WO2018077098A1 - 一种用于治疗肠道疾病的融合蛋白 - Google Patents

一种用于治疗肠道疾病的融合蛋白 Download PDF

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WO2018077098A1
WO2018077098A1 PCT/CN2017/106727 CN2017106727W WO2018077098A1 WO 2018077098 A1 WO2018077098 A1 WO 2018077098A1 CN 2017106727 W CN2017106727 W CN 2017106727W WO 2018077098 A1 WO2018077098 A1 WO 2018077098A1
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glp
fusion protein
seq
mutant
protein according
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French (fr)
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温晓芳
黄岩山
杨志愉
姚高峰
陈永露
王学莲
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浙江道尔生物科技有限公司
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Priority to US16/302,642 priority Critical patent/US10815286B2/en
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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
    • A61K47/50—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
    • A61K47/62—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 a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P39/00—General protective or antinoxious agents
    • 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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/72—Receptors; Cell surface antigens; Cell surface determinants for hormones
    • C07K14/723—G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH receptor
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • 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
    • C07K2319/31—Fusion polypeptide fusions, other than Fc, for prolonged plasma life, e.g. albumin

Definitions

  • the invention relates to the field of biomedical technology, in particular to a fusion protein for treating intestinal diseases.
  • GLP-2 is a 33 amino acid single-chain polypeptide formed by transcriptional and post-translational processing of the proglucagon gene, and has a molecular weight of about 3.9 KD. GLP-2 belongs to the gut hormone, which is secreted by the endocrine-secreting L cells in the small intestine. The proglucagon is converted to proglucagon by proglucagon prohormone convertase 1/3. obtain. In addition, some brain nerve cells also secrete GLP-2. In the gut, GLP-2 promotes the growth and development of the normal small intestine by acting on a specific G protein-coupled receptor (GLP-2 receptor). Studies have shown that GLP-2 can protect and repair damaged intestinal mucosa in various intestinal diseases and increase blood supply to the intestine.
  • GLP-2 receptor G protein-coupled receptor
  • GLP-2 is highly susceptible to degradation by in vivo protease degradation and is easily cleared by the glomerulus due to its small molecular weight.
  • the biological half-life of GLP-2 ( 1–33 ) is about 7 min, because GLP-2 can be dipeptidyl peptidase IV (DPP-IV) from the amino group in blood and tissues.
  • Alanine (Ala2) residue at position 2 is cleaved to produce 31 amino acid residues of low activity GLP-2 (3 to 33) , or is hydrolyzed by various endopeptidases into inactive small molecules.
  • Peptide Currently, GLP-2 mutant Teduglutide (trade name) developed by NPS Pharmaceuticals Inc.
  • Titreglutide replaces the second amino acid (Ala) of native GLP-2 with glycine (Gly) and is administered subcutaneously once a day (0.05 mg/kg) for short bowel syndrome in adult-dependent parenteral nutrition.
  • Al second amino acid
  • Gly glycine
  • GLP-2 small molecular weight GLP-2 still needs to be combined with various long-acting methods.
  • small peptides by simply extending the half-life, such as fusion of human serum albumin (HSA), transferrin or human immunoglobulin Fc fragments, the fusion of long-acting proteins is not significant. It reduces the degradation of the protein of interest by endogenous proteases, so it is difficult to obtain the desired results.
  • HSA human serum albumin
  • transferrin transferrin
  • human immunoglobulin Fc fragments the fusion of long-acting proteins is not significant. It reduces the degradation of the protein of interest by endogenous proteases, so it is difficult to obtain the desired results.
  • protease-sensitive amino acids are generally replaced by site-directed mutagenesis; or chemical modification methods such as Liraglutide (Novo Nordisk A/S) Liraglutide (trade name) ), the cross-linked fatty acid chain (palmitic acid) can prevent GLP-1 from degrading to a certain extent, and the fatty acid chain can bind to human serum albumin, prolonging the half-life.
  • Liraglutide Novo Nordisk A/S Liraglutide (trade name)
  • the cross-linked fatty acid chain can prevent GLP-1 from degrading to a certain extent, and the fatty acid chain can bind to human serum albumin, prolonging the half-life.
  • obtaining a stable GLP-2 mutant that is effective against protease degradation and merging it with a long-acting carrier protein or cross-linking with a polymer such as PEG is currently an effective solution.
  • a flexible linking peptide chain such as a short peptide chain formed by a combination of G and S
  • this linked peptide chain is not suitable for all cases, and simply extends the linked peptide chain. The length is likely to increase the risk of protease hydrolysis.
  • GLP-2 For GLP-2, it is imperative to obtain a long-acting GLP-2 receptor agonist with good pharmacokinetics and good pharmacokinetics by obtaining a linked peptide chain which can reduce the loss of GLP-2 activity and prevent protease hydrolysis.
  • Glucagon-like peptide-2 refers to a hormone secreted by a part of intestinal endocrine cells (L-cells), which is obtained by in vivo cleavage of proglucagon. In addition to the intestines, the brain also secretes GLP-2, the main effect of which is probably to control food absorption. GLP-2 functions by binding to the GLP-2 receptor and functions to treat or prevent intestinal diseases and intestinal damage. GLP-2 consists of 33 amino acids, and the amino acid sequence of native GLP-2 is as follows:
  • GLP-2 receptor agonists in the present invention include natural GLP-2 and GLP-2 derivatives or mutants.
  • GLP-2 receptor agonist refers to a polypeptide which binds to a GLP-2 receptor and functions to activate a GLP-2 receptor, and the physiological activity of the GLP-2 receptor agonist should be compatible with natural GLP- 2 is the same or similar.
  • GLP-2 derivative or “GLP-2 mutant” may be substituted herein to mean having at least 80% amino acid sequence homology to native GLP-2 and having the same or similar to native GLP-2.
  • Physiologically active polypeptide Some of the amino acid residues may even be chemically modified, such as ⁇ -methylation, ⁇ -hydroxylation, deamination, and the like.
  • the GLP-2 derivative of the present invention can be produced by N-terminal amino acid substitution, C-terminal amino acid addition, deletion or peptide chain modification of native GLP-2.
  • the amino acid to be added or substituted may be a natural L-amino acid or a non-natural D-amino acid or the like.
  • Long-acting carrier protein The long-acting carrier protein of the present invention refers to a protein which can prolong the half-life function of an active protein in an animal and has no or negligible effector function.
  • long-acting carrier proteins include, but are not limited to, Fc constant regions of immunoglobulins (IgG), human serum albumin or transferrin.
  • IgG immunoglobulins
  • human serum albumin human serum albumin
  • transferrin transferrin
  • PEG polyethylene glycol
  • immunoglobulin refers to a protein that participates in the protection of immunity by selective action against the action of an antigen.
  • An immunoglobulin consists of two identical light chains and two identical heavy chains. Light and heavy chains include variable and constant regions. based on There are two types of light chains in the constant region amino acid sequence: kappa and lambda (Coleman et al, Fundamental Immunology, Second Edition, 1989, 55-73). According to the characteristics of the heavy chain constant region, immunoglobulins are classified into five types: IgG, IgA, IgD, IgE, IgM. Among them, IgG is classified into IgG1, IgG2, IgG3, and IgG4 subtypes.
  • IgG1 and IgG4 are the most used antibody types. This is because the Fc fragment of IgG1 and IgG4 can circulate with high affinity binding to the FcRn receptor, and thus the half life is very long (average about 21 days).
  • Fc Mutant An Fc mutant referred to herein is a mutant of an IgG constant region Fc fragment of natural human or other mammalian origin, in which a specific amino acid is substituted or inserted into a specific amino acid.
  • the mutation site does not disrupt the FcRn binding region, so these mutations do not affect the use of the Fc mutant as a long acting protein vector.
  • the present invention provides a fusion protein having the following structure:
  • R is a GLP-2 receptor agonist
  • P is a long-acting carrier protein
  • L is a linked peptide chain and has the following formula:
  • X is selected from any one of P, GP, GGP or NGGP;
  • G/S is a peptide chain of any combination of G and S, and has a length of 5-25 amino acids;
  • W 1 and W 2 are respectively 19 kinds of any natural amino acid residues other than Cys;
  • R is a GLP-2 receptor agonist, including native GLP-2 and GLP-2 derivatives.
  • GLP-2 derivatives include GLP-2 mutants obtained by amino acid mutations, deletions, insertions or amino acid modifications, unnatural amino acid substitutions, etc., based on the native GLP-2 sequence.
  • the GLP-2 receptor agonist is a mutant in which the alanine at position 2 of the native GLP-2 sequence (SEQ ID NO: 1) is replaced by glycine (SEQ ID NO: 2).
  • the GLP-2 receptor agonist may further be selected from a GLP-2 mutant in which the alanine at position 2 of the native GLP-2 sequence is replaced by glycine and the 1-6 amino acid residues are deleted at the C-terminus. , as SEQ ID NO: 3 and SEQ ID NO: 4.
  • the GLP-2 receptor agonist is a mutant in which the alanine is replaced by glycine (SEQ ID NO: 2); in another embodiment, the The GLP-2 receptor agonist is a GLP-2 mutant in which the second alanine is replaced by glycine and the C-terminus is deleted by 6 amino acid residues (SEQ ID NO: 4).
  • the protein of the present invention may have various derivatives, which may be, but are not limited to, Different forms of salts, modified products, and the like, such as amino groups, carboxyl groups, hydroxyl groups, and sulfhydryl groups of the polypeptide are further modified.
  • the long acting carrier protein includes, but is not limited to, a constant region Fc portion of a mammalian-derived immunoglobulin IgGl or IgG4 or human serum albumin or transferrin.
  • the long-acting carrier protein is selected from the group consisting of human immunoglobulin IgG1 (SEQ ID NO: 5) or the constant region Fc portion of IgG4 (SEQ ID NO: 6) and mutants thereof, and more preferably, the long-acting effect
  • the carrier protein is selected from the constant region Fc mutant of the N297 aglycosylated human immunoglobulin IgGl or IgG4.
  • the long acting carrier protein is selected from the constant region Fc mutant of human immunoglobulin IgG1 and has the sequence set forth in SEQ ID NO: 7; in another embodiment of the invention, The long acting carrier protein is selected from the constant region Fc mutant of human immunoglobulin IgG4 and has the sequence set forth in SEQ ID NO:8.
  • a further S228P mutation is to attenuate the chain exchange phenomenon characteristic of IgG4 antibodies.
  • L is a linked peptide chain linking a GLP-2 mutant to a long acting carrier protein.
  • L is a linked peptide chain and has the following structure:
  • X is selected from any one of P, GP, GGP or NGGP; W 1 and W 2 are respectively 19 kinds of any natural amino acid residues other than Cys; u is 0 or 1; m is an integer of 1-20 .
  • the G/S is a peptide chain of any composition of G and S, and has a length of 5-25 amino acids; preferably, G/S is GGGGS (SEQ ID NO: 98), GGGGGS (SEQ ID NO: 99) or GGGGSGGGGS ( Any one of SEQ ID NO: 100).
  • the W1, W2 are each independently selected from the following amino acids A, N, D, Q, E, K, P, S, R. More preferably, W 1 , W 2 are each independently selected from the group consisting of A, P, S, E, Q, and D.
  • the L is selected from the group consisting of SEQ ID NO: 30 ((GGGGS) 2GPP GPA), SEQ ID NO: 31
  • Exendin-4 (HGDGSFSDEMNTILDNLAARDFINWLIQTKITD, SEQ ID NO: 101) still shows degradation of N-terminal dipeptide in yeast, and is significantly improved after knocking out yeast STE13 gene (Prabha L et al., Protein Expr Purif. 2009: 155-61. Identification of the dipeptidyl aminopeptidase responsible for N-terminal clipping of recombinant Exendin-4 precursor expressed in Pichia pastoris.), which indicates that for the GLP-2 mutant, the second A mutation to G may still be insufficient. Resistance to degradation by dipeptidase.
  • the inventors have found that when the GLP-2 mutant is expressed in yeast, the problem of internal degradation of the sequence far from the N-terminus is more prominent. Therefore, even if the GLP-2 mutant remains intact at the N-terminus after inactivation of the yeast STE13 gene, the sequence is internal. Degradation still reduces activity.
  • G/S sequences are flexible linker peptides well known to those skilled in the art and are commonly used to link two different proteins.
  • the inventors have found that the addition of the G/S peptide alone (most commonly the GGGGS unit) does not significantly reduce the loss of biological activity of the GLP-2 receptor agonist, but is added to the (G-W1-W2) unit. After that, the loss of activity of the GLP-2 mutant decreased in a gradient as the length of the GGGGS unit increased.
  • the addition of the (GW 1 -W 2 ) unit alone does not only reduce the loss of biological activity of the GLP-2 receptor agonist, but has a tendency to further reduce activity.
  • the inventors have found that only a combination of G/S-(GW 1 -W 2 )m can significantly reduce the loss of biological activity of the GLP-2 mutant.
  • the linked peptide chain provided by the present invention can also be added to the XSSGAPPPS unit.
  • the fusion of the XSSGAPPPS sequence at the C-terminus of the GLP-2 mutant increases the stability of a part of the GLP-2 mutant and does not affect the biological activity of the GLP-2 mutant, thereby prolonging the GLP-2.
  • the half-life of activity in the body When secreted and expressed in methanol yeast GS115, PSSGAPPPS was fused with the C-terminally deleted GLP-2 mutant, which reduced the enzymatic band of the GLP-2 mutant and increased the expression level.
  • the present inventors have found that the (XSSGAPPPS) u -G/S-(GW 1 -W 2 ) m form of the ligated peptide chain can effectively reduce GLP- compared to the GLP-2 mutant fusion protein RP without the linker peptide. 2 Enzymatic hydrolysis and loss of biological activity of the mutant. u can be 0 or 1, depending on the form of the GLP-2 mutant.
  • Another outstanding advantage of the present invention is that it overcomes the formation of multimers formed by fusion of GLP-2 and Fc fragments, such as GLP-2 MIMETIBODY (TM ), which readily forms non-covalent dimers (Baker AE et al, The dimerization of glucagon-like peptide-2 MIMETIBODY TM is linked to leucine-17 in the glucagon-like peptide-2 region.J Mol Recognit.2012 25 (3):. 155-64).
  • GLP-2 MIMETIBODY TM
  • the linker peptide acts as a bridge between two different domains and plays a critical role; and for different proteins, different linker peptides are generally required. This is because different active proteins have different high-order structures and different molecular weights, so optimization of the linker peptide is necessary in the formation of the fusion protein.
  • the flexible peptide chain composed of G and S has been successfully applied to a variety of proteins, it is not sufficient to achieve the desired effect.
  • the inventors have obtained a series of linked peptide chains for fusion between a GLP-2 mutant and a long-acting carrier protein after extensive experimental screening.
  • peptide chains are effective in reducing the loss of activity of GLP-2 receptor agonists and alleviating protease water solution.
  • GLP-2 mutants were used in combination with a linker peptide to form a fusion protein sequence (Table 1).
  • the GLP-2 mutant sequence in the table only indicates the mutated amino acid site.
  • A2G indicates that the second position in the native GLP-2 sequence (SEQ ID NO: 1) is replaced by G, and ⁇ C indicates the C-terminal deletion, ⁇ C.
  • the latter number indicates the number of amino acids deleted, such as ⁇ C6 representing a 6 amino acid deletion of the GLP-2 mutant at the C-terminus.
  • hIgG4 and hIgG1 Fc fragment mutants also label only the mutated amino acid sites, and h represents a human source.
  • Another aspect of the invention provides a nucleotide sequence encoding the fusion protein.
  • a further aspect of the invention provides a recombinant expression vector carrying a gene encoding a fusion protein encoding gene of the invention.
  • recombinant expression vectors include, but are not limited to, eukaryotic expression vectors, such as pPIC9 plasmid, pPIC9K plasmid, pPICZalpha A plasmid, pcDNA3.1, etc., prokaryotic expression vectors such as pET41a plasmid, pET32a plasmid or other self-constructed foreign genes must be expressed. Plasmids and the like of the elements required for the recombinant protein can be used to construct a preparation for expressing the present invention.
  • a further aspect of the invention provides a method of expressing the fusion protein.
  • the method for expressing the fusion protein is to introduce a recombinant expression vector containing the fusion gene-encoding gene sequence into a host cell, and to induce or constitutively express the fusion protein.
  • the expression host may be a yeast, an Escherichia coli or a mammalian cell or the like, preferably a yeast, and particularly preferably Pichi pastoris.
  • the purification treatment of the fusion protein of the present invention includes salting out, precipitation, ultrafiltration, chromatography and the like and combinations of these techniques.
  • the chromatography can be carried out by affinity chromatography, ion exchange, hydrophobic, reverse phase chromatography techniques.
  • the protein and its derivative in the present invention may be used singly or in the form of a pharmaceutical preparation in which one or more pharmaceutically acceptable excipients are added together.
  • the excipients include water, sugars such as lactose, and conventional excipients in the pharmaceutical field. Dextrose, etc., alcohols such as sorbitol, mannitol, xylitol, amino acids and the like.
  • the pharmaceutical composition of the present invention may further comprise an excipient and a bacteriostatic agent.
  • the fusion protein of the present invention can be prepared as an injection.
  • the drug of this dosage form can be prepared according to conventional methods in the pharmaceutical field.
  • the pharmaceutical preparations may be presented in single or multiple dose containers, such as sealed ampoules or vials.
  • the lyophilized preparation is prepared by freeze-drying the liquid preparation, and a sterile, pyrogen-free liquid solvent such as water for injection is added before use.
  • the fusion protein and the derivative thereof or the pharmaceutical composition thereof of the invention can be used as an intestinal protective hormone for intestinal damage repair and compensatory diseases caused by various causes, such as tumor radiotherapy and chemotherapy, tumor targeted drug therapy, severe wounding Intestinal mucosal damage caused by factors such as burns, total parenteral nutrition, inflammatory bowel disease, and treatment of patients with extensive bowel resection and small bowel transplantation.
  • the fusion protein and its derivative in the present invention can be administered by intravenous injection, subcutaneous injection or the like. Treatment includes the use of a single dose or a combined dose over a period of time.
  • Figure 1 is a SDS-PAGE electrophoresis pattern of GLP-2 mutant induction screening; wherein lanes 1-8 in A are SEQ ID NOS: 9-11, 23-27, respectively; lanes 1-11 in B are SEQ ID NO: 12-22; C is an expression-inducing sample obtained by transforming GS115 into a blank pPIC9 plasmid; M is a low molecular weight protein MARKER: 97, 66, 44, 29, 21, 14 KD.
  • Figure 2 is a SDS-PAGE electrophoresis pattern of a purified sample of GLP-2 mutant; wherein lanes 1-9 are the results of purification of SEQ ID NOS: 12-20.
  • M is a low molecular weight protein MARKER: 97, 66, 44, 29, 21, 14 KD.
  • Figure 3 is a graph showing the effect of GLP-2 mutant fusion protein on rat intestinal weight/body weight changes.
  • the various GLP-2 mutant genes, the ligated peptide chain genes, and the Fc genes of human IgG1 and IgG4 were designed by gene synthesis according to the amino acid sequence of Table 1 and the yeast preferred codon.
  • the complete fusion gene was amplified by the method of SOE-PCR (splicing by overlap extension, SOE). Those skilled in the art can easily derive the gene sequence according to the amino acid sequence of Table 1.
  • PCR reaction system 50 ⁇ L: 5 ⁇ L 10 ⁇ Pfu buffer, dNTP mix (200 ⁇ mol/L), upstream primer (0.5 ⁇ mol/L), downstream primer (0.5 ⁇ mol/L), 0.1 ⁇ g template, 0.5 ⁇ L Pfu DNA polymerase ( 5U/ ⁇ L), supplemented to 50 ⁇ L with sterile water. All PCR reaction procedures were: pre-denaturation at 94°C for 2 minutes, denaturation at 94°C for 30 seconds, annealing at 58°C for 30 seconds, extension at 72°C for 3 minutes, 27 cycles, 72°C Store at 4 ° C after 5 minutes of extension. The PCR product was detected by agarose gel electrophoresis, and the experimental results were in agreement with the theory.
  • the fusion gene was cloned into the same digested yeast expression vector pPIC9 (Life technologies, USA) by XhoI and EcoRI endonucleases at both ends to obtain a recombinant expression plasmid.
  • the linearized recombinant plasmid was transformed into GS115 by electroporation using methanol yeast Pichia pastoris GS 115 (His-) as the host strain.
  • the cells were cultured on a histidine-deficient screening plate medium at 30 ° C for 3 days until a single colony appeared.
  • the transformed recombinant yeast single colony was inoculated into 10 ml of BMGY liquid medium, cultured at 30 ° C, 250 rpm for 24 hours, left to stand overnight, the supernatant was discarded, and 10 ml of BMMY liquid medium containing 1% methanol was added, 30 ° C, 250 rpm. Inducing expression. A strain with a relatively high expression was selected as an expression strain. For details, see the instruction manual (Pichia Expression). Kit. For Expression of Recombinant Proteins in Pichia pastoris.Catalog no.K1710-01).
  • the highly expressed strain obtained by the screening was inoculated into YPD liquid medium (yeast leaching powder glucose medium), and cultured at 30 ° C, 220 rpm for 20-24 h to an OD 600 of 10 to 20, as an upper tank seed liquid.
  • the cultured seed solution was connected to a Biostat B Twin MO 5L fermentor and the medium was configured according to the Life Technologies Pichia Fermentation Process Guidelines.
  • the inoculation amount was 10%
  • the fermentation temperature was set to 30 ° C
  • the pH was 5.0.
  • methanol was added to induce expression.
  • the expression phase controlled the fermentation temperature at 25 ° C and induced the cans for 72 hours.
  • the induced expression map is shown in Figure 1.
  • a GLP-2 mutant fusion protein comprising only a G/S flexible peptide or an XSSGAPPPS unit or a combination of both, like SEQ ID NO: 10, is similar to SEQ ID NO: 9 without any linked peptide chain.
  • -11 and 23-27 have no significant effect on the degradation during fermentation, and after addition of the (G-W1-W2)m unit (Fig. 1B), as in SEQ ID NO: 12-22, during the fermentation process No significant degradation bands were detected.
  • Example 3 Isolation and purification of fusion protein
  • Example 2 The fermentation broth obtained in Example 2 was centrifuged at 8000 rpm for 30 minutes at room temperature, and the supernatant was collected and applied to a Diamond Protein A BestChrome column equilibrated with buffer A (0.5 M NaCl, 20 mM PB, pH 7.0). GLON (Shanghai) Biotechnology Co., Ltd.), once again equilibrated with buffer A, eluted with elution buffer B (0.1M Gly-HCl, pH 3.0), eluted peak plus 1/10 peak volume of neutralizing solution (1 M Tris-HCl, pH 8.0) was adjusted to pH.
  • buffer A 0.5 M NaCl, 20 mM PB, pH 7.0
  • eluted with elution buffer B 0.1M Gly-HCl, pH 3.0
  • eluted peak plus 1/10 peak volume of neutralizing solution (1 M Tris-HCl, pH 8.0
  • the purified protein was identified by physical and chemical properties such as SDS-PAGE, SEC-HPLC, RP-HPLC, and mass spectrometry. As shown in Figure 1, the recombinant protein expressed in GS115 is consistent with the theoretical molecular weight.
  • the GLP-2 mutant fusion protein to which the (XSSGAPPPS) u -G/S-(GW 1 -W 2 ) m linked peptide chain was added showed a single band on SDS-PAGE (Fig. 1B) with molecular weights between 44 and 29 KD.
  • the strip should be a degradation zone.
  • the GLP-2 mutant fusion protein (SEQ ID NOS: 12-22) with a G/S-(GW 1 -W 2 )m linked peptide chain showed a single peak on the SEC column and no significant polymer or A degradation peak with a small molecular weight, and a GLP-2 mutant fusion protein (SEQ ID NO: 9) without a linker chain, a plurality of degradation peaks and a polymer peak thereof; and SEQ ID NO: 28 further containing an XSSGAPPPS unit -29 also has a significant tendency to reduce the polymer (Table 3).
  • Monomer ⁇ Here, an active molecule formed by covalently forming two Fc chains.
  • the purified sample was subjected to mass spectrometry, and it was revealed that the GLP-2 mutant fusion protein to which the (XSSGAPPPS) u -G/S-(GW 1 -W 2 ) m linked peptide chain was added detected a main peak consistent with the theoretical molecular weight ( ⁇ 90%), other peaks contain N-terminal degradation peaks ( ⁇ 10%) that are not detectable by electrophoresis and RP-HPLC; while other GLP-2 mutant fusion proteins have a small peak content ( ⁇ 20%) consistent with theoretical molecular weight. Most of them are mass peaks smaller than the theoretical molecular weight.
  • Example 5 In vitro cytological activity assay
  • the in vitro cytological activity of the GLP-2 fusion protein was detected using a luciferase reporter assay.
  • Cloning the GLP-2R gene To the mammalian cell expression plasmid pCDNA3.1, the recombinant expression plasmid pCDNA3.1-GLP-2R was constructed, and the full-length gene of luciferase was cloned into the pCRE-EGFP plasmid, and the EGFP gene was replaced to obtain pCRE-Luc. Recombinant plasmid.
  • the pCDNA3.1-GLP-2R and pCRE-Luc plasmids were transfected into CHO cells at a ratio of 1:10, and the stably transfected expression strains were selected to obtain a recombinant GLP-2R/Luc-CHO stably transfected cell line.
  • the cells were cultured in a 10-cm cell culture dish in DMEM/F12 medium containing 10% FBS and 300 ⁇ g/ml G418. When the confluency was about 90%, the culture supernatant was discarded, and after 2 ml trypsin digestion for 2 min, Add 2 ml of DMEM/F12 medium containing 10% FBS and 300 ⁇ g/ml G418, transfer to a 15 ml centrifuge tube, centrifuge at 800 rpm for 5 min, discard the supernatant, and add 2 ml of DMEM containing 10% FBS and 300 ⁇ g/ml G418. /F12 medium was resuspended and counted.
  • DMEM/F12 medium containing 10% FBS Place 100 ⁇ l per well in a 96-well plate, ie 30,000 cells per well. After adherence, replace with DMEM/F12 medium containing 0.1% FBS. to cultivate.
  • Example 3 After discarding the supernatant in the 96-well plate, the recombinant protein purified in Example 3 was diluted with DMEM/F12 medium containing 0.1% FBS to a specified concentration, and added to the cell culture well, 100 ⁇ l/well. After 6 hours of stimulation, the test was performed. Detection was carried out according to the instructions of the lucifersae reporter kit (Ray Biotech, Cat: 68-LuciR-S200). The results are shown in Table 3. Differences in the effects of different linked peptide chains on steric hindrance and degradation of GLP-2 mutants result in differences in cellular activity. For SEQ ID NOs: 9-11 or 23-27, the production of the polymer (Example 4) may further attenuate cytological activity.
  • Example 6 In vivo animal model drug efficacy test
  • GLP-2 It has been shown to have significant anti-apoptotic effects on intestinal crypt cells and to improve intestinal mucositis caused by chemotherapy drugs. This example compares the in vivo physiological activities of various GLP-2 mutant fusion proteins by a rat model.
  • SD rats were divided into 8 groups, 6 in each group: 1) Fluorouracil (5-FU) + GLP-2 mutant 1 (SEQ ID NO: 16); 2) Fluorouracil (5-FU) + GLP -2 mutant 2 (SEQ ID NO: 17); 3) fluorouracil (5-FU) + GLP-2 mutant 3 (SEQ ID NO: 18); 4) fluorouracil (5-FU) + GLP-2 mutant 4 (SEQ ID NO: 9) 5) Fluorouracil (5-FU) + GLP-2 mutant 5 (SEQ ID NO: 10); 6) Fluorouracil (5-FU) + GLP-2 mutant 6 (SEQ ID NO) :25); 7) Fluorouracil (5-FU) + normal saline; 8) normal saline.
  • the body weight of each rat was recorded and started 7 days before the 5-FU injection, group 1) to group 6) subcutaneously injected GLP-2 mutant at a dose of 25 nmol/kg once a day; group 7) and 8) Inject the same volume of normal saline. From day 4 to day 7, group 1) to group 7) were intraperitoneally injected with 5-FU daily at a dose of 50 mg/kg, and all rats were sacrificed 24 hours after the last 5-FU injection. The rat abdominal cavity was cut, and the small intestine and large intestine of the rat were cut out in an ice bath, rinsed with physiological saline, and the length was measured and the wet weight was measured. The ratio of weight to body weight of the small intestine was calculated. The protective effect of different GLP-2 mutant fusion proteins on the intestine is shown in Fig. 3.
  • GLP-2 mutants 1 to 6 significantly attenuated 5-FU damage to the small intestine compared to the 5-FU+ saline control group, in which the efficacy of mutants 1-3 was relative to the mutant.
  • 4-6 has a better effect, indicating that the efficacy of the protein in vivo corresponds to the in vitro cytological activity.

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Abstract

本发明提供了一种用于治疗肠道疾病的融合蛋白，具有如下结构： R-L-P 其中： R为GLP-2受体激动剂； L为连接肽链； P为长效载体蛋白，本发明提供的融合蛋白具有显著的生物活性及体外稳定性。

Description

一种用于治疗肠道疾病的融合蛋白 技术领域
本发明涉及生物医药技术领域，具体涉及一种用于治疗肠道疾病的融合蛋白。
背景技术
GLP-2是胰高血糖素原(proglucagon)基因经转录、翻译后处理加工形成的33个氨基酸的单链多肽，分子量约为3.9KD。GLP-2属于肠道激素，主要由小肠内分泌L细胞在人体摄取食物后分泌，经胰高血糖素原转化酶1/3(proglucagon prohormone convertase 1/3)酶切胰高血糖素原(proglucagon)获得。另外，部分大脑神经细胞也会分泌GLP-2。在肠道中，GLP-2通过作用于特异性G蛋白偶联受体(GLP-2受体)促进正常小肠的生长和发育。研究表明，GLP-2能保护和修复各种肠道疾病中损伤的肠粘膜，增加肠道的血液供应。它在肿瘤放化疗、严重创(烧)伤、全肠外营养、炎症性肠病等因素引起的肠粘膜损伤、失血性休克、广泛肠切除、小肠移植等方面均具有很高的临床应用价值。
GLP-2极易被体内蛋白酶降解而丧失活性，并且由于分子量小容易通过肾小球而被清除。在人体血液循环中，GLP-2(1～33)的生物半衰期约为7min，原因为GLP-2在血液和组织中可被二肽基肽酶IV(dipeptidyl peptidase IV，DPP-IV)从氨基端第2位的丙氨酸(Ala2)残基处切割而产生31个氨基酸残基的低活性的GLP-2(3～33)，或被多种内肽酶水解成无活性的小分子短肽。目前，美国NPS制药公司开发的可以抵抗DPP-IV降解作用的GLP-2突变体替度鲁肽(Teduglutide，商品名为
Figure PCTCN2017106727-appb-000001
)已在美国上市。替度鲁肽将天然GLP-2的第2位氨基酸(Ala)用甘氨酸(Gly)替代，每天皮下注射给药1次(0.05mg/kg)用于成人依赖性肠外营养的短肠综合征(short bowel syndrome，SBS)的治疗(Clin Drug Investig.Teduglutide：a guide to its use in short bowel syndrome.2015：35(5)：335-40)。
替度鲁肽虽然已成功上市，但其缺点是需要每天给药，而且Ala突变为Gly虽然能降低DPP-IV对GLP-2的降解，但对于内肽酶的降解却无明显作用。小分子量的GLP-2要达到长效的目的，仍需与各种长效方法结合。对于GLP-2类小肽而言，通过单纯的常规延长半衰期的方法，如融合人血清白蛋白(HSA)、转铁蛋白或人免疫球蛋白Fc片段等，由于融合的长效蛋白并不能显著降低目的蛋白质被内源性蛋白酶降解的作用，因此很难获得理想的结果。目前一般通过定点突变的方法，将对蛋白酶敏感的氨基酸进行替换；或者采用化学修饰的方法，如诺和诺德(Novo Nordisk A/S)公司的利拉鲁肽(Liraglutide，商品名
Figure PCTCN2017106727-appb-000002
)，其上交联的脂肪酸链(棕榈酸)能在一定程度上使GLP-1避免降解，同时脂肪酸链能结合到人 血清白蛋白上，延长了半衰期。
总而言之，获得一种稳定的、能有效抵抗蛋白酶降解的GLP-2突变体并将其与长效载体蛋白融合或与PEG等高分子交联，是目前的有效解决办法。然而，小肽与长效载体蛋白融合后，最显著的一个缺点就是活性下降。常规的方法可采用柔性的连接肽链(如G和S组合形成的短肽链)来减少活性损失，然而，这种连接肽链并不适用于所有的情况，同时单纯的延长连接肽链的长度很可能会增加蛋白酶水解的风险。对于GLP-2而言，筛选获得一种既能减少GLP-2活性损失，又能防止蛋白酶水解的连接肽链，获得药代和药效良好的长效GLP-2受体激动剂成为当务之急。
发明内容
术语解释：
“胰高血糖素样肽-2(GLP-2)”指由部分肠道内分泌细胞(L-细胞)分泌的一种激素，由胰高血糖素原(proglucagon)经过体内剪切获得。除了肠道，脑部也会分泌GLP-2，主要作用很可能是控制食物吸收。GLP-2通过结合GLP-2受体起作用，起到治疗或预防肠道疾病、肠道损伤的功能。GLP-2由33个氨基酸组成，天然GLP-2的氨基酸序列如下：
GLP-2(1-33)
Figure PCTCN2017106727-appb-000003
本发明中的GLP-2受体激动剂，包括天然GLP-2及GLP-2衍生物或突变体。
在本发明中，“GLP-2受体激动剂”指可结合GLP-2受体而起到激活GLP-2受体功能的多肽，GLP-2受体激动剂的生理学活性应与天然GLP-2相同或类似。
“GLP-2衍生物”或“GLP-2突变体”在本文中可以相互替换，指与天然GLP-2相比具有至少80％氨基酸序列同源性，且具有与天然GLP-2相同或类似生理学活性的多肽。其中部分氨基酸残基甚至可经化学修饰，例如α-甲基化、α-羟化、脱氨基等。优选地，本发明的GLP-2衍生物可通过天然GLP-2的N-末端氨基酸取代、C-末端氨基酸添加、缺失或肽链修饰而制备。添加或取代的氨基酸可以是天然的L-氨基酸或者非天然的D-氨基酸等。
“长效载体蛋白”：本发明的长效载体蛋白指能达到延长活性蛋白在动物体内半衰期功能，且自身的效应功能没有或者可以忽略的蛋白。目前常用的长效载体蛋白包括但不限于：免疫球蛋白(IgG)的Fc恒定区、人血清白蛋白或转铁蛋白等。作为长效载体的另一种形式，聚乙二醇(PEG)也是常用的手段。
“免疫球蛋白”(IgG)指通过选择性针对抗原作用而参与身体保护免疫性的蛋白质。免疫球蛋白由两条相同的轻链和两条相同的重链构成。轻链和重链包括可变区和恒定区。基于 恒定区氨基酸序列的差异，有两种类型的轻链：κ和λ型(Coleman等，Fundamental Immunology，第二版，1989，55-73)。根据重链恒定区的特征，免疫球蛋白分为五种类型：IgG、IgA、IgD、IgE、IgM。其中IgG分为IgG1、IgG2、IgG3和IgG4亚型。目前的单克隆抗体药物中，IgG1和IgG4是使用最多的抗体类型。这是由于IgG1和IgG4的Fc片段能以高亲和力结合FcRn受体而获得循环，因此半衰期很长(平均约为21天)。
“Fc突变体”：本文所指的Fc突变体为天然人源或其他哺乳动物来源的IgG恒定区Fc片段中，特定氨基酸被取代或插入特定氨基酸而形成的突变体。突变位点并不破坏FcRn结合区域，因此这些突变不影响Fc突变体作为长效蛋白载体的应用。
为了克服现有技术中存在的问题，本发明提供了一种融合蛋白，具有如下结构：
R-L-P                                                  式I
其中：
R为GLP-2受体激动剂；
P为长效载体蛋白；
L为连接肽链且具有如下公式：
(XSSGAPPPS)u-G/S-(G-W1-W2)m                            式II
其中X选自P、GP，GGP或NGGP中的任一种；
G/S为G和S任意组成的肽链，长度为5-25个氨基酸；
W1，W2分别为除Cys之外的19种任意天然氨基酸残基；
u为0或1；m为1-20的整数。
式I中，R为GLP-2受体激动剂，包括天然GLP-2及GLP-2衍生物。GLP-2衍生物包括在天然GLP-2序列基础上进行氨基酸突变、缺失、插入或氨基酸修饰、非天然氨基酸替换等方式获得的GLP-2突变体。优选的，所述GLP-2受体激动剂为天然GLP-2序列(SEQ ID NO：1)的第2位丙氨酸被甘氨酸替代形成的突变体(SEQ ID NO：2)。在本发明中，所述GLP-2受体激动剂还可以选自天然GLP-2序列的第2位丙氨酸被甘氨酸替代且C末端缺失1-6个氨基酸残基的GLP-2突变体，如SEQ ID NO：3和SEQ ID NO：4。在本发明的一个实施例中，所述的GLP-2受体激动剂为第2位丙氨酸被甘氨酸替代的突变体(SEQ ID NO：2)；在另一个实施例中，所述的GLP-2受体激动剂为第2位丙氨酸被甘氨酸替代且C末端缺失6个氨基酸残基的GLP-2突变体(SEQ ID NO：4)。本发明中的蛋白质可以有各种衍生物，这些衍生物可以是但不局限于 其不同形式的盐、修饰产物等，如在多肽的氨基、羧基、羟基、巯基上再进行修饰。
式I中，所述长效载体蛋白包括但不限于哺乳动物来源的免疫球蛋白IgG1或IgG4的恒定区Fc部分或人血清白蛋白或转铁蛋白等。优选的，所述长效载体蛋白选自人免疫球蛋白IgG1(SEQ ID NO：5)或IgG4的恒定区Fc部分(SEQ ID NO：6)及其突变体，更优选的，所述长效载体蛋白选自N297位无糖基化的人免疫球蛋白IgG1或IgG4的恒定区Fc突变体。在本发明的一个实施例中，所述长效载体蛋白选自人免疫球蛋白IgG1的恒定区Fc突变体且具有SEQ ID NO：7所述序列；在本发明的另一个实施例中，所述长效载体蛋白选自人免疫球蛋白IgG4的恒定区Fc突变体且具有SEQ ID NO：8所述序列。对于IgG4抗体，进一步的S228P突变是为了减弱IgG4抗体特有的链交换现象。这些Fc片段都能明显地延长GLP-2突变体在体内的半衰期。
式I中，L为连接GLP-2突变体与长效载体蛋白的连接肽链。L为连接肽链且具有如下结构：
(XSSGAPPPS)u-G/S-(G-W1-W2)m                            式II
其中X选自P、GP，GGP或NGGP中的任一种；W1，W2分别为除Cys之外的19种任意天然氨基酸残基；u为0或1；m为1-20的整数。所述G/S为G和S任意组成的肽链，长度为5-25个氨基酸；优选的，G/S为GGGGS(SEQ ID NO：98)、GGGGGS(SEQ ID NO：99)或GGGGSGGGGS(SEQ ID NO：100)中的任一种。优选地，所述W1，W2各自独立地选自以下氨基酸A、N、D、Q、E、K、P、S、R。更优选的，W1，W2各自独立地选自A、P、S、E、Q、D。
优选的，所述L选自SEQ ID NO：30((GGGGS)2GPPGPA)，SEQ ID NO：31
((GGGGS)2GPNGAPGPS)，SEQ ID NO：32((GGGGS)2GPSGAPGPPGPEGPA)中的任一种。
一般而言，活性蛋白与其他蛋白融合后，极有可能会显著降低其生物学活性，尤其是GLP-2受体激动剂类的小肽。这是由于小肽分子量相对较小，受空间位阻的影响较大而导致活性大幅下降。
另外有文献报道，Exendin-4(HGDGSFSDEMNTILDNLAARDFINWLIQTKITD，SEQ ID NO：101)在酵母中表达仍会出现N端二肽的降解，而敲除了酵母STE13基因后则明显改善(Prabha L等，Protein Expr Purif.2009：155-61.Identification ofthe dipeptidyl aminopeptidase responsible for N-terminal clipping of recombinant Exendin-4precursor expressed in Pichia pastoris.)，这说明对于GLP-2突变体而言，第二位A突变为G也许仍不足以抵抗二肽酶的降解。然而本发明人发现，在酵母中表达GLP-2突变体时，远离N端的序列内部降解问题更为突出。因此，即便GLP-2突变体在酵母STE13基因失活后N端仍保持完整，然而序列内部 的降解仍会降低活性。
在本发明中，G/S-(G-W1-W2)m的融合可显著缓解GLP-2突变体融合蛋白的活性损失。G/S序列是本技术领域人员熟知的柔性连接肽链，常用于连接两个不同的蛋白。然而，本发明人发现，单独加入G/S肽(最常用的为GGGGS单元)并不能显著地降低GLP-2受体激动剂的生物学活性损失，但是在加入(G-W1-W2)单元后，GLP-2突变体的活性损失则随着GGGGS单元的长度的增加而呈梯度地减少。同样地，单独加入(G-W1-W2)单元不仅不能降低GLP-2受体激动剂的生物学活性损失，反而有进一步降低活性的趋势。本发明人发现，只有G/S-(G-W1-W2)m的组合形式才能显著减少GLP-2突变体的生物学活性损失。
进一步地，本发明提供的连接肽链还可以加入XSSGAPPPS单元。在本发明中，XSSGAPPPS序列融合在GLP-2突变体的C末端后，可增加部分GLP-2突变体的稳定性且并未影响GLP-2突变体的生物学活性，从而延长了GLP-2在体内的活性半衰期。在甲醇酵母GS115中分泌表达时，PSSGAPPPS与C末端缺失的GLP-2突变体融合后，减少了GLP-2突变体的酶解条带，提高表达量。
本发明人发现，与不含连接肽链的GLP-2突变体融合蛋白R-P相比，(XSSGAPPPS)u-G/S-(G-W1-W2)m形式的连接肽链能有效减少GLP-2突变体的酶解及生物学活性损失。u可以为0或1，这取决于GLP-2突变体的形式。
本发明的另一个突出优点，是克服了GLP-2与Fc片段融合后形成的多聚体形成现象，如GLP-2 MIMETIBODYTM)很容易形成非共价的二聚体(Baker AE等，The dimerization of glucagon-like peptide-2 MIMETIBODYTM is linked to leucine-17 in the glucagon-like peptide-2 region.J Mol Recognit.2012 25(3)：155-64.)。由于GLP-2 MIMETIBODYTM技术中，IgG4-Fc片段与GLP-2之间同样也含有柔性G/S连接肽链，然而本发明的实施例中，加入了(XSSGAPPPS)u-G/S-(G-W1-W2)m的GLP2突变体融合蛋白的SEC-HPLC结果显示却并未出现明显的聚体峰。这可能是由于本发明提供的连接肽链特定构象与GLP-2突变体的疏水区域作用，避免了二聚体的形成。
在由两个不同蛋白组成的融合蛋白中，连接肽链作为两个不同结构域之间的桥梁，起到极其关键的作用；而且对于不同蛋白，一般需要采用不同的连接肽链。这是因为不同活性蛋白具有不同的高级结构，分子量也不相同，因此在形成融合蛋白时，连接肽链的优化是必须的。尽管G与S组成的柔性肽链成功地应用于多种蛋白，但并不足以达到理想的效果。在本发明中，发明人经过大量的实验筛选，获得了一系列针对GLP-2突变体与长效载体蛋白之间融合的连接肽链。这些肽链能有效地减少GLP-2受体激动剂的活性损失，而且能缓解蛋白酶水 解。本发明的实施例中，采用了以下GLP-2突变体与连接肽链的组合形成融合蛋白序列(表1)。
表1 各种GLP-2融合蛋白序列
Figure PCTCN2017106727-appb-000004
Figure PCTCN2017106727-appb-000005
注：表中GLP-2突变体序列仅标注突变的氨基酸位点，如A2G表示天然GLP-2序列(SEQ ID NO：1)中第二位的A替换为G，ΔC表示C末端缺失，ΔC后的数字表示缺失的氨基酸数目，如ΔC6代表C末端缺失6个氨基酸的GLP-2突变体。同样地，hIgG4及hIgG1Fc片段突变体也仅标注突变的氨基酸位点，h代表人源。
本发明的另一方面提供了编码所述融合蛋白的核苷酸序列。
本发明的又一方面是提供携带编码本发明所述融合蛋白编码基因序列的重组表达载体。目前常用的重组表达载体包括但不限于真核表达载体，如pPIC9质粒、pPIC9K质粒、pPICZalpha A质粒、pcDNA3.1等，原核表达载体如pET41a质粒、pET32a质粒或其他自行构建的具有必须表达外源重组蛋白所需元件的质粒等，皆可用于构建表达本发明的制备。
本发明的再一方面是提供一种表达所述融合蛋白的方法。所提供的表达所述融合蛋白的方法是将含有上述融合蛋白编码基因序列的重组表达载体导入宿主细胞，诱导或组成型地表达得到所述融合蛋白。所述的表达宿主可为酵母菌、大肠杆菌或哺乳动物细胞等，优选为酵母菌，尤其优选的为毕赤酵母(Pichi pastoris)。
本发明的融合蛋白的纯化处理，其中包括盐析、沉淀、超滤、层析等技术及这些技术的组合。其中层析可以用亲和、离子交换、疏水、反相等层析技术。
本发明中的蛋白质及其衍生物可单独使用，也可以加入一种或多种药学上可以接受的辅料一起组成药物制剂的形式使用。所述的辅料包括药学领域常规的辅料为水、糖类如乳糖、 右旋糖等，醇类如山梨醇、甘露醇、木糖醇，氨基酸等。另外，本发明的药学组合物可进一步包括赋型剂和抑菌剂。
本发明的融合蛋白可以制成注射剂。该剂型的药物可以按照药学领域的常规方法制备。药物制剂可以存在于单一剂量或多剂量的容器中，如密封的安瓶或西林瓶中。冻干制剂是将液体制剂冷冻干燥制备的，使用前加入无菌、无热原的液态溶剂，如注射用水。
本发明中的融合蛋白及其衍生物或其药物组合物，作为肠道保护激素可用于各种原因引起的肠道损伤修复及代偿疾病，如肿瘤放化疗、肿瘤靶向药物治疗、严重创(烧)伤、全肠外营养、炎症性肠病等因素引起的肠粘膜损伤，以及广泛肠切除和小肠移植等病人的治疗。
本发明中的融合蛋白及其衍生物可以通过静脉注射，皮下注射等方法给药。治疗包括在一段时间内使用单一剂量或复合剂量。
附图说明
图1是GLP-2突变体诱导筛选的SDS-PAGE电泳图；其中，A中泳道1-8分别为SEQ ID NO：9-11、23-27；B中泳道1-11为SEQ ID NO：12-22；C皆为空白pPIC9质粒转化GS115获得的表达株诱导样品；M为低分子量蛋白MARKER：97、66、44、29、21、14KD。
图2是GLP-2突变体纯化样品SDS-PAGE电泳图；其中，泳道1-9为SEQ ID NO：12-20纯化后的结果。M为低分子量蛋白MARKER：97、66、44、29、21、14KD。
图3是GLP-2突变体融合蛋白对大鼠小肠重量/体重改变影响。
为了更好的理解本发明的内容下面结合具体实例对本发明作进一步的说明。
具体实施方式
下述具体的实施方式，如无特别说明，均为本领域技术人员熟知的常规方法。如重组表达质粒的构建过程，可以根据常见的分子生物学文献，如《分子克隆实验指南》第三版(Sambrook J，Russell DW，Molecular cloning：A laboratory manual.3rd edition，New York：Cold Spring Harkbor Laboratory Press，2001)或商业公司提供的操作说明书中的技术方案。
实施例1.目的基因的克隆及表达载体的构建
各种GLP-2突变体基因、连接肽链基因及人IgG1和IgG4的Fc基因根据表1中氨基酸序列及酵母偏爱密码子设计，通过基因合成。利用SOE-PCR(splicing by overlap extension，简称SOE)的方法扩增获得完整的融合基因。本领域技术人员根据表1氨基酸序列可以轻易推导其基因序 列，并设计相应引物扩增，在GLP-2突变体基因的上游引物和Fc片段的下游引物末端分别添加酶切位点XhoI和EcoRI，用于将PCR(Po1ymerase Chain Reaction，聚合酶链式反应)产物克隆至pPIC9载体，表达SEQ ID NO：9-29的融合蛋白。用于做对照的SEQ ID NO：2通过化学合成获得。表2列举了扩增表1中各个GLP-2突变体融合蛋白时使用的引物序列。
表2 GLP-2突变体融合蛋白时使用的引物序列。
SEQ ID NO： 序列(5’-3’)
54 gtactcgagaaaagacatggtgatggttctttctct
55 gggaccatatttggactcgtcagtgatcttggtctg
56 gagtccaaatatggtccc
57 accggaattcctattaacctaaagacagggaaagact
58 agaaccaccaccaccgtcagtgatcttggtctg
59 ggtggtggtggttctgagtccaaatatggtccc
60 agatcctcctcctccagaaccaccaccaccgtcagtgatcttggtctg
61 ggaggaggaggatctggcggcggcggcagtgagtccaaatatggtccc
62 ggtggtggtggttctggacctgctgagtccaaatatggtccc
63 ggaggaggatctggacctcaagacaaaactcacacatgc
64 ctagaattcctattaacccggagacagggagagaga
65 ggaggaggatctggcggcggcggcagtggacctgctgagtccaaatatggtccc
66 tctggaggaggaggatctggcggcggcggcagtggtggaggcgggtctggcggaggt
67 gggtctggcggaggtggtagtggacctgatgagtccaaatatggtccc
68 ggaggaggaggatctggcccaccaggacctgctgagtccaaatatggtccc
69 tctggaggaggaggatctggcccaccaggacctgctgacaaaactcacacatgccca
70 tctggaggaggaggatctggtccagaaggtgctccaggtccatctgagtccaaatatggtccc
71 tctggaggaggaggatctggtccatctggtgctccaggtccaccaggtccagaa
72 ggtccaccaggtccagaaggtccagctgagtccaaatatggtccc
73 tctggaggaggaggatctggtccatctggtgctccaggtccaccaggaccttcc
74 gccgggggctccggaaggaccaggaggaccaggggctccggaaggtcctggtggacc
75 ccttccggagcccccggcccgcctgagtccaaatatggtccc
76 tctggaggaggaggatctggtccagctggtgaaccaggtccatctggtcctgctgga
77 aggacctggctctccagctggtccagaaggaccaggttctccagcaggaccagatgg
78 gctggagagccaggtccttcaggccctgctggtgaacctggcccttctgggccagct
79 gggaccatatttggactcactagggccgggttcaccagctggcccagaagggcc
80 gagtccaaatatggtccc
81 aggaggaccagatcctcctcctccagaaccaccaccaccgtcagtgatcttggtctg
82 ggatctggtcctcctggtcctgctggtcctcctggtcctgctggtcctcctggtcctgctggaccacca
83 gcaggacctgggggcccggctggtcctggtggtccggctggtcctggtggtccagcagg
84 gcccccaggtcctgctggtcctcctggtcctgctggtcctcctggtcctgctggaccac
85 accatatttggactcggctggtcctggtggtccggctggtcctggtggtccagcaggac
86 gaccatatttggactcagatggtggtggagcaccagaagaagggtcagtgatcttggtc
87 gggaccatatttggactcagaaccaccaccaccaatcaaccagttgataaa
88 agatcctcctcctccagaaccaccaccacccttggtctgaatcaacca
89 tctggaggaggaggatctgagtccaaatatggtccc
90 cagaaccaccaccaccagatggtggtggagcaccagaagaaggaatcaaccagttgata
91 tctggtggtggtggttctgagtccaaatatggtccc
92 ccaccagatggtggtggagcaccagaagaaggtcctccgttcttggtctgaatcaacca
93 ccaccaccatctggtggtggtggttctggaggaggaggatctgagtccaaatatggtcc
94 caccaccagatggtggtggagcaccagaagaaggtcctccgttaatcaaccagttgata
95 ccaccatctggtggtggtggttctggaccagctggaccaaatgagtccaaatatggtcc
96 agaaccaccaccaccagatggtggtggagcaccagaagaaggcttggtctgaatcaac
97 tctggtggtggtggttctggaccagctgagtccaaatatggtccc
PCR反应体系(50μL)：5μL 10×Pfu buffer，dNTP mix(200μmol/L)，上游引物(0.5μmol/L)，下游引物(0.5μmol/L)，0.1μg模板，0.5μL Pfu DNA聚合酶(5U/μL)，无菌水补足至50μL，所有PCR反应程序均为：94℃预变性2分钟，94℃变性30秒，58℃退火30秒，72℃延伸3分钟，27个循环，72℃延伸5分钟后4℃保存。PCR产物经琼脂糖凝胶电泳检测，实验结果与理论一致。
融合基因通过两端的XhoI和EcoRI内切酶克隆至同样酶切的酵母表达载体pPIC9(Life technologies，USA)中，获得重组表达质粒。以甲醇酵母Pichia pastoris GS 115(His-)为表达宿主菌，通过电转化将线性化的重组质粒转化到GS115中。在组氨酸缺陷的筛选平板培养基上30℃培养3天，至单菌落出现。
实施例2.重组蛋白的获得
将上述转化的重组酵母单菌落接种至10ml BMGY液体培养基中，30℃，250rpm培养24小时后，静置过夜，弃上清，加入10ml含1％甲醇的BMMY液体培养基，30℃，250rpm诱导表达。选取相对表达较高的菌株作为表达株。具体步骤参见操作说明书(Pichia Expression  Kit.For Expression of Recombinant Proteins in Pichia pastoris.Catalog no.K1710-01)。
将筛选获得的高表达菌株接种至YPD液体培养基(酵母浸出粉胨葡萄糖培养基)中，30℃，220rpm培养20-24h至OD600达10～20，作为上罐种子液。将培养好的种子液接入Biostat B Twin MO 5L发酵罐，培养基按Life technologies公司Pichia Fermentation Process Guidelines配置。接种量为10％，设定发酵温度30℃，pH5.0，待甘油耗尽，开始加甲醇进行诱导表达。表达阶段控制发酵温度25℃，诱导72小时放罐。诱导表达图如图1所示。
如图1A所示，与不含任何连接肽链的SEQ ID NO：9相似，仅含G/S柔性肽或XSSGAPPPS单元或两者组合的GLP-2突变体融合蛋白，如SEQ ID NO：10-11和23-27对于发酵过程中的降解并无明显的效果，而加上了(G-W1-W2)m单元后(图1B)，如SEQ ID NO：12-22，在发酵过程中检测不到明显的降解条带。
实施例3：融合蛋白的分离纯化
将实施例2中获得的发酵液经8000rpm，室温离心30分钟收集上清，上样至经缓冲液A(0.5M NaCl，20mM PB，pH 7.0)平衡后的Diamond Protein A BestChrome层析柱(博格隆(上海)生物技术有限公司)，再次经缓冲液A平衡后用洗脱缓冲液B(0.1M Gly-HCl，pH 3.0)洗脱，洗脱峰加1/10峰体积的中和液(1M Tris-HCl，pH8.0)调节pH。加去离子水稀释至电导小于4ms/cm后，上样至经缓冲液A(20mM PB，pH 7.0)平衡后的TOSOH Super Q 650-M层析柱，再次经缓冲液A平衡后用洗脱缓冲液B(0.5M NaCl，20mM PB，pH 7.0)洗脱。Super Q洗脱样品用PBS透析置换。由于各种GLP-2突变体融合蛋白具有相似的性质，且ProteinA对Fc序列的高特异性简化了纯化步骤(然而降解带也很难彻底去除)，而Super Q则能去除酵母细胞分泌的本底杂蛋白，因此纯化步骤也相近。部分GLP-2突变体融合蛋白纯化样品如图2所示。重组蛋白浓度采用BCA法测定。
实施例4融合蛋白的理化性质分析
纯化得到的蛋白通过SDS-PAGE、SEC-HPLC、RP-HPLC、质谱等理化性质检测鉴定。如图1所示，GS115中表达的重组蛋白与理论分子量一致。加入了(XSSGAPPPS)u-G/S-(G-W1-W2)m连接肽链的GLP-2突变体融合蛋白在SDS-PAGE上呈单一条带(图1B)，分子量介于44和29KD分子量MARKER带之间，且并未见明显的降解带或共价聚体，而无连接肽链的GLP-2突变体融合蛋白或其他形式连接肽链的突变体则出现多条低于理论分子量的条带(图1A)，应为降解带。
采用Sepax SRT SEC-300(7.8*300mm，5μm，
Figure PCTCN2017106727-appb-000006
)对纯化的样品进行SEC-HPLC分析， 流动相为100mM PBS，pH6.4。结果表明，相对于仅以G/S作为连接肽的GLP-2突变体融合蛋白(SEQ ID NO：10-11及SEQ ID NO：23-27)以及无连接肽的SEQ ID NO：9，加入了G/S-(G-W1-W2)m连接肽链的GLP-2突变体融合蛋白(SEQ ID NO：12-22)在SEC柱上呈单峰，且并未见明显的聚体或分子量偏小的降解峰，而无连接肽链的GLP-2突变体融合蛋白(SEQ ID NO：9)则出现多个降解峰及其聚体峰；另外进一步含有XSSGAPPPS单元的SEQ ID NO：28-29同样具有显著的聚体减少趋势(表3)。
表3 GLP-2突变体融合蛋白在SEC-HPLC上的聚体含量分析
SEQ ID NO： 单体※(％) SEQ ID NO： 单体※(％)
9 26 21 94
10 30 22 96
11 21 23 27
12 95 24 17
16 99 25 23
17 99 26 29
18 98 27 21
19 96 28 89
20 94 29 91
单体※：此处指两条Fc链通过共价形成的活性分子。
表中仅显示单体含量，数据为3批次发酵液纯化的重组蛋白平均值。
采用Phenomenex Jupiter C4(4.6*150mm，5μm，
Figure PCTCN2017106727-appb-000007
)对纯化的样品进行RP-HPLC分析，流动相为0.1％TFA+水(A)，0.1％TFA+乙腈(B)，梯度为5％B-100％B(0-30min)。结果表明，加入了(XSSGAPPPS)u-G/S-(G-W1-W2)m连接肽链的GLP-2突变体融合蛋白在RP柱上呈单峰，而其他连接肽链或无连接肽链的GLP-2突变体融合蛋白则出现多个降解峰，与电泳结果几乎一致。
纯化后的样品进行质谱分析，结果显示加入了(XSSGAPPPS)u-G/S-(G-W1-W2)m连接肽链的GLP-2突变体融合蛋白检测到与理论分子量一致的主峰(≥90％)，其他峰包含了电泳及RP-HPLC无法检测到的N端降解峰(≤10％)；而其他的GLP-2突变体融合蛋白与理论分子量一致的峰含量小(≤20％)，大部分为小于理论分子量的质量峰。
实施例5：体外细胞学活性检测
GLP-2融合蛋白体外细胞学活性检测采用荧光素酶报告基因检测法。将GLP-2R基因克隆 至哺乳动物细胞表达质粒pCDNA3.1中，构建成重组表达质粒pCDNA3.1-GLP-2R，同时荧光素酶(luciferase)全长基因克隆至pCRE-EGFP质粒上，替换EGFP基因，得到pCRE-Luc重组质粒。pCDNA3.1-GLP-2R和pCRE-Luc质粒按摩尔比1∶10的比例转染CHO细胞，筛选稳转表达株，获得重组GLP-2R/Luc-CHO稳转细胞株。
在10-cm细胞培养皿中用含10％FBS和300μg/ml G418的DMEM/F12培养基培养细胞，等汇合度至90％左右时，弃去培养上清，加入2ml胰酶消化2min后，加入2ml含10％FBS和300μg/ml G418的DMEM/F12培养基中和，转移至15ml离心管中，800rpm离心5min后，弃去上清，加入2ml含10％FBS和300μg/ml G418的DMEM/F12培养基重悬，计数。用含10％FBS的DMEM/F12培养基稀释细胞至3*105，96孔板中每孔铺100μl，即每孔3万细胞，贴壁后换成含0.1％FBS的DMEM/F12培养基培养。
铺在96孔板的细胞弃去上清后，将实施例3中纯化的重组蛋白用含0.1％FBS的DMEM/F12培养基稀释至一系列指定浓度，加入到细胞培养孔中，100μl/孔，刺激6h后检测。根据lucifersae reporter kit(Ray Biotech，Cat：68-LuciR-S200)说明书进行检测。结果如表3所示。不同连接肽链对于空间位阻和GLP-2突变体降解的作用差异导致了细胞活性的不同。对于SEQ ID NO：9-11或23-27而言，聚体的产生(实施例4)可能进一步减弱了细胞学活性。
表3 各种GLP-2突变体融合蛋白体外细胞活性检测结果
SEQ ID NO： EC50(nM) SEQ ID NO： EC50(nM)
9 85.8 20 13.1
10 80.2 21 14.4
11 92.6 22 12.5
12 25.8 23 82.4
13 19.6 24 95.8
14 21.9 25 83.4
15 24.1 26 81.7
16 7.8 27 92.1
17 6.4 28 23.2
18 10.7 29 27.3
19 11.0 2(A2G) 1.8
实施例6：体内动物模型药效检测
癌症化疗药物通过诱导细胞凋亡和细胞周期停滞而对细胞产生毒性，尤其容易导致小肠上皮细胞的损伤，而导致胃肠道粘膜炎、腹泻及菌血症，目前并无可靠的预防措施。GLP-2 已被证明对于肠道隐窝细胞具有显著的抗凋亡效果，并改善化疗药物引起的肠道黏膜炎。本实施例通过大鼠模型比较各种GLP-2突变体融合蛋白的体内生理活性。
SD大鼠雌雄各半，分为8组，每组6只：1)氟尿嘧啶(5-FU)+GLP-2突变体1(SEQ ID NO：16)；2)氟尿嘧啶(5-FU)+GLP-2突变体2(SEQ ID NO：17)；3)氟尿嘧啶(5-FU)+GLP-2突变体3(SEQ ID NO：18)；4)氟尿嘧啶(5-FU)+GLP-2突变体4(SEQ ID NO：9)5)氟尿嘧啶(5-FU)+GLP-2突变体5(SEQ ID NO：10)；6)氟尿嘧啶(5-FU)+GLP-2突变体6(SEQ ID NO：25)；7)氟尿嘧啶(5-FU)+生理盐水；8)生理盐水。记录每只大鼠的体重，并在注射5-FU前3天开始连续7天，组1)至组6)以25nmol/kg的剂量皮下注射GLP-2突变体，每天一次；组7)和8)注射相同体积的生理盐水。第4天至第7天，组1)至组7)连续每天以50mg/kg的剂量腹腔注射5-FU，最后一次注射5-FU后24小时，所有大鼠处死。剪开大鼠腹腔，在冰浴中，截取大鼠的小肠、大肠，用生理盐水冲洗后，测量其长度并进行测湿重。计算小肠重量与体重比例，不同GLP-2突变体融合蛋白对肠道的保护作用结果如图3所示。
如图3所示，与5-FU+生理盐水对照组相比，GLP-2突变体1至6都能明显地缓解5-FU对小肠的损伤，其中突变体1-3的疗效相对于突变体4-6具有更好的效果，说明蛋白在体内的药效与体外细胞学活性相对应。每组内6只大鼠间药效学数据之间差异计算标准差(SD)：对照组，5-FU+生理盐水组，5-FU+突变体1，5-FU+突变体2，5-FU+突变体3，5-FU+突变体4，5-FU+突变体5，5-FU+突变体6的SD分别为4.21±0.79，1.88±0.24，3.85±0.53，3.77±0.22，3.58±0.73，2.48±0.44，2.76±0.27和3.41±0.14。

Claims (14)

  1. 一种融合蛋白，具有如下结构：
    R-L-P
    其中：R为GLP-2受体激动剂；
    P为长效载体蛋白；
    L为连接肽链且具有如下公式：
    (XSSGAPPPS)u-G/S-(G-W1-W2)m
    其中：X选自P、GP，GGP或NGGP中的任一种；
    G/S为G和S任意组成的肽链，长度为5-25个氨基酸；
    W1，W2分别为除Cys之外的19种任意天然氨基酸残基；
    u为0或1；m为1-20的整数。
  2. 根据权利要求1所述的融合蛋白，其中所述GLP-2受体激动剂如SEQ ID NO：2所示。
  3. 根据权利要求1所述的融合蛋白，其中所述GLP-2受体激动剂为C末端缺失1-6个氨基酸残基的GLP-2突变体。
  4. 根据权利要求1所述的融合蛋白，其中所述G/S为GGGGS、GGGGGS或GGGGSGGGGS。
  5. 根据权利要求1所述的融合蛋白，所述长效载体蛋白选自哺乳动物免疫球蛋白IgG的恒定区Fc部分、人血清白蛋白和转铁蛋白。
  6. 根据权利要求5所述的融合蛋白，所述长效载体蛋白为哺乳动物免疫球蛋白IgG1或IgG4的恒定区Fc部分或其突变体。
  7. 根据权利要求6所述的融合蛋白，所述Fc部分为SEQ ID NO：5、SEQ ID NO：6、SEQ ID NO：7或SEQ ID NO：8。
  8. 根据权利要求1所述的融合蛋白，其中所述W1，W2各自独立地选自以下氨基酸：A、D、Q、E、P、S。
  9. 根据权利要求1-8任一权利要求所述的融合蛋白，所述L选自SEQ ID NO：30、SEQ ID NO：31和SEQ ID NO：32。
  10. 一种核苷酸序列，编码权利要求1-9任一权利要求所述的融合蛋白。
  11. 一种重组表达载体，携带权利要求10所述的核苷酸序列。
  12. 一种宿主细胞，转化权利要求11所述的重组表达载体。
  13. 权利要求1-9任一权利要求所述的融合蛋白的用途，用于制备一种具有预防和/或治疗化疗中导致肠胃损伤、短肠综合症、Corhn’s肠炎的药物。
  14. 一种药物组合物，包含权利要求1-9任一权利要求所述的融合蛋白和药学上可接受的稀释剂、载体或赋形剂。
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