Ionizable polypeptides and their use in alleviating gal-10 crystal-related diseases

Abstract

The application provides an ionic polypeptide and application thereof in relieving Gal-10 crystal-related diseases, and the polypeptide is composed of arginine and tyrosine. The ionic polypeptide of the application can effectively inhibit an immune response induced by CLCs, and is an effective drug for treating related diseases targeting CLCs.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, and in particular to ion polypeptides and their use in alleviating Gal-10 crystallization-related diseases. BACKGROUND

[0002] Charcot-leyden crystals (CLCs) formed by galectin-10 were discovered by French neurologist Charcot in 1853 in the heart and spleen tissues of a dead leukemia patient, and are deposited in the extracellular double-tapered hexagonal protein crystals. German physician Leyden also discovered them in the sputum of an asthma patient in 1872, hence the name Charcot-leyden crystals. Charcot-leyden crystals appear in various tissues and organs, including the lungs, gastrointestinal tract, tumor tissue, lymph nodes, etc. They are closely related to various diseases, including but not limited to tumors, infectious diseases: pyogenic lymphadenitis, eosinophilic cystitis, liver abscess, etc., inflammatory diseases: pneumonia, sinusitis, asthma, allergic rhinitis, eosinophilic colitis, etc. Different treatment methods are usually used for different diseases, such as drugs targeting tumors, anti-inflammatory drugs for pneumonia, glucocorticoids for sinusitis, or surgical treatment.

[0003] For research targeting CLCs as a therapeutic target, there are currently reports that monoclonal antibodies of the crystal can dissolve the crystal in vitro and can inhibit lung innate immunity in mice caused by CLCs. However, monoclonal antibody drugs are high in cost, poor in stability, difficult to transport and store, and harsh in storage conditions, and the follow-up development of such drugs will be more difficult. There is an urgent need to develop a therapeutic drug that is low in cost, easy to metabolize, good in biocompatibility, and easy to produce industrially. SUMMARY

[0004] To make up for the shortcomings of the prior art, the present application uses the theory that electrostatic interaction can regulate protein assembly state to find ion polypeptides that can regulate the assembly state of CLCs, and further screens drugs that can regulate the assembly of CLCs. These small polypeptide molecules can become potential drugs for targeted treatment of CLCs-related diseases.

[0005] Further, the present application adopts the following technical solutions:

[0006] The present application provides a polypeptide, which is an ion peptide composed of arginine and tyrosine.

[0007] The second aspect of the present application provides a polypeptide derivative, characterized in that the polypeptide derivative comprises a modified product of the polypeptide according to the first aspect of the present application, a variant of the polypeptide according to the first aspect of the present application obtained by addition and / or substitution of one or more amino acids, or a conjugate of the polypeptide according to the first aspect of the present application with another substance.

[0008] The third aspect of the present application provides a nucleic acid consisting of or comprising a sequence encoding the polypeptide according to the first aspect of the present application or the polypeptide derivative according to the second aspect of the present application, or a sequence complementary thereto.

[0009] The fourth aspect of the present application provides an expression vector, characterized in that the expression vector comprises the nucleic acid according to the third aspect of the present application.

[0010] The fifth aspect of the present application provides a host cell or a cell-free expression system comprising the expression vector according to the fourth aspect of the present application.

[0011] The sixth aspect of the present application provides a pharmaceutical composition comprising the polypeptide according to the first aspect of the present application or the polypeptide derivative according to the second aspect of the present application; the nucleic acid according to the third aspect of the present application; the expression vector according to the fourth aspect of the present application; and / or the cell according to the fifth aspect of the present application.

[0012] The seventh aspect of the present application provides a kit comprising the polypeptide according to the first aspect of the present application, the polypeptide derivative according to the second aspect of the present application, the nucleic acid according to the third aspect of the present application, the expression vector according to the fourth aspect of the present application, the host cell or the cell-free expression system according to the fifth aspect of the present application, or the pharmaceutical composition according to the sixth aspect of the present application; optionally, the kit further comprises instructions for use.

[0013] The eighth aspect of the present application provides a device comprising the polypeptide according to the first aspect of the present application, the polypeptide derivative according to the second aspect of the present application, the nucleic acid according to the third aspect of the present application, the expression vector according to the fourth aspect of the present application, the host cell or the cell-free expression system according to the fifth aspect of the present application, or the pharmaceutical composition according to the sixth aspect of the present application; optionally, wherein the device is a medical device.

[0014] The ninth aspect of the present application provides use of any one of the following:

[0015] 1) Use of the polypeptide according to the first aspect of the present application, the polypeptide derivative according to the second aspect of the present application, the nucleic acid according to the third aspect of the present application, the expression vector according to the fourth aspect of the present application, the host cell or cell-free expression system according to the fifth aspect of the present application, or the pharmaceutical composition according to the sixth aspect of the present application in the preparation of a product for preventing and / or treating a disease or disorder associated with the presence or formation of galectin-10 crystals.

[0016] 2) Use of the polypeptide according to the first aspect of the present application, the polypeptide derivative according to the second aspect of the present application, the nucleic acid according to the third aspect of the present application, the expression vector according to the fourth aspect of the present application, the host cell or cell-free expression system according to the fifth aspect of the present application, or the pharmaceutical composition according to the sixth aspect of the present application in inhibiting the formation of galectin-10 crystals or promoting the dissolution of galectin-10 crystals.

[0017] The tenth aspect of the present application provides a method for inhibiting the formation of galectin-10 crystals or promoting the dissolution of galectin-10 crystals, comprising administering the polypeptide according to the first aspect of the present application, the polypeptide derivative according to the second aspect of the present application, or the pharmaceutical composition according to the sixth aspect of the present application.

[0018] The eleventh aspect of the present application provides a method for preventing and / or treating a disease or disorder associated with the presence or formation of galectin-10 crystals, comprising administering an effective amount of the polypeptide according to the first aspect of the present application, the polypeptide derivative according to the second aspect of the present application, or the pharmaceutical composition according to the sixth aspect of the present application.

[0019] Advantages of the present application:

[0020] The present application discloses an ionic polypeptide composed of arginine and tyrosine, which can regulate the assembly of CLCs and thus become a potential drug for targeted treatment of CLC-related diseases. The ionic polypeptide of the present application has the advantages of low price, simple structure, easy metabolism, good biocompatibility, easier penetration of human tissue and cell barriers, and easy industrial production. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 is a micrograph of CLCs dissolution regulated by different pH values;

[0022] Figure 2 is a standard area statistical graph of CLCs dissolution regulated by different pH values;

[0023] Figure 3 is a reaction rate graph of CLCs dissolution regulated by different pH values;

[0024] Figure 4 is a micrograph of CLCs dissolution regulated by different polypeptides;

[0025] Figure 5 This is a graph showing the effect of R12Y8 on inflammatory factors in lung injury; 5A shows the effect of R12Y8 on the inflammatory factor IL-1β in lung injury; 5B shows the effect of R12Y8 on the inflammatory factor TNF-α in lung injury.

[0026] Figure 6 This is a pathological tissue staining diagram of the effect of R12Y8 on lung injury;

[0027] Figure 7 These are micrographs showing how different ionic peptides regulate the dissolution of CLCs;

[0028] Figure 8 These are statistical charts showing the standard crystal area of ​​CLCs dissolution regulated by different ionic peptides; 8A, 8B, 8C, 8D, and 8E are statistical charts showing the standard crystal area of ​​CLCs dissolution regulated by ionic peptides R12Y8-1, R12Y8-2, R12Y8-3, R12Y8-4, and R12Y8-5, respectively.

[0029] Figure 9 This is a graph showing the reaction rate at which ionic peptides regulate the dissolution of CLCs. Detailed Implementation

[0030] In this invention, the terms "peptide" or "polypeptide" are used interchangeably in their broadest sense to refer to a compound having two or more subunit amino acids, amino acid analogs, or other peptide mimics. Thus, the term "peptide" includes short peptide sequences as well as longer polypeptides and proteins, including their variants and fusions. As used herein, the term "amino acid" refers to natural and / or non-natural or synthetic amino acids, including both D and L optical isomers, as well as amino acid analogs and peptide mimics.

[0031] Those skilled in the art will understand that the term "amino acid" as used in this invention includes the standard twenty genetically encoded amino acids and their corresponding stereoisomers of the "d" form (as opposed to the natural "l" form).

[0032] The term "galactolectin-10" (or Gal10 or Gal-10) refers to a small, hydrophobic glycan-binding protein that crystallizes spontaneously to form Charcot-Leyden crystals. Galactolectin-10 is also known as Charcot-Leyden crystal protein (CLCP), eosinophilic lysophospholipase, and lysophosphatidylcholine hydrolase. The term "galactolectin-10" is broad enough to encompass human proteins and homologs of any species. Galactolectin-10 also includes naturally occurring variants of human sequences.

[0033] "Galactin-10 crystal", "Shack-Laden crystal", "CLC" and "CLCs" are used interchangeably in the present application, and refer to crystals formed by galactin-10. The crystals formed by galactin-10 are usually bipyramidal hexagonal crystals, with a length of about 20-40 μm and a width of about 2-4 μm. These crystals are associated with eosinophilic inflammatory diseases.

[0034] The present application utilizes the theory that the assembly state of proteins can be regulated by electrostatic interaction, and through extensive and in-depth research, an ionic polypeptide that can regulate the assembly state of CLCs is found, thereby treating diseases or conditions associated with CLCs.

[0035] The present application provides a polypeptide, which is an ionic peptide consisting of amino acids arginine and tyrosine.

[0036] The amino acids of the polypeptide of the present application can be connected in any way, and exemplary connection modes are arginine-tyrosine, arginine-arginine, and tyrosine-tyrosine; the above connections can occur at any position of the polypeptide.

[0037] In some embodiments, the number of tyrosine in the polypeptide is ≥2; the number of arginine is ≥2.

[0038] In some embodiments, the number of tyrosine in the polypeptide is ≥2; the number of arginine is ≥ the number of tyrosine.

[0039] In some embodiments, the number of arginine in the polypeptide is 3:2-2:1 relative to the number of lysine.

[0040] In some embodiments, the peptide is a relatively short polypeptide, usually 2-60 amino acids in length; as a preferred embodiment, 4-40 amino acids in length.

[0041] In a specific embodiment, the length of the polypeptide is 6-20 amino acids.

[0042] In a specific embodiment, the amino acid sequence of the polypeptide is as shown in any one of SEQ ID NOs: 2-7.

[0043] In some embodiments, the present application provides a polypeptide derivative, which includes a modified product of the aforementioned polypeptide, a variant obtained by adding and / or replacing one or more amino acids to the aforementioned polypeptide, and a conjugate of the aforementioned polypeptide and other substances,

[0044] In some embodiments, the variant comprises an amino acid sequence that is at least 70%, preferably at least 80%, more preferably at least 90%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98% or 99% homologous to the amino acid sequence described above.

[0045] In some embodiments, the polypeptide derivative is a modified product of the polypeptide described above. The modification includes, but is not limited to, amino modification, methylation modification, amidation modification, hydroxylation modification, carboxylation modification, carbonylation modification, alkylation modification, acetylation modification, phosphorylation modification, sulfation modification, esterification modification, glycosylation modification, cyclization modification, biotinylation modification, fluorescent group modification, polyethylene glycol (PEG) modification, myristoylation modification, non-metal chemical element modification, immobilization modification.

[0046] The present application provides a nucleic acid consisting of or comprising a sequence encoding the polypeptide described above or the polypeptide derivative described above, or a sequence complementary to the sequence.

[0047] The term "nucleic acid", "polynucleotide" or "polynucleotide molecule" used interchangeably in the present application refers to any DNA or RNA molecule, whether single-stranded or double-stranded, and if single-stranded, the complement of the sequence thereof. In discussing nucleic acid molecules, the sequence or structure of a particular nucleic acid molecule can be described in the conventional direction of 5' to 3' sequence. In some embodiments of the present application, the nucleic acid or polynucleotide is "isolated". The term, when applied to nucleic acid molecules, means that the nucleic acid molecule is separated from sequences with which it is immediately contiguous in the naturally occurring genome of the organism from which it originated. For example, an "isolated nucleic acid" can comprise a DNA molecule inserted into a vector, such as a plasmid or virus vector, or integrated into the genomic DNA of a prokaryotic or eukaryotic cell or non-human host organism. When applied to RNA, the term "isolated polynucleotide" refers primarily to an RNA molecule encoded by an isolated DNA molecule as defined above. Alternatively, the term can refer to an RNA molecule that has been purified / isolated from other nucleic acids with which it is associated in a natural state (i.e., in a cell or tissue). An isolated polynucleotide (DNA or RNA) can further represent a molecule that is produced directly by biological or synthetic means and is separated from other components present during its production.

[0048] The present application provides an expression vector comprising the nucleic acid described above.

[0049] In some embodiments, the expression vector further comprises a regulatory sequence operably linked to the nucleic acid described above; the regulatory sequence allows expression of the polypeptide in a host cell or a cell-free expression system.

[0050] The term "expression vector" refers to a non-chromosomal nucleic acid comprising an intact replicon, such that when placed into a permissive cell, the vector can be replicated, e.g., by a process of transformation. A vector can replicate in one cell type (e.g., bacteria) but have limited ability to replicate in another cell type (e.g., mammalian cells). Vectors can be viral or non-viral. Exemplary non-viral vectors for delivering nucleic acids include naked DNA; DNA complexed with cationic lipids, alone or in combination with cationic polymers; anionic and cationic liposomes; DNA-protein complexes and particles comprising DNA condensed with cationic polymers (such as hetero-polylysine, defined oligopeptides and polyethyleneimine), in some cases also in liposomes.

[0051] Exemplary vectors include, but are not limited to, MarEx expression vectors produced by Celltrion Inc. (Korea); widely commercially available pCDNA vectors; F, Rl, RP1, Col, pBR322, ToL, Ti vectors; cosmids; bacteriophages such as lambda phage, lambda-like phage, M13 phage, Mu phage, PI phage, P22 phage, Q mu phage, T-even phage, T2 phage, T4 phage, T7 phage, etc.; plant viruses. Any one of various vectors known to those skilled in the art can be used in the present application, and the selection of the vector depends on the nature of the selected cell. Introduction of the vector into the cell can be achieved by, but not limited to, calcium phosphate transfection, viral infection, DEAE-dextran-mediated transfection, lipofection or electroporation, and any person skilled in the art can select and use an introduction method suitable for the used vector and cell.

[0052] In some embodiments, the vector comprises one or more selection markers, but is not limited thereto, and a vector not comprising a selection marker can also be used. The selection of the selection marker can depend on the selected cell (as well known to those skilled in the art), but this is not critical to the present application.

[0053] The present application provides a host cell or a cell-free expression system comprising the aforementioned expression vector.

[0054] In some embodiments, the cell comprises a prokaryotic cell, a eukaryotic cell.

[0055] In some embodiments, the prokaryotic cell comprises a bacterial cell.

[0056] In some embodiments, the eukaryotic cell comprises a protist cell, an animal cell, a plant cell, a fungal cell.

[0057] In some embodiments, the animal cell includes a mammalian cell, an avian cell, an insect cell. Exemplary mammalian cells include, but are not limited to, COS-7 cells, 293 or 293T cells, BHK cells, CHO cells, TM4 cells, CRL 1581 cells, CV1 cells, Hela cells, Vero cells, MDCK cells, BRL 3A cells, W138 cells, HepG2 cells, TRI cells, MRC 5 cells, FS4 cells.

[0058] The present application provides pharmaceutical compositions comprising the aforementioned polypeptides or derivatives thereof. Accordingly, a composition comprising a peptide can be any combination or arrangement of any one or more of the aforementioned peptides, provided that the composition retains the intended activity (or at least a portion of the intended activity). In some embodiments, the composition further comprises a pharmaceutically acceptable carrier.

[0059] In some embodiments, the pharmaceutical composition comprises the aforementioned polypeptide derivative. In some embodiments, the composition further comprises a pharmaceutically acceptable carrier.

[0060] In some embodiments, the pharmaceutical composition comprises the aforementioned nucleic acid. In some embodiments, the composition further comprises a pharmaceutically acceptable carrier.

[0061] In some embodiments, the pharmaceutical composition comprises the aforementioned host cell or cell-free expression system. In some embodiments, the composition further comprises a pharmaceutically acceptable carrier.

[0062] The pharmaceutical composition can be prepared in a manner known in the art, is sufficiently stable for storage, and is suitable for administration to humans and animals. The pharmaceutical composition can be lyophilized, for example by freeze-drying, spray-drying, spray-chilling, or by using particles from supercritical particle formation.

[0063] "Pharmaceutically acceptable" means a nontoxic material that does not reduce the effectiveness of the biological activity of the active ingredient (i.e., the polypeptide). Such pharmaceutically acceptable carriers are well known in the art. They include, but are not limited to, buffers, excipients, diluents, and / or adjuvants.

[0064] The term "buffer" means an aqueous solution containing an acid-base mixture whose purpose is to stabilize the pH. Examples of 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 lactate, PIPES, SSC, SSPE, POPSO, TAPS, TABS, TAPSO, and TES.

[0065] The term "diluent" means an aqueous or non-aqueous solution whose purpose is to dilute the peptide in a pharmaceutical formulation. The diluent can be one or more of saline, water, polyethylene glycol, propylene glycol, ethanol, or oil (e.g., safflower oil, corn oil, peanut oil, cottonseed oil, or sesame oil).

[0066] The term "adjuvant" means any compound added to a formulation to increase the biological effect of the peptide of the composition. The adjuvant can be one or more of colloidal silver or zinc, copper, or silver salts with different anions, such as, but not limited to, fluoride, chloride, bromide, iodide, thiocyanate, sulfite, hydroxide, phosphate, carbonate, lactate, glycolate, citrate, borate, tartrate, and acetate of different acyl composition. The adjuvant can also be a cationic polymer such as PHMB, cationic cellulose ethers, cationic cellulose esters, deacetylated hyaluronic acid, chitosan, cationic dendrimers, cationic synthetic polymers such as poly(vinylimidazole), and cationic polypeptides such as polyhistidine, polylysine, polyarginine, and peptides containing these amino acids.

[0067] The excipients can be one or more of a carbohydrate, a polymer, a lipid, a detergent, and a mineral. Examples of carbohydrates include lactose, sucrose, mannitol, and cyclodextrins, which are added to the composition, e.g., to facilitate lyophilization. Examples of polymers are starch, cellulose ethers, cellulose, carboxymethylcellulose, hydroxypropylmethylcellulose, hydroxyethylcellulose, ethylhydroxyethylcellulose, ethylcellulose, methylcellulose, propylcellulose, alginates, carrageenans, hyaluronic acid and its derivatives, polyacrylic acid, polysulfonates, polyethylene glycol / polyethylene oxide, polyethylene oxide / polypropylene oxide copolymers, polyvinyl alcohol / polyvinyl acetate of different degrees of hydrolysis, poly(lactic acid), poly(glycolic acid) or copolymers thereof with various compositions, and polyvinylpyrrolidone (all with different molecular weights), which are added to the composition, e.g., for viscosity control, to achieve bioadhesion or to protect the active ingredient (also applicable to A-C) from chemical and proteolytic degradation. Examples of 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, soy lecithin, hydrogenated egg and soy lecithin, which are added to the composition for similar reasons as the polymers. Examples of minerals are talc, magnesium oxide, zinc oxide, and titanium oxide, which are added to the composition to obtain benefits such as reduction of liquid accumulation or advantageous pigment properties.

[0068] The pharmaceutical composition can also contain one or more monosaccharides or disaccharides, such as xylitol, sorbitol, mannitol, lactitol, isomalt, maltitol, glycerol or xyloside, and / or monoacylglycerols, such as glycerol monolaurate.

[0069] The properties of the carrier depend on the route of administration. One route of administration is topical administration. For example, for topical administration, the preferred carrier is an emulsified cream containing the active peptide, but other common carriers can also be used, such as certain petrolatum / mineral-based and plant-based ointments, as well as polymeric gels, liquid crystal phases, and microemulsions.

[0070] The pharmaceutical composition can also include ions and a defined pH for strengthening the effect of the peptide activity.

[0071] In some embodiments, the pharmaceutical composition further comprises other drugs that treat a disease or disorder associated with CLCs.

[0072] The skilled person will appreciate that the pharmaceutical compositions and / or peptides of the application can be administered locally or systemically. Routes of administration include topical (e.g. ophthalmic), ocular, nasal, pulmonary, buccal, parenteral (intravenous, subcutaneous and intramuscular), oral, vaginal and rectal. In one embodiment, administration can be respiratory, e.g. by oral or nasal inhalation. Such administration is also referred to as inhalation or pulmonary administration. In addition, administration from an implant is possible. Suitable formulation forms are, for example, granules, powders, tablets, coated tablets, (micro-)capsules, suppositories, syrups, emulsions, microemulsions (defined as an optically isotropic thermodynamically stable system consisting of water, oil and a surfactant), liquid crystalline phases (examples include water or oil continuous lamellar phases, hexagonal phases and cubic phases) defined as systems characterised by long range order but short range disorder, or their dispersed counterparts, gels, ointments, dispersions, suspensions, creams, aerosols, wafers, droplets or injectable solutions in ampoules as well as formulations with prolonged release of the active compound using excipients, diluents or adjuvants as described above. The pharmaceutical compositions and / or peptides can also be provided in the form of a bandage, plaster or suture and the like.

[0073] In some embodiments, the pharmaceutical compositions and / or peptides are suitable for oral administration, parenteral administration and / or topical administration. For example, the pharmaceutical compositions can be suitable for topical administration (e.g. ophthalmic administration, in the form of a spray, lotion, paste or drops and the like). The route of administration can depend on the type of disease. For example, the inhalation / pulmonary route of administration is more beneficial for the treatment of infections.

[0074] In some embodiments, the pharmaceutical compositions of the application can be administered intranasally or by inhalation and are conveniently delivered in the form of a dry powder inhaler or an aerosol spray presentation from a pressurized container, pump, spray or nebulizer with the use of a suitable propellant, e.g. dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoro-ethane, a hydrofluoroalkane such as 1,1,1,2-tetrafluoroethane (HFA 134A3 or 1,1,1,2,3,3,3-heptafluoropropane (HFA 227EA3), carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve to deliver a metered amount. The pressurized container, pump, spray or nebulizer can contain a solution or suspension of the active ingredient, e.g. using a mixture of ethanol and the propellant as the solvent, which can additionally contain a lubricant, e.g. sorbitan trioleate. Capsules and cartridges (made, for example, from gelatin) for use in an inhaler or insufflator can be formulated to contain a powder mix of a polypeptide of the application and a suitable powder base such as lactose or starch.

[0075] The pharmaceutical composition will be administered to a patient in a pharmaceutically effective dose. By "pharmaceutically effective dose" is meant a dose sufficient to produce the desired effect in relation to the condition for which it is administered. The exact dose will depend on the activity of the compound, the mode of administration, the nature and severity of the condition, the age and weight of the patient, and thus can need to be adjusted in relation to these factors. Administration of the dose can be performed in a single administration in the form of a single dose unit or several smaller dose units, or by subdividing the dose into several administrations at specific intervals.

[0076] The pharmaceutical composition of the application can be administered alone or in combination with other therapeutic agents that can treat a disease or disorder associated with CLCs.

[0077] The additional therapeutic agent can be added as part of the same pharmaceutical composition or can be administered separately. The additional therapeutic agent can be administered prior to, after and / or concurrently with, simultaneously, sequentially and / or separately from the administration of the pharmaceutical composition described in the present application.

[0078] In some embodiments, the pharmaceutical composition can be in the form of a spray, aerosol, powder, injection, tablet, eye drop or eye ointment, for example for topical application.

[0079] In some embodiments, the polypeptide described in the preceding, the polypeptide derivative described in the preceding, the nucleic acid described in the preceding, the expression vector described in the preceding, the host cell or the cell-free expression system described in the preceding, or the pharmaceutical composition described in the preceding, is for use in the treatment of a disease or disorder associated with the presence or formation of crystals of galectin-10.

[0080] The disease or disorder includes, but is not limited to, infectious diseases, inflammatory diseases, tumors. The disease or disorder includes, but is not limited to, pyogenic lymphadenitis, eosinophilic cystitis, liver abscess, asthma, allergic rhinitis, eosinophilic colitis, allergic dermatitis, chronic sinusitis, allergic dermatitis, keratoconjunctivitis, celiac disease, helminth infection, gastrointestinal eosinophilic inflammation, cystic fibrosis (CF), bronchopulmonary aspergillosis (ABPA), allergic granulomatous angiitis, pneumonia, acute myelocytic leukemia, parasitic infection.

[0081] In some embodiments, the polypeptide described in the preceding, the polypeptide derivative described in the preceding, the nucleic acid described in the preceding, the expression vector described in the preceding, the host cell or the cell-free expression system described in the preceding, or the pharmaceutical composition described in the preceding, is for use in inhibiting the formation of crystals of galectin-10 or in promoting the dissolution of crystals.

[0082] The present application provides a method of inhibiting formation of galectin-10 crystals or promoting dissolution of galectin-10 crystals, comprising administering the polypeptide described above, the polypeptide derivative described above, or the pharmaceutical composition described above. Preferably, the method is a method of inhibiting formation of galectin-10 crystals or promoting dissolution of galectin-10 crystals in vitro.

[0083] The present application also provides a method of treating a subject in need thereof, wherein the method comprises administering to the subject a therapeutically effective amount of the polypeptide, polypeptide derivative, or pharmaceutical composition described above.

[0084] As used herein, "therapeutically effective amount" is intended to mean a quantity or dosage of an active ingredient (e.g., a polypeptide) sufficient to effect a therapeutic result, e.g., a quantity or dosage of an active ingredient required to eradicate or at least alleviate symptoms associated with a disease or disorder. Suitable amounts or dosages can be determined by a physician as appropriate. For example, the dosage can be adjusted based on factors such as the size or weight of the subject to be treated, the age of the subject to be treated, the general physical condition of the subject to be treated, the condition to be treated, and the route of administration.

[0085] As used herein, a method of "preventing" a disease or disorder refers to preventing the onset of the disease, preventing the worsening of symptoms, preventing the progression of the disease or disorder, or reducing the risk of a subject developing the disease or disorder. As used herein, a method of "treating" a disease or disorder refers to curing the disease or disorder and / or alleviating or eradicating symptoms associated with the disease or disorder, thereby reducing the suffering of the patient.

[0086] For a patient having a disease or disorder characterized by the presence of galectin-10 crystals, a method of treatment will typically comprise administering an active ingredient capable of dissolving galectin-10 crystals located in the patient's tissues, preferably the polypeptide or polypeptide derivative described above. For a patient identified as "at risk of developing a disease or disorder characterized by the formation of galectin-10 crystals", a method of prevention can comprise administering an active ingredient capable of inhibiting the crystallization of galectin-10, preferably the polypeptide or polypeptide derivative described above.

[0087] Galectin-10 crystals or CLCs have been observed in patients suffering from a range of diseases and disorders. It follows that the polypeptide or derivative product thereof described in the present application can be used to prevent or treat a disease or disorder selected from the group consisting of pyogenic lymphadenitis, eosinophilic cystitis, liver abscess, asthma, allergic rhinitis, eosinophilic colitis, allergic dermatitis, chronic rhinosinusitis, allergic dermatitis, keratoconjunctivitis, celiac disease, helminth infection, gastrointestinal eosinophilic inflammation, cystic fibrosis (CF), bronchopulmonary aspergillosis (ABPA), allergic granulomatous angiitis, pneumonia, acute myeloid leukemia, parasitic infection.

[0088] As mentioned above, galectin-10 crystals or CLC are especially associated with diseases or disorders characterized by eosinophilic inflammation. In a preferred embodiment, the polypeptide or polypeptide derivative according to the present application is used for treating a disease or disorder associated with eosinophilic inflammation.

[0089] The technical solutions of the present application are further illustrated below by specific examples, which do not represent a limitation on the scope of protection of the present application. Some non-essential modifications and adjustments made by others according to the concept of the present application still fall within the scope of protection of the present application.

[0090] 1. Experimental materials:

[0091] 1.1 Plasmid: After codon optimization of the Gal-10 protein sequence (MSLLPVPYTEAASLSTGSTVTIKGRPLACFLNEPYLQVDFHTEMKEESDIVF HFQVCFGRRVVMNSREYGAWKQQVESKNMPFQDGQEFELSISVLPDKYQVM VNGQSSYTFDHRIKPEAVKMVQVWRDISLTKFNVSYLKR), plus its N-terminus (MASTTHHHHHDTDIPTTGGGSRPDDDDDKENLYFQGHM) was cloned into the pET-28a vector plasmid through NcoI / XhoI double enzyme digestion sites to form pET-28a-6His-TEV-gal10.

[0092] 1.2 Reagent materials and instrument equipment

[0093] 1.2.1 Chemical reagents: Kanamycin and ampicillin were purchased from TianGen Biotech Co., Ltd.; Isopropyl-beta-D-thiogalactoside (IPTG) and Escherichia coli BL21 (DE3) were purchased from Beijing Zixiao Godun Company; SDS, Trizol and imidazole were purchased from Sigma Company; TEV enzyme was purchased from Beijing Yiqiao Godun Company; Coomassie brilliant blue staining solution (self-made); cDNA reverse transcription reagent was purchased from Takara Company; Real-time fluorescent quantitative PCR reagent was purchased from Shanghai Roche Company; Elisa kit was purchased from R&D Systems Company; NaCl was purchased from Tianjin Damao Chemical Reagent Factory; Peptide molecules were synthesized in Guoping Pharmaceutical Co., Ltd.; DEPC water, ELISA termination liquid, and TMB single-component color developing liquid were purchased from Solabio Company.

[0094] Table 1 Peptide molecules

[0095] Name Sequence Sequence No. R9 RRRRRRRRR SEQ ID NO: 1 R12Y8 YYRRRRYYRRRRYYRRRRYY SEQ ID NO: 2 R12Y8-1 YYRRRR SEQ ID NO: 3 R12Y8-2 YYRRRRYYRRRR SEQ ID NO: 4 R12Y8-3 RRRRRRRRRRRRYYYYYYYY SEQ ID NO: 5 R12Y8-4 RYYRYRRYYRRRRYRYYRRR SEQ ID NO: 6 R12Y8-5 YRYRYRYRYRYRYRRRRYRR SEQ ID NO: 7

[0096] 1.2.2 Consumables and instruments: Ni-NTA affinity chromatography column was purchased from GE company; 10 kDa concentrator tubes were purchased from Millipore company; electric heating incubator (XMTD HH.B11-600); vertical pressure steam sterilization pot (Shanghai Boxin, China); constant temperature bacterial incubator (Shanghai Boxin, China); PCR instrument (Biometra Tgradient); 384-well plate real-time fluorescent quantitative PCR instrument (Roche 480 II); table centrifuge (eppendorf, Centrifuge 5415D); electric heating constant temperature water tank (SHHW21600); micro ultraviolet spectrophotometer (NanoDrop 2000); electronic balance (Sartorius 2000S); optical inverted microscope (XDS-1B); micropipette (eppendorf Research plus); -80℃ ultra-low temperature refrigerator (SANYO, MDF-382E); high-speed refrigerated centrifuge (Beckman, Germany); pH meter (Thermo Orion 868); magnetic stirrer (IKA RH-KT / C); ultrasonic cell disruptor; pure 25 protein purification system (superdex 75, GE Healthcare); SDS-PAGE electrophoresis instrument (Bio-Rad); gel imaging instrument (Tanon); 1 mL disposable sterile syringe (purchased from Bide Medical Instrument Co., Ltd.).

[0097] 1.3 Experimental animals: C57BL / 6 strain mice were purchased from China Food and Drug Inspection Research Institute of Experimental Animal Resources.

[0098] 2, Experimental method

[0099] 2.1 Expression, purification of Gal10 recombinant protein and preparation of CLC crystal

[0100] 2.1.1 Transformation:

[0101] 1) The BL21 (DE3) competent cells and pET-28a-6His-TEV-gal10 recombinant plasmid were placed on ice to thaw;

[0102] 2) Take 0.5 μL (0.2 μg / μL) of recombinant plasmid and add it to BL21 (DE3) competent cells, incubate on ice for 15-20 min;

[0103] 3) 42℃ water bath heat shock for 90 s;

[0104] 4) quickly placed on ice, ice bath for 5 min;

[0105] 5) Add 500 μL of non-resistant LB, incubate in 37°C constant temperature incubator for 40-50 min;

[0106] 6) Take 40 μL of bacterial solution, inoculate on LB solid medium containing Kanamycin (25 μg / mL), incubate in 37°C constant temperature incubator overnight.

[0107] 2.1.2 Bacterial culture:

[0108] Take the positive monoclonal BL21 (DE3) / pET-28a-6His-TEV-gal10 on the solid medium in the above step with Kanamycin (25 μg / mL) as a selection marker, inoculate in 20 mL of LB liquid medium containing Kanamycin resistance, and incubate at 37°C, 210 r / min on a shaker for 8 h. The bacterial solution was inoculated in 1 L of LB liquid medium containing Kanamycin resistance at a ratio of 1:100, and incubated at 37°C, 210 r / min.

[0109] 2.1.3 Induction of expression of target protein and obtaining:

[0110] 1) When the optical density (OD600) of Escherichia coli at 600 nm is 0.6-0.8, add IPTG with a final concentration of 1 mM, incubate at 28°C, 210 r / min for 12-16 h to induce expression of the target protein;

[0111] 2) Enrich the overnight expression of bacterial culture, centrifuge at 6000 x g, 4°C for 20 min, discard the supernatant. Resuspend with buffer Lysis Buffer (50 mM NaH2PO4; 300 mM NaCl PH 7.4), concentrate it in a beaker with a volume of about 60-80 mL, add PMSF (final concentration 1 mM) at a ratio of 1:100 to prevent degradation of the target protein;

[0112] 3) Ultrasonic breakage of bacteria: place the beaker on ice for ultrasonic, power 25%, working time 25 min, ultrasonic on time 3 s, ultrasonic off time 9 s;

[0113] 4) Collect protein: centrifuge (4°C, 12000 x g, 30 min) to remove cell debris, and at the same time break the nucleic acid released after cell breakage, collect the supernatant, and the soluble target protein is present in the supernatant.

[0114] 2.1.4 Purification of Gal-10 protein:

[0115] 2.1.4.1 Ni-NTA affinity column chromatography:

[0116] 1) Equilibration of Ni column: Start eluting protein by running about 2-3 column volumes of Lysis Buffer through the Ni column;

[0117] 2) Centrifuge the supernatant and run 2-3 times through the Ni column;

[0118] 3) Wash the column with Lysis Buffer containing 20 mM imidazole and 0.1% Empigen detergent, which is to elute the impure proteins;

[0119] 4) Wash the column with Lysis Buffer containing 500 mM imidazole, which makes the target protein unbind from the nickel column;

[0120] 5) Concentration: Put the eluted target protein collected in step (4) into a 10 kDa concentrator tube, centrifuge at 4°C, 3000 x g for 10 min, and add a small amount of Lysis Buffer or PBS to dilute the imidazole during the concentration process to prevent the target protein from aggregating and precipitating.

[0121] 2.1.4.2 Pure 25 protein purification:

[0122] 1) Clean the column: Use sterile water to clean the column, about 8 mL. Then put the pump head into the PBS solution, repeat the above steps and perform, about 36 mL;

[0123] 2) Parameters: System flow: 0.4 mL / min; Column position: 2; Alarm delta column pressure enabled: 3.0; Alarm pre column pressure enabled: 5.0;

[0124] 3) Sample loading: First, rinse the sample loop 2-3 times with PBS, then put about 1.5 mL of concentrated sample into the sample loop, and finally slightly suck PBS and then put it into the sample loop to avoid sample residue. Place the collection tube and select inject valve: inject;

[0125] 4) Collection: When the UV 280 mark line representing the protein content rises, use the sample automatic collector to collect the target protein, and set the collection amount of each tube to 0.3 mL;

[0126] 5) Clean the column: After the sample is collected, put the pump head into PBS, continue to run until 20 mL is passed, replace it with sterile water for cleaning, run 10 mL, and then replace it with 20% ethanol solution and run 20 mL, then shut down;

[0127] 6) Protein processing: The collected protein was measured for concentration per tube and labeled, and then placed in liquid nitrogen for quick freezing and stored in a -80°C refrigerator. Before the experiment, the sample was slowly thawed at room temperature.

[0128] 2.1.4.3 SDS-PAGE identification of expression products:

[0129] 1) Sample preparation: whole bacterial sample, supernatant sample after centrifugation, protein supernatant sample after passing through a chromatography column, 20 mM imidazole elution sample, 500 mM imidazole elution sample, and protein samples collected from the head, tip, and tail of the molecular sieve peak;

[0130] 2) Gel preparation: 5% concentrated gel; according to the size of the protein, select 15% separation gel;

[0131] 3) Sample preparation: 2xLoading Buffer mixed with sample 1:1;

[0132] 4) Loading: whole bacterial sample 5 μL, marker 3 μL, and the rest of the samples 10 μL;

[0133] 5) Staining: Place the protein gel in Coomassie Brilliant Blue staining solution, microwave at high heat for 2 min;

[0134] 6) Decolorization: Place the protein gel after staining in tap water, microwave at high heat for 20-40 min.

[0135] 2.1.5 Generation and concentration determination of CLC crystals:

[0136] 1) TEV enzyme and pET-28a-6His-TEV-gal10 recombinant protein were mixed at a mass ratio of 1:10, and incubated at 4°C overnight on a shaker;

[0137] 2) Centrifuge at 600xg and 4°C for 10 min, then remove the supernatant and resuspend in PBS, repeat three times to obtain pure CLCs;

[0138] 3) 6M guanidine hydrochloride to disassemble CLCs, and use One-Drop spectrophotometer to measure CLCs concentration.

[0139] 2.2 In vitro characterization experiment of CLCs dissolution in different pH environments

[0140] 2.2.1 Dissolve CLCs in pure water, adjust the concentration to 0.3 mg / ml;

[0141] 2.2.2. Considering the buffer capacity range of different buffer solutions, citric acid-sodium citrate buffer is selected at 3.0 < pH < 7.0, PBS buffer is selected at 6.0 < pH < 8.0, and sodium carbonate-sodium bicarbonate buffer is selected at 9.0 < pH < 11.0;

[0142] 2.2.3. Mix CLCs and buffer solutions at the same ratio to disperse 0.15 mg / ml CLCs in buffer solutions with pH values of 3.5, 4.0, 4.3, 4.5, 4.8, 5.0, 5.5, 6.0, 6.5, 7.0, 8.0, 9.3, 9.5, 9.8, 10.0, and 10.3, respectively;

[0143] 2.2.4. Drop the mixed liquid on a crystal plate and observe it under an optical microscope, and take a photo every interval;

[0144] 2.2.5. Process the obtained images using PhotoShop, cut the same area, calculate the cut area using ImageJ, and normalize the data obtained at the first time point of 10 min;

[0145] 2.2.6. Calculate the initial reaction rate R according to R(t) = k·A(t)·[1-exp(βΔμ(c))]; int .

[0146] 2.3. In vitro characterization experiment of ionotropic peptide molecules dissolving CLCs

[0147] 2.3.1. Dissolve CLCs in PBS (pH 7.4) and adjust the concentration to make the CLC concentration 0.3 mg / ml;

[0148] 2.3.2. Dissolve peptide molecule R9 in PBS buffer with a molar concentration gradient of 16.0 mM, 20.0 mM, 24.0 mM, and 30.0 mM;

[0149] 2.3.3. Dissolve polypeptide molecule R12Y8 in PBS buffer with a molar concentration gradient of 2 mM, 4 mM, 8 mM, 12 mM, and 16 mM;

[0150] 2.3.4. Mix the polypeptide molecules with CLCs at a volume ratio of 1:1, drop them on a crystal plate, and observe them under an optical microscope, and take a photo every interval;

[0151] 2.3.5. Process the obtained images using PhotoShop, cut the same area, calculate the cut area using ImageJ, and normalize the data at the first time point of 10 min;

[0152] 2.3.6. Calculate the initial reaction rate R according to R(t) = k·A(t)·[1-exp(βΔμ(c))]int .

[0153] 2.4 Animal experiments

[0154] 2.4.1 Randomly divide mice (wild type, male, 6 weeks old, C57BL / 6 strain mice, body weight 20-22 g) into five groups, 6 mice in each group; and prepare perfusion drugs: PBS (negative control), CLCs (1.5 mg / ml), R12Y8 (1 mM), CLCs + R12Y8 (1 mM), CLCs + dexamethasone (0.5 mg / kg) (positive control);

[0155] 2.4.2 Drug administration stage:

[0156] 1) Use a sterile syringe to inject a tricaine anesthetic into the abdominal cavity of the mouse, the anesthetic dose is: mouse body weight (g) x 15 μL per mouse, and the mouse is fixed after being observed to be fully anesthetized (negative foot squeeze reflex);

[0157] 2) Keep the mouse airway vertical, and use ophthalmic scissors to expose a longitudinal incision of 0.5-0.8 cm in length at the middle of the skin on the ventral side of the neck 1.5 cm below the mandible, and use an ophthalmic curved forceps to bluntly separate the subcutaneous tissue, connective tissue, and neck muscles along the midline until the trachea is fully exposed;

[0158] 3) Tilt the mouse fixation device at about 30°, draw 50 μL of air into the syringe and then draw in 70 μL of the pre-prepared drug, and use the syringe to inject the needle parallel to the longitudinal axis of the trachea above the middle thyroid cartilage, with a depth of about 1 cm, and keep the position to quickly instill air and LPS into the trachea of the mouse;

[0159] 4) After the operation is completed, the mouse's breathing rate can be observed to suddenly increase, and the mouse fixation device is placed vertically on the operation table for about 1 min to help the drug distribution;

[0160] 5) Reset the mouse fixation device, use an ophthalmic curved forceps to clamp the mouse's neck tissue layer by layer according to the anatomical layers, release the mouse, and place the mouse in a lateral position and observe its state until it returns to autonomous activity.

[0161] 6) Oral administration: hold the mouse with its head, neck, and body in a straight line. Use a syringe with a gavage needle, insert the needle from the mouse's corner of the mouth, press the tongue against the upper jaw, and gently push it inwards, and after entering the esophagus, slightly penetrate and inject the drug solution.

[0162] 2.4.3 Sample collection stage: start collecting samples 6 h and 12 h after administration

[0163] 1) Use a sterile syringe to inject a tricaine anesthetic into the abdominal cavity of the mouse, the anesthetic dose is: mouse body weight (g) x 15 μL per mouse, and the mouse is fixed after being observed to be fully anesthetized (negative foot squeeze reflex);

[0164] 2) Along the trachea instillation incision re-exposed mouse trachea, cut a "V" shape under the thyroid cartilage, clean up the mouse tracheal secretions, tracheal intubation, pay attention to gentle operation to avoid the mouse tracheal disconnection;

[0165] 3) After 800 μL of sterile PBS, pipette parallel to the longitudinal axis of the trachea into the incision until it completely adheres to the inner wall of the mouse trachea, and then inject PBS into the mouse bronchial alveoli at a constant speed. During the back-suction process, a large number of bubbles will be found, which is the sign of successful operation. After the injection-back-suction process is repeated three times, the recovered liquid is placed in a 1.5 mL centrifuge tube;

[0166] 4) Repeat step (3), and place the recovered liquid in the same 1.5 mL sterile EP tube and store at -80°C;

[0167] 5) Open the mouse chest, cut the sternum, extract the heart, cut off the connective tissue and fascia between the lung tissue and the chest cavity, and then rinse the lung tissue with sterile PBS, lyse with Trizol and store at -80°C.

[0168] 2.5 Enzyme-linked immunosorbent assay (ELISA) for detecting protein changes

[0169] 2.5.1 Coating ELISA 96-well plates: Dilute the capture antibody with sterile PBS according to the working concentration, add 100 μL of the diluted capture antibody per well to the 96-well enzyme-labeled plate with high binding capacity, cover the upper surface with sealing film, and incubate overnight at room temperature;

[0170] 2.5.2 Detection process:

[0171] 1) Discard the capture antibody, add 200 μL of washing solution to each well, discard the washing solution and absorb the residual liquid with filter paper, repeat 3 times;

[0172] 2) Add 300 μL of diluent (1% BSA in PBS) to each well, incubate at room temperature for at least 1 h;

[0173] 3) Discard the diluent and repeat step 1);

[0174] 4) Prepare the standard with new diluent, add 100 μL of prepared standard or sample to each well, two replicates for each sample, shake gently, cover the plate, and incubate at 37°C for 2 h;

[0175] 5) Discard the sample and repeat step 1);

[0176] 6) Dilute the detection antibody with new diluent to the working concentration, add 100 μL of detection antibody working solution to each well, cover the plate, and incubate at room temperature for 2 h;

[0177] 7) Discard the detection antibody and repeat step 1);

[0178] 8) Dilute Streptavidin-HRP with new diluent to working solution concentration at a ratio of 1:40, add 100 μL per well, cover the plate, and incubate at room temperature for 20 min in the dark;

[0179] 9) Discard the Streptavidin-HRP working solution and repeat step 1);

[0180] 10) Add 100 μL of TMB single-component color developing solution to each well, cover the plate, and incubate at room temperature for 10-20 min in the dark;

[0181] 11) Add 50 μL of color developing termination solution to each well, shake to mix, and then use a full-featured microplate detector to detect the OD values at 450 nm and 570 nm, and subtract the OD value at 570 nm from the OD value at 450 nm;

[0182] 12) Plot in Graphpad Prism 8.0 software and perform significance difference analysis.

[0183] 2.6 H&E staining of mouse lung tissue pathological samples

[0184] 2.6.1 Divide the mice according to 2.4.1 and inject them according to 2.4.2, and carefully collect the left lung tissue of the mice (n=3) after 6 h, wash them in PBS and place them in a 10 mL tube containing 8 mL of paraformaldehyde (4%), and then place them at room temperature for 48-72 h of fixation;

[0185] 2.6.2 Dehydrate, embed, deparaffinize, stain, dehydrate and mount (this step is completed by Wuhan Sevier Biological Technology Co., Ltd.).

[0186] 2.7 Crystal structure of ionotropic polypeptide R12Y8

[0187] 2.7.1 Dilute the CLC crystal to a concentration of 0.3 mg / ml

[0188] 2.7.2 Dissolve polypeptide molecule R12Y8-1 in PBS buffer with a molar concentration gradient of 2 mM, 4 mM, 6 mM, 10 mM, and 12 mM;

[0189] 2.7.3 Dissolve polypeptide molecule R12Y8-2 in PBS buffer with a molar concentration gradient of 4 mM, 6 mM, 8 mM, 12 mM, and 16 mM;

[0190] 2.7.4 Dissolve polypeptide molecule R12Y8-3 in PBS buffer with a molar concentration gradient of 2 mM, 4 mM, 5 mM, 8 mM, and 10 mM;

[0191] 2.7.5 Dissolve polypeptide molecule R12Y8-4 in PBS buffer, molar concentration gradient is 4mM, 8mM, 12mM, 16mM;

[0192] 2.7.6 Dissolve polypeptide molecule R12Y8-5 in PBS buffer, molar concentration gradient is 4mM, 8mM, 12mM, 16mM;

[0193] 2.7.7 Mix the polypeptide molecule with CLCs at a volume ratio of 1:1, drop on the crystal plate, observe with optical microscope, and take pictures every certain time interval;

[0194] 2.7.8 Take the same area with PhotoShop, calculate the taken area with ImageJ, and take the first time point 10min for normalization;

[0195] 2.7.9 According to R(t) = k·A(t)·[1-exp(βΔμ(c))], calculate the initial reaction rate R int .

[0196] 3. Experimental results

[0197] The dissolution of CLCs under different pH regulation is shown in Figures 1-3 , and the dissolution of CLCs in acidic or alkaline environment is significant.

[0198] The dissolution of R12Y8 regulating CLCs is shown in Figure 4 , and R12Y8 can effectively dissolve CLCs.

[0199] The ELISA detection results are shown in Figure 5 , and the stimulation of CLCs causes the increase of IL-1β and TNF-α at the protein level, while the R12Y8 polypeptide treatment group can significantly reduce the inflammatory factors in the bronchoalveolar lavage fluid, and has statistical significance.

[0200] The pathological detection results are shown in Figure 6 , and R12Y8 can significantly inhibit the occurrence of lung injury.

[0201] The dissolution of CLCs regulated by different peptide molecules is shown in Figures 7-9 , and the ionic peptide molecules R12Y8-1, R12Y8-2, R12Y8-3, R12Y8-4, R12Y8-5 can regulate the assembly state of CLCs in vitro and effectively dissolve CLCs.

[0202] The above description of the embodiments is only for understanding the method of the present application and its core idea. It should be noted that, for those skilled in the art, without departing from the principles of the present application, some improvements and modifications can be made to the present application, and these improvements and modifications will also fall within the protection scope of the claims of the present application.

Claims

1. A polypeptide, characterized in that, The polypeptide is an ionic peptide consisting of arginine and tyrosine; the amino acid sequence of the polypeptide is shown in any one of SEQ ID NOs: 4-7.

2. A polypeptide derivative, characterized in that, The polypeptide derivative is a modified product of the polypeptide of claim 1.

3. A nucleic acid, characterized in that, The nucleic acid consists of a sequence encoding the polypeptide of claim 1 or the polypeptide derivative of claim 2, or a sequence complementary to the sequence.

4. An expression vector, characterized in that, The expression vector comprises the nucleic acid of claim 3.

5. The expression vector of claim 4, wherein, The expression vector further comprises a regulatory sequence operably linked to the nucleic acid of claim 3.

6. The expression vector of claim 5, wherein, The regulatory sequence allows expression of the polypeptide in a host cell or a cell-free expression system.

7. A host cell or cell-free expression system, characterized in that, The expression vector comprises any one of claims 4-6.

8. The expression system of claim 7, wherein, The cell comprises a prokaryotic cell, a eukaryotic cell.

9. The expression system of claim 8, wherein, The prokaryotic cell comprises a bacterial cell.

10. The expression system of claim 8, wherein, The eukaryotic cell comprises a protist cell, an animal cell, a fungal cell.

11. The expression system of claim 10, wherein, The animal cell comprises a mammalian cell, an avian cell, an insect cell.

12. A pharmaceutical composition, characterized by, The pharmaceutical composition comprises the polypeptide of claim 1, the polypeptide derivative of claim 2, the nucleic acid of claim 3, the expression vector of any one of claims 4-6; and / or the host cell or cell-free expression system of any one of claims 7-11.

13. The pharmaceutical composition of claim 12, wherein, The pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

14. The pharmaceutical composition of claim 12, wherein, The dosage form of the pharmaceutical composition comprises a spray, an aerosol, a powder, an injection, a tablet, an eye drop, or an eye ointment.

15. A kit comprising, The polypeptide of claim 1, the polypeptide derivative of claim 2, the nucleic acid of claim 3, the expression vector of any one of claims 4-6, the host cell or cell-free expression system of any one of claims 7-11, or the pharmaceutical composition of any one of claims 12-14.

16. The kit of claim 15, wherein The kit further comprises instructions for use.

17. An apparatus, comprising: The polypeptide of claim 1, the polypeptide derivative of claim 2, the nucleic acid of claim 3, the expression vector of any one of claims 4-6, the host cell or cell-free expression system of any one of claims 7-11, or the pharmaceutical composition of any one of claims 12-14.

18. The apparatus of claim 17, wherein, The device is a medical device.

19. Any one of the following uses: 1) Use of a polypeptide, a polypeptide derivative, a nucleic acid encoding a polypeptide, an expression vector comprising a nucleic acid encoding a polypeptide, a host cell or a cell-free expression system or a pharmaceutical composition comprising a nucleic acid encoding a polypeptide for the manufacture of a product for the prevention and / or treatment of a disease or disorder associated with the presence or formation of crystals of galectin-10; wherein, The pharmaceutical composition comprises a polypeptide, a polypeptide derivative, a nucleic acid encoding a polypeptide, an expression vector comprising a nucleic acid encoding a polypeptide, or a host cell or cell-free expression system comprising an expression vector comprising a nucleic acid encoding a polypeptide; the polypeptide is an ionic peptide consisting of arginine and tyrosine; the disease or disorder comprises asthma, allergic rhinitis, chronic rhinosinusitis, allergic dermatitis, pneumonia; the polypeptide derivative is a modified product of the polypeptide, the amino acid sequence of the polypeptide is shown in any one of SEQ ID NOs: 2-7; 2) Use of a polypeptide, a polypeptide derivative, a nucleic acid encoding a polypeptide, an expression vector comprising a nucleic acid encoding a polypeptide, a host cell or a cell-free expression system comprising an expression vector encoding a polypeptide, or a pharmaceutical composition comprising a polypeptide, a polypeptide derivative, a nucleic acid encoding a polypeptide, an expression vector comprising a nucleic acid encoding a polypeptide, or a host cell or a cell-free expression system comprising an expression vector encoding a polypeptide in the preparation of a product for inhibiting the formation of or promoting the dissolution of galectin-10 crystals in vitro; wherein the pharmaceutical composition comprises a polypeptide, a polypeptide derivative, a nucleic acid encoding a polypeptide, an expression vector comprising a nucleic acid encoding a polypeptide, or a host cell or a cell-free expression system comprising an expression vector encoding a polypeptide; the polypeptide is an ionic peptide consisting of arginine and tyrosine, the polypeptide derivative is a modified product of the polypeptide, and the amino acid sequence of the polypeptide is shown in any one of SEQ ID NOs: 2-7.

20. A method for inhibiting the formation of galactolectin-10 crystals or promoting the dissolution of galactolectin-10 crystals in vitro, characterized in that, The application also provides a method for inhibiting the formation of or promoting the dissolution of galectin-10 crystals, comprising administering a polypeptide, a polypeptide derivative, or a pharmaceutical composition comprising a polypeptide, a polypeptide derivative; the polypeptide is an ionic peptide consisting of arginine and tyrosine, the polypeptide derivative is a modified product of the polypeptide, and the amino acid sequence of the polypeptide is shown in any one of SEQ ID NOs: 2-7.

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