WO2021238302A1 - 一种白细胞介素29突变体蛋白 - Google Patents

一种白细胞介素29突变体蛋白 Download PDF

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WO2021238302A1
WO2021238302A1 PCT/CN2021/075907 CN2021075907W WO2021238302A1 WO 2021238302 A1 WO2021238302 A1 WO 2021238302A1 CN 2021075907 W CN2021075907 W CN 2021075907W WO 2021238302 A1 WO2021238302 A1 WO 2021238302A1
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protein
seq
pharmaceutical composition
mutant
fusion protein
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French (fr)
Chinese (zh)
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杨柳
李岩
冯静
郭万军
潘海
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Sciwind Biosciences Co Ltd
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Sciwind Biosciences Co Ltd
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Priority to US17/999,884 priority Critical patent/US20230265144A1/en
Priority to AU2021280623A priority patent/AU2021280623B9/en
Priority to EP21812837.9A priority patent/EP4159748A4/en
Priority to JP2022572748A priority patent/JP7741108B2/ja
Priority to CN202180037021.9A priority patent/CN115667291B/zh
Publication of WO2021238302A1 publication Critical patent/WO2021238302A1/zh
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/007—Pulmonary tract; Aromatherapy
    • A61K9/0073—Sprays or powders for inhalation; Aerolised or nebulised preparations generated by other means than thermal energy
    • A61K9/0078—Sprays or powders for inhalation; Aerolised or nebulised preparations generated by other means than thermal energy for inhalation via a nebulizer such as a jet nebulizer, ultrasonic nebulizer, e.g. in the form of aqueous drug solutions or dispersions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • 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
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • This application relates to the field of polypeptide prevention and/or treatment. Specifically, this application relates to an interleukin 29 (IL29) mutant protein, and fusion proteins, conjugates and compositions containing the mutant protein to improve the body Antiviral ability, regulate the body's immune function.
  • IL29 interleukin 29
  • Interferon is an important family of cytokines with broad-spectrum antiviral and immunomodulatory effects. So far, seven ( ⁇ , ⁇ , ⁇ , ⁇ , ⁇ , ⁇ , ⁇ ) forms of interferon have been identified, which are divided into three major groups: type I, type II, and type III.
  • type I interferons include interferon alpha, interferon beta, interferon omega, interferon delta, and interferon tau.
  • interferon gamma is the only type II interferon.
  • Type III interferon is a recently discovered family of cytokines, including interferons ⁇ 1, ⁇ 2 and ⁇ 3, also known as IL-28A, IL-28B and IL-29.
  • IL-28A, IL-28B and IL-29 have sequence homology with type I interferon, and have gene sequence homology with IL-10.
  • IL-28 and IL-29 are similar to type I interferons, both of which can induce antiviral effects of cells. Unlike type I interferons, they do not show anti-proliferation against certain B cell lines. active.
  • the wild-type IL-29 (Interferon ⁇ 1, abbreviated IFN- ⁇ 1) gene encodes a 200 amino acid protein, as shown in SEQ ID NO: 3. Among them, 1-19 amino acids in the sequence are signal peptide sequences, and the mature amino acid sequence of the protein is 181 amino acids, as shown in SEQ ID NO: 2.
  • the IL-29 molecule is composed of six protein helices A-F, among which helices A, C, D and F form a classic up-up-down-down four-helix bundle. IL-29 initiates downstream signaling pathways by interacting with its receptor complex, which is composed of IFN- ⁇ R1 and IL-10R2. It is worth noting that IFN- ⁇ Rl is unique to the IFN- ⁇ signal pathway.
  • IFN- ⁇ 1 and IFN- ⁇ R1 specifically bind to form an IFN- ⁇ 1/IFN- ⁇ R1 complex, wherein the amino acid residues of the active center of IFN- ⁇ 1 and IFN- ⁇ R1 are Pro25, Leu28, lys32, Arg35, Asp36, Glu39, respectively , Trp47, Phe152, Phe155, Arg156, Arg160.
  • type I interferon type II interferon or type III interferon
  • a protein drug due to factors such as poor stability, low activity, and short half-life in the body, its use in clinical treatment is greatly restricted. Therefore, it is desirable to obtain recombinant interferon protein drugs that are more stable and have higher specific activity through genetic engineering technology.
  • a nebulizer is required to atomize the drug solution into tiny particles, and inhale the respiratory tract and lungs to deposit the drug in the respiratory tract and lungs. This situation is for the stability of the drug Sex and activity requirements are higher, so people are more eager to obtain recombinant IL29 protein with higher stability and better activity.
  • Genetic engineering technology can change one or several amino acids in the wild protein sequence to obtain a relatively more stable and higher specific activity recombinant protein, which can solve the instability problem caused by protein drugs to a certain extent. But at the same time, the mutation of one or several amino acids may affect the folding and spatial structure of the protein, thereby affecting the activity of the protein. Therefore, whether it is possible to obtain IL29 mutant proteins with higher stability and better activity is a technical problem.
  • the present application provides an IL29 mutant protein with higher stability, better activity, fewer adverse reactions, and can be used for aerosol inhalation therapy.
  • interleukin 29 mutant protein, comprising a substitution mutation of the 161st or 162th amino acid in the amino acid sequence shown in SEQ ID NO:1, wherein the asparagus at the 161st position The amino acid (D) or glycine (G) at position 162 is replaced by other natural amino acids.
  • the interleukin 29 (IL29) mutant protein of the present application includes the aspartic acid (D) at position 161 in the amino acid sequence shown in SEQ ID NO: 1 which is replaced by glutamic acid, threonine or Serine substitution, or glycine (G) at position 162 is substituted by an aliphatic amino acid.
  • the interleukin 29 (IL29) mutant protein of the present application further includes the substitution of amino acid 165 from cysteine (C) to serine (S) in the amino acid sequence shown in SEQ ID NO: 1. mutation.
  • amino acid sequence of the full-length wild-type IL29 protein containing the signal peptide is shown in SEQ ID NO: 3, consisting of 200 amino acids, of which amino acids 1-19 are the signal peptide, and amino acids 20-200 (181aa )
  • the mature protein that constitutes IL29 is shown in SEQ ID NO: 2.
  • the interleukin 29 (IL29) mutant protein of the present application was designed starting at position 26 from the N-terminus of the wild-type protein shown in SEQ ID NO: 3.
  • the interleukin 29 (IL29) mutant protein of the present application includes an amino acid substitution mutation at position 161 or 162 of the amino acid sequence shown in SEQ ID NO:1.
  • the present application provides an interleukin 29 (IL29) mutant protein comprising a substitution mutation of the 161st or 162th amino acid in the amino acid sequence shown in SEQ ID NO: 1, wherein The aspartic acid (D) at position 161 or glycine (G) at position 162 is replaced by other natural amino acids, for example, by an amino acid selected from the group consisting of glycine, alanine, valine, leucine, Isoleucine, methionine (methionine), proline, tryptophan, serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, day Partic acid, glutamic acid, lysine, arginine or histidine.
  • IL29 interleukin 29
  • the interleukin 29 (IL29) mutant protein comprises a substitution of the 161st or 162th amino acid in the amino acid sequence shown in SEQ ID NO:1 A mutation, wherein the aspartic acid (D) at position 161 or glycine (G) at position 162 is replaced by other natural amino acids, and the starting methionine (M) is also included.
  • the aspartic acid (D) at position 161 or glycine (G) at position 162 is replaced by other natural amino acids, and the starting methionine (M) is also included.
  • the IL-29 mutant protein of the present application includes a substitution mutation of the 161st or 162th amino acid in the amino acid sequence shown in SEQ ID NO:1, that is, the mutation shown in SEQ ID NO:1 Counting from the N-terminus or amino-terminus of the protein, the mutation at position 161 or 162, for example, aspartic acid (D) at position 161 or glycine (G) at position 162 is replaced by other natural amino acids replace.
  • the IL-29 mutant protein of the present application is a mutant protein expressed in prokaryotic cells (such as E.
  • the IL-29 mutant protein of the present application is shown in SEQ ID NO: 4-9.
  • the interleukin 29 (IL29) mutant protein comprises a substitution of the 161st or 162th amino acid in the amino acid sequence shown in SEQ ID NO:1 Mutations, for example, where the aspartic acid (D) at position 161 is replaced by glutamic acid, threonine or serine, or the glycine (G) at position 162 is replaced by an aliphatic amino acid.
  • the IL-29 mutant protein of the present application is a mutant protein expressed in prokaryotic cells (such as E.
  • the interleukin 29 (IL29) mutant protein comprises or consists of the following amino acid sequence: SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8.
  • the interleukin 29 (IL29) mutant protein further comprises a cysteine residue at position 165 on the amino acid sequence shown in SEQ ID NO:1.
  • C Substitution mutation to serine (S).
  • S substitution mutation to serine
  • the interleukin 29 (IL29) mutant protein comprises or consists of the following amino acid sequence: SEQ ID NO: 5, SEQ ID NO: 7 or SEQ ID NO: 9.
  • the present application provides the interleukin 29 (IL29) mutant protein (IL29 DE) shown in the following SEQ ID NO: 4:
  • this application provides 29 (IL29) mutant protein (IL29 DE+CS) shown in the following SEQ ID NO: 5:
  • this application provides the 29 (IL29) mutant protein (IL29 DS) shown in the following SEQ ID NO: 6:
  • this application provides 29 (IL29) mutant protein (IL29 DS+CS) as shown in the following SEQ ID NO: 7:
  • this application provides 29 (IL29) mutant protein (IL29 GA) as shown in the following SEQ ID NO: 8:
  • this application provides 29 (IL29) mutant protein (IL29 GA+CS) as shown in SEQ ID NO: 9:
  • the interleukin 29 (IL29) mutant protein further comprises a short sequence (for example, a short sequence of 6 histidines) to facilitate protein purification , Or a short amino acid sequence with extended half-life.
  • a short sequence for example, a short sequence of 6 histidines
  • the application relates to a fusion protein of interleukin 29 (IL29) mutant protein, which is fused to other polypeptides or proteins at the N-terminus or C-terminus.
  • IL29 interleukin 29
  • it can be fused with human albumin, transferrin, and the Fc part of human IgG molecules to form a fusion protein to increase the half-life of the protein in vivo.
  • the interleukin 29 (IL29) mutant protein can also be fused with other proteins directed at different targets to play a combined preventive and/or therapeutic effect to improve the preventive and/or therapeutic effects of the drug.
  • it can be fused with DAS181 to enhance the activity of the mutant protein of the present invention to prevent and/or treat viruses.
  • DAS181 uses a unique host-oriented method to block respiratory virus infection by cutting off the sialic acid receptors in the human respiratory tract. These receptors bind to most major respiratory viruses and cause infections in patients. DAS181 has shown antiviral activity against four major respiratory viruses, including influenza virus (IFV), parainfluenza virus (PIV), metapneumovirus (MPV) and human enterovirus-68 (EV-68).
  • IAV influenza virus
  • PAV parainfluenza virus
  • MPV metapneumovirus
  • EV-68 human enterovirus-68
  • the application relates to a conjugate of an interleukin 29 (IL29) mutant protein, wherein the protein is conjugated to a polyalkoxy compound.
  • the polyalkoxy compound is for example polyethylene glycol (PEG), such as linear or branched polyethylene glycol, specifically monomethoxy polyethylene glycol propionaldehyde (mPEG propionaldehyde), such as 20kD, 30kD or 40kD MPEG propionaldehyde.
  • PEG polyethylene glycol
  • mPEG propionaldehyde monomethoxy polyethylene glycol propionaldehyde
  • the process of modifying the interleukin 29 mutant protein with PEG to form a conjugate is called PEGylation.
  • the PEGylation of the mutant protein in the present application can be performed by any of the PEGylation methods known in the prior art, such as using an acylation reaction or an alkylation reaction.
  • an acylation reaction or an alkylation reaction By coupling one or more PEGs that increase the overall size of the protein, the therapeutic half-life of the protein can be artificially increased to avoid rapid degradation in the body.
  • this application relates to a polynucleotide encoding any of the above-mentioned proteins or fusion proteins.
  • the application relates to a vector comprising the aforementioned polynucleotide.
  • the polynucleotide encoding the interleukin 29 (IL29) mutant protein is inserted into a suitable expression vector, so that the polynucleotide can be operably linked to the multiple cloning site to express the corresponding protein.
  • the vector can be a pET series vector such as pET-3a, pET-9a, pET-11a, pET-14b, pET-15b, pET-16b, 17b, 19b, 20b, 21a, 22b, 23a(+), 24a , 25b(+), 26b(+), 27b(+), 28a(+), 29a(+), 30a(+), 31b(+), 32a(+), 39b(+), 40b(+) , 41a(+), 42a(+), 43.1a(+), 44b, 45b, 47b, 48b, 49b(+), 50b(+), 51b(+), 52b(+); pMal series vectors such as pMal -c2X, pMal-c5X; pGEX series vectors such as pGEX-6p-1, pGEX-6P-2; pRSET series vectors such as pRSET A, B and C; pTricHis series vectors
  • the application relates to a host cell comprising the aforementioned polynucleotide or vector.
  • the host cell is used to express the IL29 mutant protein of the present invention.
  • a host cell refers to a recipient cell that receives a foreign gene through, for example, transformation or transduction.
  • Common host cells include prokaryotic receptor cells and eukaryotic receptor cells. This application preferably uses prokaryotic receptor cells as its host cells. Wherein, the host cells are E.
  • the host cell is E. coli competent cell BL21 (DE3).
  • the application relates to a pharmaceutical composition
  • a pharmaceutical composition comprising the aforementioned IL29 mutant protein, its fusion protein or its conjugate and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition can be in the form of injections, tablets, capsules, inhalants, suppositories and the like.
  • the pharmaceutical composition is an inhalant, such as a dry powder inhaler or a liquid inhalant, such as aerosol inhaler, aerosol, soft mist and spray, etc., through an inhalation device, such as an aerosol inhaler, a metered dose Inhalation device, dry powder inhalation device for drug delivery.
  • the pharmaceutical composition comprising IL29 mutant protein, fusion protein or conjugate of the present application further comprises a carrier suitable for pulmonary administration for the prevention and/or treatment of various respiratory viral infections.
  • the pharmaceutical composition containing IL29 mutant protein, fusion protein or conjugate of the present application also contains a carrier suitable for pulmonary administration for the prevention and/or treatment of diseases caused by neocoronavirus infection .
  • the application relates to a method for preparing a mutant protein of interleukin 29 (IL29), which includes: introducing a nucleic acid (polynucleotide) encoding the mutant protein of interleukin 29 (IL29) into a host cell to make
  • the host cell expresses the interleukin 29 (IL29) mutant protein, for example, inserts the nucleic acid into an expression vector; transfers the vector into a host cell (such as Escherichia coli) to obtain the corresponding expression of the mutant protein
  • the host cell engineered bacteria
  • the host cell (engineered bacteria) is cultured (for example, fermentation), the host cell (engineered bacteria) is induced to express the mutant protein; the mutant protein is harvested.
  • the above method further includes purifying and/or renaturating the harvested mutein.
  • Figure 1 and Figure 2 are SDS-PAGE electrophoresis diagrams of the IL29 mutant of Example 2 (from left to right, in Figure 1, lanes 1 to 4 are IL29 reference substance, IL29DE, IL29DS, and IL29GA in sequence; in Figure 2, lane 1 ⁇ 4 is IL29CS, IL29DE+CS, IL29DS+CS, IL29GA+CS).
  • Figures 3 to 10 are the stability reversed-phase high performance liquid phase purity maps of IL29 reference substance, IL29DE, IL29DS, IL29GA, IL29CS, IL29DE+CS, IL29DS+CS, IL29GA+CS (where the high content of the main peak represents 0 Point measurement curve, the lower main peak content represents the 14-day curve).
  • Figure 11 is a diagram of the aerosol collection device in the spray stability experiment of IL29 mutant.
  • Figure 12 shows the in vivo efficacy results of ribavirin and IL29 mutants under different dosing schedules on RSV infection mouse models.
  • Figure 13 shows the in vivo efficacy results of ribavirin and IL29 mutants under different dosing schedules on RSV infected cotton rat models.
  • Figure 14 is a graph showing changes in body weight of mice infected with influenza virus after treatment.
  • Figure 15 is a graph showing changes in survival rate of influenza virus-infected mice after treatment.
  • Figure 16 is a diagram of pathological changes of mouse lung bronchi and pulmonary arterioles injury, where A: group 1; B: group 2; C: group 3; D: group 4; E: group 5.
  • Figure 17 is a diagram of pathological changes of mouse alveolar injury, where A: group 1; B: group 2; C: group 3; D: group 4; E: group 5.
  • Figure 18a-c is a statistical diagram of the pathological damage scores of various parts of the lungs of mice. Compared with group 1, ***P ⁇ 0.001; compared with group 2, #P ⁇ 0.05, ##P ⁇ 0.01, ### P ⁇ 0.001.
  • Figure 19 shows the toxic effects of IL29 mutants at different concentrations on Vero cells.
  • Figure 20 shows the inhibition levels of the new coronavirus in the prevention group, treatment group, and positive control group.
  • Figure 21 shows the inhibitory effects of different concentrations of test drugs in the prevention group on Vero cells infected by the new coronavirus.
  • Figure 22 shows the inhibitory effects of different concentrations of test drugs in the treatment group on Vero cells infected by the new coronavirus.
  • Figure 23 shows the inhibitory effect of different concentrations of Radixivir on Vero cells infected by the new coronavirus.
  • This application provides an interleukin 29 (IL29) mutant protein, a fusion protein or conjugate containing the mutant protein, a method for preparing the mutant protein, and the mutant protein, fusion protein, Use of the conjugate or pharmaceutical composition in the prevention and/or treatment of viral infections, tumor diseases and respiratory distress syndrome.
  • the inventors screened a number of mutation sites, and finally found that the IL29 mutant protein provided by the application has a mutation of D at position 161 of the inactive center site on the amino acid sequence shown in SEQ ID NO: 1 into E or prokaryotic cells (e.g. E.
  • the resulting IL29 mutant protein activity was unexpectedly increased by about three times compared with wild-type IL29, and It is more stable and solves the problems of low activity, poor stability, and serious adverse reactions of existing antiviral protein drugs.
  • vector refers to a linear or circular DNA molecule that contains a fragment encoding a polypeptide of interest, wherein the fragment is operably linked to an additional fragment that provides for its transcription.
  • additional fragments include promoter and terminator sequences, and may also include one or more replication origins, one or more selectable markers, enhancers, polyadenylation signals, and the like.
  • Expression vectors are generally derived from plasmid or viral DNA, or may contain elements of both.
  • Polynucleotide refers to a single-stranded or double-stranded polymer of deoxyribonucleotides or ribonucleotide bases read from the 5'to the 3'end.
  • Polynucleotides include RNA and DNA, and can be isolated from natural sources, synthesized in vitro, or prepared by combining natural and synthetic molecules.
  • the size of a polynucleotide is expressed in base pairs (abbreviated "bp"), nucleotides ("nt"), or kilobases ("kb"). Where the context permits, the latter two terms can describe single-stranded or double-stranded polynucleotides.
  • bp base pairs
  • nt nucleotides
  • kb kilobases
  • Polypeptide refers to a polymer of amino acid residues connected by peptide bonds, which can be produced naturally or synthetically.
  • polypeptide, protein, and protein can be used interchangeably.
  • mutant protein refers to a wild-type protein amino acid sequence changed, for example, a protein obtained by mutating the wild-type protein amino acid sequence through genetic engineering methods.
  • mutant protein refers to a wild-type protein amino acid sequence changed, for example, a protein obtained by mutating the wild-type protein amino acid sequence through genetic engineering methods.
  • mutant protein refers to a wild-type protein amino acid sequence changed, for example, a protein obtained by mutating the wild-type protein amino acid sequence through genetic engineering methods.
  • mutant protein “mutant protein” or “mutant” have the same meaning and can be used interchangeably.
  • “Pharmaceutically acceptable” or “pharmacologically compatible” refers to materials that are not biologically or otherwise undesirable.
  • the material can be incorporated into a pharmaceutical composition administered to a patient without causing significant Adverse biological reactions or interactions with any other components contained in the composition in a harmful way.
  • the pharmaceutically acceptable carrier or excipient preferably meets the required standards for toxicology or manufacturing testing and/or is included in the inactive ingredient guide prepared by the U.S. Food and Drug Administration.
  • a method for preventing and/or treating a viral infection in a subject comprising providing the subject with a therapeutically effective amount of the interleukin 29 (IL29) mutant protein of the present invention.
  • IL29 interleukin 29
  • a method for preventing and/or treating tumors which includes providing a subject with a therapeutic amount of the interleukin 29 (IL29) mutant protein of the present invention.
  • IL29 interleukin 29
  • a method for preventing and/or treating or ameliorating respiratory distress syndrome in a subject comprises providing the subject with an effective amount of the interleukin 29 (IL29) mutant of the present invention protein.
  • IL29 interleukin 29
  • a method for preventing and/or treating diseases caused by neocoronavirus infection including providing a subject with a therapeutically effective amount of the interleukin 29 (IL29) mutant protein of the present invention .
  • IL29 interleukin 29
  • treatment refers to obtaining a desired pharmacological and/or physiological effect.
  • the effect may be to completely or partially prevent the occurrence and onset of the disease or its symptoms, partially or completely reduce the disease and/or its symptoms, and/or partially or completely cure the disease and/or its symptoms, including: (a) prevention of disease Occurs or attacks in a subject, who may have the cause of the disease, but has not yet been diagnosed as having the disease; (b) inhibit the disease, that is, block its formation; and (c) reduce the disease and/or its symptoms , That is, causing the disease and/or its symptoms to subside or disappear.
  • subject in this application refers to mammals, including but not limited to mice (rats, mice), non-human primates, humans, dogs, cats, ungulates (such as horses, cows, sheep, pigs) , Goat) etc.
  • a “therapeutically effective amount” or “effective amount” refers to an amount sufficient to achieve the prevention and/or treatment of the disease when administered to a mammal or other subject to treat the disease.
  • the “therapeutically effective amount” will vary depending on the drug used, the severity of the disease and/or symptoms of the subject to be treated, age, weight, and the like. Those skilled in the art can easily determine the appropriateness of the protein or composition of the present invention according to various parameters, especially according to the age, weight and condition of the subject to be treated, the severity of the disease or condition, and the route of administration.
  • the therapeutically effective amount and frequency of administration include, but are not limited to, enteral, topical, suppository, inhalation and parenteral administration, such as subcutaneous, intramuscular or intravenous injection.
  • IL29 interleukin 29
  • the cytopathic method refers to the use of human retinal pigment epithelial cells ARPE-19, the use of vesicular stomatitis virus (VSV) for infection, the colorimetric method of crystal violet staining to evaluate the degree of protection of cells by IL29, and then to evaluate the activity of IL29 (Kotenko Sergei V, Gallagher Grant, Baurin Vitaliy V et al. IFN-lambdas mediate antiviral protection through a distinct class II cytokine receptor complex. [J]. Nat. Immunol., 2003, 4:69-77.).
  • VSV vesicular stomatitis virus
  • the reporter gene method is to link the ISRE promoter to alkaline phosphatase cDNA and transfect it into HEK293 cells. After IL29 stimulation, the IL29 activity is evaluated by measuring alkaline phosphatase in the supernatant of HEK293 blood cells. (LaFleur D W, Nardelli B, Tsareva T et al. Interferon-kappa, a novel type I interferon expressed in human keratinocytes. [J]. J. Biol. Chem., 2001, 276: 39765-71.).
  • interleukin 29 interleukin 29
  • reversed-phase high performance liquid chromatography can be used to analyze impurities with different hydrophobicity and polarity; ion-exchange high performance liquid chromatography can be used to separate impurities. Impurities with large differences in charge; while molecular sieve exclusion chromatography is used to analyze dimers, polymers, and monomers.
  • Each detection method focuses on different points, but can be used to characterize the purity of the protein and know the stability of the protein.
  • the IL29 mutant protein gene fragment SEQ ID NO: 11-17 was obtained by chemical synthesis, and the fragment was inserted into the prokaryotic expression plasmid pET-30a(+) (Novagen) through the Node I and Xho I sites and sequenced for verification.
  • the resulting expression plasmid for transformation assay Transform the plasmid containing the target gene obtained above into E. coli BL21(DE3) competent cells (Invitrogen), place 50 ⁇ l of BL21 competent cells on an ice bath to melt, add the plasmid, shake gently, and place in the ice bath 30 minutes. Then heat shock in a 42°C water bath for 30 seconds, then quickly transfer the centrifuge tube to an ice bath and leave it for 2 minutes.
  • the lysed protein is slowly added to the refolding solution (100mM Tris-HCl, 0.5M arginine, 0.5% PEG3350 (m:V), 2mM GSH: 0.5mM GSSG, pH 8.5) to make the final protein concentration 0.2mg/ ml, stir at room temperature, overnight.
  • the pore size of the ultrafiltration membrane is 10kDa, equilibrate the ultrafiltration membrane with 20mM phosphate buffer pH 7.0, and then concentrate 1L of supernatant 10 times.
  • the concentrated solution is diluted with 5 times the volume of water for injection to be loaded, Sepharose FF packing column, 50mmol/L Tris-HCl, pH8.5, 0.1mol/L NaCl equilibration, load the sample; then use 50mmol/L Tris-HCl, pH8.5, 0.15mol/L NaCl for elution, collect the elution peak components and finally collect 600ml of sample solution; Sepharose FF packing column, 20mmol/L phosphate, pH7.4, 0.05mol/L NaCl balance, load the sample , Until the detector baseline is stable. Rinse with 20mmol/L phosphate, pH7.4, 0.2mol/L NaCl, and collect the elution peak components.
  • IL29 mutant proteins corresponding to the protein amino acid sequence SEQ ID NO: 4-10, respectively named IL29DE, IL29DE+CS, IL29DS, IL29DS+CS, IL29GA, IL29GA+CS, IL29CS .
  • the IL29 wild-type protein (IL29 reference substance for short) was also synthesized using the same method described above, which corresponds to the protein amino acid sequence SEQ ID NO:1 and has an additional M amino acid at the N-terminus.
  • the injection volume was set according to the sample concentration.
  • the injection volume was 15 ⁇ g-20 ⁇ g; finally, the purity of the main peak of the test substance was calculated by integrating according to the area normalization method.
  • the injection volume is 20 ⁇ g, and the integration is performed according to the area normalization method to calculate the purity of the main peak of the two parallel needles of the test product.
  • the HEK293-ISRE-Luc cells (purchased from China Institute for Food and Drug Control) were grown adherently in complete culture medium. Passage 1:4, 2-3 times a week, and grow in complete culture medium. Take the cultured cells and discard the culture solution, wash once with PBS, digest and collect the cells, and use the assay culture solution (BIBCO) to prepare a cell suspension containing 3.5 ⁇ 10 5 to 4.5 ⁇ 10 5 cells per 1 ml.
  • IL29 reference substance 4.783 100% IL29DE 1.697 282% IL29DS 4.316 90% IL29GA 5.264 91% IL29CS 4.639 103% IL29DE+CS 1.428 335% IL29DS+CS 4.982 96% IL29GA+CS 3.985 120%
  • the double mutant IL29DE+CS is based on the D mutation at position 162 of IL29 plus the C mutation at position 166 to S, and its biological activity is further improved on the basis of the single mutant IL29DE, but the double mutation
  • the biological activities of IL29DS+CS and IL29GA+CS mutants were not significantly improved compared with IL29 control. This result shows that the mutation of D to E, which is not located at position 162 of the active center that binds to the IL29 receptor, has an unexpected effect on improving the biological activity of the mutant protein.
  • Example 3 50°C stability test results of IL29 mutant
  • the stability of the protein in this application is mainly characterized by the purity of reversed-phase high-performance liquid phase.
  • the two test products with the largest decrease in purity are IL29 reference substance and IL29CS respectively.
  • the purity of IL29 reference substance dropped sharply from 85.82% to 34.46% in 14 days, a drop of 51.36%.
  • the purity of IL29CS dropped sharply from 97.03% to 46.20% within 14 days, a drop of up to 50.83%; the two test products with the smallest drop were IL29DE and IL29DE+CS, of which the purity of IL29DE dropped from 92.68% to 77.39% in 14 days.
  • the decrease was only 15.29%, the purity of IL29DE+CS dropped from 97.22% to 86.66% within 14 days, and the decrease was only 10.56%.
  • IL29DE has the best stability in the single-point mutant where the 162 position is mutated from D to E, and the decrease in purity is the smallest within 14 days.
  • the stability of the double-point mutant IL29DE+CS may be lower than IL29DE, and the stability of IL29DE+CS in the present invention is much higher than IL29DE, indicating that the double-point mutant IL29DE+CS has much higher stability than IL29DE.
  • the point simultaneous mutation also has a synergistic effect in improving the stability of the mutant.
  • the biological activities of the mutants were tested, and it was found that the biological activities of the mutants IL29DE and IL29DE+CS were basically unchanged within 14 days.
  • Example 6 In vitro drug efficacy determination of IL29 mutant on RSV-infected human bronchial epithelial cells (HBEC)
  • HBEC human bronchial epithelial cell
  • IL29 DE+CS IL29 DE+CS
  • BMS-433771 provided by Shanghai WuXi AppTec, Respiratory syncytial virus fusion protein inhibitor
  • Respiratory syncytial virus was inoculated at 24h before infection, and at 1h and 24h after infection. Each well was tested with RSV with a titer of 100 TCID50 (half the tissue culture infection dose).
  • RNA extraction kit (Cat. No. 74181, Qiagen) was used to extract RSV RNA from the cells and quantified by RT-qPCR.
  • GraphPad Prism software was used to analyze the compound dose-response curve and calculate the EC 50 value and the average antibacterial rate. The results are shown in Table 9 and Table 10.
  • IL29 DE+CS showed good anti-RSV efficacy in an in vitro model of human bronchial epithelial cells, with an EC 50 of 0.13ng/ml (6.5pM), which is 1/100000 of the control drug, which is far lower than the control medicine.
  • Example 7 In vivo drug efficacy determination of IL29 mutant on RSV infection mouse model
  • mice Female, 6-7 weeks old, 16-18g, SPF grade BALB/c mice (provided by Shanghai WuXi AppTec)
  • RSV Human Respiratory Syncytial Virus A2 (RSV-A2, purchased from BEI Resources, NIAID, NIHBethesda, MD) was inoculated by nasal drops, the inoculation volume was 1.1 ⁇ 105 PFU per animal, and the inoculation volume It is 50 ⁇ L.
  • the animals were administered according to the schedule in Table 11.
  • the method of administration was to spray the lungs of the mice after anesthesia, and then spray the liquid atomization device pre-filled with the drug solution (purchased from Shanghai Yuyan Instrument Co., Ltd.
  • the micro-sprinkler cannula of the company is gently inserted into the appropriate position of the mouse trachea, and the piston of the high-pressure push device of the nebulizer is quickly pressed to atomize a quantitative volume of the drug into the lungs of the mouse (reference Joseph D. Brain, Dwyn E. Knudson, Sergei P. Sorokin, Michael A.
  • the IL29 mutant used in this example is IL29 DE+CS.
  • the positive control drug ribavirin significantly inhibits the replication of RSV in mice, and the expected anti-RSV activity in vivo proves the effectiveness of this model system.
  • Ribavirin was administered 1 hour before virus infection (prevention model), and the test drug IL29 mutant was administered under the set measurement conditions (1 hour before virus infection, 1 hour after virus infection, and after virus infection). 24 hours), can significantly inhibit the replication of RSV virus in mice.
  • the virus titer in the lung tissue of mice in the first administration group 1 hour before vaccination (10 ⁇ g) and 1 hour after vaccination (10 ⁇ g) are both at the lower limit of detection The following shows the excellent anti-RSV efficacy in vivo.
  • the administration group also showed a good anti-RSV effect 24 hours after infection. Compared with the results of the prevention model in which ribavirin was administered 1 hour before infection, the antiviral effect of the treatment model in which the IL29 mutant was administered 1 hour after infection was comparable. This also fully shows that the IL29 mutant has a very good antiviral therapeutic potential.
  • Example 8 In vivo drug efficacy determination of IL29 mutant on cotton rat model of RSV infection
  • mice Sigmodon hispidus cotton rat, half male and half female, 5 weeks old, SPF grade (purchased from Envigo)
  • SPF grade purchased from Envigo
  • mice were anesthetized by intraperitoneal injection of sodium pentobarbital (75 mg/kg), followed by nasal drops
  • Method of inoculation RSV Human Respiratory Syncytial Virus A2 (RSV-A2), purchased from BEI Resources, NIAID, NIHBethesda, MD
  • the inoculation volume is 1.1 ⁇ 10 5 PFU per animal, and the inoculation volume is 50 ⁇ L.
  • the IL29 mutant used in this example is IL29 DE+CS.
  • the virus After a cotton rat is infected with respiratory syncytial virus, the virus will replicate in the bronchi of its lungs, and the viral load of the bronchial lavage fluid is a good reflection of the virus replication in the lungs. Therefore, the IL29 mutant shows very good therapeutic potential against RSV.
  • Example 9 In vitro drug efficacy determination of IL29 mutant against new coronavirus (2019-nCov)
  • Vero E6 cells African green monkey kidney cell line
  • Vero E6 cells Africann green monkey kidney cell line
  • Dilute IL29 DE+CS to 100ng/ml, set 5 multiple wells, add 100 ⁇ l to each well for 24h
  • Example 10 Determination of the efficacy of IL29 mutant on influenza virus in mice
  • Table 15 Scheme for determining the efficacy of IL29 mutant on influenza A virus in mice
  • IL29 DE+CS can significantly prolong the survival period of infected mice and protect the mice 48 hours after infection.
  • Example 11 In vivo efficacy of IL29 DE+CS in the treatment of severe respiratory distress syndrome
  • Table 16 The scheme for determining the in vivo efficacy of dexamethasone and IL29 DE+CS in the treatment of severe respiratory distress syndrome
  • Group 4 is the atomization group, the drug concentration is 1mg/ml, and the atomization is 30min.
  • the animals were given drugs before modeling, and again 12 hours after modeling.
  • Example 12 Toxic effect of IL29 mutant on Vero of African green monkey kidney cells
  • the CCK-8 method was used to detect the toxicity of different concentrations of antibodies to Vero cells.
  • Vero cells Inoculate Vero cells into a 96-well culture plate at a concentration of 5000 cells/well, culture to a cell monolayer, discard the culture medium, wash the cells twice with Hank's solution; use MEM medium to dilute the test drug IL29 mutant IL29 DE +CS and Remdesivir (Remdesivir), the initial concentration is 1000nmol/L (19.6 ⁇ g/mL) and 50 ⁇ mol/L, respectively, with 8 consecutive 3-fold dilutions.
  • Set up 3 multiple holes for each drug concentration add 150 ⁇ L of drug solution to each hole, and set up a control hole for normal growth of Vero cells at 37°C and 5% CO 2 incubator for 48 hours.
  • test drug IL29 DE+CS at a concentration of 1000 nmol/L (19.6 ⁇ g/mL) and below 1000 nmol/L had no obvious toxic effect on Vero cells, and the cell viability was greater than 90%.
  • Example 13 Inhibition of IL29 mutants on the new coronavirus in vitro
  • immunofluorescence and nucleic acid detection kits were used to detect the preventive and therapeutic effects of IL29 mutants at different concentrations on the new coronavirus.
  • this experiment sets up a prevention model group and a treatment model group.
  • Vero cells are pretreated with different concentrations of the test drug IL29 DE+CS in the above experimental group before mixing with the virus for 24h as the in vitro prevention model group, without pretreatment
  • the cells are used as an in vitro treatment model group.
  • Vero cells were seeded on a 48-well culture plate at a concentration of 10,000 cells/well, and cultured to the logarithmic growth phase of the cells. Divided into a prevention group, an experimental group, a positive control group, a negative control group (only virus liquid added) and a simple cell group (no virus added), each sample is set with 3 replicate holes.
  • the cell culture medium in the prevention group was aspirated, and the test drug IL29DE+CS samples of different concentrations were added, 500 ⁇ L per well. The remaining cells were left untreated and cultured in a 37°C, 5% CO 2 incubator for 24 hours. Then, the cell culture medium in the four groups was sucked clean, and the drug-virus mixture prepared above was added correspondingly, and the insufficient part of the negative control group and the simple cell group was filled with culture medium.
  • Pipette 200 ⁇ L of culture supernatant use magnetic bead method nucleic acid extraction kit (MVR01, Shanghai Zhijiang Biological Technology Co., Ltd.) and automatic nucleic acid extractor (EX3600, Shanghai Zhijiang Biological Technology Co., Ltd.) to extract viral nucleic acid, and finally The elution volume is 50 ⁇ L.
  • MVR01 Shanghai Zhijiang Biological Technology Co., Ltd.
  • automatic nucleic acid extractor EX3600, Shanghai Zhijiang Biological Technology Co., Ltd.
  • the elution volume is 50 ⁇ L.
  • Take 5 ⁇ L of nucleic acid extract and use one-step novel coronavirus nucleic acid detection kit fluorescent PCR method, article number Z-RR-0479-02-50, National Machinery Note 20203400057, Shanghai Zhijiang Biotechnology Co., Ltd.
  • the result is the Ct value to express the level of the virus.
  • the experimental results are as follows in Table 17 and Figure 20:
  • the test drug at a concentration of 0.0064ng/mL and 0.0064ng/mL or more in the prevention group has an inhibitory effect on the replication of new coronavirus infection
  • the test drug at a concentration of 4ng/mL and 4ng/mL or more has an inhibitory effect on the virus
  • the inhibitory rate of the drug reached 98% or more; the test drug at a concentration of 0.0064ng/mL and above in the treatment group had a certain inhibitory effect on the replication of new coronavirus infection, but the inhibitory effect was worse than that of the prevention group.
  • the inhibition rate of remdesivir with a concentration of 0.3 ⁇ M and more than 0.3 ⁇ M on the virus reached more than 90%.
  • IC test prophylaxis group 50 0.0196ng / mL
  • IC treated group 50 0.0612ng / mL
  • the positive control IC 50 0.12 ⁇ mol / L.
  • the calculation shows that the safety index SI of the test drug in the prevention group is> 10 6
  • the safety index SI of the treatment group is> 3.2 ⁇ 10 5 , showing good cytotoxicity selectivity.
  • the cell culture supernatant cultured in the above 48-well plate was aspirated and washed with PBS; 200 ⁇ L of ice 80% acetone solution was added to each well and fixed at 4°C for 30 minutes, washed with PBS and then blocked with 200 ⁇ L of 3% BSA in PBS blocking solution at room temperature After washing with PBS for 30 minutes, add 200 ⁇ L of primary antibody (rabbit anti-SARS-CoV2 NP diluted 2000 times) protein serum (Beijing Yiqiao Shenzhou Technology Co., Ltd.), and incubate overnight at 4°C.
  • primary antibody rabbit anti-SARS-CoV2 NP diluted 2000 times
  • protein serum Beijing Yiqiao Shenzhou Technology Co., Ltd.
  • Aspirate the primary antibody wash with PBS, add 200 ⁇ L FITC-labeled goat anti-rabbit IgG secondary antibody (Jackson) diluted 1500 times, and incubate for 2 hours at room temperature in the dark.
  • Aspirate the secondary antibody wash with PBS, add 200 ⁇ L of DAPI diluted 1:500 with PBS, and incubate for 5min at room temperature in the dark.
  • the immunofluorescence results are consistent with the nucleic acid test results.
  • the test drugs at concentrations of 0.032ng/mL and 0.032ng/mL in the prevention group have a certain inhibitory effect on new coronavirus infection in vitro, of which 4ng/mL and above 4ng/mL
  • the tested drug has a significant inhibitory effect on viral infections.
  • the test drug at a concentration of 0.032ng/mL and above 0.032ng/mL had a certain inhibitory effect on the new coronavirus infection in vitro, but the inhibitory effect was lower than that of the prevention group.
  • Radixivir with a concentration of 0.3 ⁇ M and more than 0.3 ⁇ M has a significant inhibitory effect on virus infection in vitro. The specific results are shown in Figure 21, Figure 22 and Figure 23.

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