Preparation and application of flagellin immunopotentiator derivative using eukaryotic cell expression system and having TLR5 activity

Abstract

The invention relates to preparation and application of a flagellin immunopotentiator derivative using a eukaryotic cell expression system and having TLR5 activity, and the flagellin variant has a high immunopotentiation effect and can be used in various vaccines and immunotherapeutic agent compositions.

Description

Technical Field

[0001] This invention was completed under the support of the Ministry of Science and ICT, Republic of Korea (Project No. 2020R1A5A2031185) (contribution rate: 60%). The specialized research management institution for this project is the National Research Foundation of Korea. The name of the research project is "Group Research Support", and the name of the research topic is "Composite Cancer Immunotherapy Research Center". Its main contractor is Jeonnam National University, and the research period is from June 1, 2018 to February 28, 2025.

[0002] This invention was completed under the support of the Ministry of Science and ICT, Republic of Korea (Project No. 2020M3A9G3080282) (contribution rate: 20%). The specialized research management institution for this project is the National Research Foundation of Korea. The name of the research project is "Biomedical Technology Development", and the name of the research topic is "Innovative Immunotherapy Research Group". Its main contractor is Jeonnam National University, and the research period is from June 1, 2020 to December 31, 2024.

[0003] This invention was completed under the support of the Ministry of Health and Welfare, Republic of Korea (Project No. HV22C0079) (contribution rate: 20%). The specialized research management institution for this project is the Korea Health Industry Development Institute. The name of the research project is "Vaccine Basic Technology Development", and the name of the research topic is "Development of a Universal Platform for Cell Immune Inducing Mucosal Immune Enhancers". Its main contractor is Jeonnam National University, and the research period is from April 1, 2022 to December 31, 2024.

[0004] The present invention relates to the preparation and application of a flagellin immunopotentiator derivative with TLR5 activity using a eukaryotic cell expression system. More specifically, it relates to a flagellin that can be expressed in eukaryotic cells and has an immunopotentiating effect equal to or higher than that of existing flagellins derived from prokaryotes.

Background Art

[0005] The structural unit protein that forms the filaments of bacterial flagella is called flagellin. Flagellins are regularly assembled to form filaments, enabling bacteria to move.

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

[0007] In addition, flagellin is a pathogen-associated molecular pattern factor that can stimulate the NLRC4-inflammasome signaling pathway present in the cytoplasm. Therefore, flagellin is an immunomodulatory substance that can activate both TLR5 present on the cell surface and the NLRC4-inflammasome present in the cytoplasm.

[0008] Taking advantage of these characteristics, it has been studied as a target for development as an immune enhancer applicable to various vaccines and immunotherapeutic agents. When multiple antigens are administered together with flagellin or in the form of a recombinant fusion protein with flagellin, it has the function of enhancing antigen-specific immune responses, and its immune enhancer efficacy has been confirmed in influenza vaccines, pneumonia vaccines, bacterial periodontal diseases, anti-cancer therapeutic vaccines, norovirus vaccines, neurodegenerative diseases, and various immune diseases. In addition, it has been reported that TLR5 activation based on flagellin can protect hematopoietic cells and gastric tissues from radiation damage and affect the survival and growth of cancer cells.

[0009] However, to date, the immunopotentiating effect of flagellin has been achieved by using recombinant proteins expressed in Escherichia coli, a prokaryotic cell. However, in order to apply the scope of the flagellin immunopotentiator to nucleic acid-based vaccines (e.g., DNA vaccines or mRNA vaccines, etc.) or antigens that require post-translational modification, flagellin expressed in a eukaryotic cell expression system needs to be prepared. However, compared with flagellin expressed in prokaryotic cells, flagellin prepared in a eukaryotic cell system exhibits significantly reduced TLR5-signaling ability. The reason for this phenomenon is speculated to be that when flagellin is expressed in eukaryotic cells, due to the steric hindrance of the post-translational modification process, TLR5-signaling cannot be effectively induced.

[0010] Therefore, there is a need to study a flagellin that has a strong immunomodulatory ability, has an effective vaccine immunopotentiator function, and can be prepared in eukaryotic cells.

Summary of the Invention

[0011]

Problems to be Solved by the Invention

[0012] Therefore, the present inventors prepared flagellin in eukaryotic cells and confirmed that it has significantly excellent immunopotentiating ability and safety.

[0013] Specifically, the present inventors confirmed that the TLR5-stimulating activity of flagellin prepared in a eukaryotic cell system is very weak compared to that of flagellin expressed in prokaryotic cells. The reason for this phenomenon is speculated to be that when flagellin is expressed in eukaryotic cells, glycosylated flagellin (glycosylated-eFlaB) is prepared through a post-translational modification process, and due to the steric hindrance of sugar molecules, glycosylated flagellin cannot effectively induce TLR5-signaling. To verify this hypothesis, bioinformatics analysis was performed, and it was confirmed that flagellin expressed in eukaryotic cells has 5 N-glycosylation sites. Therefore, a flagellin variant in which asparagine, which is presumed to be an N-glycosylation site, is replaced with alanine was prepared by introducing the site-directed mutagenesis method, and the activity of this flagellin variant was verified under in vitro and in vivo conditions. From this, eukaryotic cell-expressed flagellin immunopotentiator variants (eukFlaBvariants) that exhibit the same TLR5-stimulating activity and immunopotentiator efficacy as pFlaB were derived. Interestingly, this variant (eukFlaBvariant) exhibits an immunogenicity-loss property. By comprehensively using immunoinformatics analysis and protein modification strategies, eukaryotic cell-expressed flagellin that does not induce N-glycosylation (deglycosylated eukaryotic expressed flagellin) was established, thereby deriving a safer flagellin immunopotentiator that can be used for various clinical applications.

[0014] Accordingly, an object of the present invention is to provide a flagellin capable of being expressed in eukaryotic cells.

[0015] Another object of the present invention is to provide an immunoadjuvant composition for a vaccine, the composition comprising a flagellin capable of being expressed in eukaryotic cells.

[0016] Still another object of the present invention is to provide a composition for enhancing immune function, the composition comprising a flagellin capable of being expressed in eukaryotic cells.

[0017] Yet another object of the present invention is to provide a pharmaceutical composition for preventing or treating microbial infections or cancer, the composition comprising a flagellin capable of being expressed in eukaryotic cells.

[0018] Yet another object of the present invention is to provide an immunoadjuvant use of a flagellin capable of being expressed in eukaryotic cells.

[0019] Still yet another object of the present invention is to provide an immune function enhancing use of a flagellin capable of being expressed in eukaryotic cells.

[0020] Another object of the present invention is to provide a use of flagellin capable of being expressed in eukaryotic cells in the prevention or treatment of autoimmune diseases, inflammatory diseases, microbial infections or cancers.

[0021]

Means for Solving the Problem

[0022] The present invention relates to the preparation and application of a flagellin immunopotentiator derivative having TLR5 activity using a eukaryotic cell expression system, and the flagellin of the present invention exhibits a high immunopotentiating effect.

[0023] Hereinafter, the present invention will be described in more detail.

[0024] One embodiment of the present invention relates to a polypeptide capable of being expressed in eukaryotic cells, which is a polypeptide in which one or more asparagines (Asn, N) in the amino acid sequence of SEQ ID NO: 13 are replaced by alanine.

[0025] In one implementation example of the present invention, the polypeptide may be a polypeptide in which asparagine at one or more positions selected from the group consisting of the 83rd, 101st, 139th, 273rd, and 330th positions in the amino acid sequence of SEQ ID NO: 13 is replaced by alanine.

[0026] In one implementation example of the present invention, the polypeptide may be a polypeptide in which asparagine is replaced by alanine at the following positions: the 83rd position in the amino acid sequence of SEQ ID NO: 13; and one or more positions selected from the group consisting of the 101st, 139th, 273rd, and 330th positions in the amino acid sequence of SEQ ID NO: 13.

[0027] In one implementation example of the present invention, the polypeptide may be a polypeptide in which asparagine is replaced by alanine at the following positions: the 83rd and 101st positions in the amino acid sequence of SEQ ID NO: 13; and one or more positions selected from the group consisting of the 139th, 273rd, and 330th positions in the amino acid sequence of SEQ ID NO: 13.

[0028] In one implementation example of the present invention, the polypeptide may be a polypeptide in which asparagine is replaced by alanine at the following positions: the 83rd, 101st, and 139th positions in the amino acid sequence of SEQ ID NO: 13; and one or more positions selected from the group consisting of the 273rd and 330th positions in the amino acid sequence of SEQ ID NO: 13.

[0029] In one embodiment of the present invention, the polypeptide may comprise an amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NO: 14 to SEQ ID NO: 23.

[0030] The polypeptide of the present invention has excellent TLR-5 activity, and thus exhibits a high immune enhancement effect, and can be used as an immunoadjuvant composition for vaccines, a composition for enhancing immune function, or a pharmaceutical composition for preventing or treating autoimmune diseases, inflammatory diseases, microbial infections or cancers, but is not limited thereto.

[0031] When the polypeptide of the present invention is administered together with an influenza vaccine, since it has an excellent effect of inducing the generation of antibodies specific to influenza antigens, it can be used as an immunoadjuvant composition for influenza vaccines or an immunoadjuvant composition for treating influenza, but is not limited thereto.

[0032] Another embodiment of the present invention relates to an immunoadjuvant composition for vaccines, which comprises a polypeptide in which one or more asparagines (N) in the amino acid sequence of SEQ ID NO: 13 are replaced by alanine.

[0033] In one embodiment of the present invention, the polypeptide may be a polypeptide in which asparagine is replaced by 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.

[0034] In one embodiment of the present invention, the polypeptide may be a polypeptide in which asparagine is replaced by alanine at position 83 in 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 in the amino acid sequence of SEQ ID NO: 13.

[0035] In one embodiment of the present invention, the polypeptide may be a polypeptide in which asparagine is replaced by alanine at positions 83 and 101 in 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 in the amino acid sequence of SEQ ID NO: 13.

[0036] In one embodiment of the present invention, the polypeptide may be a polypeptide in which asparagine is replaced by alanine at positions 83, 101 and 139 in the amino acid sequence of SEQ ID NO: 13; and at one or more positions selected from the group consisting of positions 273 and 330 in the amino acid sequence of SEQ ID NO: 13.

[0037] In one embodiment of the present invention, the polypeptide may comprise an amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NO: 14 to SEQ ID NO: 23.

[0038] The immunoadjuvant composition for a vaccine of the present invention may further comprise one or more commonly used pharmaceutically and / or veterinarily acceptable carriers, diluents, antibiotics, adjuvants, etc.

[0039] The carrier may include any pharmaceutically acceptable solvent (solution), dispersion medium, coating agent, buffer, isotonic agent, suspension, colloid, stabilizer, preservative, antibiotic (antibacterial agent, antifungal agent, etc.), absorption delaying agent, adjuvant, insert, etc. during the administration to a target animal (host animal). Generally, for those skilled in the pharmaceutical art, it is well known to use chemical compounds, especially one or more delivery vehicles for an immunogen. As long as any conventional medium or agonist is incompatible with the active ingredient, it can be considered for use in compositions for diagnosis, prevention, and treatment. One or more supplementary active ingredients may also be incorporated into or co-administered with one or more of the disclosed immunogenic compositions and vaccine formulations.

[0040] The diluent may include water, saline, glucose, ethanol, glycerol, etc., and the isotonic agent may include sodium chloride, glucose, mannitol, sorbitol, lactose, etc., and the stabilizer may include albumin, but is not limited thereto.

[0041] Adjuvants include: mineral gels such as aluminum hydroxide gel; surface active substances such as lysolecithin; glycosides such as saponin derivatives such as Quil A or GPI-0100 (Galenica Pharmaceuticals, Inc., Birmingham, Alabama); Pluronic polyol; polyanions; nonionic block polymers such as Pluronic F-127 (B.A.S.F., USA); peptides; mineral oils such as Montanide ISA-206 (Seppic, France), Montanide ISA-50 (Seppic, France), Cabopol, amphigene, Amphigen Mark II (Hydronic, USA), aluminum hydrogel, emulsions of mineral oil and water such as oil-in-water emulsions (e.g., such as Bayol F / Arlacel A), or emulsions of vegetable oil and an emulsifier such as water and lecithin; alum; cholesterol; Freund's complete and incomplete adjuvants; block copolymers (CytRx, Atlanta, Georgia); SAF-M (Chiron, Emeryville, California); AMPHIGEN (registered trademark) adjuvant; monophosphoryl lipid A, Avridine lipid amine adjuvant; heat-labile enterotoxin from Escherichia coli (recombinant, etc.); cholera toxin; muramyl dipeptide; IMS1313 (Seppic, France); ISA206; Montanide 01 gel (Seppic, France); and mixtures of these adjuvants, but not limited to these. Preferably, one or more adjuvants selected from the group consisting of IMS1313, Cabopol, and Montanide 01 gel are used. The adjuvant refers to a compound or mixture that enhances the immune response and / or promotes the absorption rate after vaccination, and includes any absorption promoter.

[0042] Another embodiment of the present invention relates to a composition for enhancing immune function, which composition comprises a polypeptide in which one or more asparagines (Asn, N) in the amino acid sequence of SEQ ID NO: 13 are replaced by alanine.

[0043] In one implementation example of the present invention, the polypeptide may be a polypeptide in which asparagine is replaced by alanine at one or more positions selected from the group consisting of position 83, position 101, position 139, position 273, and position 330 in the amino acid sequence of SEQ ID NO: 13.

[0044] In one implementation example of the present invention, the polypeptide may be a polypeptide in which asparagine at the following positions is replaced by alanine: position 83 in the amino acid sequence of SEQ ID NO: 13; and one or more positions selected from the group consisting of position 101, position 139, position 273, and position 330 in the amino acid sequence of SEQ ID NO: 13.

[0045] In one implementation example of the present invention, the polypeptide may be a polypeptide in which asparagine at the following positions is replaced by alanine: position 83 and position 101 in the amino acid sequence of SEQ ID NO: 13; and one or more positions selected from the group consisting of position 139, position 273, and position 330 in the amino acid sequence of SEQ ID NO: 13.

[0046] In one implementation example of the present invention, the polypeptide may be a polypeptide in which asparagine at the following positions is replaced by alanine: position 83, position 101, and position 139 in the amino acid sequence of SEQ ID NO: 13; and one or more positions selected from the group consisting of position 273 and position 330 in the amino acid sequence of SEQ ID NO: 13.

[0047] In one implementation example of the present invention, the polypeptide may comprise an amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NO: 14 to SEQ ID NO: 23.

[0048] Another embodiment of the present invention relates to a pharmaceutical composition for preventing or treating an autoimmune disease, an inflammatory disease, a microbial infection, or cancer, the composition comprising a polypeptide in which one or more asparagines (Asn, N) in the amino acid sequence of SEQ ID NO: 13 are replaced by alanine.

[0049] In one implementation example of the present invention, the autoimmune disease can be selected from one or more of the group consisting of hemophagocytic lymphohistiocytosis, systemic lupus erythematosus, Kikuchi disease, vasculitis, adult onset Still's disease, rheumatoid arthritis, inflammatory myositis, Behcet disease, IgG4-related disease, Sjogren syndrome, giant cell arteritis, temporal arteritis, type 1 diabetes, atopic dermatitis, Crohn's disease, systemic sclerosis, psoriasis, multiple sclerosis, and Graves hyperthyroidism, but is not limited thereto.

[0050] In one implementation example of the present invention, the inflammatory disease can be selected from one or more of the group consisting of sepsis, gastritis, enteritis, nephritis, hepatitis, chronic obstructive pulmonary disease (COPD), pulmonary fibrosis, irritable bowel syndrome, inflammatory pain, migraine, headache, back pain, fibromyalgia, fascial disease, viral infection, bacterial infection, fungal infection, burns, wounds caused by surgical or dental procedures, prostaglandinosis, atherosclerosis, gout, Hodgkin's disease, pancreatitis, conjunctivitis, iritis, scleritis, uveitis, and eczema, but is not limited thereto.

[0051] In one implementation example of the present invention, the microorganism may be selected from one or more of the group consisting of Influenza virus, Corona virus, Hepatitis virus, Zika virus, Dengue virus (DENV), Hantavirus, Cytomegalovirus (CMV), Epstein Barr virus (EBV), human immunodeficiency virus (HIV), Herpes simplex virus (HSV), Chikungunya virus, Enterovirus, Marburg hemorrhagic fever virus, parvovirus, Sever Fever with Thrombocytopenia Syndrome Virus (SFTSV), Rotavirus, Norovirus, Mycobacterium tuberculosis, Group A Streptococcus (GAS), Group B Streptococcus (GBS), Staphylococcus aureus, Shigella, pathogenic E. coli, Salmonella, Chlamydia, Pseudomonas aeruginosa, Non-typeable Haemophilus influenzae, Klebsiella pneumoniae, Clostridium difficile, Plasmodium, Leishmania, Schistosoma, Trypanosoma, Brucella, Cryptosporidium, and Entamoeba, but is not limited thereto.

[0052] 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 cancer, lymphoma, rectal cancer, pancreatic cancer, esophageal cancer, gastric cancer, cervical cancer, thyroid cancer, and skin cancer, but is not limited thereto.

[0053] In one embodiment of the present invention, the polypeptide may be a polypeptide in which asparagine at one or more positions selected from the group consisting of the 83rd, 101st, 139th, 273rd, and 330th positions in the amino acid sequence of SEQ ID NO: 13 is replaced with alanine.

[0054] In one embodiment of the present invention, the polypeptide may be a polypeptide in which asparagine is replaced with alanine at the following positions: the 83rd position in the amino acid sequence of SEQ ID NO: 13; and one or more positions selected from the group consisting of the 101st, 139th, 273rd, and 330th positions in the amino acid sequence of SEQ ID NO: 13.

[0055] In one embodiment of the present invention, the polypeptide may be a polypeptide in which asparagine is replaced with alanine at the following positions: the 83rd and 101st positions in the amino acid sequence of SEQ ID NO: 13; and one or more positions selected from the group consisting of the 139th, 273rd, and 330th positions in the amino acid sequence of SEQ ID NO: 13.

[0056] In one embodiment of the present invention, the polypeptide may be a polypeptide in which asparagine is replaced with alanine at the following positions: the 83rd, 101st, and 139th positions in the amino acid sequence of SEQ ID NO: 13; and one or more positions selected from the group consisting of the 273rd and 330th positions in the amino acid sequence of SEQ ID NO: 13.

[0057] In one embodiment of the present invention, the polypeptide may comprise an amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NO: 14 to SEQ ID NO: 23.

[0058] The pharmaceutical composition of the present invention may be administered in a pharmaceutically effective amount.

[0059] In this specification, the term "pharmaceutically effective amount" refers to an amount sufficient to achieve the efficacy or activity of the above-mentioned extract.

[0060] The pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier.

[0061] The pharmaceutically acceptable carriers contained in the pharmaceutical composition of the present invention are commonly used in formulations and may also include, but are not limited to, lactose, glucose, sucrose, sorbitol, mannitol, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methyl paraben, propyl paraben, talc, magnesium stearate, and mineral oil, etc. In addition to the above components, the pharmaceutical composition of the present invention may also include lubricants, humectants, sweeteners, flavoring agents, emulsifying agents, suspending agents, and preservatives, etc.

[0062] The pharmaceutical composition of the present invention can be administered to mammals (including humans) by various routes. The administration methods can be all conventional methods. For example, it can be administered by oral, skin, intravenous, intramuscular, and subcutaneous routes, etc. Preferably, it can be administered orally.

[0063] The appropriate dosage of the pharmaceutical composition of the present invention can depend on factors such as the formulation method, administration method, patient's age, weight, gender, pathological condition, food, administration time, administration route, excretion rate, and reaction sensitivity, etc. And usually, a skilled doctor can easily determine and prescribe a dose effective for the required treatment or prevention.

[0064] The pharmaceutical composition of the present invention can be formulated using pharmaceutically acceptable carriers and / or excipients according to methods easily implemented by those of ordinary skill in the technical field to which the present invention pertains. Thus, it can be prepared in unit dosage form or placed in a multi-dose container. At this time, the dosage form can be in the form of a solution, suspension, or emulsion in an oily or aqueous medium, or can also be in the form of an extract, powder, granule, tablet, capsule, or gel (such as a hydrogel), and may also include dispersing agents or stabilizers.

[0065] Another embodiment of the present invention relates to the use of a polypeptide as a vaccine adjuvant, and the polypeptide is a polypeptide in which one or more asparagine (N) in the amino acid sequence of SEQ ID NO: 13 is replaced by alanine (A).

[0066] Another embodiment of the present invention relates to the use of enhancing the immune function of a polypeptide, and the polypeptide is a polypeptide in which one or more asparagine (N) in the amino acid sequence of SEQ ID NO: 13 is replaced by alanine (A).

[0067] Another embodiment of the present invention relates to the use of a polypeptide in the prevention or treatment of autoimmune diseases, inflammatory diseases, microbial infections or cancers, wherein the polypeptide is a polypeptide in which one or more asparagines (N) in the amino acid sequence of SEQ ID NO: 13 are replaced by alanine.

[0068]

Inventive Effect

[0069] The present invention relates to the preparation and application of a flagellin immunopotentiator derivative with TLR5 activity using a eukaryotic cell expression system. The flagellin variant of the present invention has a high immunopotentiating effect and can be used in various vaccine and immunotherapeutic agent compositions.

Brief Description of the Drawings

[0070] Figure 1 It is a diagram showing the results of confirming wild-type flagellin (prokFlaB) derived from Vibrio vulnificus expressed in Escherichia coli as a prokaryotic cell and wild-type flagellin (eukFlaB) expressed in Expi293 TM cells by Western blotting according to an embodiment of the present invention.

[0071] Figure 2 It is a graph showing the results of comparing the TLR5-stimulating activity of wild-type flagellin (prokFlaB) derived from Vibrio vulnificus expressed in Escherichia coli as a prokaryotic cell and wild-type flagellin (eukFlaB) expressed in Expi293 TM cells according to an embodiment of the present invention.

[0072] Figure 3a It is a diagram showing the results of confirming the protein state before and after treating wild-type flagellin (eukFlaB) expressed in the eukaryotic cell Expi293 TM cells with peptide N-glycosidase F (PNGase) as a deglycosylating enzyme by Western blotting according to an embodiment of the present invention.

[0073] Figure 3b It is a graph showing the results of comparing the TLR5-stimulating activity before and after treating wild-type flagellin (eukFlaB) expressed in Expi293 TM cells as a eukaryotic cell with peptide N-glycosidase F (PNGase) as a deglycosylating enzyme according to an embodiment of the present invention.

[0074] Figure 4Shows the results of predicting the glycosylated amino acid sequence of flagellin (FlaB) according to an embodiment of the present invention.

[0075] Figure 5 Is a schematic diagram of the construction of a flagellin variant that induces site-directed mutagenesis.

[0076] Figures 6 to 7 Is the result of verifying the characteristics and TLR5-stimulating activity of the flagellin variant expressed in Expi293 TM cells according to an embodiment of the present invention.

[0077] Figure 8 Is the result of determining the binding of the flagellin variant to TLR5 by the co-immunoprecipitation (Co-IP) method.

[0078] Figure 9 Shows the results of comparing the immune-enhancing efficacy of wild-type flagellin (prokFlaB) from Vibrio vulnificus expressed in Escherichia coli as a prokaryotic cell, wild-type flagellin (eukFlaB) expressed in Expi293 TM cells, and various flagellin variants in an influenza vaccine model according to an embodiment of the present invention.

[0079] Figure 10 Shows the results of comparing wild-type flagellin (prokFlaB) from Vibrio vulnificus expressed in Escherichia coli as a prokaryotic cell and wild-type flagellin (eukFlaB) expressed in Expi293 TM cells, eukFlaB N83 / 101 / 139A eukFlaB N83 / 101 / 139 / 273A eukFlaB N83 / 101 / 139 / 273 / 330A and eukFlaB

[0080] Figure 11 Shows the results of comparing wild-type flagellin (prokFlaB) from Vibrio vulnificus expressed in Escherichia coli as a prokaryotic cell, wild-type flagellin (eukFlaB) expressed in Expi293 TM cells, and the stability of various flagellin variants according to an embodiment of the present invention.

DETAILED DESCRIPTION

[0081] The present invention relates to a polypeptide capable of being expressed in eukaryotic cells, wherein one or more asparagines (N) in the amino acid sequence of SEQ ID NO: 13 are replaced with alanines.

[0082]

Embodiment

[0083] Hereinafter, the present invention will be described in more detail by the following examples. However, these examples are only for illustrating the present invention, and the scope of the present invention is not limited to these examples.

[0084] Throughout the specification, unless otherwise specified, when "%" representing the concentration of a specific substance is used, it means (weight / weight)% for solid / solid, (weight / volume)% for solid / liquid, and (volume / volume)% for liquid / liquid.

[0085]

Example 1: Construction of a plasmid for expressing wild-type flagellin in eukaryotic cells

[0086] The septicemic Vibrio vulnificus-derived FlaB (prokFlaB) expressed in prokaryotic cells was prepared by the method disclosed in the literature (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).

[0087] To express the FlaB in eukaryotic cells, the forward primer eukFlaB-F in Table 1 below including a HindIII (AAGCTT) cleavage site and the reverse primer eukFlaB-R including a NotI cleavage site were used and amplified by PCR reaction. At this time, after synthesizing an optimized FlaB according to the usage (frequency) of mammalian cell expression codons, pBHA::eukFlaB (BIONEER Corporation, Korea) cloned into the pBHA plasmid was used as a PCR reaction template.

[0088]

Table 1

[0089] SEQ ID NO: Name Sequence (5′->3′) 1 eukFlaB-F CCCAAGCTTG CCGTCAACGT GAACACCAA 2 eukFlaB-R ATAGTTTAGC GGCCGCGCCC AGCAGGGAGA GGGCGC

[0090] As the eukFlaB DNA amplification product and the eukaryotic cell expression vector pSectag2B (Invitrogen, V900 - 20), a pair of restriction enzymes (RE), HindIII and NotI, were treated overnight at 37°C. The PCR product treated with the restriction enzyme and the eukaryotic cell expression vector pSectag2B were combined with T4 DNA ligase (Enzynomics) at room temperature for 2 hours. The pSectag2B::eukFlaB plasmid, as the new construct, was transformed into Escherichia coli competent cells TOP10 (Invitrogen), and cultured using LB plates containing ampicillin. The DNA base sequence of the expression vector was confirmed by the Macrogen Online Sequencing Order System (http: / / dna.macrogen.com / kor / ) using the dideoxy - chain termination sequencing method.

[0091]

Example 2: Preparation of Recombinant Protein Using Eukaryotic Cell Expression System

[0092] Eukaryotic cell expression system cell line Expi293 TM The cells were purchased from Thermo Fisher Scientific (Cat#. A14635) and used in this invention. The transfection procedure followed the manufacturer's protocol. Expi293 TM cells were cultured in an incubator stirred at 125 rpm under the conditions of 37°C and 8% CO 2 . On day 0 (one day before transfection), Expi293 TM cells were inoculated into culture flasks (Corning, USA) at 3x10 6 / ml. On day 1 (transfection day), the number of Expi293 TM cells was measured and inoculated at 3x10 6 / ml, and the pSectag2B::eukFlaB plasmid sterilized by filtration through a 0.22 - μm filter (Millex - mil - GV, SLGVR13) was added to the cell culture system. On the next day, ExpiFectamine TM 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 through pSectag2B containing the IgK chain sequence.

[0093] On the fourth day, the medium secreting the protein was centrifuged at 17,000 rpm for 20 minutes at 4°C, and then, according to the manufacturer's guidelines, the cell-free supernatant was loaded onto a column containing Ni-NTA agarose beads (Qiagen, Hilden, Germany). To remove the remaining proteins other than the desired protein, washing buffer I (50 mM NaH 2 PO 4 、300 mM NaCl, 10 mM imidazole, pH = 8) and washing buffer II (50 mM NaH 2 PO 4 、300 mM NaCl and 20 mM imidazole, pH = 8) were used for washing. At room temperature, the target protein was dissolved and eluted with elution buffer (50 mM NaH 2 PO 4 、300 mM NaCl and 250 mM imidazole, pH = 8) for 15 minutes. The protein eluted in the above manner was dialyzed to replace the buffer with phosphate-buffered saline (PBS).

[0094] The recombinant peptide was confirmed by SDS-PAGE and protein immunoblotting with anti-FlaB antibody generated in BALB / c mice, and the results are shown in Figure 1 .

[0095]

Example 3: Verification of TLR5-stimulating activity of wild-type flagellin prepared using a eukaryotic cell expression system

[0096] To verify the TLR5-stimulating activity of wild-type flagellin prepared using a eukaryotic cell expression system, NF-κB activity was measured under various protein concentration conditions. Specifically, according to the manufacturer's protocol, HEK-Blue TM Detection (InvivoGen, hb-det2) assay system was used together with HEK-Blue TMhTLR5 cells (InvivoGen, hκb-htlr-5). For the concentration of each protein, the EC50 was calculated using the OD620nm value three times. To confirm the biological activity of deglycosylated eukFlaB, HEK293T cells purchased from Thermo Fisher Scientific were used to perform NF-κB luciferase reporter assays in vitro. HEK293T cells were maintained at 37 °C and 5% CO TM in DMEM medium (Gibco TM , cat. 15140122) containing 10% FBS and 100 units / ml of antibiotics (Gibco 2 . For NF-κB reporter assays, cells that had been passaged at least 3 times were used. HEK293T cells were seeded at 2 x 10 5 cells / well in a 24-well plate (SPL Life Sciences Co., Ltd., Korea) and cultured for 24 hours. To transfect the cells, the reporter plasmid pNF-κB-luc (100 ng / well), p3xFlag-hTLR5 (100 ng / well), pCMV-β-Gal (50 ng / well), and Effectene transfection reagent (5 μL / well) (Qiagen, 301427) were added and cultured overnight. After the transfected cells were treated with prokFlaB, eukFlaB, or mutant eukFlaB variants and cultured for 24 hours, the culture medium was removed, and the cells were lysed with cell lysis buffer (Promega, Madison, WI USA, E153A). Luciferase activation was measured from the cell lysates using a luminometer (Berthold, Lumat-Plus LB96V).

[0097] As Figure 2 shown, the experimental results confirmed that eukFlaB induced significantly lower TLR5 stimulation than prokFlaB.

[0098]

Example 4: Eukaryotic cell expression using a deglycosylase (peptide N-glycosidase F) - deglycosylation reaction of flagellin and verification of TLR5-stimulating activity of deglycosylated eukaryotic cell-expressed flagellin

[0099] According to the manufacturer's protocol, the deglycosylation reaction of eukaryotic cell-expressed eukFlaB was carried out using PNGase F enzyme (Promega, cat. V483A). PNGase F enzyme (10 U / μl) was mixed with eukFlaB (15 μg), and then incubated at a temperature of 37 °C. After 2 hours, the degree of deglycosylation was confirmed by SDS-PAGE and Western blotting with anti-FlaB antibody generated in BALB / c mice, and the results are shown in Figure 3a In. The TLR5-stimulating activity of deglycosylated eukFlaB was measured by the same method as in Example 3, and the results are shown in Figure 3b In.

[0100] As Figure 3b shown, the experimental results confirmed that deglycosylated eukFlaB restored TLR5-stimulating activity.

[0101] [Example 5: Preparation of various deglycosylated flagellin derivatives by site-directed mutagenesis and verification of TLR5-stimulating activity of various deglycosylated eukaryotic cell-expressed flagellin derivatives]

[0102] Using NetNGlyc-1.0 (https: / / services.healthtech.dtu.dk / service.php?NetNGlyc-1.0), a bioinformatics tool for determining the N-glycosylation consensus sequence Asn-Xaa-Ser / Thr, the N-glycosylation sites in the FlaB amino acid sequence were predicted.

[0103] As Figure 4 shown, five glycosylation residues were predicted in eukFlaB at positions N83, N101, N139, N273 and N330, and three residues (N83, N101 and N139) were determined to be near the TLR5 binding domain.

[0104] Thus, as Figure 5 shown, by introducing the site-directed mutagenesis method, various deglycosylated flagellins [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 ).

[0105] Site-directed mutagenesis of eukFlaB was generated according to the manufacturer's protocol (EZchange TM Site-directed Mutation, Cat.#EZ004S). Briefly, the pSectag2B::eukFlaB plasmid was used as a template for the PCR reaction. At this time, the primers in Table 2 were designed to change the amino acid asparagine (N) corresponding to the specific codon "AAC" or "AAT" to the amino acid alanine (A) corresponding to another specific codon "GCT" or "GCC" for generating site-directed mutagenesis. After separating the parental plasmid from the PCR product using the EZ-MIX enzyme, the plasmid was transformed into Escherichia coli competent cells (receptor cells) TOP10 and thinly spread on an LB plate containing ampicillin (100 μg / ml). The positive mutant sequence of eukFlaB was determined by Macrogen online sequencing (http: / / dna.macrogen.com / kor / ).

[0106]

Table 2

[0107]

[0108]

[0109] The recombinant peptides were confirmed by SDS-PAGE and Western blotting with anti-FlaB antibodies generated in BALB / c mice, and the results are shown in Figure 6 .

[0110] The eukFlaB, 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 peptide sequences are shown in Table 3 below.

[0111]

Table 3

[0112]

[0113]

[0114]

[0115] To determine the biological activities of multiple flagellins, NF-κB activity mediated by Toll-like receptor 5 (TLR5) signaling was measured by a reporter assay. HEK293T cells were maintained in DMEM (Gibco TM , cat. 16000044) containing 10% FBS (Gibco TM , cat. 15140122) and antibiotics of 100 units / ml penicillin and 100 μg / ml streptomycin (Gibco TM , cat. 11995-065) medium. HEK293T cells were seeded at 2x10 5 / well in 24-well plates (Corning costar, cat. 3526) and cultured in an incubator at 37°C and 5% CO 2 for 24 hours. To transfect the cells, the reporter plasmid pNF-κB-luc (100 ng / well), p3xFlag-hTLR5 (100 ng / well), pCMV-β-Gal (50 ng / well), and Effectene transfection reagent (5 μl / well) (Qiagen, 301427) were added and cultured overnight. After the transfected cells were treated with prokFlaB, eukFlaB, or mutant eukFlaB variants and cultured for 24 hours, the culture medium was removed, the cells were lysed with cell lysis buffer, and luciferase activation was measured from the cell lysates using a luminometer (Berthold, Lumat-Plus LB96V) to verify TLR5-signaling activity.

[0116] The experimental results, as Figure 7 shown, confirmed eukFlaB, eukFlaB N101A , eukFlaB N139A , eukFlaB N273A, eukFlaB N330A , eukFlaB N139 / 273 / 330A does not induce TLR5 activation, but eukFlaB N83A , eukFlaB N83 / 101A , eukFlaB N83 / 101 / 139A , eukFlaB N83 / 101 / 139 / 273A , eukFlaB N83 / 101 / 139 / 273 / 330A induces TLR5 activation. In particular, it was confirmed that eukFlaB N83 / 101 / 139A , eukFlaB N83 / 101 / 139 / 273A , eukFlaB N83 / 101 / 139 / 273 / 330A has the same level of TLR5-stimulating activity as flagellin (prokFlaB) derived from prokaryotic cells.

[0117]

Example 6: Determination of the Binding of Flagellin Variants to TLR5 Using Co-Immunoprecipitation (Co-IP)

[0118] As follows, co-immunoprecipitation (Co-IP) was used to determine the binding of flagellin variants to TLR5. Specifically, HEK293T cells were transfected with the p3XFlag-hTLR5 plasmid for 24 hours. After lysing the cells with RIPA buffer containing protease inhibitors, the supernatant was collected by centrifugation after sonication. The proteins prokFlaB, eukFlaB, eukFlaBN83 / 101 / 139A, and eukFlaBN83 / 101 / 139 / 273 / 330A were mixed with the supernatant and rotated overnight at 4°C (Complex I). At the same time, 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). Then, Complex II was washed 5 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 inhibitor). At 4°C, Complex I and Complex II were mixed for 6 hours and then washed 5 times with IP buffer to remove non-specific binding proteins. After adding 50 μl of 1x SDS loading buffer and heating for 10 minutes, SDS-PAGE and additional Western blot analysis were performed. The anti-FlaB eukFlaB variant was expressed together with the Myc-tag that was well detected by anti-Myc. Since the eukFlaB protein detected due to glycosylation on eukFlaB was low, two different antibodies, mouse anti-FlaB and anti-Myc-HRP ( 46-0709), were used in the Western blot analysis for detecting pFlaB and eukFlaB variants respectively.

[0119] As Figure 8 shown, the experimental results showed that prokFlaB bound to TLR5 with high affinity, but eukFlaBsms did not bind to TLR5. In addition, it was confirmed that eukFlaB N83 / 101 / 139A and eukFlaB N83 / 101 / 139 / 273 / 330AThe variant restored TLR5 binding. It was thus confirmed that glycosylation interferes with FlaB-TLR5 binding, whereas deglycosylation results in higher affinity FlaB-TLR5 binding.

[0120] [Example 7: Verification of the immunopotentiator efficacy of the eukaryotic cell-expressed flagellin variant prepared above in a mouse influenza vaccine experimental model system]

[0121] Seven-week-old female BALB / C mice (n=10) were purchased from Orient (Seongnam, Gyeonggi-do, South Korea) and acclimatized to the facility environment for 1 week before the experiment. (Virbaccorporation, Carros, France) and Lumpun TM (Bayer AG, Leverkusen, Germany) anesthetic was used for anesthesia, and then immunization was started. The following groups were used: 1) sH1N1 antigen group (sH1N1, 0.2ug / mouse); 2) sH1N1 (0.2ug / mouse) and prokFlaB (4ug / mouse); 3) or eukFlaB (4ug / 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 ), and intranasal immunization began. The vaccine antigen was diluted with PBS, and 10 μl was used in each nasal cavity of each mouse, for a total of 20 μl. The mice were immunized three times at intervals of two weeks, and serum was collected two weeks after the last immunization to determine the antibody response.

[0122] The influenza antigen-specific antibody immune response was measured by enzyme-linked immunosorbent assay (ELISA), and mouse sera were collected 2 weeks after the third immunization. After adding sH1N1 (4 μg / ml) to ELISA plates (Corning Laboratories, 3690) and smearing, they were left overnight at 4°C. The next day, the plates were washed 3 times with washing buffer PBS-T [PBS containing 0.05% Tween 20 (Thermo fisherscientific, cat. J20605-AP)], and blocking buffer [PBS-T containing 1 mM EDTA (JUNSEI, cat. 17385S0401) and 0.5% BSA (Sigma, cat. A2153-50G)] was added to block non-specific binding of antigens and antibodies at room temperature for 1 hour. The sera collected from mice were serially diluted (2-fold dilution) with blocking buffer, 40 μl was added to each well, and they were reacted at room temperature for 2 hours. The plates were washed 5 times with PBS-T, horseradish-peroxidase (HRP) conjugated anti-mouse IgG antibody was added, and they were reacted at room temperature for 1 hour, and the plates were washed with PBS-T. Next, substrate TMB (BD OptEIA, cat. 555214) was added, and when color developed, STOP buffer (1N H 2 SO 4 ) was added to terminate the reaction. Absorbance at 450 nm was measured using a microplate reader (Molecular Devices Corporation, Menlo Park, CA). The antibody titer was calculated as the absorbance value 2-fold higher than that of the well without any substance added, and the reciprocal Log2 value of the serum dilution was calculated.

[0123] As Figure 9 shown, the experimental results confirmed eukFlaBN 83A , eukFlaB N83 / 101A , eukFlaB N83 / 101 / 139A , eukFlaB N83 / 101 / 139 / 273A and eukFlaB N83 / 101 / 139 / 273 / 330AInduce the generation of influenza antigen-specific antibodies at the same level as flagellin (prokFlaB) expressed in prokaryotic cells, and thus has the efficacy of an immune enhancer.

[0124]

Example 8: Comparative verification of the antibody generation ability of prokaryotic cell-expressed flagellin and eukaryotic cell-expressed flagellin

[0125] In the influenza vaccine model of Example 7, the flagellin-specific antibody immune response was measured by enzyme-linked immunosorbent assay (ELISA). Specifically, mouse sera were collected 2 weeks after the third immunization. prokFlaB (1 μg / ml) or wild-type eukFlaB (1 μg / ml) was added to an ELISA plate (Corning Laboratories, 3690) and coated, and then left overnight at a temperature of 4°C. Enzyme-linked immunosorbent assay (ELISA) was performed in the same manner as in Example 7.

[0126] As Figure 10 shown, the experimental results confirmed that flagellin (prokFlaB) expressed in prokaryotic cells induced the generation of prokFlaB- and eukFlaB-specific antibodies, but eukFlaB, eukFlaB N83 / 101 / 139A 、eukFlaB N83 / 101 / 139 / 273A and eukFlaB N83 / 101 / 139 / 273 / 330A did not induce the generation of prokFlaB- and eukFlaB-specific antibodies. Therefore, it was confirmed that the eukFlaB variant of the present invention does not induce an immune response against flagellin itself (biological agent) during repeated administration in vivo, thus providing a safer flagellin immune enhancer.

[0127]

Example 9: Verification of the stability of deglycosylated eukaryotic cell-expressed flagellin derivatives

[0128] Purify prokFlaB, eukFlaB and N-glycosylation mutant eukFlaB, and store them at different temperatures (such as -80°C, 4°C and room temperature) for 2 weeks. The structural stability and functionality of the proteins were tested by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and NF-κB reporter analysis, respectively.

[0129] As Figure 11As shown, the experimental results confirmed that the flagellin prepared in eukaryotic cells has more excellent stability compared with the flagellin (prokFlaB) prepared in prokaryotic cells. Therefore, it is expected that the eukFlaB variant of the present invention can provide a more excellent flagellin immunopotentiator in terms of pharmacokinetics during the preparation, circulation, and in vivo administration of biopharmaceuticals.

[0130]

Industrial Applicability

[0131] The present invention relates to the preparation and application of a flagellin immunopotentiator derivative having TLR5 activity using a eukaryotic cell expression system, and more particularly, to a flagellin that can be expressed in eukaryotic cells and has an equivalent or stronger immunopotentiating effect compared to the existing flagellin derived from prokaryotes.

Claims

1. A polypeptide capable of being expressed in eukaryotic cells, wherein one or more asparagines in the amino acid sequence of SEQ ID NO: 13 are replaced by alanine.

2. The polypeptide capable of being expressed in eukaryotic cells according to claim 1, wherein the polypeptide is a polypeptide 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 replaced by alanine.

3. The polypeptide capable of being expressed in eukaryotic cells according to claim 1, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NO: 14 to SEQ ID NO:

23.

4. The polypeptide capable of being expressed in eukaryotic cells according to claim 2, wherein the polypeptide is a polypeptide in which asparagine is replaced by alanine at the following positions: 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 in the amino acid sequence of SEQ ID NO:

13.

5. The polypeptide capable of being expressed in eukaryotic cells according to claim 2, wherein the polypeptide is a polypeptide in which asparagine is replaced by alanine at the following positions: 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 in the amino acid sequence of SEQ ID NO:

13.

6. The polypeptide capable of being expressed in eukaryotic cells according to claim 2, wherein the polypeptide is a polypeptide in which asparagine is replaced by alanine at the following positions: 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 in the amino acid sequence of SEQ ID NO:

13.

7. An immunoadjuvant composition for a vaccine, which comprises a polypeptide in which one or more asparagines in the amino acid sequence of SEQ ID NO: 13 are replaced by alanine.

8. The immunoadjuvant composition for a vaccine according to claim 7, wherein the polypeptide is a polypeptide 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 replaced by alanine.

9. The immunoadjuvant composition for a vaccine according to claim 7, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NO: 14 to SEQ ID NO:

23.

10. The immunoadjuvant composition for a vaccine according to claim 8, wherein the polypeptide is a polypeptide in which asparagine is replaced by alanine at the following positions: position 83 in the amino acid sequence of SEQ ID NO: 13; and One or more positions selected from the group consisting of position 101, position 139, position 273, and position 330 in the amino acid sequence of SEQ ID NO:

13.

11. The immunoadjuvant composition for a vaccine according to claim 8, wherein, the polypeptide is a polypeptide in which asparagine at the following positions is replaced with alanine: position 83 and position 101 in the amino acid sequence of SEQ ID NO: 13; and one or more positions selected from the group consisting of position 139, position 273, and position 330 in the amino acid sequence of SEQ ID NO:

13.

12. The immunoadjuvant composition for a vaccine according to claim 8, wherein, the polypeptide is a polypeptide in which asparagine at the following positions is replaced with alanine: position 83, position 101, and position 139 in the amino acid sequence of SEQ ID NO: 13; and one or more positions selected from the group consisting of position 273 and position 330 in the amino acid sequence of SEQ ID NO:

13.

13. A composition for enhancing immune function, which comprises a polypeptide in which one or more asparagines in the amino acid sequence of SEQ ID NO: 13 are replaced with alanine.

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

15. The composition for enhancing immune function according to claim 13, wherein, the polypeptide comprises one amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NO: 14 to SEQ ID NO:

23.

16. The composition for enhancing immune function according to claim 14, wherein, the polypeptide is a polypeptide in which asparagine at the following positions is replaced with alanine: position 83 in the amino acid sequence of SEQ ID NO: 13; and one or more positions selected from the group consisting of position 101, position 139, position 273, and position 330 in the amino acid sequence of SEQ ID NO:

13.

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

13.

18. The composition for enhancing immune function according to claim 14, wherein, the polypeptide is a polypeptide in which asparagine at the following positions is replaced with alanine: position 83, position 101, and position 139 in the amino acid sequence of SEQ ID NO: 13; and One or more positions selected from the group consisting of position 273 and position 330 in the amino acid sequence of SEQ ID NO:

13.

19. A pharmaceutical composition for preventing or treating an autoimmune disease, an inflammatory disease, a microbial infection or cancer, which comprises a polypeptide in which one or more asparagines in the amino acid sequence of SEQ ID NO: 13 are replaced with alanine.

20. 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 is a polypeptide in which asparagine at one or more positions selected from the group consisting of position 83, position 101, position 139, position 273 and position 330 in the amino acid sequence of SEQ ID NO: 13 is replaced 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 an amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NO: 14 to SEQ ID NO:

23.

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

13.

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

13.

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

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