WO2023030407A1 - Polypeptide ciblant une protéine des cristaux de charcot-leyden et utilisation associée - Google Patents

Polypeptide ciblant une protéine des cristaux de charcot-leyden et utilisation associée Download PDF

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WO2023030407A1
WO2023030407A1 PCT/CN2022/116317 CN2022116317W WO2023030407A1 WO 2023030407 A1 WO2023030407 A1 WO 2023030407A1 CN 2022116317 W CN2022116317 W CN 2022116317W WO 2023030407 A1 WO2023030407 A1 WO 2023030407A1
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polypeptide
fusion
clc
allergic
seq
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PCT/CN2022/116317
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English (en)
Chinese (zh)
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张罗
王晨轩
赵妍
于兰兰
王向东
莫珊珊
李小璐
张文博
郝蕴
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首都医科大学附属北京同仁医院
中国医学科学院基础医学研究所
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Priority claimed from CN202111008517.1A external-priority patent/CN113603753B/zh
Priority claimed from CN202111008519.0A external-priority patent/CN113666999B/zh
Priority claimed from CN202111008545.3A external-priority patent/CN113667000B/zh
Application filed by 首都医科大学附属北京同仁医院, 中国医学科学院基础医学研究所 filed Critical 首都医科大学附属北京同仁医院
Publication of WO2023030407A1 publication Critical patent/WO2023030407A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • 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
    • A61P11/02—Nasal agents, e.g. decongestants
    • 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
    • A61P33/00—Antiparasitic agents
    • A61P33/14—Ectoparasiticides, e.g. scabicides
    • 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
    • A61P37/08—Antiallergic 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
    • 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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals

Definitions

  • the invention relates to the field of biomedicine, in particular to a polypeptide targeting Charcot-Leyden crystal protein and its application.
  • Type 2 immunity is a special immune response that includes innate immunity and adaptive immunity and promotes the formation of an immune barrier on the mucosal surface to clear pathogens.
  • type 2 inflammatory pathways play an important role in the development of allergic diseases.
  • Th2 cells play a key role in the type 2 inflammatory pathway by secreting type 2 cytokines.
  • DC dendritic cells
  • Galectins are carbohydrate-binding proteins involved in many physiological functions such as inflammation, immune response, cell migration, autophagy, and signal transduction, and they are also associated with fibrosis, cancer diseases. So far, a total of 16 galectins have been discovered in mammals.
  • Galectin-10 is a member of the Galectin superfamily, which exists in eosinophils, basophils, granulocytes and some T cells.
  • Charcot Leyden crystals (CLC) are non-soluble forms of Gal-10 that are only formed during eosinophil extracellular trap generation, and CLC are released extracellularly during eosinophil cell membrane disruption and cell death .
  • Staphylococcus aureus and its exotoxin are important pathogens of chronic rhinosinusitis nasal polyposis (CRSwNP), and colonization by Staphylococcus aureus induces the formation of a large number of CLCs, which can further promote natural immune responses and aggravate type 2 Immune response, and can cause the aggregation of neutrophils, screening of polypeptides that can target CLC or Gal-10 protein is expected to be applied to the diagnosis or treatment of CLC-induced diseases and/or type 2 immune diseases, for the diagnosis or treatment of CLC Induced diseases and/or type 2 immune diseases provide new approaches.
  • CCSwNP chronic rhinosinusitis nasal polyposis
  • the purpose of the present invention is to provide a polypeptide that can be used for diagnosing or treating type 2 immune diseases.
  • the first aspect of the present invention provides a polypeptide, said polypeptide comprising the amino acid sequence shown in any one of SEQ ID NO.3 or SEQ ID NO.4 or SEQ ID NO.5, or its fragment, variant, fusion or derivatives, or fusions of said fragments, variants or derivatives thereof.
  • Said fragment, variant, fusion or derivative thereof, or said fusion of said fragment, variant or derivative thereof retains the inhibitory CLCs of SEQ ID NO.3 or SEQ ID NO.4 or SEQ ID NO.5 Activity of the induced immune response.
  • the variant comprises at least 55%, 60%, 65%, 70%, 75%, 80% of the amino acid sequence shown in SEQ ID NO.3 or SEQ ID NO.4 or SEQ ID NO.5. %, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology to amino acid sequences.
  • the second aspect of the present invention provides a pharmaceutical composition for preventing or treating type 2 immune diseases, said pharmaceutical composition comprising the polypeptide described in the first aspect of the present invention.
  • type 2 immune diseases include allergic diseases and mite infections.
  • the type 2 immune disease is an allergic disease.
  • the allergic diseases include chronic sinusitis, asthma, allergic rhinitis, allergic dermatitis, and food allergy.
  • the allergic disease is chronic sinusitis.
  • the pharmaceutical composition also includes a pharmaceutically acceptable buffer, carrier or excipient.
  • the third aspect of the present invention provides the use of the polypeptide described in the first aspect of the present invention in the detection of Gal-10 protein for non-diagnostic purposes.
  • the fourth aspect of the present invention provides a nucleic acid encoding the polypeptide described in the first aspect of the present invention.
  • the fifth aspect of the present invention provides a recombinant vector comprising the nucleic acid described in the fourth aspect of the present invention.
  • the sixth aspect of the present invention provides a cell comprising the nucleic acid of the fourth aspect of the present invention or the recombinant vector of the fifth aspect of the present invention.
  • the cells include prokaryotic cells and eukaryotic cells.
  • prokaryotic cells include bacterial cells.
  • the eukaryotic cells include protozoan cells, animal cells, plant cells, and fungal cells.
  • animal cells include mammalian cells, poultry cells, and insect cells.
  • the seventh aspect of the present invention provides the polypeptide described in the first aspect of the present invention or the pharmaceutical composition described in the second aspect of the present invention or the nucleic acid described in the fourth aspect of the present invention or the recombinant vector described in the fifth aspect of the present invention or The use of the cells described in the sixth aspect of the present invention in the preparation of drugs for preventing or treating type 2 immune diseases.
  • type 2 immune diseases include allergic diseases and mite infections.
  • the type 2 immune disease is an allergic disease.
  • the allergic diseases include chronic sinusitis, asthma, allergic rhinitis, allergic dermatitis, and food allergy.
  • the allergic disease is chronic sinusitis.
  • the eighth aspect of the present invention provides a non-diagnostic method for detecting Gal-10 protein, the method comprising:
  • the method for detecting the formation of the complex comprising the polypeptide according to the first aspect of the present invention includes gel electrophoresis, chromatography techniques, western blot analysis, immunohistochemistry, mass spectrometry and/or high pressure liquid chromatography.
  • the ninth aspect of the present invention provides the polypeptide described in the first aspect of the present invention or the nucleic acid described in the fourth aspect of the present invention or the recombinant vector described in the fifth aspect of the present invention or the cell described in the sixth aspect of the present invention. Application in products for the diagnosis of type 2 immune diseases.
  • type 2 immune diseases include allergic diseases and mite infections.
  • the type 2 immune disease is an allergic disease.
  • the allergic diseases include chronic sinusitis, asthma, allergic rhinitis, allergic dermatitis, and food allergy.
  • the allergic disease is chronic sinusitis.
  • Figure 1 RT-qPCR detection results of gene expression changes in human nasal polyp mucosal epithelial cells induced by different concentrations of CLCs, in which Figure A is a statistical chart of IL-1 ⁇ expression changes, and Figure B is a statistical chart of TNF- ⁇ expression changes. C is a statistical chart of IL-6 expression changes, Figure D is a statistical chart of GM-CSF expression changes, and Figure E is a statistical chart of IL-8 expression changes;
  • FIG. 2 Statistical graph of gene expression changes in human nasal polyp mucosal epithelial cells when CLCs (100 ⁇ g/mL) were induced for 24 hours, wherein, graph A is a statistical graph of IL-1 ⁇ expression changes, graph B is a statistical graph of TNF- ⁇ expression changes, and graph C is Statistical chart of IL-6 expression change, Figure D is a statistical chart of IL-8 expression change, Figure E is a statistical chart of GM-CSF expression change;
  • Figure 3 is a diagram of the experimental results of the immune response induced by eight kinds of polypeptides at the cellular level, where Figure A is the statistical chart of IL-1 ⁇ expression, Figure B is the statistical chart of IL-6 expression, and Figure C is the expression of TNF- ⁇ Quantity statistical diagram, Figure D is a statistical diagram of IL-8 expression;
  • Figure 4 is a graph of the experimental results of the #1 polypeptide (SEQ ID NO.4) involved in the present invention to inhibit the immune response induced by CLCs at the cellular level, wherein, Figure A is a statistical graph of IL-1 ⁇ expression, and Figure B is a TNF- Statistical graph of ⁇ expression, and Figure C is a statistical graph of IL-6 expression;
  • Figure 5 is a graph of the experimental results of the #3 polypeptide (SEQ ID NO.5) involved in the present invention to inhibit the immune response induced by CLCs at the cellular level, wherein, Figure A is a statistical graph of IL-1 ⁇ expression, and Figure B is a TNF- Figure C is a statistical chart of IL-6 expression, Figure D is a statistical chart of IL-8 expression, Figure E is a statistical chart of GM-CSF expression;
  • Figure 6 is a graph of the experimental results of the #8 polypeptide (SEQ ID NO.3) involved in the present invention to inhibit the immune response induced by CLCs at the cellular level, wherein, Figure A is a statistical graph of IL-1 ⁇ expression, and Figure B is a TNF- Figure C is a statistical chart of IL-6 expression, Figure D is a statistical chart of IL-8 expression, Figure E is a statistical chart of GM-CSF expression;
  • Fig. 7 utilizes biomembrane interference technology to verify the interaction ability of #8 polypeptide (SEQ ID NO.3) involved in the present invention and soluble Gal-10 protein;
  • Figure 8 uses FITC-labeled polypeptides to verify the affinity of #8 polypeptide (SEQ ID NO.3) involved in the present invention with CLC;
  • Fig. 9 is the dynamic process of dissolving CLC by polypeptides of different concentrations, in order to verify that #8 polypeptide (SEQ ID NO.3) involved in the present invention has the ability to dissolve CLC;
  • Figure 10 is a mouse lung injury model induced by CLC was established, and the inflammation-inducing ability of exogenous CLC was verified in mice;
  • Figure 11 verifies the detection results of inflammatory factors that #8 polypeptide (SEQ ID NO.3) involved in the present invention alleviates lung injury in mice in the CLC-induced acute lung injury model in mice;
  • FIG. 12 verifies that #8 polypeptide (SEQ ID NO.3) involved in the present invention alleviates the lung pathological results of mouse lung injury in the mouse CLC-induced acute lung injury model.
  • amino acid includes the standard 20 genetically encoded amino acids and their corresponding stereoisomers in the "D” form (as compared to the natural "L” form), omega-amino acids, other naturally occurring amino acids, unconventional amino acids (eg, ⁇ , ⁇ -disubstituted amino acids, N-hydrocarbyl amino acids, etc.) and chemically derivatized amino acids.
  • amino acid refers to both L-alanine and D-alanine, unless expressly stated otherwise.
  • amino acid residue is (where appropriate) represented by a one-letter designation corresponding to the conventional amino acid common name.
  • “Variants” of polypeptides include insertions, deletions and substitutions, which are either conservative or non-conservative.
  • a conservative substitution refers to the substitution of an amino acid within the same general class (eg, acidic amino acid, basic amino acid, non-polar amino acid, polar amino acid, or aromatic amino acid) with another amino acid within the same class.
  • conservative and non-conservative amino acid substitutions is well known in the art.
  • variants of polypeptides that exhibit activity that binds to CLCs and/or that inhibit immune responses induced by CLCs are included.
  • the variant comprises at least 55%, 60%, 65%, 70% of the amino acid sequence shown in any of SEQ ID NO.3 or SEQ ID NO.4 or SEQ ID NO.5. , 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid sequence homology.
  • a "fusion" of a polypeptide includes an amino acid sequence corresponding to a reference sequence (eg, SEQ ID NO. 3 or SEQ ID NO. 4 or SEQ ID NO. 5, or a fragment or variant thereof) fused to any other polypeptide.
  • the polypeptide can be fused to a polypeptide such as glutathione-S-transferase (GST) or protein A to facilitate purification of the polypeptide. Examples of such fusions are well known to those skilled in the art.
  • GST glutathione-S-transferase
  • the polypeptide can be fused to an oligohistidine tag such as His6 or an epitope recognized by an antibody such as the well known Myc tag epitope.
  • fusions comprising hydrophobic oligopeptide end tags can be used. Fusions to any variant or derivative of said polypeptide are also included within the scope of the invention.
  • Fusions may comprise additional moieties that confer desired characteristics on the polypeptides of the invention; for example, such moieties may be used to detect or isolate the polypeptides, or to facilitate cellular uptake of the polypeptides.
  • the moiety may for example be a biotin moiety, a streptavidin moiety, a radioactive moiety, a fluorescent moiety, eg a small fluorophore or a green fluorescent protein (GFP) fluorophore, as known to those skilled in the art.
  • GFP green fluorescent protein
  • a module may be an immunogenic tag, such as a Myc tag, as known to those skilled in the art, or may be a lipophilic molecule or polypeptide domain capable of facilitating cellular uptake of the polypeptide, as known to those skilled in the art.
  • polypeptide of the invention may comprise one or more amino acids modified or derivatized, eg, by PEGylation, amidation, esterification, acylation, acetylation and/or alkylation.
  • PEGylated proteins can exhibit reduced renal clearance and proteolysis, reduced toxicity, reduced immunogenicity, and increased solubility.
  • PEG molecules can vary, and PEG variants that have been used for PEGylation of proteins include PEG and monomethoxy-PEG. Additionally, they can be either linear or branched.
  • PEG can be coupled at naturally occurring disulfide bonds as described in WO 2005/007197. Disulfide bonds can be stabilized through the addition of chemical bridges that do not damage the structure of the polypeptide. This allows the creation of bridges for site-specific attachment of PEG using the conjugation thiol selectivity of the two sulfurs that make up the disulfide bond. Thus, the need to engineer residues into the peptide for attachment to the target molecule is circumvented.
  • Polypeptides for use in the invention may be prepared in any suitable manner.
  • Such polypeptides include isolated naturally occurring polypeptides, recombinantly produced polypeptides, synthetically produced polypeptides or polypeptides produced by combinations of these methods. Methods for preparing such polypeptides are well known in the art.
  • nucleic acid sequence encoding the polypeptide of the present invention can be obtained completely through chemical synthesis.
  • the nucleic acid sequence can then be introduced into various existing DNA molecules (or eg vectors) and cells known in the art.
  • mutations can also be introduced into the sequence of the polypeptides of the invention by chemical synthesis.
  • nucleic acid encoding a polypeptide includes a nucleic acid comprising a sequence encoding a polypeptide of the present invention, especially a polypeptide having any amino acid sequence shown in SEQ ID NO.3 or SEQ ID NO.4 or SEQ ID NO.5 .
  • the term also includes nucleic acids comprising a single contiguous region or multiple discontinuous regions encoding the polypeptide (e.g., due to integrating phage, integrating insert sequences, integrating vector sequences, integrating transposon sequences, or due to RNA editing or genomic DNA reconstruction) and additional regions, which may also comprise coding and/or non-coding sequences.
  • the vectors for constructing the recombinant vectors of the present invention include (but are not limited to) the MarEx expression vectors produced by Celltrion Inc. (Korea); pCDNA vectors widely available on the market; F, R1, RP1, Col, pBR322, ToL, Ti Vectors; cosmids; bacteriophages such as lambda phages, lambda phages, M13 phages, Mu phages, P1 phages, P22 phages, Q ⁇ phages, T-even phages, T2 phages, T4 phages, T7 phages, etc.; plant viruses.
  • MarEx expression vectors produced by Celltrion Inc. (Korea); pCDNA vectors widely available on the market; F, R1, RP1, Col, pBR322, ToL, Ti Vectors; cosmids; bacteriophages such as lambda phages, lambda phages,
  • any of various vectors known to those skilled in the art can be used in the present invention, and the choice of the vector depends on the properties of the selected cells.
  • the introduction of the vector into the cell can be achieved by (but not limited to) calcium phosphate transfection, virus infection, DEAE-dextran mediated transfection, lipofection or electroporation, and anyone skilled in the art can Choose and use an introduction method appropriate for the vector and cells used.
  • the above-mentioned vectors contain one or more selection markers, but are not limited thereto, and vectors that do not contain selection markers can also be used.
  • the choice of selectable marker may depend on the cells selected (as is well known to those skilled in the art), but is not critical to the invention.
  • Polypeptides of the invention can be prepared by any of a variety of techniques.
  • polypeptides can be produced by cell culture techniques, including production of polypeptides by conventional techniques, or by transfection of nucleic acid molecules of the polypeptides into suitable bacterial or mammalian cell hosts to allow production of the polypeptides, which can be recombinant of.
  • transfection are intended to include various techniques commonly used to introduce exogenous DNA into prokaryotic or eukaryotic cells, such as electroporation, calcium phosphate precipitation, DEAE-dextran transfection, and the like.
  • polypeptides of the invention may be expressed in prokaryotic or eukaryotic cells
  • expression of the polypeptides in eukaryotic cells is preferred, and most preferably in mammalian cells, since such eukaryotic cells (especially mammalian cells) are more likely to Assemble and secrete correctly folded polypeptides than prokaryotic cells.
  • a recombinant expression vector of a nucleic acid molecule encoding a polypeptide is introduced into a mammalian cell, the polypeptide is secreted by culturing the cell for a period of time sufficient to allow expression of the polypeptide in the cell, or more preferably, the medium in which the cell is cultured.
  • Polypeptides can be recovered from the culture medium using standard protein purification methods.
  • “Pharmaceutically acceptable” means a non-toxic material that does not reduce the active ingredient.
  • Such pharmaceutically acceptable buffers, carriers or excipients are well known in the art (see Remington's Pharmaceutical Sciences, 18th edition, edited by A.R Gennaro, Mack Publishing Company (1990) and handbook of Pharmaceutical Excipients, 3rd edition, A ed. Kibbe, Pharmaceutical Press (2000)).
  • buffer is intended to mean an aqueous solution containing an acid-base mixture for the purpose of stabilizing the pH.
  • buffers are Trizma, Bicine, Tricine, MOPS, MOPSO, MOBS, Tris, Hepes, HEPBS, MES, phosphate, carbonate, acetate, citrate, glycolate, lactate , borate, ACES, ADA, tartrate, AMP, AMPD, AMPSO, BES, CABS, cacodylate, CHES, DIPSO, EPPS, ethanolamine, glycine, HEPPSO, imidazole, imidazole lactic acid, PIPES, SSC , SSPE, POPSO, TAPS, TABS, TAPSO and TES.
  • the carriers of the present invention include antimicrobial agents, isotonic agents, antioxidants, local anesthetics, suspending agents, dispersing agents, emulsifying agents, chelating agents, thickening agents or solubilizing agents.
  • Excipients can be one or more of the following: carbohydrates, polymers, lipids and minerals.
  • carbohydrates include lactose, sucrose, mannitol, and cyclodextrins, which are added to compositions, eg, to facilitate lyophilization.
  • polymers are various degrees of hydrolyzed starch, cellulose ethers, cellulose, carboxymethylcellulose, hydroxypropylmethylcellulose, hydroxyethylcellulose, ethylhydroxyethylcellulose, ethylcellulose , methylcellulose, propylcellulose, alginates, carrageenans, hyaluronic acid and its derivatives, polyacrylic acid, polysulphonate, polyethylene glycol/ Polyethylene oxide, polyethylene oxide/polypropylene oxide copolymer, polyvinyl alcohol/polyvinyl acetate, poly(lactic acid), poly(glycolic acid) or copolymers thereof with various compositions, and polyvinylpyrrolidone ( They all have different molecular weights), which are added to the composition, for example, to control viscosity, to achieve bioadhesion, or to protect the active ingredient from chemical and proteolytic degradation.
  • lipids are fatty acids, phospholipids, mono-, di-, and triglycerides, ceramides, sphingolipids, and glycolipids (all with different acyl chain lengths and degrees of saturation), egg lecithin, soybean lecithin, hydrogenated lecithin and soy lecithin, which are added to the composition for similar reasons as the polymer.
  • minerals are talc, magnesium oxide, zinc oxide and titanium oxide, which are added to compositions for benefits such as reduced liquid build-up or favorable pigment properties.
  • compositions of the invention must be sterile and stable under the conditions of manufacture and storage.
  • the preferred methods of preparation are vacuum drying and freeze-drying, which yield active ingredients from previously sterile-filtered solutions of the active ingredient and other desired ingredients. ingredients and powders of other desired ingredients.
  • the compositions of the invention may be in solution, and suitable pharmaceutically acceptable excipients may be added and/or mixed before or at the time of delivery to provide an injectable unit dosage form.
  • the pharmaceutically acceptable excipients used in the present invention are suitable for high drug concentrations, maintain proper fluidity, and delay absorption if necessary.
  • polypeptides of the invention can be prepared with carriers that will protect them against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems.
  • a controlled release formulation including implants, transdermal patches, and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers may be used in the present invention, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid.
  • the polypeptide may be coated with, or administered simultaneously with, a material or compound that prevents the inactivation of the polypeptide.
  • polypeptides can be administered with a suitable carrier such as liposomes or diluents.
  • the route of administration of the pharmaceutical composition of the present invention can be divided into oral administration and parenteral administration.
  • Oral dosage forms can be formulated as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard capsules, soft gelatin capsules, syrups or elixirs, pills, dragees, liquids, gels or ointment.
  • These formulations may contain pharmaceutical excipients, which include but are not limited to: granulating and disintegrating agents, binders, lubricants, preservatives, coloring, flavoring or sweetening agents, vegetable oils or minerals Oils, humectants, and thickeners.
  • Preparations for parenteral administration may be in the form of aqueous or nonaqueous isotonic sterile nontoxic injection or perfusion solutions or suspensions.
  • the solutions or suspensions may include agents such as 1,3-butanediol, Ringer's solution, Hank's solution, isotonic solutions, such as 1,3-butanediol, Ringer's solution, Hank's solution, etc. sodium chloride solutions, oils, fatty acids, local anesthetics, preservatives, buffers, viscosity or solubility increasing agents, water-soluble antioxidants, oil-soluble antioxidants, and metal chelating agents.
  • CLCs Charge-Leyden crystals
  • CLC crystals CLC crystals
  • CLC Charge-Leyden crystals
  • Galectin-10 refers to a small hydrophobic glycan-binding protein that self-crystallizes to form Charcot-Leyden crystals. Galectin-10 is also known as Charcot-Leyden crystal protein (CLCP), eosinophil lysophospholipase, and lysophosphatidyl hydrolase.
  • CLCP Charcot-Leyden crystal protein
  • eosinophil lysophospholipase eosinophil lysophospholipase
  • lysophosphatidyl hydrolase lysophosphatidyl hydrolase
  • polypeptide refers to the ability to bind a given target (eg CLC crystals).
  • a polypeptide may be monospecific and contain one or more binding sites that specifically bind a target, or a polypeptide may be multispecific and contain two or more binding sites that specifically bind the same or different targets.
  • isolated refers to an "artificial" alteration of the natural state. If an “isolated” composition or substance exists in nature, it has been altered from its original environment, or has been removed from its original environment, or both. As this term is used herein, for example, a polypeptide naturally occurring in a living animal is not “isolated”, but said polypeptide is “isolated” when it is separated from the coexisting substances of its natural state.
  • Plasmid After the Gal-10 protein sequence is humanized and codon-optimized, a solubilizing fragment is added to its N-terminus, and cloned into the pET-28a vector plasmid through the NcoI/XhoI double restriction site to form pET-28a -Gal10-TEV-6XHis.
  • the Gal-10 protein sequence is shown in Table 1, and the Gal-10 protein sequence with a solubilizing fragment is shown in Table 2.
  • Kanamycin and Ampicillin were purchased from Tiangen Biotechnology Co., Ltd.; Isopropyl-beta-D-thiogalactoside (IPTG ) and Escherichia coli BL21 (DE3) were purchased from Beijing Quanshijin Company; SDS, Trizol and imidazole were purchased from Sigma Company; TEV enzyme was purchased from Beijing Yiqiao Shenzhou Co., Ltd.; Coomassie Brilliant Blue staining solution (self-made); cDNA reverse transcription reagent Purchased from Takara Company; real-time fluorescent quantitative PCR reagents were purchased from Aibotec Biotechnology; the peptide was synthesized in Guoping Pharmaceutical Co., Ltd., the sequence is shown in Table 3, wherein the first amino acid at the C-terminal of #3 polypeptide (SEQ ID NO.5) An amino group is connected, and the 8th amino acid at the N-terminal is citrulline.
  • Electric constant temperature incubator (XMTD HH.B11-600); PCR instrument (Biometra Tgradient); desktop centrifuge (eppendorf, Centrifuge 5415D); electric constant temperature water tank (SHH W21 600); trace ultraviolet spectrophotometer (NanoDrop 2000); Balance (Sartorius 2000S); electronic analytical balance (Sartorius, BS110S); optical inverted microscope (XDS-1B); micropipette (eppendorf Research plus); cell CO2 incubator (SANYO); , MDF-382E); pH meter (Thermo Orion 868); magnetic stirrer (IKA RH-KT/C); microplate reader (Bio-Rad, 680); ultrasonic breaker; pure 25 protein purification system (superdex 75, GE Healthcare); SDS-PAGE electrophoresis instrument (Bio-Rad); gel imager (Molecular Imager Gel Doc XR, Bio-Rad); ABI PCR instrument (ABI 7500).
  • Heat shock heat shock in 42°C water bath for 90s
  • Kanamycin 25mg/mL
  • the bacterial liquid was inoculated into 1L LB liquid medium containing kanamycin at a ratio of 1:100, and cultured at 37°C and 210r/min shaking.
  • Ultrasonic disruption of bacteria power 25%, working time 25min, ultrasonic on time 3s, ultrasonic off time 9s; centrifuge (4°C, 13,000 ⁇ g, 30min) to remove cell debris, and at the same time break up the nucleic acid released after cell disruption, Centrifugal sedimentation makes the cell lysate less viscous and facilitates subsequent processing. Collect the supernatant, and the soluble protein of interest exists in the supernatant.
  • Concentration Put the eluted and collected target protein into a 10kDa concentrator tube, centrifuge at 2000 ⁇ g for 10 min at 4°C, add a small amount of Lysis Buffer or PBS to dilute the imidazole during the concentration process to prevent the target protein from aggregation and precipitation.
  • Clean the chromatography column Use sterile water to clean the chromatography column, about 8mL. Then put the pump head in the PBS solution, repeat the above steps and execute, and clean about 36mL;
  • Sample loading Rinse the sample loop two or three times with PBS first, then inject 2mL of a sample with a concentration of about 6.35mg/mL into the sample loop, and finally absorb PBS slightly before injecting into the sample loop to avoid sample residue. Arrange the collection tube and select inject valve:inject;
  • Protein treatment Measure the concentration of each tube of the collected protein and mark it, place it in liquid nitrogen for quick freezing, and store it in a -80°C refrigerator. Let stand at room temperature and melt slowly before the experiment.
  • Sample preparation Whole bacteria sample, centrifuged supernatant sample, protein supernatant passed through the chromatographic column sample, 20mM imidazole passed through the sample, 500mM imidazole passed through the sample, collected at the peak head, peak peak and peak tail in the molecular sieve protein samples;
  • Sample loading 5 ⁇ L of whole bacteria sample, 3 ⁇ L of marker, and 10 ⁇ L of other samples;
  • TEV enzyme and pET-28a-gal10-TEV-6His recombinant protein were mixed at a mass ratio of 1:10, and incubated overnight at 4°C for enzyme digestion on a shaker;
  • the laboratory self-made polypeptide chip (containing 400 different polypeptide sequences), by respectively incubating the CLCs labeled with the control group-EGFP fluorescent protein and the experimental group-EGFP protein with the chip, to screen out the polypeptide sequences that can interact with CLCs.
  • Blocking buffer prepared now
  • TBST TBS+0.1% Tween-20
  • elution buffer (contains BSA, ready-to-use) to elute the bound phage, shake gently for no more than 10min, transfer the eluate into a microcentrifuge tube, and dilute with 150 ⁇ L 1M Tris-HCL (PH 9.1) neutralize.
  • the polypeptides that may interact with the Gal-10 with the solubilizing tag can be obtained.
  • step (1) of step 3 in Example 1 were co-stimulated with polypeptide (50 ⁇ M) and CLCs (100 ⁇ g/ml), and cellular RNA was collected after 24 hours for reverse transcription and qPCR.
  • the present invention has screened 8 polypeptides that may bind to Gal10 protein, and the polypeptides (#1, #3, #8) involved in the present invention can bind to CLCs.
  • the experimental results of these eight polypeptides inhibiting the immune response induced by CLCs were verified at the cellular level, as shown in Figure 3 (in the figure, * indicates P ⁇ 0.05, ** indicates P ⁇ 0.01, *** indicates P ⁇ 0.001, ** ** indicates P ⁇ 0.0001).
  • the polypeptide (#1) involved in the present invention can effectively inhibit the increased expression of human nasal mucosal epithelial cells IL-1 ⁇ , TNF- ⁇ , and IL-6 caused by CLCs (100 ⁇ g/mL); 5, the polypeptide (#3) involved in the present invention can effectively inhibit the human nasal mucosal epithelial cells IL-1 ⁇ , TNF- ⁇ , IL-6, IL-8 and GM-CSF induced by CLC crystals (100 ⁇ g/mL). As shown in Figure 6, the polypeptide (#8) involved in the present invention can effectively inhibit the human nasal mucosal epithelial cells IL-1 ⁇ , TNF- ⁇ , IL-6, Expression of IL-8 and GM-CSF was increased. The above experimental results prove that the polypeptide involved in the present invention can effectively inhibit the natural immune response activated by CLC crystals.
  • Example 3 CLC-induced nasal polyp-derived human nasal mucosal epithelial cell innate immune response activation model
  • tissue washing solution physiological saline containing 200 ⁇ g/mL of penicillin and streptomycin
  • CLCs stimulate human nasal mucosal epithelial cells
  • the primary nasal mucosal epithelial cells of nasal polyps grow to about 85-90%, discard the medium, wash with 2mL of sterile PBS, digest with 2mL of 0.25% trypsin at 37°C for 4-5min, and then add 1mL of FBS-containing medium Complete medium was terminated.
  • the 12-well plate was cultured for about 2 days. When the primary nasal mucosal epithelial cells grew to about 80%, the medium was discarded, and the culture medium was continued for 24 hours with BEGE medium containing different concentrations of CLC.
  • RNA sample After the RNA sample is completely thawed, take the sample to room temperature for 10 minutes to fully dissolve the RNA.
  • RNA sample Take 1 ⁇ L RNA sample and measure the RNA concentration and purity with a micro-volume ultraviolet spectrophotometer NanoDrop2000 (Thermo Scientific). And take 1 ⁇ L (just greater than 100ng) mixed with 7 ⁇ L of DEPC-treated H 2 O and 2 ⁇ L of 5 ⁇ RNA Loading Buffer, and use 1% agarose gel electrophoresis to detect the integrity of the RNA. If the integrity of the RNA is good, the brightness ratio of 28S to 18S is about 2:1.
  • RT-qPCR real-time fluorescent quantitative PCR
  • reaction system was added to a 384-well plate, and the sealing film was evenly pasted on the 384-well plate, and centrifuged at 1500 r ⁇ min ⁇ 1 for 2 min.
  • relative gene expression value 2 (-(Ct target gene-Ct internal reference)) , and finally set the control expression value to 1 to calculate the relative expression value of the gene in other samples.
  • polypeptide No. 8 As shown in Figure 7 and Figure 8, taking polypeptide No. 8 as an example, it can be directly combined with Gal-10 protein and CLC crystals, which proves that the polypeptide involved in the present invention can be used as a diagnostic agent for the diagnosis of CLC-induced diseases and/or type 2 immunity Reagents of disease. As shown in Figure 9, taking polypeptide No. 8 as an example, it can directly dissolve CLC crystals, which proves that the polypeptide involved in the present invention can target and dissolve CLC, and provide a basis for the treatment of CLC-induced diseases and/or type 2 immune diseases .
  • Example 5 The polypeptide can alleviate the inflammatory response of CLC-induced lung injury in mice
  • mice were randomly divided into five groups, 6 in each group;
  • mice were intraperitoneally injected with tribromoethanol anesthesia, the anesthesia dose was: mouse body weight (g) ⁇ 15 ⁇ L/mouse, and the mice were fully anesthetized (foot squeeze reflex negative) and then immobilized;
  • mice were intraperitoneally injected with tribromoethanol anesthesia, the anesthesia dose was: mouse body weight (g) ⁇ 15 ⁇ L/mouse, and the mice were fully anesthetized (foot squeeze reflex negative) and then immobilized;
  • a 1000 ⁇ L tip is covered with a 200 ⁇ L tip to obtain alveolar lavage fluid from mice.
  • step 4 put the lavage solution into the same 1.5mL microcentrifuge tube, freeze it at -196°C, and store it in a -80°C refrigerator;
  • RT-qPCR real-time fluorescent quantitative PCR
  • RNA reversed to cDNA and real-time fluorescent quantitative PCR (RT-qPCR) to detect changes in gene expression.
  • RT-qPCR real-time fluorescent quantitative PCR
  • a CLC-induced mouse lung injury model was successfully established, and the inflammation-inducing ability of exogenous CLC was verified in mice.
  • FIG. 11 in the acute lung injury model induced by CLC in mice, the detection results of the inflammatory factors of #8 polypeptide (SEQ ID NO.3) involved in the present invention in alleviating lung injury in mice were verified.
  • FIG. 12 in the CLC-induced acute lung injury model in mice, it was verified that #8 polypeptide (SEQ ID NO.3) involved in the present invention alleviates the lung pathological results of lung injury in mice.
  • the results of animal experiments prove the therapeutic effect of the polypeptide on CLC-induced diseases and/or type 2 immune diseases.

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Abstract

La présente invention concerne un polypeptide ciblant une protéine des cristaux de Charcot-Leyden et une utilisation associée. Le polypeptide contient au moins l'une d'une séquence d'acides aminés telle que représentée par SEQ ID NO : 3 ou SEQ ID NO : 4 ou SEQ ID NO : 5, ou un fragment, une variante, une fusion ou un dérivé de celle-ci, ou une fusion du fragment, de la variante ou du dérivé de celle-ci. La présente invention concerne également un acide nucléique codant pour le polypeptide, un vecteur recombinant et une cellule pour l'expression du polypeptide. L'invention concerne en outre l'utilisation du polypeptide dans la préparation d'un médicament pour la prévention ou le traitement d'une maladie immunitaire de type 2, et un procédé de détection d'une protéine Gal-10 à des fins non diagnostiques.
PCT/CN2022/116317 2021-08-31 2022-08-31 Polypeptide ciblant une protéine des cristaux de charcot-leyden et utilisation associée WO2023030407A1 (fr)

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CN202111008517.1A CN113603753B (zh) 2021-08-31 2021-08-31 靶向夏科-莱登结晶蛋白的多肽及其应用
CN202111008519.0A CN113666999B (zh) 2021-08-31 2021-08-31 用于治疗2型免疫疾病的多肽
CN202111008545.3A CN113667000B (zh) 2021-08-31 2021-08-31 多肽在制备治疗2型免疫疾病的药物中的应用
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