An interleukin-29 mutant protein

Abstract

Provided are an interleukin-29 (IL29) mutant protein, a fusion protein comprising the mutant protein, a conjugate, a pharmaceutical composition, a product, and a kit. The provided IL29 mutant protein has good activity and stability and can effectively prevent and / or treat viral infections, tumors, and other diseases requiring immune function regulation.

Description

Technical Field

[0001] The present application relates to the field of polypeptide prevention and / or treatment. Specifically, the present application relates to an interleukin-29 (IL29) mutant protein, a fusion protein, a conjugate and a composition comprising the mutant protein, which are used to improve the body's antiviral ability and regulate the body's immune function. Background Art

[0002] Interferon is an important family of cytokines with broad-spectrum antiviral and immunomodulatory effects. To date, seven forms of interferon have been identified (α, β, ω, δ, τ, γ, λ), which are divided into three major groups: type I, type II, and type III. The so-called "type I" interferons include interferon α, interferon β, interferon ω, interferon δ, and interferon τ. Currently, interferon γ is the only type II interferon. Type III interferons are a recently discovered family of cytokines, including interferon λ1, λ2, and λ3, also known as IL-28A, IL-28B, and IL-29.

[0003] IL-28A, IL-28B, and IL-29 share sequence homology with type I interferons and genetic sequence homology with IL-10. Functionally, IL-28 and IL-29 are similar to type I interferons in that they can induce antiviral responses in cells. However, unlike type I interferons, they do not exhibit antiproliferative activity against certain B cell lines.

[0004] The wild-type IL-29 (interferon λ1, abbreviated as IFN-λ1) gene encodes a 200-amino acid protein, as shown in SEQ ID NO: 3. Amino acids 1-19 of this sequence constitute a signal peptide sequence, and the mature amino acid sequence of the protein consists of 181 amino acids, as shown in SEQ ID NO: 2. The IL-29 molecule is composed of six protein helices, A to F, with helices A, C, D, and F forming 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-λR1 and IL-10R2. Notably, IFN-λR1 is unique to the IFN-λ signaling pathway. IFN-λ1 specifically binds to IFN-λR1 to form an IFN-λ1 / IFN-λR1 complex, in which the amino acid residues in the active center of IFN-λ1 binding to IFN-λR1 are Pro25, Leu28, lys32, Arg35, Asp36, Glu39, Trp47, Phe152, Phe155, Arg156, and Arg160.

[0005] Whether type I, type II, or type III interferons, as protein drugs, their use in clinical treatment is significantly limited due to factors such as poor stability, low activity, and a short in vivo half-life. Therefore, the use of genetic engineering techniques to obtain more stable and more specifically active recombinant interferon protein drugs is highly desired. In particular, when using nebulized inhalation therapy to prevent and / or treat respiratory diseases, a nebulizer is required to atomize the drug solution into tiny particles, which are then inhaled into the respiratory tract and lungs for deposition. This situation places even higher demands on drug stability and activity, leading to a desire for recombinant IL29 proteins with greater stability and activity. Genetic engineering techniques can, to a certain extent, address the instability issues associated with protein drugs by altering one or more amino acids in the wild-type protein sequence to produce relatively more stable recombinant proteins with higher specific activity. However, these mutations in one or more amino acids can affect the protein's folding and spatial structure, thereby affecting its activity. Therefore, the ability to obtain more stable and more active IL29 mutant proteins remains a technical challenge. Summary of the Invention

[0006] In summary, in order to solve the problems of the prior art, the present application provides an IL29 mutant protein with higher stability, better activity, fewer adverse reactions and can be used for aerosol inhalation therapy.

[0007] On the one hand, the present application provides an interleukin 29 (IL29) mutant protein, comprising a substitution mutation of the amino acid at position 161 or 162 in the amino acid sequence shown in SEQ ID NO: 1, wherein the aspartic acid (D) at position 161 or the glycine (G) at position 162 is substituted by other natural amino acids.

[0008] In some embodiments, the interleukin 29 (IL29) mutant protein of the present application comprises that the aspartic acid (D) at position 161 of the amino acid sequence shown in SEQ ID NO: 1 is replaced by glutamic acid, threonine or serine, or the glycine (G) at position 162 is replaced by an aliphatic amino acid.

[0009] In some embodiments, the interleukin 29 (IL29) mutant protein of the present application further comprises a substitution mutation of amino acid 165 from cysteine (C) to serine (S) in the amino acid sequence shown in SEQ ID NO: 1.

[0010] In the present application, the amino acid sequence of the full-length wild-type IL29 protein including the signal peptide is shown in SEQ ID NO: 3, which consists of 200 amino acids, of which amino acids 1-19 are the signal peptide and amino acids 20-200 (181 aa) constitute the mature protein of IL29 (amino acid sequence shown in SEQ ID NO: 2).

[0011] The interleukin 29 (IL29) mutant protein of the present application is designed starting from position 26 starting 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 comprises an amino acid substitution mutation at position 161 or 162 of the amino acid sequence shown in SEQ ID NO: 1.

[0012] In some embodiments of the present application, the present application provides an interleukin 29 (IL29) mutant protein, comprising a substitution mutation of the amino acid at position 161 or 162 on the amino acid sequence shown in SEQ ID NO: 1, wherein the aspartic acid (D) at position 161 or the glycine (G) at position 162 is substituted by other natural amino acids, for example, by an amino acid selected from the following: glycine, alanine, valine, leucine, isoleucine, methionine (methionine), proline, tryptophan, serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine or histidine.

[0013] Those skilled in the art will appreciate that positions 161 and 162 of SEQ ID NO: 1 correspond to the corresponding positions of SEQ ID NO: 2 (wild-type interleukin-29 mature protein) and SEQ ID NO: 3 (wild-type interleukin-29 full-length protein including a signal peptide). Therefore, amino acid mutations at the corresponding positions of SEQ ID NO: 2 or SEQ ID NO: 3 (or amino acid sequences of different lengths derived from SEQ ID NO: 2 or SEQ ID NO: 3) are also encompassed within the scope of protection of this application. The scope of protection of this application also encompasses interleukin-29 mutant proteins derived from SEQ ID NO: 2 or SEQ ID NO: 3 but having amino acid sequences of different lengths that contain substitution mutations at the corresponding positions of positions 161 and 162.

[0014] In some embodiments of the interleukin-29 (IL29) mutant protein described above, the interleukin-29 (IL29) mutant protein comprises a substitution mutation at amino acid position 161 or 162 of the amino acid sequence set forth in SEQ ID NO: 1, wherein the aspartic acid (D) at position 161 or the glycine (G) at position 162 is substituted with another naturally occurring amino acid, and further comprises an initial methionine (M). This is because when IL29 is expressed in prokaryotic cells (e.g., E. coli), the expressed IL29 protein contains a methionine at the N-terminus or amino terminus.

[0015] In some embodiments of the present application, the IL-29 mutant protein of the present application comprises a substitution mutation at amino acid position 161 or 162 of the amino acid sequence set forth in SEQ ID NO: 1, i.e., a mutation at position 161 or 162, counting from the N-terminus or amino-terminus of the protein set forth in SEQ ID NO: 1, such as a substitution of aspartic acid (D) at position 161 or glycine (G) at position 162 with another naturally occurring amino acid. In some embodiments, the IL-29 mutant protein of the present application is a mutant protein expressed in prokaryotic cells (e.g., E. coli) and comprises a substitution mutation at position 162 or 163 (counting from M due to the addition of an N-terminal M), such as a substitution of aspartic acid (D) at position 162 or glycine (G) at position 163 with another naturally occurring amino acid. In a specific embodiment, the IL-29 mutant protein of the present application is as set forth in SEQ ID NOs: 4-9.

[0016] In some embodiments of the interleukin-29 (IL29) mutant protein, the interleukin-29 (IL29) mutant protein comprises a substitution mutation at amino acid position 161 or 162 of the amino acid sequence of SEQ ID NO: 1, for example, wherein the aspartic acid (D) at position 161 is substituted with glutamic acid, threonine, or serine, or the glycine (G) at position 162 is substituted with an aliphatic amino acid. In some embodiments, the IL-29 mutant protein of the present application is a mutant protein expressed in prokaryotic cells (e.g., E. coli) and comprises a substitution mutation at position 162 or 163 (counted from M due to the addition of an N-terminal M), for example, wherein the aspartic acid (D) at position 162 is substituted with glutamic acid, threonine, or serine, or the glycine (G) at position 163 is substituted with an aliphatic amino acid.

[0017] In some embodiments of the above-mentioned interleukin 29 (IL29) mutant protein, 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.

[0018] In some embodiments of the interleukin-29 (IL29) mutant protein, the interleukin-29 (IL29) mutant protein further comprises a cysteine (C) to serine (S) substitution mutation at position 165 of the amino acid sequence set forth in SEQ ID NO: 1. As described above, when the IL29 mutant protein is expressed in prokaryotic cells (e.g., E. coli), the cysteine (C) to serine (S) substitution mutation at position 165 appears at position 166.

[0019] In some embodiments of the above-mentioned interleukin 29 (IL29) mutant protein, 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.

[0020] In some embodiments of the present application, the present application provides an interleukin 29 (IL29) mutant protein (IL29 DE) as shown in the following SEQ ID NO: 4:

[0021]

[0022]

[0023] In some embodiments, the present application provides a 29 (IL29) mutant protein (IL29 DE+CS) as shown in the following SEQ ID NO: 5:

[0024]

[0025] In some embodiments, the present application provides an IL29 (IL29) mutant protein (IL29 DS) as shown in the following SEQ ID NO: 6:

[0026]

[0027] In some embodiments, the present application provides a 29 (IL29) mutant protein (IL29 DS+CS) as shown in the following SEQ ID NO: 7:

[0028]

[0029] In some embodiments, the present application provides an IL29 (IL29) mutant protein (IL29 GA) as shown in the following SEQ ID NO: 8:

[0030]

[0031]

[0032] In some embodiments, the present application provides a 29 (IL29) mutant protein (IL29 GA+CS) as shown in SEQ ID NO: 9:

[0033]

[0034] In some embodiments of the above-mentioned interleukin 29 (IL29) mutant protein, the interleukin 29 (IL29) mutant protein further comprises a short sequence for facilitating protein purification (eg, a short sequence of 6 histidines), or a short amino acid sequence for extending half-life.

[0035] In some embodiments, the present application relates to a fusion protein of an interleukin 29 (IL29) mutant protein, in which the interleukin 29 (IL29) mutant protein is fused to other polypeptides or proteins at the N-terminus or C-terminus. For example, it can be fused with human albumin, transferrin, the Fc portion of a human IgG molecule, etc. to form a fusion protein to increase the half-life of the protein in the body. The interleukin 29 (IL29) mutant protein can also be fused with other proteins targeting different targets to play a combined preventive and / or therapeutic role to improve the preventive and / or therapeutic effects of the drug. For example, it can be fused with DAS181 to enhance the activity of the mutant protein of the present invention in preventing and / or treating viruses. DAS181 uses a unique host-oriented approach to block respiratory viral infection by cutting off sialic acid receptors in the human respiratory tract. These receptors bind to most major respiratory viruses, causing infection 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).

[0036] In some embodiments, the present application relates to conjugates of interleukin 29 (IL29) mutant proteins, 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 monomethoxypolyethylene glycol propionaldehyde (mPEG propionaldehyde), such as 20kD, 30kD or 40kD mPEG propionaldehyde. The process of modifying interleukin 29 mutant proteins with PEG to form conjugates is called PEGylation. The PEGylation of the mutant proteins in the present application can be carried out by any of the PEGylation methods known in the art, such as using an acylation reaction or an alkylation reaction. By conjugating 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.

[0037] In some embodiments, the present application relates to a polynucleotide encoding any one of the above proteins or fusion proteins.

[0038] In some embodiments, the present application relates to a vector comprising the above-mentioned polynucleotide. Briefly, a polynucleotide encoding an interleukin-29 (IL29) mutant protein is inserted into a suitable expression vector such that the polynucleotide is operably linked to a multiple cloning site to express the corresponding protein.

[0039] 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(+), 3 9b(+), 40b(+), 41a(+), 42a(+), 43.1a(+), 44b, 45b, 47b, 48b, 49b(+), 50b(+), 51b(+), 52b(+); pMal series vectors such as pMal-c2X and pMal-c5X; pGEX series vectors such as pGEX-6p-1 and pGEX-6P-2; pRSET series vectors such as pRSET A, B and C; pTricHis series vectors such as pTrcHis A, B and C. Preferably, the vector is pET-30a(+).

[0040] In some embodiments, the present application relates to host cells comprising the above-mentioned polynucleotides or vectors. The host cells are used to express the IL29 mutant proteins of the present invention. A host cell refers to a recipient cell that accepts an exogenous gene through, for example, transformation or transduction. Common host cells include prokaryotic recipient cells and eukaryotic recipient cells. The present application prefers prokaryotic recipient cells as its host cells. The host cells are Escherichia coli competent cells BL21(DE3), Tuner(DE3), Origami(DE3), Rosetta(DE3), JM109(DE3), BL21star(DE3), Rosetta-gami(DE3), Rosetta-gami B(DE3), BL21(DE3)pLysS, or BL21star(DE3)pLysS. Preferably, the host cells are Escherichia coli competent cells BL21(DE3).

[0041] In some embodiments, the present application relates to a pharmaceutical composition comprising the above-mentioned IL29 mutant protein, its fusion protein or conjugate and a pharmaceutically acceptable carrier.

[0042] The pharmaceutical composition may be in the form of an injection, tablet, capsule, inhaler, suppository, etc. Preferably, the pharmaceutical composition is an inhaler, such as a dry powder inhaler or a liquid inhaler, such as a nebulizer inhaler, aerosol, soft mist, and spray, and is administered through an inhalation device, such as a nebulizer inhaler, a metered dose inhaler, or a dry powder inhaler.

[0043] In some embodiments, the pharmaceutical composition comprising an 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.

[0044] In some embodiments, the pharmaceutical composition of the present application comprising an IL29 mutant protein, fusion protein or conjugate further comprises a carrier suitable for pulmonary administration for the prevention and / or treatment of diseases caused by infection with the new coronavirus.

[0045] In some embodiments, the present application relates to a method for preparing an interleukin 29 (IL29) mutant protein, comprising: introducing a nucleic acid (polynucleotide) encoding the above-mentioned interleukin 29 (IL29) mutant protein into a host cell, so that the host cell expresses the interleukin 29 (IL29) mutant protein, for example, inserting the nucleic acid into an expression vector; transferring the vector into a host cell (such as Escherichia coli) to obtain a corresponding host cell (engineered bacteria) expressing the mutant protein; culturing (such as fermentation) the host cell (engineered bacteria) to induce the host cell (engineered bacteria) to express the mutant protein; and harvesting the mutant protein.

[0046] In some embodiments, the above method further comprises purifying the harvested mutant protein and / or renaturing the mutant protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 and Figure 2 The SDS-PAGE electrophoresis diagram of the IL29 mutants in Example 2 (from left to right, Figure 1 In the middle, lanes 1 to 4 are IL29 control, IL29DE, IL29DS, and IL29GA; Figure 2 In the figure, lanes 5 to 8 are IL29CS, IL29DE+CS, IL29DS+CS, and IL29GA+CS, respectively).

[0048] Figures 3 to 10 They are the stability reversed-phase HPLC purity graphs of IL29 control, IL29DE, IL29DS, IL29GA, IL29CS, IL29DE+CS, IL29DS+CS, and IL29GA+CS (the one with high main peak content represents the 0-point determination curve, and the one with low main peak content represents the 14-day curve).

[0049] Figure 11 A diagram of the aerosol collection device used in the aerosol stability experiment of IL29 mutants.

[0050] Figure 12 The results are the in vivo efficacy of ribavirin and IL29 mutants under different dosing regimens in RSV-infected mouse models.

[0051] Figure 13 The results are the in vivo efficacy of ribavirin and IL29 mutants under different dosing regimens in the RSV-infected cotton rat model.

[0052] Figure 14 Figure 2 shows the weight changes of mice infected with influenza virus after treatment.

[0053] Figure 15 This is a graph showing the changes in survival rate of mice infected with influenza virus after treatment.

[0054] Figure 16 These are pictures showing the pathological changes of injury in the small bronchi and pulmonary arterioles of mice, where A: Group 1; B: Group 2; C: Group 3; D: Group 4; E: Group 5.

[0055] Figure 17 These are pictures showing the pathological changes of alveolar damage in mice, where A: Group 1; B: Group 2; C: Group 3; D: Group 4; E: Group 5.

[0056] Figure 18a-c is the statistical graph of pathological injury scores of various parts of the mouse lungs. Compared with group 1, ***P<0.001; compared with group 2, #P<0.05, ##P<0.01, ###P<0.001.

[0057] Figure 19 The cytotoxic effects of different concentrations of IL29 mutants on Vero cells.

[0058] Figure 20 It is the inhibition level of the prevention group, treatment group, and positive control group against the new coronavirus.

[0059] Figure 21 This is the inhibitory effect of different concentrations of test drugs in the prevention group on the infection of Vero cells with the new coronavirus.

[0060] Figure 22 This is the inhibitory effect of different concentrations of the test drugs in the treatment group on the infection of Vero cells with the new coronavirus.

[0061] Figure 23 The figure shows the inhibitory effect of different concentrations of remdesivir on Vero cells infected with the new coronavirus. DETAILED DESCRIPTION

[0062] The present application provides an interleukin-29 (IL29) mutant protein, a fusion protein or conjugate comprising the mutant protein, a method for preparing the mutant protein, and the use of the mutant protein, fusion protein, conjugate or pharmaceutical composition in preventing and / or treating viral infections, tumor diseases and respiratory distress syndrome. The inventors screened multiple mutation sites and finally found that the IL29 mutant protein provided by the present application, after the D at position 161 of the inactive central site on the amino acid sequence shown in SEQ ID NO: 1 was mutated to E, or after the D at position 162 of the mutant protein of SEQ ID NO: 1 obtained by expression in prokaryotic cells (e.g., E. coli) was mutated to E, the activity of the IL29 mutant protein obtained was unexpectedly increased by about three times compared with the wild-type IL29, and it was more stable, thus solving the problems of low activity, poor stability and severe adverse reactions of existing antiviral protein drugs.

[0063] definition

[0064] The term "recombinant expression vector" or "vector" is used to refer to a linear or circular DNA molecule that contains a segment encoding a polypeptide of interest, wherein the segment is operably linked to additional segments that provide for its transcription. These additional segments include promoter and terminator sequences and may also include one or more origins of replication, one or more selectable markers, enhancers, polyadenylation signals, etc. Expression vectors are generally derived from plasmid or viral DNA, or may contain elements of both.

[0065] "Polynucleotide" refers to a single-stranded or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5' to the 3' end. Polynucleotides include RNA and DNA, which 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. Polynucleotide and nucleic acid are used interchangeably in the present invention.

[0066] "Polypeptide" refers to a polymer of amino acid residues linked by peptide bonds, and may be naturally occurring or synthetically produced. In this application, polypeptide, protein, and albumen are used interchangeably.

[0067] A "mutant" protein refers to a protein in which the amino acid sequence of a wild-type protein is altered, for example, a protein obtained by genetically engineering the wild-type protein's amino acid sequence. In this application, "mutant protein," "mutant protein," or "mutant" are synonymous and are used interchangeably.

[0068] "Pharmaceutically acceptable" or "pharmacologically compatible" refers to a material that is not biologically or otherwise undesirable, e.g., a material that can be incorporated into a pharmaceutical composition administered to a patient without causing a significant adverse biological reaction or interacting in a deleterious manner with any other component of the composition. Pharmaceutically acceptable carriers or excipients preferably meet the required standards for toxicology or manufacturing testing and / or are included in the inactive ingredient guide compiled by the U.S. Food and Drug Administration.

[0069] In some embodiments of the present application, a method for preventing and / or treating viral infection in a subject is provided, comprising providing the subject with a therapeutically effective amount of the interleukin 29 (IL29) mutant protein of the present invention.

[0070] In some embodiments of the present application, a method for preventing and / or treating tumors is provided, comprising providing a subject with a therapeutic amount of the interleukin 29 (IL29) mutant protein of the present invention.

[0071] In some embodiments of the present application, a method for preventing and / or treating or improving respiratory distress syndrome in a subject is provided, comprising providing the subject with an effective amount of the interleukin 29 (IL29) mutant protein of the present invention.

[0072] In some embodiments of the present application, a method for preventing and / or treating diseases caused by novel coronavirus infection is provided, comprising providing a therapeutically effective amount of the interleukin 29 (IL29) mutant protein of the present invention to a subject.

[0073] As used herein, the term "treating" refers to obtaining a desired pharmacological and / or physiological effect. The effect may be complete or partial prevention of the occurrence or onset of a disease or its symptoms, partial or complete alleviation of a disease and / or its symptoms, and / or partial or complete cure of a disease and / or its symptoms, including: (a) preventing the occurrence or onset of a disease in a subject who may have a predisposition to the disease but has not yet been diagnosed with the disease; (b) inhibiting the disease, i.e., blocking its development; and (c) alleviating the disease and / or its symptoms, i.e., causing the disease and / or its symptoms to subside or disappear.

[0074] The term "subject" in this application refers to mammals, including but not limited to murines (rats, mice), non-human primates, humans, dogs, cats, ungulates (e.g., horses, cows, sheep, pigs, goats), etc.

[0075] "Therapeutically effective amount" or "effective amount" refers to an amount sufficient to achieve the described prevention and / or treatment of the disease when administered to a mammal or other subject for the treatment of a disease. The "therapeutically effective amount" will vary depending on the drug used, the severity of the disease and / or its symptoms, and the age, weight, etc. of the subject to be treated. A person skilled in the art can readily determine the appropriate therapeutically effective amount and frequency of administration of the protein or composition of the present invention based on various parameters, particularly the age, weight and condition of the subject to be treated, the severity of the disease or condition, and the route of administration. Routes of administration include, but are not limited to, enteral, topical, suppository, inhalation, and parenteral administration, such as subcutaneous, intramuscular, or intravenous injection.

[0076] Common methods for detecting interleukin 29 (IL29) protein activity include cytopathic assay, reporter gene assay, etc.

[0077] The cytopathic effect assay uses human retinal pigment epithelial (ARPE) cells infected with vesicular stomatitis virus (VSV) and uses crystal violet staining to colorimetrically assess the degree of IL-29 protection, thereby evaluating IL-29 activity (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).

[0078] The reporter gene method involves linking the interferon-kappa response element (ISRE) promoter to alkaline phosphatase cDNA and transfecting the cells into HEK293 cells. After IL29 stimulation, IL29 activity is assessed by measuring alkaline phosphatase in the supernatant of HEK293 blood cells (LaFleur DW, Nardelli B, Tsareva T et al. Interferon-kappa, a novel type Iinterferon expressed in human keratinocytes. [J]. J. Biol. Chem., 2001, 276: 39765-71.).

[0079] There are many methods for testing the stability of interleukin-29 (IL29) proteins. For example, reversed-phase HPLC can be used to analyze impurities with different hydrophobicity and polarity; ion-exchange HPLC can be used to separate impurities with large charge differences; and molecular sieve exclusion chromatography can be used to analyze dimers, polymers, and monomers. Each testing method focuses on different aspects, but all can be used to characterize protein purity and thus determine protein stability.

[0080] This is further illustrated by the following non-limiting examples.

[0081] Example

[0082] Example 1: Preparation of IL29 mutant protein

[0083] 1.1 Construction of mutant protein expression engineering bacteria

[0084] IL29 mutant protein gene fragments (SEQ ID NOs: 11-17) were obtained by chemical synthesis. These fragments were inserted into the prokaryotic expression plasmid pET-30a(+) (Novagen) via the NodeI and XhoI sites and verified by sequencing. The resulting expression plasmids were used for transformation assays. The plasmids containing the target gene were transformed into Escherichia coli BL21(DE3) competent cells (Invitrogen). 50 μl of the BL21 competent cells were thawed in an ice bath, the plasmids were added, the cells were gently shaken, and the cells were placed in an ice bath for 30 minutes. The cells were then heat-shocked in a 42°C water bath for 30 seconds, and the centrifuge tubes were quickly transferred to an ice bath and placed for 2 minutes without shaking. 500 μl of sterile LB medium (without antibiotics) was added to the centrifuge tubes, mixed thoroughly, and incubated at 37°C, 180 rpm, for 1 hour to allow the bacteria to recover. 200 μl of the transformed competent cells were aspirated and applied to a plate containing kanamycin-resistant LB agar, and the cells were evenly spread. Place the plate at 37°C until the liquid is absorbed, invert the plate, and incubate overnight at 37°C. The next day, use an inoculation loop to pick a single colony from the transformation plate and inoculate it into 15 ml of sterile LB medium (containing kanamycin) and incubate overnight at 30°C.

[0085] 1.2 Expression and purification of IL29 mutant proteins

[0086] To 50 ml of LB medium, add 50 μl of the above bacterial suspension and 50 μl of kanamycin. Mix thoroughly, incubate overnight in a 30°C shaker. Add 10 ml of the overnight inoculated suspension to 1000 ml of LB medium and add 1000 μl of kanamycin. Shake thoroughly, incubate at 37°C, 200 rpm, and incubate until the OD600 reaches 0.4-0.6. Induce with IPTG at a final concentration of 0.5 mM. Continue incubating for 4 hours, then harvest the cells. The expressed IL29 mutant accounts for approximately 30-50% of the total bacterial protein, primarily as inclusion bodies.

[0087] The fermented cells were washed three times with TE (10 mmol / L Tris-HCl, 1 mmol / L EDTA, pH 6.5) solution (m:V = 1:10), then disrupted by high-pressure homogenization at 60 MPa. After homogenization, the cell disruption rate was examined microscopically. When the cell disruption rate reached approximately 95% (approximately 2-3 cell disruptions), the cells were centrifuged at 8000 rpm for 15 minutes, and the pellet was collected. The pellet was placed in a beaker and added with inclusion body washing solution (10 mM Tris-HCl + 1 mM EDTA + 0.5% Triton-X100, pH 6.5, m:V = 1:10). The mixture was stirred on a magnetic stirrer for 30 minutes and washed three to five times. Inclusion bodies were lysed with inclusion body lysis solution (7 M guanidine hydrochloride + 50 mM Tris-HCl + 10 mM DTT, pH 6.5, m:V = 1:10) and stirred at room temperature overnight. The cleaved protein was slowly added to a renaturation solution (100 mM Tris-HCl, 0.5 M arginine, 0.5% PEG3350 (m:V), 2 mM GSH: 0.5 mM GSSG, pH 8.5) to a final protein concentration of 0.2 mg / ml, and stirred at room temperature overnight.

[0088] The refolded solution was centrifuged at 8000 rpm for 5 minutes, and the supernatant was collected. Using an ultrafiltration membrane with a pore size of 10 kDa, the membrane was equilibrated in 20 mM phosphate buffer, pH 7.0. One liter of supernatant was concentrated 10-fold. The concentrate was diluted with 5 volumes of water for injection before loading. The column was then packed with Sepharose FF medium, equilibrated with 50 mmol / L Tris-HCl, pH 8.5, 0.1 mol / L NaCl, and the sample was loaded. Elution was then performed with 50 mmol / L Tris-HCl, pH 8.5, 0.15 mol / L NaCl, and the peak fractions were collected. Finally, 600 ml of sample solution was collected. The column was then packed with Sepharose FF medium, equilibrated with 20 mmol / L phosphate, pH 7.4, 0.05 mol / L NaCl, and the sample was loaded until the detector baseline stabilized. The column was rinsed with 20 mmol / L phosphate, pH 7.4, 0.2 mol / L NaCl, and the peak fractions were collected.

[0089] Seven IL29 mutant proteins (referred to as IL29 mutants) were obtained, corresponding to the protein amino acid sequences SEQ ID NOs: 4-10, and were named IL29DE, IL29DE+CS, IL29DS, IL29DS+CS, IL29GA, IL29GA+CS, and IL29CS, respectively.

[0090] At the same time, the IL29 wild-type protein (referred to as IL29 control) was also synthesized using the same method as above, corresponding to the protein amino acid sequence SEQ ID NO: 1 and having an additional M amino acid at the N-terminus.

[0091] Example 2 Detection of various indicators of the obtained IL29 mutants

[0092] 2.1 SDS-PAGE electrophoresis to determine the molecular weight and purity of IL29 mutants:

[0093] Using SDS-PAGE electrophoresis loading buffer, in the presence of mercaptoethanol, the marker and 10 μg of the protein obtained above were loaded and electrophoresed at a constant voltage of 200 V for 45 minutes. The protein was stained with Coomassie Brilliant Blue G-25 to detect its molecular weight and purity. Figure 1 and 2 shown.

[0094] Depend on Figure 1 and 2 It can be seen that the molecular weights of the IL29 mutant protein and the control are 20 kDa respectively, indicating that the target protein obtained is correct, with only one band and no other impurities, and the purity can reach 100%.

[0095] 2.2 In vitro reversed-phase HPLC purity of IL29 mutants using chromatography

[0096] 2.2.1 Reversed-phase liquid chromatography

[0097] Prepare mobile phase A: acetonitrile: water: trifluoroacetic acid in a volume ratio of 20:80:0.1, and mobile phase B: acetonitrile: water: isopropanol: trifluoroacetic acid in a volume ratio of 70:20:10:0.1. Analyze each protein sample using an XBridge BEH C18 column (130A, 5 μm, 4.6 mm x 100 mm). The analysis conditions are: flow rate 1.0 ml / min, acquisition time 65 min, acquisition wavelength 214 nm, column temperature 45°C, and elution gradient as shown in Table 1:

[0098] Table 1

[0099] Time (min) 0 5 5.01 45 55 55.01 65 B％ 27 27 27 49 70 27 27

[0100] Two injections of the test sample solution were injected in parallel, and the injection volume was set according to the sample concentration, with the injection volume being 15 μg to 20 μg. Finally, the purity of the main peak of the test sample was calculated by integrating according to the area normalization method.

[0101] 2.2.2 Ion exchange chromatography

[0102] Prepare mobile phase A: 25 mmol / L phosphate buffer (pH 7.0); mobile phase B: 25 mmol / L phosphate buffer, 0.5 mol / L sodium chloride (pH 6.7). Analyze each protein sample using a Thermo ProPac WCX-10 4.0*250 mm column. The analysis conditions were a flow rate of 0.8 ml / min, a collection time of 55 min, a collection wavelength of 214 nm, a column temperature of 25°C, and an elution gradient as shown in Table 2 below:

[0103] Table 2

[0104] Time (min) 0 30 31 40 41 55 A％ 80 70 10 10 80 80 B％ 200 30 90 90 20 20

[0105] The injection volume was 20 μg, and the purity of the main peak of two parallel injections of the test sample was calculated according to the area normalization method.

[0106] 2.3 Determination of in vitro cellular biological activity of IL29 mutants using reporter gene assay

[0107] HEK293-ISRE-Luc cells (purchased from the China Food and Drug Administration) were grown in complete culture medium. The cells were passaged at a ratio of 1:4 and grown 2-3 times per week in complete culture medium. The culture medium was discarded, the cells were washed once with PBS, and then digested and collected. The cells were then prepared with assay medium (BIBCO) to contain 3.5×10 5 ~4.5×10 5 Prepare a cell suspension of 100 cells. Transfer the prepared IL29 mutant protein and IL29 control into a 96-well plate that can be used for cell culture and chemiluminescence microplate reader (Molecular Devices) reading, add 100 μl to each well, and then inoculate the above cell suspension into the same 96-well plate, 100 μl per well. Culture at 37°C and 5% carbon dioxide for 19 to 23 hours. Carefully aspirate the supernatant in the 96-well plate, add cell lysate and luciferase substrate according to the instructions of the luciferase assay kit (Bright-GloTM Luciferase Assay System, Promega), and measure with a chemiluminescence microplate reader, and record the EC values of the IL29 mutant and IL29 control, respectively. 50 The relative biological activity was calculated as follows: IL29 control was used as the standard, its activity at 0 point was defined as "100%", and the relative biological activity = IL29 control EC50 (0 point) / IL29 mutant EC50.

[0108] The EC50 of each test product of IL29 mutants was determined, and the relative biological activity of each mutant was calculated using the IL29 control as a reference. The specific results are shown in Table 3 below:

[0109] Table 3 In vitro cell biological activity data of IL29 mutants

[0110] Protein type <![CDATA[EC 50 (ng / ml)]]> Relative biological activity (%) IL29 control 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％

[0111] As shown above, although the D at position 162 of IL29 is not located in the active site for IL29 receptor binding, the experiments unexpectedly found that the single mutation IL29DE had an EC50 of only 1.697, far lower than the 4.783 of the IL29 control, and surprisingly increased its biological activity by nearly threefold. The other single mutation mutants, IL29DS, IL29GA, and IL29CS, showed essentially no difference in EC50 / biological activity compared to the IL29 control. The double mutation IL29DE+CS, which incorporates a D mutation at position 162 and a C-to-S mutation at position 166, further improved its biological activity compared to the single mutation IL29DE. However, the double mutations IL29DS+CS and IL29GA+CS showed no significant improvement in biological activity compared to the IL29 control. These results suggest that the D-to-E mutation at position 162, which is not located in the active site for IL29 receptor binding, has an unexpected effect on enhancing the biological activity of the mutated protein.

[0112] Example 3: Results of 50°C stability test of IL29 mutants

[0113] The stability of the protein in this application is mainly characterized by the reversed-phase high-performance liquid chromatography purity.

[0114] Under the conditions of 50°C ± 2°C / 75% relative humidity ± 5% relative humidity, samples were taken according to Table 4, and the purity of each IL29 mutant sample was determined by reverse-phase high-performance liquid chromatography using the same method as in Section 2.2.1 of Example 2. The biological activity of each IL29 mutant sample was determined using the same method as in Section 2.3 of Example 2. See Table 5 for details. The purity values at day 0 and day 14 in Table 5 were compared in a chromatogram. Figures 3 to 10 .

[0115] Table 4 Stability verification scheme of IL29 mutants at 50°C

[0116]

[0117] Table 5 RP-HPLC purity determination results of IL29 mutants at 50°C

[0118]

[0119] From Table 5 and Figures 3 to 10The results showed that the two test samples with the largest decrease in purity were IL29 control and IL29CS. The purity of IL29 control dropped sharply from 85.82% to 34.46% within 14 days, a decrease of 51.36%. The purity of IL29CS dropped sharply from 97.03% to 46.20% within 14 days, a decrease of 50.83%. The two test samples with the smallest decreases were IL29DE and IL29DE+CS. The purity of IL29DE dropped from 92.68% to 77.39% within 14 days, a decrease of only 15.29%. The purity of IL29DE+CS dropped from 97.22% to 86.66% within 14 days, an even lower decrease of only 10.56%. The purity of other single-point mutants IL29DS and IL29GA decreased by 24.22% and 19.65% within 14 days, respectively; the purity of other double-point mutants IL29DS+CS and IL29GA+CS decreased by 18.25% and 14.97% within 14 days, respectively.

[0120] From the above results, it can be inferred that among the single-point mutants with a D-to-E mutation at position 162, IL29DE showed the greatest stability and the smallest decrease in purity over 14 days. Furthermore, since IL29CS is much less stable than IL29DE, conventional reasoning suggests that the double-point mutant IL29DE+CS would be less stable than IL29DE. However, the stability of IL29DE+CS in the present invention was significantly higher than IL29DE, indicating that the simultaneous double-point mutations have a synergistic effect in improving mutant stability. Simultaneously, the biological activities of the mutants were tested, revealing that the biological activities of the mutants IL29DE and IL29DE+CS remained essentially unchanged over 14 days.

[0121] Example 4: Results of 25°C Light Stability Test of IL29 Mutants

[0122] Under the conditions of 25°C ± 2°C / 60% ± 5% relative humidity / 5000 ± 500 lux, samples were taken as shown in Table 6, and the stability of IL29 mutants was determined using the same method as in Example 2. The results are shown in Table 7, where IL29CS was used as a control.

[0123] Table 6 IL29 mutants 25°C light stability scheme

[0124]

[0125] Table 7 Stability determination results of IL29 mutants under 25°C light irradiation

[0126]

[0127]

[0128] From the above results, it can be seen that the stability of IL29 mutant IL29DE+CS is better than that of IL29CS under 25℃ light conditions.

[0129] Example 5: Aerosol Stability Experiment of IL29 Mutants

[0130] The atomization experiment was carried out using a German PARI LCD jet nebulizer and a TurboBOY N atomizer pump. A total of 2 ml of sample was atomized and the following Figure 11 Aerosols were collected in a similar manner. The collected samples were subjected to reverse-phase HPLC purity determination of each IL29 mutant test sample using the same method as in 2.2.1 of Example 2 to verify the aerosol stability of each mutant. IL29CS was used as a control. The results are shown in Table 8 below.

[0131] Table 8 Determination results of aerosol stability of IL29 mutants

[0132]

[0133] The results in the table above show that the purity of the IL29 mutants IL29 DE+CS and IL29 GA+CS decreased by only approximately 3% after nebulization, while the purity of IL29 DS+CS decreased by 8.1%. However, the purity of IL29CS decreased significantly after nebulization, dropping by nearly 11%. This indicates that the nebulization stability of the IL29 mutants is significantly enhanced compared to that of IL29CS. The IL29 mutants IL29 DE+CS and IL29 GA+CS exhibit particularly excellent nebulization stability, making them particularly suitable for the preparation of nebulized inhalation formulations.

[0134] Example 6: In vitro efficacy determination of IL29 mutants on RSV-infected human bronchial epithelial cells (HBEC)

[0135] After the cell differentiation (Cell Application) of HBEC (human bronchial epithelial cell) was completed, serially diluted IL29 mutants (IL29 DE+CS) and positive control drugs (BMS-433771, provided by Shanghai WuXi AppTec, a respiratory syncytial virus fusion protein inhibitor) were added to the differentiated HBEC cells and incubated in a 37°C and 5% CO2 incubator. Respiratory syncytial virus (respiratory syncytial virus, provided by Shanghai WuXi AppTec) was inoculated 24 hours before infection, and 1 hour and 24 hours after infection. RSV with a titer of 100 TCID50 (half tissue culture infectious dose) was added to each well of the activity test well. After the cells were cultured in a 37°C and 5% CO2 incubator for 3 days, RSV RNA in the cells was extracted using an RNA extraction kit (Cat. No. 74181, Qiagen) and quantified by RT-qPCR. GraphPad Prism software was used to analyze the compound dose-response curve and calculate the EC 50 The values and average inhibition rates are shown in Tables 9 and 10.

[0136] Table 9 Inhibition of RSV infection by the positive control drug BMS-433771 in the HBEC cell model

[0137]

[0138] Table 10 Inhibition of RSV infection by IL29 mutants in HBEC cell model

[0139]

[0140] The results showed that IL29 DE+CS showed good anti-RSV efficacy in the in vitro model of human bronchial epithelial cells. 50 It is 0.13ng / ml (6.5pM), which is 1 / 100000 of the control drug and is much lower than the control drug.

[0141] Example 7: In vivo efficacy determination of IL29 mutants in RSV-infected mouse models

[0142] On day 0, all mice (female, 6-7 weeks old, 16-18 g, specific pathogen-free (SPF) grade BALB / c mice (provided by Shanghai WuXi AppTec)) were anesthetized by intraperitoneal injection of sodium pentobarbital (75 mg / kg) and inoculated intranasally with RSV (human respiratory syncytial virus A2 (RSV-A2, purchased from BEI Resources, NIAID, NIH, Bethesda, MD) at an inoculum of 1.1 × 105 PFU per animal in a 50 μL inoculation volume.

[0143] From day 0 to day 3, animals were dosed according to the scheme in Table 11. The method of administration was pulmonary spray. After the mice were anesthetized, the micro-spray nozzle cannula of the liquid atomizer (purchased from Shanghai Yuyan Instrument Co., Ltd.) pre-filled with the drug solution was gently inserted into the appropriate position of the mouse trachea, and the piston of the nebulizer high-pressure push device was quickly pressed to aerosolize a quantitative volume of the drug into the mouse lungs (reference Joseph D. Brain, Dwyn E. Knudson, Sergei P. Sorokin, Michael A. Davis, Pulmonary distribution of particles given by intratracheal instillation or by aerosol inhalation, Environmental research 11, Volume 11, Issue 1, 1976, Pages 13-33 of the dosing regimen), with a frequency of once a day. The 5th day after infection was the end point of the in vivo experiment. All animals were euthanized and lung tissues were collected to detect the virus titer (plaque assay). The results are shown in Table 12 and Figure 12 .

[0144] Table 11 In vivo dosing regimen in mice

[0145]

[0146] Note: The IL29 mutant used in this example is IL29 DE+CS.

[0147] Table 12 In vivo efficacy results of different drugs on RSV-infected mouse model

[0148]

[0149]

[0150] Note: Compared with the vehicle (normal saline) group, ***P<0.001, **P<0.01

[0151] The data showed that RSV could replicate in large quantities in mice after inoculation. The positive control drug ribavirin significantly inhibited RSV replication in mice, showing the expected in vivo anti-RSV activity and proving the effectiveness of this model system. Ribavirin was administered 1 hour before viral infection (prevention model), and the test drug IL29 mutant was able to significantly inhibit RSV replication in mice under the set assay conditions (1 hour before viral infection, 1 hour after viral infection, and 24 hours after viral infection). The viral titers in the lung tissues of mice in the first-dose groups 1 hour before inoculation (10 μg) and 1 hour after inoculation (10 μg) were both below the detection limit, demonstrating excellent in vivo anti-RSV efficacy. The group administered 24 hours after infection also showed good anti-RSV effects. 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 demonstrates that the IL29 mutant has excellent antiviral therapeutic potential.

[0152] Example 8: In vivo efficacy determination of IL29 mutants in RSV-infected cotton rat model

[0153] On day 0, all mice (Sigmodon hispidus cotton rats, half male and half female, 5 weeks old, SPF grade (purchased from Envigo)) were anesthetized by intraperitoneal injection of sodium pentobarbital (75 mg / kg) and inoculated intranasally with RSV (human respiratory syncytial virus A2 (RSV-A2), purchased from BEI Resources, NIAID, NIH, Bethesda, MD) at an inoculum of 1.1 × 10 5 PFU per animal, with an inoculation volume of 50 μL.

[0154] From day 0 to day 3, animals were dosed according to the assay protocol in Table 13. The method of administration was pulmonary spray (reference to the dosing protocol of Joseph D. Brain, Dwyn E. Knudson, Sergei P. Sorokin, Michael A. Davis, Pulmonary distribution of particles given by intratracheal instillation or by aerosolinhalation, Environmental research 11, Volume 11, Issue 1, 1976, Pages 13-33), with a frequency of once a day. A 3 ml syringe with a 22 G needle was inserted into the trachea, and 2 ml of 0.9% saline was injected into the lungs to collect bronchoalveolar lavage fluid (BALF). The collected BALF was divided into sterile 1.5 ml EP tubes, frozen on dry ice, and stored at -80°C until plaque assay analysis. The results are shown in Tables 14 and Figure 13 .

[0155] Table 13 In vivo dosing regimen in cotton rats

[0156]

[0157] Note: The IL29 mutant used in this example is IL29 DE+CS.

[0158] Table 14 In vivo efficacy results of different drugs on RSV-infected cotton rat model

[0159]

[0160] Table 14 and Figure 13 The data showed that RSV can replicate in large quantities in cotton rats after inoculation. The test drug IL29 mutant can significantly inhibit the replication of RSV virus in mice under the set conditions (therapeutic model of administration 1 hour after inoculation). The virus titer in the bronchial lavage fluid of cotton rats in the first-dose group (Groups 2, 3, and 4) 1 hour after inoculation (1μg) was statistically different from that in the control group, and the three dose groups (Groups 2-4) showed dose-related effects, indicating excellent in vivo anti-RSV efficacy. After cotton rats are infected with respiratory syncytial virus, the virus will replicate in large quantities in the bronchi of their lungs, and the viral load in the bronchial lavage fluid very well reflects the viral replication in the lungs. Therefore, the IL29 mutant shows excellent therapeutic potential for respiratory syncytial virus.

[0161] Example 9: In vitro efficacy determination of IL29 mutants against the new coronavirus (2019-nCov)

[0162] Add 200 μl of 5 × 10 4 Vero E6 cells (African green monkey kidney cell line) were cultured at 37°C with 5% CO2 for 24 hours; the test drug IL29 DE+CS was diluted to 100 ng / ml, 5 replicate wells were set up, 100 μl was added to each well, and the effect was 24 hours; 100 μl of the virus 2019-nCoV with a titer of 100 TCID50 / ml (stored at -80°C by the Pathogen Center of the Institute of Medical Laboratory Animals, Chinese Academy of Medical Sciences, with a titer of 10 5 TCID 50 / ml), blank control (solvent control) and virus control (negative control); the cells were incubated in a 37°C, 5% CO2 incubator for 4-5 days; the cytopathic effect (CPE) was observed under an optical microscope, and complete cell lesions were recorded as "++++", 75% lesions were recorded as "+++", 50% lesions were recorded as "++", 25% lesions were recorded as "+", and no lesions were recorded as "-".

[0163] The results showed that at a concentration of 100 ng / ml (5 nmol / L), the IL29 mutant recorded a lesion as "-", and 100% protected Vero E6 cells from infection by the new coronavirus, showing a very good effect against the infection of the new coronavirus (2019-nCov).

[0164] Example 10: Determination of the efficacy of IL29 mutants against influenza virus in mice

[0165] According to the dosing regimen in Table 15, the efficacy of influenza virus in BALB / c mice (provided by Shanghai Lingchang Biotechnology Co., Ltd.) was determined, with 8 mice in each group. Figure 14 and Figure 15 .

[0166] Table 15 Protocol for determining the efficacy of IL29 mutants against influenza A virus in mice

[0167]

[0168]

[0169] It can be seen that in the mouse infection model of influenza A virus WSN / 33, when administered 48 hours after infection, IL29 DE+CS can significantly prolong the survival of infected mice and play a protective role in mice.

[0170] Example 11: In vivo efficacy of IL29 DE+CS in treating severe respiratory distress syndrome

[0171] Male C57 mice (18-22 g, 30 mice, Shanghai Lingchang Biotechnology Co., Ltd.) were anesthetized, and the skin was cut to expose the trachea. Lipopolysaccharide (LPS) solution (50 μl / mouse, 0.3 mg / kg) was slowly injected through the trachea to replicate an acute lung injury model. The animal weight and health status were monitored for a total of 24 h from the start of modeling to the end of the measurement.

[0172] The efficacy of dexamethasone (DEX) and IL29 DE+CS in a severe respiratory distress syndrome mouse model was determined according to the dosing regimen in Table 16. Mice were euthanized 24 hours after modeling, and lung tissue was fixed for subsequent pathological examination. The results are shown in Table 16. Figure 16-18a -c.

[0173] Table 16 Protocol for determining the in vivo efficacy of dexamethasone and IL29 DE+CS in the treatment of severe respiratory distress syndrome

[0174] Grouping Number of animals Model drug Dosage Group 1 6 no Normal saline Atomization Group 2 6 yes Normal saline Atomization Group 3 6 yes DEX-3mg / kg oral Group 4 6 yes IL29 DE+CS-240μg / kg* Atomization Group 5 6 yes IL29 DE+CS-10mg / kg subcutaneous

[0175] *Group 4 was the nebulized group, with a drug concentration of 1 mg / ml and nebulization for 30 minutes. The drug was administered before modeling and again 12 hours after modeling.

[0176] It can be seen that the study in the LPS-induced acute lung injury model in mice showed that in histological evaluation, the IL29 DE+CS nebulization and subcutaneous administration groups had a significant therapeutic effect in reducing lung injury and inflammatory response compared with the model group.

[0177] Example 12: Toxic effects of IL29 mutants on African green monkey kidney Vero cells

[0178] In the Vero cell culture system, the CCK-8 method was used to detect the toxicity of different concentrations of antibodies to Vero cells.

[0179] Vero cells were seeded into 96-well culture plates at a concentration of 5000 cells / well, cultured to a cell monolayer, the culture medium discarded, and the cells washed twice with Hank's solution; the test drugs IL29 mutant IL29 DE+CS and Remdesivir were diluted in MEM medium, with starting concentrations of 1000nmol / L (19.6μg / mL) and 50μmol / L, respectively, and serially diluted 3-fold to 8 concentrations. Three replicate wells were set for each drug concentration, and 150μL of drug solution was added to each well. At the same time, a control well for normal growth of Vero cells was set up and cultured in a 37°C, 5% CO2 incubator for 48h. Then 15μL of CCK-8 reagent was added to each well. After 3 hours, the OD value at 450nm was measured using a microplate reader. The dose toxicity effect and maximum non-toxic concentration of the drug on the cells were calculated, and the drug-cytotoxicity reaction curve was drawn.

[0180] The results showed that the test drug IL29 DE+CS at a concentration of 1000nmol / L (19.6μg / mL) and below had no significant toxic effect on Vero cells, and the cell viability was greater than 90%. Remdesivir at a concentration of 50μmol / L and below had no significant toxic effect on Vero cells, and the cell viability was greater than 90%. Test drug IL29 DE+CS CC 50 >1000nmol / L (19.6μg / mL), specifically Figure 19 As shown, Remdesivir CC 50 >50μmol / L.

[0181] Example 13: Inhibitory effect of IL29 mutants on novel coronavirus in vitro

[0182] In the Vero cell culture model, immunofluorescence and nucleic acid detection kits (fluorescence PCR method) were used to detect the preventive and therapeutic effects of different concentrations of IL29 mutants against the new coronavirus.

[0183] 2.1 Preparation of drug-virus mixture

[0184] Experimental group: The test drug IL29 mutant IL29 DE+CS was diluted 5-fold in MEM medium containing 10% FBS starting from the highest concentration of 100 ng / mL, and then mixed with the new coronavirus virus liquid (preserved by the State Key Laboratory of Infectious Diseases Diagnosis and Treatment of Zhejiang University, TCID50 of 10 -5.5 / mL) were mixed at an MOI of 100.

[0185] Positive control group: The remdesivir positive control group was diluted to 6 final concentrations of 10 μM, 3 μM, 1 μM, 0.3 μM, 0.1 μM, and 0.03 μM using 10% FBS MEM culture medium, and mixed with the new coronavirus strain virus liquid at an MOI = 100 ratio.

[0186] This experiment set up a prevention model group and a treatment model group. Vero cells were pretreated with different concentrations of the test drug IL29 DE+CS samples in the above experimental group before mixing with the virus for 24 hours as the in vitro prevention model group, and cells without pretreatment were used as the in vitro treatment model group.

[0187] 2.2 Experimental process

[0188] Vero cells were seeded into 48-well culture plates at a concentration of 10,000 cells / well and cultured until the cells reached the logarithmic growth phase. The cells were divided into a prevention group, an experimental group, a positive control group, a negative control group (only virus solution was added), and a cell-only group (no virus was added). Each sample was plated in triplicate.

[0189] The cell culture medium in the prevention group was aspirated, and 500 μL of the test drug IL29DE+CS sample at varying concentrations was added to each well. The remaining cells remained untreated and incubated at 37°C, 5% CO2 for 24 hours. The cell culture medium in all four groups was then aspirated, and the drug-virus mixture prepared above was added to each well. The negative control group and the cell-only group were incubated to the same volume with culture medium. The cells were incubated at 35°C, 5% CO2 for 3 hours. The culture medium was aspirated, and the cells were washed with PBS. Then, 500 μL of the drug diluent at the corresponding concentration was added to each well. The cells were incubated at 35°C, 5% CO2 for 48 hours.

[0190] 2.2.1 Novel Coronavirus Nucleic Acid Testing

[0191] 200 μL of culture supernatant was aspirated and viral nucleic acid was extracted using a magnetic bead-based nucleic acid extraction kit (MVR01, Shanghai Zhijiang Biotechnology Co., Ltd.) and a fully automated nucleic acid extractor (EX3600, Shanghai Zhijiang Biotechnology Co., Ltd.), with a final elution volume of 50 μL. Five μL of the nucleic acid extract was then used to detect viral nucleic acid levels using a one-step novel coronavirus nucleic acid detection kit (fluorescence PCR method, catalog number Z-RR-0479-02-50, National Medical Device Registration No. 20203400057, Shanghai Zhijiang Biotechnology Co., Ltd.).

[0192] The results are expressed as Ct values to indicate the level of the virus. The relationship between Ct value and viral copy number is: y = -3.33x + 48.69, where y is the Ct value and x is the logarithm of the number of viruses with a base of 10. The inhibition rate is calculated as: Inhibition rate = [1-10^((y 病毒对照 -y) / 3.33)]×100%. The experimental results are shown in Table 17 and Figure 20 :

[0193] Table 17 Results of inhibition rate determination of each group against novel coronavirus

[0194]

[0195] The above results show that in the prevention group, the test drug at concentrations of 0.0064 ng / mL and above had an inhibitory effect on the infection and replication of the new coronavirus, among which the test drug at concentrations of 4 ng / mL and above had an inhibition rate of over 98%; in the treatment group, the test drug at concentrations of 0.0064 ng / mL and above had a certain inhibitory effect on the infection and replication of the new coronavirus, but the inhibitory effect was worse than that in the prevention group. Except for 0.8 ng / mL, the inhibitory effect was less than 75%. In the positive control group, the inhibition rate of remdesivir at concentrations of 0.3 μM and above was over 90%.

[0196] After calculation, the IC of the test drug prevention group 50 =0.0196 ng / mL, IC of the treatment group 50 =0.0612ng / mL, positive control group IC 50 =0.12μmol / L. Calculation shows that the safety index SI of the preventive group of the test drug is >10 6 The safety index SI of the treatment group was >3.2×10 5 , showing good cytotoxic selectivity.

[0197] 2.2.2 Immunofluorescence detection

[0198] The cell culture supernatant from the 48-well plate was aspirated and washed with PBS. Each well was fixed with 200 μL of ice-cold 80% acetone at 4°C for 30 minutes, washed with PBS, and then blocked with 200 μL of blocking buffer containing 3% BSA in PBS for 30 minutes at room temperature. After washing with PBS, 200 μL of primary antibody (rabbit anti-SARS-CoV2 NP serum diluted 2000-fold (Beijing Sino Biological Science and Technology Co., Ltd.) was added and incubated overnight at 4°C. The primary antibody was aspirated, the plates were washed with PBS, and 200 μL of FITC-conjugated goat anti-rabbit IgG secondary antibody (Jackson) diluted 1500-fold was added and incubated at room temperature in the dark for 2 hours. The secondary antibody was aspirated, the plates were washed with PBS, and 200 μL of DAPI diluted 1:500 in PBS was added and incubated at room temperature in the dark for 5 minutes. The DAPI stain was aspirated, the plates were washed with PBS, and the plates were examined under a microscope.

[0199] The immunofluorescence results were consistent with the nucleic acid test results. In the prevention group, the test drugs at a concentration of 0.032ng / mL and above had a certain inhibitory effect on the in vitro infection of the new coronavirus, among which the test drugs at a concentration of 4ng / mL and above had a significant inhibitory effect on viral infection. In the treatment group, the test drugs at a concentration of 0.032ng / mL and above had a certain inhibitory effect on the in vitro infection of the new coronavirus, but the inhibitory effect was lower than that of the prevention group. In the positive control group, remdesivir at a concentration of 0.3μM and above had a significant inhibitory effect on in vitro viral infection. For specific results, see the results. Figure 21 、 Figure 22 and Figure 23 .

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Claims

1. Use of a mutant protein as shown in SEQ ID NO: 5 in the preparation of a medicament for preventing and / or treating a viral infectious disease, wherein the viral infectious disease is a respiratory disease caused by viral infection, wherein the virus is influenza A virus, wherein the mutant protein as shown in SEQ ID NO: 5 is administered via the respiratory tract, and wherein the mutant protein as shown in SEQ ID NO: 5 is administered by aerosol inhalation.

2. Use of an aerosol inhalation formulation of the mutant protein as shown in SEQ ID NO: 5 in the preparation of a medicament for preventing and / or treating a viral infectious disease, wherein the viral infectious disease is a respiratory disease caused by a viral infection, and wherein the virus is influenza A virus.

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