Novel anti-tuberculosis engineered proteins and uses thereof

Abstract

The present application relates to a novel anti-tuberculosis engineered protein, and more specifically, to an anti-tuberculosis use of an engineered protein based on human tryptophanyl-tRNA synthetase 1 containing one or more amino acid variations. The engineered protein of the present application not only has high thermal stability, but also has an effect of significantly inhibiting the growth of Mycobacterium tuberculosis. Furthermore, it is confirmed through experiments that the engineered protein of the present application promotes intragranuloma infiltration of immune cells. This indicates that the engineered protein of the present application has excellent anti-tuberculosis efficacy and immune-enhancing efficacy, and can be variously utilized in the field of tuberculosis treatment and the field of immune enhancement.

Description

Technical Field

[0001] This invention relates to novel anti-tuberculosis engineered proteins, and more specifically, to the anti-tuberculosis use of engineered proteins based on human tryptophanyl-tRNA synthetase 1 containing one or more amino acid variations. Background Technology

[0002] Tuberculosis is a disease transmitted through coughing, sneezing, or talking. It is usually caused by Mycobacterium tuberculosis (Mycobacterium tuberculosis). Mytobacterium tuberculosis Infection with Mtb can result in either active or inactive forms of tuberculosis. Active tuberculosis is infectious to healthy individuals, but inactive tuberculosis is not infectious.

[0003] Regardless of these types, it is known that one-third of the world's population is infected. Currently, mutations frequently occur in mycobacteria, including Mtb and BCG (bacillus Calmette-Guerin), leading to antibiotic resistance. Therefore, there is a real need to develop appropriate technologies for the diagnosis, prevention, and treatment of tuberculosis. Summary of the Invention

[0004] Technical issues Therefore, the inventors developed an engineered protein containing WARS1 with one or more amino acid variations, and confirmed that the engineered protein has improved thermal stability and tuberculosis growth inhibition effect, thus completing the present invention.

[0005] Therefore, the object of the present invention is to provide an engineered protein comprising a polypeptide represented by the amino acid sequence of SEQ ID NO: 2, said polypeptide comprising one or more amino acid variations.

[0006] Another object of the present invention is to provide a nucleic acid encoding the engineered protein.

[0007] Another object of the present invention is to provide a recombinant vector comprising a nucleic acid encoding the engineered protein.

[0008] Another object of the present invention is to provide a composition comprising the engineered protein for the prevention, improvement or treatment of tuberculosis.

[0009] Another object of the present invention is to provide an immune-enhancing composition comprising the engineered protein.

[0010] Another object of the present invention is to provide a combined treatment method comprising the step of administering the engineered protein to an individual in need.

[0011] Technical solution To achieve the aforementioned objective, the present invention provides an engineered protein comprising a polypeptide represented by the amino acid sequence of SEQ ID NO: 2, wherein the polypeptide comprises one or more amino acid variations.

[0012] Furthermore, the present invention provides a pharmaceutical composition comprising the engineered protein for the prevention or treatment of tuberculosis.

[0013] Furthermore, the present invention provides a food composition comprising the engineered protein for the prevention or improvement of tuberculosis.

[0014] Furthermore, the present invention provides a health food composition comprising the engineered protein for the prevention or improvement of tuberculosis.

[0015] Furthermore, the present invention provides a composition for enhancing immunity comprising the engineered protein.

[0016] Furthermore, the present invention provides a treatment for tuberculosis, comprising administering the engineered protein to an individual in need.

[0017] The effects of the invention The engineered protein of this invention not only exhibits high thermal stability but also significantly inhibits the growth of Mycobacterium tuberculosis. Furthermore, experiments have confirmed that the engineered protein of this invention promotes the infiltration of immune cells into tissues. This indicates that the engineered protein of this invention possesses excellent anti-tuberculosis and immune-enhancing effects, and can be utilized in a variety of ways in the fields of tuberculosis treatment and immune enhancement. Attached Figure Description

[0018] Figure 1 A graph illustrating the results of evaluating the in vitro efficacy of the single engineered protein of the present invention through cytokine and chemokine expression analysis.

[0019] Figure 2 A graph showing the results of evaluating the in vitro efficacy of the composite engineered protein of the present invention through cytokine and chemokine expression analysis.

[0020] Figure 3 A graph showing the results of evaluating the in vitro stability of the single engineered protein and the composite engineered protein of the present invention.

[0021] Figure 4 The figure shows the results of evaluating the in vitro anti-tuberculosis efficacy when the composite engineered protein L046I / A376C / V398C of the present invention was administered alone, or when the wild type was administered alone or in combination with the composite engineered protein L046I / A376C / V398C and the anti-tuberculosis agent (INH) was administered (P < 0.001, P < 0.01 vs INH (administered alone)).

[0022] Figure 5A graph showing the results of analyzing the distribution of immune cells in lung tissue following the administration route of the composite engineered protein L046I / A376C / V398C of the present invention.

[0023] Figure 6 To demonstrate the in vivo efficacy of the composite engineered protein L046I / A376C / V398C of the present invention in terms of dosage and route of administration ( In vivo The results of anti-tuberculosis efficacy are shown in the figure (P < 0.001, P < 0.05 vs solvent group (Vehicle or Wild type), P < 0.01 vs intranasal administration).

[0024] Best practice The present invention will now be described in detail.

[0025] According to one embodiment of the present invention, the present invention provides an engineered protein comprising a polypeptide represented by the amino acid sequence of SEQ ID NO: 2, wherein the polypeptide comprises one or more amino acid variations.

[0026] In a specific example of the present invention, the engineered protein can be used to treat tuberculosis. In embodiments of the present invention, it has been confirmed that the engineered protein functions in vitro (…). in vitro It also significantly inhibits the growth of Mycobacterium tuberculosis in vivo.

[0027] The polypeptide of the present invention may consist of the amino acid sequence represented by SEQ ID NO: 2 and may be encoded by the base sequence represented by SEQ ID NO: 1, including functional equivalents of the protein.

[0028] The term "functional equivalent" refers to a peptide of SEQ ID NO: 2 that, as a result of the addition, substitution, or deletion of amino acids, has at least 80% sequence homology (i.e., identity) with the peptide, preferably at least 90%, and more preferably at least 95%, including, for example, sequences with 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100% sequence homology, indicating a peptide exhibiting physiological activity substantially homogeneous with the peptide of SEQ ID NO: 2. In this specification, sequence homology or homogeneity is defined as the percentage of amino acid residues in the candidate sequence relative to the amino acid sequence of SEQ ID NO: 2 after aligning the amino acid sequence of SEQ ID NO: 2 with the candidate sequence and introducing gaps. Where necessary, to obtain the maximum percentage of sequence homology, conserved substitutions are not considered as part of sequence homology. Furthermore, extensions, deletions, or insertions at the N-terminus, C-terminus, or internal portion of the amino acid sequence of SEQ ID NO: 1 are not interpreted as affecting sequence homology or homogeneity.

[0029] Furthermore, the sequence homogeneity can be determined using standard methods for comparing similar portions of the amino acid sequences of two polypeptides. Computer programs such as BLAST or FASTA align the respective amino acids of two polypeptides in an optimal matching manner (along the full length of one or both sequences or along predicted portions of one or both sequences). These programs provide default opening penalties and default gap penalties, and provide scoring matrices such as PAM250 (Standard Scoring Matrix) that can be used in conjunction with the computer program. For example, the percentage of sequence homogeneity can be calculated as follows: multiply the total number of identical matches by 100, and divide by the length of the longer sequence within the corresponding made span and the sum of gaps introduced into the longer sequence to align the two sequences.

[0030] In the foregoing, "substantially identical physiological activity" refers to anti-tuberculosis or immune-enhancing activity. The scope of "functional equivalents" in this invention includes derivatives that, while maintaining the basic backbone and anti-tuberculosis or immune-enhancing activity of the peptide of SEQ ID NO: 2, involve modifications to the chemical structure of a portion of the peptide. For example, these modifications include structural alterations to change the peptide's stability, storage properties, volatility, or solubility.

[0031] In this invention, "amino acid variation" refers to a protein with a different sequence formed by the deletion, insertion, non-conservative or conserved substitution of one or more amino acid residues, or a combination thereof. Amino acid substitutions in proteins and peptides that do not alter their overall molecular activity are well known in the art to which this invention pertains. The polypeptide or its variants can be extracted or synthesized from nature, or prepared by gene recombination methods using a DNA sequence as a template.

[0032] In a specific example of the present invention, the polypeptide may contain variations at one or more positions selected from the group consisting of P006, A007, S008, E011, F013, T018, S032, V044, S045, L046, K047, S049, A054, T089, R127, L132, R133, I136, F137, A168, F360, A376, and V398 of the amino acid sequence of SEQ ID NO: 2. The polypeptide of the present invention may undergo conservative substitutions at the aforementioned positions.

[0033] In this invention, conservative substitution refers to replacing one amino acid with another amino acid having similar structure and / or chemical properties. Such amino acid substitutions typically occur based on similarities in the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic nature of the residues. For example, positively charged (basic) amino acids include arginine, lysine, and histidine; negatively charged (acidic) amino acids include glutamic acid and aspartic acid; aromatic amino acids include phenylalanine, tryptophan, and tyrosine; and hydrophobic amino acids include alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, and tryptophan. Furthermore, amino acids can be divided into amino acids with electrically charged side chains and amino acids with uncharged side chains. Amino acids with charged side chains include aspartic acid, glutamic acid, lysine, arginine, and histidine. Amino acids with uncharged side chains can be further divided into nonpolar amino acids and polar amino acids. Nonpolar amino acids include glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, and proline. Polar amino acids include serine, threonine, cysteine, tyrosine, asparagine, and glutamine. Generally, conserved substitutions have little or no effect on the activity of proteins or peptides.

[0034] In a preferred embodiment of the present invention, the polypeptide may comprise one or more amino acid variations selected from the group consisting of P006C, A007C, A007G, S008T, E011D, F013Y, T018A, S032P, V044L, V044M, S045Q, L046I, K047R, S049T, A054T, T089N, R127K, L132M, R133H, I136M, F137Y, A168C, F360C, A376C, and V398C. Preferably, the polypeptide may comprise two or more amino acid variations selected from the group consisting of A168C, A376C, V398C, L046I, F360C, and K047R. More preferably, the polypeptide may further comprise amino acid variations of L046I or K047R.

[0035] More specifically, a polypeptide containing one amino acid variation can be P006C, A007C, A007G, S008T, E011D, F013Y, T018A, S032P, V044L, V044M, S045Q, L046I, K047R, S049T, A054T, T089N, R127K, L132M, R133H, I136M, F137Y, A168C, F360C, A376C, or V398C. Furthermore, examples of polypeptides containing two amino acid variations in this invention may be L046I / K047R, L046I / R133H, L046I / I136M, L046I / F137Y, K047R / R133H, K047R / I136M, K047R / F137Y, R133H / I136M, R133H / F137Y, I136M / F137Y, A168C / F360C, or A376C / V398C. Furthermore, examples of the polypeptides comprising three amino acid variations of the present invention may be L046I / K047R / R133H, L046I / K047R / I136M, L046I / K047R / F137Y, K047R / R133H / I136M, K047R / R133H / F137Y, R133H / I136M / F137Y, L046I / A168C / F360C, L046I / A376C / V398C, K047R / A168C / F360C, or K047R / A376C / V398C. The polypeptides of the present invention may comprise combinations of the aforementioned amino acid variations, but the scope of the present invention is not limited thereto.

[0036] In a preferred embodiment of the invention, the polypeptide may comprise amino acid variations of (a) A376C and V398C; or (b) A168C and F360C. In this case, the thermal stability of the engineered protein can be significantly improved.

[0037] In a more preferred embodiment of the invention, the polypeptide comprises the following amino acid variations: (a) A376C and V398C; or (b) A168C and F360C. It may also comprise amino acid variations of L046I or K047R. Thus, it is confirmed that the composite engineered protein not only has improved thermal stability but also excellent anti-tuberculosis efficacy.

[0038] Specific examples of the engineered proteins of the present invention are shown in Table 2 of Example 1 described below.

[0039] The engineered protein of this invention not only exhibits high thermal stability but also significantly inhibits the growth of Mycobacterium tuberculosis. Furthermore, experiments have confirmed that the engineered protein of this invention promotes the infiltration of immune cells into tissues. This indicates that the engineered protein of this invention possesses excellent anti-tuberculosis and immune-enhancing effects, and can be utilized in a variety of ways in the fields of tuberculosis treatment and immune enhancement.

[0040] According to another embodiment of the present invention, the present invention provides a nucleic acid encoding the engineered protein and a recombinant vector comprising the nucleic acid.

[0041] The nucleic acid of the present invention may comprise a nucleic acid encoding a polypeptide represented by the amino acid sequence of SEQ ID NO: 2, preferably, it may comprise a nucleic acid represented by the base sequence of SEQ ID NO: 1. Furthermore, the nucleic acid of the present invention may be a nucleic acid encoding a polypeptide represented by the amino acid sequence of SEQ ID NO: 2 containing one or more amino acid variations, and more specifically, it may be a nucleic acid encoding a polypeptide variant described in Table 2.

[0042] Furthermore, variants of the base sequence are included within the scope of this invention. Specifically, the gene having more than 70% sequence homology with the base sequence of SEQ ID NO: 1, more preferably, more than 80% sequence homology, even more preferably, more than 90% sequence homology, and most preferably, more than 95% sequence homology, refers to a sequence exhibiting substantially the same physiological activity as the base sequence represented by SEQ ID NO: 1. The "% sequence homology" of the polynucleotide is confirmed by comparing two best-aligned sequences and a comparison region, where the polynucleotide may include additions or deletions (i.e., vacancies) compared to a reference sequence (excluding additions or deletions) for best alignment of the two sequences.

[0043] In this invention, a vector refers to a tool used to express a target gene in a host cell. Examples include plasmid vectors, granular vectors, phage vectors, and viral vectors such as adenovirus vectors, retrovirus vectors, and adeno-associated virus vectors. Vectors that can be used as recombinant vectors can be prepared by editing plasmids (e.g., pGLS, pSC101, pGV1106, pACYC177, ColE1, pKT230, ME290, pBR322, pUC8 / 9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14, pGEX series, pET series, and pUC19, etc.), phages (e.g., λgt4λB, λCharon, λΔz1, and M13, etc.), or viruses (e.g., CMV, SV40, etc.) commonly used in the field of this invention.

[0044] The nucleic acid encoding the engineered protein in the recombinant vector can be operatively linked to a promoter. The term "operatively linked" refers to the functional binding between a nucleotide expression regulatory sequence (e.g., a promoter sequence) and other nucleotide sequences. Therefore, the regulatory sequence can thereby regulate the transcription and / or translation of the other nucleotide sequences.

[0045] The recombinant vector can be constructed as a typical vector for cloning or for expression. The expression vector can be a conventional expression vector used in the art to which this invention pertains for expressing exogenous proteins in plants, animals, or microorganisms. The recombinant vector can be constructed using a variety of methods known in the art to which this invention pertains.

[0046] The recombinant vector can be constructed using prokaryotic or eukaryotic cells as hosts. For example, when using eukaryotic cells as hosts, the origin of replication initiated in the eukaryotic cells containing the vector includes, but is not limited to, the f1 origin of replication, SV40 origin of replication, pMB1 origin of replication, adenovirus origin of replication, AAV origin of replication, CMV origin of replication, and BBV origin of replication. Furthermore, promoters derived from mammalian cell genomes (e.g., metallothionein promoters) or from mammalian viruses (e.g., adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus (CMV) promoter, and HSV tk promoter) can be used, typically possessing a polyadenylated sequence as a transcription termination sequence.

[0047] According to another embodiment of the present invention, the present invention provides a composition comprising the engineered protein for the prevention, improvement, or treatment of tuberculosis. The compositions of the present invention may be pharmaceutical compositions, food compositions, or health functional food compositions.

[0048] In a specific embodiment of the present invention, the composition may further comprise an antituberculosis agent, which may be one or more selected from the group consisting of rifampicin, isoniazid, pyrazinamide, and ethambutol. When the engineered protein of the present invention is administered co-administered with the antituberculosis agent, the therapeutic effect of the antituberculosis agent may be enhanced.

[0049] In this invention, the pharmaceutical composition can be formulated into various forms for use according to conventional methods. For example, it can be formulated into oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, and syrups, and into external preparations, suppositories, sterile injectable solutions, transdermal administration agents, and nasal inhalers.

[0050] In order to administer the pharmaceutical composition of the present invention, in addition to the active ingredient, it may also contain one or more pharmaceutically acceptable carriers. The pharmaceutically acceptable carriers contained in the pharmaceutical composition of the present invention are those commonly used in formulation, including lactose, glucose, sucrose, sorbitol, mannitol, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylparaben, propylparaben, talc, magnesium stearate, and mineral oil, but are not limited thereto. In addition to the aforementioned components, the pharmaceutical composition of the present invention may also contain lubricants, humectants, sweeteners, flavorings, emulsifiers, suspending agents, preservatives, etc.

[0051] The dosage of the pharmaceutical composition of the present invention will vary depending on the preparation method, administration method, administration time and / or route of administration of the pharmaceutical composition, and will vary with factors including the type and extent of the response to be achieved by administration of the pharmaceutical composition, the type of individual being administered, age, weight, general health status, symptoms or severity of disease, sex, diet, metabolism, drugs used simultaneously or sequentially by related individuals, other compositional components, and similar factors well known in the medical field. Those skilled in the art to which this invention pertains can easily determine and prescribe the effective dosage required for the target treatment.

[0052] For example, the dosage of the pharmaceutical composition of the present invention can be from 1 mg / kg to 1000 mg / kg per day, but in no way does the dosage limit the scope of the present invention.

[0053] The route of administration and method of administration of the pharmaceutical composition of the present invention can be independent of each other, and the method is not particularly limited. It can be carried out through any route of administration and method of administration that enables the pharmaceutical composition to reach the relevant target location.

[0054] The pharmaceutical composition can be administered orally or via parenteral administration. For example, parenteral administration methods include inhalation, intravenous administration, intraperitoneal administration, intramuscular administration, transdermal administration, or subcutaneous administration.

[0055] The food composition of the present invention can be prepared by methods commonly used in the technical field to which this invention pertains, wherein the preparation can be carried out by adding raw materials and ingredients commonly added in the technical field to which this invention pertains. Furthermore, the food composition can be prepared without limitation into dosage forms recognized as food compositions.

[0056] The food products of this invention include various food categories, beverages, chewing gum, tea, vitamin complexes, functional foods, etc. Furthermore, the food products include, but are not limited to, special nutritional foods (e.g., modified dairy products, infant formula, etc.), processed meat products, processed fish products, tofu products, frozen products, noodle products (e.g., ramen, noodles, etc.), bread products, health supplements, condiments (e.g., soy sauce, soybean paste, chili sauce, mixed sauces, etc.), sauces, biscuits (e.g., snacks), candies, chocolates, chewing gum, ice cream, dairy products (e.g., fermented milk, cheese, etc.), other processed foods, pickles, preserved foods (various types of pickles, preserved vegetables, etc.), beverages (e.g., fruit drinks, vegetable drinks, fermented beverages, etc.), natural seasonings (e.g., ramen broth), and food additives. The food products, beverages, or food additives can be prepared using conventional preparation methods.

[0057] When using the composition of the present invention as a health functional food additive, it can be added directly or used together with other health functional foods, and can be used appropriately according to conventional methods. The mixing amount of the active ingredients can be appropriately determined according to the purpose of use. Generally, when preparing it as a food or beverage, preferably, no more than 50 parts by weight of the composition of the present invention can be added relative to the raw materials, more preferably, no more than 25 parts by weight of the composition of the present invention can be added. However, in the case of long-term intake for the purpose of regulating health and hygiene, the amount can be below the range without any safety issues, and therefore, it is also possible to use it in amounts above the range.

[0058] In addition to engineered proteins as active ingredients, the food compositions of the present invention, like conventional food compositions, may also contain various sweeteners or natural carbohydrates as additional ingredients. For example, the aforementioned natural carbohydrates may be: monosaccharides, such as glucose, fructose, etc.; disaccharides, such as maltose, sucrose, etc.; polysaccharides, such as dextrin, cyclodextrin, and other common sugars; and sugar alcohols such as xylitol, sorbitol, erythritol, etc. The aforementioned sweeteners may advantageously be natural sweeteners (such as sematrandrine, stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.) and / or synthetic sweeteners (such as saccharin, aspartame, etc.).

[0059] Furthermore, in addition to engineered proteins, the food composition may contain various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic and natural flavoring agents, coloring agents, fillers (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, and / or carbonating agents used in carbonated beverages. Moreover, the food composition of the present invention may also contain natural fruit juice and fruit pulp used in the preparation of fruit juice beverages and vegetable beverages.

[0060] According to another embodiment of the present invention, the present invention provides a composition comprising engineered proteins for enhancing immunity.

[0061] In this invention, immunostimulation is an important therapeutic strategy for enhancing the body's defense mechanisms against various diseases, including infectious diseases, cancer, and inflammatory diseases. This enhancement can be achieved by increasing the activity of immune cells to stimulate the immune response. For example, phagocytosis plays a crucial role in the immune response. Macrophages, as the primary cells involved in phagocytosis, absorb microorganisms and other pyrogenic particles and stimulate the immune response by secreting various cytokines such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-12 (IL-12), as well as cytotoxic and inflammatory substances such as nitric oxide (NO). Therefore, increasing macrophage activity can be a means of enhancing immunity. Infections and diseases primarily occur in states of weakened immune function; therefore, various studies are actively underway on how immunostimulatory substances can enhance the immune response when the body's immune system function is impaired.

[0062] The immune-enhancing compositions of the present invention may contain pharmaceutically effective amounts of engineered proteins and excipients or diluents.

[0063] In the above context, the pharmaceutically effective amount refers to the sufficient amount to exert an immune-enhancing effect. "Pharmaceutically acceptable" means a composition that is physiologically acceptable and, when administered to an individual, generally does not cause gastrointestinal disturbances, vertigo, or other allergic reactions or similar reactions.

[0064] According to another embodiment of the present invention, a treatment method for tuberculosis is provided, comprising the step of administering an engineered protein to an individual in need.

[0065] In a specific example of the present invention, the individual may be an individual predicted to develop tuberculosis, an individual who has already developed the disease, or an individual who has been determined to be cured, but is not limited thereto.

[0066] Furthermore, the treatment method of the present invention administers, simultaneously or sequentially with the engineered protein, a known antituberculosis agent in the art to which this invention pertains. Examples of such antituberculosis agents include rifampin, isoniazid, pyrazinamide, and ethambutol, but the scope of the present invention is not limited thereto. Detailed Implementation

[0067] The present invention will now be described in more detail through embodiments. These embodiments are merely illustrative of the invention, and it should be understood by those skilled in the art that the scope of the invention should not be construed as being limited by these embodiments.

[0068] Example 1. Design of engineered proteins containing WARS1 This relates to engineered proteins of human tryptophanyl-tRNA synthetase I (WARS1) (hereinafter referred to as "engineered proteins") that have therapeutic effects on tuberculosis. Specifically, it relates to engineered proteins containing one to three amino acid variations.

[0069] The sequence information of wild-type WARS1 (WARS1 WT) used in this embodiment is shown in Table 1.

[0070] Table 1

[0071]

[0072] The engineered proteins designed with 1 to 3 amino acid variations are shown in Table 2.

[0073] Table 2

[0074] The engineered proteins containing one to three amino acid variations, as described in Table 2, are used in the examples described later.

[0075] Example 2. In vitro efficacy evaluation of single and complex engineered proteins The in vitro efficacy of the single engineered proteins (numbers 1 to 25 in Table 2) and the composite engineered proteins (numbers 26 to 47 in Table 2) designed in Example 2 was evaluated. Specifically, 2 × 10⁶ proteins were seeded per well in a 96-well culture plate. 4 Following the J774a.1 (ATCC, TIB-67) assay, cells were cultured for 24 hours in Duchenne Modified Igor Medium (DMEM) containing fetal bovine serum (FBS). Cells were then treated with different concentrations of WARS1 WT and single or multiple engineered proteins. After 18 hours, cell culture was collected for enzyme-linked immunosorbent assay (ELISA) of mCXCL2 and mTNFα (R&D Systems, DY452, Biolegend, #430904). Results of ELISA assessment of the in vitro efficacy of single and multiple engineered proteins are as follows: Figure 1 and Figure 2 As shown.

[0076] like Figure 1 and Figure 2 As shown, compared with wild-type WARS1, the engineered protein L046I, K047R, R133H, I136M and F137Y significantly increased mCXCL2 / MIP2 and mTNFα, which are homologues of IL-8.

[0077] And, as Figure 2 As shown, the engineered proteins A168C / F360C, A376C / V398C, L046I / A168C / F360C, and L046I / A376C / V398C were found to increase the expression of mTNF and mCXCL2 / MIP2.

[0078] Example 3. In vitro stability assessment of single engineered proteins and composite engineered proteins In this embodiment, the in vitro stability of single engineered proteins (L046I, K047R, R133H, I136M, and F137Y) and composite engineered proteins (A168C / F360C, A376C / V398C, L046I / A168C / F360C, L046I / A376C / V398C, K047R / A168C / F360C, and K047R / A376C / V398C) was evaluated. Specifically, the temperature resistance of the composite engineered proteins was assessed using a protein thermal stability (PTS) test. Specifically, 5 μg of wild-type WARS1, a single engineered protein, or a composite engineered protein was added to Protein Thermal Shift™ dye (Thermo Fisher Scientific, #4461146) and distilled water to prepare the analytical sample. The prepared analytical samples were analyzed using the methods shown by the manufacturing company (Thermo Fisher Scientific, Protein Thermal Shift™ software v1.4). The melting point (Tm, °C) of each engineered protein was analyzed using this method; an increase in Tm is interpreted as an increase in material stability. Results evaluating the in vitro stability of single engineered proteins and composite engineered proteins are as follows: Figure 3 As shown.

[0079] like Figure 3 As shown, the results of analyzing the Tm of proteins under harsh conditions confirmed that the Tm of single engineered proteins R133H and I136M decreased, while the Tm of L046I, K047R, and F137Y increased. The Tm of composite engineered proteins containing the A168C / F360C combination or the A376C / V398C combination increased.

[0080] Example 4. In vitro anti-tuberculosis efficacy evaluation of the engineered protein complex L046I / A376C / V398C In the foregoing embodiments, it was confirmed that the engineered protein L046I / A376C / V398C increased the expression of mTNF and mCXCL2 / MIP2, and exhibited high stability. Therefore, this embodiment evaluates the in vitro anti-tuberculosis efficacy of the engineered protein L046I / A376C / V398C. Specifically, Mtb-infected mouse macrophages J774.1A were treated alone with wild-type WARS1 and the engineered protein (L046I / A376C / V398C), or simultaneously with isoniazid (INH), a first-line anti-tuberculosis agent. The anti-tuberculosis efficacy of the candidate substance was then confirmed by measuring the CFU of Mycobacterium tuberculosis.

[0081] - Dosage concentration: H37Rv was infected with J774.1A, a mouse macrophage cell line, at 10 Moi. Each cell was then treated with the following substances: (i) 0.1 μg / ml INH; (ii) 100 nM wild-type WARS1; (iii) 100 nM engineered protein L046I / A376C / V398C; or (iv) 100 nM engineered protein L046I / A376C / V398C + 0.1 μg / ml INH. The number of tuberculosis bacteria was then measured on day 1 and day 3.

[0082] - Method for measuring the number of tuberculosis bacteria: After diluting the homogenized suspension of whole organs sequentially on Middlebrook 7H11 agar medium and spreading it onto plates, the plates were incubated at 37°C for 3-4 weeks and the colonies were counted. The number of viable tuberculosis bacteria in the cells on the first and third days was measured in this way and expressed as a percentage compared with the control group.

[0083] Results confirming the anti-tuberculosis efficacy of the candidate substances are as follows Figure 4 As shown.

[0084] like Figure 4 As shown, compared with the INH-only treatment group, the wild-type WARS1-only treatment group and the combined engineered protein L046I / A376C / V398C-only treatment group showed higher tuberculosis-killing ability. Furthermore, compared with the combined engineered protein L046I / A376C / V398C-only treatment group, the combined engineered protein L046I / A376C / V398C+INH treatment group showed significantly higher tuberculosis-killing ability. These results indicate that combined administration of the combined engineered protein L046I / A376C / V398C with anti-tuberculosis agents can significantly improve the anti-tuberculosis effect.

[0085] Example 5. Analysis of the distribution of immune cells in lung tissue following the administration route of the engineered protein L046I / A376C / V398C. The infiltration of immune cells in lung tissue was compared depending on the administration route of the engineered protein L046I / A376C / V398C. Specifically, the engineered protein L046I / A376C / V398C was administered to tuberculosis-infected C57BL / 6 mice via tail vein (10 mpk) or intranasal administration (1.5 mpk). Phosphate-buffered saline (PBS) was administered via tail vein at 10 mpk with minimal or no bleeding. Four hours after administration, the lungs of the mice were excised and inoculated with 1 mg / mL collagenase D and 10 units / mL DNase I, and isolated into single cells using GentleMACS. FACS staining was then performed using antibodies against FACS, live / dead staining solution, and FcγR blocking agent. Intracellular staining of iNOS and CD206 was performed using fixation / perm buffer (ebioscience, #00-5523-00). Neutrophil analysis of intracellular CD45 + CD11b + SiglecF - Ly6G + % of eosinophils analyzed intracellular CD45 in live cells + CD11b int SiglecF + Ly6G - % of alveolar macrophages, as lung tissue-specific macrophages, were analyzed for intracellular CD45 content in living cells. + CD11b int SiglecF + Ly6G + CD64 + % of. Antibody information used in the analysis is shown in Table 3.

[0086] Table 3

[0087] The results of the analysis of the distribution of immune cells in lung tissue following the administration route of the engineered protein complex L046I / A376C / V398C are as follows: Figure 5 As shown.

[0088] like Figure 5As shown, tail vein administration of the engineered protein complex L046I / A376C / V398C significantly increased the infiltration of macrophages, neutrophils, and eosinophils, and exhibited increased polarization towards iNOS-positive M1 macrophages. Furthermore, it was confirmed that compared to the tail vein administration group, the nasal administration group showed a 1.4–2.2-fold increase in macrophage and neutrophil infiltration into lung tissue. These results indicate that even low-concentration nasal administration of the engineered protein complex L046I / A376C / V398C can effectively infiltrate immune cells into lung tissue.

[0089] Example 6. In vivo anti-tuberculosis efficacy evaluation of the engineered protein complex L046I / A376C / V398C The anti-tuberculosis efficacy was evaluated after intravenous administration of either engineered protein L046I / A376C / V398C or wild-type WARS1 to mice with active tuberculosis infection. Specifically, mice with active tuberculosis infection were infected with 624 Mycobacterium tuberculosis (M. tb Erdman) via airborne infection. Starting two weeks later, mice were treated with either engineered protein L046I / A376C / V398C or wild-type WARS1 three times weekly for a total of 13 weeks. During treatment, after intravenous administration of either engineered protein L046I / A376C / V398C (0.25 mpk) or wild-type WARS1 (0.25 mpk), Mycobacterium tuberculosis Cfu was confirmed in lung tissue and spleen, and the degree of inflammation was analyzed by lung tissue staining.

[0090] The Cfu (cluster fibrosis) of Mycobacterium tuberculosis was measured by excising lung and spleen tissues from mice in each experimental group and determining the number of Mycobacterium tuberculosis. The number of viable Mycobacterium tuberculosis in the lung and spleen tissues was measured by sequentially diluting the homogenized suspension of the whole organs onto Middlebrook 7H11 agar medium, plating it, culturing it at 37°C for 3–4 weeks, and counting the colonies. The results were expressed as a mean logarithmic value for each tissue from the entire lung and spleen. 10 It is expressed as CFU ± standard deviation.

[0091] The results of evaluating the in vivo anti-tuberculosis efficacy of the engineered protein complex L046I / A376C / V398C are as follows: Figure 6 As shown.

[0092] like Figure 6 As shown, the tail vein administration group of the engineered protein L046I / A376C / V398C significantly inhibited the growth of Mycobacterium tuberculosis compared with the wild-type WARS1 tail vein administration group. Furthermore, both the tail vein administration group and the nasal administration group of the engineered protein L046I / A376C / V398C significantly inhibited the growth of Mycobacterium tuberculosis at low concentrations.

[0093] In summary, the inventors have developed an engineered protein comprising WARS1 with one to three amino acid variations. The engineered protein was confirmed to not only have high thermostability but also significantly inhibit the growth of Mycobacterium tuberculosis. This indicates that the engineered protein of this invention has excellent anti-tuberculosis efficacy and can be utilized in the prevention, improvement, or treatment of tuberculosis.

[0094] The foregoing has described specific aspects of the present invention in detail. However, those skilled in the art will understand that these specific descriptions are merely preferred embodiments, and the scope of the present invention is not limited thereto. Therefore, the essential scope of the present invention should be defined by the scope of the claims and their equivalents.

Claims

1. An engineered protein, characterized in that, Contains a polypeptide represented by the amino acid sequence of SEQ ID NO:

2. The polypeptide contains one or more amino acid variations.

2. The engineered protein according to claim 1, characterized in that, The engineered protein is used to treat tuberculosis.

3. The engineered protein according to claim 1, characterized in that, The polypeptide contains variations at one or more positions selected from the amino acid sequence of SEQ ID NO: 2, consisting of P006, A007, S008, E011, F013, T018, S032, V044, S045, L046, K047, S049, A054, T089, R127, L132, R133, I136, F137, A168, F360, A376, and V398.

4. The engineered protein according to claim 3, characterized in that, It includes one or more amino acid variations selected from the group consisting of P006C, A007C, A007G, S008T, E011D, F013Y, T018A, S032P, V044L, V044M, S045Q, L046I, K047R, S049T, A054T, T089N, R127K, L132M, R133H, I136M, F137Y, A168C, F360C, A376C, and V398C.

5. The engineered protein according to claim 4, characterized in that, The polypeptide comprises two or more amino acid variations selected from the group consisting of A168C, A376C, V398C, L046I, F360C, and K047R.

6. The engineered protein according to claim 5, characterized in that, The polypeptide also contains amino acid variations of L046I or K047R.

7. A nucleic acid, characterized in that, Encodes the engineered protein according to any one of claims 1 to 6.

8. A recombinant vector, characterized in that, It includes the nucleic acid as described in claim 7.

9. A pharmaceutical composition for the prevention or treatment of tuberculosis, characterized in that, It comprises the engineered protein according to any one of claims 1 to 6.

10. The pharmaceutical composition for the prevention or treatment of tuberculosis according to claim 9, characterized in that, The composition further comprises one or more antituberculosis agents selected from the group consisting of rifampin, isoniazid, pyrazinamide and ethambutol.

11. A food composition for the prevention or improvement of tuberculosis, characterized in that, It comprises the engineered protein according to any one of claims 1 to 6.

12. A health food composition for preventing or improving tuberculosis, characterized in that, It comprises the engineered protein according to any one of claims 1 to 6.

13. A composition for enhancing immunity, characterized in that, It comprises the engineered protein according to any one of claims 1 to 6.

14. The composition for enhancing immunity according to claim 13, characterized in that, The engineered protein promotes tissue infiltration of neutrophils, eosinophils, or macrophages.

15. A treatment method for tuberculosis, characterized in that, The step includes administering the engineered protein of any one of claims 1 to 6 to an individual in need.

Images