WO2022247740A1 - 一种多肽及其在制备免疫调节药物中的应用 - Google Patents

一种多肽及其在制备免疫调节药物中的应用 Download PDF

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WO2022247740A1
WO2022247740A1 PCT/CN2022/094056 CN2022094056W WO2022247740A1 WO 2022247740 A1 WO2022247740 A1 WO 2022247740A1 CN 2022094056 W CN2022094056 W CN 2022094056W WO 2022247740 A1 WO2022247740 A1 WO 2022247740A1
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polypeptide
cells
pep20
present
polynucleotide
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PCT/CN2022/094056
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English (en)
French (fr)
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王宏林
孙洋
楼方舟
孙力波
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上海市第一人民医院
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • 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
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/06—Antiasthmatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/06—Antipsoriatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to the field of biomedicine, more specifically, the present invention relates to a polypeptide and its application in the preparation of immunomodulatory drugs.
  • cytokine IL-17A Since its discovery in 1993, the pro-inflammatory cytokine IL-17A has been the focus of research on autoimmune inflammatory diseases. It was originally found to be produced by a specific subset of CD4 + T cells (so-called Th17 cells), however, other immune cell subsets are also known to synthesize and express IL-17, including CD8 + T cells. The presence of CD8 + T cells (also known as Tc17 cells) expressing IL-17 has been described in several human inflammatory diseases.
  • Psoriasis is a common chronic immune inflammatory disease with approximately 125 million patients worldwide. The main symptoms of psoriasis are skin diseases with erythema and scales. Psoriasis is a disease caused by systemic inflammation. Immune cells in patients excessively release pro-inflammatory factors such as IL23 and TNF, and activate effector T cells to release inflammatory factors such as IL17 and IL26 to induce abnormal differentiation and proliferation of keratinocytes, and trigger Sustained damage to multiple tissues and organs. Complications of psoriasis include psoriatic arthritis, autoimmune disease, cardiovascular disease, diabetes, etc.
  • MS Multiple sclerosis
  • IL-17 plays a key "prime” role in initiating the pathogenic immune response that mediates damage in experimental autoimmune encephalomyelitis (EAE) and MS.
  • EAE experimental autoimmune encephalomyelitis
  • CNS central nervous system
  • drugs that block IL-17 may not need to cross the blood-brain barrier to be effective in treating MS.
  • IL-17 as a drug target for relapsing-remitting MS is elucidated, and drugs that block IL-17 have great potential in the treatment of multiple sclerosis, among others.
  • RA Rheumatoid arthritis
  • RA Rheumatoid arthritis
  • the disease affects 1-2% of the world's population, most commonly middle-aged women.
  • the etiology of RA is unknown, but pro-inflammatory cytokines play a central role in RA. Modulation of cytokine imbalance may represent a reliable approach to control this disease.
  • the pro-inflammatory cytokines TNF- ⁇ and IL-1 play a vital role in the pathogenesis of arthritis. Clinical studies have shown that blocking TNF- ⁇ has curative effect, but some patients are ineffective against TNF- ⁇ .
  • cytokines other than IL-1 and TNF- ⁇ may be involved in the pro-inflammatory cytokine cascade.
  • Trm tissue memory T cells
  • IL-17A induces the production of pro-inflammatory mediators by several articular cells including synovial fibroblasts, macrophages, and chondrocytes.
  • IL-17 receptor signaling has been identified as a key pathway in the transition from acute synovitis to chronic destructive arthritis, and these observations strongly implicate IL-17A as an important mediator of arthritis.
  • bioblockers targeting IL-17 factors have shown excellent efficacy in the treatment of various autoimmune diseases, but they also show different degrees of adverse reactions, including Nasopharyngitis, upper respiratory tract infection, injection site reactions, and headaches; some cases also have severe candida infection (a specific risk associated with IL-17 blockers); in addition, a large number of studies have shown that patients with autoimmune diseases have used it for a long time Severe drug tolerance reactions often occur after biological inhibitors. Therefore, it is still necessary to continue to develop highly active immunotherapeutic drugs with good curative effect and small side effects.
  • IL-17 factors such as Secukinumab, Ixekizumab, and Brodalumab
  • Polypeptide drugs generally refer to peptide chains consisting of no more than 50 amino acids (aa).
  • the size of peptide drugs is between small molecule drugs (MW ⁇ 500) and protein drugs (MW>5000), which well bridges the gap between the two and is a new class of drugs.
  • aa small molecule drugs
  • protein drugs MW>5000
  • peptides have demonstrated their superiority in clinical application and production. In clinical application, it is very similar to monoclonal antibody drugs and recombinant protein drugs, with strong specificity and good curative effect; the production and preparation have the advantages of high quality controllability, high purity, easy modification and stable structure.
  • the global peptide drug market has exceeded 20 billion US dollars, and has maintained an annual growth rate of 10%.
  • the purpose of the present invention is to provide a novel polypeptide PEP20 encoded by the host gene of long-chain non-coding RNA LINC01871, which can specifically target CD8 + T cells in an inflammatory environment and inhibit the differentiation of CD8 + T cells into Tc17 cells , can be applied to the preparation of immunomodulatory drugs to solve the problems of low safety of existing immunomodulatory drugs and scarcity of polypeptide immunomodulatory drugs.
  • the first object of the present invention is to provide an isolated polypeptide, the amino acid sequence of which is shown in SEQ ID NO:2.
  • the polypeptide is encoded by the host gene of long-chain non-coding RNALINC01871.
  • the polypeptide is translated by the nucleotide sequence whose nucleotide sequence is 58th to 117th in SEQ ID NO: 1 or its degenerate sequence.
  • Another object of the present invention is to provide an isolated polynucleotide encoding said polypeptide.
  • its nucleotide sequence is as the 58th to 117th positions in SEQ ID NO: 1 or its degenerate sequence.
  • Another object of the present invention is to provide an expression vector containing said polynucleotide.
  • Another object of the present invention is to provide a recombinant cell containing the expression vector or the polynucleotide in its genome.
  • Another object of the present invention is to provide the application of the polypeptide or polynucleotide encoding it, or the expression vector, or the recombinant cell in the preparation of immunomodulatory drugs;
  • the immunomodulatory drug is a drug for preventing, alleviating or treating autoimmune diseases
  • the autoimmune diseases include: psoriasis, dermatitis, multiple sclerosis, asthma, rheumatoid arthritis, enteritis.
  • the immunoregulatory drug is a drug that inhibits the differentiation of CD8 + T cells into Tc17 cells, and inhibits the over-differentiation, over-proliferation and over-expression of IL-17A of Tc17 cells.
  • Another object of the present invention is to provide a method for preparing said polypeptide
  • the method includes: cultivating the recombinant cell, thereby recombinantly expressing the polypeptide;
  • the method includes: preparing the polypeptide by in vitro artificial synthesis or biological synthesis.
  • Another object of the present invention is to provide a pharmaceutical composition for immune regulation, said pharmaceutical composition comprising: any of the aforementioned polypeptides or polynucleotides encoding them, or said expression vectors, or The recombinant cell; and a pharmaceutically or physiologically acceptable carrier.
  • Another object of the present invention is to provide a kit for immune regulation, which includes: any of the aforementioned polypeptides or polynucleotides encoding them; or the expression vector; or the said recombinant cell; or said pharmaceutical composition.
  • the PEP20 polypeptide provided by the present invention can specifically target CD8 + effector T cells, inhibit the differentiation of CD8 + T cells into Tc17 cells, thereby treating autoimmune inflammatory diseases such as psoriasis, and the PEP20 polypeptide has a small molecular weight and is easy to treat. Enter the cell to play a role;
  • the PEP20 polypeptide provided by the present invention is biologically synthesized and chemically synthesized through prokaryotic expression, which is easy to produce in large quantities and has good stability. Compared with existing antibodies that are effective for the treatment of autoimmune inflammatory diseases such as psoriasis, the price is lower, safer.
  • Figure 1 is a detection and analysis diagram after PEP20 is obtained;
  • A is the mass spectrometry diagram of PEP20
  • FIG. 2 shows the effect of PEP20 on the differentiation of Tc17 cells.
  • Fig. 3 is the influence of control group and PEP20, PEP20-PEG group to treat the skin inflammation of psoriasis model mice;
  • A is the comparison of the skin lesion symptoms of the control group and the PEP20, PEP20-PEG group treated psoriasis model mice;
  • B is the comparison of epidermal thickness of psoriasis-like model mice treated with PEP20 and PEP20-PEG groups.
  • C is the H&E staining results of psoriasis-like model mice treated with control group, PEP20, and PEP20-PEG group.
  • Figure 4 is a comparison of the disease severity before and after the control group and PEP20 treated EAE model mice; wherein:
  • A is the disease score comparison between the control group and PEP20-treated EAE model mice
  • LLB Luxol Fast Blue
  • IL-17 is now recognized as a key cytokine in the pathogenesis of human autoimmune and autoinflammatory diseases, and there is increasing evidence that IL-17-producing CD8 + T cells (Tc17) are mediators.
  • Tc17 CD8 + T cells
  • Key inflammatory T cells that induce a variety of autoimmune inflammatory diseases. Including: multiple sclerosis, psoriasis, psoriatic arthritis, rheumatoid arthritis, dermatitis, enteritis, etc. Therefore, the applicant of the present invention has gone through a lot of research in order to find a drug for treating the aforementioned autoimmune inflammatory diseases, and finally discovered a brand-new polypeptide derived from human cells, called PEP20.
  • the PEP20 polypeptide is a polypeptide with 20 amino acids encoded by the open reading frame nucleotide sequence of the long-chain non-coding RNALINC01871 host gene, which can specifically regulate the differentiation of CD8 + effector T cells, has immune regulation function, and can be used for It can prevent or treat autoimmune diseases, such as psoriasis, rheumatoid arthritis and multiple sclerosis, and has the advantages of low toxicity, stable effect, long-lasting effect, and easy synthesis.
  • autoimmune diseases such as psoriasis, rheumatoid arthritis and multiple sclerosis
  • polypeptide encoded by the host gene of LINC01871 can be used interchangeably.
  • the regulation includes “up-regulation” and “down-regulation”, wherein “up-regulation” includes but not limited to: promotion, increase, increase, enhancement, etc.; said “down-regulation” includes but not limited to: reduction, Inhibit, antagonize, block, block, etc.
  • a "pharmaceutically acceptable” ingredient is one that is suitable for use in humans and/or mammals without undue adverse side effects (eg, toxicity), ie, has a reasonable benefit/risk ratio.
  • pharmaceutically acceptable carrier refers to a carrier for the administration of a therapeutic agent, including various excipients and diluents.
  • pharmaceutical carriers which, by themselves, are not essential active ingredients and which are not unduly toxic upon administration.
  • the PEP20 polypeptide of the present invention may be a recombinant polypeptide or a synthetic polypeptide. It may be a product of chemical synthesis, and the methods of chemical synthesis are familiar to those skilled in the art, such as solid-phase polypeptide synthesis methods.
  • the sequence of the PEP20 polypeptide of the present invention is: MVEEIQASLMWQQAREREGE (SEQ ID NO: 2).
  • the present invention also includes fragments, derivatives and analogs of PEP20 polypeptides.
  • fragment refers to a polypeptide that substantially maintains the same biological function or activity of the PEP20 polypeptide of the present invention. Fragments, derivatives or analogs of PEP20 polypeptides may be:
  • a polypeptide formed by fusing a mature polypeptide with another compound such as a compound that prolongs the half-life of the polypeptide, such as polyethylene glycol
  • another compound such as a compound that prolongs the half-life of the polypeptide, such as polyethylene glycol
  • a polypeptide formed by fusing an additional amino acid sequence to this polypeptide sequence (such as a leader sequence or a secretory sequence or a sequence for purifying the polypeptide or a proprotein sequence, or a fusion protein).
  • an additional amino acid sequence such as a leader sequence or a secretory sequence or a sequence for purifying the polypeptide or a proprotein sequence, or a fusion protein.
  • PEP20 may refer to the polypeptide having the sequence shown in SEQ ID NO:2.
  • the term also includes one or several (for example, within 300, preferably within 200, more preferably within 100, more preferably within 50) of the C-terminus and/or N-terminus that have the same function as the PEP20 polypeptide.
  • substitutions with amino acids with similar or similar properties generally do not change the function of the protein.
  • adding one or several amino acids at the C-terminus and/or N-terminus usually does not change the function of the protein.
  • the term also includes active fragments and active derivatives of PEP20 polypeptides.
  • polypeptides formed by modifying one or several amino acids for the purpose of increasing the stability, half-life, and promoting efficacy of the polypeptide (usually without changing the primary structure), including: polypeptides in vivo or in vitro Chemically derivatized forms such as acetylation or carboxylation. Modification also includes glycosylation. Modified forms also include sequences with phosphorylated amino acid residues (eg, phosphotyrosine, phosphoserine, phosphothreonine). Also included are polypeptides that have been modified to increase resistance to hydrolysis or to optimize solubility.
  • the PEP20 disclosed by the invention is easy to synthesize, has good stability, low cost, does not have immune rejection, can specifically inhibit the differentiation of Tc17 cells in an inflammatory environment, has a very significant therapeutic effect, is not easy to relapse after treatment, and has few side effects.
  • the polypeptide of the present invention can also form a complex with other functional molecules, and the complex includes: the polypeptide of the present invention, and a functional molecule linked to the polypeptide.
  • the functional molecules are markers with tracking function, including but not limited to fluorescent dyes, MRI contrast agents, radioactive imaging agents, magnetic particles or chemical reagents with coloring functions.
  • the marker or functional small molecule with tracking function may be fluorescein isothiocyanate (FITC).
  • the functional molecules are small functional molecules, including small inorganic molecules and small organic molecules, and the molecular weight is less than 1000 Daltons.
  • the functional molecules are functional macromolecules, such as functional polypeptides (such as antibodies), functional nucleic acids; preferably, the functional nucleic acids include but are not limited to: plasmids, siRNA, DNA, Oligonucleotides, miRNA, antisense nucleic acid, etc.
  • the functional molecules are preparations with the function of molecular packaging and loading, including but not limited to: liposomes, polymers, dendritic molecules, nano-packaging preparations, and the like.
  • the functional molecules are viral vectors capable of carrying genetic material, including but not limited to retroviral vectors, lentiviral vectors, or adenoviral vectors.
  • the functional molecule can also be a membrane-penetrating peptide, after the polypeptide of the present invention is linked to it, the ability of the polypeptide to penetrate cells and enter cells can be improved.
  • the membrane-penetrating peptide can use any molecule known in the art that can guide the peptide or its coding gene into cells, or use any molecule that improves the ability of the peptide to penetrate cells.
  • Some peptides with membrane-penetrating function include: 1protein derived CPPs, such as penetratin, TAT, and pVEC; 2model peptides, such as MAP and (Arg)7; 3designed CPPs Such as MPG and Transportan etc.
  • PaCPPs Amphiphilic CPPs
  • SaCPPs Moderately amphipathic CPPs
  • NaCPPs Non-amphipathic CPPs
  • the polypeptide described in the present invention can be connected to the functional molecule by covalent connection or non-covalent connection. It should be understood that as long as the functions of the polypeptide and functional molecules can be retained, any connection method can be included in the present invention.
  • Covalent linkage usually connects two molecules by forming a covalent bond, and some non-covalent linkages (without forming a covalent bond), such as coupling, adsorption, binding, etc., are also applicable.
  • the polypeptide is connected to the functional molecule through a chemical bond; more preferably, the chemical bond is a peptide bond.
  • the present invention also provides a polynucleotide sequence encoding the PEP20 polypeptide of the present invention.
  • a polynucleotide of the invention may be in the form of DNA or RNA.
  • DNA may be a coding strand or a non-coding strand, and a "polynucleotide encoding a polypeptide" may include a polynucleotide that encodes the polypeptide, or a polynucleotide that also includes additional coding and/or non-coding sequences.
  • the present invention also relates to a vector comprising the polynucleotide of the present invention, and a host cell (recombinant cell) produced by genetic engineering with the coding sequence of the vector of the present invention or the C-terminal three amino acid truncation of the PEP20 polypeptide, as well as the host cell produced by recombinant technology Methods for the polypeptides of the invention.
  • a vector comprising the polynucleotide of the present invention, and a host cell (recombinant cell) produced by genetic engineering with the coding sequence of the vector of the present invention or the C-terminal three amino acid truncation of the PEP20 polypeptide, as well as the host cell produced by recombinant technology Methods for the polypeptides of the invention.
  • expression vector refers to bacterial plasmid, bacteriophage, yeast plasmid, plant cell virus, mammalian cell virus or other vectors well known in the art. In short, any plasmid and vector can be used as long as it can be replicated and stabilized in the host.
  • An important feature of expression vectors is that they usually contain an origin of replication, a promoter, marker genes, and translational control elements.
  • the vector containing the above-mentioned appropriate polynucleotide sequence and appropriate promoter or control sequence can be used to transform appropriate host cells so that they can express the polypeptide.
  • the host cell may be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a plant cell.
  • Representative examples are: Escherichia coli; Streptomyces, Agrobacterium; fungal cells such as yeast; plant cells and the like.
  • the main contribution of the present invention is not only to obtain the PEP20 polypeptide, but also to verify the function of the polypeptide.
  • the PEP20 of the present invention can specifically target CD8 + T cells in an inflammatory environment, inhibit the differentiation of CD8 + T cells into Tc17 cells, and inhibit the over-differentiation, over-proliferation and over-expression of IL-17A of Tc17 cells.
  • PEP20 can effectively inhibit the differentiation of Tc17 cells, and both PEP20 and its PEG-modified PEP20-PEG can reduce the number of inflammatory cells in the skin of IMQ-induced psoriasis model mice, and reduce the Proliferation of keratinocytes in the skin, thereby effectively attenuating the onset of IMQ-induced psoriasis in mice.
  • the exogenously synthesized PEP20 can effectively reduce the severity of the EAE model in mice and alleviate the demyelination of the mouse brain tissue.
  • the above research results show that the PEP20 polypeptide can be used to prepare drugs for autoimmune diseases, or to prepare drugs for inhibiting excessive differentiation, excessive proliferation, and overexpression of IL-17A of Tc17 cells.
  • the autoimmune disease is an autoimmune disease characterized by excessive differentiation of CD8 + T cells to Tc17 cells and excessive secretion of IL-17A.
  • the autoimmune disease is an autoimmune inflammatory disease such as psoriasis, multiple sclerosis, and rheumatoid arthritis.
  • it also has potential preventive or therapeutic effects on other diseases or symptoms related to Tc17 cell immune regulation dysfunction.
  • the diseases or symptoms known to be related to the immune regulation dysfunction of Tc17 cells are selected from: tumor or viral infection, inflammatory response, rheumatoid arthritis, organ transplantation, systemic lupus erythematosus, psoriasis, multiple sclerosis, etc. .
  • the present invention also provides a pharmaceutical composition for immune regulation, said pharmaceutical composition comprising: the polypeptide of the present invention or a polynucleotide encoding it, or an expression vector containing the polynucleotide or expressing the polypeptide recombinant cells; and a pharmaceutically or physiologically acceptable carrier.
  • Suitable pharmaceutically acceptable carriers are well known to those of ordinary skill in the art. A full description of pharmaceutically acceptable carriers can be found in Remington's Pharmaceutical Sciences.
  • the pharmaceutically acceptable carrier in the composition may contain liquids, such as water, phosphate buffer saline, ringer's solution, physiological saline, balanced salt solution, glycerin or sorbitol and the like.
  • auxiliary substances in these carriers such as lubricants, glidants, wetting agents or emulsifiers, pH buffer substances and stabilizers, such as albumin.
  • a safe and effective amount of the polypeptide of the present invention or the polynucleotide encoding it, or an expression vector containing the polynucleotide or a recombinant cell expressing the polypeptide is administered to a mammal (such as a human), wherein Such a safe and effective amount will generally be at least about 0.01 microgram/kg body weight, and in most cases will not exceed about 10 mg/kg body weight. Of course, factors such as the route of administration and the health status of the patient should also be considered for the specific dosage, which are within the skill of skilled physicians.
  • the precise effective amount for a subject will depend on the size and health of the subject, the nature and extent of the disorder, and the therapeutic agents and/or combination of therapeutic agents chosen for administration.
  • the effective amount can be determined by routine experimentation and is within the judgment of the clinician for a given situation.
  • the present invention also provides a kit or kit, comprising: the polypeptide of the present invention or a polynucleotide encoding it, or an expression vector containing the polynucleotide or a recombinant cell expressing the polypeptide, or the pharmaceutical composition.
  • the pharmaceutical composition of the present invention can be contained in an injector (such as a needle for injection), and the injector can contain a dosage of the pharmaceutical composition once.
  • the injectable drug delivery device can be contained in a medicine box for convenient storage and use.
  • kit or kit of the present invention Instructions for use may also be included in the kit or kit of the present invention, so as to facilitate correct use by those skilled in the art.
  • sequence of the host gene of LINC 01871 is as follows (SEQ ID NO: 1):
  • the PEP20 obtained by the solid-phase polypeptide synthesis method in Example 1 was tested for its effect on the differentiation of Tc17 cells.
  • mice Spleen cells of mice were obtained, and initial mouse CD8 + T cells were isolated by immunomagnetic beads, cultured in RPMI-1640 medium at 37°C, and anti-CD3 (5 ⁇ g/ml), anti- CD28 (2 ⁇ g/ml), TFG- ⁇ (10ng/ml), IL-23 (20ng/ml), anti-IL-4 (5 ⁇ g/ml) and anti-IFN- ⁇ (10 ⁇ g/ml) were cultured for 3 days, To obtain Tc17 cells.
  • Tc17 cells were cultured in RPMI-1640 medium at 37°C and divided into three culture groups: Untreated group, Scramble group and PEP20 group, in which PEP20 group was added with 10 ⁇ M concentration of PEP20, and Scramble group was added with 10 ⁇ M concentration of Scramble Peptides (randomly scrambled sequences with the same 20 amino acids as PEP20), the Untreated group did not receive any treatment.
  • Untreated group was added with 10 ⁇ M concentration of PEP20
  • Scramble group was added with 10 ⁇ M concentration of Scramble Peptides (randomly scrambled sequences with the same 20 amino acids as PEP20)
  • PEP20 can significantly inhibit the differentiation of CD8 + T cells into Tc17 cells induced in vitro.
  • Example 3 PEP20 and PEP20-PEG can effectively alleviate the signs of psoriasis in mice induced by IMQ
  • SPF level mice were randomly divided into 6 groups, including Vehicle group (i.e. solvent group), 3 positive control groups, PEP20 group and PEP20-PEG group, and IMQ was used to smear the inside and outside of mouse ear skin, 30mg/mouse, total After smearing for 7 days, an IMQ-induced psoriasis model in mice was established, and solvent, ainti-IL17, ainti-IL23p19, ainti-TNF ⁇ , PEP20 and PEP20-PEG were respectively performed on the second day after IMQ administration (a small amount of dissolution first In HBSS, then dilute with normal saline to use the concentration of 80 ⁇ g/200 ⁇ l) tail vein administration, the dose is 80 ⁇ g/20g mice, once a day, wherein antibody drugs ainti-IL17, ainti-IL23p19, ainti-TNF ⁇ are used as positive control drugs .
  • Vehicle group
  • Ear skin thickness was measured regularly every day from the day of induction.
  • the mice were treated on the 7th day of IMQ induction, and part of the ear skin was obtained, fixed with 4% paraformaldehyde for 24 hours, dehydrated, embedded, cut into tissue sections with a thickness of 5 ⁇ m, stained by H&E, and observed under a microscope.
  • Example 4 PEP20 can effectively reduce the signs of experimental autoimmune encephalomyelitis (EAE) mice
  • mice were treated with EAE model, and heat-inactivated Mycobacterium nucleatum was added to the incomplete Freund's adjuvant, that is, the complete Freund's adjuvant, and the final concentration of MOG35-55 was 10mg/ml.
  • Mice were subcutaneously injected with MOG35-55 and complete Freund's adjuvant at 1:1 mixed emulsified antigen at 2 points on both sides of the dorsal midline; on the day of immunization and the next day, mice were injected with pertussis toxin 200ng/mouse into the tail vein to induce small Murine EAE phenotype.
  • Each model mouse was injected with 80 ⁇ g of PEP20 (dissolved in HBSS) into the tail vein on the 10th day, and injected once every 3 days.
  • Copaxone a current clinical drug for MS, as the positive control drug group.
  • the incidence and scoring of the model mice were observed and recorded daily, and 30 days was the complete scoring cycle, and the demyelination of the brain tissue of the mice was observed by Luxol Fast Blue (LFB) staining.
  • LLB Luxol Fast Blue
  • the EAE scoring criteria are as follows:
  • exogenously synthesized PEP20 can significantly reduce the disease phenotype of EAE model mice, specifically as follows: Compared with the solvent control group (Vehicle group), the EAE clinical score in the PEP20 group was significantly reduced ( Figure 4). A); and the demyelination of the brain tissue was significantly relieved (B of Figure 4).
  • the PEP20 polypeptide provided by the present invention can effectively inhibit the differentiation of CD8 + T cells into Tc17 cells, inhibit the over-differentiation, over-proliferation and over-expression of IL-17A of Tc17 cells, and can be used to prepare drugs for autoimmune diseases and easy to prepare in large quantities, compared with existing medicines, the present invention has lower cost, better curative effect and higher safety.

Abstract

本发明提供了一种多肽及其在制备免疫调节药物中的应用。所述的功能性多肽为长链非编码RNA LINC01871的host gene编码的新型多肽PEP20，其可在炎症环境中特异性靶向CD8 +T细胞，抑制CD8 +T细胞向Tc17细胞的分化，抑制Tc17细胞过度分化、过度增殖以及IL-17A的过度表达，具有免疫调节功能，可用于预防或治疗自身免疫性疾病，如银屑病、类风湿性关节炎与多发性硬化症等。本发明的PEP20分子量小，容易进入细胞发挥作用；且可通过化学方法合成，易于大量制备，稳定性好，相对于现有的治疗银屑病等自身免疫炎症性疾病的抗体价格更低、更安全。

Description

一种多肽及其在制备免疫调节药物中的应用 技术领域
本发明涉及生物医药领域，更具体地说，本发明涉及一种多肽及其在制备免疫调节药物中的应用。
背景技术
促炎性细胞因子白介素IL-17A自1993年被发现以来，一直是研究自身免疫性炎症性疾病的重点。起初发现它是由CD4 +T细胞的一个特定子集(所谓的Th17细胞)产生，但是，众所周知，其他免疫细胞亚群也可以合成和表达IL-17，包括CD8 +T细胞。表达IL-17的CD8 +T细胞(也称为Tc17细胞)的存在已在几种人类炎症性疾病中得到描述。
一项早期研究报道了银屑病病人皮损部位CD8 +T细胞克隆中IL-17mRNA的存在。后来使用流式细胞仪进行检测后发现，银屑病病人的皮肤斑块含有数量增加的IL-17的CD8 +T细胞，而在对照皮肤样本中未观察到。近年来有研究表明在多发性硬化症患者的脑组织活动性病变中，在CD8 +T细胞也检测到了IL-17表达，且分布均匀，与非活动性病变相比，分泌IL17因子的细胞比例较高。总之，这些数据表明IL-17 +CD8 +T细胞存在于各种人类炎症性疾病的组织中，表明这些细胞可能有助于免疫病理的发生发展。
银屑病(Psoriasis)是一种常见的慢性免疫炎症性疾病，全球约有1.25亿患者。银屑病主要症状表现为以红斑、鳞屑为主的皮肤疾病。银屑病为系统性炎症所致疾病，患者体内免疫细胞过度释放IL23、TNF等促炎症因子，激活效应性T细胞释放IL17、IL26等炎性因子诱导角质形成细胞的异常分化、增殖，并且引发多组织及器官的持续性损伤。银屑病会带来包括银屑病性关节炎、自身免疫性疾病、心血管疾病、糖尿病等并发症。
多发性硬化症(MS)是中枢神经系统的一种不可治愈的炎症性自身免疫疾病，影响全球数百万人。Kingston Mills教授团队发现，IL-17在启动介导 实验性自身免疫性脑脊髓炎(EAE)和MS损害的致病性免疫反应中起着关键的“启动”作用。IL-17的主要作用是动员并激活淋巴结中的致病免疫细胞，然后迁移至中枢神经系统(CNS)以引起神经损伤。至关重要的是，科学家发现，阻断IL-17的药物可能不需要穿过血脑屏障即可有效治疗MS。因此，阐明了IL-17作为复发-缓解MS药物靶标的重要性，阻断IL-17的药物在治疗多发性硬化症等中的潜力巨大。
类风湿关节炎(RA)是一种慢性全身性疾病，其特征是自身免疫，活化的炎性细胞浸润关节滑膜、滑膜增生、新血管生成以及软骨和骨骼的逐步破坏。该疾病影响全世界1-2％的人口，最常见的是中年妇女。RA的病因尚不清楚，但促炎性细胞因子在RA中起着核心作用。细胞因子失衡的调节可能代表了控制这种疾病的可靠方法。促炎性细胞因子TNF-α和IL-1在关节炎的发病机制中起着至关重要的作用，临床研究表明，阻断TNF-α后具有疗效，但是，一部分患者对抗TNF-α无效，并且这种治疗方法无法治愈该疾病。因此，IL-1和TNF-α以外的细胞因子可能参与促炎性细胞因子级联反应。现在有大量证据支持T细胞在RA中普遍存在的慢性炎症的发生和延续中的作用，且经发现绝大多数是Trm(组织记忆性T细胞)产生的IL-17在早期疾病中被上调，并被认为会导致与RA相关的炎症。IL-17A可诱导滑膜成纤维细胞、巨噬细胞和软骨细胞等几种关节细胞产生促炎性介质。因此，IL-17受体信号转导已被确定为将急性滑膜炎转变为慢性破坏性关节炎的关键途径，这些观察结果强烈暗示IL-17A是关节炎的重要介质。
目前，市场上针对于IL-17因子的生物阻断剂(如Secukinumab、Ixekizumab和Brodalumab)已在多种自身免疫性疾病的治疗中发挥卓越的功效，但是也显示出不同程度的不良反应，包括鼻咽炎、上呼吸道感染及注射部位反应及头疼等；部分病例也存在严重的念珠菌感染(是与IL-17阻滞剂相关的特定风险)；另外，大量研究表明自身免疫性疾病病人长期使用生物抑制剂后常出现严重的药物耐受反应。因此仍要继续研发疗效好、副作用小的高活性的免疫治疗药物。
多肽类药物通常是指不超过50个氨基酸(aa)构成的肽链。多肽药物其大小介于小分子化药(MW<500)和蛋白药物(MW>5000)之间，很好地弥补了两者之间的缺口，是新一类药物。多肽作为新一类药物，在临床应用 上和生产制备上体现了自己的优越性。在临床应用上跟单抗药物、重组蛋白药物很相似，特异性强、疗效好；生产制备具有质量可控性高、纯度高、易修饰结构稳定等优点。市场方面，全球多肽药物的市场已经超过200亿美元，并且保持每年10％的增速。目前全球多肽类药物的研发已广泛涉足疫苗、抗肿瘤药物、心脑血管药物、抗病毒多肽以及抗菌性活性肽、诊断试剂盒的研究。目前全球已经批准了近60个多肽产品上市。免疫性疾病的有效多肽类药物从1952年至今只有4种，在免疫性疾病治疗领域的多肽类药物的开发整体规模尚小，具有较大的发展空间和应用价值。
发明的公开
本发明的目的在于提供了一种由长链非编码RNA LINC01871的host gene编码的新型多肽PEP20，其可在炎症环境中特异性靶向CD8 +T细胞，抑制CD8 +T细胞向Tc17细胞的分化，可应用于制备免疫调节药物，以解决现有免疫调节药物安全性低、多肽类免疫调节药物稀少的问题。
基于上述，本发明的第一个目的是提供一种分离的多肽，所述的多肽的氨基酸序列如SEQ ID NO：2所示。
优选地，所述的多肽由长链非编码RNALINC01871的host gene编码。
优选地，所述的多肽由核苷酸序列为SEQ ID NO：1中第58～117位的核苷酸序列或其简并序列所翻译。
本发明的另一个目的是提供一种分离的多核苷酸，其编码所述的多肽。优选地，其核苷酸序列如SEQ ID NO：1中第58～117位或其简并序列。
本发明的另一个目的是提供一种表达载体，其含有所述的多核苷酸。
本发明的另一个目的是提供一种重组细胞，其含有所述的表达载体或其基因组中包含所述的多核苷酸。
本发明的另一个目的是提供所述的多肽或编码其的多核苷酸，或所述的表达载体，或所述的重组细胞在制备免疫调节药物中的应用；
优选地，所述的免疫调节药物是预防、缓解或治疗自身免疫性疾病的药物；
进一步地，所述的自身免疫性疾病包括：银屑病、皮炎、多发性硬化症、哮喘与类风湿性关节炎、肠炎。
所述的免疫调节药物是抑制CD8 +T细胞向Tc17细胞的分化，抑制Tc17细胞过度分化、过度增殖以及IL-17A的过度表达的药物。
本发明的另一个目的是提供一种制备所述的多肽的方法；
一些实施例中，所述方法包括：培养所述的重组细胞，从而重组表达所述的多肽；
另一些实施例中，所述方法包括：通过体外人工合成的方法或生物学合成方法制备所述的多肽。
本发明的另一个目的是提供一种用于免疫调节的药物组合物，所述的药物组合物包括：前面任一所述的多肽或编码其的多核苷酸，或所述的表达载体，或所述的重组细胞；以及药学上或生理学上可接受的载体。
本发明的另一个目的是提供一种用于免疫调节的药盒，所述的药盒中包括：前面任一所述的多肽或编码其的多核苷酸；或所述的表达载体；或所述的重组细胞；或所述的药物组合物。
相对于现有技术，本发明的有益效果是：
1.本发明提供的PEP20多肽能够特异性靶向CD8 +的效应T细胞，抑制CD8 +T细胞向Tc17细胞的分化从而治疗银屑病等自身免疫性炎症性疾病，且PEP20多肽分子量小，容易进入细胞发挥作用；
2.本发明提供的PEP20多肽通过原核表达进行生物学合成和化学合成，易于大量制备，稳定性好，相对于现有的对于治疗银屑病等自身免疫炎症性疾病有效的抗体价格更低、更安全。
附图的简要说明
图1为PEP20获得后的检测分析图；其中：
A为PEP20的质谱分析图；
B为PEP20的HPLC纯度分析图。
图2为PEP20对Tc17细胞分化的影响。
图3为对照组与PEP20、PEP20-PEG组处理类银屑病模型鼠皮肤炎症的影响；其中：
A为对照组与PEP20、PEP20-PEG组处理类银屑病模型鼠的皮损症状对比；
B为对照组与PEP20、PEP20-PEG组处理类银屑病模型鼠的表皮厚度对比。
C为对照组与PEP20、PEP20-PEG组处理类银屑病模型鼠的H&E染色结果。
图4为对照组与PEP20处理EAE模型小鼠前后发病程度的对比；其中：
A为对照组与PEP20处理EAE模型小鼠的疾病评分对比；
B为对照组与PEP20处理EAE模型小鼠的脑组织Luxol Fast Blue(LFB)染色结果。
实现本发明的最佳方式
以下将结合附图和实施例对本发明的技术方案做进一步说明。
基于现有研究可知，IL-17现在被认为是人类自身免疫和自身炎症性疾病发病机制中的关键细胞因子，越来越多的证据表明产生IL-17的CD8 +T细胞(Tc17)是介导多种自身免疫炎症性疾病的关键炎性T细胞。包括：多发性硬化症、银屑病、银屑病性关节炎、类风湿性关节炎、皮炎、肠炎等。因此，本发明申请人为了寻找治疗前述自身免疫炎症性疾病的药物，经过了大量研究，最终发现了一种全新的来源于人细胞的多肽，称为PEP20。所述PEP20多肽为长链非编码RNALINC01871 host gene的开放阅读框核苷酸序列编码产生的具有20个氨基酸的多肽，可特异性调节CD8 +效应性T细胞的分化，具有免疫调节功能，可用于预防或治疗自身免疫性疾病，如银屑病、类风湿性关节炎与多发性硬化症等，且具有毒性小、作用稳定、效应持久、易于合成等优势。
术语
如本发明所用，所述的“LINC01871的host gene编码的多肽”、“PEP20”、“PEP20多肽”、“所述的多肽”、“功能性多肽”可以互换使用。
如本发明所用，所述的调节包括“上调”和“下调”，其中“上调”包括但不限于：促进、提高、升高、增强等；所述的“下调”包括但不限于：降低、抑制、拮抗、阻滞、阻断等。
如本发明所用，“药学上可接受的”的成分是适用于人和/或哺乳动物而无过度不良副反应(如毒性)的，即具有合理的效益/风险比的物质。术语“药 学上可接受的载体”指用于治疗剂给药的载体，包括各种赋形剂和稀释剂。该术语指这样一些药剂载体：它们本身并不是必要的活性成分，且施用后没有过分的毒性。
PEP20多肽及其获得
本发明人发现微小LINC01871的host gene的部分核苷酸序列能够编码产生一段功能性多肽，并命名为PEP20。
本发明的PEP20多肽可以是重组多肽或合成多肽。其可以是化学合成的产物，化学合成的方法对于本领域技术人员而言是熟悉的，例如固相多肽合成方法。
本发明的PEP20多肽的序列为：MVEEIQASLMWQQAREREGE(SEQ ID NO：2)。
本发明还包括PEP20多肽的片段、衍生物和类似物。如本发明所用，术语“片段”、“衍生物”和“类似物”是指基本上保持本发明的PEP20多肽相同的生物学功能或活性的多肽。PEP20多肽的片段、衍生物或类似物可以是：
(1)有一个或多个(如1-5个、1-3个或1-2个)保守或非保守性氨基酸残基(优选保守性氨基酸残基)被取代的多肽，而这样的取代的氨基酸残基可以是也可以不是由遗传密码编码的，或
(2)在一个或多个氨基酸残基中具有取代基团的多肽，或
(3)成熟多肽与另一个化合物(比如延长多肽半衰期的化合物，例如聚乙二醇)融合所形成的多肽，或
(4)附加的氨基酸序列融合到此多肽序列而形成的多肽(如前导序列或分泌序列或用来纯化此多肽的序列或蛋白原序列，或融合蛋白)。根据本发明的定义这些片段、衍生物和类似物属于本领域熟练技术人员公知的范围。
在本发明中，PEP20可以指具有SEQ ID NO：2所示序列的多肽。该术语还包括具有与PEP20多肽相同功能的、在C末端和/或N末端添加一个或数个(例如为300个以内，较佳地200个以内，更佳地100个以内，更佳地50个以内，例如40、30、20、10、5、3、2、1)氨基酸。例如，在本领域中，用性能相近或相似的氨基酸进行取代时，通常不会改变蛋白质的功能。又比如，在C末端和/或N末端添加一个或数个氨基酸通常也不会改变蛋白质的功能。该术语还包括PEP20多肽的活性片段和活性衍生物。
本发明中，也包括为了增加多肽的稳定性、半衰期、促进功效而对一个或几个氨基酸加以修饰后构成的修饰形式的多肽(通常不改变一级结构)，包括：体内或体外的多肽的化学衍生形式如乙酰化或羧基化。修饰还包括糖基化。修饰形式还包括具有磷酸化氨基酸残基(如磷酸酪氨酸、磷酸丝氨酸、磷酸苏氨酸)的序列。还包括被修饰从而提高了抗水解性能或优化了溶解性能的多肽。
本发明公开的PEP20合成简易、稳定性好、成本低、不具免疫排斥性、能够在炎症环境中特异性抑制Tc17细胞的分化，治疗效果极为显著，治疗后疾病不易复发且副作用小。
本发明的多肽还能与其它的功能性分子构成复合体，所述的复合体包括：本发明所述的多肽，以及与所述多肽连接的功能性分子。
一些实施例中，所述的功能性分子是具有示踪功能的标志物，包括但不限于荧光染料、MRI造影剂、放射性显影剂、磁性粒子或具有着色功能的化学试剂。例如，所述的具有示踪功能的标志物或功能性小分子可以为异硫氰酸荧光素(FITC)。
一些实施例中，所述的功能性分子是功能性小分子，包括无机小分子和有机小分子，其分子量小于1000道尔顿。
一些实施例中，所述的功能性分子是功能性大分子，例如功能性多肽(如抗体)、功能性核酸；优选地，所述的功能性核酸包括但不限于：质粒、siRNA、DNA、寡核苷酸、miRNA、反义核酸等。
一些实施例中，所述功能性分子为具有分子包装载物功能的制剂，包括但不限于：脂质体、多聚体、树突状分子、纳米包装制剂等。
一些实施例中，所述的功能性分子为可携带遗传物质的病毒载体，包括但不限于逆转录病毒载体、慢病毒载体或腺病毒载体等。
作为本发明的优选方式，所述的功能性分子还可以是一种穿膜肽，本发明的多肽与之连接后，能提高多肽穿透细胞、进入到细胞中的能力。所述的穿膜肽可以采用本领域已知的可引导肽或其编码基因进入到细胞内的任何分子，或采用提高肽穿透细胞能力的任何分子。一些具有穿膜功能的肽包括：①蛋白衍生肽(protein derived CPPs)，如penetratin、TAT和pVEC等；②模型肽(model peptides)如MAP和(Arg)7等；③设计肽(designed CPPs) 如MPG和Transportan等。从其两亲性性质也可将其分为3类：①两亲性CPPs(PaCPPs)，如MPG、transportan、TP10、Pep-1；②中等两亲性CPPs(SaCPPs)，如penetratin，RL16；③非两亲性CPPs(NaCPPs)，如R9。
本发明所述的多肽与功能性分子的连接方式可以为共价连接或非共价连接。应理解，只要能够保留多肽及功能性分子的功能，任何连接方式均可包含在本发明中。共价连接通常以形成共价键的方式将两个分子进行连接，而一些非共价连接(不形成共价键)，例如偶联、吸附、结合等也可应用。
作为本发明的优选方式，所述的多肽与功能性分子之间通过化学键相连；更佳地，所述的化学键是肽键。
本发明还提供了编码本发明PEP20多肽的多核苷酸序列。本发明的多核苷酸可以是DNA形式或RNA形式。DNA可以是编码链或非编码链，“编码多肽的多核苷酸”可以是包括编码此多肽的多核苷酸，也可以是还包括附加编码和/或非编码序列的多核苷酸。
本发明也涉及包含本发明的多核苷酸的载体，以及用本发明的载体或PEP20多肽C端三氨基酸截短体的编码序列经基因工程产生的宿主细胞(重组细胞)，以及经重组技术产生本发明所述多肽的方法。
术语“表达载体”指本领域熟知的细菌质粒、噬菌体、酵母质粒、植物细胞病毒、哺乳动物细胞病毒或其他载体。总之，只要能在宿主体内复制和稳定，任何质粒和载体都可以用。表达载体的一个重要特征是通常含有复制起点、启动子、标记基因和翻译控制元件。
包含上述的适当多核苷酸序列以及适当启动子或者控制序列的载体，可以用于转化适当的宿主细胞，以使其能够表达多肽。宿主细胞可以是原核细胞，如细菌细胞；或是低等真核细胞，如酵母细胞；或是高等真核细胞，如植物细胞。代表性例子有：大肠杆菌；链霉菌属、农杆菌；真菌细胞如酵母；植物细胞等。
PEP20多肽的应用
本发明的主要贡献不仅在于获得所述的PEP20多肽，同时还在于验证了该多肽的功能。
本发明所述的PEP20可在炎症环境中特异性靶向CD8 +T细胞，抑制CD8 +T细胞向Tc17细胞的分化，抑制Tc17细胞过度分化、过度增殖以及 IL-17A的过度表达。
在本发明的具体实施例中，确定了PEP20能够有效抑制Tc17细胞分化，并且PEP20与其PEG修饰的PEP20-PEG都能够减少IMQ诱导的类银屑病模型鼠皮肤中炎症细胞的数量，减少耳部皮肤角质形成细胞的增殖，从而有效地减轻IMQ诱导的小鼠类银屑病动物模型的发病。
在本发明的另一具体实施例中，外源合成的PEP20能够有效减轻小鼠EAE模型的发病程度，缓解小鼠脑组织脱髓鞘情况。
上述研究结果表明，所述的PEP20多肽可用于制备自身免疫性疾病药物，或用于制备抑制Tc17细胞过度分化、过度增殖、IL-17A过度表达的药物。
所述的自身免疫性疾病是CD8 +T细胞向Tc17细胞的过度分化、IL-17A过度分泌为特征的自身免疫性疾病。较佳地，所述的自身免疫性疾病是银屑病、多发性硬化症、类风湿性关节炎等自身免疫性炎症性疾病。此外，对其它一些与Tc17细胞免疫调节功能失调相关的疾病或症状也具有潜在的预防或治疗作用。
目前，已知与Tc17细胞的免疫调节功能失调相关的疾病或症状选自：肿瘤或病毒感染，炎症反应、类风湿关节炎、器官移植、系统性红斑狼疮、银屑病、多发性硬化症等。
药物组合物及药盒
本发明还提供一种用于免疫调节的药物组合物，所述的药物组合物包括：本发明所述的多肽或编码其的多核苷酸，或含有该多核苷酸的表达载体或表达该多肽的重组细胞；以及药学上或生理学上可接受的载体。
合适的药学上可接受的载体是本领域普通技术人员所熟知的。在Remington’s Pharmaceutical Sciences中可找到关于药学上可接受的载体的充分说明。在组合物中药学上可接受的载体可含有液体，如水、磷酸盐缓冲液、ringer溶液、生理盐水、平衡盐溶液、甘油或山梨醇等。另外，这些载体中还可能存在辅助性的物质，如润滑剂、助流剂、润湿剂或乳化剂、pH缓冲物质和稳定剂，如白蛋白等。
在使用时，是将安全有效量的本发明所述的多肽或编码其的多核苷酸，或含有该多核苷酸的表达载体或表达该多肽的重组细胞施用于哺乳动物(如人)，其中该安全有效量通常至少约0.01微克/千克体重，而且在大多数情况 下不超过约10毫克/千克体重。当然，具体剂量还应考虑给药途径、病人健康状况等因素，这些都是熟练医师技能范围之内的。
对于某一对象的精确有效量取决于该对象的体型和健康状况、病症的性质和程度、以及选择给予的治疗剂和/或治疗剂的组合。对于某给定的状况而言，可以用常规实验来确定该有效量，临床医师是能够判断出来的。
本发明还提供了一种药盒或试剂盒，其中包括：本发明所述的多肽或编码其的多核苷酸，或含有该多核苷酸的表达载体或表达该多肽的重组细胞，或所述的药物组合物。
为了便于临床应用，本发明的药物组合物可以包含在注射用给药器(如注射用针)中，所述的注射用给药器中，可以包含一次给药量的所述的药物组合物。所述的注射用给药器可以被包含在药盒中，以方便储存、使用。
本发明所述的药盒或试剂盒中，还可包括使用说明书，以利于本领域技术人员按照正确的方式使用。
下面将结合附图1至图4以及具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件如J.萨姆布鲁克等编著，分子克隆实验指南，第三版，科学出版社，2002中所述的条件，或按照制造厂商所建议的条件。
实施例1、PEP20序列分析和体外合成
1、PEP20序列分析
LINC 01871的host gene的序列如下(SEQ ID NO：1)：
Figure PCTCN2022094056-appb-000001
Figure PCTCN2022094056-appb-000002
Figure PCTCN2022094056-appb-000003
Figure PCTCN2022094056-appb-000004
上述序列中，下划线粗体部分为PEP20核苷酸序列。翻译为氨基酸的序列为：MVEEIQASLMWQQAREREGE(SEQ ID NO：2)。
2、PEP20的体外合成
使用常规的固相多肽合成方法，按照SEQ ID NO：2氨基酸序列合成多肽，并通过质谱检测和HPLC检测分析所得多肽的正确性与纯度。结果如图1所示，质谱分析确定氨基酸正确，其分子量为2420.69(图1的A)；HPLC检测所得多肽的纯度为98.65％(图1的B)。使用前溶于HBSS待用。
实施例2、PEP20抑制Tc17细胞分化
实施例1中固相多肽合成方法得到的PEP20，测定其对Tc17细胞分化的影响。
获取小鼠的脾脏细胞，利用免疫磁珠分离小鼠初始CD8 +T细胞，在37℃条件下培养于RPMI-1640培养基中，在培养基中加入anti-CD3(5μg/ml)、anti-CD28(2μg/ml)、TFG-β(10ng/ml)、IL-23(20ng/ml)、anti-IL-4(5μg/ml)和anti-IFN-γ(10μg/ml)培养3天，以得到Tc17细胞。
在37℃条件下、RPMI-1640培养基中培养Tc17细胞，分为3个培养组，分别为Untreated组、Scramble组和PEP20组，其中PEP20组加入10μM浓度的PEP20，Scramble组加入10μM浓度的Scramble多肽(具有与PEP20相同20个氨基酸的随机打乱序列)，Untreated组不做任何处理。通过流式细胞术观察PEP20对Tc17分化的影响。
结果如图2所示，PEP20可以明显地抑制体外诱导CD8 +T细胞向Tc17细胞的分化。
实施例3、PEP20与PEP20-PEG能够有效减轻IMQ诱导的小鼠类银屑病体征
将SPF级别小鼠随机分为6组，包括Vehicle组(即溶剂组)、3个阳性 对照组、PEP20组及PEP20-PEG组，用IMQ涂抹小鼠耳部皮肤内外侧，30mg/只，共计涂抹7天，建立IMQ诱导的小鼠类银屑病模型，并于IMQ给药后第2天分别进行溶剂、ainti-IL17、ainti-IL23p19、ainti-TNFα、PEP20及PEP20-PEG(先少量溶解于HBSS，再用生理盐水稀释为使用浓度为80μg/200μl)尾静脉给药，剂量为80μg/20g小鼠，每天一次，其中抗体药物ainti-IL17、ainti-IL23p19、ainti-TNFα作为阳性对照药物。从诱导当天开始每天定时测量耳部皮肤厚度。IMQ诱导7天时处理小鼠，获取部分耳部皮肤，4％多聚甲醛固定24h，常规脱水、包埋、将组织块切成厚度为5μm的组织切片、H&E染色后，显微镜下观察IMQ诱导的类银屑病模型鼠耳部皮肤的病理情况。
结果如图3所示，观测模型鼠的表皮以及H&E染色结果可见，外源合成的PEP20及PEP20-PEG能够明显减轻类银屑病模型鼠的皮损症状，使小鼠表皮鳞屑减少、炎细胞浸润减少(图3的A、C)；统计结果显示，PEP20及PEP20-PEG能够明显使小鼠的耳部表皮厚度降低(图3的B)。
实施例4、PEP20能够有效减轻实验性自身免疫性脑脊髓炎(EAE)小鼠体征
对小鼠进行EAE模型造模处理，在不完全弗氏佐剂中加入热灭活核分枝杆菌，即为完全弗氏佐剂，MOG35-55配成终浓度为10mg/ml。小鼠分别于背侧中线两侧2点皮下注射由MOG35-55与完全弗氏佐剂按1：1混合乳化抗原；免疫当天及第二天给小鼠尾静脉注射百日咳毒素200ng/只诱导小鼠EAE表型。
每只造模小鼠第10天开始尾静脉注射80μg的PEP20(溶于HBSS)，每3天注射一次，我们将现MS临床用药Copaxone作为阳性对照药物组。每日观察与记录模型鼠发病情况与评分，30天为评分完整周期，利用Luxol Fast Blue(LFB)染色观测小鼠脑组织脱髓鞘情况。
EAE评分标准如下：
0分：不发病；
1分：尾巴无力；
2分：轻微后肢无力；
3分：严重后肢瘫痪；
4分：四肢瘫痪；
5分：濒临死亡或死亡。
结果如图4所示，外源合成的PEP20能够明显减轻EAE模型鼠的疾病表型，具体表现为：与溶剂对照组(即Vehicle组)相比，PEP20组EAE临床评分显著降低(图4的A)；且脑组织脱髓鞘情况有明显缓解(图4的B)。
综上所述，本发明提供的PEP20多肽能有效抑制CD8 +T细胞向Tc17细胞的分化，抑制Tc17细胞过度分化、过度增殖以及IL-17A的过度表达，能够用于制备自身免疫性疾病的药物；且易于大量制备，较现有药物，本发明成本更低，且疗效好、安全性更高。
尽管本发明的内容已经通过上述优选实施例作了详细介绍，但应当认识到上述的描述不应被认为是对本发明的限制。在本领域技术人员阅读了上述内容后，对于本发明的多种修改和替代都将是显而易见的。因此，本发明的保护范围应由所附的权利要求来限定。

Claims (10)

  1. 一种分离的多肽，其特征在于，所述的多肽的氨基酸序列如SEQ ID NO：2所示。
  2. 一种分离的多核苷酸，其特征在于，所述的核苷酸编码权利要求1所述的多肽。
  3. 如权利要求2所述的分离的多核苷酸，其特征在于，核苷酸的序列为SEQ ID NO：1中第58～117位或其简并序列。
  4. 一种表达载体，其特征在于，其含有权利要求2或3所述的多核苷酸。
  5. 一种重组细胞，其特征在于，其含有权利要求4所述的表达载体或其基因组中包含权利要求2或3所述的多核苷酸。
  6. 权利要求1所述的多肽，或权利要求2或3所述的多核苷酸，或权利要求4所述的表达载体，或权利要求5所述的重组细胞在制备免疫调节药物中的应用；所述的免疫调节药物是缓解或治疗自身免疫性疾病的药物；所述的自身免疫性疾病为银屑病、多发性硬化症与类风湿性关节炎。
  7. 如权利要求6所述的应用，其特征在于，所述的免疫调节药物是抑制CD8 +T细胞向Tc17细胞的分化，抑制Tc17细胞过度分化、过度增殖以及IL-17A过度表达的药物。
  8. 一种制备权利要求1所述的多肽的方法，其特征在于，所述方法包括：培养权利要求5所述的重组细胞，从而重组表达权利要求1所述的多肽；或所述方法包括：通过体外人工合成的方法或生物学合成方法制备权利要求1所述的多肽。
  9. 一种用于免疫调节的药物组合物，其特征在于，所述的药物组合物包括：权利要求1所述的多肽，或权利要求2或3所述的多核苷酸，或权利要求4所述的表达载体，或权利要求5所述的重组细胞；以及药学上或生理学上可接受的载体。
  10. 一种用于免疫调节的药盒，其特征在于，所述的药盒中包括：
    权利要求1所述的多肽；或
    权利要求2或3所述的多核苷酸；或
    权利要求4所述的表达载体；或
    权利要求5所述的重组细胞；或
    权利要求9所述的药物组合物。
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