CN111777667B

CN111777667B - Small peptide and application thereof in preparation of immunoregulation medicine

Info

Publication number: CN111777667B
Application number: CN202010661884.0A
Authority: CN
Inventors: 王宏林; 孙力波
Original assignee: Shanghai Jiaotong University School of Medicine
Current assignee: Shanghai Jiaotong University School of Medicine
Priority date: 2020-07-10
Filing date: 2020-07-10
Publication date: 2022-09-09
Anticipated expiration: 2040-07-10
Also published as: CN111777667A

Abstract

The invention provides a small peptide and application thereof in preparing an immunoregulation medicament. The small peptide is a C-terminal three-amino-acid truncated body of miPEP 155. The mippe 155C-terminal three-amino-acid truncated body can more effectively inhibit dendritic cells from presenting antigens to T cells, reduces the number of Th17 cells, has an excellent immune regulation function, and can be used for preventing or treating autoimmune diseases, such as psoriasis.

Description

Small peptide and application thereof in preparation of immunoregulation medicine

Technical Field

The invention belongs to the field of biological medicines, and particularly relates to a C-terminal three-amino-acid truncation of a small peptide (miPEP155) coded by a micro RNA155hostgene and application thereof in preparation of immunoregulation medicaments.

Background

Dendritic Cells (DCs) are the most powerful Antigen Presenting Cells (APCs) in vivo, linking innate immunity to adaptive immunity. DCs recognize pathogens via pattern recognition receptors and migrate to T cell regions of lymphoid organs, presenting antigens to specific T cells. Autoimmune disease is mainly caused by disruption of the balance of self tolerance, and DCs are closely related to the development of autoimmune disease. Under the influence of genetic and environmental factors, the innate immunity and acquired immune dysfunction and the body's tolerance to self-antigenic determinants are reduced or lost, so that the activation of autoreactive lymphocytes causes pathological damage to single or multiple organs. In this process, DC plays an important role.

Th17 cells (T helper cell 17, Th17) are a newly discovered subset of T cells capable of secreting interleukin 17 (IL-17). In the stimulation signal and inflammatory factor environment of DC, Th17 can be a helper T cell formed by differentiating TH0 cell under the stimulation of IL-6 and IL-23, and ROR gamma is its important transcription factor. Th17 cell can secrete IL-17A, IL-17F, IL-6 and Tumor necrosis factor a (TNF-a), etc., and these cytokines can mobilize, recruit and activate neutrophils collectively, thus effectively mediating the inflammatory response of tissue, therefore Th17 has important meaning in autoimmune diseases and body defense response.

Studies have shown that Th17 is closely related to autoimmune diseases such as psoriasis, autoimmune encephalitis, asthma, rheumatoid arthritis, etc. In addition, it is also closely related to colitis, brain tumor, diabetes and other diseases. Therefore, the Th17 has important significance for the occurrence and development of autoimmune diseases, and the deep research on the Th17 is helpful for understanding the pathogenesis of autoimmune diseases and has profound significance for disease prognosis judgment and further treatment.

Psoriasis is a chronic inflammatory disease, and causes thereof mainly include: infection, immunity, genetics, spirit and environmental factors. Major pathological changes in psoriasis include: keratinocyte parakeratosis or hyperkeratosis, dermal massive inflammatory cell infiltration and abnormal vascular hyperplasia are often accompanied with systemic diseases such as diabetes, coronary heart disease, hypertension, depression, metabolic syndrome and the like. The Chinese people have more psoriasis in young and strong years of 25-45 years, the incidence rate of psoriasis in children is gradually increased in recent years, and the psoriasis is not easy to cure and easy to relapse, so that the physical and mental health of patients is seriously harmed, and the life quality of the patients is seriously influenced. At present, the clinical methods for treating psoriasis comprise medication, physical therapy, immunobiological therapy, traditional Chinese medicine therapy and the like. Chinese imports medicines for treating psoriasis every year, especially small molecular biological medicines with high technological content and strong targeting property have huge cost, but most of psoriasis treatment methods based on biological preparation medicines such as antibodies and the like easily cause toxic and side effects to a certain extent, and organisms can generate drug resistance to medicines, so that some psoriasis patients have relapse and rebound after stopping using some molecular antibody medicines. Therefore, the development of a new generation of new medicine for treating psoriasis, which has strong targeting property, good long-term curative effect, high safety and low price, of the independent intellectual property rights of China is imperative.

Polypeptide drugs are customarily referred to as polypeptide hormones. Generally, compounds consisting of 50 or less amino acid residues are included in the polypeptides. It is known that a large number of hormones and active polypeptides are contained and secreted in an organism, and nearly 40 active polypeptide substances exist in the brain alone, and new active polypeptide substances are continuously discovered, isolated and purified.

The polypeptide medicine aiming at the autoimmune diseases has important clinical application value. In the previous research work of the inventor, a small peptide (miPEP155) coded by the micro RNA155hostgene is obtained, the small peptide has a remarkable effect of preventing and treating autoimmune diseases such as psoriasis, and further research of the inventor shows that the efficacy of the small peptide in the aspect of immune regulation has room for improvement.

Disclosure of Invention

The invention aims to provide a C-terminal three-amino-acid truncation of a small peptide (miPEP155) coded by a micro RNA155hostgene and application thereof in preparation of immunomodulatory drugs. The small peptide can specifically target Dendritic Cells (DC) in an inflammatory environment, inhibit antigen presentation of the DC, reduce the expression level of mRNA and protein of IL-17A in the skin lesion area of psoriasis patients and psoriasis (psoriasis-like) models, and reduce the expression level of mRNA and protein of transcription factor C/EBP beta of Th17, and animal in vivo experiments can effectively reduce IMQ-induced animal psoriasis models, can be obtained in large quantities through artificial synthesis or biological synthesis, and can be applied to preparation of immunoregulatory medicaments.

In a first aspect of the invention, an isolated small peptide is provided, wherein the amino acid sequence of the small peptide is shown as SEQ ID NO. 2.

In another preferred embodiment, the small peptide is encoded by the nucleotide sequence as shown in SEQ ID NO. 1 at positions 181-222 or a degenerate sequence thereof.

In another aspect of the invention, an isolated polynucleotide is provided that encodes the small peptide.

In another preferred embodiment, the nucleotide sequence of the polynucleotide is as shown in SEQ ID NO. 1 at positions 181-222 or a degenerate sequence thereof.

In another aspect of the invention, an expression vector is provided comprising the polynucleotide encoding the small peptide.

In another preferred embodiment, the expression vector comprises a viral vector or a non-viral vector.

In another aspect of the present invention, there is provided a recombinant cell comprising said expression vector or a genome thereof comprising said polynucleotide.

In another aspect of the invention, there is provided the use of said small peptide or polynucleotide encoding the same, or said expression vector or said recombinant cell in the preparation of an immunomodulatory drug.

In a preferred embodiment, the immunomodulatory drug comprises: drugs that inhibit dendritic cells from presenting antigen to T cells (inhibit dendritic cell antigen presentation from being overactivated); preferably, it is immunomodulating by inhibiting antigen presentation by DCs.

In a preferred embodiment, the immunomodulatory drug comprises: an agent that inhibits the overexpression of IL-17A.

In another preferred embodiment, the immunomodulatory drug comprises: a medicament for preventing, alleviating or treating autoimmune diseases.

In another preferred embodiment, the autoimmune disease includes (and is not limited to): preventing, alleviating or treating psoriasis, vitiligo, dermatitis, experimental autoimmune encephalitis, asthma, rheumatoid arthritis or Absidial reaction.

In another aspect of the invention, there is provided a method of preparing the small peptide, the method comprising: culturing said recombinant cell, thereby recombinantly expressing said small peptide.

In another aspect of the invention, there is provided a method of making the small peptide, the method comprising: the small peptide is prepared by an in vitro artificial synthesis method.

In another aspect of the present invention, there is provided a pharmaceutical composition for immunomodulation, comprising: any one of the small peptides described above or a polynucleotide encoding the same, or said expression vector or said recombinant cell; and a pharmaceutically or physiologically acceptable carrier.

In another aspect of the present invention, there is provided a kit for immunomodulation comprising: any of the foregoing small peptides or polynucleotides encoding same, expression vectors, recombinant cells or pharmaceutical compositions.

In another preferred embodiment, the polynucleotide, expression vector, recombinant cell or pharmaceutical composition is placed in a container and prepared into a kit.

In another preferred embodiment, the kit further comprises instructions for use to guide the application to a person skilled in the art or a clinician.

In another preferred embodiment, the container includes but is not limited to: vials, syringes, and the like.

Other aspects of the invention will be apparent to those skilled in the art in view of the disclosure herein.

Drawings

FIG. 1A, mass spectrometry is carried out to determine the correctness of the amino acid of the terminal three-amino-acid truncation of miPEP155C, and the molecular weight detected by mass spectrometry is 1593.9 (theoretical value: 1593.99).

HPLC purity analysis of the 1B, miPEP155C terminal three amino acid truncation of FIG. 1 indicated 95.5% purity.

FIG. 2 (A-D), exogenously synthesized miPEP155 and miPEP 155C-terminal three-amino-acid truncations inhibit DC antigen presentation. Mu μm miPEP155 or miPEP 155C-terminal three-amino-acid truncation (miPEP155-C3) is added into the R848-stimulated BMDC, and detection is carried out by flow cytometry, so that the antigen presentation of the DC can be inhibited by exogenously synthesized miPEP155 and miPEP 155C-terminal three-amino-acid truncation, but the inhibition effect of the miPEP 155C-terminal three-amino-acid truncation is obviously improved.

FIG. 3 shows that miPEP155 synthesized by external source can effectively reduce the sign of mouse psoriasis induced by IMQ.

A. The miPEP155 synthesized by the external source can obviously reduce the attack of the psoriasis-like disease of the model mouse (D1-D7 show the 1 st day-7 th day).

B. The miPEP155 synthesized by the external source can effectively inhibit the multiplication of keratinocytes in the epidermis of an IMQ-induced psoriasis-like model mouse.

FIG. 4, the exogenously synthesized miPEP 155C-terminal three-amino-acid truncation can reduce the amount of Th17 in the epidermis and dermis of a psoriasis-like model mouse. miPEP 155C-terminal three-amino-acid truncation is injected into tail veins to treat IMQ-induced psoriasis-like model mice, after 7 days, the mice are sacrificed, ear skins are taken for fixation, and immunohistochemical staining detects that the number of Th17 in the ear epidermis and dermis of the mice in the miPEP 155C-terminal three-amino-acid truncation treatment group is reduced.

FIG. 5, exogenously synthesized miPEP155 was able to reduce the expression level of IL-17AmRNA in psoriasis patients' peripheral blood mononuclear cells.

Detailed Description

The present inventors have conducted extensive studies to find that a part of the nucleotide sequence of hostgene of microRNA 155 can encode a polypeptide (miPEP155) that produces a small peptide and is called microRNA 155 hostgene. Through further structural optimization of mippe 155, the inventors constructed a C-terminal three-amino acid truncation of mippe 155 (mippe 155-C3). The miPEP 155C-terminal three-amino-acid truncated body can more effectively inhibit dendritic cells from presenting antigens to T cells, reduces the number of Th17 cells, has an excellent regulation-free function, and can be used for preventing or treating autoimmune diseases, such as psoriasis.

As used herein, the terms "C-terminal three-amino acid truncation of the polypeptide encoded by the microRNA 155 hostgene", "C-terminal three-amino acid truncation of the polypeptide encoded by the miRNA-155 precursor", "C-terminal three-amino acid truncation of the miPEP155 polypeptide", "truncation", "small peptide" and "miPEP 155-C3" are used interchangeably.

As used herein, a "pharmaceutically acceptable" component is one that is suitable for use in humans and/or mammals without undue adverse side effects (such as toxicity), i.e., with a reasonable benefit/risk ratio. The term "pharmaceutically acceptable carrier" refers to a carrier for administration of a therapeutic agent, including various excipients and diluents. The term refers to such pharmaceutical carriers: they are not necessary active ingredients per se and are not excessively toxic after administration.

As used herein, "effective amount" refers to an amount of a therapeutic agent that treats, ameliorates, or prevents a disease or condition of interest, or that exhibits a detectable therapeutic or prophylactic effect.

Small peptides

The inventors found that the partial nucleotide sequence of hostgene of microRNA 155 encodes a C-terminal three amino acid truncation producing a small peptide, named miPEP 155-C3.

The C-terminal three-amino-acid truncation of the miPEP155 polypeptide can be recombinant peptide or synthetic peptide. It can be a product of chemical synthesis or produced from prokaryotic or eukaryotic hosts (e.g., bacterial, yeast, higher plant, insect, and mammalian cells) using recombinant techniques. Methods of chemical synthesis are familiar to those skilled in the art, for example, solid phase polypeptide synthesis methods.

The sequence of the miPEP 155C-terminal three-amino-acid truncation is as follows: MEMALMVAQTRKGK (SEQ ID NO: 2).

The invention also includes fragments, derivatives and analogs of the C-terminal three amino acid truncation of the mippe 155 polypeptide. As used herein, the terms "fragment," "derivative," and "analog" refer to a polypeptide that retains substantially the same biological function or activity as the C-terminal three amino acid truncation of the mippe 155 polypeptide of the present invention. The fragment, derivative or analogue of the C-terminal three amino acid truncation of the mippe 155 polypeptide may be:

(i) polypeptides in which one or more (e.g., 1-10, 1-5, 1-3, or 1-2) conserved or non-conserved amino acid residues (preferably conserved amino acid residues) are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code, or

(ii) Polypeptides having substituent groups in one or more amino acid residues, or

(iii) The mature polypeptide is fused to another compound (e.g., a compound that increases the half-life of the polypeptide, such as polyethylene glycol), or

(iv) Additional amino acid sequences are fused to the polypeptide sequence to form a polypeptide (e.g., a leader or secretory sequence or a sequence used to purify the polypeptide or a proprotein sequence, or a fusion protein). Such fragments, derivatives and analogs are within the purview of those skilled in the art in view of the definitions herein.

In the present invention, the C-terminal three-amino acid truncation of the miPEP155 polypeptide may refer to a polypeptide having the sequence shown in SEQ ID No. 2. The term also includes the addition of one or several (e.g., within 300, preferably within 200, more preferably within 100, more preferably within 50, e.g., 40, 30, 20, 10, 5, 3, 2, 1) amino acids at the C-terminus and/or N-terminus that have the same function as the C-terminal three amino acid truncations of the mippe 155 polypeptide. For example, in the art, substitutions with amino acids of similar or similar properties will not generally alter the function of the protein. Also, for example, the addition of one or several amino acids at the C-terminus and/or N-terminus does not generally alter the function of the protein. The term also includes active fragments and active derivatives of C-terminal three amino acid truncations of the mippe 155 polypeptide. However, when one or several amino acids are added to the C-terminal and/or N-terminal of the small peptide of the present invention, the peptide formed by adding SVV as the amino acid to the C-terminal is not included.

In the present invention, modified forms of polypeptides (usually without changing the primary structure) comprising one or more amino acids modified to increase the stability, half-life, or efficacy of the polypeptide are also included, including: chemically derivatized forms of the polypeptide, such as acetylation or carboxylation, in vivo or in vitro. Modifications also include glycosylation. Modified forms also include sequences having phosphorylated amino acid residues (e.g., phosphotyrosine, phosphoserine, phosphothreonine). Also included are polypeptides modified to improve resistance to hydrolysis or to optimize solubility. For example, in the truncation, a portion of the amino acids are modified to form a new polypeptide that retains its original function.

The main contribution of the invention is not only to obtain the miPEP155 polypeptide C-terminal three-amino-acid truncation, but also to demonstrate that the truncation has more ideal functions compared with the miPEP155 polypeptide. The miPEP155C terminal three-amino-acid truncation disclosed by the invention is simple to synthesize, low in cost, free from immunological rejection, capable of specifically inhibiting antigen presentation (presentation) of DC in an inflammatory environment, good in curative effect, less prone to relapse of diseases after treatment, small in side effect and extremely obvious in treatment effect.

The invention also provides a polynucleotide sequence encoding the C-terminal three-amino-acid truncation of the miPEP155 polypeptide. The polynucleotide of the present invention may be in the form of DNA or RNA. The DNA may be a coding strand or a non-coding strand, and the "polynucleotide encoding a polypeptide" may include a polynucleotide encoding the polypeptide, or may further include additional coding and/or non-coding sequences.

The invention also relates to a vector comprising the polynucleotide of the invention, a genetically engineered host cell (recombinant cell) engineered with the coding sequence of the vector of the invention or the C-terminal three amino acid truncation of the mippe 155 peptide, and a method for producing the polypeptide of the invention by recombinant techniques.

The term "expression vector" refers to a bacterial plasmid, bacteriophage, yeast plasmid, plant cell virus, mammalian cell virus, or other vector well known in the art. In general, any plasmid or vector can be used as long as it can replicate and is stable in the host. An important feature of expression vectors is that they generally contain an origin of replication, a promoter, a marker gene and translation control elements.

Vectors comprising the appropriate polynucleotide sequences described above, together with appropriate promoter or control sequences, may be used to transform an appropriate host cell so that it can express the polypeptide. The host cell may be a prokaryotic cell, such as a bacterial cell; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as plant cells. Representative examples are: escherichia coli, Streptomyces, Agrobacterium; fungal cells such as yeast; plant cells, and the like.

Applications of

The invention also provides a method for preparing an immunoregulation medicament by using the miPEP155 polypeptide C-terminal three-amino-acid truncation, or a medicament for inhibiting DC antigen presentation. Preferably, the immunomodulatory drug is: a medicine for preventing and treating psoriasis.

In a specific embodiment of the invention, it was determined that the mippe 155C-terminal three amino acid truncation is capable of inhibiting DC antigen presentation. In addition, the miPEP 155C-terminal three-amino-acid truncation can reduce the number of Th17 cells in the skin and spleen of an animal with a psoriasis model, reduce the proliferation of ear skin keratinocytes and effectively reduce the expression level of IL-17A protein and mRNA, thereby effectively reducing the morbidity of the animal with the psoriasis model. In the detection of the peripheral blood of psoriasis patients, the exogenously administered miPEP 155C-terminal three-amino-acid truncation can effectively reduce the expression level of IL-17A. The research result shows that the C-terminal three-amino-acid truncation of the miPEP155 polypeptide can be applied to inhibiting the antigen presentation of DC and can be used for preparing the immunoregulation medicament.

For example, the autoimmune diseases include: multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus and psoriasis. In addition, the compound also has potential prevention or treatment effects on other diseases or symptoms related to DC antigen presentation and Th17 immune regulation dysfunction. At present, the diseases or symptoms known to be associated with the immunoregulatory dysfunction of Th17 are selected from: tumor or virus infection, inflammatory reaction, rheumatoid arthritis, organ transplantation, systemic lupus erythematosus, psoriasis, Crohn's disease or ulcerative colitis, infectious diseases, etc.

For example, the tumor includes: prostate cancer, breast cancer, liver cancer, glioma, intestinal cancer, cervical cancer, non-small cell lung cancer, pancreatic cancer, gastric cancer, bladder cancer, skin cancer, striated muscle cancer, squamous cell carcinoma of the tongue, nasopharyngeal carcinoma, ovarian cancer, placental villus cancer, glioma, lymphoma, leukemia, rectal adenocarcinoma, or melanoma, and the like.

For example, the inflammatory response includes: allergic inflammation, folliculitis, tonsillitis, pneumonia, hepatitis, nephritis, acne, asthma, autoimmune diseases, chronic inflammation, chronic prostatitis, glomerulonephritis, hypersensitivity, inflammatory bowel disease, pelvic inflammatory disease, reperfusion injury, rheumatoid arthritis, transplant rejection, vasculitis or interstitial cystitis, etc.

For example, the infectious disease includes: plague, cholera, infectious atypical pneumonia, aids, viral hepatitis, poliomyelitis, human infection with highly pathogenic avian influenza, measles, epidemic hemorrhagic fever, rabies, epidemic encephalitis b, hand-foot-and-mouth disease, dengue fever, anthrax, bacterial and amoebic dysentery, tuberculosis, typhoid and paratyphoid fever, epidemic cerebrospinal meningitis, pertussis, diphtheria, neonatal tetanus, scarlet fever, brucellosis, gonorrhea, syphilis, leptospirosis, schistosomiasis, malaria, influenza, mumps, rubella, acute hemorrhagic conjunctivitis, leprosy, epidemic and endemic typhus, black fever, echinococcosis, filariasis, infectious diarrhea diseases other than cholera, bacterial and amoebic dysentery, typhoid and paratyphoid fever, fungal infection, and the like.

The small peptides of the invention can also form complexes with other functional molecules, said complexes comprising: the small peptide of the invention and a functional molecule operatively linked to the small peptide. Such functional molecules include, but are not limited to: functional biological macromolecules, functional small molecules, fluorescent tracers, imaging agents, liposomes, nano-preparations, polymers or viral vectors; preferably, the functional biological macromolecules include, but are not limited to: functional polypeptide, functional nucleic acid.

As an embodiment of the present invention, the functional molecule may be a marker having a tracing function, including but not limited to a fluorescent dye, an MRI contrast agent, a radioactive contrast agent, a magnetic particle, or a chemical agent having a coloring function. For example, the marker or functional small molecule with tracer function may be FITC.

As an embodiment of the invention, the functional molecule can be a functional small molecule, including inorganic small molecules and organic small molecules, and the molecular weight of the functional molecule is less than 1000 daltons.

As an embodiment of the present invention, the functional molecule may be a functional macromolecule, for example, a functional polypeptide (such as an antibody), a functional nucleic acid.

As another embodiment of the present invention, the functional molecule is a functional nucleic acid fragment, including but not limited to plasmid, siRNA, DNA, oligonucleotide, miRNA, antisense nucleic acid, etc.

As a preferred mode of the invention, the functional molecule may be a cell-penetrating peptide to which the small peptide of the invention is linked to facilitate its entry into the cell. The cell-penetrating peptide is a peptide for guiding the small peptide into the cell, and the cell-penetrating peptide can adopt any molecule which can guide the peptide or a coding gene thereof into the cell and is known in the field, or adopt any molecule which can improve the cell penetrating capacity of the peptide.

Some peptides with membrane penetration function include:

protein derived peptides (protein derived CPPs), such as pendatin, TAT, pVEC and the like;

model peptides (models peptides) such as MAP and (Arg)7, etc.; design peptides (designed CPPs) such as MPG and Transportan, etc.

Cell-penetrating peptides can also be classified into 3 classes from their amphiphilic nature:

amphipathic CPPs (PaCPPs), such as MPG, transportan, TP10, Pep-1;

(sacpps) moderate amphipathic cpps, such as pendatin, RL 16;

(NaCPPs) of non-amphiphilic CPPs, e.g., R9.

As an embodiment of the present invention, the functional molecule is a preparation having a function of molecular packaging carrier, including but not limited to liposome, polymer, dendritic molecule, nano-packaging preparation, etc.

As an embodiment of the present invention, the functional molecule is a viral vector that can carry genetic material, including but not limited to retroviral, lentiviral, or adenoviral vectors, and the like.

The connection mode of the small peptide and the functional molecule can be covalent connection or non-covalent connection. It is understood that any means of attachment can be included in the invention so long as the function of the small peptide and functional molecule is retained. Covalent attachment two molecules are typically attached in a manner that forms a covalent bond. While some non-covalent attachment (without formation of covalent bonds) such as coupling, adsorption, conjugation, etc. may also be applied.

As a preferable mode of the invention, the small peptide is connected with the functional molecule through a chemical bond; more preferably, the chemical bond is a peptide bond.

The small peptide and the functional molecule can be directly connected or connected through a polypeptide linker (connecting peptide). The linker comprises, for example, 1-30 amino acids; preferably 1-20 amino acids; e.g. 15, 10, 8, 6, 5, 4, 3, 2, 1 amino acids. The linker peptide is positioned so as not to substantially affect the function of the small peptide and the functional molecule, respectively. The connecting peptide can also comprise at least one specific enzyme cutting site. The enzyme cutting site is selected from (but not limited to): an enterokinase cleavage site, a thrombin cleavage site, or a trypsin cleavage site. The arrangement of the enzyme cutting site is convenient for separating the small peptide from the functional molecule subsequently. The connection between the small peptide and the functional molecule can be performed by peptide bond, and the functional molecule can be positioned at the amino terminal of the small peptide or the carboxyl terminal of the small peptide according to requirements.

In one embodiment of the present invention, the small peptide can be linked to a functional molecule through a chemical reaction such as amino, carboxyl or thiol, including but not limited to linking between the polypeptide and a polymer, covalent modification of the polypeptide on the surface of the liposome or nanoparticle, esterification, sulfurization, etc.

Pharmaceutical composition and kit

The present invention also provides a pharmaceutical composition for immunomodulation, comprising: the polypeptide or the polynucleotide for coding the polypeptide, or an expression vector containing the polynucleotide or a recombinant cell for expressing the polypeptide; and a pharmaceutically or physiologically acceptable carrier.

Suitable pharmaceutically acceptable carriers are well known to those of ordinary skill in the art. Sufficient information about pharmaceutically acceptable carriers can be found in Remington's Pharmaceutical Sciences. Pharmaceutically acceptable carriers in the compositions may comprise liquids such as water, phosphate buffered saline, ringer's solution, physiological saline, balanced salt solution, glycerol or sorbitol, and the like. In addition, auxiliary substances, such as lubricants, glidants, wetting or emulsifying agents, pH buffering substances and stabilizers, such as albumin and the like, may also be present in these carriers.

In use, a safe and effective amount of a polypeptide of the invention, or a polynucleotide encoding it, or an expression vector comprising the polynucleotide, or a recombinant cell expressing the polypeptide, is administered to a mammal (e.g., a human), wherein the safe and effective amount is typically at least about 0.01 micrograms/kg body weight, and in most cases no more than about 10 milligrams/kg body weight. Of course, the particular dosage will also take into account such factors as the route of administration, the health of the patient, and the like, which are within the skill of the skilled practitioner.

The precise effective amount for a subject will depend upon the size and health of the subject, the nature and extent of the disorder, and the therapeutic agent and/or combination of therapeutic agents selected for administration. The effective amount can be determined by routine experimentation for a given condition, as will be appreciated by a clinician.

The invention also provides a kit or kit comprising: the small peptide or the polynucleotide for coding the small peptide, or the expression vector containing the polynucleotide or the recombinant cell for expressing the polypeptide; or the pharmaceutical composition.

For convenience of clinical application, the pharmaceutical composition of the present invention may be contained in an administration syringe (e.g., a needle for injection) in which the pharmaceutical composition may be contained in an amount administered at one time. The administration device for injection may be contained in a cartridge for convenient storage and use.

The kit or kit of the present invention may further comprise instructions for use, which will facilitate the use of the kit or kit in a proper manner by those skilled in the art.

The excellent technical effects of the invention are mainly as follows:

the invention discloses that a C-terminal three-amino-acid truncation of a small peptide (miPEP155) coded by a micro RNA155hostgene has the function of inhibiting dendritic cell antigen presentation, can reduce the expression level of IL-17A protein and mRNA in a skin lesion region of a psoriasis model animal, can obviously reduce the morbidity of the psoriasis model animal, and can reduce the expression level of the mRNA of IL-17A in the peripheral blood of the psoriasis patient by the miPEP 155C-terminal three-amino-acid truncation. The miPEP 155C-terminal three-amino-acid truncation can be used for specifically targeting DC cells; the molecular weight is small, and the protein can easily enter cells to play a role; the small peptide is biologically synthesized through prokaryotic expression, is easy to prepare in large quantity, has good stability and lower price compared with the existing effective antibody for treating psoriasis; the animal living body has good effect of treating the psoriasis-like model, and can reduce the mRNA expression level of the IL-17A of the peripheral blood cells and the transcription factors thereof of psoriasis patients, thereby being applied to preventing, relieving or treating human psoriasis and other autoimmune diseases.

In general, as the length of a polypeptide increases, its stability deteriorates and its half-life in vivo is shortened accordingly. In physiological situations, not all sequences of a polypeptide are necessarily required for its function. Therefore, by exploring the shortest functional domain of the polypeptide to exert the function, the stability and half-life period of the polypeptide medicament can be increased, the synthesis cost is reduced, and the biological toxicity is reduced. The polypeptide has low concentration in the organism, but strong physiological activity, and plays a very important role in regulating physiological functions. The peptide has the unique advantages of low toxicity, high specificity, small molecular weight and the like, and can be obtained in large quantity through in vitro synthesis and biological synthesis.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, for which specific conditions are not noted in the following examples, are generally performed according to conventional conditions such as those described in J. SammBruk et al, molecular cloning protocols, third edition, scientific Press, 2002, or according to the manufacturer's recommendations.

Example 1 sequence analysis and in vitro Synthesis of C-terminal three amino acid truncations of miPEP155

1. Sequence analysis of miPEP 155C-terminal three-amino-acid truncation

The sequence of miR-155host gene is as follows, wherein the coding sequence of miR-155C-terminal three amino acid truncation is not underlined:

in the above sequences, the predicted sequence of the C-terminal three-amino acid truncation of miPEP155 is underlined in italics. The sequence translated into amino acids is: MEMALMVAQTRKGK (SEQ ID NO: 2).

In the above sequence "

"(SEQ ID NO:4) is a non-truncated miPEP155 encoding a peptide having the amino acid sequence: MEMALMVAQTRKGKSVV (SEQ ID NO: 3).

2. In vitro synthesis of miPEP 155C-terminal three-amino-acid truncation

The miPEP 155C-terminal three-amino-acid truncation is synthesized according to the amino acid sequence of SEQ ID NO. 2 by using a conventional solid-phase polypeptide synthesis method, and the amino acid correctness is determined by mass spectrometry as shown in figure 1A.

According to the HPLC analysis result, the purity of the obtained truncated small peptide was 95.1%, as shown in fig. 1B. Dissolving in ddH before use ₂ And O is standby.

Example 2 inhibition of DC antigen presentation by miPEP 155C-terminal three-amino acid truncation

The influence of the miPEP 155C-terminal three-amino-acid truncation (miPEP155-C3) obtained by the solid-phase polypeptide synthesis method in example 1 on DC antigen presentation was determined.

Obtaining bone marrow cells of mice, and separating the mice by using immunomagnetic beads

CD4+ T cells were cultured in 1640 complete medium at 37 ℃ and GM-CSF (20ng/ml) and IL-4(10ng/ml) were added to the medium, and 3/4 medium changes were made every two days and cultured for 6 days to obtain Th 17.

The DC is cultured in RPMI 1640 medium at the temperature of 37 ℃ and divided into a plurality of culture groups, physiological saline with the concentration of 50 mu M (CTRL) and the miPEP 155C-terminal three-amino-acid truncation (miPEP155-C3) are respectively added, and the influence of the miPEP 155C-terminal three-amino-acid truncation on the DC antigen presentation is observed by flow cytometry. As a result, as shown in FIGS. 2A and C, the miPEP 155C-terminal three-amino-acid truncation can significantly inhibit the antigen presentation of DC induced in vitro.

Under the same treatment conditions as above, the C-terminal three-amino acid truncation of miPEP155 was replaced with non-truncated miPEP155 and flow cytometric observation was performed. As a result, as shown in fig. 2B and D, the effect of mippe 155 on down-regulation of MHC-II inhibition of antigen presentation was greatly diminished compared to the C-terminal three-amino acid truncation of mippe 155.

Example 3 MiPEP 155C-terminal three-amino-acid truncation can reduce the signs of animal models of psoriasis

Randomly dividing SPF level mice into an Imiquimod (IMQ) group and an IMQ + miPEP 155C-terminal three-amino-acid truncation (miPEP155-C3) group, coating IMQ on the inner side and the outer side of the ear skin of the mice, wherein 25 mg/mouse is coated for a week in total, establishing an IMQ-induced mouse psoriasis-like disease model, and simultaneously performing physiological saline and miPEP 155C-terminal three-amino-acid truncation (dissolved in ddH) ₂ And O, diluting with normal saline, wherein the concentration is as follows: 100. mu.g/200. mu.l) was administered into the tail vein at a dose of 100. mu.g/mouse once a day, and the ear skin thickness was measured and the mouse body weight was weighed periodically every day from the day of induction. Treating mice after 7 days of IMQ induction, collecting ear skin, fixing with 4% paraformaldehyde for 24 hr, dehydrating, embedding, cutting tissue block into tissue slices with thickness of 5 μm, and culturing&E, staining, and observing pathological conditions of ear skins of the IMQ-induced psoriasis-like model mice under a microscope.

The results are shown in fig. 3A-B, and the exogenously synthesized miPEP 155C-terminal three-amino-acid truncation can obviously reduce the psoriasis-like phenotype of the model mouse, which is specifically shown as follows: compared with a control group, the miPEP 155C-terminal three-amino-acid truncation (C3) can obviously thin the epidermis of a mouse, reduce scales and reduce inflammatory cell infiltration.

From the left panel of fig. 3A (thickness of the otic cortex), the difference between the two groups reached "x, P < 0.0001", showing that the effect of the miPEP 155C-terminal three amino acid truncation was extremely superior; according to the right graph of fig. 3A, the improvement degree of the miPEP 155C-terminal three-amino-acid truncation (C3) on the acanthosis pachynsis is also very significant, and the difference of the two groups reaches "x, P is < 0.001"; these effects are unexpected.

Example 4. miPEP 155C-terminal three-amino-acid truncation can reduce the expression level of IL-17A mRNA and protein in animal models of psoriasis

1. Effect of miPEP 155C-terminal three-amino acid truncation on IL-17A mRNA

An IMQ-induced mouse psoriasis-like model and a miPEP 155C-terminal three-amino-acid truncation body treatment model mouse are prepared according to the steps of example 3, a part of ear skin is obtained, Trizol is added to extract RNA, and the expression level of IL-17A mRNA in a mouse psoriasis-like model skin lesion area induced by IMQ is detected through reverse transcription and qPCR.

Results as shown in fig. 4, exogenous administration of miPEP 155C-terminal three amino acid truncation (miPEP155-C3) was able to very significantly reduce the expression level of IL-17A mRNA in the lesion area of psoriatic mice (. star, P < 0.001).

2. Effect of miPEP 155C-terminal three-amino-acid truncation on IL-17 protein levels

An IMQ-induced mouse psoriasis-like model and a miPEP 155C-terminal three-amino-acid truncation-treated model mouse were prepared according to the procedure of example 3, a part of ear skin was obtained, the skin was pulverized by a tissue homogenizer, a mixture of Total protein extract buffer and protease phosphatase inhibitor (Total protein extract buffer: 1: 100) was added, and the mixture was shaken and mixed once every 5min on ice for 20min to sufficiently cleave the protein. And detecting the expression of the IL-17A protein according to a mouse IL-17A kit.

The result is shown in figure 5, and the exogenously administered miPEP 155C-terminal three-amino acid truncation (miPEP155-C3) can obviously reduce the level of IL-17 protein in the skin lesion area of psoriasis-like model mice.

Example 5 MiPEP 155C-terminal three-amino-acid truncation can effectively reduce the expression of IL-17A mRNA of peripheral blood mononuclear cells of psoriasis patients

Obtaining peripheral blood of a psoriasis patient, attaching to the wall for 4h to remove mononuclear cells, culturing in a primary culture solution, setting an NC group and a miPEP 155C-terminal three-amino-acid truncation body administration group, stimulating for 24h to collect cells, centrifuging at 3000rpm for 20min, discarding supernatant, then re-suspending the cells with 1ml of PBS, centrifuging again, adding Trizol to obtain RNA, and detecting the expression level of IL-17A mRNA in the peripheral blood of the psoriasis patient by reverse transcription and qPCR.

As a result, as shown in FIG. 5, exogenous administration of miPEP 155C-terminal three-amino acid truncation (miPEP155-C3) can significantly reduce the expression level of IL-17A mRNA in the psoriasis patients peripheral blood mononuclear cells. Since IL-17A is an important factor causing psoriasis, the miPEP 155C-terminal three-amino acid truncation disclosed by the invention has a relieving and treating effect on psoriasis.

All documents mentioned in this application are incorporated by reference in this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Sequence listing

<110> Shanghai college of medicine of transportation university

<120> small peptide and application thereof in preparation of immunoregulation drugs

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atggagatgg ctctaatggt ggcacaaacc aggaagggga aatctgtggt ttaaattctt 240

tatgcctcat cctctgagtg ctgaaggctt gctgtaggct gtatgctgtt aatgctaatc 300

gtgatagggg tttttgcctc caactgactc ctacatatta gcattaacag tgtatgatgc 360

ctgttactag cattcacatg gaacaaattg ctgccgtggg aggatgacaa agaagcatga 420

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atctggtcac tgggatgttc aaccttaaac taagttttga aagtaaggtt atttaaaaga 540

tttatcagta gtatcctaaa tgcaaacatt ttcatttaaa tgtcaagccc atgtttgttt 600

ttatcattaa cagaaaatat attcatgtca ttcttaattg caggttttgg cttgttcatt 660

ataatgttca taaacacctt tgattcaact gttagaaatg tgggctaaac acaaatttct 720

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tctggcaacg ttttttaaaa gatttagaaa ctggtacttt cccccaggta acgattttct 840

gttcaggcaa cttcagttta aaattaatac ttttatttga ctcttaaagg gaaactgaaa 900

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Claims

1. An isolated small peptide, wherein the amino acid sequence of the small peptide is shown in SEQ ID NO 2.

2. The small peptide of claim 1, which is encoded by the nucleotide sequence of SEQ ID No. 1 from position 181 to 222 or a degenerate sequence thereof.

3. An isolated polynucleotide encoding the small peptide of claim 1 or 2.

4. The polynucleotide of claim 3, wherein the nucleotide sequence is from position 181 to 222 of SEQ ID NO. 1 or a degenerate sequence thereof.

5. An expression vector comprising the polynucleotide of claim 3; and the small peptide encoded by the polynucleotide does not include a peptide formed after the addition of the amino acid SVV at the C-terminus.

6. A recombinant cell comprising the expression vector of claim 5 or comprising the polynucleotide of claim 3 in its genome, wherein the polynucleotide encodes a small peptide that excludes a peptide formed upon addition of the amino acids SVV at the C-terminus.

7. Use of a small peptide or polynucleotide encoding it according to claim 1, or an expression vector according to claim 5 or a recombinant cell according to claim 6 in the manufacture of an immunomodulatory medicament for the alleviation or treatment of an autoimmune disease, said autoimmune disease being psoriasis.

8. A method of making the small peptide of claim 1, comprising: culturing the recombinant cell of claim 6, thereby recombinantly expressing the small peptide of claim 1.

9. A method of making the small peptide of claim 1, comprising: the small peptide of claim 1 is prepared by in vitro synthetic methods or biological synthetic methods.

10. A pharmaceutical composition for immunomodulation for the alleviation or treatment of psoriasis, said pharmaceutical composition comprising: a small peptide according to claim 1 or a polynucleotide encoding same; and a pharmaceutically or physiologically acceptable carrier.

11. A pharmaceutical composition for the treatment of psoriasis, comprising:

the expression vector of claim 5; or

The recombinant cell of claim 6.