Production and application of flagellin immunopotentiator derivatives with TLR5 activity using a eukaryotic cell expression system

Abstract

The present invention relates to the production and application of flagellin immunopotentiator derivatives with TLR5 activity using a eukaryotic cell expression system. The flagellin variants of the present invention have a high immunopotentiating effect and can be used in various vaccines and immunotherapeutic compositions.

Description

[Technical Field]

[0001] This invention was made under the support of the Ministry of Science and ICT under project number 2020R1A5A2031185 (contribution rate 60%), the research management organization for this project is the Korea Research Foundation, the research project name is "Collective Research Support", the research topic name is "Complex Cancer Immunotherapy Research Center", the sponsoring organization is Jeongnam University, and the research period is from 2018.06.01 to 2025.02.28.

[0002] This invention was made under the support of the Ministry of Science and ICT under project number 2020M3A9G3080282 (contribution rate 20%), the research management organization for this project is the Korea Research Foundation, the research project name is "Biomedicine Technology Development", the research topic name is "Immunotherapy Innovation Research Group", the lead organization is Jeongnam University, and the research period is from 2020.06.01 to 2024.12.31.

[0003] This invention was made under the support of the Ministry of Health and Welfare under project number HV22C0079 (contribution rate 20%), the research management specialist for this project is the Korea Health Industry Development Institute, the research project name is "Vaccine-based technology development", the research topic name is "Development of a general-purpose platform for cellular immunity-inducing mucosal immune enhancers", the lead institution is Jeongnam University, and the research period is from 2022.04.01 to 2024.12.31.

[0004] The present invention relates to the production and application of flagellin immunopotentiator derivatives with TLR5 activity using a eukaryotic cell expression system, and more specifically to flagellin proteins that can be expressed in eukaryotic cells and have immunopotentiating effects equivalent to or greater than those of existing prokaryotic flagellin proteins. [Background technology]

[0005] The unit proteins that make up the filaments of bacterial flagella are called flagellins, and flagellins are regularly combined to form filaments that enable bacteria to move.

[0006] TLR5 is a pattern recognition receptor expressed on the surface of various immune cells, including macrophages and dendritic cells. When TLR5 recognizes flagellin, it activates NF-kB and induces the production of proinflammatory cytokines. NF-kB is a transcription factor that regulates the expression of genes involved in immune and inflammatory responses. It exists as a cytoplasmic protein complex containing an inhibitory protein called IkB (NF-kB inhibitor). When TLR5 is stimulated and activated, IkB is degraded and NF-kB activation is induced. NF-kB activation induces the production of proinflammatory cytokines such as TNF-α (Tumor Necrosis Factor-α) and IL-1β (Interleukin-1β).

[0007] Flagellin is also a pathogen-associated molecular pattern factor that can stimulate the NLRC4-inflammasome signaling system present in the cytoplasm. Therefore, flagellin is an immunomodulator that can activate both TLR5 present on the cell surface and the NLRC4-inflammasome present in the cytoplasm.

[0008] Because of these characteristics, flagellin has been studied as a potential immune enhancer for various vaccines and immunotherapeutics. When various antigens are administered together with flagellin or in the form of a recombinant fusion protein, flagellin enhances antigen-specific immune responses, demonstrating its immune-enhancing efficacy in influenza vaccines, pneumonia vaccines, bacterial periodontal disease, anti-cancer therapeutic vaccines, norovirus vaccines, neurodegenerative diseases, and various immune disorders. Furthermore, flagellin-mediated TLR5 activation has been reported to protect hematopoietic cells and gastrointestinal tissues from radiation and to affect the survival and growth of cancer cells.

[0009] However, research into the effects of flagellin as an immunopotentiator has been conducted using recombinant proteins expressed in the prokaryotic cell Escherichia coli. However, to extend the application of flagellin as an immunopotentiator to nucleic acid-based vaccines (e.g., DNA vaccines or mRNA vaccines) or antigens requiring post-translational modification, it is necessary to produce flagellin expressed in a eukaryotic cell expression system. However, flagellin produced in a eukaryotic cell system exhibits significantly reduced TLR5 signaling activity compared to flagellin expressed in prokaryotic cells. This phenomenon is presumed to be due to the inefficient induction of TLR5 signaling when flagellin is expressed in eukaryotic cells, due to steric hindrance caused by post-translational modification.

[0010] Therefore, there is a need for research into flagellin proteins that can be produced in eukaryotic cells and have potent immunomodulatory properties as well as effective vaccine immunopotentiator functions. Summary of the Invention [Problem to be solved by the invention]

[0011] Therefore, the present inventors produced flagellin protein in eukaryotic cells and confirmed that it has particularly excellent immune enhancing ability and safety.

[0012] Specifically, the inventors confirmed that flagellin produced in a eukaryotic cell system exhibited significantly weaker TLR5-stimulating activity than flagellin expressed in prokaryotic cells. We speculate that this phenomenon is due to the production of glycosylated flagellin (glycosylated-eFlaB) through post-translational modification when flagellin is expressed in eukaryotic cells, and that this glycosylated flagellin was unable to efficiently induce TLR5 signaling due to steric hindrance caused by the sugar molecules. To support this hypothesis, bioinformatics analysis confirmed that flagellin expressed in eukaryotic cells contains five N-glycosylation sites. Therefore, we used site-directed mutagenesis to create flagellin variants in which asparagine, the predicted N-glycosylation site, was replaced with alanine, and the activity of these variants was examined in vitro and in vivo. As a result, we derived eukaryotic cell-expressed flagellin immunopotentiator variants (eukFlaB variants) that exhibit the same TLR5 stimulatory activity and immunopotentiator efficacy as pFlaB. Interestingly, these variants (eukFlaB variants) also exhibited deglycosylated properties. In summary, by using immunoinformatic analysis and protein modification strategies to establish a deglycosylated eukaryotic-expressed flagellin that does not induce N-glycosylation, we have derived a flagellin immunopotentiator that is safer and has various clinical applications.

[0013] It is therefore an object of the present invention to provide a flagellin protein that can be expressed in eukaryotic cells.

[0014] Another object of the present invention is to provide an adjuvant composition for a vaccine comprising a flagellin protein that can be expressed in eukaryotic cells.

[0015] It is still another object of the present invention to provide a composition for enhancing immune function, which comprises a flagellin protein that can be expressed in eukaryotic cells.

[0016] It is yet another object of the present invention to provide a pharmaceutical composition for preventing or treating microbial infection or cancer, comprising a flagellin protein that can be expressed in eukaryotic cells.

[0017] It is yet another object of the present invention to provide an immunoadjuvant use of a flagellin protein that can be expressed in eukaryotic cells.

[0018] Yet another object of the present invention is to provide an immune function enhancing use of a flagellin protein that can be expressed in eukaryotic cells.

[0019] It is yet another object of the present invention to provide a use of a flagellin protein expressible in eukaryotic cells for the prevention or treatment of autoimmune diseases, inflammatory diseases, microbial infections, or cancer. [Means for solving the problem]

[0020] The present invention relates to the production and application of flagellin immunopotentiator derivatives having TLR5 activity using a eukaryotic cell expression system, and the flagellin protein of the present invention exhibits a high immunopotentiating effect.

[0021] The present invention will now be described in more detail.

[0022] One aspect of the present invention relates to a polypeptide expressible in a eukaryotic cell, in which one or more asparagines (Asparagine, N) in the amino acid sequence of SEQ ID NO: 13 have been substituted with alanines (Alanine).

[0023] In one embodiment of the present invention, the polypeptide may have asparagine substituted with alanine at one or more positions selected from the group consisting of positions 83, 101, 139, 273, and 330 in the amino acid sequence of SEQ ID NO: 13.

[0024] In one embodiment of the present invention, the polypeptide may have asparagine substituted with alanine at position 83 of the amino acid sequence of SEQ ID NO: 13; and at one or more positions selected from the group consisting of positions 101, 139, 273, and 330.

[0025] In one embodiment of the present invention, the polypeptide may have asparagine substituted with alanine at positions 83 and 101 of the amino acid sequence of SEQ ID NO: 13; and at one or more positions selected from the group consisting of positions 139, 273, and 330.

[0026] In one embodiment of the present invention, the polypeptide may have asparagine substituted with alanine at one or more positions selected from the group consisting of positions 83, 101, and 139, and positions 273 and 330 in the amino acid sequence of SEQ ID NO: 13.

[0027] In one embodiment of the present invention, the polypeptide may comprise one amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs: 14-23.

[0028] The polypeptide of the present invention has excellent TLR-5 activation ability and exhibits a high immune enhancing effect, and therefore can be used as, but is not limited to, an immune adjuvant composition for vaccines, a composition for enhancing immune function, or a pharmaceutical composition for preventing or treating autoimmune diseases, inflammatory diseases, microbial infections, or cancer.

[0029] The polypeptide of the present invention has an excellent effect of inducing the production of antibodies specific to influenza antigens when administered together with an influenza vaccine, and can therefore be used as, but not limited to, an immune adjuvant composition for influenza vaccines or an immune adjuvant composition for influenza treatment.

[0030] Another aspect of the present invention relates to an adjuvant composition for a vaccine, comprising a polypeptide in which one or more asparagines (Asparagine, N) in the amino acid sequence of SEQ ID NO: 13 are substituted with alanine (Alanine).

[0031] In one embodiment of the present invention, the polypeptide may have asparagine substituted with alanine at one or more positions selected from the group consisting of positions 83, 101, 139, 273, and 330 in the amino acid sequence of SEQ ID NO: 13.

[0032] In one embodiment of the present invention, the polypeptide may have asparagine substituted with alanine at position 83 of the amino acid sequence of SEQ ID NO: 13; and at one or more positions selected from the group consisting of positions 101, 139, 273, and 330.

[0033] In one embodiment of the present invention, the polypeptide may have asparagine substituted with alanine at positions 83 and 101 of the amino acid sequence of SEQ ID NO: 13; and at one or more positions selected from the group consisting of positions 139, 273, and 330.

[0034] In one embodiment of the present invention, the polypeptide may have asparagine substituted with alanine at one or more positions selected from the group consisting of positions 83, 101, and 139, and positions 273 and 330 in the amino acid sequence of SEQ ID NO: 13.

[0035] In one embodiment of the present invention, the polypeptide may comprise one amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs: 14-23.

[0036] The immune adjuvant composition for vaccines according to the present invention may further comprise one or more commonly used pharmaceutically and / or veterinarily acceptable carriers, diluents, antibiotics, adjuvants, etc.

[0037] Carriers may include any solvents (solutions), dispersion media, coatings, buffers, isotonic agents, suspensions, colloids, stabilizers, preservatives, antibiotics (antibacterial agents, antifungal agents, etc.), absorption retardants, adjuvants, inserts, and the like, that are pharmaceutically acceptable for administration to a target animal (host animal). In general, the use of one or more delivery vehicles for chemical compounds, particularly immunogens, is well known to those skilled in the pharmaceutical industry. Insofar as any conventional media or agent is incompatible with the active ingredient, its use in diagnostic, prophylactic, and therapeutic compositions is contemplated. One or more supplementary active ingredients may also be incorporated into or administered together with one or more of the disclosed immunogenic compositions and vaccine formulations.

[0038] Diluents may include water, saline, dextrose, ethanol, glycerol, etc., isotonic agents may include sodium chloride, dextrose, mannitol, sorbitol, lactose, etc., and stabilizers may include, but are not limited to, albumin.

[0039] Adjuvants include mineral gels, such as aluminum hydroxide gels; surface-active substances, such as lysolecithin; glycosides, such as saponin derivatives, for example, Quil A or GPI-0100 (Galenica Pharmaceuticals, Inc., Birmingham, Alabama); pluron polyols; polyanions; nonionic block polymers, such as Pluron F-127 (BASF, USA); peptides; mineral oils, such as Montanide ISA-206 (Seppic, France), Montanide ISA-50 (Seppic, France), Cabopol, Amphigen, Amphigen Mark II (Hydronic, USA), alhydrogel, oil emulsions (e.g., BayolF / Arlacel). Examples of adjuvants include, but are not limited to, emulsions of mineral oil and water such as PEG-100 (a mineral oil and water emulsion such as PEG-100), or emulsions of vegetable oil, water, and an emulsifier such as lecithin; alum; cholesterol; Freund complete and incomplete adjuvants; block copolymers (CytRx, Atlanta, GA); SAF-M (Chiron, Emeryville, CA); AMPHIGEN® adjuvant; monophosphoryl lipid A, Avridine lipid-amine adjuvant; heat-labile enterotoxin from E. coli (e.g., recombinant); cholera toxin; muramyl dipeptide; IMS 1313 (Seppic, France); ISA206; Montanide 01 gel (Seppic, France); and mixtures of these adjuvants, preferably one or more adjuvants selected from the group consisting of IMS 1313, Cabopol, and Montanide 01 gel. The adjuvant refers to a compound or mixture that enhances the immune response and / or speeds up absorption after inoculation, and includes any absorption enhancer.

[0040] Yet another aspect of the present invention relates to a composition for enhancing immune function, comprising a polypeptide in which one or more asparagines (Asparagine, N) in the amino acid sequence of SEQ ID NO: 13 are substituted with alanine (Alanine).

[0041] In one embodiment of the present invention, the polypeptide may have asparagine substituted with alanine at one or more positions selected from the group consisting of positions 83, 101, 139, 273, and 330 in the amino acid sequence of SEQ ID NO: 13.

[0042] In one embodiment of the present invention, the polypeptide may have asparagine substituted with alanine at position 83 of the amino acid sequence of SEQ ID NO: 13; and at one or more positions selected from the group consisting of positions 101, 139, 273, and 330.

[0043] In one embodiment of the present invention, the polypeptide may have asparagine substituted with alanine at positions 83 and 101 of the amino acid sequence of SEQ ID NO: 13; and at one or more positions selected from the group consisting of positions 139, 273, and 330.

[0044] In one embodiment of the present invention, the polypeptide may have asparagine substituted with alanine at one or more positions selected from the group consisting of positions 83, 101, and 139, and positions 273 and 330 in the amino acid sequence of SEQ ID NO: 13.

[0045] In one embodiment of the present invention, the polypeptide may comprise one amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs: 14-23.

[0046] Yet another aspect of the present invention relates to a pharmaceutical composition for preventing or treating an autoimmune disease, an inflammatory disease, a microbial infection, or cancer, comprising a polypeptide in which one or more asparagines (Asparagine, N) in the amino acid sequence of SEQ ID NO: 13 are substituted with alanine.

[0047] In one embodiment of the present invention, the autoimmune disease is selected from the group consisting of hemophagocytic lymphohistiocytosis, systemic lupus erythematosus, Kikuchi's disease, vasculitis, adult onset Still's disease, rheumatoid arthritis, inflammatory myositis, Behcet's disease, IgG4-linked disease, Sjogren's syndrome, giant cell arteritis, temporal arteritis, type 1 diabetes, atopic dermatitis, Crohn's disease, systemic sclerosis, psoriasis, multiple sclerosis, and Graves' hyperthyroidism. The present invention may be, but is not limited to, one or more selected from the group consisting of:

[0048] In one embodiment of the present invention, the inflammatory disease may be one or more selected from the group consisting of sepsis, gastritis, enteritis, nephritis, hepatitis, chronic obstructive pulmonary disease (COPD), pulmonary fibrosis, irritable bowel syndrome, inflammatory pain, migraine, headache, lower back pain, fibromyalgia, fascial disease, viral infection, bacterial infection, fungal infection, burns, wounds from surgical or dental operations, prostaglandin excess syndrome, atherosclerosis, gout, Hodgkin's disease, pancreatitis, conjunctivitis, iritis, sclera, uveitis, and eczema, but is not limited thereto.

[0049] In one embodiment of the present invention, the microorganism is selected from the group consisting of influenza virus, coronavirus, hepatitis virus, Zika virus, dengue virus (DENV), hanta virus, cytomegalovirus (CMV), Epstein-Barr virus (EBV), human immunodeficiency virus (HIV), herpes simplex virus (HSV), chikungunya virus, enterovirus, Marburg hemorrhagic fever virus, parvovirus, severe fever with thrombocytopenia syndrome virus (SFTSV), rotavirus, norovirus, and mycobacterium tuberculosis. tuberculosis), Group A Streptococcus (GAS), Group B Streptococcus (GBS), Staphylococcus aureus, Shigella, and pathogenic E. coli.The bacterial pathogen may be one or more selected from the group consisting of, but is not limited to, Salmonella, Chlamydia, Pseudomonas aeruginosa, Non-typeable Haemophilus influenzae, Klebsiella pneumoniae, Clostridium difficile, Plasmodium, Leishmania, Schistosoma, Trypanosoma, Brucella, Cryptosporidium, and Entamoeba.

[0050] In one embodiment of the present invention, the cancer may be one or more selected from the group consisting of breast cancer, lung cancer, colon cancer, kidney cancer, liver cancer, ovarian cancer, prostate cancer, testicular cancer, urogenital tract cancer, lymphatic system cancer, rectal cancer, pancreatic cancer, esophageal cancer, gastric cancer, cervical cancer, thyroid cancer, and skin cancer, but is not limited thereto.

[0051] In one embodiment of the present invention, the polypeptide may have asparagine substituted with alanine at one or more positions selected from the group consisting of positions 83, 101, 139, 273, and 330 in the amino acid sequence of SEQ ID NO: 13.

[0052] In one embodiment of the present invention, the polypeptide may have asparagine substituted with alanine at position 83 of the amino acid sequence of SEQ ID NO: 13; and at one or more positions selected from the group consisting of positions 101, 139, 273, and 330.

[0053] In one embodiment of the present invention, the polypeptide may have asparagine substituted with alanine at positions 83 and 101 of the amino acid sequence of SEQ ID NO: 13; and at one or more positions selected from the group consisting of positions 139, 273, and 330.

[0054] In one embodiment of the present invention, the polypeptide may have asparagine substituted with alanine at one or more positions selected from the group consisting of positions 83, 101, and 139, and positions 273 and 330 in the amino acid sequence of SEQ ID NO: 13.

[0055] In one embodiment of the present invention, the polypeptide may comprise one amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs: 14-23.

[0056] The pharmaceutical compositions of the present invention may be administered in a pharmaceutically effective amount.

[0057] As used herein, the term "pharmaceutically effective amount" refers to an amount sufficient to achieve the efficacy or activity of the composition described above.

[0058] The pharmaceutical composition of the present invention may contain a pharmaceutically acceptable carrier.

[0059] The pharmaceutically acceptable carriers contained in the pharmaceutical composition of the present invention are those commonly used in formulations, including, but not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil. In addition to the above ingredients, the pharmaceutical composition of the present invention may further contain lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc.

[0060] The pharmaceutical composition of the present invention may be administered to mammals, including humans, via various routes, including any conventional route, such as oral, cutaneous, intravenous, intramuscular, or subcutaneous, preferably oral.

[0061] The appropriate dosage of the pharmaceutical composition of the present invention varies depending on factors such as the formulation method, administration method, age, weight, sex, pathological condition, diet of the patient, administration time, administration route, excretion rate, and reaction sensitivity, and an ordinary skilled physician can easily determine and prescribe an effective dosage for the desired treatment or prevention.

[0062] The pharmaceutical compositions of the present invention may be prepared in unit dose form or in multi-dose containers by formulating them with pharmaceutically acceptable carriers and / or excipients by a method readily practiced by those skilled in the art to which this invention pertains. In this case, the dosage form may be in the form of a solution, suspension, or emulsion in an oil or aqueous medium, or in the form of an extract, powder, granules, tablets, capsules, or gel (e.g., hydrogel), and may further contain a dispersing agent or stabilizer.

[0063] Yet another aspect of the present invention relates to the use of a polypeptide in which one or more asparagines (Asparagine, N) in the amino acid sequence of SEQ ID NO: 13 are substituted with alanine (Alanine) as a vaccine immunoadjuvant.

[0064] Yet another aspect of the present invention relates to the use of a polypeptide in which one or more asparagines (Asparagine, N) in the amino acid sequence of SEQ ID NO: 13 are substituted with alanine (Alanine) for enhancing immune function.

[0065] Yet another aspect of the present invention relates to the use of a polypeptide in which one or more asparagines (Asparagine, N) in the amino acid sequence of SEQ ID NO: 13 are substituted with alanine (Alanine) for the prevention or treatment of autoimmune diseases, inflammatory diseases, microbial infections, or cancer. [Effects of the Invention]

[0066] The present invention relates to the production and application of flagellin immunopotentiator derivatives with TLR5 activity using a eukaryotic cell expression system, and the flagellin variants of the present invention have high immunopotentiating effects and can be used in various vaccines and immunotherapeutic compositions. [Brief explanation of the drawings]

[0067] [Figure 1] FIG. 1 shows the results of Western blot analysis of wild-type flagellin (prokFlaB) derived from Vibrio septicemicus expressed in Escherichia coli, a prokaryotic cell, and wild-type flagellin (eukFlaB) expressed in Expi293™ cells, a eukaryotic cell, according to one embodiment of the present invention. [Figure 2] 1 is a graph showing the results of a comparison of the TLR5-stimulating activity of wild-type flagellin (prokFlaB) derived from Vibrio septicemicus expressed in Escherichia coli, a prokaryotic cell, and wild-type flagellin (eukFlaB) expressed in Expi293™ cells, a eukaryotic cell, according to one embodiment of the present invention. [Figure 3A] FIG. 1 shows the results of Western blotting to confirm the protein state before and after treatment of wild-type flagellin (eukFlaB) expressed in eukaryotic cells, Expi293™ cells, with PNGase (Peptide N-glycosidase F), a deglycosylating enzyme, in accordance with one embodiment of the present invention. [Figure 3B] 1 is a graph showing the results of comparing the TLR5 stimulating activity of wild-type flagellin (eukFlaB) expressed in eukaryotic cells, Expi293™ cells, before and after treatment with PNGase (Peptide N-glycosidase F), a deglycosylating enzyme, according to one embodiment of the present invention. [Figure 4] 1 shows the results of predicting the glycated amino acid sequence of flagellin (FlaB) according to one embodiment of the present invention. [Figure 5] FIG. 1 shows the construction of flagellin variants induced by site-directed mutagenesis. [Figure 6] 6 and 7 show the results of examining the characteristics and TLR5 stimulating activity of flagellin variants expressed in eukaryotic cells, Expi293™ cells, according to one embodiment of the present invention. [Figure 7] 6 and 7 show the results of examining the characteristics and TLR5 stimulating activity of flagellin variants expressed in eukaryotic cells, Expi293™ cells, according to one embodiment of the present invention. [Figure 8] The results show the binding of flagellin variants to TLR5 measured by Co-IP (Co-immunoprecipitation). [Figure 9] This shows the results of a comparison of the immune-enhancing efficacy of wild-type flagellin (prokFlaB) derived from Vibrio septicemicus expressed in Escherichia coli, a prokaryotic cell, wild-type flagellin (eukFlaB) expressed in Expi293™ cells, a eukaryotic cell, and various flagellin variants in an influenza vaccine model according to one embodiment of the present invention. [Figure 10] In one embodiment of the present invention, wild-type flagellin (prokFlaB) derived from Vibrio septicemicus expressed in Escherichia coli, a prokaryotic cell, and wild-type flagellin (eukFlaB), eukFlaBN83 / 101 / 139A, eukFlaBN83 / 101 / 139 / 273A, and eukFlaBN83 / 101 / 139 / 273 / 330A expressed in Expi293™ cells, a eukaryotic cell, were used as immune enhancers in an influenza vaccine model, and the results show that prokFlaB- or eukFlaB-selective antibody production is comparable. [Figure 11] 1 shows the results of a comparison of the stability of wild-type flagellin (prokFlaB) derived from Vibrio septicemicus expressed in Escherichia coli, a prokaryotic cell, wild-type flagellin (eukFlaB) expressed in Expi293™ cells, a eukaryotic cell, and various flagellin variants according to one embodiment of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0068] The present invention relates to a polypeptide that can be expressed in a eukaryotic cell, in which one or more asparagines (Asparagine, N) in the amino acid sequence of SEQ ID NO: 13 have been substituted with alanines (Alanine). [Example]

[0069] The present invention will now be described in more detail with reference to the following examples, which are merely for illustrative purposes and are not intended to limit the scope of the present invention.

[0070] Throughout this specification, "%" used to indicate the concentration of a particular substance is (wt / wt)% for solid / solid, (wt / vol)% for solid / liquid, and (vol / vol)% for liquid / liquid, unless otherwise specified.

[0071] Example 1: Construction of a plasmid for producing wild-type flagellin expressed in eukaryotic cells

[0072] Vibrio vulnificus FlaB (prokFlaB) expressed in prokaryotic cells was produced by the method disclosed in Lee SE, et al. A bacterial flagellin, Vibrio vulnificus FlaB, has a strong mucosal adjuvant activity to induce protective immunity. Infect Immun. 2006;74(1):694-702.

[0073] To express FlaB in eukaryotic cells, it was amplified by PCR using the forward primer eukFlaB-F, which contains a HindIII (AAGCTT) cleavage site, and the reverse primer eukFlaB-R, which contains a NotI cleavage site, as shown in Table 1. FlaB was synthesized based on codon usage (frequency) optimized for mammalian cell expression and cloned into the pBHA plasmid, pBHA::eukFlaB (Bioneer, Korea), which was used as the PCR template.

[0074] [Table 1]

[0075] The eukFlaB DNA amplification product and the eukaryotic expression vector pSectag2B (Invitrogen, V900-20) were digested overnight at 37°C with a pair of restriction enzymes (REs) HindIII and NotI. The digested PCR product and the eukaryotic expression vector pSectag2B were ligated at room temperature for 2 hours using T4 DNA ligase (Enzynomics). The new construct, pSectag2B::eukFlaB plasmid, was transformed into E. coli competent cells TOP10 (Invitrogen) and cultured on LB plates containing ampicillin. The DNA sequence of the expression vector was confirmed by dideoxy-chain termination sequencing using the Macrogen Online Sequencing Order System (http: / / dna.macrogen.com / kor / ).

[0076] Example 2: Recombinant protein production using eukaryotic cell expression systems

[0077] Expi293™ cells, a eukaryotic expression cell line, were purchased from Thermofish Scientific (Thermofish Scientific, Cat#. A14635) and used in the present invention. The transfection procedure followed the manufacturer's protocol. Expi293™ cells were cultured in an incubator at 37°C, 8% CO2, and agitated at 125 rpm. On day 0 (the day before transfection), Expi293™ cells were cultured in a corning culture flask at 3x10 6 On day 1 (the day of transfection), Expi293™ cells were counted and 3x10 6 The pSectag2B::eukFlaB plasmid was inoculated at 1 / ml and sterilized by filtration through a 0.22 μm filter (Millexmil-GV, SLGVR13) and added to the cell culture. On day 2, ExpiFectamine™ 293 transfection enhancer 1 and enhancer 2 were added, and the culture was continued until day 4. At this time, the recombinant protein was secreted into the culture medium by pSectag2B, which contains the IgK chain sequence.

[0078] On day 4, the culture medium containing the secreted protein was centrifuged at 17,000 rpm for 20 min at 4°C, and the cell-free supernatant was loaded onto a column containing Ni-NTA agarose beads (Qiagen, Hilden, Germany) according to the manufacturer's instructions. To remove residual proteins except for the desired protein, wash buffer I (50 mM NaHPO 4、 The column was washed with washing buffer II (50 mM NaH2PO4, 300 mM NaCl, and 20 mM imidazole, pH = 8) and washing buffer II (50 mM NaH2PO4, 300 mM NaCl, and 20 mM imidazole, pH = 8). The target protein was dissolved and isolated with elution buffer (50 mM NaH2PO4, 300 mM NaCl, and 250 mM imidazole, pH = 8) at room temperature for 15 minutes. The eluted protein was then dialyzed into phosphate-buffered saline (PBS).

[0079] The recombinant peptide was confirmed by SDS-PAGE and Western blot using anti-FlaB antibody raised in BALB / c mice, and the results are shown in Figure 1 .

[0080] Example 3: Verification of TLR5 stimulating activity of wild-type flagellin produced using a eukaryotic cell expression system

[0081] To verify the TLR5-stimulating activity of wild-type flagellin produced using a eukaryotic cell expression system, NF-κB activity was measured at various protein concentrations. Specifically, HEK-Blue™ hTLR5 cells (InvivoGen, hκb-htlr-5) were used with the HEK-Blue™ detection (InvivoGen, hb-det2) assay system according to the manufacturer's protocol. EC50 values ​​were calculated using triplicate OD 620 nm values ​​for each protein concentration. To confirm the biological activity of deglycosylated eukFlaB, in vitro NF-κB luciferase reporter assays were performed using HEK293T cells purchased from Thermo Fisher Scientific. HEK293T cells were maintained at 37°C and 5% CO2 in DMEM medium (Gibco™, cat. 11995-065) containing 10% FBS and 100 units / ml of antibiotics (Gibco™, cat. 15140122). For NF-κB reporter analysis, cells were passaged at least three times. HEK293T cells were cultured in 24-well plates (SPL Life Sciences Co., Ltd., Korea) at 2 × 10 5Cells were seeded at 1 / well and cultured for 24 hours. To transfect cells, reporter plasmids pNF-κB-luc (100 ng / well), p3xFlag-hTLR5 (100 ng / well), pCMV-β-Gal (50 ng / well), and Effectene (5 μl / well) (Qiagen, 301427) were added and cultured overnight. Transfected cells were treated with prokFlaB, eukFlaB, or mutant eukFlaB variants and cultured for 24 hours. After 24 hours, the culture medium was removed, and cells were lysed with cell lysis buffer (Promega, Madison, WI, USA, E153A). Fluorescent enzyme activity was measured in the cell lysates using a photometer (Berthold, Lumat-Plus LB96V).

[0082] As a result of the experiment, as shown in Figure 2, it was confirmed that eukFlaB induced significantly lower TLR5 stimulation than prokFlaB.

[0083] Example 4: Deglycosylation of eukaryotic cell-expressed flagellin using deglycosylation enzyme (Peptide N-glycosidase F) and verification of TLR5 stimulating activity of deglycosylated eukaryotic cell-expressed flagellin

[0084] Deglycosylation of eukFlaB expressed in eukaryotic cells was performed using PNGase F enzyme (Promega, cat. V483A) according to the manufacturer's protocol. PNGase F enzyme (10 U / μl) and eukFlaB (15 μg) were mixed and incubated at 37°C. After 2 hours, the degree of deglycosylation was confirmed by SDS-PAGE and Western blotting using an anti-FlaB antibody produced in BALB / c mice. The results are shown in Figure 3A. The TLR5-stimulating activity of deglycosylated eukFlaB was measured using the same method as in Example 3. The results are shown in Figure 3B.

[0085] As a result of the experiment, as shown in Figure 3B, it was confirmed that deglycosylated eukFlaB restored its TLR5-stimulating activity.

[0086] Example 5: Construction of various deglycosylated flagellin derivatives using site-directed mutagenesis and verification of the TLR5 stimulating activity of various deglycosylated eukaryotic cell-expressed flagellin derivatives

[0087] N-glycosylation sites in the FlaB amino acid sequence were predicted using NetNGlyc-1.0 ( https: / / services.healthtech.dtu.dk / service.php?NetNGlyc-1.0 ), a bioinformatic tool that determines the N-glycosylation consensus sequence Asn-Xaa-Ser / Thr.

[0088] As shown in Figure 4 , eukFlaB has five predicted glycosylated residues at N83, N101, N139, N273, and N330, and three residues (N83, N101, and N139) were confirmed to be located near the TLR5-binding domain.

[0089] Therefore, as shown in Figure 5, we introduced site-directed mutagenesis to generate various deglycosylated flagellin proteins [eukFlaB N83A , eukFlaB N101A , eukFlaB N139A , eukFlaB N273A , eukFlaB N330A , eukFlaB N83A,N101A (eukFlaB N83 / 101A ), eukFlaB N83A,N101A,N139A (eukFlaB N83 / 101 / 139A ), eukFlaB N83A,N101A,N139A,N273A (eukFlaB N83 / 101 / 139 / 273A ), eukFlaB N83A,N101A,N139A,N273A,N330A (eukFlaB N83 / 101 / 139 / 273 / 330A ), and eukFlaB N139A,N273A,N330A (eukFlaB N139 / 273 / 330A ) was produced.

[0090] Site-directed mutations of eukFlaB were generated according to the manufacturer's protocol (EZchange™ Site-directed Mutation, Cat. # EZ004S). Briefly, the pSectag2B::eukFlaB plasmid was used as a template for PCR reactions. The primers used (Table 2) were designed to generate site-directed mutations by changing the amino acid asparagine (N) corresponding to the specific codons "AAC" or "AAT" to the amino acid alanine (A) corresponding to the other specific codons "GCT" or "GCC." After isolating the parent plasmid from the PCR product using EZ-MIX enzyme, the plasmid was transformed into E. coli competent cells (water-soluble cells) TOP10 and spread onto LB plates containing ampicillin (100 μg / ml). The sequences of the positive eukFlaB mutants were confirmed using Microgen Online Sequencing (http: / / dna.macrogen.com / kor / ).

[0091] [Table 2]

[0092] The recombinant peptide was confirmed by SDS-PAGE and Western blot using anti-FlaB antibody raised in BALB / c mice, and the results are shown in FIG.

[0093] eukFlaB produced by the above method, eukFlaB N83A , eukFlaB N101A , eukFlaB N139A , eukFlaB N273A , eukFlaB N330A , eukFlaB N83 / 101A , eukFlaB N83 / 101 / 139A , eukFlaB N83 / 101 / 139 / 273A , eukFlaB N83 / 101 / 139 / 273 / 330A , and eukFlaB N139 / 273 / 330A The peptide sequences are shown in Table 3 below.

[0094] [Table 3] TIFF2025537485000005.tif182170

[0095] To determine the biological activity of various flagellins, we measured Toll-like receptor 5 (TLR5) signal-mediated NF-κB activation by a reporter assay. HEK293T cells were maintained in DMEM (Gibco™, cat. 11995-065) medium containing 10% FBS (Gibco™, cat. 16000044) and 100 units / ml penicillin and 100 μg / ml streptomycin antibiotics (Gibco™, cat. 15140122). HEK293T cells were plated at 2 x 10 cells per well in a 24-well plate (Corning Costar, cat. 3526). 5 Cells were seeded at 1 / well and cultured for 24 hours in a 37°C, 5% CO2 incubator. To transfect the cells, reporter plasmids pNF-κB-luc (100 ng / well), p3xFlag-hTLR5 (100 ng / well), pCMV-β-Gal (50 ng / well), and Effectene (5 μl / well) (Qiagen, 301427) were added and cultured overnight. Transfected cells were treated with prokFlaB, eukFlaB, or mutant eukFlaB variants and cultured for 24 hours. After 24 hours of culture, the medium was removed, the cells were lysed in cell lysis buffer, and the fluorescent enzyme activation of the cell lysates was measured using a photometer (Berthold, Lumat-Plus LB96V) to verify TLR5 signaling activity.

[0096] As a result of the experiment, as shown in Figure 7, eukFlaB, eukFlaB N101A , eukFlaB N139A , eukFlaB N273A , eukFlaB N330A , eukFlaB N139 / 273 / 330A does not induce TLR5 activation, but eukFlaBN83A , eukFlaB N83 / 101A , eukFlaB N83 / 101 / 139A , eukFlaB N83 / 101 / 139 / 273A , eukFlaB N83 / 101 / 139 / 273 / 330A We confirmed that eukFlaB induces TLR5 activation. N83 / 101 / 139A , eukFlaB N83 / 101 / 139 / 273A , eukFlaB N83 / 101 / 139 / 273 / 330A confirmed that it has the same level of TLR5-stimulating activity as prokaryotic flagellin (prokFlaB).

[0097] Example 6: Measuring the binding of flagellin variants to TLR5 using co-immunoprecipitation (Co-IP)

[0098] Co-immunoprecipitation (Co-IP) was used to measure the binding of flagellin variants to TLR5. Specifically, HEK293T cells were transfected with the p3XFlag-hTLR5 plasmid for 24 hours. Cells were lysed using RIPA buffer containing protease inhibitors, sonicated, and centrifuged to collect the supernatant. Proteins prokFlaB, eukFlaB, and eukFlaB were also detected. N83 / 101 / 139A , and eukFlaB N83 / 101 / 139 / 273 / 330AThe supernatant was mixed with the supernatant and rotated overnight at 4°C (Complex I). Simultaneously, Protein A / G plus-agarose (Santa Cruz Biotechnology, sc-2003) and anti-Flag tag antibody (Abcam, ab1162) were mixed and rotated overnight at 4°C (Complex II). Complex II was then washed five times with IP buffer (1% Triton X-100, 25 mM Tris pH 7.5, 10% glycerol, 150 mM NaCl, 1 mM DTT, 1 mM EDTA, and protease inhibitors). Complex I and II were mixed at 4°C for 6 hours, then washed five times with IP buffer to remove nonspecifically bound proteins. After heating for 10 minutes in 50 μl of 1x SDS loading buffer, the mixture was subjected to SDS-PAGE and additional Western blot analysis. Because the anti-FlaB eukFlaB variants are expressed with a Myc-tag that is well detected by anti-Myc, the amount of eukFlaB protein detected by glycosylation on eukFlaB is low. Therefore, two different antibodies, mouse anti-FlaB and anti-Myc-HRP (Novex®, 46-0709), were used in Western blot analysis to detect pFlaB and eukFlaB variants, respectively.

[0099] As a result of the experiment, as shown in Figure 8, it was observed that prokFlaB bound to TLR5 with high affinity, but eukFlaBsms did not bind to TLR5. 83 / 101 / 139A and eukFlaBN 83 / 101 / 139 / 273 / 330A The transformed form was confirmed to restore TLR5 binding, confirming that glycosylation interferes with FlaB-TLR5 binding and that deglycosylation results in higher affinity FlaB-TLR5 binding.

[0100] Example 7: Verification of the immune-enhancing efficacy of the eukaryotic cell-expressed flagellin variants prepared above in an influenza vaccine experimental model system using mice

[0101] Seven-week-old female BALB / C mice (n=10) were purchased from Orient (Seongnam-si, Kyunggi-do, Korea) and allowed to adapt to the facility environment for one week before the experiment. Mice were anesthetized with a mixture of Zoletil® 50 (Virbac Corporation, Carros, France) and Rumpun™ (Bayer AG, Leverkusen, Germany) administered intraperitoneally, followed by immunization. The groups were: 1) sH1N1 antigen group (sH1N1, 0.2 μg / mouse), 2) sH1N1 (0.2 μg / mouse) and prokFlaB (4 μg / mouse), or 3) eukFlaB (4 μg / mouse) mixture (sH1N1 + prokFlaB, sH1N1 + eukFlaB, sH1N1 + eukFlaB). N83A , sH1N1+eukFlaB N83 / 101A , sH1N1+eukFlaB N83 / 101 / 139A , sH1N1+eukFlaB N83 / 101 / 139 / 273A , and sH1N1+eukFlaB N83 / 101 / 139 / 273 / 330A Intranasal immunization was initiated using 10 μl of the vaccine antigen diluted in PBS and administered to each nostril of the mice, for a total of 20 μl. Mice were immunized three times at two-week intervals, and serum was collected two weeks after the final immunization to determine antibody responses.

[0102] Influenza antigen-specific antibody immune responses were measured by enzyme-linked immunosorbent assay (ELISA). Mouse sera were collected two weeks after the third immunization. ELISA plates (Corning Laboratories, 3690) were coated with sH1N1 (4 μg / ml) and incubated overnight at 4°C. The next day, the plates were washed three times with PBS-T [PBS containing 0.05% Tween 20 (Thermo Fisher Scientific, cat. J20605-AP)] and blocked with blocking buffer [1 mM EDTA (JUNSEI, cat. 17385S0401) and 0.5% BSA in PBS-T (Sigma, cat. A2153-50G)] for 1 hour at room temperature to block nonspecific binding of antigen and antibody. Mouse sera were serially diluted (2-fold) in blocking buffer and added at 40 μl per well for 2 hours at room temperature. The plate was washed five times with PBS-T, and horseradish peroxidase (HRP)-conjugated anti-mouse IgG antibody was added and incubated at room temperature for 1 hour. The plate was then washed with PBS-T. The substrate TMB (BD OptEIA, cat. 555214) was added to develop color, and the reaction was stopped by adding STOP buffer (1N H2SO4). Absorbance was measured at 450 nm using a microplate reader (Molecular Devices Corporation, Menlo Park, CA). Antibody titers were calculated as absorbance values ​​that were 2-fold higher than those of control wells, and the reciprocal Log2 values ​​of serum dilutions were calculated.

[0103] As a result of the experiment, as shown in Figure 9, eukFlaB N83A , eukFlaB N83 / 101A , eukFlaB N83 / 101 / 139A , eukFlaB N83 / 101 / 139 / 273A , and eukFlaB N83 / 101 / 139 / 273 / 330Ademonstrated that it induces the production of influenza antigen-specific antibodies at the same level as flagellin (prokFlaB) expressed in prokaryotic cells, and has immune-enhancing properties.

[0104] Example 8: Comparative verification of antibody production ability against prokaryotic cell-expressed flagellin and eukaryotic cell-expressed flagellin

[0105] In the influenza vaccine model of Example 7, flagellin-specific antibody immune responses were measured using enzyme-linked immunosorbent assay (ELISA). Specifically, mouse sera were collected two weeks after the third immunization. ELISA plates (Corning Laboratories, 3690) were coated with prokFlaB (1 μg / ml) or wild-type eukFlaB (1 μg / ml) and then incubated overnight at 4°C. ELISA was performed using the same method as in Example 7.

[0106] As a result of the experiment, as shown in Figure 10, flagellin (prokFlaB) expressed in prokaryotic cells induced the production of prokFlaB- and eukFlaB-specific antibodies, but not eukFlaB. N83 / 101 / 139A , eukFlaB N83 / 101 / 139 / 273A , and eukFlaB N83 / 101 / 139 / 273 / 330A confirmed that the eukFlaB variant of the present invention does not induce the production of prokFlaB- and eukFlaB-specific antibodies. Therefore, it was confirmed that the eukFlaB variant of the present invention can provide a safer flagellin immune enhancer by repeatedly administering it to the body without inducing an immune response against flagellin itself, which is a biological product.

[0107] Example 9: Stability verification of deglycosylated eukaryotic cell-expressed flagellin derivatives

[0108] ProkFlaB, eukFlaB, and N-glycosylated mutant eukFlaB were purified and stored at different temperatures, such as -80°C, 4°C, and room temperature, for 2 weeks. The structural stability and functionality of the proteins were examined by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and NF-κB reporter assay, respectively.

[0109] As a result of the experiment, it was confirmed that flagellin produced in eukaryotic cells has superior stability compared to flagellin produced in prokaryotic cells (prokFlaB), as shown in Figure 11. Therefore, it is expected that the eukFlaB variant of the present invention can provide a flagellin immunopotentiator with superior pharmacokinetics during the production, distribution, and in vivo administration of biological products. [Industrial Applicability]

[0110] The present invention relates to the production and application of flagellin immunopotentiator derivatives with TLR5 activity using a eukaryotic cell expression system, and more specifically to flagellin proteins that can be expressed in eukaryotic cells and have immunopotentiating effects equivalent to or greater than those of existing prokaryotic flagellin proteins.

Claims

1. A polypeptide that can be expressed in a eukaryotic cell, in which one or more asparagine (Asparagine, N) in the amino acid sequence of SEQ ID NO: 13 is replaced with alanine (Alanine).

2. The polypeptide is a polypeptide expressible in a eukaryotic cell as described in claim 1, in which asparagine at one or more positions selected from the group consisting of positions 83, 101, 139, 273, and 330 in the amino acid sequence of SEQ ID NO: 13 is substituted with alanine.

3. The polypeptide expressible in a eukaryotic cell according to claim 1, wherein the polypeptide comprises one amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs: 14 to 23.

4. The polypeptide is Position 83 in the amino acid sequence of SEQ ID NO: 13; and one or more positions selected from the group consisting of positions 101, 139, 273, and 330; 3. The polypeptide expressible in a eukaryotic cell according to claim 2, wherein asparagine in the formula (I) is substituted with alanine.

5. The polypeptide is Positions 83 and 101 in the amino acid sequence of SEQ ID NO: 13; and one or more positions selected from the group consisting of positions 139, 273, and 330; 3. The polypeptide expressible in a eukaryotic cell according to claim 2, wherein asparagine in the formula (I) is substituted with alanine.

6. The polypeptide is Positions 83, 101, and 139 in the amino acid sequence of SEQ ID NO: 13; and one or more positions selected from the group consisting of positions 273 and 330; 3. The polypeptide expressible in a eukaryotic cell according to claim 2, wherein asparagine in the formula (I) is substituted with alanine.

7. An immunoadjuvant composition for vaccines, comprising a polypeptide in which one or more asparagines (Asparagine, N) in the amino acid sequence of SEQ ID NO: 13 are substituted with alanine (Alanine).

8. The immunoadjuvant composition for vaccines according to claim 7, wherein the polypeptide has asparagine at one or more positions selected from the group consisting of positions 83, 101, 139, 273, and 330 in the amino acid sequence of SEQ ID NO: 13 substituted with alanine.

9. The immunoadjuvant composition for vaccines according to claim 7, wherein the polypeptide comprises one amino acid sequence selected from the group consisting of amino acid sequences of SEQ ID NOs: 14 to 23.

10. The polypeptide is Position 83 in the amino acid sequence of SEQ ID NO: 13; and one or more positions selected from the group consisting of positions 101, 139, 273, and 330; 9. The immunoadjuvant composition for vaccines according to claim 8, wherein asparagine in the formula (I) is substituted with alanine.

11. The polypeptide is Positions 83 and 101 in the amino acid sequence of SEQ ID NO: 13; and one or more positions selected from the group consisting of positions 139, 273, and 330; 9. The immunoadjuvant composition for vaccines according to claim 8, wherein asparagine in the formula (I) is substituted with alanine.

12. The polypeptide is Positions 83, 101, and 139 in the amino acid sequence of SEQ ID NO: 13; and one or more positions selected from the group consisting of positions 273 and 330; 9. The immunoadjuvant composition for vaccines according to claim 8, wherein asparagine in the formula (I) is substituted with alanine.

13. A composition for enhancing immune function, comprising a polypeptide in which one or more asparagines (Asparagine, N) in the amino acid sequence of SEQ ID NO: 13 are substituted with alanine (Alanine).

14. The composition for enhancing immune function described in claim 13, wherein the polypeptide has asparagine at one or more positions selected from the group consisting of positions 83, 101, 139, 273, and 330 in the amino acid sequence of SEQ ID NO: 13 substituted with alanine.

15. The composition for enhancing immune function described in claim 13, wherein the polypeptide comprises one amino acid sequence selected from the group consisting of amino acid sequences of SEQ ID NOs: 14 to 23.

16. The polypeptide is Position 83 in the amino acid sequence of SEQ ID NO: 13; and one or more positions selected from the group consisting of positions 101, 139, 273, and 330; The composition for enhancing immune function according to claim 14, wherein asparagine in the formula (I) is substituted with alanine.

17. The polypeptide is positions 83 and 101 in the amino acid sequence of SEQ ID NO: 13; and one or more positions selected from the group consisting of positions 139, 273, and 330; The composition for enhancing immune function according to claim 14, wherein asparagine in the formula (I) is substituted with alanine.

18. The polypeptide is positions 83, 101, and 139 in the amino acid sequence of SEQ ID NO: 13; and one or more positions selected from the group consisting of positions 273 and 330; The composition for enhancing immune function according to claim 14, wherein asparagine in the formula (I) is substituted with alanine.

19. A pharmaceutical composition for preventing or treating autoimmune diseases, inflammatory diseases, microbial infections or cancer, comprising a polypeptide in which one or more asparagines (Asparagine, N) in the amino acid sequence of SEQ ID NO: 13 are substituted with alanine (Alanine).

20. 20. The pharmaceutical composition for preventing or treating an autoimmune disease, inflammatory disease, microbial infection, or cancer according to claim 19, wherein the polypeptide has asparagine at one or more positions selected from the group consisting of positions 83, 101, 139, 273, and 330 in the amino acid sequence of SEQ ID NO: 13 substituted with alanine.

21. The pharmaceutical composition for preventing or treating an autoimmune disease, an inflammatory disease, a microbial infection, or cancer according to claim 19, wherein the polypeptide comprises one amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs: 14 to 23.

22. The polypeptide is Position 83 in the amino acid sequence of SEQ ID NO: 13; and one or more positions selected from the group consisting of positions 101, 139, 273, and 330; 21. The pharmaceutical composition for preventing or treating an autoimmune disease, an inflammatory disease, a microbial infection or cancer according to claim 20, wherein asparagine in the formula (I) is substituted with alanine.

23. The polypeptide is positions 83 and 101 in the amino acid sequence of SEQ ID NO: 13; and one or more positions selected from the group consisting of positions 139, 273, and 330; 21. The pharmaceutical composition for preventing or treating an autoimmune disease, an inflammatory disease, a microbial infection or cancer according to claim 20, wherein asparagine in the formula (I) is substituted with alanine.

24. The polypeptide is Positions 83, 101, and 139 in the amino acid sequence of SEQ ID NO: 13; and one or more positions selected from the group consisting of positions 273 and 330; 21. The pharmaceutical composition for preventing or treating an autoimmune disease, an inflammatory disease, a microbial infection or cancer according to claim 20, wherein asparagine in the formula (I) is substituted with alanine.

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