WO2014171721A1

WO2014171721A1 - Ik factor and pharmaceutical use of nucleic acid encoding ik factor

Info

Publication number: WO2014171721A1
Application number: PCT/KR2014/003282
Authority: WO
Inventors: 남재환; 박혜림; 이상명; 이동희
Original assignee: 가톨릭대학교 산학협력단; (주)셀인바이오
Priority date: 2013-04-16
Filing date: 2014-04-16
Publication date: 2014-10-23

Abstract

The present invention relates to a pharmaceutical composition for treating and/or preventing arthritis, containing, as an active ingredient, a gene carrier in which an IK factor or a fragment thereof, or a nucleic acid encoding the same is inserted. The IK factor or the fragment thereof, and the nucleic acid encoding the same, which are the active ingredients of the pharmaceutical composition according to the present invention, are derived from an organism, have no side effects if administered for a long time, thereby ensuring safety, and are expected to effectively treat arthritis by being involved in the high level mechanism for arthritis treatment.

Description

Pharmaceutical Use of IK Factor and Nucleic Acids Encoding IK Factor

FIELD OF THE INVENTION The present invention relates to pharmaceutical uses of peptides derived from living organisms and nucleic acids encoding them, and more particularly to pharmaceutical uses of IK factors or fragments thereof and / or nucleic acids encoding them.

Arthritis is a disease involving joint stiffness and persistent pain around the joints due to inflammation of one or more joints. Arthritis can be largely divided into acute arthritis and chronic arthritis. Chronic arthritis, such as gouty arthritis, polyarthritis, rheumatoid arthritis, and osteoarthritis due to aging of bones or joints, causes persistent pain in the joint area.

Among chronic arthritis, rheumatoid arthritis is an unexplained chronic inflammatory disease characterized by multiple arthritis. To date, the exact cause of rheumatoid arthritis is unknown. However, genetic predisposition, bacterial and viral infections are generally thought to be the cause of rheumatoid arthritis, and hormones are also believed to be involved in rheumatoid arthritis. Rheumatoid arthritis not only affects the joints, but also adversely affects other organs of the body. Thus, patients with rheumatoid arthritis are not only unable to use or walk their hands or feet, but also frequently feel tired and uncomfortable. Furthermore, rheumatoid arthritis causes symptoms such as weight loss and sleep deprivation, and if rheumatoid arthritis persists, physical activity, such as muscle weakness, may be restricted, which may greatly affect personal and social activities. When physical activity is limited by rheumatoid arthritis, the likelihood of obesity increases, can lead to heart disease due to cholesterol levels, and can lead to depression.

Rheumatoid arthritis occurs in 0.2 men per 1,000 men and 0.4 women per 1,000 women per year, and the prevalence of rheumatoid arthritis is 0.4-1.4% worldwide. Moreover, the market for rheumatoid arthritis drugs is expected to grow rapidly as the elderly population grows.

To date, drugs for treating rheumatoid arthritis include nonsteroidal anti-inflammatory drugs (NSAIDs); steroid; Non-biologic disease-modifying antirheumatic drug, DMARD such as antimalarial agent, hydroxychloroquinone (HCQ), sulfasalazine, methotrexate (MTX), leflunomide ); Biological antirheumatic drugs such as tumor necrosis factor (TNF) inhibitors or IL-6 inhibitors are used.

As biological antirheumatic agents, tumor necrosis factor (TNF) inhibitors such as etanercept, adalimumab, infliximab, golomumab, certolizumab; IL-6 inhibitors such as Tocilizumab; Rituximab, a monoclonal antibody that is a B-cell scavenger; Abatacept, which blocks an immune response between antigen presenting cells and T-cells; As small molecule inhibitors, tofacitinib, which selectively blocks JAK (Janue activated kinase), has been developed.

However, most of these rheumatoid arthritis drugs cause serious side effects. For example, nonsteroidal anti-inflammatory drugs cause gastrointestinal side effects, and nonsteroidal anti-inflammatory drugs, including selective COX-2 inhibitors, can cause serious cardiovascular side effects. In addition, nonsteroidal anti-inflammatory drugs are known to have side effects such as renal failure, interstitial nephritis, and acute renal failure. Steroids can cause hypothalamic-pituitary-adrenal atrophy, resulting in adrenal insufficiency, and side effects such as glaucoma, cataracts, osteonecrosis, osteoporosis, hypertension and hypokalemia. In particular, nonsteroidal anti-inflammatory drugs and steroids are not only to relieve inflammation, it does not inhibit the progress of the actual arthritis, and there is a disadvantage that the prevention effect of the joint damage is insufficient.

Meanwhile, the anti-rheumatic drugs currently developed may inhibit the progression of early rheumatoid arthritis, but do not cure rheumatoid arthritis and some side effects have been reported. For example, abiotic antirheumatic drugs have side effects such as vision disorders, retinal lesions, skin rashes, abnormal liver function levels, and nausea (nausea). In addition, tumor necrosis factor inhibitors among biological antirheumatic drugs are problematic for blood abnormalities, heart failure, liver dysfunction, and opportunistic infections, and IL-6 inhibitors may cause side effects such as liver dysfunction and white blood cell reduction. Other biological antirheumatic drugs have been reported with side effects such as infection problems, tumor potential, gastrointestinal perforation and hyperlipidemia.

In particular, most biological antirheumatic drugs have a fatal disadvantage that they can only be administered by subcutaneous administration or by vascular injection, and over time, the immunogenicity of the human body causes a decrease in response to the drug. In addition, the currently developed biological antirheumatic drugs expose the vulnerability to opportunistic infection and cancer suppression as a result of suppressing the overall immune system. In addition, these biological antirheumatic drugs are quite expensive, resulting in significant economic burden on patients. In particular, antibodies, which are representative biological anti-rheumatic drugs, which have been used in the past, have a problem of not responding to many patients with rheumatoid arthritis.

The present invention has been proposed to solve the above-mentioned problems of the prior art, and an object of the present invention is a pharmaceutical for treating and / or preventing arthritis in which safety is ensured due to no side effects and toxicity to a living body despite prolonged administration. It is to provide a composition.

It is another object of the present invention to provide a pharmaceutical composition for treating and / or preventing arthritis, which can be prepared in large quantities at low cost without avoiding efficacy, thereby reducing the burden on the patient.

The present invention provides a pharmaceutical composition for treating or preventing arthritis, containing a pharmaceutically effective amount of an IK factor or fragment of an IK factor.

In one exemplary embodiment, the fragment of IK factor comprises a peptide having the amino acid sequence of SEQ ID NO: 4 or a partial peptide of SEQ ID NO: 4.

For example, the fragment of the IK factor consists of at least one amino acid residue selected from 382 th serine, 489 tyrosine, and 492 tyrosine of the amino acid sequence of SEQ ID NO: 2 and adjacent amino acids of these selected amino acid residues. It may consist of peptides having 10 or more amino acids.

In exemplary embodiments, the fragment of the IK factor is phosphorylated with at least one amino acid consisting of 382 th serine, 489 th tyrosine, and 492 th tyrosine of the amino acid sequence of SEQ ID NO: 2.

In one optional embodiment, the fragment of IK factor is a fragment of a peptide having an amino acid sequence of SEQ ID NO: 2, wherein at least one selected from among 382 th serine and 489 th tyrosine of the amino acid sequence of SEQ ID NO: 2 It may consist of a peptide having at least 10 amino acids consisting of one amino acid residue and contiguous amino acids of these selected amino acid residues.

In a specific embodiment, the fragment of the IK factor may be composed of a peptide having 10 or more amino acids in which two or more adjacent amino acids are linked to the N-terminus and C-terminus of the selected amino acid, respectively.

In an exemplary embodiment, the fragment of the IK factor may be composed of a peptide having at least one amino acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 47, SEQ ID NO: 49.

Pharmaceutical compositions containing the IK factor or fragments of IK factor can be used to treat rheumatoid arthritis.

In an exemplary embodiment, the IK factor or fragment of IK factor may be contained in the pharmaceutical composition at a concentration of 1.0 ng / mL to 10 μg / mL.

The present invention also provides a pharmaceutical composition for treating or preventing arthritis, comprising a gene carrier comprising a nucleic acid molecule encoding an IK factor or fragment of an IK factor.

For example, the nucleic acid encoding a fragment of the IK factor includes a nucleic acid having a nucleotide sequence of SEQ ID NO: 3 or a partial nucleic acid of SEQ ID NO: 3.

In an exemplary embodiment, the nucleic acid encoding a fragment of the IK factor is at least one selected from nucleotide residues 1144-1146, 1465-1467 nucleotide residues, and 1474-1476 nucleotide residues of the nucleotide sequence of SEQ ID NO: 1. It may consist of a nucleic acid having at least 30 nucleotides consisting of one nucleotide residue and adjacent nucleotides of these selected nucleotide residues.

In one exemplary embodiment, the nucleic acid encoding a fragment of the IK factor comprises at least one nucleotide residue selected from the set of 1144-1146 nucleotides and the set of 1465-1467 nucleotides of the nucleotide sequence of SEQ ID NO: 1. And nucleic acids having 30 or more nucleotides consisting of contiguous nucleotides of these selected nucleotide residues.

In one specific embodiment, the nucleic acid encoding a fragment of the IK factor consists of a nucleic acid having at least 30 nucleotides each having at least 6 contiguous nucleotides linked to the 5 'and 3' ends of the selected nucleotide residue. Can be.

In an exemplary embodiment, the nucleic acid encoding the fragment of the IK factor encodes a peptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 47 and SEQ ID NO: 49. And a nucleic acid having a nucleotide sequence.

For example, the nucleic acid encoding the fragment of the IK factor may be composed of a nucleic acid having at least one nucleotide sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 46 and SEQ ID NO: 48. have.

Nucleic acids encoding the IK factor or fragments of the IK factor can be used to treat rheumatoid arthritis.

In an exemplary embodiment, the nucleic acid encoding the IK factor or fragment of IK factor may be contained in the pharmaceutical composition at a concentration of 1.0 ng / mL to 10 μg / mL.

For example, the gene carrier may have the form of a naked nucleic acid molecule, a plasmid, a viral vector and a liposome or niosome containing the plasmid or the viral vector.

At this time, for example, the viral vector may be selected from the group consisting of adenovirus, adeno-associated virus, retrovirus, lentivirus, baculovirus, herpes simplex virus, vaccinia virus.

The present invention also relates to an IK factor or fragment thereof for treating arthritis, a nucleic acid encoding an IK factor or a fragment thereof, and / or a vector or gene carrier having these nucleic acids.

The present invention also provides a step of administering an IK factor or fragment of IK factor, a nucleic acid encoding IK factor or a fragment thereof, and / or a vector or gene delivery carrier having these nucleic acids in a therapeutically effective amount or in a pharmaceutically effective amount. It includes a method of treating arthritis, which includes.

According to the present invention, the IK factor or fragments thereof and / or nucleic acids encoding these peptides are involved in the upstream level of reaction mechanisms associated with arthritis, for example rheumatoid arthritis, thereby promoting the development of rheumatoid arthritis. Suppress

The IK factor or fragment thereof and / or nucleic acid encoding them used as an active ingredient according to the present invention can be secured because it is an immune balance modulator in the human body that overall regulates the activity of immune cells affecting arthritis. have.

In particular, since the IK factor or fragment thereof is an endogenous substance produced in the human body, it has low immunogenicity and does not significantly affect the entire population of immune cells, and therefore, long-term administration is expected to have no side effects.

It can also be manufactured at a much lower cost than, for example, conventional rheumatoid arthritis therapies being developed as monoclonal antibodies. In addition, it is expected to solve the problem of avoiding the efficacy of the existing arthritis treatments.

1 is a schematic diagram showing the location of a full-length nucleic acid encoding an IK factor and a truncated nucleic acid fragment (tIK nucleic acid fragment) consisting of only the C-terminal nucleotide except for the N-terminal nucleotide of the full-length nucleic acid. to be. "HA" in Figure 1 represents a hemagglutinin tag (haemagglutinin) tag.

The top part of FIG. 2 schematically shows the position of nucleotides constituting a nucleic acid fragment (tIK nucleic acid fragment) in which some nucleotides are cleaved relative to a nucleotide encoding a full-length IK factor according to an exemplary embodiment of the present invention. It is a schematic diagram. The remainder of FIG. 2 shows the positions of the amino acids and substituted amino acids of the active sites predicted to be involved in the physiological function of the IK factor relative to the construction of the variant IK factor relative to the exemplary amino acid sequence of the full length IK factor. It is a schematic diagram which shows the kind schematically. "HA" in Figure 2 represents a hemagglutinin (haemagglutinin) tag (tag).

Figure 3 shows MHC by expression of a truncated nucleic acid fragment of IK factor (tIK nucleic acid fragment) and mutant nucleic acid fragments (S382A, Y489F, Y492F, Y489492F, S382AY489492F) in which some nucleotides of the tIK nucleic acid fragment are substituted with other nucleotides. This is a graph measuring the degree of inhibition of the expression of CNOT1, CDCA3 and MAPK1, which are higher regulators that inhibit the expression of CIITA, a class II transactivator. In FIG. 3, * means P <0.05 as compared to negative control, and # means P <0.05 when mutant nucleic acid fragments are expressed as compared with wild type tIK.

Figure 4a is a mouse produced by crossing the IL-1 Receptor antagonist knock-out (IL1RaKO) mouse with arthritis naturally induced, IL1RaKO mouse and transgenic mice expressing some cleaved IK factor (tIK) according to the present invention (tIK-IL1RaKO) is a graph measuring arthritis index over time. In FIG. 4A, * means P <0.05 compared to negative control, ** means P <0.01 compared to negative control, and *** means P <0.001.

Figure 4b is a graph measuring the incidence of arthritis over time in IL1RaKO mice and tIK-IL1RaKO mice.

5 is a photograph of the joint region of IL1RaKO mice naturally induced arthritis, and tIK-IL1RaKO mouse produced according to the present invention.

Figure 6a shows the results of measurement of arthritis-derived joint tissue from IL1RaKO mice and tIK-IL1RaKO mice produced according to the present invention using H & E staining. The left side is a micrograph of the joint tissue, and the right side is a graph analyzing the degree of inflammation in the joint tissue. In FIG. 6A, * means P <0.05 as compared to negative control.

FIG. 6B shows the results of measurement of joint tissue derived from IL1RaKO mice naturally induced arthritis and tIK-IL1RaKO mice prepared according to the present invention using safranin O staining. The left side is a micrograph of the joint tissue and the right side is a graph analyzing the degree of cartilage erosion in the joint tissue. In FIG. 6B, * means P <0.05 as compared to negative control.

Figure 7 is a photograph of the degree of bone damage in the joint area of IL1RaKO mice naturally induced arthritis, and tIK-IL1RaKO mouse produced according to the present invention.

Figure 8a is a graph measuring the mRNA expression level of inflammatory cytokines in the joint region of IL1RaKO mice naturally induced arthritis and tIK-IL1RaKO mouse prepared according to the present invention. In FIG. 8A, * denotes P <0.05 and ** denotes P <0.01 compared to negative control.

Figure 8b is a photograph of the degree of expression of inflammatory cytokines after staining joint tissue of IL1RaKO mice naturally induced arthritis, and tIK-IL1RaKO mice prepared according to the present invention.

9 is a graph measuring the mRNA expression level of inflammatory cytokines in serum of IL1RaKO mice naturally induced arthritis and tIK-IL1RaKO mice prepared according to the present invention. ** in FIG. 9 means P <0.01 compared to negative control.

Figure 10a is to examine the degree of differentiation of etiological T cells in spleen cells of IL-1 Receptor antagonist knock-out (IL1RaKO) mice and arthritis induced naturally, according to the invention (tIK-IL1RaKO), Plots and graphs measuring the amount of Th1 cells, which are pathogenic T cells.

Figure 10b is to examine the degree of differentiation of etiological T cells in spleen cells of arthritis induced IL-1 Receptor antagonist knock-out (IL1RaKO) mouse, and mouse (tIK-IL1RaKO) produced according to the present invention, Plots and graphs measuring the amount of Th17 cells, the etiological T cells.

Figure 11 is a graph measuring the amount of macrophage activator factor in splenocytes of IL-1 Receptor antagonist knock-out (IL1RaKO) mice in which arthritis is naturally induced and mice prepared according to the present invention (tIK-IL1RaKO). . In FIG. 11, * means P <0.05 as compared to negative control.

Figure 12a is a graph measuring the expression level of inflammatory cytokines in cells after transfection of macrophages with peptides containing amino acids involved in the activity of IK factor in accordance with the present invention. In FIG. 12A, * denotes P <0.05 and ** denotes P <0.01 compared to negative control.

Figure 12b is a graph measuring the expression level of inflammatory cytokines in cell culture after transfection of macrophages with peptides containing amino acids involved in the activity of IK factor according to the present invention. In FIG. 12B, ** denotes P <0.01 and *** denotes P <0.001 as compared to the negative control.

Figure 13a is a plot and a graph analyzing the degree of differentiation of Th0 cells in spleen cells of wild-type mice injected with peptides containing amino acids involved in the activity of IK factor according to the present invention.

Figure 13b is a plot and graph analyzing the degree of differentiation of Th1 cells in spleen cells of wild-type mice injected with a peptide containing amino acids involved in the activity of tIK according to the present invention.

Figure 13c is a plot and graph analyzing the degree of differentiation of Th17 cells in spleen cells of wild-type mice injected with a peptide containing amino acids involved in the activity of tIK according to the present invention.

Figure 14a is a graph measuring the arthritis index over time after injecting the IK factor-derived peptide into IL1RaKO mice, a naturally occurring arthritis model according to the present invention, Figure 14b is arthritis over time in the mouse It is a graph measuring the incidence rate.

Figure 15a is a plot and graph of the differentiation of Th17 cells in spleen cells of the mouse after injecting a peptide derived from IK factor in accordance with the present invention into a mouse that is a naturally occurring arthritis model. In FIG. 15A, * means P <0.05 as compared to negative control.

Figure 15b is a photograph of the degree of expression of inflammatory cytokines after staining the joint tissue of a mouse, a naturally occurring arthritis model injected with IK factor-derived peptides according to the present invention.

Figure 16 is a SDS-PAGE measurement photograph confirming that the IK factor was expressed after injecting a truncated IK gene fragment (tIK) having the Fc tag sequence added to the 3 'end into insect cells using a Baculovirus expression system. .

17 shows the IK factor in recombinant adeno-associated virus (tIK-AAV) in which a truncated IK gene fragment (a gene fragment encoding tIK) having an HA tag sequence added to its 5 'end was inserted into an adeno-associated virus. Western-blotting results show expression.

Figure 18a is a graph measuring the arthritis index over time after injecting the adeno-associated virus vector inserted with the truncated IK gene fragment (gene fragment encoding tIK) into IL1RaKO mice, a naturally occurring arthritis model, 18b is a graph measuring the incidence of arthritis over time in the mouse.

19 is a Western-blotting result showing that the plasmid inserted with the truncated IK gene fragment (tIK) having the HA tag sequence added to the 5 'end was injected into CHO cells, and that the IK factor was expressed in CHO cells. .

The present inventors have studied new functions of IK factor, a type of cytokine expressed in vivo, so that gene carriers having IK factors or fragments thereof and / or nucleic acids encoding them are for example rheumatoid arthritis. The present invention was completed by clarifying that the progression of arthritis as described above. EMBODIMENT OF THE INVENTION Hereinafter, this invention is demonstrated in detail, referring an accompanying drawing.

Justice

The term "amino acid" is used herein in its broadest sense and is intended to include naturally-occurring L-amino acids or residues. 1- and 3-letter abbreviations commonly used for naturally-occurring amino acids are used herein (Lehninger, Biochemistry, 2d ed., Pp. 71-92, (Worth Publishers: New York, 1975)). Amino acids are chemicals having properties known in the art to be characteristic of not only D-amino acids but also chemically-modified amino acids such as amino acid analogs, naturally-occurring amino acids such as norleucine, and amino acids that are not commonly incorporated into proteins. And synthetically-synthesized compounds include, for example, analogs or mimetics of phenylalanine or proline that allow conformational limitations of the same peptide compound as natural phenylalanine (Phe) or proline (Pro). Analogs and mimetics are referred to herein as "functional equivalents" of amino acids Other examples of amino acids are described in Roberts and Vellaccio, The Peptide. s: Analysis, Synthesis, Biology, Eds. Gross and Meiehofer, Vol. 5, p. 341 (Academic Press, Inc .: N.Y. 1983).

For example, synthetic peptides synthesized by standard solid-phase synthesis techniques are not limited to amino acids encoded by genes, thus allowing a wider variety of substitutions for a given amino acid. Amino acids not encoded by the genetic code are referred to herein as "amino acid analogs" and are described, for example, in WO 90/01940. For example, amino acid analogs include 2-amino adipic acid (Aad) for Glu and Asp; 2-aminopimelic acid (Apm) for Glu and Asp; 2-aminobutyric acid (Abu) for Met, Leu and other aliphatic amino acids; 2-aminoheptanoic acid (Ahe) for Met, Leu and other aliphatic amino acids; 2-aminobutyric acid (Aib) for Gly; Cyclohexylalanine (Cha) for Val, Leu and Ile; Homoarginine (Har) for Arg and Lys; 2,3-diaminopropionic acid (Dap) for Lys, Arg and His; N-ethylglycine (EtGly) for Gly, Pro and Ala; N-ethylglycine (EtGly) for Gly, Pro and Ala; N-ethylasparagine (EtAsn) for Asn and Gln; Hydroxylysine (Hyl) for Lys; Allohydroxylysine (AHyl) for Lys; 3- (and 4-) hydroxyproline (3Hyp, 4Hyp) for Pro, Ser and Thr; Allo-isoleucine (AIle) for Ile, Leu and Val; 4-amidinophenylalanine for Arg; N-methylglycine (MeGly, Sarcosine) for Gly, Pro and Ala; N-methylisoleucine (MeIle) for Ile; Norvaline (Nva) for Met and other aliphatic amino acids; Norleucine (Nle) for Met and other aliphatic amino acids; Ornithine (Orn) for Lys, Arg and His; Citrulline (Cit) and methionine sulfoxide (MSO) for Thr, Asn and Gln; And N-methylphenylalanine (MePhe), trimethylphenylalanine, halo- (F-, Cl-, Br- or I-) phenylalanine or trifluorylphenylalanine for Phe.

As used herein, the term "peptide" includes all proteins, protein fragments and peptides that have been isolated from naturally occurring, synthesized by a recombinant technique or chemically synthesized. For example, the peptide of the present invention consists of at least 5, preferably 10 amino acids.

In certain embodiments, variants of a compound are provided, such as peptide variants having one or more amino acid substitutions. As used herein, "peptide variants" refers to the original amino acid while one or more amino acids are substituted, deleted, added and / or inserted into the amino acid sequence of the peptide. It is said to exert almost the same biological function as the peptide consisting of. Peptide variants should have at least 70% identity, preferably at least 90% and more preferably at least 95% identity with the original peptide. Such substituents may include amino acid substituents known as "conservatives". Variants may also include nonconservative changes. In one exemplary embodiment, the sequence of the variant polypeptide differs from the original sequence by replacing, deleting, adding or inserting five or fewer amino acids. Variants may also be altered by deletion or addition of amino acids with minimal impact on the immunogenicity, secondary structure and hydropathic nature of the peptide.

Unless stated otherwise, “conservative” substitutions herein means that there is no significant change in properties such as secondary structure and hydropathic nature of the polypeptide even when one amino acid is substituted with another amino acid. Amino acid variations with respect to such conservative substitutions may be attributed to the relative similarity of amino acid side chain substituents such as polarity, charge, solubility, hydrophobicity, hydrophilicity and / or amphipathic nature. Can be obtained based on similarity.

For example, amino acids are based on common side chain properties: 1) hydrophobic (leucine, methionine, alanine, valine, leucine, isoleucine) 2) neutral hydrophilic (cysteine, serine, threonine, asparagine, glutamine), 3) acidic (aspartic acid, Glutamic acid), 4) basic (histidine, lysine, arginine), 5) residues that affect chain orientation (glycine, proline), and 6) aromatics (tryptophan, tyrosine, phenylalanine). Conservative substitutions will entail exchanging a member of one of each of these classes for another member of the same class.

By analysis of the size, shape and type of amino acid side chain substituents, arginine, lysine and histidine are all positively charged residues; Alanine, glycine and serine have similar sizes; It can be seen that phenylalanine, tryptophan and tyrosine have a similar shape. Thus, based on these considerations, arginine, lysine and histidine; Alanine, glycine and serine; Phenylalanine, tryptophan, and tyrosine are biologically equivalent functions.

In introducing variants, hydropathic idex of amino acids can be considered. Each amino acid is assigned a hydrophobicity index depending on its hydrophobicity and charge: isoleucine (+4.5); Valine (+4.2); Leucine (+3.8); Phenylalanine (+2.8); Cysteine / cysteine (+2.5); Methionine (+1.9); Alanine (+1.8); Glycine (-0.4); Threonine (-0.7); Serine (-0.8); Tryptophan (-0.9); Tyrosine (-1.3); Proline (-1.6); Histidine (-3.2); Glutamic acid (-3.5); Glutamine (-3.5); Aspartic acid (-3.5); Asparagine (-3.5); Lysine (-3.9); And arginine (-4.5).

The hydrophobic amino acid index is very important in conferring the interactive biological function of proteins. It is known that substitution with amino acids having similar hydrophobicity indexes can retain similar biological activity. When introducing mutations with reference to the hydrophobicity index, substitutions are made between amino acids which exhibit a hydrophobicity index difference of preferably within ± 2, more preferably within ± 1, even more preferably within ± 0.5.

On the other hand, it is also well known that substitutions between amino acids having similar hydrophilicity values result in proteins with equivalent biological activity. As disclosed in US Pat. No. 4,554,101, the following hydrophilicity values are assigned to each amino acid residue: arginine (+3.0); Lysine (+3.0); Aspartic acid (+ 3.0 ± 1); Glutamic acid (+ 3.0 ± 1); Serine (+0.3); Asparagine (+0.2); Glutamine (+0.2); Glycine (0); Threonine (-0.4); Proline (-0.5 ± 1); Alanine (-0.5); Histidine (-0.5); Cysteine (-1.0); Methionine (-1.3); Valine (-1.5); Lysine (-1.8); Isoleucine (-1.8); Tyrosine (-2.3); Phenylalanine (-2.5); Tryptophan (-3.4). When introducing mutations with reference to hydrophilicity values, substitutions are made between amino acids which exhibit a hydrophilicity value difference of preferably within ± 2, more preferably within ± 1 and even more preferably within ± 0.5.

Amino acid exchange in proteins that do not alter the activity of the molecule as a whole is known in the art (H. Neurode, R. L. Hill, The Proteins, Academic Press, New York, 1979). The most commonly occurring exchanges are amino acid residues Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Thy / Phe, Ala / Exchange between Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, Asp / Gly.

As used herein, “polynucleotide” or “nucleic acid” is used interchangeably and refers to a polymer of nucleotides of any length and encompasses DNA (such as cDNA) and RNA molecules inclusively. The “nucleotide”, a structural unit of a nucleic acid molecule, can be incorporated into a polymer by deoxyribonucleotides, ribonucleotides, modified nucleotides or nucleotides, and / or analogs thereof, or by DNA or RNA polymerase, or by synthetic reactions. May be any substrate present. Polynucleotides may include modified nucleotides, sugars or analogs in which the nucleotides are modified, such as methylated nucleotides and analogs thereof (Scheit, Nucleotide Analogs, John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews , 90: 543-584 (1990).

Variations in nucleotides do not result in mutations in proteins. Such nucleic acids include nucleic acid molecules comprising a codon that is functionally equivalent or a codon that encodes the same amino acid, or a codon that encodes a biologically equivalent amino acid. In addition, variations in nucleotides may result in changes in the protein itself. Even in the case of mutations that bring about changes in amino acids of proteins, those exhibiting almost the same activity as the proteins of the present invention can be obtained.

In the scope of having the effect of the IK factor of the present invention and fragments thereof and / or nucleic acids encoding them, such as arthritis, the peptides and nucleic acid molecules of the present invention are amino acid sequences or nucleotide sequences listed in the sequence listing. It should be appreciated by those skilled in the art that the present invention is not limited thereto. For example, the biological functional equivalents that may be included in the peptide of the present invention may be a peptide having a variation in the amino acid sequence exhibiting biological activity equivalent to that of the peptide of the present invention.

In general, the peptides (including fusion proteins) and polynucleotides mentioned herein are isolated. An “isolated” peptide or polynucleotide is one that is removed from its original environment. For example, proteins that exist in nature are separated by removing some or all of the substances that exist together in that state. Such polypeptides should be at least 90% pure, preferably at least 95%, more preferably at least 99% pure. Polynucleotides are isolated by cloning in a vector.

As used herein, the term "vector" refers to a construct that is capable of delivery to a host cell and preferably enables expression of one or more desired genes or sequences. For example, a vector may include viral vectors, DNA or RNA expression vectors, plasmids, cosmids or phage vectors, DNA linked to cationic condensing agents, or RNA expression vectors, DNA or RNA expression vectors packaged in liposomes, specific eukaryotic cells such as producer cells, and the like.

As used herein, "expression control sequence" refers to a nucleic acid sequence that controls the transcription of a nucleic acid. Expression control sequences include promoters or enhancers, such as constitutive promoters or inducible promoters. The expression control sequence is linked to the nucleic acid sequence to be transcribed.

As used herein, the term “operatively linked” refers to a functional binding between a nucleic acid expression control sequence (eg, an array of promoters, signal sequences, or transcriptional regulator binding sites) and other nucleic acid sequences, The regulatory sequence thereby controls the transcription and / or translation of the other nucleic acid sequence.

As used herein, the term "pharmaceutically effective amount" or "therapeutically effective amount" means an amount sufficient to achieve the efficacy or activity of the active ingredient peptide or fragments thereof and / or nucleic acids encoding them. For example, a pharmaceutical composition containing a gene carrier comprising a peptide according to the present invention or a nucleic acid encoding these peptides can be used to treat and / or treat chronic arthritis, including gouty arthritis, polyarthritis, deformable arthritis, including rheumatoid arthritis. Can be applied to prevent.

Peptides, Nucleic Acids and Vectors

IK factor is a cytokine that inhibits the expression of major histocompatibility complex II (MHC II) on the surface of antigen presenting cells (APCs) capable of presenting antigens to CD4 T cells. Cain. IK factor is known to activate cAMP to inhibit the expression of MHC class II expressed by interferon-gamma (IFN-γ, interferon-γ) induced by viral infection.

As exemplarily shown in FIG. 1, the full-nucleic acid encoding the IK factor has a Nuclear Localization Signal Sequence (NLS) and a trimeric coiled-coil motif in the N-terminal (5 'end) region. have. In addition, the C-terminal (3 'end) region of the full-length nucleic acid encoding the IK factor repeats the Arg (R) -Asp (D) and Arg (R) -Glu (E) sequences found mainly in nuclear proteins. It has a RED domain and three NLSs. Exemplary full-nucleic acid encoding an IK factor is a polynucleotide consisting of 1,674 nucleotides represented by SEQ ID NO: 1. The IK factor may be expressed except for the last transcription termination codon of SEQ ID NO: 1, and may consist of 557 amino acids of SEQ ID NO: 2. However, those skilled in the art will recognize that the IK factor that can be used in the present invention and the IK nucleic acid encoding the same are not limited to those listed in the sequence listing.

Joints are organically connected between the bone and bone of the synovial membrane, cartilage, bone, synovial fluid, and is a complex tissue that affects each other through cytokines. Symptoms such as chronic inflammation, tissue necrosis, cell infiltration, neovascularization, and joint destruction in joints are known as the major etiology of rheumatoid arthritis. Synovial tissue called pannus, known as an essential pathology for the development of rheumatoid arthritis, invades tissue around the joint and causes bone resorption. In Pannus, antigen-presenting cells and T cells are observed at a very high density. The interaction of these cells is known to initiate and widen the immune response induced by T cells. In fact, many antigen-presenting cells expressing MHC class II are observed in Pannus patients with rheumatoid arthritis, and HLA-DR-positive cells and T cells are located at the same site. Therefore, to treat rheumatoid arthritis, it may be effective to suppress the interaction between antigen-presenting cells expressing MHC class II and T cells.

In particular, there are IL-17 + T cells and functionally activated IL-17 in the synovial membrane, a lesion of rheumatoid arthritis. IL-17 is highly detected in the serum of rheumatoid arthritis patients, among which IL-17A induces the expression of IL-1β and IL-6 in synovial cells of rheumatoid arthritis patients. In connection with arthritis, IL-17 inhibits the production of matrix in chondrocytes and osteoblasts, causing joint damage and deficient tissue regeneration. IL-17 also activates the expression and function of matrix metalloproteinases (MMPs) and causes irreversible cartilage damage in mouse models with tumor necrosis factor (TNF). Local inflammation of Th17 cells and synovial cells in the inflammatory response promotes the secretion of MMPs and expression of IL-1β and IL-6 in synovial cells. In connection with bone destruction, IL-17 amplifies RANK signals in osteoclasts by increasing the expression of receptor activator of NF-κB ligand (RANKL) on osteoblasts. In particular, Th17 cells expressing RANKL are known to play an important role in the osteoclast differentiation process.

According to the present invention, the nucleic acid encoding the IK factor or fragment thereof can inhibit and prevent the progression of arthritis. In particular, these nucleic acids inhibit the secretion of cytokines associated with rheumatoid arthritis and block the differentiation of immune cells.

Also in an exemplary embodiment of the invention, a nucleic acid fragment encoding a fragment of an IK factor, eg, an N-terminally truncated fragment (which may be abbreviated herein as "tIK factor" or "tIK") tIK nucleic acid fragments) can be expressed in an appropriate expression system. These nucleic acid fragments are expressed to inhibit the expression of class II MHC (MHC II) involved in the inflammatory immune response, one of the main symptoms of arthritis. Specifically, according to an exemplary embodiment of the present invention, a tITA nucleic acid fragment in which some nucleotides are cleaved and / or a mutant tIK nucleic acid fragment in which some nucleotides are substituted is involved in the expression of MHC II (class II, major histocompatibility complex). Increasing the expression of cell division cycle-associated protein 3 (CDCA3), CCR4-NOT transcription complex subunit 1 (CNOT1) and miogen-activated protein kinase 1 (MAPK1), which are high regulatory factors that inhibit the expression of transactivator (FIG. 3). In addition, the mutant tIK substituted with some nucleotides was found to be able to halve this effect, confirming that a specific site in the IK factor is a physiologically active site involved in the expression of a higher regulatory factor of MHC II.

According to exemplary embodiments of the invention, compared to mice naturally induced by arthritis (IL1RaKO), metastatic mice induced to express fragments of the IK factor, e.g., tIK, a truncated fragment of the N-terminus (see In the specification, "tIK-IL1RaKO") significantly reduced the incidence of arthritis (see FIGS. 4A, 4B and 5). In addition, the degree of inflammation or cartilage damage in the articular tissue of tIK-IL1RaKO mice was significantly reduced in the articular tissues of IK1RaKO mice (see FIGS. 6A and 6B), and the degree of bone damage was also greatly reduced (see FIG. 7).

In particular, tIK-IL1RaKO mice have significantly reduced secretion of cytokines associated with arthritis in joint tissues and serum compared to IL1RaKO mice. Specifically, the metastatic mouse (tIK-IL1RaKO) induced to express the cleaved IK factor (tIK factor) is compared to the arthritis-induced mice (IL-1 antagonist-receptor knockout mouse, IL1RaKO mouse). Of course (see FIGS. 8A and 8B), the expression of cytokine causing inflammation also decreases significantly in serum (see FIG. 9).

In an exemplary embodiment of the invention, the nucleic acid encoding a fragment of the IK factor is expressed through an appropriate expression system and is a cytokine involved in the inflammatory immune response associated with arthritis Interleukin-1 beta, IL-1β. ), Expression of monocyte chemotactic protein-1 (MCP-1), also known as interleukin-6 (IL-6), interleukin-17 (IL-7), and CCL2 (Chemokine (CC motif) ligand 2). Suppress These results indicate that the injection of nucleic acid encoding an IK factor or fragment thereof into the human body through an appropriate vector or gene transporter can inhibit the progression of arthritis, and in particular can affect the systemic immune response. In addition, the metastatic mouse (tIK-IL1RaKO) induced to express the IK factor suppresses the differentiation of T cells, which are arthritis diseases, as compared to naturally induced arthritis-induced mice (IL1RaKO) mice (see FIGS. 10A and 10B). It also affects the activator of macrophages (see FIG. 11).

In addition, in exemplary embodiments of the present invention, relatively small size IK factor fragments comprising an active site, such as peptides before and after the active site and 10 amino acids adjacent thereto, can also inhibit the progression of arthritis. These peptides inhibit the expression of tumor necrosis factor-alpha (TNF-α), IL-6, IL-17, inflammatory cytokines in macrophages (see FIGS. 12A, 12B and 15B) and are involved in arthritis It also inhibits the differentiation of Th1 cells and Th17 cells, which are pathogenic cells (see FIGS. 13A-13C). These peptides also inhibit arthritis in IL1RaKO mice, naturally occurring arthritis induced mice (see FIGS. 14A and 14B), as well as inhibit the differentiation of Th17 cells in arthritis induced mice (see FIG. 15A).

In an exemplary embodiment, the nucleic acid encoding the IK factor or fragment thereof can be injected using an appropriate expression system or gene delivery vehicle, which can prevent the progression of arthritis or treat the induced arthritis (FIGS. 19A and 19B). Reference).

As mentioned above, IL-17, which is secreted according to the differentiation of Th17 cells, plays an important role in rheumatoid arthritis. In accordance with the present invention, the IK factor or fragment thereof and / or nucleic acid encoding them inhibits the differentiation of Th17 cells, which has been noted as the root cause of rheumatoid arthritis, which makes treatment much more efficient than conventional arthritis therapeutics. .

In addition, most biopharmaceuticals represented by monoclonal antibodies are quite expensive because they can cause an immune response when injected into the human body, and because of the complexity of the manufacturing process. On the contrary, in the present invention, the IK factor, whose efficacy has been confirmed, is safe since it is almost impossible to induce an immune response in that it is a cytokine inherent in the human body, and thus it can be manufactured at relatively low cost.

The present invention therefore relates to an IK factor or fragment thereof for treating arthritis. As an active ingredient for treating arthritis, preferably, some amino acids of the full-length IK factor are fragments of the cleaved IK factor, more preferably fragments of the N-terminal amino acids of the full-length IK factor are cleaved.

In exemplary embodiments, the full-length IK factor may be a peptide having an amino acid sequence of SEQ ID NO: 2. As an exemplary form of a fragment of the IK factor, 315 amino acids constituting the N-terminus of the full-length IK factor of SEQ ID NO: 2 are cleaved, and the sequence of SEQ ID NO: 4 having 242 amino acids of the C-terminal It may be a peptide such as truncated IK (tIK) having an amino acid sequence or a partial peptide of SEQ ID NO: 4. As an example of fragments of the IK factor, peptide fragments whose N-terminus are cut out among the amino acids constituting the full-length IK factor are not only easy to manufacture because they are relatively small, but also in vivo and / or in vivo. It may be advantageous to exert biological functions in comparison to full-length IK factor ex vivo.

According to an exemplary embodiment of the present invention, the amino acid serine (S382), 489 th (SEQ ID NO: 4 peptide) of the 382th (67th in the peptide of SEQ ID NO: 4), the peptide of SEQ ID NO: 174th) amino acid tyrosine (Y489) and / or 492th (177th in peptide of SEQ ID NO: 4) amino acid tyrosine (Y492) is involved in the biological activity of the IK factor and / or fragments thereof. In particular, the 382th amino acid serine (S382) and / or the 489th amino acid tyrosine (Y489) in the peptide of SEQ ID NO: 2 is predicted to be the active site of the IK factor and / or fragment of the IK factor.

Thus, in addition to fragments of IK factor having the amino acid sequence of SEQ ID NO: 4, fragments of relatively short length IK factor containing amino acids of these active sites can be used. Illustratively, fragments of the IK factor that can be used in accordance with the present invention include the serine (S382), which is the 382th amino acid, the tyrosine (Y489), which is the 489th amino acid, and the tyrosine (Y489), which is the 492th amino acid, in the peptide of SEQ ID NO: 2. And at least one amino acid residue selected from C) and at least 10, eg, at least 100 or at least 200 amino acids, consisting of contiguous amino acids of these selected amino acid residues. In an exemplary embodiment the fragment of the IK factor is at least one selected from the 82nd amino acid serine (S382), the 489th amino acid tyrosine (Y489) and the 492th amino acid tyrosine (Y489) in the peptide of SEQ ID NO: 2 And a peptide consisting of 10 or more and 100 or less, preferably 10 or more and 50 or less, more preferably 10 or more and 30 or less amino acids, each consisting of the amino acid residues of the selected amino acid residues and adjacent amino acids of these selected amino acid residues. do. At least one amino acid of these selected amino acid residues may be a modified amino acid such as phosphorylation.

Exemplary fragments of the IK factor that may be used in accordance with the present invention are peptides or partial peptides thereof having the amino acid sequence of SEQ ID NO: 2. In an exemplary embodiment, the fragment of the IK factor consists of at least one amino acid residue selected from serine, which is the 382th amino acid, and tyrosine, the 489th amino acid, in the peptide of SEQ ID NO: 2 and adjacent amino acids of these selected amino acid residues. It is a peptide which has 10 or more, for example 10 or more and 100 or less, preferably 10 or more and 50 or less, more preferably 10 or more and 30 or less amino acids.

For example, peptides that can be used as fragments of the IK factor include the serine (S382), the 382th amino acid, the tyrosine (Y489), the 489th amino acid, and the tyrosine (Y492), the 492th amino acid, in the peptide of SEQ ID NO: 2. An active amino acid such as may not be located at the N-terminus or C-terminus of the peptide. Peptides may be used in which two or more adjacent amino acids are linked to each of the N-terminus and C-terminus of these selected active amino acids so that these selected amino acids that are active amino acids can be arranged in the middle of the peptide. For example, 2 to 8, preferably 3 to 7 or more adjacent amino acids may be linked to the N-terminus and C-terminus of the active amino acid, respectively. For example, 2 to 100, such as 3 to 70, preferably 3 to 30, more preferably 3 to 20 contiguous amino acids may be linked to the N-terminus and C-terminus of the active amino acid, respectively.

In one exemplary embodiment, the fragment of the IK factor is a peptide of SEQ ID NO: 47 consisting of 377 amino acid glutamic acid-391 amino acid aspartic acid in the peptide of SEQ ID NO: 2 and / or the 484th Also included is a peptide of SEQ ID NO: 49 consisting of the amino acid aspartic acid—the 496th amino acid lysine.

According to an exemplary embodiment, the full length IK factor of SEQ ID NO: 2 consists of 557 amino acids, and the fragment of the IK factor of SEQ ID NO: 4, wherein the N-terminus of the IK factor is cleaved, is 242 amino acids. It consists of. In consideration of this point, the fragment of IK factor according to the present invention may be composed of up to about 300 to 500 amino acids of the amino acid of SEQ ID NO: 2. However, fragments of IK factor that can be used in accordance with the present invention are not limited thereto.

Also, for example, except for active amino acids such as S382, Y489 and Y492 in the IK factor and / or fragments of IK factor, the remaining amino acids may be substituted with other amino acids, for example by conservative substitutions, If necessary some amino acids may be deleted, added and / or inserted.

Peptides that are IK factors or fragments thereof can be isolated by preparation through recombinant means or chemical synthesis. By way of example, peptides expressed by the nucleic acid sequences mentioned herein can be readily prepared by known methods, using any of a number of known expression vectors. Expression can be realized in a suitable host cell transformed with an expression vector comprising a DNA sequence encoding the peptide. Suitable host cells include prokaryotes, yeasts and eukaryotes. E. coli, yeast or mammalian cell lines (such as Cos or CHO) are preferably used as host cells. For purification of the protein, the supernatant containing recombinant protein secreted into the culture, first obtained in a water-soluble host / vector system, is concentrated using a commercially available filter. In the next step, the concentrate obtained above is purified using an appropriate purification matrix such as an affinity matrix or an ion exchange resin. Finally, pure protein can be obtained by performing one or several reverse phase HPLC.

Fragments or variants consisting of up to 100, typically up to 50 amino acids can be prepared synthetically. For example, such polypeptides can be synthesized by commercially available solid-phase techniques, the Merrifield solid-phase synthesis method, which sequentially adds amino acids to growing amino acid chains ( Merrifield, 1963, J. Am. Chem. Soc. 85: 2146-2149. Equipment for automatic synthesis of polypeptides can be purchased from the supplier and can be manipulated according to the supplier's manual.

For example, peptides described herein expressed in a microorganism can be secreted into and recovered from the periplasm of the host cell. Typically, protein recovery generally involves grinding the microorganism by means such as osmotic shock, sonication or lysis. Once the cells are destroyed, cell debris or whole cells can be removed by centrifugation or filtration. Proteins can be further purified, for example, by affinity resin chromatography. Alternatively, the protein can be transferred to and separated from the culture medium. The cells can be removed from the culture and the culture supernatant filtered and concentrated to further purify the protein produced. Expressed polypeptides are commonly known methods, such as fractional distillation on immunoaffinity or ion-exchange columns; Ethanol precipitation; Reverse phase HPLC; Chromatography on silica or cation exchange resins such as DEAE; Chromatofocusing; SDS-PAGE; Ammonium sulfate precipitation; Gel filtration using, for example, Sephadex G-75; Hydrophobic affinity resins, ligand affinity using suitable antigens immobilized on a matrix, and Western blot assays can be further isolated and identified.

In addition, the peptides produced can be purified to obtain a substantially homogeneous formulation for further assays and uses. Standard protein purification methods known in the art can be used. The following procedures are examples of suitable purification procedures: fractional distillation on immunoaffinity or ion-exchange columns, ethanol precipitation, reverse phase HPLC, silica or cation-exchange resins, such as chromatography on DEAE, chromatographic focusing, SDS-PAGE, ammonium sulfate Precipitation, and gel filtration using, for example, Sephadex G-75.

The invention also relates to a nucleic acid or polynucleotide encoding an IK factor or fragment thereof for treating arthritis. Similar to peptides, preferably the IK nucleic acid fragments from which some nucleotides of the nucleic acid encoding the full length IK are cleaved are preferred, and particularly preferably the IK nucleic acid fragments from which the nucleotides of the 5 'end are cleaved.

For example, the nucleic acid encoding the full-length IK factor may be a nucleic acid having a nucleotide sequence of SEQ ID NO: 1. For example, a nucleic acid encoding a fragment of an IK factor is a truncated IK factor having a nucleotide sequence of SEQ ID NO: 3 wherein 945 nucleotides constituting the 5 'end of the nucleic acid of SEQ ID NO: 1 are cleaved. It may be a nucleic acid (tIK nucleic acid fragment) encoding a portion or a nucleic acid consisting of some nucleotides of SEQ ID NO: 3.

In the peptide of SEQ ID NO: 2, the serine (S382), 389 amino acid tyrosine (Y489) and the 492 amino acid tyrosine (Y492), respectively, were 1144-1146 (SEQ ID NO: 1). 199-201) nucleotide residue of 3, 1465-1467 (520-522 of SEQ ID NO: 3) of SEQ ID NO: 1, 1474-1476 of SEQ ID NO: 1 (SEQ ID NO: 529-531) nucleotide residues of 3 can be expressed.

Thus, in addition to IK fragment nucleic acids having the nucleotide sequence of SEQ ID NO: 3, IK fragment nucleic acids consisting of nucleotides of relatively short length can also be used. For example, the IK fragment nucleic acid according to the present invention is a nucleotide residue of 1144-1146 (SEQ ID NO: 199-201) of SEQ ID NO: 1, 1465-1467 (SEQ ID NO: 1) 520-522) nucleotide residues of No. 3, 1474-1476 (529-531 of SEQ ID NO: 3) of SEQ ID NO: 1 and at least 30 adjacent to these selected nucleotide residues, eg For example, it may consist of more than 300 or more than 600 nucleotides. In an exemplary embodiment, the nucleic acid encoding a fragment of the IK factor comprises a 1144-1146th nucleotide residue of SEQ ID NO: 1, a 1465-1467th nucleotide residue of SEQ ID NO: 1, Nucleotide residues of 1474-1476 and 30 or more and 300 or less, preferably 30 or more and 150 or less, more preferably 30 or more and 90 or less nucleotides each consisting of adjacent nucleotides of these selected nucleotide residues Nucleic acids to be included.

Illustratively, the IK fragment nucleic acid is a nucleic acid having a nucleotide sequence of SEQ ID NO: 1 or a partial fragment thereof. In an exemplary embodiment the nucleic acid encoding the partial fragment of the IK factor is selected from at least one nucleotide residue selected from nucleotide residues 1144-1146 and 1465-1467 nucleotide residues of the nucleotide sequence of SEQ ID NO: 1; It is a nucleic acid having 30 or more, for example, 30 or more and 300 or less, preferably 30 or more and 150 or less, more preferably 30 or more and 90 or less nucleotides composed of contiguous nucleotides of a nucleotide set.

Six or more contiguous nucleotides may each be linked to the 5 'and 3' ends of these selected nucleotide sets so that the selected nucleotide residues can be arranged in the middle of the polynucleotide. For example, 6 to 24, preferably 9 to 21 or more contiguous nucleotides may be linked to the 5 'end and 3' end of the selected nucleotide residue. For example, 6-300, such as 9-210, preferably 9-90, more preferably 9-60 contiguous nucleotides can be linked to the 5 'end and 3' end of the selected nucleotide residue, respectively.

In one exemplary embodiment, the nucleic acid encoding a fragment of the IK factor may be a nucleic acid having the nucleotide sequence of SEQ ID NO: 46 and / or a nucleic acid having the nucleotide sequence of SEQ ID NO: 48. For example, the nucleic acid encoding a fragment of the IK factor may be composed of up to about 900 to 1500 nucleotides among the nucleotides of SEQ ID NO: 1, but the present invention is not limited thereto. Of the amino acids of SEQ ID NO: 2, 1144-1146 of SEQ ID NO: 1 coding for serine (S382), 489 amino acid tyrosine (Y489), and / or tyrosine (Y492) 492 amino acid, respectively The set of nucleotides, the set of 1465-1467 nucleotides and / or the set of 1474-1476 nucleotides may have different nucleotide sequences encoding serine and tyrosine, respectively.

In accordance with the present invention, nucleic acids encoding an IK factor or fragment thereof can be included in a suitable vector. The vector also includes an expression control sequence linked to the polynucleotide of the present invention. In an exemplary embodiment of the invention, the vector may also comprise one or more polynucleotides encoding other molecules of interest. Polynucleotides of the present invention may also bind to other polynucleotides to encode fusion proteins.

Illustratively, the polynucleotides of the invention are formulated to enter into and express in mammalian cells. Such compositions are particularly useful for use for therapeutic purposes. There are many ways to express polynucleotides in host cells, and any suitable method can be used. For example, one polynucleotide is an adenovirus, an adeno-associated virus, a retrovirus, a vaccinia, a lentivirus baculovirus or another. It can be inserted into viral vectors such as pox virus (eq, avian pox virus). Techniques for inserting DNA into such vectors are well known. Retroviral vectors include targeting sites such as genes for selectable markers that facilitate the identification or selection of transduced cells and / or genes that encode ligands that act as receptors for specific target cells. can additionally insert a targeting moiety. Targeting can also be accomplished by known methods using antibodies.

Many vectors available and known in the art can be used for the purposes of the present invention. Selection of the appropriate vector will depend primarily on the size of the nucleic acid inserted into the vector and the particular host cell transformed with the vector. Each vector contains various components depending on its function (amplification or expression of heterologous polynucleotides, or both) and compatibility with the particular host cell in which the vector is present. Vector components generally include, but are not limited to, origin of replication (especially when the vector is inserted into prokaryotic cells), selection marker genes, promoters, ribosomal binding sites (RBS), signal sequences, heterologous nucleic acid inserts, and transcription termination sequences. .

For example, recombinant vectors of the present invention may be used to express expression control sequences that may affect the expression of proteins, such as initiation codons, termination codons, polyadenylation signals, enhancers, signal sequences for membrane targeting or secretion, and the like. It may include. Polyadenylation signals increase the stability of transcripts or facilitate cellular transport. Enhancer sequences are nucleic acid sequences that are located at various sites in the promoter and increase transcriptional activity as compared to the transcriptional activity by the promoter in the absence of the enhancer sequence. As a signal sequence, when the host is Escherichia (Escherichia) in gyunin include PhoA signal sequence, OmpA signal sequence, etc., the host is Bacillus (Bacillus) when in gyunin include α- amylase signal sequence, subtilisin signal sequence, etc. Shin, If the host is a yeast, MF-α signal sequence, SUC2 signal sequence, etc., if the host is an animal cell, insulin signal sequence, a-interferon signal sequence, antibody molecular signal sequence, etc. may be used, but the present invention is limited thereto. It doesn't work.

One type of vector is a "plasmid" which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated therein. Another type of vector is a phage vector. Another type of vector is a viral vector in which additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in the host cell into which they are introduced (eg, bacterial vectors and episomal mammalian vectors with bacterial origins of replication). Other vectors (eg, non-episomal mammalian vectors) can be integrated into the genome of the host cell upon introduction into the host cell and thereby replicate with the host genome. In addition, certain vectors may direct expression of the gene to which the vector is operably linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply, "recombinant vectors"). In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.

The vector system of the present invention may be constructed through various methods known in the art, and specific methods thereof are disclosed in Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press (2001), This document is incorporated herein by reference. For example, vectors that can be used in the present invention include plasmids often used in the art (eg pSC101, ColE1, pBR322, pUC8 / 9, pHC79, pUC19, pET, etc.), phage (eg λgt4λB, λ-Charon , λΔz1, λGEM.TM.-11 and M13, etc.) or viruses (eg SV40, etc.).

In a specific embodiment of the invention, the nucleic acid is inserted into a host cell using a virus expression system (vaccinia or other pox virus, retrovirus, lentivirus, baculovirus, adenovirus or adeno-associated virus). Exemplary viral vectors include retroviral vectors derived from HIV, SIV, murine retroviruses, gibbon ape leukemia virus, adeno-associate viruses, and adenoviruses, and the like. But not limited thereto (Miller et al., 1990, Mol. Cell Biol. 10: 4239; J. Kolberg 1992, NIH Res. 4:43; Cornetta et al., 1991, Hum. Gene Ther. 2: 215). Retroviruses derived from rodent leukemia virus (MulVe), basic leukemia virus (GIBBON ape leukemia virus, GaLV), ecotropic retroviruses, simian immunodeficiency virus (SIV), human immunodeficiency virus (HIV), etc. Vectors are widely used (Buchscher et al., 1992, J. Virol, 66 (5): 2731-2739; Johann et al., 1992, J. Virol. 66 (5): 1635-1640; Sommerfelt et al. , 1990, Virol. 176: 58-59; Wilson et al., 1989, J. Virol. 63: 2374-2378; Miller et al., 1991, J. Virol. 65: 2220-2224; Rosenberg and Fauci 1993 in Fundermental Immunology, Third Edition, WE Paul (ed.) Raven Press, Ltd., New York and the references therein; Miller et al., 1990, Mol. Cell.Biol. 10: 4239; R. Kolberg 1992, J. NIH Res. 4:43; Cornetta et al., 1991, Hum. Gene Ther. 2: 215).

Constitutive or inducible promoters can be used in the present invention depending on the needs of a particular situation that can be identified by one skilled in the art. Many promoters that are recognized by a variety of possible host cells are well known. The selected promoter may be operably linked to the cistron DNA encoding the polypeptides described herein by removing the promoter from the source DNA via restriction enzyme digestion and inserting the isolated promoter sequence into the selection vector. Both native promoter sequences and multiple heterologous promoters can be used to direct amplification and / or expression of a target gene. However, heterologous promoters are generally preferred because they allow greater transcription and higher yield of expressed target genes as compared to native target polypeptide promoters.

On the other hand, when the vector of the present invention is an expression vector and the eukaryotic cell is a host, a promoter derived from the mammalian cell genome (for example, metallothionine promoter) or a promoter derived from a mammalian virus (for example, adeno) Late viral promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus promoter and tk promoter of HSV) can be used and generally have a polyadenylation sequence as a transcription termination sequence.

In addition, when the recombinant vector of the present invention is a replicable expression vector, it may include a replication origin, which is a specific nucleic acid sequence from which replication is initiated. In addition, the recombinant vector may include a selection marker. The selection marker is for selecting cells transformed with the vector, and markers conferring a selectable phenotype such as drug resistance, nutritional requirements, resistance to cytotoxic agents or expression of surface proteins can be used. Vectors of the invention, as markers, include antibiotic resistance genes commonly used in the art and include, for example, ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin and There is a resistance gene for tetracycline. Since only cells expressing a selection marker survive in an environment treated with a selective agent, transformed cells can be selected. Representative examples of selection markers include ura4, leu1, his3, and the like, which are nutritional markers, but the types of selection markers that can be used in the present invention are not limited by the above examples.

Various in vitro amplification techniques are known for amplifying a subcloned sequence in an expression vector. Such techniques include polymerase chain reaction (PCR), ligand chain reaction (LCR), Qβ-replicase amplification, and other RNA polymerases (Sambrook et al., 1989, Molecular Cloning-A Laboratory Manual). (2nd Ed) 1-3; US Patent No. 4,683,202; PCR protocols A Guide to Methods and Applications, Innis et al., Eds.Academic Press Inc. San Diego, CA 1990. Improved methods of cloning in vitro amplified nucleic acids are described in US Patent No. 5,426,039.

Vectors of the present invention may also be fused with other sequences to facilitate purification of the peptides expressed therefrom. Sequences to be fused include, for example, glutathione S-transferase (Pharmacia, USA), maltose binding protein (NEB, USA), FLAG (IBI, USA) and 6x His (hexahistidine; Quiagen, USA), and most preferably Is 6 × His. Because of the additional sequence for this purification, the protein expressed in the host is purified quickly and easily through affinity chromatography. If desired, sequences encoding Fc fragments may be fused to facilitate extracellular secretion of these peptides.

According to an exemplary embodiment of the present invention, peptides expressed by a vector containing a nucleotide sequence encoding an IK factor or partial fragment thereof are purified by affinity chromatography. For example, when glutathione-S-transferase is fused, glutathione, which is a substrate of this enzyme, can be used, and when 6x His is used, a desired peptide can be obtained using a Ni-NTA His-binding resin column (Novagen, USA). Can be obtained quickly and easily.

Host cells capable of continuously cloning and expressing the above-described vectors while being stable are known in the art and can use any host cell. For example, E. coli JM109, E. coli BL21 (DE3), E. coli RR1, E. coli LE392, E. coli B, E. coli X 1776, E. coli W3110, Bacillus subtilis, Bacillus thuringiensis Strains of the genus Bacillus, and enterobacteria and strains such as Salmonella typhimurium, Serratia marsons and various Pseudomonas species.

In addition, when transforming a vector of the present invention into eukaryotic cells, as host cells, yeast ( Saccharomyce cerevisiae ), insect cells (such as SF9 cells) and human cells (such as CHO cell lines (Chinese hamster ovary), W138, BHK, COS-7, 293, HepG2, 3T3, RIN and MDCK cell lines) and the like.

The vectors of the invention can be used to genetically modify cells in vitro, ex vivo or in vitro . Methods for genetically modifying cells include infection or transduction of cells with viral vectors, calcium phosphate precipitation, bacterial protoplasts containing DNA of receptor cells. Fusion methods, treatment of liposomes or microspheres containing DNA in receptor cells, DEAE dextran, receptor-mediated endocytosis, electroporation, microinjection Various methods are known, including micro-injection and gene bombardment.

Pharmaceutical Compositions and Administration

According to another aspect of the present invention, the present invention provides a therapeutically effective amount of the aforementioned IK factor or fragment thereof; And it relates to a pharmaceutical composition for the treatment of arthritis comprising a pharmaceutically acceptable carrier as needed. In this case, the IK factor or fragment thereof peptide can be administered directly to the subject.

According to another aspect of the present invention, the present invention provides a gene carrier having a nucleic acid molecule encoding an IK factor or fragment thereof; And it relates to a pharmaceutical composition for the treatment of arthritis comprising a pharmaceutically acceptable carrier as needed. The use of gene carriers is intended for so-called gene therapy.

Accordingly, the present invention provides a method of administering to a subject a pharmaceutical composition or a pharmaceutical composition containing as an active ingredient a gene carrier comprising a nucleic acid encoding an IK factor or a fragment thereof and a nucleic acid encoding the IK factor or a fragment thereof. It relates to a method of treating arthritis having a step. In this case, the active ingredient may be contained in the pharmaceutical composition at a concentration of 1.0 ng / mL to 10 μg / mL.

In one embodiment, there is provided a pharmaceutical composition or medicament containing a compound of the invention and a therapeutically inert carrier, diluent or excipient, as well as a method of using the compound of the invention to make such a composition and medicament. In one example, the compound may be formulated by mixing in a desired degree of purity at ambient temperature, appropriate pH, together with a physiologically acceptable carrier, that is, a carrier that is nontoxic to the recipient at the dosages and concentrations employed in the herbal dosage form. . The pH of the formulation mainly depends on the specific use and concentration of the compound, but is preferably in the range of about 3 to about 8. In one example, the compound is formulated in acetate buffer at pH 5. In another embodiment, the compound is sterile. The compound may be stored, for example, as a solid or amorphous composition, lyophilized formulation or aqueous solution.

The compositions are formulated, dosed, and administered in a fashion consistent with good medical practice. Factors to consider in this regard include the particular disorder to be treated, the particular patient to be treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the dosing schedule, and other factors known to the practitioner.

If necessary, sustained-release preparations may be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the compound, which matrices are in the form of shaped articles, eg films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (eg, poly (2-hydroxyethyl-methacrylate), or poly (vinyl alcohol)), polylactide, L-glutamic acid, and gamma-ethyl- Copolymers of L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers and poly-D-(-)-3-hydroxybutyric acid.

In one example, a pharmaceutically effective amount of a compound of the invention administered parenterally per dose will range from about 0.01-100 mg / kg, alternatively about 0.1 to 20 mg / kg, based on the patient's body weight per day, Typical initial ranges for compounds are 0.3 to 15 mg / kg / day. In another embodiment, oral unit dosage forms such as tablets and capsules preferably contain about 5-100 mg of the compound of the present invention.

The compounds used as active ingredients according to the invention can be used in any suitable means, for example oral, topical (including buccal and sublingual), rectal, vaginal, transdermal, parenteral, subcutaneous, intraperitoneal, intrapulmonary, intradermal, intradural and Epidural and intranasal, and if desired, by topical treatment, by means for intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration.

Compounds which are active ingredients of the present invention can be administered in any convenient dosage form, for example tablets, powders, capsules, solutions, dispersions, suspensions, syrups, sprays, suppositories, gels, emulsions, patches and the like. Such compositions may contain components conventional to pharmaceutical formulations, such as diluents, carriers, pH adjusters, sweeteners, bulking agents and additional active agents.

Typical formulations are prepared by mixing the compound of the invention with a carrier or excipient. Suitable carriers and excipients are well known to those skilled in the art and are described, for example, in Ansel, Howard C., et al., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems. Philadelphia: Lippincott, Williams & Wilkins, 2004; Gennaro, Alfonso R., et al. Remington: The Science and Practice of Pharmacy. Philadelphia: Lippincott, Williams & Wilkins, 2000; And Rowe, Raymond C. Handbook of Pharmaceutical Excipients. Chicago, Pharmaceutical Press, 2005.

For example, the pharmaceutically acceptable carrier included in the pharmaceutical composition of the present invention is conventionally used in the preparation, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, know Nate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oils However, the present invention is not limited thereto.

The formulations also contain one or more buffers, stabilizers, surfactants, wetting agents, lubricants, emulsifiers, suspending agents, preservatives, antioxidants, opacifiers, glidants, processing aids, colorants, sweeteners, flavorings, flavoring agents, diluents, and drugs (Ie, compounds of the present invention or pharmaceutical compositions thereof) may include other known additives to provide a nice appearance or to aid in the manufacture of a pharmaceutical product (ie, a medicament).

The pharmaceutical compositions of the present invention may be prepared in unit dose form by formulating with a pharmaceutically acceptable carrier and / or excipient according to methods which can be easily carried out by those skilled in the art. Or may be prepared by incorporating into a multi-dose container. In this case, the formulation may be in the form of a solution, suspension or emulsion in an oil or an aqueous medium, or may be in the form of extracts, powders, granules, tablets or capsules, and may further include a dispersant or stabilizer.

The resulting composition is dried, granulated, mixed with magnesium stearate and compressed into tablet form using conventional equipment. Examples of aerosol formulations can be prepared, for example, by dissolving 5-400 mg of the compound of the invention in a suitable buffer solution such as phosphate buffer and, if desired, by adding a salt such as isotonic agent, for example sodium chloride. have. Impurities and contaminants can be removed by filtering the solution using, for example, a 0.2 micron filter.

Thus one embodiment includes a pharmaceutical composition comprising a compound or stereoisomer or pharmaceutically acceptable salt thereof. Further embodiments include pharmaceutical compositions comprising a compound or stereoisomer or pharmaceutically acceptable salt thereof together with a pharmaceutically acceptable carrier or excipient.

The gene therapy agent of the present invention described above includes a gene carrier comprising a nucleic acid molecule of interest as an active ingredient. The gene carrier is designed to transport and express the nucleic acid molecule of interest, and details of the nucleic acid molecule of interest as a carrier object are omitted in order to avoid overlapping with the above description.

To prepare a gene carrier, the nucleic acid molecule of interest is preferably present in a suitable expression construct. In the expression construct, the nucleic acid molecule of interest is preferably operably linked to a promoter. As used herein, the term “operably linked” means functional binding between a nucleic acid expression control sequence (eg, an array of promoters, signal sequences, or transcriptional regulator binding sites) and other nucleic acid sequences, thereby The regulatory sequence will control the transcription and / or translation of said other nucleic acid sequence. In the present invention, a promoter bound to a nucleic acid molecule of interest is preferably a promoter derived from a mammalian virus that can operate in animal cells, more preferably mammalian cells to regulate transcription of the nucleic acid molecule of interest. And promoters derived from the genome of mammalian cells, such as, for example, the cytomegalo virus (CMV) promoter, the adenovirus late promoter, the vaccinia virus 7.5K promoter, the SV40 promoter, the tk promoter of HSV, the RSV promoter, the EF1 alpha promoter, Metallothionine promoter, beta-actin promoter, promoter of human IL-2 gene, promoter of human IFN gene, promoter of human IL-4 gene, promoter of human lymphotoxin gene, promoter of human GM-CSF gene, cancer cell specific Red promoters (eg, TERT promoter, PSA promoter, PSMA promoter, CEA promoter, E2F Promoters and AFP promoters) and tissue specific promoters (eg, albumin promoters). Preferably, the expression construct used in the present invention comprises a polyadenylation sequence (e.g., a calcined hormone hormone terminator and an SV40 derived polyadenylation sequence).

Gene carriers can be produced in a variety of forms, for example, 1) naked (recombinant) DNA molecules, 2) plasmids, 3) viral vectors, and 4) the naked (recombinant) DNA molecules or plasmids. It can be prepared in the form of nesting liposomes or niosomes. When DNA is naked (Ulmer et al., 1993, Science 259: 1745-1749; Cohen, 1993, Science 259: 1691-1692), DNA is coated into biodegradable beads and further into the cell. Can be delivered efficiently.

The nucleic acid molecule of interest can be applied to all gene delivery systems used in conventional gene therapy, preferably plasmids, adenoviruses (Lockett LJ, et al., Clin. Cancer Res. 3: 2075-2080, 1997), Adeno-associated viruses (AAV, Lashford LS., Et al., Gene Therapy Technologies, Applications and Regulations Ed.A. Meager, 1999), retroviruses (Gunzburg WH, et al., Retroviral vectors. Therapy Technologies, Applications and Regulations Ed.A. Meager, 1999), lentiviruses (Wang G. et al., J. Clin. Invest. 104 (11): R55-62 (1999)), herpes simplex virus (Chamber R., et al., Proc. Natl. Acad. Sci USA 92: 1411-1415, 1995), vaccinia virus (Puhlmann M. et al., Human Gene Therapy 10: 649-657, 1999), baculovirus (King LA. And Possee RD., The Baculovirus Expression System, Springer, 1992), Methods in Molecular Biology, Vol 199, SC Basu and M. Basu (Eds.) , Human Press 2002) or niosomes.

The method of introducing the above-described gene carrier into a cell may be carried out through various methods known in the art. In the present invention, when the gene carrier is produced based on a viral vector, it is carried out according to a virus infection method known in the art. Infection of host cells with viral vectors is described in the references cited above.

In the present invention, when the gene delivery system is naked recombinant DNA molecules or plasmids, micro-injection (Capecchi, M.R., Cell, 22: 479, 1980); And Harland and Weintraub, J. Cell Biol. 101: 1094-1099, 1985)), calcium phosphate precipitation (Graham, F.L. et al., Virology, 52: 456, 1973); And Chen and Okayama, Mol. Cell. Biol. 7: 2745-2752, 1987), electroporation (Neumann, E. et al., EMBO J., 1: 841, 1982; and Tur-Kaspa et al., Mol. Cell Biol., 6: 716-718 (1986)), liposome-mediated transfection (Wong, TK et al., Gene, 10:87, 1980); Nicolau and Sene, Biochim. Biophys. Acta, 721: 185-190, 1982; And Nicolau et al., Methods Enzymol., 149: 157-176, 1987)), DEAE-dextran treatment (Gopal, Mol. Cell Biol., 5: 1188-1190, 1985)), and gene bombardment ( Yang et al., Proc. Natl. Acad. Sci., 87: 9568-9572, 1990)) can be used to introduce the gene into the cell.

Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to the invention described in the following examples.

실시예 1 : 아이케이 인자 단편의 CIITA 상위 조절 인자 발현 측정Example 1 Measurement of CIITA Higher Regulatory Factor Expression of IK Factor Fragments

1) Selection of active site of IK factor

Computer programs were used to screen the active sites of IK factor. In silico program 'scanlite' provided on the website http://scansite.mit.edu/motifscan_seq.phtml, the fragment fragment (tIK) amino acid sequence of the IK factor of SEQ ID NO: 3 was input and set and selected. . All motif analysis data was based on the SWISS-PROT database. Table 1 shows the results of screening the active site of IK factor. In Table 1, the score quantifies the probability that the site of tIK actually interacts with the kinase, and the percnetile is the highest percentage of kinase motifs associated with tIK among the kinase motifs in the SWISS-PROT database. A number that indicates whether or not The S382 site and the Y482 site are motif candidates presented under all selection conditions of high / medium / low stringency, and the Y489 site is motif candidates presented under medium / low stringency conditions.

Table 1

Active site screening of IK factor

Score	percentile	Motif	Motif group	site	order
0.482	0.416%	Aurora A (AuroA)	Baso_ST_kin ^a	S382	EKKRH S YFEKPKV
0.392	0.273%	PKC epsion (PKC-epsion)	Baso_ST_kin ^a	S382	EKKRH S YFEKPKV
0.323	0.177%	(PKA_kin) ^b	Baso_ST_kin ^a	S382	EKKRH S YFEKPKV
0.408	0.163%	Grb2 SH2 (Grb2_SH2)	SH2 ^c	Y492	TQEEYSE Y MNNKEAL
0.574	0.378%	InsR_Kin ^d	Y_Kin ^e	Y489	DFDTQEE Y SEYMNNK
0.484	0.638%	Src_Kin ^f	Y_Kin ^e	Y489	DFDTQEE Y SEYMNNK

^a : Basophilic serine / threonine kinase; ^b : Protein Kinase A; ^c : Src homology 2 group; ^d : Insulin Receptor Kinase; ^e : Tyrosine kinase group; ^f : Src Kinase

2) Design of Nucleic Acid Fragments with Some Nucleotide Truncated

As schematically shown in FIG. 2, based on the selection result in 1), a mutant tIK nucleic acid fragment having a mutation in which a tIK nucleic acid fragment and an amino acid of an active site were replaced with another amino acid was designed. As the tIK nucleic acid fragment, a gene fragment of SEQ ID NO: 3 was used, and a point mutant nucleic acid fragment in which amino acids (S382, Y489, Y492) predicted to play an important role in the function of tIK as kinase motifs were replaced with structurally similar amino acids. Was produced.

A mutant gene fragment in which the nucleotide encoding serine (the amino acid of SEQ ID NO: 2), which is the 3rd amino acid (AGC, which is the 1144-1146 nucleotide among the nucleotides of SEQ ID NO: 1), is replaced with the nucleotide (GCC) encoding alanine ( S382A) was produced. A mutant gene in which the nucleotide encoding tyrosine, which is the 489th amino acid among the amino acids of SEQ ID NO: 2 (TAC, which is the 1465-1467 nucleotide among the nucleotides of SEQ ID NO: 1), was substituted with the nucleotide (TTC) encoding phenylalanine A fragment (Y489F) and a nucleotide encoding tyrosine, which is the 492th amino acid among the amino acids of SEQ ID NO: 2, as a nucleotide (TTC) encoding phenylalanine A substituted double mutant gene fragment (Y492F) was produced.

A mutant gene fragment (Y489492F) in which the nucleotides encoding the 489th and the 492th tyrosine among the amino acids of SEQ ID NO: 2 were replaced with the nucleotides encoding the phenylalanine (Y489492F), and the 389th serine and the 489th amino acids of the amino acid of SEQ ID NO: 2; And a triple mutant gene fragment (S382AY489492F) in which the 492th tyrosine was substituted with nucleotides encoding alanine and phenylalanine, respectively. HA-tag sequence (hemagglutinin sequence; SEQ ID NO: 54) was added to the 5 'end of each gene fragment for detection, isolation and purification of the expressed protein. The tIK nucleic acid fragments used in this example and the nucleotide sequences of the mutant nucleic acid fragments are shown in Table 2 below.

TABLE 2

IK nucleic acid fragments and mutant nucleic acid fragments

snippet	Nucleotide Sequence	Amino acid sequence	Remarks
tIK	SEQ ID NO: 3	SEQ ID NO: 4
S382A	SEQ ID NO: 5	SEQ ID NO: 6	Ser → Ala
Y489F	SEQ ID NO: 7	SEQ ID NO: 8	Tyr → Phe
Y492F	SEQ ID NO: 9	SEQ ID NO: 10	Tyr → Phe
Y489492F	SEQ ID NO: 11	SEQ ID NO: 12	Tyr → Phe
S382AY489492F	SEQ ID NO: 13	SEQ ID NO: 14	Ser → Ala; Tyr → Phe

The tIK fragment and the mutant nucleic acid fragment were prepared by inserting the amplified insert into the pcDNA 3.1 vector using fusion PCR. In the case of the S382A mutant, a PCR primer was prepared using a reverse primer set having a forward primer complementary to the 5 'portion of the tIK nucleic acid from the HA tag, and a nucleotide sequence capable of encoding alanine to replace the 382 th serine with alanine. It was. Using this primer set, the first fragment from HA tag to alanine (Ala) 382 was amplified using the tIK gene as a template. Subsequently, a set of forward primers containing nucleotides encoding alanine and complementary reverse primer sets at the 3 ′ end of the tIK gene were used to replace the 382 th serine with alanine. The first section was amplified. 1μl each of the two fragments obtained by PCR was added to 500μl microtube, and 1μl of complementary forward primer complementary to the front of the tIK gene and 1μl of complementary reverse primer to the terminal of the tIK gene. And fusion PCR was performed by adding dNTP, polymerase, and distilled water. By cutting the ends of the amplification fragments obtained by PCR with restriction enzymes and inserting them into pcDNA 3.1 and transforming, the plasmid containing the amino acid-substituted insert was selected, and the amino acid substitution was finally confirmed by sequencing. Other mutations were made in the same way as above. Table 3 below shows the primer sets used for amplification of tIK nucleic acid fragments and mutant tIK nucleic acid fragments according to this example.

TABLE 3

Primer sets used for amplification of tIK nucleic acid fragments and mutant nucleic acid fragments

plasmid (HA tagged)	Section 1 Primer	2nd section primer
tIK	-Forward: SEQ ID NO: 15 ^a -Reverse: SEQ ID NO: 16 ^b
S382A	-Forward: SEQ ID NO: 17 ^* -Reverse: SEQ ID NO: 19	-Forward: SEQ ID NO: 20- Reverse: SEQ ID NO: 16
Y489F	-Forward: SEQ ID NO: 18 ^c -Reverse: SEQ ID NO: 21	-Forward: SEQ ID NO: 22- Reverse: SEQ ID NO: 16
Y492F	-Forward: SEQ ID NO: 17- Reverse: SEQ ID NO: 23	-Forward: SEQ ID NO: 24- Reverse: SEQ ID NO: 16
Y489492F	-Forward: SEQ ID NO: 18 ^c -Reverse: SEQ ID NO: 25	-Forward: SEQ ID NO: 26- Reverse: SEQ ID NO: 16
S382Y489492F	Using the S67A fragment and the Y174_177F fragment as a template, three mutated inserts were obtained using SEQ ID NO: 18 ^c as the forward primer and SEQ ID NO: 16 as the reverse primer.

^a : including EcoRI recognition site; ^b : including XhoI recognition site; ^c : HindIII recognition site included

The function of tIK and mutant nucleic acids was confirmed by the mRNA expression level of the upper regulators that regulate the expression of CIITA, a class II transactivator that promotes MHC class II expression. CNOT1 (CCR4-NOT transcription complex subunit 1), CDCA3 (Cell division cycle-associated protein 3), and Mitogen-activated protein kinase 1 (MAPK1) were selected as top regulators that regulate the expression of CIITA. Transfection of tIK-inserted plasmids into human Raji B cells without endogenous tIK increased expression of factors that inhibit the expression of CIITA (CNOT1, CDCA3, MAPK1) compared to the control vector-transfected group. It was confirmed. tIK nucleic acid fragments were thought to ultimately influence the inhibition of expression of MHC class II by increasing the factors that inhibit CIITA. Based on these results, it was confirmed whether the same phenomenon occurs in the tIK mutant nucleic acid fragment in which the kinase motif is replaced with another amino acid.

Specifically, cDNA was prepared by transfection of Raji B cells with electroplasmic plasmids in which the tIK mutants S382A, Y489F, Y492F, Y489492F, and S382AY489492F were inserted. Plasmids in which tIK nucleic acid fragments were inserted were also used, and cDNAs obtained by transfection of the pcDNA 3.1 plasmids into which these nucleic acid fragments were not inserted were used as control groups. Quantitative real-time PCR was performed using the prepared cDNA, and the measured result value (Ct value) was calculated as a relative value based on the result of the group transfected with ^pcDNA 3.1 plasmid using the 2 ^(-ΔΔCt) method. . CDNA of the factors that regulate the expression of CIITA (CDCA3, CNOT1, MAPK1) was used as a template, and the primer sequences used to confirm the expression of these factors are shown in Table 4.

Table 4

CIITA regulator	Forward primer	Reverse primer
CDCA3	SEQ ID NO: 27	SEQ ID NO: 28
CNOT1	SEQ ID NO: 29	SEQ ID NO: 30
MAPK1	SEQ ID NO: 31	SEQ ID NO: 32

The results of measuring the expression of factors regulating the expression of CIITA according to the present embodiment are shown in FIG. 3. Based on the group transfected with pcDNA 3.1, a negative control vector, it was found that CDCA3, CNOT1, and MAPK1, which are factors that inhibit CIITA expression, were increased in the tIK transfected group. In addition, the mutation of Y492F, Y489492F, and S382AY489492F was replaced by another amino acid at the corresponding sequence position, and the expression of the factor that inhibits the expression of CIITA was reduced as compared with the positive control tIK. In particular, the S382 and Y489F mutations significantly reduced the expression of the CIITA expression inhibitory gene, which was increased due to tIK expression. Therefore, it was confirmed that the 382th and 492th amino acids in the full-length IK gene can act as an active site in particular responsible for the physiological function of IK.

실시예 2 : IL-1 Receptor antagonist knock-out mice 및 IK Transgenic mice를 활용한 동물 모델 개발Example 2 Animal Model Development Using IL-1 Receptor Antagonist Knock-out Mice and IK Transgenic Mice

IL-1 receptor antagonist knock out mice (IL1RaKO, from Balb / c), an animal model that is immunologically sensitive and spontaneously develops arthritis to increase the sensitivity to arthritis inducing substances, and the nucleotides of SEQ ID NO: 3 A new autoimmune arthritis-induced animal mouse (tIK-IL1RaKO) by cross-breeding tIK-Transgenic mice (tIK, Balb / c) capable of expressing a nucleic acid encoding a truncated IK factor with sequence (tIK nucleic acid fragment) Developed a model. To prepare truncated IK (tIK) transgenic mice, tIK-pcDNA 3.1 clone containing the nucleic acid of SEQ ID NO: 3 was micro-injected into fertilized eggs obtained from Balb / c female. The baby was implanted 3 weeks after implantation into the uterus of Balb / c female mouse of fertility. To obtain the gene of mice obtained through cross-breeding, after 3 weeks of age, the tail was cut to 0.3 mm and tail lysis buffer (pH8.0 Tris buffer 50mM, EDTA 50mM, 0.5% SDS in distilled water, 20mg / ml proteinase K) Incubated for 18 hours in a 55 ℃ constant temperature water bath to obtain a tail lysate.

This was transferred to a vacutainer, phenol / chloroform / isoamylalcohol (25: 24: 1) was added and inverted 50 times, followed by centrifugation (2000 rpm, 5 min, room temperature) to remove the remaining debris except genomic DNA. The chloroform was added to the inverting and centrifugation (2000 rpm, 5 min, room temperature) to separate only the genomic DNA layer. The transparent supernatant obtained after the separation was transferred to a new micro tube, and 100% ethanol was added thereto, followed by inverting to obtain a white thread-like genomic DNA. The pipette tip was lifted up, washed with 75% ethanol, air dried for 10 minutes, and dissolved in distilled water. Amplified genomic DNA derived from a transgenic mouse into which tIK nucleic acid fragment was inserted was amplified by PCR using a primer set for tIK genotyping (forward primer is SEQ ID NO: 33; the reverse primer is SEQ ID NO: 34). . On the other hand, amplification by PCR method using a primer set for IL1RaKO genotyping (forward primer is SEQ ID NO: 35, the reverse primer is SEQ ID NO: 36 and 37) and then amplified by electrophoresis of two amplification products in 1% Agarose The degree was compared. Based on the results of electrophoresis, both amplification products were identified as tIK-IL1RaKO double positive transgenic mice obtained through cross-breeding.

실시예 3 : 자가면역 관절염 동물 모델을 이용하여 IK의 효능 분석Example 3 Efficacy Analysis of IK Using an Autoimmune Arthritis Animal Model

The incidence of arthritis was observed in tIK-IL1RaKO mice and IL1RaKO mice, which are new autoimmune arthritis animal models obtained through cross-breeding of Example 2. To compare the incidence of arthritis in the natural state, 10 numbers of tIK-IL1RaKO mice and 10 numbers of IL1RaKO mice were bred in cages under the same conditions. During breeding, the degree of arthritis was confirmed by checking the degree of swelling of the mouse ankle area every week. The detailed score counting method is as follows. Arthritis evaluation was recorded based on the mean arthritic index by Rosoliniec, etc., and the monitoring was performed three times a week in the range that the maximum score of one ankle was 4 points and the four feet of the mice did not exceed 16 points. Continued. In addition, the incidence rate of arthritis was expressed in% by monitoring period. Figure 4a is a graph measuring the arthritis index, Figure 4b is a graph measuring the incidence of arthritis. In addition, Figure 5 is a photograph taken a joint picture observed in the mouse of each group. As shown, the tIK-IL1RaKO mouse group showed a significantly lower arthritis index than the IL1RaKO mouse group, and the incidence rate also showed a 60% lower incidence of arthritis. In addition, it was confirmed that the degree of inflammation of the mouse ankle region was significantly alleviated in the tIK-IL1RaKO group compared to the IL1RaKO group.

실시예 4 : 마우스의 관절 부위 병인 상태 확인Example 4 Checking the Pathogenesis of Mouse Joints

In order to confirm the pathogenesis of arthritis-induced mouse joints, the joint sites were obtained by monitoring the arthritis spontaneously occurring group at the 16th week of each group (tIK-IL1RaKO group; IL1RaKO group). Joint sites were fixed in 10% neutral formalin and demineralized by soaking in demineralization solution for 7 hours. The demineralized joint area was washed with water for 16 hours, and a paraffin block was prepared by dehydration, clarity, and infiltration. Paraffin blocks were cut using a slicer, 7 μm slices were prepared, attached to slides, and stained with hematoxylin and eosin (H & E). The process of H & E staining is as follows. Paraffin was removed from the paraffin sections (deparaffinization), followed by hydration, and nuclei stained with hematoxylin. After washing, the cytoplasm was stained with eogene. After dehydration, transparency, and encapsulation, the optical status of the tarsal bone in the ankle and the infiltration of various immune cells were confirmed. The results are shown in Figure 6a. Articular tissues derived from tIK-IL1RaKO mice had smaller structural changes in tarsal bone and less infiltration of immune cells than those from IL1RaKO mice. It was confirmed that synovial inflammation was low in the tIK-IL1RaKO group.

In addition, cartilage erosion was confirmed by safranin O staining. Safranin O staining was followed by deparaffinization of tissue sections, followed by water treatment, hematoxylin staining, safranin O staining, fast green staining, dehydration, clearing and encapsulation. The results are shown in Figure 6b. The degree of erosion of cartilage can be confirmed by changing the color of the stained area.In the articular tissue derived from IL1RaKO, the cartilage is destroyed in a large part, but the tissue of the joint derived from tIK-IL1RaKO is well maintained. .

실시예 5: 관절염에 의한 골 손상 정도 분석Example 5 Analysis of Bone Damage by Arthritis

In order to compare the bone damage due to inflammation in arthritis-induced mice in the IL1RaKO group and the tIK-IL1RaKO group, the joint sites were obtained at the 16th week of monitoring of the arthritis spontaneous group. Tissues were fixed in 10% neutral formalin for 18 hours and micro-CT was photographed using skyscan1172. X-ray sources of 50 kV and 200 uA were used, and a cross-sectional image of the tissue was taken at a pixel resolution of 15 μm using a filter of 0,5 mm. Joints derived from IL1RaKO mice were found to have many bone damages and structural deformations due to inflammation, but the joints derived from tIK-IL1RaKO mice maintained their original structure with little bone damage (FIG. 7). It was confirmed that IK expression is also involved in suppressing bone destruction caused by arthritis.

실시예 6 : 관절부위 염증 사이토카인의 발현 확인Example 6 Expression of Inflammatory Cytokines at the Joint Site

(1) mRNA expression confirmation

In order to confirm the expression level of inflammatory cytokines in arthritis-induced mouse joints, joints were obtained by autopsy of mice in each group at the 16th week of monitoring of arthritis naturally occurring groups. After the joints were ground in LN ₂ , 400 μl of Trizol reagent was used for RNA isolation. Chloroform was added and strongly vortexed and then incubated at room temperature for 5 minutes. After centrifugation (12500 rpm, 15 min, 4 ° C.), the supernatant was transferred to a new micro tube. After isopropanol and glycoblue in the supernatant was incubated at room temperature for 5 minutes. The supernatant was removed after centrifugation (12500 rpm, 10 min, 4 ° C.). After removing the supernatant, the remaining pellet was washed with 70% ethanol, centrifuged (9500 rpm, 5 min, 4 ℃), the pellet was air dried and dissolved in DEPC-water. Isolated RNA is QuantiTect CDNA was prepared using Reverse Transcription Kit (Qiagen). Quantitative real-time PCR was performed using the prepared cDNA (cDNA 1 μl, primer set 1 μl, 2XSYBR green mix 12.5 μl, Distilled water 9.5 μl, 40 cycles).

The measured result value (Ct value) for each group was calculated as a relative value based on the results of the wild type mice group using the 2 ^(-ΔΔCt) method. Using cDNA as a template, the expression of inflammatory cytokines (IL-1β, IL-6, IL-17A), which are known to increase expression in the onset of arthritis, was measured. Primer sequences used to amplify inflammatory cytokines (IL-1β, IL-6, IL-17A) using cDNA as a template are shown in Table 5, and the measurement results are shown in FIG. 8A.

Table 5

Inflammatory cytokines	Forward primer	Reverse primer
IL-1β	SEQ ID NO: 38	SEQ ID NO: 39
IL-6	SEQ ID NO: 40	SEQ ID NO: 41
IL-17A	SEQ ID NO: 42	SEQ ID NO: 43

Inflammatory cytokines (IL-1β, IL-6), which are involved in inflamed joints, showed higher expression levels in IL1RaKO mice than in tIK-IL1RaKO mice. In addition, IL-17, a major factor that inhibits tissue production in chondrocytes and osteoblasts, induces joint damage, and depletes tissue regeneration, was significantly lowered in joint tissues derived from tIK-IL1RaKO mice. These results confirm that IK-derived nucleic acid fragments can regulate the expression of inflammatory cytokines expressed when arthritis is induced, thereby lowering arthritis levels.

(2) Confirmation of cytokine expression using tissue staining

In order to confirm the pathogenesis of arthritis-induced mouse joints, the joint sites were obtained by autopsy of mice in each group (tIK-IL-1RaKO group; IL-1RaKO group) at week 16 of monitoring the arthritis naturally occurring group. Joint area was fixed by 10% neutral formalin and then demineralized by immersion in demineralization solution for 7 hours. The demineralized joint area was washed with water for 16 hours, and a paraffin block was prepared by dehydration, clarity, and infiltration. Paraffin blocks were cut using a slicer to make 7 μm sections, attached to slides, and stained with inflammatory cytokines (IL-17, IL-1β, TNFα). The paraffin fragment was deparaffinized and then functioned. After attaching the primary antibody against IL-17, IL-1β, and TNFα for 16 hours, the secondary antibody labeled with biotin was attached. After incubation with ABC reagent, the cells were colored with peroxidase substrate solution and washed with water to confirm the expression of inflammatory cytokines around synovial joints. The results are shown in Figure 8b. Joint tissues derived from tIK-IL1RaKO mice showed less infiltration of immune cells in the synovial site than joint tissues derived from IL1RaKO mice, and showed particularly low expression of IL-17. It was confirmed that synovial inflammation was low in the tIK-IL1RaKO group.

실시예 7 : 혈청 내 염증 사이토카인의 발현 확인Example 7 Expression of Inflammatory Cytokines in Serum

In order to confirm the expression of inflammatory cytokines in the blood of arthritis-induced mice, the mice of each group were autopsied at week 16 of monitoring the arthritis spontaneous generation, and the blood was obtained by heart blood collection. The obtained blood was centrifuged (4000 rpm, 15 min, 4 ° C) to separate only serum, and the expression of inflammatory cytokines in serum was confirmed by ELISA.

Using the MCP-1 ELISA kit (R & D system) as an example, the standard provided by the kit is placed on a plate pre-coated with anti-MCP-1 primary antibody, and the serum stock solution is added for 2 hours at room temperature. incubation. After incubation, all the samples in the wells were removed and washed four times with 0.05% PBST (Phosphate Buffered Saline Tween-20). A conjugate solution capable of binding to MCP-1 bound to the anti-MCP-1 antibody in the well was added and incubated at room temperature for 2 hours. After incorporation, the substrate solution was added and monitored for color development by reacting with the conjugate. When the color development degree of the serum sample to be measured within the color development range of the standard was added to the stop solution (2N H ₂ SO ₄ ) and the absorbance was measured at 450 nm wavelength using an ELISA reader. Based on this, the concentration of MCP-1 indicating the absorbance of the sample was determined based on the absorbance of the standard concentration, and cytokines other than MCP-1 were measured using the same method as above. In order to amplify the MCP-1 cytokine, a forward primer of SEQ ID NO: 44 and a reverse primer of SEQ ID NO: 45 were used, and the remaining cytokines were amplified using the same primers as in Example 6.

The analysis results are shown in FIG. Similar to the results of mRNA expression of inflammatory cytokines in the joint region, Example 6, the chemokine MCP-1, IL-known to be involved in the differentiation of pathogenic T cells in the serum of tIK-IL1RaKO mice than IL1RaKO mice 6, and the expression of IL-17, a major factor in the development of arthritis, was found to show a significantly low pattern. It is expected that tIK may also affect systemic immune responses and contribute to lower arthritis levels.

실시예 8 : 병인 T-helper 세포의 발현 측정Example 8 Measurement of Expression of Pathogenic T-helper Cells

T cells, one of the immune cells, have been found to be important cells that cause disturbance of the immune system. Activated T cells differentiate into effector T cells, Th1, Th2, Th17 and regulatory T cells, respectively. The incidence and course of the disease depends on which way the differentiation pathway is promoted. Th1 and Th17 cells secrete IFN-γ and IL-17, respectively, and are known to form the central axis of inflammatory bowel diseases such as diabetes, rheumatoid arthritis and Crohn's disease.

In this example, when arthritis is inhibited by the nucleic acid fragment derived from IK, it was confirmed whether the expression pattern of the pathogenic T hepler cells is involved. Monitoring of Spontaneous Arthritis Groups At week 16, mice in each group were autopsied to obtain spleens. A 100 μm strainer was placed in a 100 mm petri dish containing Serum free RPMI media, and the spleen was placed on the strainer. The spleen was then torn using a syringe needle to allow immune cells in the spleen to exit the dish containing the media through the stainer. . After removing all the spleens, the cells spread on the media were collected in a tube and centrifuged at 1500 rpm, 4 ° C for 5 minutes to obtain only pellets. Red blood cell lysis buffer was added to the pellet and incubated at 4 ° C. for 5 minutes. Again, centrifuged at 1500rpm, 4 ℃, 5 minutes to remove the red blood cells to finally obtain the splenocytes. After splenocytes were suspended in RPMI media containing FBS, only 1 × 10 ⁶ cells were taken and stained for flow cytometry analysis of the etiology of Th17 cells. PMA (100 ng / ml) and ionomycin (200 ng / ml) were added to the splenocytes obtained by each group, and incubated for 4 hours in a 37 ° C incubator, and golgi stop reagent for staining intracellular IL-17 was added. Cells stimulated with PMA and ionomycin were washed with cold PBS and reacted for 30 minutes at 4 ° C. with an antibody against CD4 (PE-Cy5 conjugated), a surface molecule of etiological Th17 cells. After washing with cold PBS, to increase the permeability of the cells so that the antibody can penetrate the cells to stain the intracellular IL-17, the antibody against IL-17 (PE conjugated) was added and reacted at room temperature for 30 minutes. . After completion of the reaction, the cells were washed with a fixative solution and finally stained with 1% paraformaldehyde solution, and used for flow cytometry analysis.

Th1 cells secreting IFN-γ were 6.09% in IL1RaKO mice, but 3.24% in tIK-IL1RaKO mice, which was lower than that of IL1RaKO (FIG. 10A). Th17 cells secreting IL-17 were also shown to be 1.78% in IL1RaKO mice but 1.2% in tIK-IL1RaKO mice, which showed a 1.5-fold reduction in the expression of etiological Th17 cells compared to IL1RaKO (FIG. 10B).

The results of this analysis are consistent with the results of expression analysis of inflammatory cytokines known to be involved in the differentiation of Th17 cells identified in the joints and serum of mice in Examples 6 and 7. Therefore, when arthritis was induced, it was confirmed that IK-derived nucleic acid fragments could suppress the inflammatory response of arthritis by inhibiting the expression of Th17 cells and Th1 cells, which are diseases that contribute greatly to the inflammatory response.

실시예 9 : 대식세포 활성 인자 발현 분석Example 9 Macrophage Activator Factor Expression Analysis

Macrophages activated in rheumatoid arthritis secrete inflammatory cytokines and interact with various immune cells to initiate and maintain joint inflammation. Macrophages not only induce T cell activity by presenting antigens to T cells, but also induce osteoclast activation along with T cells by releasing various cytokines. Macrophages play an important role in maintaining the inflammatory response of arthritis, so in this example it was confirmed whether macrophages are involved in the arthritis inhibition phenomenon by IK.

Spleen cells of IL1RaKO group mice and tIK-IL1RaKO group were obtained at week 16 of spontaneous arthritis monitoring, and splenocytes were obtained by the same procedure as in Example 8. The obtained splenocytes had an antibody against macrophage factor CD11b (APC conjugated), a pathogenous macrophage factor F4 / 80 antibody (PE-cy7 conjugated), and a macrophage active factor CD86 antibody (FITC conjugated). Spleen cells were stained. Stained cells were fixed in 1% paraformaldehyde solution and used for flow cytometry analysis. As shown in Figure 11, the expression of macrophage activator in the spik cells derived from tIK-IL1RaKO was reduced compared to IL1RaKO. Therefore, it was confirmed that IK expression affects the inhibition of macrophage activity as well as etiological Th17 cells.

실시예 10 : 활성 펩타이드의 합성 및 대식세포 활성 억제 확인Example 10 Synthesis of Active Peptides and Confirmation of Inhibition of Macrophage Activity

Compared to tIK, the active peptide, which is a partial fragment of the short-length IK factor, which is easier to express and purify, was prepared and confirmed to function when applied to the arthritis model. Through Example 1 a peptide comprising two amino acids (S382, Y489) expected to play a major role in the function of the IK factor was synthesized. A peptide consisting of 14 amino acids including S382 (SEQ ID NO: 47, S peptide), and a peptide consisting of 13 amino acids including Y489 (SEQ ID NO: 49, Y peptide) were synthesized. S382 and Y489 were synthesized to be substituted with phosphoric acid groups, respectively. Each active peptide was synthesized by Fmoc solid phase synthesis. The remaining reagents were removed by repeating the coupling-wash-deprotection-wash procedure, and a multi-channel automated synthesizer, a COMDEL instrument, was used. Through this synthesis, peptides were synthesized from the C-terminus to the N-terminus of each active peptide. After all reactions were completed, the synthesized peptide was separated from the resin using reagent K. The separated peptide was precipitated by addition of cold diethyl ether, and once again washed with diethyl ether and dried under vacuum. The molecular weight of the synthesized peptide was measured using a mass spectrometer, and purification was performed when the molecular weight was the same as the expected molecular weight. Purification was performed under reverse-HPLC, C18 column, and 220nm wavelength purification conditions.

Purified S peptide and Y peptide were confirmed the degree of macrophage activation under the conditions of stimulation of lipopolysaccharide (lipspolysaccharide, LPS). In the J774a.1 macrophage line, S and Y peptides were simultaneously transfected at a concentration of 500 ng / mL and treated with LPS (O111: B4) to confirm the expression of inflammatory cytokines. In macrophages obtained 24 hours after LPS treatment, mRNA expression of inflammatory cytokines (TNF-α, IL-6) was low in the group treated with the active peptide (FIG. 12A). In the cell culture solution, inflammatory cytokines were expressed lower than that of the control group in the condition of tIK active peptide (FIG. 12B). Therefore, it was confirmed that the active peptide inhibits the expression of inflammatory cytokines associated with arthritis.

실시예 11 : 활성 펩타이드에 의한 병인 T helper 세포 분화 억제 확인Example 11: Confirmation of inhibition of T helper cell differentiation of etiology by active peptide

In order to confirm the effect of the IK factor active peptide on the differentiation of pathogenic T helper cells, splenocytes were obtained by the method shown in Example 6 using spleens of wild-type mice. Only naive CD4 T cells were selectively isolated from splenocytes, and pretreated with S peptide and Y peptide (100 ng / mL or 500 ng / mL) for 1 hour. A culture medium containing various cytokines and neutralizing antibodies capable of differentiating into etiological T helper cells was added, and when the condition medium was added, the same concentration of S peptide and Y peptide was again obtained (100 ng / mL or 500 ng / mL). Was added. For differentiation into Th0 cells, culture medium containing anti-CD3, anti-CD28, IL-2 was added to the cells. For the differentiation into Th1 cells, the culture medium containing anti-CD3, anti-CD28, IL-2, IL-12 and anti-IL-4 was added. In addition, for the differentiation into Th17 cells, culture medium containing anti-CD3, anti-CD28, IL-6, TGF-β, anti-IL-4, and anti-IFNγ was added to the cells. After 2 days of culture, the culture medium containing various cytokines and neutralizing antibodies was added to differentiate into T helper cells under the same conditions. After 2 days, the pathogenic T helper cells were stained for flow cytometry analysis.

First, phorbol 12-myristate 13-acetate (PMA, 50 ng / mL) and ionomycin (200 ng / mL) were added to the cultured cells. While incubating for 4 hours in a 37 ° C. incubator, golgi stop (monensin) was added to stain intracellular IL-17. After incubation the cells were washed with cold PBS. In order to stain intracellular cytokine (IL-17, IFNγ), the cell permeability was increased to allow the antibody to penetrate into the cell, and the antibody against IL-17 and IFNγ was added and reacted on ice for 30 minutes. After the reaction, the cells were washed with a fixative solution, and finally, cells stained with 1% paraformaldehyde solution were used for flow cytometry analysis.

As shown in FIGS. 13A and 13B, the treatment of the active peptide showed a tendency to decrease the differentiation of CD4 + IFNγ + T cells under Th0 cell differentiation conditions and Th1 cell differentiation conditions. In addition, as shown in Figure 13c, the differentiation of CD4 + IL-17 + T cells was inhibited by the treatment of the active peptide. These results indicate that the short active peptide having the active site amino acid of IK factor can inhibit the differentiation into Th1 cells and Th17 cells, which act as T helper cells, which are pathogenesis in inflammatory diseases.

실시예 12 : 활성 펩타이드의 주입을 통한 관절염 억제Example 12 Inhibition of Arthritis by Infusion of Active Peptides

In order to examine the arthritis inhibitory effects of S peptides and Y peptides, active peptides of IK factor, these peptides were injected into IL1RaKO mice, a model of naturally occurring arthritis. Seven IL1RaKO mice were used in each group, and S peptide and Y peptide were simultaneously intraperitoneally injected at a concentration of 10 mg / kg (S peptide 5 mg / kg, Y peptide 5 mg / kg) as active peptides. . The control group was intraperitoneally injected with the same volume of phosphate buffered saline (PBS). Peptides were injected at intervals of 2 to 3 days, and the weight of mice, the incidence of arthritis, and the index were recorded for each Peptide injection. Arthritis index of mice 7 weeks after the peptide injection was lower than the control group injected with PBS (Fig. 14a), the incidence of arthritis in the peptide injection group (Fig. 14b).

실시예 13 : 활성 펩타이드 주입후 관절염 모델에서 병인 세포 분석Example 13: Pathogenesis Cell Analysis in Arthritis Models After Active Peptide Injection

(1) flow cytometry analysis

By injecting the active peptide of IK factor, the distribution of Th17 cells, a major factor involved in the initiation and maintenance of arthritis inflammatory response, was measured. According to Example 12, mice were necropsied at week 8 of the active peptide injection to obtain spleens of mice in each group. The spleens were ground to obtain splenocytes, and PMA (50 ng / mL) and ionomycin (200 ng / mL) were added. While incubating for 4 hours in a 37 ° C. incubator, golgi stop (monensin) was added to stain intracellular IL-17. After incubation, the cells were washed with cold PBS, and an antibody (APC conjugated) to CD4, a surface molecule of etiological Th17 cells, was added and reacted at 4 ° C. for 30 minutes. When the reaction is complete, the cells are washed, the cell permeability is increased to allow the antibody to penetrate into the cells to stain the IL-17, and then the antibody to the IL-17 (PE conjugated) is added and the reaction is performed at room temperature for 30 minutes. Reacted. After the reaction, the cells were washed with a fixative solution and finally stained with 1% paraformaldehyde solution, and used for flow cytometry analysis.

As a result of analysis of etiological Th17 cells in splenocytes of arthritis-induced mice in the peptide injection group and the PBS injection group, it can be seen that fewer Th17 cells appear in the spleen of IL1RaKO injected with the active peptide (FIG. 15A). This means that the active peptide of IK factor has the effect of inhibiting the differentiation and proliferation of Th17 cells, which play a major role in the arthritis etiology.

(2) Confirmation of cytokine expression using tissue staining

After injection of IK active peptide, the expression of inflammatory cytokines in the synovial area of the joint was confirmed. According to Example 12, mice were autopsied at week 8 of the active peptide injection to obtain joint sites. Joint area was fixed by 10% neutral formalin and demineralized by soaking in demineralized solution for 7 hours. The demineralized joint area was washed with water for 16 hours, and a paraffin block was prepared by dehydration, clarity, and infiltration. Paraffin blocks were cut using a slicer to make 7 μm sections, attached to slides, and stained with inflammatory cytokines (IL-17, IL-1β, TNFα). The paraffin fragment was deparaffinized and then functioned. After attaching the primary antibody against IL-17, IL-1β, and TNFα for 16 hours, the secondary antibody labeled with biotin was attached. After incubation with ABC reagent, the cells were colored with peroxidase substrate solution and washed with water to confirm the expression of inflammatory cytokines around synovial joints. The results are shown in FIG. 15B. In the case of mice injected with the active peptide, it was confirmed that the expression of inflammatory cytokines in the joint region was low and the infiltration of inflammatory cells was significantly reduced.

실시예 14 : Baculovirus expression system을 이용한 tIK 발현 및 정제Example 14 tIK Expression and Purification Using a Baculovirus Expression System

For mass expression of IK fragment (tIK) protein, the tIK plasmid tagged with Fc was transfected into baculovirus and then expressed in insect cells (SF9 cells). A nucleic acid (SEQ ID NO: 50) encoding a thrombin recognition site was linked to the 3 ′ end of the tIK sequence and an Fc tag sequence (SEQ ID NO: 51). After the virus seed was produced, baculovirus expressing tIK-Fc was gradually infected to a larger number of insect cells over the first (100 mm dish scale), the second (T75 flask scale), and the third (1 L conical flask). Finally, 3 days after tertiary virus infection, 1 L volume of cell culture containing secreted tIK-Fc was obtained. TIK-Fc protein present in the cell culture was purified using immunoprecipitation method. When rProtein A resin was added to the Sepharose column and the culture solution was injected, the Fc portion of the tIK-Fc protein contained in the culture medium was combined with the resin, thereby remaining in the column. Expression of tIK-Fc protein (approximately 55 kDa) was primarily confirmed by Commassie blue staining (result not shown). To obtain the pure tIK-Fc protein, we tried to isolate the tIK and Fc region conjugate from the resin by breaking the non-covalent bond between rProtein A and Fc region. In order to break the non-covalent bond, the protein was eluted with Elute buffer (0.1M Glycine buffer, pH2.8) and then neutralized with Neutralization buffer (1M Tris-HCl, pH8.0) to prevent protein denaturation. Column washing was performed using washing buffer (0.15M NaCl-20mM Na ₂ HPO ₄ , pH8.0). As a result of confirming the solution separated into a total of seven fractions through affinity chromatography through SDS-PAGE, the protein was eluted. As a result of checking the size of the protein, 55 kDa, which is the size of the tIK-Fc protein, was bound. This confirmed that the Fc region and the fusion protein can separate the conjugate (FIG. 16).

실시예 15 : tIK 유전자 전달을 위한 재조합 아데노-연관바이러스(rAAV) 벡터 시스템 확립Example 15: Establishment of a Recombinant Adeno-associated Virus (rAAV) Vector System for tIK Gene Delivery

Adeno-associated viruses (AAV), unlike retroviruses, do not cause disease and are selectively integrated into chromosome 19 of human cells, and thus are used as gene therapy vectors for chronic diseases. Using this property, in the present embodiment, a rAAV2 capable of delivering a tIK nucleic acid fragment into a cell is subjected to a gene cloning step in which the tIK nucleic acid fragment represented by SEQ ID NO: 3 is surrounded by an inverted terminal repeat (ITR) form of AAV. -tIK vector system was established. PSP72-XP7-scAAV2-CMV-GFP, a rAAV viral vector capable of GFP expression, was used as an expression vector. a HA tag sequence (SEQ ID NO: 54) is linked to the 5 'end of the tIK nucleic acid fragment, and a primer complementary to both ends of the tIK nucleic acid fragment (forward primer is SEQ ID NO: 55; the reverse primer is SEQ ID NO: 56 PCR was performed using). The ends of the amplified fragments thus obtained were cut with restriction enzymes and inserted into expression vectors to prepare tIK-HA-sp72-XP7-scAAV2 recombinant expression vectors. After transforming the prepared recombinant expression vector into Escherichia coli, the plasmid into which the tIK amplification fragment was inserted could be selected. The tIK gene linked to adeno-associated virus was injected into 293T cells by transfection with polyethylenimine (PEI). Viruses that were found to express tIK using PEI were again infected with 293T cells and confirmed by Western blotting (FIG. 17).

실시예 16 : AAV2-tIK 주입을 통한 관절염 억제 확인Example 16: Confirmation of Arthritis Inhibition through AAV2-tIK Injection

RAAV2-tIK prepared in Example 15 was applied to an in vivo disease model to confirm the therapeutic effect of arthritis according to the expression of tIK nucleic acid. AAV2-tIK and AAV2-GFP viruses were injected intravenously through the tail of IL1RaKO, a naturally occurring arthritis at a concentration of 1 × 10 ¹¹ vg / 100 ul, respectively. RAAV2-tIK and rAAV-GFP were injected at two week intervals, and once a week mouse joint condition and incidence were measured. IL1RaKO injected with rAAV2-tIK showed lower arthritis incidence and arthritis index than IL1RaKO injected with rAAV2-GFP (FIG. 18A), and the incidence of arthritis was low (FIG. 18B). Through this, IK nucleic acid was delivered using a virus vector system, which confirmed the possibility of lowering the incidence of arthritis and arthritis index.

실시예 17 : CHO cell line에서 tIK 발현 시스템 구축Example 17 tIK expression system construction in CHO cell line

Chinese hamster ovary (CHO) cells have high expression efficiency during gene injection and are widely used as animal cell lines for producing recombinant protein pharmaceutical products. To confirm the efficiency of tIK expression in CHO cells, tIK plasmids (tIK-PcDNA3.1) having a HA tag sequence (SEQ ID NO: 54) linked to the 5 'end were injected into CHO cells. For amplification of tIK, a forward primer of SEQ ID NO: 55 and a reverse primer of SEQ ID NO: 57 were used. Expression of tIK in CHO cells was confirmed by western blotting (FIG. 19).

Claims

A pharmaceutical composition for treating or preventing arthritis containing a pharmaceutically effective amount of an IK factor or fragment of an IK factor.
The pharmaceutical composition of claim 1, wherein the fragment of IK factor comprises a peptide having an amino acid sequence of SEQ ID NO: 4 or a partial peptide of SEQ ID NO: 4.
The fragment of claim 1, wherein the fragment of the IK factor comprises at least one amino acid residue selected from 382 th serine, 489 tyrosine, and 492 tyrosine of the amino acid sequence of SEQ ID NO: 2, and a contiguous amino acid of these selected amino acid residues. Pharmaceutical composition consisting of a peptide having at least 10 amino acids consisting of.
4. The pharmaceutical composition according to claim 3, wherein the fragment of IK factor is phosphorylated at least one amino acid consisting of 382 th serine, 489 th tyrosine and 492 th tyrosine of the amino acid sequence of SEQ ID NO: 2. Composition.
The fragment of claim 3, wherein the fragment of the IK factor is a fragment of a peptide having an amino acid sequence of SEQ ID NO: 2, wherein at least one selected from 382 th serine and 489 th tyrosine of the amino acid sequence of SEQ ID NO: 2. A pharmaceutical composition consisting of a peptide having at least 10 amino acids consisting of amino acid residues and contiguous amino acids of these selected amino acid residues.
The pharmaceutical composition according to claim 5, wherein the fragment of the IK factor is composed of a peptide having 10 or more amino acids each having two or more adjacent amino acids linked to the N-terminus and C-terminus of the selected amino acid.
The pharmaceutical composition of claim 1, wherein the fragment of IK factor is composed of a peptide having an amino acid sequence selected from at least one group consisting of SEQ ID NO: 4, SEQ ID NO: 47, and SEQ ID NO: 49. .
The pharmaceutical composition of claim 1, wherein the pharmaceutical composition is used to treat rheumatoid arthritis.
The pharmaceutical composition of claim 1, wherein the IK factor or fragment of IK factor is contained in a pharmaceutical composition at a concentration of 1.0 ng / mL to 10 μg / mL.
A pharmaceutical composition for treating or preventing arthritis, comprising a gene carrier comprising a nucleic acid molecule encoding an IK factor or fragment of an IK factor.
The pharmaceutical composition of claim 10, wherein the nucleic acid encoding the fragment of IK factor comprises a nucleic acid having a nucleotide sequence of SEQ ID NO: 3 or a partial nucleic acid of SEQ ID NO: 3.
The nucleic acid encoding a fragment of the IK factor of claim 10, wherein the nucleic acid encoding a fragment of the IK factor is at least one selected from nucleotide residues 1144-1146, 1465-1467 nucleotide residues, and 1474-1476 nucleotide residues of the nucleotide sequence of SEQ ID NO: 1. A pharmaceutical composition consisting of a nucleic acid having one nucleotide residue and at least 30 nucleotides consisting of adjacent nucleotides of these selected nucleotide residues.
13. The nucleic acid encoding a fragment of the IK factor according to claim 12, wherein the nucleic acid encoding the fragment of IK factor comprises at least one nucleotide residue selected from nucleotide residues 1144-1146 and 1465-1467 nucleotide residues of the nucleotide sequence of SEQ ID NO: 1; A pharmaceutical composition consisting of a nucleic acid having at least 30 nucleotides consisting of contiguous nucleotides of a selected nucleotide residue.
The nucleic acid encoding a fragment of the IK factor is composed of nucleic acids having 30 or more nucleotides each having at least 6 adjacent nucleotides linked to the 5 'and 3' ends of the selected nucleotide residue. Pharmaceutical compositions.
The method of claim 10, wherein the nucleic acid encoding a fragment of the IK factor encodes a peptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 47 and SEQ ID NO: 49 Pharmaceutical composition consisting of a nucleic acid having a nucleotide sequence.
The nucleic acid encoding a fragment of the IK factor is composed of a nucleic acid having at least one nucleotide sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 46 and SEQ ID NO: 48. Pharmaceutical compositions.
The pharmaceutical composition of claim 10, wherein the pharmaceutical composition is used to treat rheumatoid arthritis.
The pharmaceutical composition of claim 10, wherein the nucleic acid encoding the IK factor or a fragment of the IK factor is contained in the pharmaceutical composition at a concentration of 1.0 ng / mL to 10 μg / mL.
The pharmaceutical composition of claim 10, wherein the gene carrier is in the form of a naked nucleic acid molecule, a plasmid, a viral vector and a liposome or niosome containing the plasmid or the viral vector.
The pharmaceutical composition of claim 19, wherein the viral vector is selected from the group consisting of adenovirus, adeno-associated virus, retrovirus, lentivirus, baculovirus, herpes simplex virus, vaccinia virus.