WO2020063628A1 - GLP1-Fc融合蛋白及其缀合物 - Google Patents

GLP1-Fc融合蛋白及其缀合物 Download PDF

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WO2020063628A1
WO2020063628A1 PCT/CN2019/107709 CN2019107709W WO2020063628A1 WO 2020063628 A1 WO2020063628 A1 WO 2020063628A1 CN 2019107709 W CN2019107709 W CN 2019107709W WO 2020063628 A1 WO2020063628 A1 WO 2020063628A1
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Prior art keywords
glp
fusion protein
seq
diabetes
glp1
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PCT/CN2019/107709
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English (en)
French (fr)
Inventor
王亚里
陈宪
朱鹿燕
刘宾
王晓山
任子甲
周婷婷
阎海霞
徐莺莺
高慧慧
王锦
徐阳
刘亚辉
莫炜川
陈昕
高洁
苏鸿声
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北京辅仁瑞辉生物医药研究院有限公司
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Priority to AU2019349201A priority Critical patent/AU2019349201B2/en
Priority to JP2021517699A priority patent/JP7174149B2/ja
Priority to KR1020217011644A priority patent/KR102649941B1/ko
Priority to MX2021003349A priority patent/MX2021003349A/es
Priority to US17/279,174 priority patent/US20220000984A1/en
Priority to CA3113738A priority patent/CA3113738A1/en
Priority to EP19866716.4A priority patent/EP3858866A4/en
Priority to EA202190744A priority patent/EA202190744A1/ru
Priority to BR112021005419-7A priority patent/BR112021005419A2/pt
Publication of WO2020063628A1 publication Critical patent/WO2020063628A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/605—Glucagons
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/61—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule the organic macromolecular compound being a polysaccharide or a derivative thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
    • 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
    • A61P3/08—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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/90—Fusion polypeptide containing a motif for post-translational modification
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/90—Fusion polypeptide containing a motif for post-translational modification
    • C07K2319/915—Fusion polypeptide containing a motif for post-translational modification containing a motif for acylation

Definitions

  • the present invention relates to a biologically active polypeptide fusion protein that prolongs the half-life in vivo and its conjugate with a polymer, in particular a GLP-1 receptor agonist that prolongs the half-life in vivo, a pharmaceutical composition containing them, and a For the treatment of diabetes mellitus, especially type II diabetes) and for weight control.
  • type 1 diabetes insulin-dependent diabetes mellitus
  • type 2 diabetes non-insulin-dependent diabetes mellitus
  • type 2 diabetes accounts for more than 90% of diabetic patients.
  • Type II diabetes is characterized by insulin secretion or dysfunction and ⁇ -cell dysfunction, which causes fat, carbohydrate, and protein metabolism disorders, resulting in chronic hyperglycemia, and eventually leads to various microvascular, macrovascular, and various organ complications.
  • insulin secretion-promoting drugs such as sulfonylureas, meglitinides, dipeptidyl peptidase inhibitors, and GLP-1 analogs
  • non-insulin-promoting drugs Such as insulin, ⁇ -glucosidase inhibitors, biguanides, thiazolidinediones, insulin analogs and the like.
  • insulin secretion-promoting drugs such as sulfonylureas, meglitinides, dipeptidyl peptidase inhibitors, and GLP-1 analogs
  • non-insulin-promoting drugs Such as insulin, ⁇ -glucosidase inhibitors, biguanides, thiazolidinediones, insulin analogs and the like.
  • HbA1c glycated hemoglobin
  • GLP-1 enteric hormone Glucagon-like peptide-1
  • GLP-1 has the following functions: acting on islet ⁇ -cells in a glucose-dependent manner, promoting the transcription of insulin genes, increasing insulin biosynthesis and secretion; stimulating the proliferation and differentiation of ⁇ -cells, inhibiting ⁇ -cell apoptosis and thereby increasing islets ⁇ -cell number; Inhibition of glucagon secretion; Increased sensitivity of peripheral cell insulin receptors; Reduce HbA1c; Inhibit appetite and food intake; Delay gastric emptying (Diabetic Med, 18, 144-149, 2001; Diabetes, 51 , 1443-1452, 2002; Diabetologia, 45, 1263-1273, 2002; Diabetes, 50, 525-529, 2001; Diabetes, 50, 725, 2001; Diabetes, 52, 365-371, 2003; Recent Prog.
  • GLP-1 is easily degraded by dipeptidyl peptidase (DPPIV) in vivo, and its half-life is less than 2 minutes, which makes it almost impossible to become an effective anti-diabetic drug.
  • DPPIV dipeptidyl peptidase
  • peptide drugs generally have short half-life in vivo, poor physical and chemical stability, and are easily degraded by various proteases in the body, these drugs usually require multiple injections within a day. It imposes a greater burden on the patient's physical, mental, and economic, and limits patient compliance with medication. Therefore, there is an urgent need in the art for drugs with a new structure, thereby prolonging their plasma cycle and increasing their systemic drug exposure.
  • the inventors found that the polypeptide (protein) is fused with a fusion partner (such as an Fc fragment, albumin, XTEN or transferrin) that can extend the half-life, and further with a hydrophilic polymer (such as poly Alkyl glycols, such as PEG), can effectively improve the stability of biologically active polypeptides in vivo and improve the efficacy of drugs, thus completing the present invention.
  • a fusion partner such as an Fc fragment, albumin, XTEN or transferrin
  • a hydrophilic polymer such as poly Alkyl glycols, such as PEG
  • the present invention provides a fusion protein of a GLP-1 receptor agonist, which comprises a GLP-1 receptor agonist and a fusion partner (FP) that increases the half-life in vivo connected thereto; wherein said The GLP-1 receptor agonist is directly connected to the fusion partner or connected through a first linker L1; preferably, the first linker L1 comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 , 10 or more amino acid peptides; more preferably, the first linker L1 is a flexible peptide containing A, T, G, and / or S, such as (GS) n, where n is 1-50 Integers such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;
  • the first joint L1 is selected from:
  • PRFQDSSSSKAPPPSLPSPSRLPGPSDTPILPQ (SEQ ID NO: 14);
  • SSSSKAPPPS (SEQ ID NO: 16);
  • the GLP-1 receptor agonist is a full-length, truncated, or variant form of GLP-1 or a GLP-1 analog, such as Exendin-4, GLP-1 (1-36 ), GLP-1 (1-37), GLP-1 (7-36), GLP-1 (7-37), liraglutide, lixisenatide, aprilide, doxyl, or solanum Marupide.
  • GLP-1 receptor agonist is a full-length, truncated, or variant form of GLP-1 or a GLP-1 analog, such as Exendin-4, GLP-1 (1-36 ), GLP-1 (1-37), GLP-1 (7-36), GLP-1 (7-37), liraglutide, lixisenatide, aprilide, doxyl, or solanum Marupide.
  • the Fc segment is human An immunoglobulin Fc segment; preferably, the immunoglobulin Fc segment is composed of one to four domains selected from the group consisting of a CH1 domain, a CH2 domain, a CH3 domain, and a CH4 domain; also preferably, the The immunoglobulin Fc segment is an Fc segment from IgG, IgA, IgD, IgE or IgM, more preferably an IgG Fc segment; further preferably, the Fc segment is from IgG1, IgG2, IgG3 or IgG4 Fc segment; It is also preferred that the IgG Fc segment has reduced ADCC effect and / or CDC effect and / or enhanced binding affinity to the FcRn receptor.
  • the fusion partner is further linked directly to a stretch of amino acid sequence ST containing a recognition site for Sortase enzyme, or is linked through a second linker L2, such as Sortase A or Sortase B;
  • the ST comprises the core recognition site LPXTG of Sortase A, wherein X is any amino acid, such as LPETG, LPETGG or LPETGGG;
  • the ST further comprises an affinity tag linked to the Sortase enzyme recognition sequence, such that The ST sequence is, for example, LPETGGHHHHHH or LPETGGWSHPQFEK;
  • the second linker L2 is a peptide containing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acids; more preferably, the second linker L2 is Flexible peptides containing A, T, G, and / or S, such as (GS) n where n is an integer from 1 to 50, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;
  • the second linker L2 is selected from:
  • the fusion protein has the structure: GLP-1-L1-FP-L2-ST, wherein
  • GLP-1 represents a GLP-1 receptor agonist
  • GLP-1 receptor agonist, L1, L2, FP, and ST have the same definitions as in the previous embodiment, wherein either or both of L1 and L2 may be absent .
  • the amino acid sequence of the fusion protein is shown in SEQ ID NO: 1, 3, 5, 7, or 22; or the fusion protein is represented by SEQ ID NO: 2, 4, 6, 8, or 23
  • the nucleic acid sequence shown below is encoded.
  • a conjugate in which one, two or more hydrophilic polymer molecules are attached to the terminus of the fusion protein in the first aspect, preferably via transfection
  • a peptide reaction is linked to the sortase enzyme recognition site; in particular, the fusion protein may be in the form of a monomer or a dimer.
  • the third aspect of the present invention provides a method for preparing the conjugate according to the second aspect, which comprises combining the fusion protein described in the first aspect with a Sortase enzyme (such as Sortase A or Sortase B) and a hydrophilic A polymer molecule is contacted, wherein the hydrophilic polymer molecule bears an amino group at one end that can be amidated by Sortase.
  • a Sortase enzyme such as Sortase A or Sortase B
  • hydrophilic A polymer molecule bears an amino group at one end that can be amidated by Sortase.
  • hydrophilic polymer molecule is provided with polyGly at one end, for example with GGGAA.
  • a fourth aspect of the invention provides a pharmaceutical composition comprising an effective amount of the fusion protein in the first aspect and / or the conjugate described in the second aspect, and optionally a pharmaceutically acceptable Accepted vectors.
  • the pharmaceutical composition is used to reduce blood sugar or body weight, for example for the treatment of diabetes, more specifically for the treatment of type I diabetes and / or type II diabetes, and in particular for the treatment of type II diabetes.
  • the pharmaceutical composition is in the form of a liquid formulation or a lyophilized formulation.
  • a fifth aspect of the present invention provides the use of the fusion protein according to the first aspect or the conjugate according to the second aspect in the preparation of a medicament for reducing blood sugar or body weight, for example, the medicament is used for treatment Diabetes, including type 1 diabetes and type 2 diabetes, especially type 2 diabetes.
  • a sixth aspect of the present invention provides a method for reducing blood sugar or body weight, which comprises administering to the subject in need thereof the fusion protein described in the first aspect or the conjugate described in the second aspect, for example, with a For the treatment of diabetes, including type 1 diabetes and type 2 diabetes, especially type 2 diabetes.
  • a seventh aspect of the present invention provides a kit comprising the fusion protein according to the first aspect, the conjugate according to the second aspect, and / or the pharmaceutical composition according to the fourth aspect. , And optional instruction manuals.
  • the GLP-1-Fc conjugate of the present invention not only can provide a longer-term hypoglycemic effect, but also can significantly reduce the rapid weight loss caused by taking hypoglycemic drugs compared with existing similar drugs, Therefore, the clinical safety of such drugs is greatly improved, and therefore has great clinical application value.
  • Figure 1A shows the structural diagram of the eukaryotic expression vector of GLP-1 fusion protein
  • Figure 1B shows the growth curve and cells of GLP1-L1-Fc2-L2-ST stable cell line (clone number: P1-3G3C2) during fermentation Liveness test results.
  • Figure 2 shows the results of microscopic examination of GLP1-L1-Fc2-L2-ST stable cell lines (clone number: P1-3G3C2) at different time points during the fermentation process.
  • Fig. 2A shows the cells at the 5th day of culture
  • Fig. 2B shows the cells at the time of sample collection (day 13).
  • Figure 3 shows the glucose metabolism in the culture supernatant of GLP1-L1-Fc2-L2-ST stable cell line (clone number: P1-3G3C2) during fermentation.
  • Figure 4 shows the NH4 + and lactose (Lac) metabolism in the culture supernatant of GLP1-L1-Fc2-L2-ST stable cell line (clone number: P1-3G3C2) during fermentation.
  • Figure 5 shows the non-reduced (M / 1/2/3 on the left) and reduced (4/5/6 / M on the right) electrophoresis results of GLP1-L1-Fc2-L2-ST protein (1 and 6: terminal fermentation Liquid supernatant; 3 and 4 are purified samples, 2 and 5 are fermentation broth samples on day 10).
  • FIG. 6 shows the results of SDS electrophoresis (A) and SEC analysis (B) of the purified GLP1-L1-Fc2-L2-ST protein.
  • Figure 7 shows the detection results of cross-linking rate of two proteins by SDS electrophoresis and RPC analysis.
  • a and C are SDS detection of GLP1-L1-Fc2-L2-ST protein cross-linking 20kDa and 40kDa PEG;
  • B and D are RPC detection of GLP1-L1-Fc2-L2-ST protein cross-linking 20kDa and 40kDa PEG,
  • E RPC detection of GLP1-L1-Fc2-ST protein cross-linking 40 kDa PEG.
  • Figure 8 shows the anion exchange chromatogram (A) and RPC detection (B) results of the sample after GLP1-L1-Fc2-L2-ST cross-linking.
  • Figure 9 shows the results of the activity detection of GLP1-L1-Fc2-L2-ST after cross-linking with 20 kDa PEG and 40 kDa PEG, respectively.
  • the relative activity of the uncrosslinked substrate (GLP1-Fc) was 98.10%.
  • the activity decreased.
  • the relative activity of the 20 kDa cross-linked product (PEG (20 kDa) -GLP1-Fc) was 40.38%; the relative activity of the 40 kDa cross-linked product (PEG (40 kDa) -GLP1-Fc) was 32.85%.
  • Figure 10A shows the glucose tolerance test curve of C57BL / 6 mice after 24 hours of administration
  • Figure 10B shows the effect of glucose tolerance (AUC) on C57BL / 6 mice at 24 hours after administration
  • Figure 10D shows the glucose tolerance test curve after administration of 144h
  • Figure 10D shows the effect of 144h after administration on glucose tolerance (AUC) of C57BL / 6 mice
  • Figure 10E shows the glucose tolerance test curve of C57BL / 6 mice after 168h of administration
  • Figure 10F Shows the effect of 168h on glucose tolerance (AUC) in C57BL / 6 mice after administration
  • Figure 10G shows the glucose tolerance test curve for C57BL / 6 mice after 192h administration
  • Figure 10H shows the C57BL / 6 mice after 192h administration.
  • FIG. 10I shows the glucose tolerance test curve of C57BL / 6 mice after 216 h administration
  • Figure 10J effect of 216 h on glucose tolerance (AUC) of C57BL / 6 mice after administration
  • Figure 10K Shows the glucose tolerance test curve of C57BL / 6 mice after 240 h administration
  • Figure 10L shows the effect of 240 hours after administration on glucose tolerance (AUC) of C57BL / 6 mice
  • Fig. 10M shows C57BL / 6 mice after administration Weight change.
  • Figure 11 shows the growth curve and cell viability results of GLP1-L1-Fc4-L2-STG3 stable cell line (clone number: P1-P132C7) during fed-batch fermentation.
  • FIG. 12 shows the results of microscopic examination of GLP1-L1-Fc4-L2-STG3 stable cell line (clone number: P1-P132C7) at different time points during fed-batch fermentation.
  • Fig. 12A shows the cells at the 5th day of culture
  • Fig. 12B shows the cells at the time of sample collection (day 13).
  • Figure 13 shows the glucose metabolism in the culture supernatant of GLP1-L1-Fc4-L2-STG3 stable cell line (clone number: P1-P132C7) during fed-batch fermentation.
  • FIG. 14 shows the pH change trend of a GLP1-L1-Fc4-L2-STG3 stable cell line (clone number: P1-P132C7) during a fed-batch fermentation in a 15L bioreactor.
  • Figure 15 shows the SDS-PAGE electrophoresis diagram of the target protein of the 15L fed-batch fermentation supernatant of the GLP1-L1-Fc4-L2-STG3 protein.
  • Channel 1 is the purified sample
  • channel 2 is the protein marker
  • channels 3 and 4 And 5 are the fermentation broth supernatants on days 12, 13, and 14, respectively.
  • FIG. 16 shows the results of SDS electrophoresis (A) and SEC analysis (B) of the purified GLP1-L1-Fc4-L2-STG3 protein.
  • Figure 17 shows the results of the detection of the cross-linking rate of the two proteins by SDS electrophoresis and RPC analysis.
  • A is SDS detection of GLP1-L1-Fc4-L2-STG3 protein cross-linking 40 kDa and PEG;
  • B is RPC detection of GLP1-L1-Fc4-L2-STG3 protein cross-linking 40 kDa PEG.
  • FIG. 18 shows anion exchange chromatogram (A) and SEC detection (B, C) results of the sample after GLP1-L1-Fc4-L2-STG3 cross-linking.
  • Figure 19 shows the results of the activity detection of GLP1-L1-Fc4-L2-STG3 after crosslinking with 40 kDa PEG.
  • the relative activity of the uncrosslinked substrate (GLP1-Fc) was 112.24%.
  • the activity decreased.
  • the relative activity of the 40kDa cross-linked GLP1-L1-Fc4-L2-STG3 product (PEG (40kDa) -GLP1-Fc) was 21.51%.
  • Figure 20A shows the effect on glucose tolerance (AUC) of C57BL / 6 mice 1-8 days after dosing;
  • Figure 20B shows the weight change of C57BL / 6 mice after dosing.
  • Figure 21A shows the effect on blood glucose of type 2 diabetic mice induced by STZ 1-12 days after dosing
  • Figure 21B shows the weight change of type 2 diabetic mice after dosing.
  • amino acid as used herein includes natural amino acids, unnatural amino acids, and amino acid analogs and all of their D and L stereoisomers.
  • Unnatural amino acids include, but are not limited to, azetidine carboxylic acid, 2-aminoadipate, 3-aminoadipate, ⁇ -alanine, aminopropionic acid, 2-aminobutyric acid, 4-aminobutyric acid , 6-aminocaproic acid, 2-aminoheptanoic acid, 2-aminoisobutyric acid, 3-aminoisobutyric acid, 2-aminopimelate, tert-butylglycine, 2,4-diaminoisobutyric acid, 2 , 2'-diaminopimelate, 2,3-diaminopropionic acid, N-ethylglycine, N-ethylasparagine, homoproline, hydroxylysine, all-hydroxylysine, 3-hydroxyproline, 4-hydroxyproline,
  • Amino acid analogs include natural and unnatural amino acids that are reversibly or irreversibly chemically blocked at their C-terminal carboxyl group, N-terminal amino group or side chain group, or chemically modified to another functional group, such as methionine Sulfone, methionine sulfone, S- (carboxymethyl) -cysteine, S- (carboxymethyl) -cysteine sulfoxide and S- (carboxymethyl) -cysteine sulfone.
  • polypeptide or “protein” as used herein interchangeably refer to a series of at least two amino acid residues connected to each other by a covalent bond (eg, a peptide bond), and may be a recombinant polypeptide, a natural polypeptide, or a synthetic polypeptide.
  • the "polypeptide” or “protein” according to the present invention also includes variants or analogues thereof, that is, the amino acid sequence is changed by one or more substitutions, deletions, insertions, fusions, truncations, or any combination thereof. Different peptides.
  • a variant polypeptide may be fully functional or may lack one or more active functions.
  • Fully functional variants may contain, for example, only conservative changes or changes in non-critical residues or non-critical regions. Functional variants may also include substitutions of similar amino acids, which result in unchanged or insignificant changes in function. Amino acids that are important for function can be identified by methods known in the art, such as site-directed mutagenesis or glycine scanning mutagenesis (Cunningham, B. and Wells, J., Science, 244: 1081-1085, 1989) . Sites critical for polypeptide activity can be determined, for example, by structural analysis such as crystallization, nuclear magnetic resonance, or photoaffinity labeling (Smith, L. et al., J. Mol. Biol., 224: 899-904, 1992; de Vos A. et al. Science, 255: 306-312, 1992).
  • conjugate refers to the product formed by a polypeptide or a polypeptide variant covalently or non-covalently linked to a modifying group described herein.
  • polymer used herein has the meaning commonly understood by those of ordinary skill in the art, and includes both the polymer itself and its terminally modified derivatives, unless explicitly stated otherwise.
  • the average molecular weight is generally used to represent the molecular weight of the polymer, and specifically, it can be a number average molecular weight or a weight average molecular weight. Although there may be some deviations between the number average molecular weight and the weight average molecular weight when there is a large difference in the degree of polymerization of the polymers, for polymers with a narrower distribution range, the two tend to be equal.
  • the polymers mentioned herein, such as polyethylene glycol when referring to its molecular weight, it can be either a weight average molecular weight or a number average molecular weight.
  • Glucagon-like peptides including glucagon-like peptide-1 (GLP-1) and glucagon-like peptide-2 (GLP-2), both of which are derived from glucagon Proglucagon, a proglucagon consisting of 158 amino acids, can be cut into different peptide chains, because GLP-1 has the ability to promote insulin secretion, protect islet ⁇ cells, inhibit glucagon secretion, and inhibit gastric emptying
  • GLP-1 has the ability to promote insulin secretion, protect islet ⁇ cells, inhibit glucagon secretion, and inhibit gastric emptying
  • the pharmacological effect of reducing appetite can be used clinically for the treatment of type 2 diabetes and obesity;
  • GLP-2 is a nutritional factor that can promote the growth of the small intestine, inhibit apoptosis, promote gastric emptying, and increase the pharmacological effect of appetite.
  • the biologically active GLP-1 in the human body is mainly GLP-1 (7-36) amide and GLP-1 (7-37).
  • Natural GLP-1 is easily hydrolyzed by dipeptidyl peptidase IV (DPP-IV). (Half life is less than 5min), so it is difficult to use in clinical applications.
  • GLP-1 receptor agonist refers to a molecule capable of activating GLP-1 receptor activity, such as naturally occurring GLP-1, which can be derived from different sources such as humans or animals, and also includes natural GLP-1 receptors.
  • Agonists such as GLP-1 and exendin-4 have one or more wild-type sequences (such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 17, 18, 19, 20 or more) functional variants, fragments and analogs formed from amino acid substitutions, deletions and / or added amino acid sequences, such as liraglutide, Risenatide, Abiluotide, Doxyl, Somalutide, etc.
  • GLP-1 receptor agonists such as GLP-1 and its functional variants and fragments
  • a fusion partner such as the Fc segment of human immunoglobulin
  • GLP-1 fusion protein a fusion protein having GLP-1 receptor agonist activity
  • GLP-1 fusion protein a fusion protein having GLP-1 receptor agonist activity
  • Human immunoglobulin IgG consists of four polypeptides (two identical copies of the light and heavy chains) covalently linked by disulfide bonds. Proteolytic IgG molecules by papain produce two Fab fragments and one Fc segment. The Fc segment consists of two polypeptides linked together by a disulfide bond. Each polypeptide, from N to C-terminus, consists of a hinge region, a CH2 domain, and a CH3 domain. The Fc segment structure is almost identical in all subtypes of human immunoglobulin. IgG is one of the most abundant proteins in human blood, which makes up 70 to 75% of the total immunoglobulins in human serum.
  • IgG immunoglobulins
  • Fusion regions of IgG with other proteins have been reported to form fusion proteins (see, for example, Capon et al., Nature, 337: 525-531, 1989; Chamow et al., Trends Biotechnol., 14: 52-60, 1996; U.S. Patent Nos. 5,116,964 and 5,541,087).
  • a typical fusion protein is a heavy dimeric protein, which is linked to the protein through cysteine residues in the IgG hinge chain region to form a molecule similar to IgG but lacking the CH1 region and light chain.
  • the Fc fusion protein Due to structural homology, the Fc fusion protein exhibits in vitro pharmacokinetic properties that are quite similar to those of the isotype of human IgG. Therefore, manufacturing a hGH fusion protein containing a link to the Fc region of a human IgG protein will help extend the circulating half-life of hGH and / or increase its biological activity.
  • the Fc region of an immunoglobulin is safe for use as a drug carrier because it is a biodegradable polypeptide that is metabolized in vivo.
  • the Fc region of an immunoglobulin has a relatively low molecular weight compared to the entire immunoglobulin molecule, so The preparation, purification, and production of the compounds are advantageous. Since the immunoglobulin Fc region does not contain Fab fragments (whose amino acid sequence differs depending on the antibody subclass and is therefore highly heterogeneous), it is expected that the immunoglobulin Fc region can greatly increase the homogeneity of the substance and have low antigenicity .
  • Fc region of an immunoglobulin refers to a heavy chain constant region 2 (CH2) and a heavy chain constant region 3 (CH3) that contain immunoglobulins, but not an immunoglobulin heavy chain and light chain.
  • the immunoglobulin Fc region of the present invention may contain a part of the Fc region containing the heavy chain constant region 1 (CH1) and / or the light chain constant region 1 (CL1) in addition to the variable regions of the heavy and light chains, or All, as long as it has a physiological function that is basically similar to or better than the natural protein. Further, it may be a fragment having a deletion in a relatively long portion of the amino acid sequence of CH2 and / or CH3.
  • the Fc region of the immunoglobulin of the present invention may include 1) a CH1 domain, a CH2 domain, a CH3 domain, and a CH4 domain; 2) a CH1 domain and a CH2 domain; 3) a CH1 domain and a CH3 domain; 4) CH2 domain and CH3 domain; 5) Combination of one or more domains with the immunoglobulin hinge region (or a part of the hinge region) and 6) Each structure of the constant region of the heavy and light chains Dimer of the domain.
  • the immunoglobulin Fc region of the present invention includes a natural amino acid sequence and a sequence derivative (mutant) thereof.
  • the amino acid sequence derivative has a sequence different from the natural amino acid sequence due to the deletion, insertion, non-conservative or conservative substitution of one or more amino acid residues, or a combination thereof.
  • amino acid residues known at positions 214 to 238, 297 to 299, 318 to 322, or 327 to 331 that are important for binding can be used as suitable targets for modification.
  • the most common replacements are Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Thy / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu and Asp / Gly can be used in both directions.
  • the Fc region can be modified by phosphorylation, sulfation, acrylate, glycosylation, methylation, farnesylation, acetylation, amidation, and the like.
  • the Fc derivative is a derivative having the same biological activity or improved structural stability (for example, structural stability to heat, pH, etc.) as the Fc region of the present invention.
  • these Fc regions can be obtained from natural forms isolated from humans and other animals including cattle, goats, pigs, mice, rabbits, hamsters, rats, and guinea pigs, or they can be derived from transformed animal cells or microorganisms Recombinant or derivative.
  • they can be obtained from natural immunoglobulins by isolating intact immunoglobulins from human or animal organisms and treating them with proteolytic enzymes. Papain digests natural immunoglobulins into Fab and Fc regions, while pepsin treatment results in the production of pFc 'and F (ab') 2 fragments.
  • these fragments can be subjected to size exclusion chromatography to isolate Fc or pFc '.
  • the immunoglobulin Fc region of the present invention may be a form having a natural sugar chain, an increased sugar chain compared to a natural form, or a reduced sugar chain compared to a natural form, or may be a deglycosylated form.
  • the increase, decrease, or removal of the immunoglobulin Fc sugar chain can be accomplished by methods commonly used in the art, such as chemical methods, enzymatic methods, and genetic engineering methods using microorganisms. Removal of the sugar chain from the Fc fragment results in a significantly reduced binding affinity to complement (C1q) and a reduction or loss of antibody-dependent cell-mediated cytotoxicity or complement-dependent cytotoxicity, thereby not inducing an unnecessary in vivo immune response.
  • the deglycosylated or unglycosylated form of the immunoglobulin Fc region may be more suitable for the purpose of the present invention for use as a medicament.
  • deglycosylation means the enzymatic removal of sugar moieties from the Fc region
  • unglycosylated means that the Fc region is in an aglycosylated form (e.g., in eukaryotic cells such as Produced in mammalian cells or in prokaryotes such as E. coli).
  • the immunoglobulin Fc region may be an Fc region derived from IgG, IgA, IgD, IgE, and IgM, or may be prepared by a combination or hybrid thereof.
  • it is derived from IgG or IgM (they are one of the most abundant proteins in human blood), and most preferably it is derived from IgG (which is known to extend the half-life of ligand binding proteins).
  • dimer or multimer may be formed of two or more fragments selected from the group consisting of an IgG fragment, an IgA fragment, an IgM fragment, an IgD fragment, and an IgE fragment.
  • the GLP-1 fusion protein of the invention is in the form of a homodimer.
  • the GLP-1 fusion protein is further linked to a fragment containing a recognition site for Sortase enzyme (ie, the "ST" fragment described in the present invention).
  • Sortase enzyme including Sortase A and Sortase B, was first discovered to have the function of anchoring bacterial surface proteins to the cell wall. It was found that the "sorting signal" of the surface protein consists of three key parts-the LPXTG sequence, the hydrophobic sequence, and a tail with a positively charged residue in most cases. The LPXTG sequence is very conserved. Can be recognized by Sortase.
  • cysteine (Cys) of the transpeptidase attacked as a nucleophilic group, acting on the peptide bond between the threonine and glycine of the C-terminal motif LPXTG of the substrate (such as a surface protein), causing it to cleave (Acylation), which in turn produces an acylase intermediate.
  • the polypeptide is then covalently attached to the cell wall or pilus subunit to function (deacylation).
  • Sortase A is used for site-directed coupling of PEG.
  • the core recognition sites LPETG, LPETGG, LPETGGG of Sortase A are used.
  • different affinity tags such as: LPETGGHHHHHH, LPETGGWSHPQFEK, etc. can be added after the recognition site.
  • the GLP-1 fusion protein of the invention is produced by recombinant methods, including, for example, expression in a suitable prokaryotic or eukaryotic host cell, and the GLP-1 fusion protein of the invention is then isolated therefrom by conventional techniques.
  • the nucleotide sequence encoding the peptide can be synthesized first by chemical synthesis, and then the sequence can be cloned into a suitable expression vector for expression under the control of a suitable promoter.
  • a mutagenesis method such as PCR mutagenesis can also be used to obtain the nucleotide sequence encoding GLP-1 from wild-type GLP-1, and then clone the sequence and the sequence of other elements for constructing the fusion protein to appropriate expression Expression in the vector is under the control of a suitable promoter.
  • Suitable eukaryotic host cells are mammalian cells, such as CHO, COS, HEK 293, BHK, SK-Hep and HepG2.
  • the cells are preferably grown under conditions suitable for expressing a GLP-1 fusion protein of the invention.
  • the reagents and conditions for producing or isolating the GLP-1 fusion protein of the present invention there are no particular restrictions, and any system known in the art or commercially available can be applied.
  • the GLP-1 fusion protein is obtained by methods that have been described in the art.
  • GLP-1 fusion proteins are available for the preparation of GLP-1 fusion proteins, which can be selected from eukaryotic and prokaryotic expression vectors.
  • Prokaryotic expression vectors can include, for example, plasmids such as pRSET, pET, and pBAD, and the like, among which promoters that can be used are, for example, lac, trc, trp, recA, or araBAD.
  • Eukaryotic expression vectors include: (i) vectors for expression in yeast such as pAO, pPIC, pYES, pMET, among which promoters such as AOX1, GAP, GAL1, AUG1, etc.
  • insects expressed in cells such as pMT, pAc [delta], plB, pMIB, pBAC, etc., where promoters such as PH, p10, MT, Ac5, OplE2, gp64, poh, etc.
  • the GLP-1 fusion protein is expressed in a prokaryotic or eukaryotic cell system and uses a codon-optimized coding sequence.
  • the sequence expressing the GLP-1 fusion protein comprises a leader peptide and / or a signal peptide, so as to facilitate the secretion of the GLP-1 fusion protein from the cell to the outside of the cell for isolation and purification.
  • the sequence expressing the GLP-1 fusion protein does not include a leader peptide and / or a signal peptide, which is not secreted outside the cell, and is isolated and purified by lysing the cell.
  • the GLP-1 protein in the fusion protein of the present invention, is directly connected to the Fc segment. In another embodiment, the GLP-1 protein and the Fc segment are connected through the first linker L1. Ligated; in yet another embodiment, the Fc segment is directly linked to the fragment containing the Sortase recognition sequence, and in another embodiment, the Fc segment is linked to the fragment containing the Sortase recognition sequence via a second linker L (2 )connected.
  • the first and / or second linker L1 and / or L2 are peptides containing one, two, or three or more different amino acids. More preferably, the first linker L1 and / or L2 is a flexible peptide containing A, T, G, and / or S, such as (GS) n, where n is an integer from 1 to 50, such as 1, 2, 3 , 4, 5, 6, 7, 8, 9 or 10;
  • the first joint L1 is selected from:
  • PRFQDSSSSKAPPPSLPSPSRLPGPSDTPILPQ (SEQ ID NO: 14);
  • SSSSKAPPPS (SEQ ID NO: 16);
  • the second linker L2 is selected from:
  • the GLP-1 fusion protein of the present invention can be conjugated with one or more polymer groups to form a GLP-1 fusion protein conjugate, wherein the polymer is conjugated to the terminus of the fusion protein, such as the N-terminus or C End.
  • a "polymer” is preferably physiologically acceptable, which includes being soluble in an aqueous solution or suspension, and administering the GLP-1 fusion protein conjugate in a pharmaceutically effective amount without adversely affecting mammals such as side effects. polymer.
  • the polymer that can be used in the present invention is not particularly limited.
  • the polymer usually preferably has 2 to about 3000 repeating units.
  • the polymer group may be selected from natural or synthetic polymers, examples of which include, but are not limited to, for example, polysaccharides, polyalkylene glycols, such as polyethylene glycol (PEG), polypropylene glycol (PPG), polyethylene oxide ( PEO), copolymers of ethylene glycol and propylene glycol, polyvinyl alcohol, etc., or any combination thereof.
  • polysaccharides such as polyethylene glycol (PEG), polypropylene glycol (PPG), polyethylene oxide ( PEO), copolymers of ethylene glycol and propylene glycol, polyvinyl alcohol, etc., or any combination thereof.
  • PEG polyethylene glycol
  • PPG polypropylene glycol
  • PEO polyethylene oxide
  • copolymers of ethylene glycol and propylene glycol polyvinyl alcohol, etc., or any combination thereof.
  • the GLP-1 fusion protein conjugate of the invention is conjugated using one or more PEG groups.
  • the polymer is not limited to a specific structure, and it may be linear (such as alkoxyPEG or bifunctional PEG), branched or multi-armed (such as branched PEG or linked to a polyol core) PEG), dendritic or may have degradable linkages.
  • the internal structure of the polymer can be organized in any number of different modes, which can be selected from the group consisting of homopolymers, alternating copolymers, random copolymers, block copolymers, alternating terpolymers, random terpolymers, and embedded polymers. Segment trimer and so on.
  • the polymers may also include poly (alkylene oxide) polymers, polymaleic acid, poly (D, L-alanine), and the like.
  • the polymer is polyethylene glycol (PEG) or a derivative thereof such as methoxy polyethylene glycol (mPEG).
  • PEG polyethylene glycol
  • the polyethylene glycol (PEG) includes both a type in which the terminal group is a hydroxyl group and another terminal in the terminal group.
  • the other groups include, but are not limited to, alkoxy, cycloalkoxy, cycloalkyloxy, alkenyl, aryloxy, or aralkyloxy.
  • PEG molecular types are all known in the art and are routinely used in polypeptide modification.
  • the PEG side chain can be linear, branched, branched, or composed of multiple arms. Different polyethylene glycols can have different polymer chain lengths and polymer structures.
  • the molecular weight of the PEG used in the present invention is not particularly limited, and its molecular weight range may be 0.1 to 200 kDa, for example, 1 to 150 kDa, 2 to 100 kDa, 3 to 80 kDa, or 4 to 50 kDa, or 5 to 40 kDa.
  • Other useful PEG molecules include, for example, those disclosed in WO 03/040211, US 6,566,506, US 6,864,350, and US 6,455,639.
  • the PEG has the general formula HO-CH2CH2O- (CH2CH2O) n-CH2CH2-OH, where n ranges from about 5 to 4000.
  • the PEG of the present invention includes PEG with other terminal groups, such as methoxy PEG, branched PEG, branched PEG, and the like.
  • Suitable branched PEGs can be prepared as described in US Patent No. 5,932,462, the entire disclosure of which is incorporated herein by reference.
  • the branched PEG refers to a PEG having a branch near a polymer chain end, and the main chain of the branched PEG may be linear or branched.
  • the invention provides a GLP-1 fusion protein conjugate having an extended biological half-life and significant GLP-1 receptor agonist activity.
  • conjugate is calculated The sum of the molecular weights of all conjugated polymer groups in the composition, unless otherwise specified.
  • the polymers used in the present invention are known in the art and can be obtained through a variety of routes, including, for example, through commercial routes, such as CarboMer, Inc., JTBaker, The Dow Chemical Company, etc., or according to Methods known in the art are self-made, for example as described in EP1245608.
  • the invention is not limited to polymers made by any particular method.
  • the conjugate can be isolated by a suitable method. Suitable methods include, for example, ultrafiltration, dialysis, or chromatography, which are within the capabilities of those of ordinary skill in the art.
  • the GLP-1 fusion protein or GLP-1 fusion protein conjugate of the present invention may have a variety of uses, including, for example, for lowering blood glucose.
  • the present invention also provides a pharmaceutical composition for lowering blood glucose, comprising a therapeutically effective amount of a fusion protein or conjugate of the present invention, and optionally a pharmaceutically acceptable carrier.
  • the pharmaceutical composition can be used for the treatment of diabetes, more preferably for the treatment of type I and / or type II diabetes, and particularly preferably for the treatment of type II diabetes.
  • the therapeutically effective amount of the fusion protein or conjugate of the present invention depends on the route of administration, the type of subject, and the physical characteristics of the particular mammal under consideration. These factors and their relationship to determining this amount are well known to those skilled in the art of medicine. This amount and method of administration can be adjusted for optimal efficacy to deliver the peptide to the subject, but will depend on factors well known to those skilled in the medical arts such as weight, diet, co-administration, and other factors.
  • the pharmaceutical composition of the present invention can be administered in a combination therapy, that is, in combination with one or more other agents, wherein the therapeutic agents are administered together, or sequentially.
  • the other agents may be administered before, during, or after the administration of one or more fusion proteins or conjugates of the invention, or a pharmaceutical composition thereof.
  • the other agents that can be used in the present invention include, for example, agents that lower blood sugar, such as insulin, insulin analogs, dextrin agonists, and cholecystokinin, and / or other compounds or compositions for treating diseases.
  • such a combination can achieve a combined, even synergistic effect.
  • pharmaceutically acceptable carrier As used herein, “pharmaceutically acceptable carrier”, “pharmaceutically acceptable carrier” or “physiologically acceptable carrier” is used interchangeably and includes any and all physiologically compatible salts, solvents, dispersion media, coatings , Antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like.
  • the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or perfusion).
  • the therapeutic agent may be coated with a material to protect the therapeutic agent from acids and other natural conditions that may inactivate the therapeutic agent.
  • the pharmaceutical formulation of the present invention is administered in a pharmaceutically acceptable amount in a pharmaceutically acceptable amount.
  • pharmaceutically acceptable means a non-toxic substance that does not interfere with the biologically active efficacy of the active ingredient.
  • Such formulations typically contain salts, buffers, preservatives, compatible carriers, and optionally other therapeutic agents, such as supplemental immune enhancers, including adjuvants, chemokines and cytokines.
  • the salts should be pharmaceutically acceptable, but non-pharmaceutically acceptable salts can be conveniently used to prepare their pharmaceutically acceptable salts, and they are not excluded from the scope of the present invention.
  • the GLP-1 fusion protein or GLP-1 fusion protein conjugate of the present invention may be combined with a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier refers to one or more compatible solid or liquid fillers, diluents or encapsulating substances, which are suitable for administration to mammals such as humans.
  • carrier means an organic or inorganic, natural or synthetic ingredient that is combined with an active ingredient to facilitate application.
  • the components of the pharmaceutical composition can also be blended in the absence of interactions that can significantly disrupt the efficacy of the desired drug.
  • the pharmaceutical composition of the present invention may comprise a buffer system, preferably the buffer system is an acetate buffer solution having a pH of about 3.0 to about 6.0, or a phosphate buffer solution having a pH of about 5.0 to about 9.0.
  • suitable buffering agents include acetate; citrate; borate; and phosphate.
  • the pharmaceutical composition may also contain suitable preservatives, such as: benzalkonium chloride; tert-butyl alcohol; parabens and thimerosal.
  • suitable preservatives such as: benzalkonium chloride; tert-butyl alcohol; parabens and thimerosal.
  • the pharmaceutical composition may conveniently be presented in unit dosage form and may be prepared by any method known in the pharmaceutical arts. All methods include the step of combining the active agent with a carrier that contains one or more accessory ingredients. Generally, the composition is prepared by closely combining the active compound with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product.
  • a pharmaceutical composition suitable for parenteral administration may be a sterile aqueous or non-aqueous formulation comprising one or more fusion proteins or conjugates.
  • the formulation is isotonic with the blood of the subject.
  • This formulation can be formulated according to known methods using suitable dispersing or wetting agents and suspending agents.
  • the sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, such as a solution in 1,3-butanediol.
  • the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution.
  • sterile, non-volatile oils are conventionally used as solvents or suspension media.
  • any mild, non-volatile oil can be used, including synthetic mono- or diglycerides.
  • fatty acids such as oleic acid can be used in injectable preparations.
  • Carrier formulations suitable for oral, subcutaneous, intravenous, intramuscular administration can be found in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA.
  • the fusion proteins or conjugates of the invention can be prepared with carriers that protect them from rapid release, such as controlled release formulations, including implants, transdermal patches, and microencapsulated delivery systems.
  • controlled release formulations including implants, transdermal patches, and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for preparing such formulations are known in the prior art, see, for example, Sustained Controlled Release Drug Delivery Systems, J.R. Robinson, ed., Marcel Dekker, Inc., New York, 1978 and the like.
  • the pharmaceutical composition of the present invention can be administered by any conventional route, including injection or gradual infusion over time.
  • the administration can be oral, intravenous, intraperitoneal, intramuscular, intraluminal, intratumoral, or transdermal.
  • the pharmaceutical composition of the present invention is administered in an effective amount.
  • An "effective amount” is the amount of any fusion protein or conjugate provided herein that, alone or in combination with a further dose and / or other therapeutic agent, produces a desired response (e.g., lowering blood glucose levels in a subject). This may include only temporarily slowing the development of diabetes, or in some embodiments, permanently stopping the development of diabetes.
  • the pharmaceutical composition used in the foregoing method is preferably sterile and contains an effective amount of a fusion protein or conjugate alone or in combination with another formulation in a weight unit or volume unit suitable for administration to a patient, To produce a desired response, such as a reduction in blood glucose.
  • the dosage of the fusion protein or conjugate to be administered to a subject can be selected according to different parameters, especially according to the mode of administration used and the condition of the subject. Other factors include the period of treatment required. If the response in the subject is insufficient at the initial dose applied, a higher dose (or an effective higher dose achieved by a different, more local delivery route) can be applied to the extent that patient tolerance allows.
  • the pharmaceutical composition of the invention comprises 0.20 mg / ml to 5 mg / ml GLP-1 fusion protein or / and comprises 4 mg / ml to 40 mg / ml GLP-1 fusion protein conjugate, preferably 0.20 mg / ml ⁇ 5mg / ml GLP-1 fusion protein and / or containing 4mg / ml ⁇ 40mg / ml GLP-1 fusion protein conjugate, more preferably 0.5mg / ml ⁇ 2mg / ml GLP-1 fusion protein and / Or it contains 10mg / ml ⁇ 20mg / ml GLP-1 fusion protein conjugate.
  • the dosage of the GLP-1 fusion protein or GLP-1 fusion protein conjugate of the present invention may range from about 10 ⁇ g / kg of patient weight to about 100,000 ⁇ g / kg of patient weight. In some embodiments, the dosage may range from about 0.1 mg / kg to about 20 mg / kg. In other embodiments, the dosage may range from about 0.1 mg / kg to 5 mg / kg, 0.1 mg / kg to 10 mg / kg, or 0.1 mg / kg to 15 mg / kg. In other embodiments, the dosage may range from about 1 mg / kg to 5 mg / kg, 5 mg / kg to 10 mg / kg, 10 mg / kg to 15 mg / kg, or 15 mg / kg to 20 mg / kg.
  • the dose is about 0.1 mg / kg, 0.5 mg / kg, 1 mg / kg, 2 mg / kg, 3 mg / kg, 5 mg / kg, 7 mg / kg, 10 mg / kg, 12 mg / kg, 15mg / kg, 17mg / kg, 20mg / kg, 25mg / kg or 30mg / kg.
  • the dose is about 1 mg / kg, 3 mg / kg, 5 mg / kg, or 6 mg / kg. Based on the composition, the dose may be delivered continuously (e.g., by a continuous pump), or at periodic intervals.
  • the dose of the GLP-1 fusion protein or GLP-1 fusion protein conjugate of the invention when administered intravenously, may be 0.1 to 20 mg / kg or any value therein.
  • the ideal time interval for multiple administrations of a particular composition can be determined by one skilled in the art without undue experimentation.
  • Other dosing regimens of the provided compositions are known to those skilled in the art, wherein the dosage, schedule of administration, site of administration, mode of administration, etc. may be different from the foregoing.
  • the dose is administered intravenously.
  • the dosage regimen is a single intravenous administration.
  • kits comprising a GLP-1 fusion protein or a GLP-1 fusion protein conjugate (eg, in a pharmaceutical composition) and instructions for use is also within the scope of the invention.
  • the kit may additionally contain at least one other agent, such as one or more other blood glucose lowering agents.
  • the kit may include a carrier compartmented to tightly hold one or more container devices or a series of container devices (e.g., test tubes, tubes, flasks, bottles, syringes) therein. Wait).
  • the components of the kit can be packaged in an aqueous medium or in a lyophilized form.
  • compositions provided herein may be in a lyophilized form or provided in an aqueous medium.
  • the subject is a vertebrate, more preferably a mammal, and most preferably a human, but may also be other animals, such as domestic animals (such as dogs, cats, etc.), and domestic animals (such as cattle, sheep, pigs, Horses, etc.) or experimental animals (such as monkeys, rats, mice, rabbits, guinea pigs, etc.).
  • domestic animals such as dogs, cats, etc.
  • domestic animals such as cattle, sheep, pigs, Horses, etc.
  • experimental animals such as monkeys, rats, mice, rabbits, guinea pigs, etc.
  • the GLP-1 fusion protein and / or GLP-1 fusion protein conjugate in the present invention can be administered alone, but is preferably administered as a pharmaceutical composition, which usually includes a suitable pharmaceutical excipient selected according to the planned manner of administration , Diluent or carrier. It can be applied to a patient / subject in need of treatment by any suitable means. The precise dose will depend on a number of factors, including the precise nature of the GLP-1 fusion protein and the GLP-1 fusion protein conjugate.
  • Some suitable modes of administration include, but are not limited to, oral, rectal, nasal, topical (including oral and sublingual), subcutaneous, vaginal or parenteral (including subcutaneous, muscular, intravenous, intradermal, intrathecal, Extrameningeal).
  • the pharmaceutical composition of the present invention comprises an isotonicity regulator and / or a preservative, preferably the isotonicity regulator is one or more of sucrose, mannitol, sodium chloride, and glycerol.
  • the preservative is selected from the group consisting of m-cresol, benzyl alcohol, methyl paraben, ethyl paraben, propyl paraben and butyl paraben.
  • Those skilled in the art can well prepare the GLP-1 fusion protein or GLP-1 fusion protein of the present invention by using, for example, isotonic excipients such as physiological saline, Ringer's injection or lactated Ringer's injection.
  • compositions for oral administration may be in the form of tablets, capsules, powders, or oral solutions.
  • a tablet may include a solid carrier such as gelatin or an adjuvant.
  • Liquid pharmaceutical compositions typically include a liquid carrier, such as water, petroleum, animal or vegetable oils, mineral or synthetic oils.
  • a physiological saline solution, glucose or other sugar solutions or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may also be included.
  • the pharmaceutical composition is in the form of a liquid formulation and / or a lyophilized formulation, preferably the lyophilized formulation contains a lyophilized protective agent, and more preferably the lyophilized protective agent is selected from the group consisting of sucrose, lactose , Mannitol, trehalose and other sugars.
  • the GLP-1 fusion proteins and / or GLP-1 fusion protein conjugates described herein are preferably administered to a subject in a "therapeutically effective amount” or "effective amount”.
  • the composition is preferably administered to a subject in a "therapeutically effective amount” which is sufficient to demonstrate its benefit to the subject.
  • the actual amount administered, as well as the rate and time course of administration, will depend on the condition and severity of the person being treated.
  • the prescription of treatment e.g., determining the dosage, etc.
  • the dosage range of the GLP-1 fusion protein and / or GLP-1 fusion protein conjugate may be 30 mg / kg body weight / day to 0.00001 mg / kg body weight / day, or 3 mg / kg / Days to 0.0001 mg / kg / day, or 0.3 mg / kg / day to 0.01 mg / kg / day.
  • the invention also provides a method of treating a disease, the method comprising administering to a subject in need thereof a therapeutically effective amount of a GLP-1 fusion protein and / or a GLP-1 fusion protein conjugate.
  • the disease is selected from the group consisting of postprandial dumping syndrome, postprandial hyperglycemia, impaired glucose tolerance, obesity, eating disorders, insulin resistance syndrome, diabetes, and hyperglycemia.
  • the disease is type II diabetes.
  • the GLP-1 fusion protein constructed in this example has the following structural characteristics: GLP-1-L1-Fc-L2-ST.
  • GLP-1 represents various forms of GLP-1 or its analogues, preferably the sequence of GLP-1 is the same as that of Duraglutide (Duraglutide, Trulicity TM );
  • L1 and L2 represent the first and second linkers, respectively Sequence, the sequence of L1 is GGGGSGGGGSGGGGSA, the sequence of L2 is GGGGSGGGGS, GGGGSGGGGSGGGGS, or GSGGGSGGGGSGGGGS or no L2;
  • Fc represents the immunoglobulin Fc region, and the Fc region is derived from human IgG2 (represented by Fc2 in the present invention) or IgG4 (represented by (Represented by Fc4), preferably the Fc region is consistent with duraglutide (Duraglutide, Trulicity TM ) and is a variant of human IgG4 (Fc4);
  • the amino acid sequences of different GLP-1 fusion proteins are reversely translated into nucleotide sequences according to the codon bias of CHO cells.
  • the full-length DNA fragment of the GLP-1 fusion protein was obtained by whole gene synthesis and PCR amplification, and cloned into the eukaryotic expression vector pFRL-DHFR through the HindIII-EcoRI restriction sites at both ends.
  • the vector pFRL-DHFR contains elements such as the human cytomegalovirus promoter (hCMV-MIE Promoter), SV40, PolyA, and DHFR (dihydrofolate reductase), which can assist the efficient and stable expression of foreign genes in eukaryotic cells (attached) Figure 1A).
  • Table 1 lists the different GLP-1 fusion proteins specifically constructed in the examples.
  • GLP1-L1-Fc4-ST nucleotide sequence N-(GLP1-L1-Fc4-ST nucleotide sequence:
  • GLP1-L1-Fc4-L2-ST nucleotide sequence SLP1-L1-Fc4-L2-ST nucleotide sequence:
  • GLP1-L1-Fc2-L2-ST nucleotide sequence SLP1-L1-Fc2-L2-ST nucleotide sequence:
  • GLP1-L1-Fc2-ST nucleotide sequence SLP1-L1-Fc2-ST nucleotide sequence:
  • GLP1-L1-Fc4-L2-STG3 nucleotide sequence
  • the plasmid constructed above was transfected into the host cell -CHO DG44 by electrotransfection. During transfection, the cell density was adjusted to 6 ⁇ 10 6 cells / mL, the volume was 0.8 mL, and the amount of plasmid was 40 ⁇ g. The plasmid and cells were gently mixed and added to an electric rotor (Bio-Rad, 4mm). The electrophoresis instrument was Bio -Rad (GENE PULSER XCELL), electrical conversion parameters: voltage: 290V, pulse duration: 20ms.
  • cells were transferred to a petri dish containing 15 mL of recovery medium. After 48 hours of incubation, 96-well plates were inoculated, and the medium was replaced with a screening medium containing 50 nM MTX. The confluence rate of clones to be cloned reached more than 50%, and high-expressing clones were screened by dot blot method.
  • the antibody was goat anti-human IgG Fc.
  • the selected clones with relatively high expression levels were sequentially transferred to 24-well plates, 6-well plates, T25 cell culture flasks and cell culture shake flasks for expansion culture.
  • cells were cultured using a pressurized method with increasing MTX concentration.
  • MTX By inhibiting the DHFR gene by MTX, co-amplification of the DHFR gene and the fusion protein gene is achieved.
  • a 15L bioreactor (Applikon, Biobundle 15L) was seeded in a volume of 8L and the seeding density was 0.8-1.6 ⁇ 10 6 cells / mL.
  • Set the culture parameters as follows: pH 6.95 ⁇ 0.15; DO45%; large bubble aeration and oxygen supply; speed 80-140rpm.
  • the temperature was lowered to 33 ° C. and cultured.
  • Feeding was started on the fourth day of the culture, and the residual sugar content was measured daily. The sugar was added to 3g / L daily before cooling and 4g / L daily after cooling.
  • cell counts and viability were measured daily. On day 13, the cell viability was collected at around 90%.
  • Figures 1B to 4 show the growth curve, cell viability, microscopic examination results, and various process parameters of the GLP1-L1-Fc2-L2-ST stable cell line (clone number: P1-3G3C2) during fermentation. It can be seen from FIG. 1B that the cell density was the highest at 20.5 ⁇ 10 6 / mL from the fermentation to the ninth day; when the samples were collected on the 13th day, the cell density was 15.5 ⁇ 10 6 / mL and the viability was 86.6%. Microscopic examination showed good cell morphology, with no apparent dead cells (Figure 2A and Figure 2B). Figures 3 and 4 show the metabolism of sugar, NH 4 + and lactic acid (Lac) during fermentation.
  • Figures 11 to 14 show the growth curve, cell viability, microscopic examination results, and various process parameters of GLP1-L1-Fc4-L2-STG3 during fed-batch fermentation. It can be seen from FIG. 11B that the cell density was highest at 19.9 ⁇ 10 6 cells / mL from the fermentation to the eighth day; when the samples were collected on the 14th day, the cell density was 16.0 ⁇ 10 6 cells / mL and the viability was 92%. Microscopic examination showed good cell morphology, with no apparent dead cells ( Figures 12A and 12B). Figures 13 and 14 show sugar metabolism and pH changes during fermentation.
  • GLP1-L1-Fc2-L2-ST The content of GLP1-L1-Fc2-L2-ST protein in the fermentation supernatant was detected by molecular interaction instrument (Pall Life Science, Qke) was 1.8 g / L, and the content of GLP1-L1-Fc4-L2-STG3 was 1.19 g / L. SDS-PAGE electrophoresis was used to detect the integrity of the GLP1-L1-Fc2-L2-ST molecule.
  • GLP1-L1-Fc2-L2-ST is a double-stranded Fc fusion protein with a complete molecular weight of 61.6 kDa (without sugar). As shown in Fig.
  • the molecular weight of GLP1-L1-Fc2-L2-ST in the non-reduced and reduced states is basically consistent with the theoretical molecular weight.
  • the changes of GLP1-L1-Fc4-L2-STG3 protein were detected by SDS-PAGE electrophoresis ( Figure 15). It can be seen that the yield of the target protein increased significantly on the 12th to 14th days of fermentation, and the target protein was the main component in the supernatant. , Less protein content. The expressed proteins were identified by ELISA experiments and the results showed positive.
  • the supernatants of the GLP1-L1-Fc2-L2-ST and GLP1-L1-Fc4-L2-STG3 fermentation broths were purified in two steps: affinity chromatography (Unimab), anion exchange chromatography (QHP, BXK).
  • the purified protein was detected by SDS-PAGE and SEC (size exclusion chromatography).
  • the size of the target protein is basically consistent with the theoretical molecular weight;
  • SEC uses Agilent Advancebio SEC 300A, 2.7 ⁇ m, 7.8 * 300mm column, 50mM Tris + 150mM NaCl + 10% acetonitrile , Isocratic elution at pH 7.0 (column temperature is 30 ° C, flow rate is 0.5 mL / min, sampler temperature is 4.0 ° C, injection volume is 50 ⁇ g, and sample analysis time is 35 min). It can be seen from FIG.
  • the retention time of the GLP1-L1-Fc2-L2-ST protein is 14.6min, and the purity after the two-step purification is 97.2%.
  • the retention time of the GLP1-L1-Fc4-L2-STG3 protein is between At 14.49 min, the purity was 98.412% after two steps of purification.
  • the C-terminus of all constructed GLP1-Fc fusion proteins contains the corease sequence of SortaseA enzyme-LPETG, and therefore can be specifically recognized by SortaseA enzyme.
  • Sortase A the amide bond between T and G is cleaved.
  • T reacts with the thiol group at 184 C of Sortase to form a thioester bond intermediate, and is subsequently attacked by GGGAA-PEG with poly Gly at the N-terminus.
  • PEGs of different molecular weights were linked to the C-terminus of the protein.
  • the PEGylation reaction system of GLP1-L1-Fc2-L2-ST and GLP1-L1-Fc2-ST proteins is as follows: 20kDa- or 40kDa GGGAA-PEG (GGGAA was purchased from Shanghai Gill Biochemical Co., Ltd., 20kDa- or 40kDa GGGAA-PEG (Synthesized by Beijing Jiankai Technology Co., Ltd.) Dissolve and adjust the pH to 8.0 with a buffer solution, and then add the protein to a 50mM Tris 150mM NaCl pH8.0 buffer system at a ratio of 1:15 and 1:10, and then add Sortase A enzyme and 10 mM CaCl 2 were sampled at different time points of the reaction. After 90 min, EDTA was added to stop the reaction.
  • the PEGylation reaction system of GLP1-L1-Fc4-L2-STG3 protein is as follows: 40 kDa GGGAA-PEG (GGGAA was purchased from Shanghai Gill Biochemical Co., Ltd., 40 kDa GGGAA-PEG was synthesized by Beijing Key Kai Technology Co., Ltd.) with a buffer solution and dissolved Adjust the pH to 7.0, and then add protein: PEG to 20mM Tris150mM NaCl pH7.0 buffer system at a ratio of 1:15 (molar ratio), and then add Sortase A and CaCl2 (10mM), which is added per milligram of the target protein 50IU of the enzyme, the final concentration of the protein of interest is 6mg / mL. After 30 min, the reaction was stopped by adding EDTA.
  • the detection results of GLP1-L1-Fc4-L2-STG3 protein are shown in FIG. 17.
  • 40 kDa PEGylation products gradually increased.
  • the PEGylation products reached the maximum and the reaction was terminated.
  • the total cross-linking rate of the 40 kDa PEG with the GLP1-L1-Fc4-L2-STG3 protein was 80.86. %, Of which single cross-linking is 44.087%, peak time is 10.543min, double-crosslinking is 36.782%, and peak time is 9.698min.
  • GLP1-L1-Fc2-L2-ST was cross-linked with PEG at 40 kDa
  • the sample was subjected to Butyl HP Hydrophobic Chromatography to remove unreacted GGGAA-PEG and Sortase enzymes, and anion exchange chromatography was performed to remove uncross-linked substrate proteins. The residual amount of the substrate is controlled within 1%.
  • FIG. 8A after the GLP1-L1-Fc2-L2-ST is crosslinked with 40 kDa and PEG, the sample can be divided into two peaks P1 and P2 by anion exchange chromatography.
  • the RPC test results show (Figure 8B) that the P1 peak is mainly a double crosslinked product (double crosslinked 90.84%, single crosslinked 8.4%, substrate 0.15%), and the P2 peak is mainly a single crosslinked product (double crosslinked 8.56%). , 90.12% single cross-linked, 0.73% substrate).
  • the retention time of the uncrosslinked GLP1-L1-Fc2-L2-ST was 17.3 minutes, and the retention times of the single and double crosslinked products were 22.5 and 23.2 minutes, respectively.
  • GLP1-L1-Fc4-L2-STG3 was cross-linked with 40kDa and PEG, and the sample was anion exchange chromatography (SuperQ 5PW, TOSOH) to separate single and double cross-links and substrates.
  • the chromatogram is shown in Figure 18A.
  • the P1 peak is mainly a double crosslinked product (97.172% of double crosslinks, 2.766% of single crosslinks, and substrate 0%)
  • the peak of P2 is mainly a single crosslink product (double crosslinks). (19.722% crosslinked, 79.754% crosslinked, 0.158% substrate).
  • the retention times of the single and double crosslinked products were 10.63 and 9.8min, respectively.
  • GLP-1 can bind to GLP-1 receptor (GLP-1R), activate adenylate cyclase (AC), generate cAMP, and then activate cAMP-dependent transcription factor cAMP response element binding protein CREB), which activates transcription of downstream genes after binding to the cAMP response element (CRE).
  • GLP-1R GLP-1 receptor
  • AC activate adenylate cyclase
  • CREB cAMP-dependent transcription factor cAMP response element binding protein
  • the detection in this example includes the following steps: HEK293 / CRE-Luc / GLP1R cells (purchased from GenScript, # M00562) are cultured to the logarithmic growth phase, and trypsinized and resuspended in complete culture medium to adjust the cell density to 1 ⁇ 10 6 cells / mL, 50 ⁇ L / well were added to a white 96-well plate; duraglutide and the test product were diluted to 400 ng / mL with complete medium, and diluted 4 times, a total of 9 gradients, 50 ⁇ L / well Add cells, incubate at 37 ° C, 5% CO 2 for 5 h; add 100 ⁇ L of Bright-Glo Luciferase (promega) to each well, and develop light for 10 min in the dark. A full-band fluorescence microplate reader (TECAN, 200PRO) was used to measure the fluorescence value.
  • Figure 9 shows the results of the activity detection of GLP1-L1-Fc2-L2-ST after cross-linking with 20 kDa PEG and 40 kDa PEG, respectively.
  • the relative activity of the uncrosslinked substrate (GLP1-Fc) was 98.10%.
  • the activity decreased, in which the relative activity of the 20 kDa cross-linked product (represented as PEG (20 kDa) -GLP1-Fc in the figure) was 40.38%;
  • the relative activity of GLP1-Fc) was 32.85%.
  • FIG. 19 shows the results of activity detection of GLP1-L1-Fc4-L2-STG3 and the product after cross-linking with 40 kDa PEG. Similar to the PEG conjugated product of GLP1-L1-Fc2-L2-ST, compared to Duraglutide, the product of GLP1-L1-Fc4-L2-STG3 after 40 kDa PEG conjugation (represented as PEG (40 kDa in the figure) The activity of) -GLP1-Fc) was reduced to some extent, and the relative activity was 21.51%.
  • mice C57BL / 6 (purchased from Beijing Weitong Lihua Co., Ltd.) mice were housed in five cages, fed with common mouse growth feed, and freely drinking water. Light time is 12h (08: 00 am-20:00pm) and dark time is 12h (20:00 pm-08:am).
  • mice Forty healthy male C57BL / 6 mice aged 6-8 weeks were divided into 5 groups of 8 mice each. They are: (1) GLP1-Fc group (GLP-1-L1-Fc2-L2-ST substrate before cross-linking); (2) Duraglutide group; (3) 40kDa PEG GLP1-Fc group (GLP-1 -L1-Fc2-L2-ST cross-linked 40 kDa PEG); (4) 20 kDa PEG GLP1-Fc group (GLP-1-L1-Fc2-L2-ST cross-linked 20 kDa PEG); (5) control group.
  • the groups (1) to (4) were administered at 3 mg / kg, and the control group was administered with physiological saline.
  • mice were fasted for 6 hours, blood glucose and body weight were measured, and free water was consumed during fasting; kg glucose solution, the volume is 10ml / kg, the blood glucose value at 10, 30, 60, 90, 120min after the glucose load is measured, the glucose tolerance curve is drawn, and the area under the blood glucose curve (AUC) is calculated; the data are expressed as mean ⁇ standard error (Mean ⁇ SEM), the data was analyzed using Graphpad 7.0 7.0 statistical software, and the differences were analyzed using Mann-whiteney statistics.
  • Figure 10M shows the daily weight change of C57BL / 6J mice after administration. Compared with the control group, the weight of each group decreased in 24 hours. Among them, the weight loss of the GLP1-Fc group, especially duraglutide and uncrosslinked PEG, was significant. It is worth noting that both PEG (20kDa) -GLP1-Fc and PEG (40kDa) -GLP1-Fc can significantly reduce the risk of rapid weight loss caused by drugs, indicating that PEGylation modification can significantly improve the side effects of clinical application of this class of drugs , With better security.
  • mice Eighteen healthy male C57BL / 6 mice aged 8-10 weeks were divided into 3 groups of 6 mice each. They are: (1) control group; (2) duraglutide group; (3) PEG (40kDa) -GLP1-Fc group (GLP1-L1-Fc4-L2-STG3 cross-linked 40kDa PEG).
  • the (2) and (3) groups were administered according to equal activity (that is, based on the mass conversion of the activity measured in vitro in Figure 19), the dosage of duraglutide was 0.76 mg / kg, and PEG (40kDa) -GLP1- The dose of Fc was 3.5 mg / kg, and the control group was given physiological saline. IPGTT experiments were performed 1, 5, 6, 7, and 8 days after administration.
  • mice were fasted for 6 hours, blood glucose and body weight were measured, and water was free during the fasting period. After 6 hours, 2.0 g / kg was given intraperitoneally.
  • Glucose solution with a volume of 10ml / kg, measuring the blood glucose value at 10, 30, 60, 90, 120min after glucose load, and calculating the area under the blood glucose curve (AUC); the data is expressed as mean ⁇ standard error (Mean ⁇ SEM), The data was analyzed using Graphpad prism 7.0 statistical software, and the differences were analyzed using Mann-whiteney statistics.
  • Figure 20B shows the change in body weight of C57BL / 6 mice after administration. Compared with the control group, both groups lost weight within 24 hours. Among them, the weight loss was more obvious in the duraglutide group. It is worth noting that PEG (40kDa) -GLP1-Fc can also significantly reduce the risk of rapid weight loss caused by the drug, further illustrating that PEGylation modification can significantly improve the side effects of clinical application of such GLP-1 receptor agonists .
  • C57BL / 6 purchased from Beijing Weitong Lihua Co., Ltd. mice were housed in five cages, fed with common mouse growth feed, and freely drinking water. Light time is 12h (08: 00 am-20:00pm), and dark time is 12h (20:00 pm-08:00am).
  • mice Twenty-four type II diabetic mice were divided into 4 groups of 6 mice each. They are: (1) control group; (2) duraglutide-1 mg / kg group; (3) PEG (40kDa) -GLP1-Fc (GLP1-L1-Fc4-L2-STG3 cross-linked 40kDa PEG) -1mg / kg group; (4) PEG (40kDa) -GLP1-Fc (GLP1-L1-Fc4-L2-STG3 cross-linked 40kDa PEG) -3mg / kg group.
  • the dosage of duraglutide was 1 mg / kg
  • the dosage of PEG (40kDa) -GLP1-Fc was 1 mg / kg and 3 mg / kg
  • the control group was given normal saline.
  • Blood glucose values were measured, weights were measured on 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12 days without fasting, and drinking water was free.
  • Data are shown as mean ⁇ SD (Mean ⁇ SEM) means that the data was analyzed with Graphpad and 7.0 statistical software, and the difference was analyzed with Mann-whiteney statistics.
  • the weight change of diabetic mice after administration was also observed. As shown in FIG. 21B, compared with the control group, the weight of all experimental groups of mice decreased. Among them, the weight loss of the duraglutide group was more obvious, and the weight loss reached 2.5 g compared to the control group at the most.
  • the two dose groups of PEG (40kDa) -GLP1-Fc had a smaller effect on body weight.
  • the body weight of the PEG (40kDa) -GLP1-Fc-1mg / kg group was reduced by up to 0.3g compared to the control group, and the weight of the PEG (40kDa) -GLP1-3mg / kg group was reduced by up to 1.1g compared to the control group.
  • PEG (40kDa) -GLP1-Fc can also significantly improve the rapid weight loss caused by drugs when used in the treatment of diabetes at various doses, indicating that PEGylation modification can significantly improve the side effects of clinical application of such drugs, and Effectively improve patient compliance.
  • PEGylated GLP1-Fc can promote insulin secretion from islet ⁇ cells, inhibit the temporary increase in blood glucose caused by exogenous glucose uptake, and promote glucose utilization; GLP1-Fc protein appears more durable after PEG modification The effect of lowering blood sugar and the side effects of the drug are smaller, avoiding the problem of rapid weight loss often caused by GLP-1 weight loss drugs, and has a good prospect of clinical application.

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Abstract

本发明提供了一种新型GLP-1融合蛋白及其缀合物，含有它们的药物组合物以及它们在降低血糖或体重，尤其是治疗糖尿病、特别是II型糖尿病中的用途。

Description

GLP1-Fc融合蛋白及其缀合物
交叉引用
本申请要求发明名称为“GLP1-Fc融合蛋白及其缀合物”于2018年9月26日提交到中国专利局的中国专利申请201811125762.9的优先权，其内容通过引用以整体并入本文。
技术领域
本发明涉及延长体内半衰期的生物活性多肽融合蛋白及其与聚合物的缀合物，特别是延长体内半衰期的GLP-1受体激动剂，含有它们的药物组合物以及它们在降低血糖(尤其是治疗糖尿病，特别是II型糖尿病)以及控制体重中的用途。
背景技术
随着经济社会的发展、人口平均寿命的延长和生活方式的变化，糖尿病已经成为世界各国主要的卫生保健问题。无论在发达国家或是发展中国家，糖尿病的发病率都在急剧上升。2007年全球约有2.46亿糖尿病患者，全球范围内每10秒钟就有一个糖尿病患者死亡，预计到2025年全世界糖尿病患者将达到3.33亿。我国已成为糖尿病发病的“重灾区”，目前有糖尿病患者4000万，糖尿病的发病率约为5％，是全球糖尿病第二大国(第一为印度)。糖尿病患者分为两种，一是胰岛素依赖型糖尿病(Ⅰ型糖尿病)和非胰岛素依赖型糖尿病(Ⅱ型糖尿病)。其中，Ⅱ型糖尿病占糖尿病患者的90％以上。Ⅱ型糖尿病特点是胰岛素分泌或作用失调及β-细胞功能障碍，致使脂肪、碳水化合物以及蛋白质代谢紊乱，造成慢性血糖过高，最终导致各种微血管、大血管及各种脏器并发症出现。如今，控制糖尿病的药物有2类：1)促胰岛素分泌类，如磺酰脲类，氯茴苯酸类，二肽基肽酶抑制剂，GLP-1类似物；2)非促胰岛素分泌药物，如胰岛素，α-葡萄糖苷酶抑制剂，双胍类，噻唑烷二酮类，胰岛素类似物等。目前，临床上使用较多的传统糖尿病治疗药物对Ⅱ型糖尿病患者皆无能为力，不能遏制胰脏β-细胞的不断进行性的恶化，不能降低血液中糖化血红蛋白(HbA1c)水平，也不能阻止糖尿病的并发症如心脏病、肾衰竭，且都伴 随着不同程度的毒副作用。因此，需要研究新的Ⅱ型糖尿病治疗药物。
1985年发现了一种肠激素胰高血糖素样肽-1(Glucagon-like peptide-1，GLP-1)，其是进食后由胰高血糖素原基因表达，主要在肠道黏膜L-细胞分泌的一种产物，它能刺激胰岛β-细胞分泌胰岛素(J Med Chem,47,4128-4134,2004)，对稳定血糖水平有重要作用。外源给予GLP-1能使Ⅱ型糖尿病患者的血糖水平正常化(Diabetes Care,15,270-276,1992；Lancet,359,824-830,2002；Endoer.Rev,16,390-410,1996；Diabetologia,28,565-573,1985)。GLP-1具有下列功能：以葡萄糖依赖方式作用于胰岛β-细胞，促进胰岛素基因的转录，增加胰岛素的生物合成和分泌；刺激β-细胞的增殖和分化，抑制β-细胞凋亡从而增加胰岛β-细胞数量；抑制胰高血糖素的分泌；增加周边细胞胰岛素受体的敏感性；降低HbA1c；抑制食欲及摄食；延缓胃内容物排空(Diabetic Med,18,144-149,2001；Diabetes,51,1443-1452,2002；Diabetologia,45,1263-1273,2002；Diabetes,50,525-529,2001；Diabetes,50,725,2001；Diabetes,52,365-371,2003；Recent Prog.Hormne Res.56,377-399,2001；Disbetologia,39,1546-1553,1996；Am.J.Physicl Endocrinol.Metab,281,E242-247,2001；U.S.patent 477967,478017,478425；Diabetes Care,22,403-408,1999；J.Clin.Endocrinology and Metabolism,88,3082-3089,2003；Diabetes,44,1295,1995)。但GLP-1在体内很容易被二肽基肽酶(DPPIV)降解，半衰期不足2分钟，几乎不能成为有效的抗糖尿病药物。
由于多肽类药物普遍存在体内半衰期较短，物理、化学稳定性较差，易被体内各种蛋白酶降解等特性，使得这些药物通常需要在一天之内多次注射。给患者身体、心理和经济带来较大的负担，限制了患者用药依从性。因此，本领域迫切需要具有新结构的药物，从而延长其血浆周期和增加其***性药物暴露(systemic drug exposure)。
发明内容
发明人经过多年的研究和长期的试验发现，将多肽(蛋白)与能够延长半衰期的融合伴侣(例如Fc片段、白蛋白、XTEN或转铁蛋白)融合，进一步与亲水聚合物(例如聚亚烷基二醇，更例如PEG)缀合的手段，可以有效提高生物活性多肽的体内稳定性，提高药物疗效，从而完成了本发明。
在第一个方面中，本发明提供了一种GLP-1受体激动剂的融合蛋白，其包括GLP-1受体激动剂以及与其连接的增加体内半衰期的融合伴侣(FP)；其中所述GLP-1受体激动剂与所述融合伴侣直接连接或者通过第一接头L1连接；优选地，所述第一接头L1为包含1、2、3、4、5、6、7、8、9、10个或更多个氨基酸的肽段；更优选地，所述第一接头L1为包含A、T、G和/或S的柔性肽段，例如(GS)n，其中n为1-50的整数，如1、2、3、4、5、6、7、8、9或10；
进一步优选地，所述第一接头L1选自：
GSGGGSGGGGSGGGGS(SEQ ID NO:9)；
GSGGGGSGGGGSGGGGSGGGGSGGGGS(SEQ ID NO:10)；
GGGGSGGGGSGGGGSGGGGS(SEQ ID NO:11)；
GSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS(SEQ ID NO:12)；
GGGSGGGSGGGSGGGSGGGS(SEQ ID NO:13)；
PRFQDSSSSKAPPPSLPSPSRLPGPSDTPILPQ(SEQ ID NO:14)；
SSSSKAPPPSLPSPSRLPGPSDTPILPQ(SEQ ID NO:15)；
SSSSKAPPPS(SEQ ID NO:16)；
SRLPGPSDTPILPQ(SEQ ID NO:17)和
GGGGSGGGGSGGGGSA(SEQ ID NO:18)。
在一个实施方案中，其中所述GLP-1受体激动剂是全长、截短或变体形式的GLP-1或GLP-1类似物，例如是Exendin-4、GLP-1(1-36)、GLP-1(1-37)、GLP-1(7-36)、GLP-1(7-37)、利拉鲁肽、利司那肽、阿必鲁肽、度拉糖肽或索玛鲁肽。
在另一个实施方案中，其中所述融合伴侣(FP)为全长、截短或变体形式的免疫球蛋白Fc区段、白蛋白、XTEN或转铁蛋白，优选所述Fc区段是人免疫球蛋白Fc区段；优选地，所述免疫球蛋白Fc区段由选自CH1结构域、CH2结构域、CH3结构域和CH4结构域的一至四个结构域构成；还优选地，所述免疫球蛋白Fc区段是来自于IgG、IgA、IgD、IgE或IgM的Fc区段，更优选为IgG Fc区段；进一步优选地，所述Fc区段是来自IgG1、IgG2、IgG3或IgG4的Fc区段；此外还优选所述IgG  Fc区段具有降低的ADCC效应和/或CDC效应和/或与FcRn受体增强的结合亲和力。
在又一个实施方案中，其中所述融合伴侣(FP)还与包含Sortase酶识别位点的一段氨基酸序列ST直接连接或者通过第二接头L2连接，所述Sortase酶例如是Sortase A或Sortase B；优选地，所述ST包含Sortase A的核心识别位点LPXTG，其中X是任意氨基酸，例如LPETG、LPETGG或LPETGGG；还优选地，所述ST还包含与Sortase酶识别序列连接的亲和标签，这样的ST序列例如是LPETGGHHHHHH或LPETGGWSHPQFEK；
其中，所述第二接头L2为包含1、2、3、4、5、6、7、8、9、10个或更多个氨基酸的肽段；更优选地，所述第二接头L2为包含A、T、G和/或S的柔性肽段，例如(GS)n其中n为1-50的整数，如1、2、3、4、5、6、7、8、9或10；
进一步优选地，所述第二接头L2选自：
GSGGGSGGGGSGGGGS(SEQ ID NO:9)；
GGGGSGGGGSGGGGSA(SEQ ID NO:18)；
GGGGS(SEQ ID NO:19)；
GGGGSGGGGS(SEQ ID NO:20)和
GGGGSGGGGSGGGGS(SEQ ID NO:21)。
在另一个实施方案中，所述融合蛋白具有如下结构：GLP-1-L1-FP-L2-ST，其中
GLP-1表示GLP-1受体激动剂，并且GLP-1受体激动剂、L1、L2、FP和ST具有如前述实施方案中相同的定义，其中L1和L2任一个或两者可以不存在。
在一个具体方案中，所述融合蛋白的氨基酸序列如SEQ ID NO：1、3、5、7或22所示；或者所述融合蛋白由SEQ ID NO：2、4、6、8或23所示的核酸序列所编码。
在本发明的第二个方面中，提供了一种缀合物，其中一个、二个或更多个亲水聚合物分子连接至第一个方面中所述融合蛋白的末端，优选地经转肽反应连接至所述sortase酶识别位点；特别地，所述融合蛋白可以是单体形式或二聚体形式。
在一个实施方案中，其中所述一个、二个或多个亲水聚合物分子各自独立地选自多糖和聚亚烷基二醇，如聚丙二醇和聚乙二醇；所述聚亚烷基二醇可以是末端封端的，例如经烷氧基如甲氧基封端的；和/或所述聚亚烷基二醇是直链的或支链的；例如所述聚亚烷基二醇是支链的，例如是支链的聚乙二醇，尤其是经甲氧基封端的支链聚乙二醇；所述聚亚烷基二醇的分子量可以是>＝1、>＝10、>＝20、>＝30、>＝40、>＝50、>＝60、>＝70、>＝80、>＝90、>＝100、>＝110、>＝120、>＝130、>＝140、>＝150或>＝160kDa，例如是5、10、20、30、40、50、60、70、80、90或100kDa，或者以上任意二数值之间的值；优选地所述亲水性聚合物分子的分子量是20kDa或40kDa。
本发明第三个方面提供了一种制备第二个方面所述缀合物的方法，其中包括使第一个方面中所述的融合蛋白与Sortase酶(如Sortase A或Sortase B)和亲水聚合物分子接触，其中所述亲水聚合物分子一端带有可被Sortase酶酰胺化的氨基。
在一个实施方案中，其中所述亲水聚合物分子的一端带有聚Gly，例如带有GGGAA。
本发明的第四个方面提供了一种药物组合物，其包含有效量的第一个方面中的融合蛋白和/或第二个方面中所述的缀合物，以及任选地药学上可接受的载体。
在一个实施方案中，所述药物组合物用于降低血糖或体重，例如用于治疗糖尿病，更具体地用于治疗I型糖尿病和/或Ⅱ型糖尿病，尤其是用于治疗II型糖尿病。
在另一个实施方案中，所述药物组合物为液体制剂形式或冻干制剂形式。
本发明的第五个方面提供了第一个方面所述的融合蛋白或者第二个方面所述的缀合物在制备用于降低血糖或体重的药物中的用途，例如所述药物用于治疗糖尿病，包括I型糖尿病和II型糖尿病，尤其是II型糖尿病。
本发明的第六个方面提供了一种降低血糖或体重的方法，其包括对有此需要的对象施用第一个方面所述的融合蛋白或者第二个方面所述的缀合物，例如用于治疗糖尿病，包括I型糖尿病和II型糖尿病，尤其是 II型糖尿病。
本发明的第七个方面提供了一种试剂盒，其包含第一个方面所述的融合蛋白、第二个方面所述的缀合物、和/或第四个方面所述的药物组合物，以及任选的使用说明书。
申请人惊奇的发现，本发明的GLP-1-Fc缀合物不但能够提供更长期的降糖效果，而且比起现有的类似药物，能够显著降低服用降糖药物所引起的快速体重下降，因此极大改善了这类药物的临床安全性，因此具有极大的临床应用价值。
附图说明
图1A显示了GLP-1融合蛋白真核表达载体的结构图；图1B显示了GLP1-L1-Fc2-L2-ST稳定细胞株(克隆号：P1-3G3C2)在发酵过程中的生长曲线和细胞活率的检测结果。
图2显示了GLP1-L1-Fc2-L2-ST稳定细胞株(克隆号：P1-3G3C2)在发酵过程中不同时间点的镜检结果。图2A显示的是培养第5天时的细胞，图2B显示了收样(第13天)时的细胞。
图3显示了GLP1-L1-Fc2-L2-ST稳定细胞株(克隆号：P1-3G3C2)在发酵过程中培养上清中的糖代谢情况。
图4显示了GLP1-L1-Fc2-L2-ST稳定细胞株(克隆号：P1-3G3C2)在发酵过程中培养上清中的NH4+和乳糖(Lac)代谢情况。
图5显示了GLP1-L1-Fc2-L2-ST蛋白的非还原(左侧M/1/2/3)和还原(右侧4/5/6/M)电泳结果(1和6：末端发酵液上清；3和4是纯化后样品，2和5是第10天发酵液样品)。
图6显示了GLP1-L1-Fc2-L2-ST蛋白经纯化后的SDS电泳(A)及SEC分析(B)检测结果。
图7显示了两种蛋白通过SDS电泳及RPC分析对交联率的检测结果。A和C分别为GLP1-L1-Fc2-L2-ST蛋白交联20kDa和40kDa PEG的SDS检测；B和D分别为GLP1-L1-Fc2-L2-ST蛋白交联20kDa和40kDaPEG的RPC检测，E为GLP1-L1-Fc2-ST蛋白交联40kDa PEG的RPC检测。
图8显示GLP1-L1-Fc2-L2-ST交联后样品的阴离子交换层析色谱图 (A)及RPC检测(B)结果。
图9显示了GLP1-L1-Fc2-L2-ST分别与20kDa PEG和40kDa PEG交联后的活性检测结果。与杜拉鲁肽相比，未交联底物(GLP1-Fc)的相对活性为98.10％。PEG交联后活性均有下降，其中，20kDa交联产物(PEG(20kDa)-GLP1-Fc)的相对活性为40.38％；40kDa交联产物(PEG(40kDa)-GLP1-Fc)的相对活性为32.85％。
图10A显示了C57BL/6小鼠给药24h后糖耐量试验曲线；图10B显示了给药后24h对C57BL/6小鼠糖耐量(AUC)的影响；图10C显示了C57BL/6小鼠给药144h后糖耐量试验曲线；图10D显示了给药后144h对C57BL/6小鼠糖耐量(AUC)的影响；图10E显示了C57BL/6小鼠给药168h后糖耐量试验曲线；图10F显示了给药后168h对C57BL/6小鼠糖耐量(AUC)的影响；图10G显示了C57BL/6小鼠给药192h后糖耐量试验曲线；图10H显示了给药后192h对C57BL/6小鼠糖耐量(AUC)的影响；图10I显示了C57BL/6小鼠给药216h后糖耐量试验曲线；图10J给药后216h对C57BL/6小鼠糖耐量(AUC)的影响；图10K显示了C57BL/6小鼠给药240h后糖耐量试验曲线；图10L显示了给药后240h对C57BL/6小鼠糖耐量(AUC)的影响；图10M显示了给药后C57BL/6小鼠体重变化。
图11显示了GLP1-L1-Fc4-L2-STG3稳定细胞株(克隆号：P1-P132C7)在补料分批发酵过程中的生长曲线和细胞活率的检测结果。
图12显示了GLP1-L1-Fc4-L2-STG3稳定细胞株(克隆号：P1-P132C7)在补料分批发酵过程中不同时间点的镜检结果。图12A显示的是培养第5天时的细胞，图12B显示了收样(第13天)时的细胞。
图13显示了GLP1-L1-Fc4-L2-STG3稳定细胞株(克隆号：P1-P132C7)在补料分批发酵过程中培养上清中的糖代谢情况。
图14显示了GLP1-L1-Fc4-L2-STG3稳定细胞株(克隆号：P1-P132C7)在15L生物反应器里补料分批发酵过程中的pH变化趋势。
图15显示了GLP1-L1-Fc4-L2-STG3蛋白的15L补料分批发酵上清液目的蛋白的SDS-PAGE电泳图，通道1是纯化后样品，通道2是蛋白Marker，通道3、4和5依次是第12天、13天和14天的发酵液上清。
图16显示了GLP1-L1-Fc4-L2-STG3蛋白经纯化后的SDS电泳(A)及SEC分析(B)检测结果。
图17显示了两种蛋白通过SDS电泳及RPC分析对交联率的检测结果。A为GLP1-L1-Fc4-L2-STG3蛋白交联40kDa PEG的SDS检测；B为GLP1-L1-Fc4-L2-STG3蛋白交联40kDaPEG的RPC检测。
图18显示GLP1-L1-Fc4-L2-STG3交联后样品的阴离子交换层析色谱图(A)及SEC检测(B、C)结果。
图19显示了GLP1-L1-Fc4-L2-STG3与40kDa PEG交联后的活性检测结果。与杜拉鲁肽相比，未交联底物(GLP1-Fc)的相对活性为112.24％。PEG交联后活性有所下降，40kDa交联GLP1-L1-Fc4-L2-STG3产物(PEG(40kDa)-GLP1-Fc)的相对活性为21.51％。
图20A显示了给药后1-8天对C57BL/6小鼠糖耐量(AUC)的影响；图20B显示了给药后C57BL/6小鼠体重变化。
图21A显示了给药后1-12天对STZ诱导的II型糖尿病小鼠血糖的影响；图21B显示了给药后II型糖尿病小鼠体重变化。
具体实施方式
本申请所用术语具有与现有技术中该术语相同的含义。为了清楚地表明所用术语的含义，以下给出一些术语在本申请中的具体含义。当本文定义与该术语的常规含义有冲突时，以本文定义为准。
本文中使用的术语“氨基酸”包括天然氨基酸、非天然氨基酸、和氨基酸类似物以及所有它们的D和L立体异构体。非天然氨基酸包括但不限于氮杂环丁烷羧酸、2-氨基己二酸、3-氨基己二酸、β-丙氨酸、氨基丙酸、2-氨基丁酸、4-氨基丁酸、6-氨基已酸、2-氨基庚酸、2-氨基异丁酸、3-氨基异丁酸、2-氨基庚二酸、叔丁基甘氨酸、2，4-二氨基异丁酸、2，2’-二氨基庚二酸、2，3-二氨基丙酸、N-乙基甘氨酸、N-乙基天冬酰胺、高脯氨酸、羟赖氨酸、别-羟赖氨酸、3-羟脯氨酸、4-羟脯氨酸、异锁链赖氨素、别-异亮氨酸、N-甲基丙氨酸、N-甲基甘氨酸、N-甲基异亮氨酸、N-甲基戊基甘氨酸、N-甲基缬氨酸、萘丙氨酸、正缬氨酸、正亮氨酸、鸟氨酸、戊基甘氨酸、2-哌啶酸和硫代脯氨酸。氨基酸类似物包括在其C-末端羧基、N末端氨基或其侧链基团可逆或不可逆地化学封闭的、或被化学修饰成另一官能团的天然氨基酸和非天然氨基酸，例如甲硫氨酸亚砜、甲硫氨酸砜、S-(羧甲基)-半胱氨酸、S-(羧甲基)-半胱氨酸亚砜和S-(羧甲基)-半 胱氨酸砜。
本文中使用的术语“多肽”或“蛋白”可互换地指通过共价键(例如肽键)相互连接的一串至少两个氨基酸残基，可以是重组多肽、天然多肽或合成多肽。此外，本发明所述的“多肽”或“蛋白”还包括其变体或类似物形式，即通过一个或多个取代、缺失、***、融合、截短或其任意组合在氨基酸序列上有所不同的多肽。变体多肽可以是完全功能性的或者可缺乏一种或多种活性的功能。完全功能性变体可含有例如仅仅保守性改变或非关键残基或非关键区域的改变。功能性变体还可包含相似氨基酸的替换，其导致功能未改变或不显著的改变。可以通过本领域已知的方法鉴定对于功能来说重要的氨基酸，所述方法例如定点诱变或甘氨酸扫描诱变(Cunningham，B.和Wells，J.，Science，244：1081-1085，1989)。可以例如通过结构分析如结晶、核磁共振或光亲和标记来确定对于多肽活性来说关键的位点(Smith，L.等，J.Mol.Biol.，224：899-904，1992；de Vos,A.等，Science，255：306-312，1992)。
本文中使用的术语“缀合物”是指多肽或多肽变体与本文所述的修饰基团共价或非共价连接后形成的产物。
一般地，本文中使用的术语“聚合物”具有本领域普通技术人员通常理解的含义，既包括聚合物本身，也包括其末端修饰的衍生物，除非另有明确说明。
此外，对于聚合物如聚乙二醇来说，有多种方法测定其分子量。由于聚合物是由一定分布范围内的不同聚合度的分子构成，一般用平均分子量来表示聚合物的分子量，具体来说可以是数均分子量或重均分子量。尽管在聚合物的聚合度差异较大时，数均分子量和重均分子量可能有一些偏差，但是对于分布范围较窄的聚合物来说，二者趋于相等。除非特别指出，对于本文提及的聚合物如聚乙二醇，在提及其分子量时，既可以是重均分子量，也可以是数均分子量。
GLP-1受体激动剂
胰高血糖素样肽(Glucagon-like peptide)，包括胰高血糖素样肽-1(GLP-1)和胰高血糖素样肽-2(GLP-2)，二者均来源于胰高血糖素原(Proglucagon)，胰高血糖素原由158个氨基酸组成，可被切割成不同的肽 链，因为GLP-1具有促进胰岛素分泌，保护胰岛β细胞，抑制胰高血糖素分泌，抑制胃排空，降低食欲的药理作用，临床可用于二型糖尿病和肥胖症的治疗；GLP-2是一种营养因子，具有促进小肠生长，抑制细胞凋亡，促进胃排空，增加食欲的药理作用，临床上可用于治疗短肠综合症。人体内具有生物活性的GLP-1主要是GLP-1(7-36)酰胺和GLP-1(7-37)，天然GLP-1易被二肽基肽酶IV(DPP-IV)迅速水解失活(半衰期小于5min)，因此难以用于临床应用。
术语“GLP-1受体激动剂”是指能够激活GLP-1受体活性的分子，例如天然存在的GLP-1，其可以来源于人或动物等不同来源，也包括与天然GLP-1受体激动剂(如GLP-1和exendin-4)野生型序列相比有一个或更多个(如1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20或更多个)氨基酸置换、缺失和/或添加的氨基酸序列所形成的功能性变体、片段和类似物，例如利拉鲁肽、利司那肽、阿必鲁肽、度拉糖肽、索玛鲁肽等。
GLP-1融合蛋白
GLP-1受体激动剂(例如GLP-1及其功能性变体和片段)与能够增加半衰期的融合伴侣(例如人免疫球蛋白的Fc区段)连接形成一种融合蛋白，所述融合蛋白在本发明中可以被称为“具有GLP-1受体激动剂活性的融合蛋白”或者“GLP-1融合蛋白”。
人免疫球蛋白IgG由4个通过二硫键共价连接的多肽(轻链和重链的两个相同拷贝)组成。通过木瓜蛋白酶来蛋白水解IgG分子产生两个Fab片段和一个Fc区段。所述Fc区段由通过二硫键连接在一起的两个多肽组成。每个多肽，从N至C末端，由铰链区、CH2结构域和CH3结构域组成。所述Fc区段结构在所有亚型的人免疫球蛋白中几乎相同。IgG是人血液中最丰富的蛋白之一，其在组成了人血清中总免疫球蛋白的70至75％。
IgG在循环中的半衰期在所有5种类型的免疫球蛋白中是最长的，并且可达到21天。已有报导将IgG的Fc区域与其它蛋白质(如各种细胞因子和可溶性受体)结合而形成融合蛋白(参见，例如Capon等人，Nature，337：525-531，1989；Chamow等人，Trends Biotechnol.，14：52-60，1996；美国专利No.5,116,964和5,541,087)。典型的融合蛋白是一重二聚体蛋白 质，系通过IgG Fc绞链区中的半胱氨酸残基与蛋白连接，而形成类似IgG、但缺少CH1区域和轻链的分子。由于结构上的同源，Fc融合蛋白表现出的体外药物动力学特性与同种型的人IgG相当类似。因此制造含有与人IgG蛋白质的Fc区域相连的hGH融合蛋白，将有助于延长hGH的循环半衰期和/或增加它的生物活性。
免疫球蛋白的Fc区用作药物载体是安全的，因为它是体内代谢的可生物降解多肽，此外，与整个免疫球蛋白分子相比，免疫球蛋白Fc区具有相对低的分子量，因此在缀合物的制备、纯化和生产上是有利的。由于免疫球蛋白Fc区不包含Fab片段(其氨基酸序列根据抗体亚类而不同并且因此是高度不均一的)，因此可以预期免疫球蛋白Fc区可极大地增加物质的均一性并具有低抗原性。
本发明所使用的术语“免疫球蛋白的Fc区”是指含有免疫球蛋白的重链恒定区2(CH2)和重链恒定区3(CH3)，而不含免疫球蛋白的重链和轻链的可变区、重链恒定区1(CH1)和轻链恒定区1(CL1)的蛋白质。它还可包含重链恒定区处的铰链区。此外，本发明的免疫球蛋白Fc区可含有包含除了重链和轻链的可变区以外的重链恒定区1(CH1)和/或轻链恒定区1(CL1)的Fc区的一部分或全部，只要它具有与天然蛋白质基本相似或更好的生理功能即可。此外，它可以是在CH2和/或CH3的氨基酸序列的相对长的部分中具有缺失的片段。即，本发明的免疫球蛋白Fc区可包含1)CH1结构域、CH2结构域、CH3结构域和CH4结构域；2)CH1结构域和CH2结构域；3)CH1结构域和CH3结构域；4)CH2结构域和CH3结构域；5)一种或更多种结构域与免疫球蛋白铰链区(或铰链区的一部分)的组合以及6)重链恒定区和轻链恒定区的各个结构域的二聚体。
此外，本发明的免疫球蛋白Fc区包括天然氨基酸序列及其序列衍生物(突变体)。由于一个或更多个氨基酸残基的缺失、***、非保守或保守替换或其组合，氨基酸序列衍生物具有与天然氨基酸序列不同的序列。例如，在IgG Fc中，第214至238、297至299、318至322或327至331位处的已知对于结合重要的氨基酸残基可用作修饰的适合靶点。此外，其它多种衍生物也可以，包括其中缺失了能够形成二硫键的区域的、缺失了天然Fc形式N-端处数个氨基酸残基的或者向天然Fc形式的N-端添加了甲硫氨酸残基的衍生物。此外，为了去除效应子功能，缺失可发生于补体 结合位点，如C1q结合位点和ADCC位点。制备这样的免疫球蛋白Fc区的序列衍生物的技术公开于WO 97/34631WO 96/32478中。
蛋白质和肽中一般不改变分子活性的氨基酸替换是本领域已知的(H.Neurath蛋白质和肽中一般不改变分子活性的氨基酸替换是本领域已知的(H.Neurath，R.L.Hill，The Proteins，Academic Press，New York，1979)。最常发生的替换是Ala/Ser、Val/Ile、Asp/Glu、Thr/Ser、Ala/Gly、Ala/Thr、Ser/Asn、Ala/Val、Ser/Gly、Thy/Phe、Ala/Pro、Lys/Arg、Asp/Asn、Leu/Ile、Leu/Val、Ala/Glu和Asp/Gly，双向皆可。
如果需要，Fc区可以通过磷酸化、硫酸化、丙烯酸酯化、糖基化、甲基化、法尼基化(farnesylation)、乙酰化、酰胺化等进行修饰。
上述Fc衍生物是与本发明的Fc区具有相同生物活性或者改善的结构稳定性(例如对热、pH等的结构稳定性)的衍生物。
此外，这些Fc区可以得自从人和包括牛、山羊、猪、小鼠、兔、仓鼠、大鼠和豚鼠的其他动物中分离的天然形式，或者可以是得自转化的动物细胞或微生物的其重组体或衍生物。在本文中，它们可通过从人或动物有机体分离完整的免疫球蛋白并用蛋白水解酶对它们进行处理而从天然免疫球蛋白获得。木瓜蛋白酶将天然免疫球蛋白消化成Fab区和Fc区，而胃蛋白酶处理则导致产生pFc’和F(ab’)2片段。例如，可对这些片段进行尺寸排阻层析以分离Fc或pFc’。
此外，本发明的免疫球蛋白Fc区可以是具有天然糖链、与天然形式相比糖链增加或与天然形式相比糖链减少的形式，或者可以是去糖基化的形式。免疫球蛋白Fc糖链的增加、减少或去除可以通过本领域中常用方法完成，如化学法、酶促法和利用微生物的遗传工程方法。从Fc片段去除糖链导致与补体(C1q)的结合亲和力明显降低和抗体依赖性细胞介导的细胞毒性或补体依赖性细胞毒性的降低或丧失，从而不会诱导不必要的体内免疫应答。鉴于此，去糖基化或未糖基化形式的免疫球蛋白Fc区可更适于本发明的目的以作为药物使用。
本发明所使用的术语“去糖基化”意指从Fc区酶促地去除糖部分，而术语“未糖基化”意指Fc区是以无糖基化形式(例如在真核细胞如哺乳动物细胞中或者在原核生物如大肠杆菌中)产生的。
此外，免疫球蛋白Fc区可以是衍生自IgG、IgA、IgD、IgE和IgM的 Fc区，或者通过其组合或杂合制备而成。优选地，其衍生自IgG或IgM(它们是人类血液中最丰富的蛋白质之一)，最优选衍生自IgG(已知其延长配体结合蛋白质的半衰期)。
本发明所使用的术语“组合”意指编码相同来源的单链免疫球蛋白Fc区的多肽与不同来源的单链多肽连接从而形成二聚体或多聚体。即，二聚体或多聚体可以由选自以下的两种或更多种片段形成：IgG Fc片段、IgA Fc区段、IgM Fc区段、IgD Fc区段和IgE Fc区段。
在另一个实施方案中，本发明的GLP-1融合蛋白以同源二聚体的形式存在。
在另一个实施方案中，所述GLP-1融合蛋白进一步连接包含Sortase酶识别位点的片段(即本发明中所述的“ST”片段)。
术语“Sortase酶”，包括Sortase A、Sortase B，最早发现其具有将细菌的表面蛋白锚定在细胞壁上的作用。经鉴定发现，表面蛋白的“锚定信号(sorting signal)”由三个关键的部分组成——LPXTG序列、疏水序列和一个大多数情况下带正电荷的残基尾巴，其中LPXTG序列非常保守，能够被Sortase所识别。随后转肽酶的半胱氨酸(Cys)作为亲核基团进行攻击，作用在底物(如表面蛋白)C末端基序LPXTG的苏氨酸和甘氨酸之间的肽键，使其裂开(酰化作用)，进而产生一个酰基酶中间物。随后把多肽共价联结到细胞壁或菌毛亚基上发挥功能(脱酰作用)。
在本发明的一个实施方案中，利用Sortase A进行PEG的定点偶联。在一个具体实施方案中，使用了Sortase A的核心识别位点LPETG、LPETGG、LPETGGG。在另一个具体实施方案中，还可以在识别位点后添加不同亲和标签如:LPETGGHHHHHH、LPETGGWSHPQFEK等。
在另一个实施方案中，本发明的GLP-1融合蛋白通过重组方法产生，包括例如在合适的原核或真核宿主细胞中进行表达，然后通过常规技术从中分离出本发明的GLP-1融合蛋白。例如，可以首先通过化学合成法合成编码所述肽的核苷酸序列，随后将所述序列克隆到合适的表达载体中在合适的启动子控制下进行表达。或者，还可以采用诱变法如PCR诱变法从野生型GLP-1获得编码GLP-1的核苷酸序列、并且随后将所述序列以及构建融合蛋白的其他元件的序列克隆到合适的表达载体中在合适的启动子控制下进行表达。这些技术完全在本领域普通技术人员的能力范围之内，并 且在现有技术中有众多的教导。
合适的真核宿主细胞有哺乳动物细胞，例如CHO、COS、HEK 293、BHK、SK-Hep和HepG2。所述细胞优选地生长于适合表达本发明GLP-1融合蛋白的条件下。至于用于生产或分离本发明的GLP-1融合蛋白的试剂和条件，则没有任何特别的限制，本领域已知的或商业上可得到的任何体系均可应用。在一个优选的实施方案中，所述GLP-1融合蛋白通过本领域中已描述的方法获得。
有多种表达载体可用于制备GLP-1融合蛋白，其可选自真核和原核表达载体。原核表达载体可包括例如质粒如pRSET、pET和pBAD等，其中可采用的启动子有例如lac、trc、trp、recA或araBAD等。真核表达载体包括有：(i)用于在酵母中表达的载体如pAO,pPIC,pYES,pMET,其中可使用诸如AOX1,GAP,GAL1,AUG1等的启动子；(ii)用于在昆虫细胞中表达的载体如pMT,pAc[delta],plB,pMIB,pBAC等，其中可使用诸如PH,p10,MT,Ac5,OplE2,gp64,polh等的启动子；和(iii)用于在哺乳动物细胞中表达的载体如pSVL,pCMV,pRc/RSV,pcDNA3,pBPV等，以及源自病毒体系的载体如痘苗病毒、腺伴随病毒、疱疹病毒、逆转录病毒等，其中可使用诸如CMV,SV40,EF-1,UbC,RSV,ADV,BPV和β肌动蛋白等的启动子。在一个优选的实施方案中，所述GLP-1融合蛋白在原核或真核细胞体系中进行表达，并且使用经过密码子优化的编码序列。在一个优选的实施方案中，表达所述GLP-1融合蛋白的序列包含前导肽和/或信号肽，以利于所述GLP-1融合蛋白从细胞中分泌到细胞外，从而进行分离纯化。在另一个优选的实施方案中，表达所述GLP-1融合蛋白的序列不包含前导肽和/或信号肽，其不分泌到细胞外，通过裂解细胞对其进行分离纯化。
在另一个实施方案中，本发明所述的融合蛋白中，GLP-1蛋白与Fc区段是直接连接的，在另一个实施方案中，GLP-1蛋白与Fc区段是通过第一接头L1连接的；在又一个实施方案中，Fc区段与包含Sortase识别序列的片段是直接连接的，在另一个实施方案中，Fc区段与包含Sortase识别序列的片段是通过第二接头L(2)连接的。
在一个优选实施方案中，所述第一和/或第二接头L1和/或L2为包含1个、2个或3个以上不同的氨基酸组成的肽段。更优选地，所述第一接头L1和/或L2为包含A、T、G和/或S的柔性肽段，例如(GS)n，n为1至50的 整数，例如1、2、3、4、5、6、7、8、9或10；
例如所述第一接头L1选自：
GSGGGSGGGGSGGGGS(SEQ ID NO:9)；
GSGGGGSGGGGSGGGGSGGGGSGGGGS(SEQ ID NO:10)；
GGGGSGGGGSGGGGSGGGGS(SEQ ID NO:11)；
GSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS(SEQ ID NO:12)；
GGGSGGGSGGGSGGGSGGGS(SEQ ID NO:13)；
PRFQDSSSSKAPPPSLPSPSRLPGPSDTPILPQ(SEQ ID NO:14)；
SSSSKAPPPSLPSPSRLPGPSDTPILPQ(SEQ ID NO:15)；
SSSSKAPPPS(SEQ ID NO:16)；
SRLPGPSDTPILPQ(SEQ ID NO:17)和
GGGGSGGGGSGGGGSA(SEQ ID NO:18)。
又例如，所述第二接头L2选自：
GSGGGSGGGGSGGGGS(SEQ ID NO:9)；
GGGGSGGGGSGGGGSA(SEQ ID NO:18)；
GGGGS(SEQ ID NO:19)；
GGGGSGGGGS(SEQ ID NO:20)和
GGGGSGGGGSGGGGS(SEQ ID NO:21)。
GLP-1融合蛋白缀合物
本发明的GLP-1融合蛋白可以与一个或多个聚合物基团相缀合，形成GLP-1融合蛋白缀合物，其中所述聚合物缀合至融合蛋白的末端，例如N末端或者C末端。本文所用的“聚合物”优选是生理可接受的，其包括在水溶液或悬液中可溶、并且以药学有效量施用该GLP-1融合蛋白缀合物后对哺乳动物没有负面影响如副作用的聚合物。可在本发明中使用的聚合物没有特别的限制。所述聚合物通常优选具有2到约3000个重复单元。该聚 合物基团可以选自天然或合成聚合物，其实例包括、但不限于例如多糖、聚亚烷基二醇，如聚乙二醇(PEG)、聚丙二醇(PPG)、聚氧化乙烯(PEO)、乙二醇与丙二醇的共聚物、聚乙烯醇等或者其任何组合。在一个优选实施方案中，本发明GLP-1融合蛋白缀合物中使用一个或多个PEG基团进行缀合修饰。
在本发明中，所述聚合物并不限于特别的结构，其可以是线性的(如烷氧基PEG或双功能PEG)、分支或多臂的(如分叉PEG或连接到多元醇核心的PEG)、树枝状的或者可以具有可降解的连键。此外，聚合物的内部结构可以以任意数目的不同模式组织，其可选自均聚物、交替共聚物、无规共聚物、嵌段共聚物、交替三聚物、无规三聚物和嵌段三聚物等。所述聚合物还可包括聚(环氧烷)聚合物、聚马来酸、聚(D,L-丙氨酸)等。
在一些实施方案中，所述聚合物是聚乙二醇(PEG)或其衍生物例如甲氧基聚乙二醇(mPEG)。在本文中，如果没有特别指明，所述聚乙二醇(PEG)既包括末端基团为羟基也包括末端为其它基团的类型。所述其它基团包括但不限于烷氧基、环烷氧基、环烷基氧基、烯基、芳氧基或芳烷基氧基。这些PEG分子类型都是现有技术中已知的，并且在多肽修饰中常规使用。PEG侧链可以是线性的、分枝的、分叉的或者由多个臂组成，不同的聚乙二醇可以具有不同的聚合链长度和聚合结构。
本发明中对于所用PEG的分子量没有特别的限制，其分子量范围可以是0.1至200kDa，例如1至150kDa、2至100kDa、3至80kDa或4至50kDa，还可以是5至40kDa。另一些有用的PEG分子包括例如WO 03/040211、US 6,566,506、US 6,864,350和US 6,455,639中公开的那些。特别地，所述PEG具有通式HO-CH2CH2O-(CH2CH2O)n-CH2CH2-OH，其中n的范围是约5至4000。如上所述，本发明的PEG包括带其它末端基团的PEG，例如甲氧基PEG、分支PEG、分叉PEG等。合适的分支PEG可按照美国专利No.5,932,462中所述进行制备，该专利的全部公开内容通过参考并入本文。所述分叉PEG是指在靠近聚合物链一端的地方具有分支的PEG，分叉PEG的主链可以是直链或支链的。
本领域技术人员已知的是，在缀合了聚合物基团的生物活性分子中，随着所述聚合物基团分子量的增加，该缀合物分子的生物活性逐渐降低(Bailon et al.Rational design of potent,long-lasting form of interferon:A40kDa branched polyethylene glycol-conjugated interferonα-2a for  the treatment of hepatitis C.Bioconjugate Chem 2001；12:195-202；Bowen et al.Relationship between molecular mass and duration of activity of polyethylene glycol conjugated granulocyte colony-stimulating factor mutein.Experimental Hematology 1999；27:425-32；Bailon et al.PEG-modified biopharmaceuticals.Expert Opin Deliv.2009；6:1-16)。本领域技术人员还已知的是，随着所述聚合物基团分子量的增加，该缀合物分子的生物半衰期和/或血浆半衰期逐渐延长。
为了在长时间内提供稳定的治疗作用，另外为了减少给药频率，以提高患者依从性，在保留显著的GLP-1受体激动剂活性的同时，希望尽可能延长所述GLP-1融合蛋白缀合物的生物半衰期。因此，在一个实施方案中，本发明提供了具有延长的生物半衰期和显著的GLP-1受体激动剂活性的GLP-1融合蛋白缀合物。
在一个具体实施方案中，在本发明的GLP-1融合蛋白缀合物中，所述一个或多个聚合物基团(如PEG)的分子量为>＝1、>＝10、>＝20、>＝30、>＝40、>＝50、>＝60、>＝70、>＝80、>＝90、>＝100、>＝110、>＝120、>＝130、>＝140、>＝150或>＝160kDa，例如是5、10、20、30、40、50、60、70、80、90或100kDa，或者以上任意二数值之间的值。需要指出的是，当描述一种GLP-1融合蛋白缀合物中缀合聚合物基团的分子量时，如果该缀合物中有多个缀合聚合物基团，计算的是该缀合物中所有缀合聚合物基团的分子量总和，除非另外指明。
用于本发明中的聚合物是现有技术中已知的，其可通过多种途径得到，包括例如通过商业途径获得，如CarboMer,Inc.，J.T.Baker，The Dow Chemical Company等等，或者根据本领域中已知的方法自行制备，例如EP1245608中所述的。本发明并不局限于通过任何具体方法制得的聚合物。
缀合反应之后，可以通过合适的方法将缀合物分离出来。适用的方法包括例如超滤法、透析法或色谱法等，这些均在本领域普通技术人员的能力范围之内。
药物组合物
本发明的GLP-1融合蛋白或GLP-1融合蛋白缀合物可以有多种用途， 包括例如用于降低血糖。因此，本发明还提供了一种用于降低血糖的药物组合物，其中包含治疗有效量的本发明的融合蛋白或缀合物，以及任选地药学上可接受的载体。优选地，所述药物组合物可用于治疗糖尿病，更优选地用于治疗I型糖尿病和/或II糖尿病，尤其优选地用于治疗II型糖尿病。
本发明的融合蛋白或缀合物的治疗有效量取决于给药途径、受试者类型以及所考虑的具体哺乳动物的身体特征。这些因素及其与确定该量之间的关系是医药领域中技术人员熟知的。可调整该量和施用方法以达到最佳效力，从而将肽递送到受试者，但是将取决于医药领域技术人员熟知的因素例如体重、饮食、同时用药和其它因素。
本发明的药物组合物可在联合疗法中施用，即与一种或多种其它药剂联合应用，其中所述治疗剂一起施用，或者依次施用。在另一些实施方案中，所述其它药剂可在施用一种或多种本发明的融合蛋白或缀合物或者其药物组合物之前、期间或之后施用。可用于本发明的所述其它药剂包括例如可降低血糖的药剂，例如胰岛素、胰岛素类似物、糊精激动剂和缩胆囊素，和/或其它用于治疗疾病的化合物或组合物。优选地，这样的联合用药可以实现组合的、甚至协同的效果。
本文所使用的“可药用载体”、“药学上可接受的载体”或“生理可接受载体”可互换使用，包括任何和所有的生理上相容的盐、溶剂、分散介质、包衣、抗菌剂和抗真菌剂、等渗和吸收延迟剂等等中的一种或多种。在一些实施方案中，所述载体适于静脉内、肌内、皮下、胃肠外、脊髓或表皮给药(例如通过注射或灌注)。取决于给药途径，可用一定材料包被所述治疗剂，以保护该治疗剂免受酸和其它可能使该治疗剂失活的天然条件的作用。
施用时，本发明的药物制剂以可药用量在可药用组合物中施用。术语“可药用的”意为不干扰活性成分的生物活性效力的无毒物质。这样的制剂通常含有盐、缓冲剂、防腐剂、相容载体和任选的其它治疗剂，例如补充性免疫增强剂，包括佐剂，趋化因子和细胞因子。当使用在药物中时，所述盐应该是可药用的，但是非可药用盐可方便的用于制备其可药用盐，它们并不排除在本发明的范围之外。
如果需要，本发明的GLP-1融合蛋白或GLP-1融合蛋白缀合物可与可药用载体组合。本文所使用的术语“可药用载体”指一种或多种相容的固 体或液体填充剂、稀释剂或封装物质，其适于施用于哺乳动物例如人。术语“载体”表示有机或无机、天然或合成的成分，其与活性成分组合以便于应用。药物组合物的组分也能够以不存在可显著破坏所需药物疗效的相互作用的形式共混合。
优选地，本发明的药物组合物可以包含缓冲体系，优选地所述缓冲体系为pH为约3.0至约6.0的醋酸盐缓冲溶液，或者pH为约5.0至约9.0的磷酸盐缓冲溶液。在一些具体实施方案中，合适的缓冲剂包括乙酸盐；柠檬酸盐；硼酸盐以及磷酸盐。
任选地，所述药用组合物也可含有合适的防腐剂，例如：苯扎氯铵；氯叔丁醇；对羟基苯甲酸酯类和硫柳汞。
所述药用组合物可方便地以单位剂量形式存在，并可通过药学领域任何公知方法制备。所有方法包括将所述活性剂与载体联合的步骤，所述载体包含一种或多种辅助成分。通常，通过将所述活性化合物与液体载体、精细分割的固体载体或以上两者均一并密切地联合来制备所述组合物，必要时接着使产品成形。
适于胃肠外给药的药物组合物可以是包含一种或多种融合蛋白或缀合物的无菌水性或非水性制剂。在一些实施方案中，所述制剂与受试者的血液等渗。可根据已知方法使用合适的分散剂或润湿剂以及助悬剂配制此制剂。所述无菌注射制剂也可以是在无毒胃肠外可接受稀释剂或溶剂中的无菌注射溶液或悬液，例如1,3-丁二醇中的溶液。可使用的可接受载体和溶剂包括水、林格溶液和等渗氯化钠溶液。另外，无菌的不挥发性油常规用作溶剂或悬浮介质。为此，可使用任何温和的不挥发性油，包括合成的甘油单酯或甘油二酯。另外，脂肪酸如油酸可用于注射制剂。适于经口、皮下、静脉内、肌内等施用的载体配方可在Remington’s Pharmaceutical Sciences,Mack Publishing Co.,Easton,PA中找到。
本发明的融合蛋白或缀合物可与保护其避免快速释放的载体制备在一起，例如受控释放配方，包括植入物、透皮贴剂和微胶囊递送***。可使用生物可降解、生物相容性聚合物，例如乙烯乙酸乙烯酯、聚酐、聚乙醇酸、胶原、聚原酸酯和聚乳酸。现有技术中已知许多制备这样的配方的方法，参阅如Sustained and Controlled Release Drug Delivery Systems,J.R.Robinson,ed.,Marcel Dekker,Inc.,New York,1978等。
本发明的药物组合物可通过任何常规途径施用，包括注射或随时间逐渐输注。例如，所述施用可以是经口、静脉内、腹膜内、肌内、腔内、肿瘤内、或透皮。
本发明的药物组合物以有效量施用。“有效量”是本文提供的任何融合蛋白或缀合物的量，其单独或者与进一步的剂量和/或其它治疗剂一起产生期望的应答(例如在受试者中降低血糖水平)。这可包括仅仅暂时减缓糖尿病的发展，或者在一些实施方案中，还包括使糖尿病的发展永久性停止。
当然这样的量将取决于受治疗的具体疾病、所述疾病的严重程度、患者个体参数(包括年龄、生理状况、身高和体重)、治疗持续时间、同时进行的治疗的性质(如果有的话)、具体的给药途径以及在医疗卫生工作者知识范围内的类似因素。这些因素对本领域技术人员来说是公知的，仅仅使用常规实验就可获知。一般优选使用各个成分或其组合的最大剂量，也就是说根据合理医学判断的最高安全剂量。但是，本领域技术人员可以理解，患者可由于医学原因、心理原因或基本上任何其它原因而要求较低剂量或可容许的剂量。
在前述方法中所使用的药物组合物优选是无菌的，且在适于施用给患者的重量单位或体积单位中含有有效量的单独或与另一种制剂组合的融合蛋白或缀合物，以产生期望的应答，例如血糖的降低。
施用于受试者的融合蛋白或缀合物的剂量可根据不同参数进行选择，特别是根据所使用的给药模式和受试者的状态。其它因素包括所需的治疗时期。如果在所应用的最初剂量下受试者中的应答不足，可应用更高的剂量(或通过不同的更局部的递送途径实现的有效更高剂量)到患者容忍度允许的范围。
在一些实施方案中，本发明的药物组合物包含0.20mg/ml～5mg/ml GLP-1融合蛋白或和/或包含4mg/ml～40mg/ml GLP-1融合蛋白缀合物，优选地0.20mg/ml～5mg/ml GLP-1融合蛋白和/或包含4mg/ml～40mg/ml GLP-1融合蛋白缀合物，更优选地0.5mg/ml～2mg/ml GLP-1融合蛋白和/或包含10mg/ml～20mg/ml GLP-1融合蛋白缀合物。一般地，本发明的GLP-1融合蛋白或GLP-1融合蛋白缀合物的剂量范围可从约10μg/kg患者体重到约100,000μg/kg患者体重。在一些实施方案中，所述剂 量范围可从约0.1mg/kg到约20mg/kg。在另一些实施方案中，所述剂量范围可从约0.1mg/kg到5mg/kg、0.1mg/kg到10mg/kg或0.1mg/kg到15mg/kg。在另一些实施方案中，所述剂量范围可从约1mg/kg到5mg/kg、5mg/kg到10mg/kg、10mg/kg到15mg/kg或15mg/kg到20mg/kg。在另一些实施方案中，所述剂量约为0.1mg/kg、0.5mg/kg、1mg/kg、2mg/kg、3mg/kg、5mg/kg、7mg/kg、10mg/kg、12mg/kg、15mg/kg、17mg/kg、20mg/kg、25mg/kg或30mg/kg。在另一个实施方案中，所述剂量约为1mg/kg、3mg/kg、5mg/kg或6mg/kg。基于所述组合物，所述剂量可连续递送(例如通过连续泵)，或者以周期性间隔递送。在一些实施方案中，当静脉内给药时，本发明的GLP-1融合蛋白或GLP-1融合蛋白缀合物的剂量可以为0.1至20mg/kg或其中任何值。特定组合物多次给药的理想时间间隔可由本领域技术人员确定，而不需过度实验。所提供组合物的其它给药方案是本领域技术人员已知的，其中剂量、施用时间表、给药部位、给药模式等可以与前述有所不同。在一个实施方案中，所述剂量以静脉内给药。在另一个实施方案中，药剂施用方案是单次静脉内给药。
包含GLP-1融合蛋白或GLP-1融合蛋白缀合物(例如在药物组合物中)和使用说明的药盒也在本发明的范围内。所述药盒可另外含有至少一种其它的试剂，例如一种或多种其它降低血糖的药剂。在另一个实施方案中，药盒可包含载体，所述载体经区室化，以在其中紧密固定地容纳一个或多个容器装置或一系列容器装置(例如试管、管、烧瓶、瓶、注射器等)。所述药盒的成分可包装在水性介质中，或者为冻干形式。
本文提供的组合物可以是冻干形式或在水性介质中提供。
优选地，所述受试者是脊椎动物，更优选是哺乳动物，最优选是人，但也可以是其它动物，如家养动物(如狗、猫等)，家畜(如牛、羊、猪、马等)或实验动物(如猴子、大鼠、小鼠、兔子、豚鼠等)。
本发明中的GLP-1融合蛋白和/或GLP-1融合蛋白缀合物可以单独施用，但优选地作为药物组合物施用，其通常包括根据施用的计划方式所选的适合的药物赋形剂、稀释剂或载体。其可以通过任何适合的方式适用于需要治疗的患者/受试者。精确的剂量将取决于多个因素，包括该GLP-1融合蛋白和GLP-1融合蛋白缀合物的精确性质。
一些合适的施用方式包括(但不限于)口服、直肠、鼻部、局部(包 括口腔和舌下)、皮下、***或胃肠外的(包括皮下、肌肉、静脉、皮内、鞘内和硬脑膜外)施用。
在一些实施方案中，本发明的药物组合物包含等渗调节剂和/或防腐剂，优选地所述等渗调节剂为蔗糖、甘露醇、氯化钠和丙三醇中的一种或多种，以及所述防腐剂选自间甲酚、苄醇、对羟基苯甲酸甲酯、对羟基苯甲酸乙酯、对羟基苯甲酸丙酯和对羟基苯甲酸丁酯。本领域的技术人员通过使用例如等张赋形剂如生理盐水、林格氏注射液或乳酸林格氏注射液等，能够很好的制备本发明的GLP-1融合蛋白或GLP-1融合蛋白缀合物的适合的溶液。根据要求，还可以加入稳定剂、缓冲剂、抗氧化剂和/或其它一些添加剂。口服施用的药物组合物可以是片剂、胶囊、粉剂或口服液等形式。片剂可以包括固体载体，如明胶或辅剂。液体药物组合物通常包括液体载体，如水、石油、动物或植物油、矿物油或合成油。也可以包括生理盐水溶液、葡萄糖或其它糖溶液或二醇类，如乙二醇、丙二醇或聚乙二醇。在一些实施方案中，所述药物组合物为液体制剂和/或冻干制剂的形式，优选地所述冻干制剂含有冻干保护剂，更优选地所述冻干保护剂选自蔗糖、乳糖、甘露醇、海藻糖等糖。
本文所述的GLP-1融合蛋白和/或GLP-1融合蛋白缀合物优选以“治疗有效量”或“有效量”施用给受试者。所述组合物优选以“治疗有效量”施用给受试者，所述治疗有效量或有效量足以显示其对于所述受试者的益处。施用的实际量，以及施用的速率和时间过程会取决于所治疗者的自身情况和严重程度。治疗的处方(例如确定剂量等)由医护人员决定，并且通常考虑所治疗的疾病、患者个体的情况、递送部位、施用方法以及对于医生来说已知的其它因素。
在一些实施方案中，所述的GLP-1融合蛋白和/或GLP-1融合蛋白缀合物的剂量范围可以是30mg/kg体重/天至0.00001mg/kg体重/天，或者3mg/kg/天至0.0001mg/kg/天，或者0.3mg/kg/天至0.01mg/kg/天。
本发明还提供一种治疗疾病的方法，所述方法包括向有此需要的受试者施用治疗有效量的GLP-1融合蛋白和/或GLP-1融合蛋白缀合物。在一些实施方案中，所述疾病选自下述之中：餐后倾倒综合征、餐后高血糖、葡萄糖耐量降低、肥胖、进食紊乱、胰岛素耐受综合征、糖尿病和高血糖症。在一个优选的实施方案中，所述疾病是II型糖尿病。
本发明将通过以下实施例进行进一步说明，其不应以任何方式解释为进一步限制。本申请中所有引用的参考文献的全部内容(包含文章参考、授权的专利、公开的专利申请和共同未决的专利申请)均明确地通过引用并入本文。以下实施例中，若未具体指出，所用试剂和材料是能够商业获得的至少分析纯或与此相当级别的产品。
实施例
实施例1 GLP-1融合蛋白的制备
1.GLP-1融合蛋白真核表达载体的构建
本实施例中构建的GLP-1融合蛋白具有如下结构特征：GLP-1-L1-Fc-L2-ST。其中，GLP-1代表各种形式的GLP-1或其类似物，优选GLP-1序列同杜拉鲁肽(杜拉鲁肽，Trulicity TM)一致；L1和L2分别表示第一和第二接头序列，L1的序列是GGGGSGGGGSGGGGSA，L2的序列是GGGGSGGGGS、GGGGSGGGGSGGGGS、GSGGGSGGGGSGGGGS或无L2；Fc表示免疫球蛋白Fc区，Fc区来源于人IgG2(本发明中以Fc2表示)或IgG4(本发明中以Fc4表示)，优选Fc区与杜拉鲁肽(杜拉鲁肽，Trulicity TM)一致，为人IgG4的变体(Fc4)；ST表示Sortase A识别位点，ST序列为LPETG或LPETGGG。
不同GLP-1融合蛋白的氨基酸序列依据CHO细胞的密码子偏嗜性反向翻译成核苷酸序列。以全基因合成结合PCR扩增方法得到GLP-1融合蛋白的全长DNA片段，并通过两端HindIII-EcoRI酶切位点克隆至真核表达载体pFRL-DHFR上。载体pFRL-DHFR中包含人类巨细胞病毒启动子(hCMV-MIE Promoter)、SV40 PolyA、DHFR(二氢叶酸还原酶)等元件，可协助外源基因在真核细胞中高效、稳定地表达(附图1A)。
表1列举了实施例具体所构建的不同GLP-1融合蛋白。
表1.不同GLP-1融合蛋白的氨基酸序列特征
Figure PCTCN2019107709-appb-000001
1.GLP1-L1-Fc4-ST氨基酸序列
Figure PCTCN2019107709-appb-000002
GLP1-L1-Fc4-ST核苷酸序列:
Figure PCTCN2019107709-appb-000003
Figure PCTCN2019107709-appb-000004
2.GLP1-L1-Fc4-L2-ST氨基酸序列
Figure PCTCN2019107709-appb-000005
GLP1-L1-Fc4-L2-ST核苷酸序列:
Figure PCTCN2019107709-appb-000006
Figure PCTCN2019107709-appb-000007
3.GLP1-L1-Fc2-L2-ST氨基酸序列
Figure PCTCN2019107709-appb-000008
GLP1-L1-Fc2-L2-ST核苷酸序列:
Figure PCTCN2019107709-appb-000009
Figure PCTCN2019107709-appb-000010
4.GLP1-L1-Fc2-ST氨基酸序列
Figure PCTCN2019107709-appb-000011
GLP1-L1-Fc2-ST核苷酸序列:
Figure PCTCN2019107709-appb-000012
Figure PCTCN2019107709-appb-000013
5.GLP1-L1-Fc4-L2-STG3氨基酸序列:
Figure PCTCN2019107709-appb-000014
GLP1-L1-Fc4-L2-STG3核苷酸序列:
Figure PCTCN2019107709-appb-000015
Figure PCTCN2019107709-appb-000016
2.稳定细胞株筛选与发酵
2.1稳定细胞株筛选
采用电转染的方法将上述构建的质粒转染到宿主细胞-CHO DG44中。转染时将细胞密度调整到6×10 6cells/mL，体积0.8mL，质粒用量为40μg，质粒和细胞轻轻混匀后加入到电转杯中(Bio-Rad，4mm)，电转仪为Bio-Rad(GENE PULSER XCELL)，电转参数：电压：290V，脉冲时长：20ms。
电转后将细胞转入含有15mL恢复培养基的培养皿，培养48小时后接种96孔板，培养基换成含50nM MTX的筛选培养基。待克隆汇合率达到50％以上，采用点印迹方法筛选高表达克隆株。抗体为山羊抗人IgG Fc。将筛选出的表达量相对较高的克隆，依次转入24孔板、6孔板、T25细胞培养瓶和细胞培养摇瓶中扩大培养。
为了提高融合蛋白产量，采用递增MTX浓度的加压方法培养细胞。通过MTX对DHFR基因的抑制，实现DHFR基因和融合蛋白基因的共扩增。
2.2发酵
15L生物反应器(Applikon，Biobundle 15L)接种体积8L，接种密度为0.8-1.6×10 6个细胞/mL。设定培养参数如下：pH 6.95±0.15；DO45％；大泡通气供氧；转速80-140rpm。当培养到第5天、细胞密度约为10-12×10 6个细胞/mL时，降温至33℃培养。从培养的第四天开始补料，并每天检测残糖含量，降温前每天补糖至3g/L，降温后每天补至4g/L。发酵过程中，每天检测细胞计数和活力。在第13天，细胞活力在90％左右收样。
图1B至图4显示了GLP1-L1-Fc2-L2-ST稳定细胞株(克隆号：P1-3G3C2)在发酵过程中的生长曲线、细胞活力、镜检结果及各项工艺参数。从图1B可见，在发酵到第九天时，细胞密度最高，为20.5×10 6/mL；第13天收样时，细胞密度为15.5×10 6/mL，活力为86.6％。镜检 细胞形态较好，没有明显死细胞(图2A和图2B)。图3和图4显示了发酵时糖、NH 4 +和乳酸(Lac)代谢情况。
图11至图14显示了GLP1-L1-Fc4-L2-STG3在补料分批发酵过程中的生长曲线、细胞活力、镜检结果及各项工艺参数。从图11B可见，在发酵到第八天时，细胞密度最高，为19.9×10 6个细胞/mL；第14天收样时，细胞密度为16.0×10 6个细胞/mL，活力为92％。镜检细胞形态较好，没有明显死细胞(图12A和图12B)。图13和图14显示了发酵时糖代谢和pH变化情况。
通过分子互作仪(Pall Life Science，Qke)检测发酵上清GLP1-L1-Fc2-L2-ST蛋白的含量为1.8g/L，GLP1-L1-Fc4-L2-STG3的含量为1.19g/L；SDS-PAGE电泳检测GLP1-L1-Fc2-L2-ST分子的完整性，GLP1-L1-Fc2-L2-ST为双链Fc融合蛋白，其完整分子量为61.6kDa(不含糖)。如图5所示，在非还原和还原状态下，GLP1-L1-Fc2-L2-ST分子量与理论分子量基本一致。同时，利用SDS-PAGE电泳检测GLP1-L1-Fc4-L2-STG3蛋白变动情况(图15)，可以看出发酵第12到14天目的蛋白产量有明显增加，上清液中目的蛋白为主要成分，杂蛋白含量少。表达的蛋白通过ELISA实验鉴别，结果显示为阳性。
2.3纯化与检测
GLP1-L1-Fc2-L2-ST和GLP1-L1-Fc4-L2-STG3发酵液上清经两步纯化：亲和层析(Uni mab，纳微)、阴离子交换层析(QHP，BXK)。纯化后蛋白经SDS-PAGE和SEC(体积排阻色谱)检测。根据SDS-PAGE的结果(图6A和图16A)，目的蛋白大小与理论分子量基本保持一致；SEC使用Agilent Advancebio SEC 300A，2.7μm，7.8*300mm色谱柱，利用50mM Tris+150mM NaCl+10％乙腈，pH7.0进行等度洗脱(柱温为30℃，流速0.5mL/min，进样器温度4.0℃，进样体积50μg，样品分析时间为35min)。从图6B可见，GLP1-L1-Fc2-L2-ST蛋白的保留时间在14.6min，两步纯化后纯度为97.2％；从图16B可见，GLP1-L1-Fc4-L2-STG3蛋白的保留时间在14.49min，两步纯化后纯度为98.412％。
实施例2 20kDa PEG和40kDa PEG与GLP1-Fc融合蛋白的连接
所有构建的GLP1-Fc融合蛋白的C端均含有Sortase A酶核心识别 序列-LPETG，因此可被Sortase A酶特异性识别。在Sortase A酶的作用下，T和G之间的酰胺键被切断，T与Sortase酶184位C的巯基反应形成硫酯键中间体，随后被N端带有聚Gly的GGGAA-PEG攻击，最终将不同分子量大小的PEG连接到蛋白的C端。
GLP1-L1-Fc2-L2-ST和GLP1-L1-Fc2-ST蛋白的PEG化反应体系如下：将20kDa-或40kDa GGGAA-PEG(GGGAA购自上海吉尔生化有限公司，20kDa-或40kDa GGGAA-PEG由北京键凯科技有限公司合成)用缓冲液溶解并调节pH到8.0，然后将蛋白分别以1:15和1:10的投料比加入到50mM Tris 150mM NaCl pH8.0缓冲体系中，再加入Sortase A酶和10mM CaCl 2，分别在反应的不同时间点取样，90min后加入EDTA终止反应。
GLP1-L1-Fc4-L2-STG3蛋白的PEG化反应体系如下：将40kDa GGGAA-PEG(GGGAA购自上海吉尔生化有限公司，40kDa GGGAA-PEG由北京键凯科技有限公司合成)用缓冲液溶解并调节pH到7.0，然后将蛋白：PEG以1:15(摩尔比)的投料比加入到20mM Tris150mM NaCl pH7.0缓冲体系中，再加入Sortase A酶和CaCl2(10mM)，其中每毫克目的蛋白加入50IU所述酶，所述目的蛋白终浓度为6mg/mL。30min后加入EDTA终止反应。
上述连接产物通过SDS-PAGE和反相色谱法(RPC)检测连接率(交联率)，其中单交联是指融合蛋白二聚体中仅一个单体连接上PEG分子，双交联是指融合蛋白二聚体中两个单体均连接上PEG分子。反相色谱法检测使用Waters
Figure PCTCN2019107709-appb-000017
BEH C4，3.5μm，4.6*250mm色谱柱，通过0.1％TFA水和0.1％TFA乙腈进行梯度洗脱，柱温为50℃，检测波长280nm，流速0.5mL/min，进样器温度4.0℃，进样体积20μg，样品的分析时间为50min。GLP1-L1-Fc2-L2-ST和GLP1-L1-Fc2-ST蛋白的检测结果如图7所示：随着反应时间的延长，20kDa和40kDa的PEGylation化产物逐渐增多，到90min时，PEGylation化产物达到最大并进行终止反应，此时GLP1-L1-Fc2-L2-ST蛋白20kDa的总交联率为75.62％，其中单交联55.57％，双交联20.05％；40kDa的总交联率为40.15％，其中单交联36.36％，双交联3.79％；GLP1-L1-Fc2-ST蛋白40kDa的总交联率为60.83％，其中单交联率为48.46％，双交联率为12.37％。从40kDa PEG交联结果可以看出，在相同的反应体系里，GLP1-L1-Fc2-ST的交联效率要远高于 GLP1-L1-Fc2-L2-ST。
GLP1-L1-Fc4-L2-STG3蛋白的检测结果如图17所示。随着反应时间的延长，40kDa的PEGylation化产物逐渐增多，到30min时，PEGylation化产物达到最大并终止反应，此时GLP1-L1-Fc4-L2-STG3蛋白交联40kDaPEG的总交联率为80.86％，其中单交联44.087％，出峰时间10.543min，双交联36.782％，出峰时间9.698min。
交联后纯化和检测
GLP1-L1-Fc2-L2-ST经40kDa PEG交联后样品经Butyl HP疏水层析除去未反应的GGGAA-PEG、Sortase A酶并通过阴离子交换层析，去除未交联的底物蛋白,将底物的残余量控制在1％以内。如图8A所示，GLP1-L1-Fc2-L2-ST经40kDa PEG交联后样品通过阴离子交换层析可分为两个峰P1、P2。RPC检测结果显示(图8B)，P1峰主要为双交联产物(双交联90.84％，单交联8.4％，底物0.15％)，P2峰主要为单交联产物(双交联8.56％，单交联90.12％，底物0.73％)。未交联GLP1-L1-Fc2-L2-ST的保留时间为17.3min，单、双交联产物的保留时间分别为22.5和23.2min。
GLP1-L1-Fc4-L2-STG3经40kDa PEG交联后样品通过阴离子交换层析(SuperQ 5PW，TOSOH)可将单双交联以及底物分开，层析图如图18A所示。根据SEC检测结果显示(图18B和C)，P1峰主要为双交联产物(双交联97.172％，单交联2.766％，底物0％)，P2峰主要为单交联产物(双交联19.722％，单交联79.754％，底物0.158％)。单、双交联产物的保留时间分别为10.63和9.8min。
实施例3 GLP1-L1-Fc2-L2-ST交联蛋白的活性检测
GLP-1可通过与GLP-1受体(GLP-1R)结合，激活腺苷酸环化酶(AC)，产生cAMP，进而激活cAMP依赖的转录因子cAMP反应元件结合蛋白(cAMP response element binding protein，CREB)，后者与cAMP反应元件(cAMP response element，CRE)结合后激活下游基因的转录。
本实施例的检测包含下列步骤：将HEK293/CRE-Luc/GLP1R细胞(购自GenScript，#M00562)培养至对数生长期，胰酶消化后用完全培养基重悬，调整细胞密度至1×10 6个/mL，50μL/孔加入白色96孔 板中；杜拉鲁肽及供试品用完全培养基稀释至400ng/mL，并按4倍倍比稀释，共9个梯度，50μL/孔加入细胞，37℃，5％CO 2孵育5h；加入Bright-Glo Luciferase(promega)每孔100μL，避光显色10min。使用全波段荧光酶标仪(TECAN，200PRO)，测荧光值。
以标准品和供试品的荧光值为纵坐标，以浓度的对数值为横坐标，通过Softmax分析软件进行四参数方程拟合，处理数据。根据以下公式计算样品的相对活性。
Figure PCTCN2019107709-appb-000018
图9显示了GLP1-L1-Fc2-L2-ST分别与20kDa PEG和40kDa PEG交联后的活性检测结果。与杜拉鲁肽相比，未交联底物(GLP1-Fc)的相对活性为98.10％。PEG交联后活性均有下降，其中，20kDa交联产物(图中表示为PEG(20kDa)-GLP1-Fc)的相对活性为40.38％；40kDa交联产物(图中表示为PEG(40kDa)-GLP1-Fc)的相对活性为32.85％。
图19显示了GLP1-L1-Fc4-L2-STG3及与40kDa PEG交联后产物的活性检测结果。与GLP1-L1-Fc2-L2-ST的PEG缀合产物类似，与杜拉鲁肽相比，GLP1-L1-Fc4-L2-STG3经40kDa PEG缀合后的产物(图中表示为PEG(40kDa)-GLP1-Fc)的活性有一定程度的降低，相对活性为21.51％。
实施例4 GLP-1融合蛋白或其PEG缀合物的体内降糖活性
C57BL/6(购自北京维通利华有限公司)小鼠每笼饲养5只，喂以普通小鼠生长饲料，自由饮水。光照时间12h(08：00am-20:00pm)，黑暗时间12h(20:00pm-08:am)。
(1)GLP1-L1-Fc2-L2-ST的实验结果：
将40只6-8周龄健康雄性C57BL/6小鼠，分为5组，每组8只。分别为：(1)GLP1-Fc组(交联前GLP-1-L1-Fc2-L2-ST底物)；(2)杜拉鲁肽组；(3)40kDaPEG GLP1-Fc组(GLP-1-L1-Fc2-L2-ST交联40kDa PEG)；(4)20kDa PEG GLP1-Fc组(GLP-1-L1-Fc2-L2-ST交联20kDa PEG)；(5)对照组。其中，(1)至(4)组按3mg/kg给药，对照组给予生理盐水。分别于给药后24、144、168、192、216、240h进 行IPGTT实验，实验步棸如下：小鼠禁食6h，检测血糖值、体重，禁食期间自由饮水；6h后腹腔给予2.0g/kg葡萄糖溶液，体积为10ml/kg，检测葡萄糖负荷后10、30、60、90、120min的血糖值，绘制糖耐量曲线，计算血糖曲线下面积(AUC)；数据以均数±标准误(Mean±SEM)形式表示，采用Graphpad prism7.0统计软件分析数据，并用Mann-whiteney统计分析差异性。
如图10A、10B、10C、10D、10E和10F所示，在单次给药后6天内，各实验组小鼠血糖和糖耐量实验曲线下面积(AUC)相对于对照组均有显著降低。7d后，杜拉鲁肽和GLP1-Fc组与对照组比较无明显差异，说明GLP1-Fc和杜拉鲁肽的药效能够维持6d；如图10G、10H、10I、10J、10K和10L所示，PEG(20kDa)-GLP1-Fc在单次给药后第8天仍具有显著的糖耐受作用，9天后糖耐量实验曲线下面积(AUC)与对照组比较无差异，药效能够维持8天；PEG(40kDa)-GLP1-Fc在单次给药后第9天仍具有显著的糖耐受作用，10天后糖耐量实验曲线下面积(AUC)与对照组比较无差异，药效能够维持9天。
图10M显示了C57BL/6J小鼠给药后每天的体重变化。与对照组对比，在24h内各组体重均有下降。其中尤以杜拉鲁肽和未交联PEG的GLP1-Fc组体重下降明显。值得注意的是，无论PEG(20kDa)-GLP1-Fc还是PEG(40kDa)-GLP1-Fc均可显著降低药物引起的体重快速下降的风险，说明PEG化修饰可明显改善该类药物临床应用的副作用，具有更好的安全性。
(2)GLP1-L1-Fc4-L2-STG3的实验结果：
将18只8-10周龄健康雄性C57BL/6小鼠，分为3组，每组6只。分别为：(1)对照组；(2)杜拉鲁肽组；(3)PEG(40kDa)-GLP1-Fc组(GLP1-L1-Fc4-L2-STG3交联40kDa PEG)。其中，(2)和(3)组按等活性给药(即根据图19体外检测的活性进行质量换算)，杜拉鲁肽的给药剂量是0.76mg/kg，PEG(40kDa)-GLP1-Fc的给药剂量是3.5mg/kg，对照组给予生理盐水。分别于给药后1、5、6、7、8天进行IPGTT实验，实验步棸如下：小鼠禁食6h，检测血糖值、体重，禁食期间自由饮水；6h后腹腔给予2.0g/kg葡萄糖溶液，体积为10ml/kg，检测葡萄糖负荷后10、30、60、90、120min的血糖值，计算血糖曲线下面积(AUC)；数据以均数±标准误(Mean±SEM)形式表示，采用Graphpad prism7.0统计软件分析数据，并用Mann-whiteney统计分析 差异性。
如图20A所示，在单次给药后5天内，各实验组小鼠血糖和糖耐量实验曲线下面积(AUC)相对于对照组均显著降低。5d后，杜拉鲁肽组与对照组比较无明显差异，说明杜拉鲁肽的药效能够维持5d；PEG(40kDa)-GLP1-Fc在单次给药后第7天仍具有显著的糖耐受作用，8天后糖耐量实验曲线下面积(AUC)与对照组比较无差异，这表明PEG(40kDa)-GLP1-Fc的药效能够维持7天。
图20B显示了C57BL/6小鼠给药后的体重变化。与对照组对比，在24h内两组体重均有所下降。其中尤以杜拉鲁肽组体重下降较明显。值得注意的是，PEG(40kDa)-GLP1-Fc也可以显著降低药物引起的体重快速下降的风险，进一步说明了PEG化修饰可明显改善该类GLP-1受体激动剂类药物临床应用的副作用。
实施例5 GLP1-L1-Fc4-L2-STG3交联产物在STZ诱导的II型糖尿病小鼠体内的降糖活性检测
II型糖尿病小鼠模型构建
C57BL/6(购自北京维通利华有限公司)小鼠每笼饲养5只，喂以普通小鼠生长饲料，自由饮水。光照时间12h(08：00am-20:00pm)，黑暗时间12h(20:00pm-08:00am)。将30只8-10周龄健康雌性C57BL/6小鼠进行60mg/kg STZ诱导，连续给药4天，2周后进行血糖值检测，非空腹血糖值≧16.7mmol/L为诱导成功II型糖尿病小鼠。
过程
将24只II型糖尿病小鼠，分为4组，每组6只。分别为：(1)对照组；(2)杜拉鲁肽-1mg/kg组；(3)PEG(40kDa)-GLP1-Fc(GLP1-L1-Fc4-L2-STG3交联40kDa PEG)-1mg/kg组；(4)PEG(40kDa)-GLP1-Fc(GLP1-L1-Fc4-L2-STG3交联40kDa PEG)-3mg/kg组。其中，杜拉鲁肽的给药剂量是1mg/kg，PEG(40kDa)-GLP1-Fc的给药剂量分别是1mg/kg和3mg/kg，对照组给予生理盐水。分别于0、1、2、3、4、5、6、7、8、9、10、11、12天检测血糖值、称量体重，不禁食，自由饮水；数据以均数±标准误(Mean±SEM)形式表示，采用Graphpad prism7.0统计软件分析数据，并用Mann-whiteney统计分析差异性。
结果
如图21A所示，在单次给药后，各实验组小鼠血糖相对于对照组均有显著降低。在第6天时，杜拉鲁肽-1mg/kg组与对照组相比，血糖差异仍然很显著(P＝0.0022)，但到了7天时，血糖值与对照组相比已经无明显差异(P＝0.9654)。这表明杜拉鲁肽的降血糖效果最多能够维持6d。
相反，在单次给药PEG(40kDa)-GLP1-Fc-1mg/kg后，第10天仍具有显著降血糖作用(P＝0.0381)，直到第11天时才与对照组相近(P＝0.1143)；而PEG(40kDa)-GLP1-Fc-3mg/kg则在单次给药后第11天仍具有显著降血糖作用(P＝0.0381)，直到第12天时才与对照组相比无明显差异(P＝0.0762)。以上结果表明PEG(40kDa)-GLP1-Fc具有显著更好的长效降糖效果，能够有效用于II型糖尿病的治疗。
进一步的还观察了给药后糖尿病小鼠的体重变化，如图21B所示，与对照组对比，所有实验组小鼠的体重均有下降。其中尤以杜拉鲁肽组体重下降较明显，其中最多时相比对照组小鼠体重减少达到2.5g，PEG(40kDa)-GLP1-Fc的两个剂量组则对体重的影响明显较小，PEG(40kDa)-GLP1-Fc-1mg/kg组小鼠体重最多与对照组相比降低0.3g，PEG(40kDa)-GLP1-3mg/kg组小鼠体重最多与对照组相比降低1.1g。由此进一步说明，PEG(40kDa)-GLP1-Fc在各剂量下用于糖尿病的治疗时同样能显著改善药物引起的体重快速下降，说明PEG化修饰可明显改善该类药物临床应用的副作用，同时有效提高患者的依从性。
综上，PEG化的GLP1-Fc能促进胰岛β细胞分泌胰岛素，抑制外源性葡萄糖摄入所引起的暂时性血糖升高，促进葡萄糖利用；经PEG修饰后，GLP1-Fc蛋白表现出更持久的降血糖作用，并且药物的副作用更小，避免了GLP-1类降体重药常常导致的体重快速下降的问题，具有良好的临床应用前景。
本发明已通过各个具体实施例作了举例说明。但是，本领域普通技术人员能够理解，本发明并不限于各个具体实施方式，普通技术人员在本发明的范围内可以作出各种改动或变型，而仍不背离本发明的精神和范围。这样的改动和变型均在本发明的范围之内。

Claims (15)

  1. 一种GLP-1受体激动剂的融合蛋白，其包括GLP-1受体激动剂以及与其连接的增加体内半衰期的融合伴侣(FP)；其中所述GLP-1受体激动剂与所述融合伴侣直接连接或者通过第一接头L1连接；优选地，所述第一接头L1为包含1、2、3、4、5、6、7、8、9、10个或更多个氨基酸的肽段；更优选地，所述第一接头L1为包含A、T、G和/或S的柔性肽段，例如(GS)n，其中n为1-50的整数，如1、2、3、4、5、6、7、8、9或10；
    进一步优选地，所述第一接头L1选自：
    GSGGGSGGGGSGGGGS(SEQ ID NO:9)；
    GSGGGGSGGGGSGGGGSGGGGSGGGGS(SEQ ID NO:10)；
    GGGGSGGGGSGGGGSGGGGS(SEQ ID NO:11)；
    GSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS(SEQ ID NO:12)；
    GGGSGGGSGGGSGGGSGGGS(SEQ ID NO:13)；
    PRFQDSSSSKAPPPSLPSPSRLPGPSDTPILPQ(SEQ ID NO:14)；
    SSSSKAPPPSLPSPSRLPGPSDTPILPQ(SEQ ID NO:15)；
    SSSSKAPPPS(SEQ ID NO:16)；
    SRLPGPSDTPILPQ(SEQ ID NO:17)和
    GGGGSGGGGSGGGGSA(SEQ ID NO:18)。
  2. 根据权利要求1所述的融合蛋白，其中所述GLP-1受体激动剂是全长、截短或变体形式的GLP-1或GLP-1类似物，例如是Exendin-4、GLP-1(1-36)、GLP-1(1-37)、GLP-1(7-36)、GLP-1(7-37)、利拉鲁肽、利司那肽、阿必鲁肽、度拉糖肽或索玛鲁肽。
  3. 根据权利要求1或2所述的融合蛋白，其中所述融合伴侣(FP)为全长、截短或变体形式的免疫球蛋白Fc区段、白蛋白、XTEN或转铁蛋白，优选所述Fc区段是人免疫球蛋白Fc区段；优选地，所述免疫球蛋白Fc区段由选自CH1结构域、CH2结构域、CH3结构域和CH4 结构域的一至四个结构域构成；还优选地，所述免疫球蛋白Fc区段是来自于IgG、IgA、IgD、IgE或IgM的Fc区段，更优选为IgG Fc区段；进一步优选地，所述Fc区段是来自IgG1、IgG2、IgG3或IgG4的Fc区段；此外还优选所述IgG Fc区段具有降低的ADCC效应和/或CDC效应和/或与FcRn受体增强的结合亲和力。
  4. 根据权利要求1至3中任一项所述的融合蛋白，其中所述融合伴侣(FP)还与包含Sortase酶识别位点的一段氨基酸序列ST直接连接或者通过第二接头L2连接，所述Sortase酶例如是Sortase A或Sortase B；优选地，所述ST包含Sortase A的核心识别位点LPXTG，其中X是任意氨基酸，例如LPETG、LPETGG或LPETGGG；还优选地，所述ST还包含与Sortase酶识别序列连接的亲和标签，这样的ST序列例如是LPETGGHHHHHH或LPETGGWSHPQFEK；
    其中，所述第二接头L2为包含1、2、3、4、5、6、7、8、9、10个或更多个氨基酸的肽段；更优选地，所述第二接头L2为包含A、T、G和/或S的柔性肽段，例如(GS)n，其中n为1-50的整数，如1、2、3、4、5、6、7、8、9或10；
    进一步优选地，所述第二接头L2选自：
    GSGGGSGGGGSGGGGS(SEQ ID NO:9)；
    GGGGSGGGGSGGGGSA(SEQ ID NO:18)；
    GGGGS(SEQ ID NO:19)；
    GGGGSGGGGS(SEQ ID NO:20)；和
    GGGGSGGGGSGGGGS(SEQ ID NO:21)。
  5. 根据权利要求4所述的融合蛋白，其具有如下结构：GLP-1-L1-FP-L2-ST，其中
    GLP-1表示GLP-1受体激动剂，并且GLP-1受体激动剂、L1、L2、FP和ST具有如权利要求4中相同的定义，其中L1和L2任一个或两者可以不存在。
  6. 一种缀合物，其中一个、二个或更多个亲水聚合物分子连接至权利要求1至5中任一项所述融合蛋白的末端，优选地经转肽反应连接至所述Sortase酶识别位点；特别地，所述融合蛋白可以是单体形式或二聚体形式。
  7. 权利要求6的缀合物，其中所述一个、二个或多个亲水聚合物分子各自独立地选自多糖和聚亚烷基二醇，如聚丙二醇和聚乙二醇；所述聚亚烷基二醇可以是末端封端的，例如经烷氧基如甲氧基封端的；和/或所述聚亚烷基二醇是直链的或支链的；例如所述聚亚烷基二醇是支链的，例如是支链的聚乙二醇，尤其是经甲氧基封端的支链聚乙二醇；所述聚亚烷基二醇的分子量可以是>＝1、>＝10、>＝20、>＝30、>＝40、>＝50、>＝60、>＝70、>＝80、>＝90、>＝100、>＝110、>＝120、>＝130、>＝140、>＝150或>＝160kDa，例如是5、10、20、30、40、50、60、70、80、90或100kDa，或者以上任意二数值之间的值；优选地所述亲水性聚合物分子的分子量是20kDa或40kDa。
  8. 一种制备权利要求6或7所述缀合物的方法，其中包括使权利要求1至5中任一项所述的融合蛋白与Sortase酶(如Sortase A或Sortase B)和亲水聚合物分子接触，其中所述亲水聚合物分子一端带有可被Sortase酶酰胺化的氨基。
  9. 根据权利要求8所述的方法，其中所述亲水聚合物分子的一端带有聚Gly，例如带有GGGAA。
  10. 一种药物组合物，其包含有效量的权利要求1至5中任一项所述的融合蛋白和/或权利要求6或7所述的缀合物，以及任选地药学上可接受的载体。
  11. 根据权利要求10的药物组合物，其用于降低血糖或体重，例如用于治疗糖尿病，更具体地用于治疗I型糖尿病和/或Ⅱ型糖尿病，尤其是用于治疗II型糖尿病。
  12. 根据权利要求10或11所述的药物组合物，其为液体制剂形式或冻干制剂形式。
  13. 权利要求1至5中任一项所述的融合蛋白或者权利要求6或7所述的缀合物在制备用于降低血糖或体重的药物中的用途，例如所述药物用于治疗糖尿病，包括I型糖尿病和II型糖尿病，尤其是II型糖尿病。
  14. 一种降低血糖或体重的方法，其包括对有此需要的对象施用权利要求1至5中任一项所述的融合蛋白或者权利要求6或7所述的缀合物，例如用于治疗糖尿病，包括I型糖尿病和II型糖尿病，尤其是II 型糖尿病。
  15. 一种试剂盒，其包含权利要求1至5中任一项的融合蛋白、权利要求6或7所述的缀合物、和/或权利要求10至12中任一项所述的药物组合物，以及任选的使用说明书。
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