一；X 18除选自P、F、H和W外的氨基酸之任一；另外X 17与X 18不能同时为R，X 29为T或缺失，X z选自GGPSSGAPPPS、GPSSGAPPPS、PSSGAPPPS、SSGAPPPS、GGPSSGAPPS、GPSSGAPPS、PSSGAPPS或SSGAPPS之任一。 The multiplex active protein according to claim 1, wherein the structure of A includes a structure represented by Formula II, and the structure shown in Formula II is: HSQGTFTSDYSKYLD X 16 X 17 X 18 AQDFVQWLMN X 29 X z wherein, wherein any one selected II X 16 in addition to Y, N, W, and H of an amino acid; X 17 is selected from any one of amino acid except P, L, T, F and H a; X 18 except Any one of amino acids selected from P, F, H and W; in addition, X 17 and X 18 cannot be R at the same time, X 29 is T or missing, and X z is selected from GGPSSGAPPPS, GPSSGAPPPS, PSSGAPPPS, SSGAPPPS, GGPSSGAPPS, GPSSGAPPS, Any of PSSGAPPS or SSGAPPS.
  3. 根据权利要求1所述的多重活性蛋白，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)所述L为富含G、S和/或A的连接链。 The multiplex active protein according to claim 1, further comprising any one or more of the following features: (1) said F is a F C moiety derived from a mammalian immunoglobulin; (2) L is a linking chain rich in G, S and/or A.
  4. 根据权利要求1所述的多重活性蛋白，其特征在于，还包括以下特征的任一项或多项：(1)所述A的氨基酸酸序列如SEQ ID NO.46、SEQ ID NO.55、SEQ ID NO.59、SEQ ID NO.68、SEQ ID NO.74之任一所示；(2)所述F的氨基酸序列如SEQ ID NO.11～20之任一所示；(3)所述L的氨基酸序列如SEQ ID NO.21～43之任一所示。The multiplex active protein according to claim 1, further comprising any one or more of the following features: (1) the amino acid sequence of said A is SEQ ID NO. 46, SEQ ID NO. SEQ ID NO. 59, SEQ ID NO. 68, SEQ ID NO. 74; (2) the amino acid sequence of F is as shown in any one of SEQ ID NO. 11-20; (3) The amino acid sequence of L is as shown in any one of SEQ ID NOS. 21 to 43.
  5. 根据权利要求1所述的多重活性蛋白，其特征在于，所述多重活性蛋白的氨基酸序列如SEQ ID NO.87、SEQ ID NO.89、SEQ ID NO.100之任一所示。The multiplex active protein according to claim 1, wherein the multiplexed active protein has an amino acid sequence as shown in any one of SEQ ID NO. 87, SEQ ID NO. 89, and SEQ ID NO.
  6. 一种多重活性蛋白，所述多重活蛋白的结构中包括如式III所示结构，如式III所示结构为：A-L 1-F-L 2-B，其中，A为GCGR/GLP-1R双效激动剂，F为长效蛋白单元，B为天然FGF21或FGF21类似物，L 1为连接A与F的连接链，L 2不存在或为连接B与F的连接链。 A multiplex active protein comprising a structure as shown in Formula III, wherein the structure shown in Formula III is: AL 1 -FL 2 -B, wherein A is a GCGR/GLP-1R double-acting agonist Agent, F is a long-acting protein unit, B is a natural FGF21 or FGF21 analog, L 1 is a linking chain connecting A and F, L 2 is absent or is a linking chain connecting B and F.
  7. 根据权利要求6所述的多重活性蛋白，其特征在于，所述A的结构中包括如式II所示结构，如式II所示结构为：HSQGTFTSDYSKYLD X 16X 17X 18 AQDFVQWLMN X 29 X z式II，其中，X 16选自除Y、N、W、和H外的氨基酸之任一；X 17选自除P、L、T、F和H外的氨基酸之任一；X 18选自除P、F、H和W外的氨基酸之任一；另外X 17与X 18不能同时为R，X 29为T或缺失，X z选自GGPSSGAPPPS、GPSSGAPPPS、PSSGAPPPS、SSGAPPPS、GGPSSGAPPS、GPSSGAPPS、PSSGAPPS或SSGAPPS之任一。 The multiplex active protein according to claim 6, wherein the structure of A includes a structure represented by Formula II, and the structure shown in Formula II is: HSQGTFTSDYSKYLD X 16 X 17 X 18 AQDFVQWLMN X 29 X z II, wherein X 16 is selected from any one of amino acids other than Y, N, W, and H; X 17 is selected from any one of amino acids other than P, L, T, F, and H; X 18 is selected from Any of the amino acids other than P, F, H and W; in addition, X 17 and X 18 cannot be R at the same time, X 29 is T or missing, and X z is selected from GGPSSGAPPPS, GPSSGAPPPS, PSSGAPPPS, SSGAPPPS, GGPSSGAPPS, GPSSGAPPS, PSSGAPPS or Any of SSGAPPS.
  8. 根据权利要求6所述的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)所述L 1为富含G、S和/或A的连接链；(3)所述L 2为富含G、S和/或A的连接链。 The method according to claim 6 further comprising any one or more of the following features: (1) said F is a F C moiety derived from a mammalian immunoglobulin; (2) said L 1 is rich in G, a linking chain of S and/or A; (3) said L 2 is a linking chain rich in G, S and/or A.
  9. 根据权利要求6所述的还包括以下特征的任一项或多项：The method of claim 6 further comprising any one or more of the following features:
    (1)所述A的氨基酸酸序列如SEQ ID NO.46、SEQ ID NO.54、SEQ ID NO.55、SEQ ID NO.68之任一所示；(2)所述F的氨基酸序列如SEQ ID NO.11～20之任一所示；(3)所述L 1的氨基酸序列如SEQ ID NO.21～43之任一所示；(4)所述L 2的氨基酸序列如SEQ ID NO.21～43之任一所示；(5)所述B氨基酸序列如SEQ ID NO.143-146、SEQ ID NO.155之任一所示。 (1) The amino acid sequence of the A is as shown in any one of SEQ ID NO. 46, SEQ ID NO. 54, SEQ ID NO. 55, and SEQ ID NO. 68; (2) the amino acid sequence of the F is as SEQ ID NO. 11 to 20; (3) the amino acid sequence of L 1 is as shown in any one of SEQ ID NOS. 21 to 43; (4) the amino acid sequence of L 2 is SEQ ID Any one of NO. 21 to 43; (5) the B amino acid sequence is as shown in any one of SEQ ID NO. 143-146 and SEQ ID NO.
  10. 根据权利要求6所述的还包括以下特征的任一项或多项：所述多重活性蛋白的氨基酸序列如SEQ ID NO.147-154或SEQ ID NO.156-160之任一所示。Further, according to claim 6, further comprising any one or more of the following features: the amino acid sequence of said multiplex active protein is as set forth in any one of SEQ ID NO. 147-154 or SEQ ID NO. 156-160.
  11. 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1-10之任一项所述多重活性蛋白。An isolated polynucleotide encoding the multiplex active protein of any one of claims 1-10.
  12. 一种重组表达载体，包含如权利要求11所述分离的多核苷酸。A recombinant expression vector comprising the polynucleotide isolated according to claim 11.
  13. 一种宿主细胞，所述细胞含有如权利要求12所述重组表达载体或基因组中整合有外源的如权利要求11所述分离的多核苷酸。A host cell comprising the recombinant expression vector of claim 12 or a polynucleotide isolated according to claim 11 integrated in the genome.
  14. 如权利要求1-10之任一项所述多重活性蛋白的制备方法，其特征在于，A method for producing a multiplex active protein according to any one of claims 1 to 10, characterized in that
    在合适的条件下培养如权利要求13所述宿主细胞，使之表达所述多重活性蛋白，而后分离及纯化获得所述多重活性蛋白。The host cell of claim 13 is cultured under suitable conditions to express the multiplex active protein, followed by isolation and purification to obtain the multiplex active protein.
  15. 如权利要求1-10之任一项所述多重活性蛋白在制备治疗代谢相关疾病的药物中的用途。Use of the multiplex active protein according to any one of claims 1 to 10 for the preparation of a medicament for treating a metabolic related disease.
  16. 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1-10之任一项所述多重活性蛋白。A method of promoting weight loss or preventing weight gain, comprising administering a multiplex active protein according to any one of claims 1 to 10 in a subject.
  17. 一种组合物，含有如权利要求1-10之任一项所述多重活性蛋白或如权利要求13所述宿主细胞的培养物，以及药学上可接受的载体。A composition comprising a multiplex active protein according to any one of claims 1 to 10 or a culture of a host cell according to claim 13, and a pharmaceutically acceptable carrier.
  18. 如权利要求1-10之任一项所述多重活性蛋白在制备融合蛋白中的用途。Use of the multiplex active protein according to any one of claims 1 to 10 for the preparation of a fusion protein.
  19. 一种融合蛋白，其结构中含有如权利要求1-10之任一项所述多重活性蛋白。A fusion protein comprising the multiplex active protein of any one of claims 1-10.
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