Patent Application
20170209541
The present invention relates to a pharmaceutical use of a peptide derived from a living body and a nucleic acid encoding the peptide, more particularly to a pharmaceutical use of IK factor or fragments of IK factor and/or a nucleic acid encoding IK factor or the fragments of IK factor.
Arthritis is a disorder accompanied by joint stiffness and continuous periarticular pains caused by one or more joint inflammations. Arthritis can be classified into an acute arthritis and a chronic arthritis. The chronic arthritis such as gouty arthritis, polyarthritis, rheumatoid arthritis and osteoarthritis deformans caused by aging of bones or joints results in continuous periarticular pains.
Rheumatoid arthritis among the chronic arthritis is a chronic inflammatory disorder of unknown cause characterized by polyarthritis. The exact cause of the rheumatoid arthritis has not been known up to date. But, it has been generally considered that a hereditary tendency, bacterial or viral infections and the likes induce the rheumatoid arthritis to develop and inferred that hormones are concerned in developing the rheumatoid arthritis. The rheumatoid arthritis has harmful influences on not only joints but also other organs of the body. Accordingly, patients who have induced rheumatoid arthritis cannot walk and use their own hands or feet, and frequently feel tired or unpleasant. Furthermore, rheumatoid arthritis cause weight loss, lack of sleep and the likes, and continuous rheumatoid arthritis results in limitation of physical activity of the patients caused by muscular weakening, and thereby, considerably influencing on private and social activities of the patients. Also, when the physical activity of the patients is limited caused by rheumatoid arthritis, it is highly likely that the patients become obese and suffer from heart disease resulted from high cholesterol level and get the blues.
Rheumatoid arthritis has been occurred 0.2 cases for every 1,000 people for men and 0.4 cases per 1,000 persons for women per year, and prevalence of rheumatoid arthritis is 0.4-1.4%, which shows relatively even distribution in worldwide. As the aging population has increased gradually, it is expected that the market of therapeutic agents for rheumatoid arthritis grow sharply.
Currently, non-steroidal anti-inflammatory drugs(NSAID); steroids; non-biological disease-modifying anti-rheumatoid drugs(DMARD) such as anti-malarial drugs, hydroxychloroquinone(HCQ), sulfasalazine, methotrexate(MTX), leflunomide; and biological anti-rheumatoid drugs such as tumor necrosis factor(TNF) inhibitors or IL-6 inhibitors have been used as therapeutic agents for rheumatoid arthritis.
As the biological anti-rheumatoid drugs, tumor necrosis factor(TNE) inhibitors such as etanercept, adalimumab, infliximab, golimumab and certolizumab; monoclonal antibody Rituximab as a B-cell inhibitor; Abatacept blocking immune responses between antigen-presenting cells and T-cell; Tofacitinib blocking selectively JAK(Janus activated kinase) as a small molecule inhibitor and the likes have been developed.
But, most of those therapeutic agents for rheumatoid arthritis induce severe side effects. For example, non-steroidal anti-inflammatory drugs have caused gastrointestinal side effects, and some non-steroidal anti-inflammatory drugs including selective COX-2 inhibitors may induce severe side effects in cardiovascular system. Besides, it has been known that non-steroidal anti-inflammatory drugs induce side effects such as decline in renal function, interstitial nephritis, an acute renal failure and the likes. When administering steroids for a long term, they cause atrophy of hypothalamus-pituitary gland-adrenal gland so that adrenal insufficiency may be occurred, and they induce other side effects such as glaucoma, cataract, osteonecrosis, osteoporosis, hypertension, hypokalemia, and the likes. Particularly, because the non-steroidal anti-inflammatory drugs and steroids do not suppress of the progression of arthritis in reality, but only alleviates inflammation, they show little effects with regard to preventing joint damages.
Meanwhile, currently developed anti-rheumatoid drugs can suppress of the progression of early rheumatoid arthritis, but they cannot cure completely rheumatoid arthritis. Also, it has been reported that anti-rheumatoid drugs can induce some side effects. For example, some non-biological anti-rheumatoid drugs can induce side effects such as visual disturbance, retinopathy, skin rash, hepatic dysfunction, nausea and the likes. Also, tumor necrosis factor inhibitors of biological anti-rheumatoid drugs have problems such as blood abnormality, heart failure, hepatic dysfunction and opportunistic infection and IL-6 inhibitors can cause side effects such as hepatic dysfunction, leucopenia and the likes. It has been reported that other biological anti-rheumatoid drugs can induce infection problems as well as side effects such as a risk of tumorigenesis, gastrointestinal perforation and hyperlipidemia.
Particularly, most of the biological anti-rheumatoid drugs have a fatal defect that they can be administered only through subcutaneous injection or intravascular injection, and induce response decline to the drugs owing to immunogenicity by the human body by lapse of time. Also, since the currently developed biological anti-rheumatoid drugs suppress general immune system, they are exposed to the vulnerability of opportunistic infections and cancer suppression. Furthermore, those biological anti-rheumatoid drugs are very expensive, which causes financial burdens of patients considerably. Particularly, not a few patients with rheumatoid arthritis do no react to monoclonal antibodies which have been used as a representative biological anti-rheumatoid drug.
The present invention is proposed for solving the problem of the prior art, and an object of the present invention is to provide a pharmaceutical composition for treating or preventing arthritis that assures safety since no side effects and toxicity to a living body in spite of long-term administering.
Another object of the present invention is to provide a pharmaceutical composition for treating or preventing arthritis that shows no efficiency avoidances, and facilitate cheap manufacture in large quantity so as to lower patients' burden.
The present invention provides a pharmaceutical composition for treating or preventing arthritis comprises a pharmaceutically effective amount of IK factor or fragments of IK factor.
In an exemplary embodiment, the fragments 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.
For example, the fragments of IK factor may comprise a peptide having 10 or more amino acids that comprises at least one amino acid residue selected from Serine 382, Tyrosine 489 and Tyrosine 492 of SEQ ID NO: 2 and amino acids adjacent to the selected amino acid residue.
For example, the at least one amino acid residue consisting of Serine 382, Tyrosine 489 and Tyrosine 492 of SEQ. ID NO: 2 consisting of the fragments of IK factor is phosphorylated.
In an alternative embodiment, the fragments of IK factor are fragments of a peptide having amino acids of SEQ ID NO: 2, wherein the fragment of the peptide has 10 or more amino acids that comprises at least one amino acid residue selected from Serine 382 and Tyrosine 489 of SEQ ID NO: 2 and amino acids adjacent to the selected amino acid residue.
In a concrete embodiment, the fragments of IK factor comprise a peptide having 10 or more amino acids that comprise two or more adjacent amino acids bonded to each of N-terminal and C-terminal of the selected amino acid residue.
In an exemplary embodiment, the fragments of IK factor comprise a peptide having amino acids selected from the group consisting of at least one of SEQ ID NO: 4, SEQ ID NO: 47 and SEQ ID NO: 49.
The pharmaceutical composition containing IK factor or the fragments of IK factor may be used in treating rheumatoid arthritis.
In an exemplary embodiment, IK factor or the fragments of IK factor may be contained in an amount of 1.0 ng/mL to 10 μg/mL in the pharmaceutical composition.
Besides, the present invention provides a pharmaceutical composition for treating or preventing arthritis comprises a pharmaceutically effective amount of a gene delivery vehicle that comprises a nucleic acid molecule encoding IK factor or fragments of IK factor.
For example, the nucleic acid molecule encoding the fragments of IK factor comprises a nucleic acid having a base sequence of SEQ ID NO: 3 or a partial nucleic acid of SEQ ID NO: 3.
In an exemplary embodiment, the nucleic acid molecule encoding the fragments of IK factor may comprise a nucleic acid having 30 or more nucleotides that comprises at least one nucleotide residue set selected from nucleotide residues 1144-1166, nucleotide residues 1465-1467 and nucleotide residues 1474-1476 of a base sequence of SEQ ID NO: 1 and nucleotides adjacent to the selected nucleotide residue set.
In one exemplary embodiment, the nucleic acid molecule encoding the fragments of IK factor may comprise a nucleic acid having 30 or more nucleotides that comprises at least one nucleotide residue set selected from nucleotide residues 1144-1166 and nucleotide residues 1465-1467 of SEQ ID NO: 1 and nucleotides adjacent to the selected nucleotide residue set.
In a concrete embodiment, the nucleic acid molecule encoding the fragments of IK factor may comprise a nucleic acid having 30 or more nucleotides that comprises at least six adjacent nucleotides bonded to each of 5′-end and 3′-end of the selected nucleic acid residue set.
In an exemplary embodiment, the nucleic acid molecule encoding the fragments of IK factor comprises a nucleotide sequence encoding a peptide having an amino acid sequence selected from the group consisting of at least one of SEQ ID NO: 4, SEQ ID NO: 47 and SEQ ID NO: 49.
For example, the nucleic acid molecule encoding the fragments of IK factor may comprise a nucleotide sequence selected from the group consisting of at least one of SEQ ID NO: 3, SEQ ID NO: 46 and SEQ ID NO: 48.
The nucleic acid molecule encoding IK factor or the fragments of IK factor may be used in treating rheumatoid arthritis.
in an exemplary embodiment, the nucleic acid molecule encoding IK factor or the fragments of IK factor may be contained in an amount of 1.0 ng/mL to 10 μg/mL in the pharmaceutical composition.
For example, the gene delivery vehicle may have a naked nucleic acid molecule, a plasmid, a viral vector, or a liposome or a niosome containing the viral vector.
In this case, the viral vector may be selected from the group consisting of Adenovirus, Adeno associated virus, Retrovirus, Lentivirus, Baculovirus, herpes simplex virus and vaccinia virus.
Also, the present invention relates to IK factor or fragments of IK factor, a nucleic acid encoding IK factor or the fragments of IK factor and/or a vector or a gene delivery vehicle having the nucleic acid for treating arthritis.
Furthermore, the present invention relates to a method of treating arthritis comprises administering a therapeutically effective amount or a pharmaceutically effective amount of IK factor or the fragments of IK factor, a nucleic acid encoding IK factor or the fragments of IK factor, and/or a vector or a gene delivery vehicle having the nucleic acid to a subject.
According to the present invention, IK factor or the fragments of IK factor and/or a nucleic acid encoding IK factor or the fragments of IK factor are involved in reaction mechanism in an upstream level with relation to arthritis, for example rheumatoid arthritis, and suppress the progression of rheumatoid arthritis.
IK factor or the fragments of IK factor and/or a nucleic acid encoding thereof used as an active ingredient in the composition are immune-regulatory molecules in a human body that regulates generally activities of immunocytes having effects on arthritis so that they can be administered safely into the human body.
Particularly, because IK factor and/or the fragments thereof is an intrinsic molecule generated in the human body, and therefore, they shows little immunogenicity and are not involved with whole population of the immunocytes, it is expected that no side effects are occurred in spite of administering them for a long time.
Besides, it is possible to manufacture the pharmaceutical composition cheaply compared to the existing therapeutic agents for rheumatoid arthritis, for example, monoclonal antibodies. Furthermore, it is expected that the pharmaceutical composition of the invention can solve the problem of efficiency avoidance that is induced by the existing therapeutic drugs for rheumatoid arthritis.
Upper portion of
The inventors examined a new function of IK factor that is a sort of cytokine expressed in living organisms and found that IK factor or fragments thereof or gene delivery vehicles having nucleic acids encoding IK factor or the fragments suppress the progression of arthritis, for example, rheumatoid arthritis and completed the present invention. Hereinafter, the present invention will be described referring to the accompanying drawings.
Definition
As used herein, the term “amino acid” is used in the broadest sense and is intended to include naturally occurring L-amino acids or residues thereof. Conventionally used one-character or three-character abbreviation for the naturally occurring amino acid is used herein (See documents [Lehninger, Biochemistry, 2d ed., pp. 71-92, Worth Publishers: New York, 1975]). Amino acid includes not only D-amino acid but also chemically-modified amino acids, for example, amino acid analogs, naturally occurring amino acids that is typically incorporated to proteins, for example, norleucine, and chemically-synthesized compounds with amino acid-like characteristics such as properties known to a relevant art. For example, phenylalanine or proline analogs or mimetics that allows conformational limitations of peptide compounds identically to natural phenylalanine (Phe) or proline (Pro) is included in the definition of amino acid. Such analogs and mimetics are referred as “functional equivalences” of amino acids as used herein. Other examples of amino acids are illustrated in documents (See [Roberts and Vellaccio, The peptides: 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 technique are not limited to amino acids encoded by genes, and therefore, allows a more widely various substitutions for the given amino acids. Amino acids that are not encoded by the genetic code are referred as “amino acid analog” as used herein, and for example, are described in WO 90/01940. For example, amino acid analogs include 2-amino adipic acid (Aad) to Glu and Asp; 2-amino pimelic acid (Apm) to Glu and Asp; 2-amino butyric acid (Abu) to Met, Leu and other aliphatic amino acids; 2-amino heptanoic acid (Ahe) to Met, Leu and other aliphatic amino acids; 2-amino iso-butyric acid (Aib) to Gly; cyclohexyl alanine (Cha) to Val, Leu and Ile; homo arginine (Har) to Arg and Lys; 2,3-diamino propionic acid (Dap) to Arg and His; N-ethyl glycine (EtGly) to Gly, Pro and Ala; N-ethyl asparagine (EtAsn) to Asn and Gln; hydroxyl lysine (Hyl) to Lys; allo hydroxyl lysine (AHyl) to Lys; 3-(and 4-) hydroxyl proline (3Hyp, 4Hyp) to Pro, Ser and Thr; alto-isoleucine (AIle) to Ile, Leu and Val; 4-amidino phenyl alanine to Arg; N-methyl glycine (MeGly, sarcosine) to Gly, Pro and Ala; N-methyl isoleucine (MeIle) to Ile; norvaline (Nva) to Met and other aliphatic amino acids; ornithine (Orn) to Lys, Arg and His; citrulline (Cit) and methionine sulfoxide (MSO) to Thr, Asn and Gln; and N-methyl phenyl alanine (MePhe), trimethyl phenyl alanine, halo-(F—, Cl—, Br— or I—) phenyl alanine or trifluoryl phenyl alanine to Phe.
As used herein, the term ‘peptide’ includes any of proteins, fragments of the proteins and peptides that are isolated from naturally-occurring environment or synthesized by recombinant technique or chemical synthesis. For example, the peptides of the present invention may comprise at least 5, preferably 10 amino acids.
In a particular embodiment, compounds variants, for example peptide variants with one or more amino acid substitutions are provided. As used herein, the term “peptide variants” is intended to mean that peptides of which amino acid sequence has one or more amino acid substitutions, deletions, additions and/or insertions, and which exhibits virtually the identical biological functions as the original peptide. The peptide variants should have the identity of 70% or more, preferably 90% of more, more preferably 95% or more as the original peptide. As such substituents, amino acid substituents as known “conservative” may be included. Also, variants may include nonconservative changes. In one exemplary embodiment, the sequence of the variant polypeptides become different from original sequence by substitutions, deletions, additions or insertions of 5 or less amino acids. Besides, variants may be changed by deletions or additions of amino acids that have minimal effects upon immunogenicity, a secondary structure, and hydropathic nature of a peptide.
As used herein, the term “conservative” substitution means that there are little changes in the secondary structure and hydropathic nature of polypeptides in case amino acids of the polypeptides changed to other amino acids, unless mentioned otherwise. Such amino acid variations with regard to the conservative substitutions may be obtained based upon relative similarity of side chain substituents of amino acids, for example, polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or amphipathic nature.
For example, amino acids may be classified as 1) hydrophobic (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 having influence on the chain directions (glycine, proline), and 6) aromatic (tryptophane, tyrosine, phenylalanine) based upon the common side chains properties. Conservative variation will accompany an exchange of one member in each of the classes for another member in the same class.
It is possible to know that any of arginine, lysine and histidine is positively charged residue; alanine, glycine and serine has similar sizes; phenylalanine, tryptophane and tyrosine has similar shapes by analysis for the sizes, shapes and kinds of the side chain substitutions of amino acids. Accordingly, based upon those considerations, arginine, lysine and histidine; alanine, glycine and serine; and phenylalanine, tryptophane and tyrosine can be biologically functional equivalents.
Hydropathic index may be considered in introducing variations. Each amino acid is given hydropathic index based upon its own hydrophobicity and charge: Isoleucine (+4.5); Valine (+4.2); Leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (−0.4); threonine (−0.7); serine (−0.8); tryptophane (−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).
Hydropathic index of amino acids is very important in according interactive biological function of peptides. It has been known that similar biological activities may be maintained in only substituting amino acids having similar hydropathic indices. In case of introducing variations considering the hydropathic index, reciprocal substitutions among amino acids having hydropathic index value differences within preferably ±2, more preferably ±1, further more preferably ±0.5 are done.
Also, it is well known that reciprocal substitutions among amino acids having similar hydrophilicity induce proteins having equivalently biological activities. As disclosed in U.S. Pat. No. 4,554,101, following hydrophilicity value are accorded 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); leucine (−1.8); isoleucine (−1.8); tyrosine (−2.3); phenylalanine (−2.5); and tryptophane (−3.4). In case of introducing variations considering the hydrophilicity value, reciprocal substitutions among amino acids having hydrophilicity value differences within preferably ±2, more preferably ±1, further more preferably ±0.5 are done.
Amino acid exchanges in proteins that do not generally modify the molecular activities are known in the art (See, H. Neurath, R. L. Hill, The proteins, Academic Press, New York, 1979). The most commonly occurred exchanges are inter-exchange of amino acid residues between Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Thy/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, and Asp/Gly.
The term “polynucleotide” or “nucleic acid” are used inter-changeably herein, refers to polymers of nucleotides of any length, and includes comprehensibly DNA (i.e., cDNA) and RAN molecules. “Nucleotide”, which is a subunit of nucleic acid molecules, may be deoxyribonucleotide, ribonucleotide, modified deoxyribonucleotide or ribonucleotide, and/or analogs thereof, or any substrates that can be incorporated into polymers by DNA or RNA polymerase or synthetic reactions. Polynucleotide may comprise modified nucleotides, analogues modified polysaccharides or nucleotides, for example methylated nucleotides and analogues thereof (See, Scheit, Nucleotide Analogs, John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews, 90:543-584 (1990)).
Some variations in nucleotides do not result in variations in proteins. Such nucleic acids include nucleic acid molecules having codons encoding functionally equivalent or identical amino acids or encoding biologically equivalent amino acids. On the other hand, other variations in nucleotides may induce changes in proteins. In spite of variations causing changes of amino acids of proteins, it is possible to obtain variant proteins that show substantially the same activities as the proteins of the present invention.
A person having ordinary skill in the art will appreciate easily that peptides and nucleic acids according to the present invention are not limited to the amino acid sequences or nucleotide (base) sequences described in Sequence Listing, within the scope that they have characteristics of IK factor or fragments thereof and/or nucleic acids encoding IK factor or fragment thereof of the present invention, for example, they have an effect of treating arthritis. For example, biologically functional equivalents, which can be included in the scope of the present invention, may be peptides having amino acid sequence variations that exhibit equivalent biological activities as the peptides of the present invention.
Generally, the peptides (including fusion proteins) and polynucleotides described herein are isolated. “Isolated” peptides or polynucleotides are separated from the original environment. For example, naturally occurring proteins are isolated by removing whole or part of co-existent material in a natural state. Such polypeptides should have purity of 90% or more, preferably 95% or more, more preferably 99% or more. Polynucleotides are isolated by cloning in the vectors.
As used herein, the term “vector” means a construct that can be transfected or delivered into host cells, and preferably prepared in order to enable one or more target genes or sequences to be expressed. For example, the vector includes viral vectors, DNA or RNA expression vectors, plasmid, cosmid, or phage vectors, DNA or RAN expression vectors linked to CCA (cationic condensing agents), DNA or RNA expression vectors packaged with liposomes, specific eukaryotic cells such as producer cells and the likes.
As used herein, the term “expression control sequence” means a nucleic acid sequence which regulates a transcription of nucleic acid molecules. The expression control sequence includes promoters such as a constitutive promoter or an inducible promoter, enhancers and the likes. The expression control sequence is linked to nucleic acid sequence to be transcribed.
As used herein, the term “operatively linked” means a functional linkage between nucleic acid control sequences (for example, promoters, signal sequences, or array at transcription regulatory factor linkage sites) and other nucleic acid sequences, and thereby, the control sequences regulates transcriptions and/or translations of the other nucleic acid sequences.
As used herein, the term “pharmaceutically effective amount” or “therapeutically effective amount” means an amount of sufficiently accomplishing efficacy or activation of an active ingredient, a peptide or fragments thereof and/or nucleic acids encoding the peptide or fragments thereof. For example, the pharmaceutical composition containing peptides or gene delivery vehicles including nucleic acids encoding the peptides may be applied to treat and/or prevent gouty arthritis, polyarthritis, arthritis deformans as well as rheumatoid arthritis.
Peptides, Nucleic Acids and Vectors
IK factor is a cytokine that suppresses the expression of Major histocompatibility complex II (MHC class II) on the surface of antigen presenting cell (APC) that presents antigens with CD4 T cells. it was reported that IK factor activates cAMP and suppresses expressions of IFN-γ (interfereon-gamma) that is induced by viral infections and of MHC class II which is induced by IFN-γ.
As exemplarily shown in
A joint is a composite tissue that synovial membranes, cartilages, bones and synovial fluids are linked each other systematically and have reciprocal influences on each other through cytokines. It was reported that phenomena such as chronic inflammation, tissue necrosis, cellular infiltration, neovascularization, joint destructions and the likes within joints are major etiology of rheumatoid arthritis. Synovial membrane tissues, which are referred as pannus and known as essentially pathological findings in rheumatoid arthritis, infiltrates around the joints and thereby, inducing bone resorption. It was reported that antigen presenting cells and T cells with a high density are found in pannus and interactions among such cells cause initiation and expansion of immune responses induced by T cells. Actually, much antigen presenting cells expressing MHC class II are found in pannus of patients with rheumatoid arthritis and HLA-DR positive cells and T cells are located at the same site. Accordingly, inhibiting interactions between antigen presenting cells expressing MHC class II and T cells may be effective for treating efficiently rheumatoid arthritis.
Particularly, there are IL-17+T cells and functionally activated IL-17 in the pathogenic lesion, synovial membrane in case of developing rheumatoid arthritis. IL-17 is highly detected in serum of patients with rheumatoid arthritis, and IL-17A induces expressions of IL-1β and IL-6 within synovial membranes of patients with rheumatoid arthritis. With regard to arthritis, IL-17 suppresses substrate production within chondrocytes and osteoblasts and causes joint damages, and thereby, making a lack of tissue regenerations. Also, IL-17A activates expression and functions of matrix metalloproteinase (MMPs) and causes irrevocable cartilage erosions within the mouse model together with tumor necrosis factor (TNF). Local interaction between Th17 cells and synovial membrane cells in inflammatory responses facilitates secretion of MMP and expressions of IL-1β and IL-6 in the synovial membranes. With regard to osteoclasia, IL-17 causes receptor activator of NF-κB ligand (RANKL) on the osteoblasts to express so that it makes an amplification of RANK signaling in the osteoclasts. Particularly, it was known that Th17 cells that makes an expression of RANKL plays an important role in differentiation process of osteoclasts.
According to the present invention, nucleic acid molecules encoding IK factor or fragments of IK factor can suppress and prevent arthritis progression. Especially, such nucleic acids suppress secretion of rheumatoid-arthritis related cytokines and block differentiation of rheumatoid-arthritis related immune cells.
Besides, in an exemplary embodiment of the present invention, nucleic acid fragments (tIK nucleic acid fragment) encoding fragments of IK factor, for example, encoding truncated fragments having only C-terminal fragments without excised N-terminal fragments (referred as “tIK” factor or “tIK” herein) may be expressed within appropriate expression systems. Those nucleic acids are expressed to peptides which suppress expression of MHC class II associated with an inflammatory immune response which is one of major indications of arthritis. More concretely, tIK nucleic acid fragments, which are truncated nucleic acid fragments compared to the full-length IK nucleic acid, and/or mutant tIK nucleic acid fragments, which are variants that some nucleotides are substituted for original nucleotides of tIK nucleic acids, increases expressions of CDCA3 (Cell division cycle-associated protein 3), CNOT1 (CCR4-NOT transcription complex subunit 1) and MAPKI (Mitogen-activated protein kinase I) each of which is an upstream regulatory factor suppressing the expression of CHTA (class II, major histocompatibility complex, transactivator) which is involved in the expression of MHC class II (See
According to an exemplary embodiment of the present invention, the transgenic mice, which are induced to express fragments of IK factor such as fragments without excised N-terminal (“tIK-IL1RaKO” mice) show less arthritis inducement level compared to spontaneously-induced arthritis mouse model (“IL1RaKO” mice) (See,
Particularly, tIK-IL1RaKO mice exhibit reduced secretion of cytokines that is involved in arthritis progression in joint tissue and serum compared to IL1RaKO mice. More concretely, there are reduced expression of cytokines which induce inflammation in the joints (See
In an exemplary example of the present invention, the nucleic acid molecules encoding fragments of IK factor are expressed through an appropriate expression system, and suppresses expressions of interleukin-1 beta (IL-1β), interleukin-6 (IL-6), interleukin-17 and monocyte chemotactic protein-1 (MCP-1), also known as CCL2 (Chemokine (C-C motif) ligand 2), each of which are involved in inflammatory immune response. Such results means that injecting nucleic acid molecules encoding IK factor or fragments of IK factor using appropriate vectors or gene delivery vehicles into a human body can suppress the progressions of arthritis and may influences upon systemic immune responses especially. In addition, tIK-IL1RaKO mice, which are induced to express IK factor, show suppressive T cell differentiation (See
Furthermore, in an exemplary embodiment of the present invention, relatively small sized fragments of IK factor containing active sites, for example peptides consisting of about 10 amino acids having an amino acid at the active sites and adjacent amino acids can suppress the progression of arthritis. Such peptides suppress the expressions of inflammatory cytokines in macrophages such as tumor necrosis factor alpha (TNF-α), IL-6 and IL-17 (See
In an exemplary embodiment, the nucleic acid molecules encoding IK factor or fragments of IK factor may be transfected into a living body using appropriated expression systems or gene delivery vehicles so as to suppress the progression of arthritis or treat induced arthritis (See
As described above, IL-17 which is secreted depending upon the differentiation of Th17 cell plays an important role with regard to rheumatoid arthritis, Sine IK factor or fragment of IK factor and/or nucleic acids encoding IK factor or fragments of IK factor suppress the differentiation of Th17 cell, which receives attentions as a basic cause of rheumatoid arthritis, it is possible to treat rheumatoid arthritis more effectively compared to the conventionally used arthritis drugs using peptides and/or nucleic acid molecules of the present invention.
In addition, because most of the biologics as represented by monoclonal antibodies may induce immune responses in case they are injected into human bodies and prepared through complex manufacturing processes, they are sold at a high price. In contrast, IK factor of which efficiencies are certified in the present invention is an intrinsic cytokine in a human body so that there is little chance of immune response induced by IK factor, and therefore, IK factor is very safe. Also, it is possible to prepare IK factor at a relatively lower price.
Accordingly, the present invention relates to IK factor or fragments of IK factor for treating arthritis. As an active ingredient for treating arthritis, fragments of IK factor having truncated some amino acids of full-length IK factor are preferable, and fragments of IK factor without excised N-terminal amino acids of full-length IK factor are more preferable.
Exemplarily, the full-length IK factor may be a peptide having an amino acid sequence of SEQ ID NO: 2. As an example of the fragments of IK factor, a peptide such as a truncated IK factor (tIK factor) which has a amino acid sequence of SEQ ID NO: 4 which does not have excised 315 amino acids consisting of N-terminus from the full-length IK factor and has only 242 amino acids consisting of C-terminus of the full-length IK factor of SEQ ID NO: 2 or partial peptides of SEQ ID NO: 4. As an example of the fragments of IK factor, the peptide fragments without excised amino acids at N-terminal region among the amino acids consisting of the full-length IK factor is preferable because the fragments is relatively small so that they are easy to prepare. Also, the peptide fragments without excised amino acids at N-terminal may be more advantageous to exhibit biological functions in in vivo and/or ex vivo compared to the full-length IK factor.
According to exemplary examples of the present invention, the 382nd amino acid Serine of SEQ ID NO: 2 (67th amino acid of SEQ ID NO: 4) Serine (S382), the 489th amino acid of SEQ ID NO: 2 (174th amino acid of SEQ ID NO: 4) Tyrosine (Y489) and/or the 492nd amino acid of SEQ ID NO: 2 (177th amino acid of SEQ ID NO: 4) Tyrosine (Y492) are concerned with the biological functions of IK factor and/or fragments of IK factor. Particularly, it is expected that the 382nd amino acid Serine of SEQ ID NO: 2 (S382) and/or the 489th amino acid Tyrosine of SEQ ID NO: 2 (Y489) are active sites of IK factor and/or the fragments of IK factor.
Accordingly, in addition to the fragment of IK factor having an amino acid sequence of SEQ ID NO: 4, it is possible to use other fragments of IK factor comprising the amino acids at the active sites with relatively short length as fragments of IK factor. Exemplarily, the fragments of IK factor according to the present invention may comprise peptides that include 10 or more, for example 100 or more, or 200 or more, amino acids which has at least one amino acid residue selected from the group consisting of the 382nd amino acid Serine (S382), the 489th amino acid Tyrosine (Y489) and the 492nd amino acid Tyrosine (Y492) of SEQ ID NO: 2, and amino acids adjacent to the selected amino acid residue. In an exemplary embodiments, the fragments of IK factor comprises peptides that includes 10 to 100, preferably 10 to 50, more preferably 10 to 30 amino acids which has at least one amino acid residue selected from the 382nd amino acid Serine (S382), the 489th amino acid Tyrosine (Y489) and the 492nd amino acid Tyrosine (Y492) of SEQ ID NO: 2, and amino acids adjacent to the selected amino acid residue. At least one amino acid of such selected amino acid residues may be a modified amino acid such as a phosphorylated amino acid.
Exemplarily, the fragments of IK factor according to the present invention are a peptide having an amino acid sequence of SEQ ID NO: 2 or partial peptides of SEQ ID NO: 2. In an exemplary embodiment, the fragments of IK factor are peptides that include 10 or more, for example 10 to 100, preferably 10 to 50, more preferably 10 to 30 amino acids which has at least one amino acid residue selected from the 382nd amino acid Serine (S382) and the 489th amino acid Tyrosine (Y489) of SEQ ID NO: 2, and amino acids adjacent to the selected amino acid residue.
Exemplarily, the active amino acids such as the 382nd amino acid Serine (S382), the 489th amino acid Tyrosine (Y489) and the 492nd amino acid Tyrosine (Y492) are not located at N-terminal or C-terminal of the peptides that may be used as the fragments of IK factor. Peptides having 2 or more adjacent amino acids bonded respectively to each of the N-terminal and C-terminal of the selected active amino acid residue may be used in order to align the active amino acid, the selected amino acid residue in the center of the peptides. Exemplarily, 2 to 8 or more, preferably 3 to 7 or more adjacent amino acids may be bonded respectively to each of the N-terminal and C-terminal of the active amino acid. For example, 2 to 100 such as 3 to 70, preferably 3 to 30, more preferably 3 to 20 adjacent amino acids may be bonded respectively to each terminal of the active amino acid.
In one exemplary embodiment, the fragments of IK factor also comprise a peptide of SEQ ID NO: 47 that consists of amino acids from the 377th amino acid Glutamic acid to the 391st amino acid Aspartic acid of SEQ ID NO: 2, and/or a peptide of SEQ ID NO: 49 that consists of amino acids from the 484th amino acid Aspartic acid to the 496th amino acid Lysine of SEQ ID NO: 2.
According to an exemplary example, the full-length IK factor consists of 557 amino acids of SEQ ID NO: 2 and the fragment of IK factor of SEQ ID NO: 4, which does not include the excised N-terminal region compared to the full-length IK factor, consists of 242 amino acids. Considering those points, the fragments of IK factor according to the present invention may be consisted of maximally 300-500 amino acids of SEQ ID NO: 2. But, the fragments of IK which can be used in the present invention is not limited to such fragments.
Also, for example, except the active amino acids such as S382, Y489 and Y492 in the IK factor and/or fragments of the IK factor, the rest other amino acids may be substituted for other amino acids by a conservative substitution, and some amino acids may be deleted, added and/or inserted if necessary.
IK factor or the peptides as fragments of IK factor may be isolated by manufacturing through recombinant means or chemical synthesis. Exemplarily, the peptides which are expressed by nucleic acid sequences referred herein may be manufactured easily by known processes using any of known may expression vectors. The expression can be accomplished in appropriated host cells that are transformed by expression vectors comprising DNA sequences encoding the peptides. The appropriated host cells include prokarytoes, yeast cells and eukaryotes. It is preferable to use E. coli, yeast or mammalian cell lines (such as Cos or CHO cell) as a host cell. A supernatant containing a recombinant protein which is obtained by water soluble host/vector system and secreted into culture solution is concentrated using a commercially available filter for the purification of proteins. In the next step, the concentrated solution obtained by the above-mentioned procedure is purified using appropriate purification matrix such as affinity matrix or ion exchange matrix. Lastly, pure protein can be obtained by performing a single step or multiple steps of reverse phase HPLC assay.
The fragments or variants with 100 or less, generally 50 or more amino acids can be manufactured by chemical synthesis. For example, such polypeptides can be synthesized commercially available solid-phase techniques, i.e., Merrifield solid-phase synthesis method which adds subsequently amino acids to a growing amino acid chain (See, Merrifield, 1963, J. Am. Chem. Soc. 85:2146-2149). The apparatus for automatic synthesizing polypeptides may be available from supplying companies and can be operated pursuant to the supplier's manual.
For example, the peptides according to the present invention expressed in bacteria may be secreted to a periplasm of the host and be recovered therefrom. Typically, recovering proteins comprises homogenizing the bacteria by means of osmotic shock, ultra-sonication or dissolution. If the cells are destroyed, it is possible to remove them by centrifugating, or filtering cell debris or the whole cells. The proteins may be further isolated by, for example, an affinity resin chromatography assay. Alternatively, proteins may be transferred to a culture medium from where they may be isolated. The produced proteins by separating cells from the culture media, filtering and concentrating the culture supernatant may be further isolated. The expressed polypeptides may be further isolated and characterized by conventionally known processes, for example, fractional distillation on a immunoaffinity or ion-exchange column; ethanol precipitation, reverse-phase chromatography; silica or cation exchange resins for example chromatography on DEAE; chromatofocusing; SDS-PAGE; ammonium phosphate precipitation; gel filtration chromatography, for example using sephadex G75; ligand affinity or western-blotting assay using hydrophobic affinity resins or appropriate antigens immobilized on a matrix.
Besides, the produced peptides may be purified in order to obtain a substantially homogenizing agent for further assay and uses. Standard protein purification methods which have been known to the art may be used. Following procedures are examples of an appropriate purification: fractional distillation on immunoaffinity or cation-exchange resins, for example, chromatography on DEAE, chromatofocusing, SDA-PAGE, ammonium phosphate precipitation, and gel filtration chromatography, for example using sephadex G75.
Also, the present invention relates to a nucleic acid molecule or a polynucleotide encoding IK factor or fragments of IK for treating arthritis. Similarly to the peptides, IK nucleic acid fragments, which some nucleotides encoding the fill-length IK factor are excised, are preferable, and IK nucleic acid fragments, which nucleotides encoding 5′-end the full-length IK factor are excised, are more preferable as the IK nucleic acid fragments.
Exemplarily, the nucleic acid molecule encoding the full-length IK factor may be a nucleic acid having a nucleotide sequence of SEQ ID NO: 1. Exemplarily, the nucleic acid molecule encoding the fragment of IK factor may be a nucleic acid encoding the truncated IK factor (tIK nucleic acid fragment) having a nucleotide sequence of SEQ ID NO: 3, which 945 nucleotides at 5′-end of SEQ ID NO: 1 are out of excised, or nucleic acids consisting a part of the nucleotides of SEQ ID NO: 3.
Each of the 382nd amino acid Serine (S382), the 489th amino acid Tyrosine (Y489) and the 492nd amino acid Tyrosine (Y492) of SEQ ID NO: 2 may be expressed respectively the 1444-1146th nucleotide residues set of SEQ 111) NO: 1 (the 199-201st nucleotide residues set of SEQ ID NO: 3), the 1465-1467th nucleotide residues set of SEQ ID NO: 1 (the 520-522nd nucleotide residues set of SEQ ID NO: 3) and the 1474-1476th nucleotide residues set of SEQ ID NO: 1 (the 529-531st nucleotide residues set of SEQ ID NO: 3).
Accordingly, in addition to the IK nucleic acid fragment having a nucleotide sequence of SEQ ID NO: 3, other IK nucleic acid fragments comprising nucleotides with relatively short length as the IK nucleic acid fragments. Exemplarily, the IK nucleic acid fragments according to the present invention may comprise 30 or more, for example 300 or more, or 600 or more nucleic acids that has at least one nucleotide residues set selected from the group consisting of the 1144-1146th nucleotide residues set of SEQ ID NO: 1 (199-201stnucleotide residues set of SEQ ID NO: 3), the 1465-1467th nucleotide residues set of SEQ ID NO: 1 (the 520-522nd nucleotide residues set of SEQ ID NO: 3) and the 1474-1476th nucleotide residues set of SEQ ID NO: 1 (the 529-531st nucleotide residues set of SEQ ID NO: 3), and nucleotides adjacent to the selected nucleotide residues set. In an exemplary embodiment, the nucleic acids encoding fragment of IK factor comprise 30 to 300, preferably 30 to 150, more preferably 30 to 90 nucleotides which has at least one nucleotide residues set selected from the group consisting of the 1144-1146th nucleotide residues set of SEQ ID NO: 1, the 1464-1467th nucleotide residues set of SEQ ID NO: 1 and the 1474-1476th nucleotide residues set of SEQ ID NO: 1, and nucleotides adjacent to the selected nucleotide residues set.
Exemplarily, the IK nucleic acid fragments is a nucleic acid molecule having a nucleotide sequence of SEQ ID NO: I or partial fragments of the nucleic acid molecule. In an exemplary embodiment, the nucleic acid molecule encoding the partial fragments of IK factor is nucleic acids which has 30 to 300, preferably 30 to 300, more preferably 30 to 90 nucleotides that comprises at least one nucleotide residues set selected from the group consisting of the 1144-1146th nucleotide residues set of SEQ ID NO: 1 and the 1465-1467th nucleotide residues set of SEQ ID NO: 1, and nucleotides adjacent to the selected nucleotide residues set.
6 or more nucleotides may be bonded respectively to each of 5′-end and 3′-end of the selected nucleotide residues set in order to align the selected nucleotide residues set in the center of the nucleic acids. Exemplarily, 6 to 24 or more, preferably 9 to 21 or more adjacent nucleotides may be bonded respectively each of 5′-end and 3′-end of the selected nucleotide residues set. For example, 6 to 300 such as 9 to 210, preferably 9 to 90, more preferably 9 to 60 adjacent nucleotides may be bonded respectively to each of 5′-end and 3′-end of the selected nucleotide residues set.
In one exemplary embodiment, the nucleic acid molecules encoding the fragments of IK factor may a nucleic acid having a nucleotide sequence of SEQ ID NO: 46 and/or a nucleic acid having a nucleotide sequence of SEQ ID NO: 48. Exemplarily, the nucleic acid molecule encoding the fragments of IK factor may comprise, but are not limited to, maximally about 900-1,500 nucleotides in the nucleotides of SEQ ID NO: 1. Each of the 1144-1146th nucleotide residues set of SEQ ID NO: 1 encoding the 382nd amino acid Serine (S382) of SEQ ID NO: 2, the 1465-1467th nucleotide residues and/or the 1474-1476th nucleotide residues set of SEQ ID NO: 1 each of which encoding the 489th amino acid Tyrosine (Y489) and the 492nd amino acid Tyrosine (Y492) of SEQ ID NO: 2 may have other nucleotide sequences encoding serine or tyrosine.
The nucleic acid molecules encoding IK factor or the fragments of IK factor may be included into appropriate vectors. The vectors include expression control sequences linked to the polynucleotide of the present invention. In an exemplary embodiment of the present invention, the vectors may comprise one or more polynucleotides encoding interesting target molecules. The polynucleotide of the present invention may combine other polynucleotides so as to encode fusion proteins.
Exemplarily, the polynucleotides of the present invention are constructed in order to transfect into mammalian cells and expressed within the cells. Such a construction is particularly useful for the purposes of treatment. There are many processes to express the polynucleotides in the host cells and it is possible to adopt any appropriate processes. For example, one polynucleotide may be inserted into viral vectors such as adenovirus, adeno-associated virus, retrovirus, vaccinia virus, Lentivirus, baculovirus or other pox viruses (e.g. avian pox virus), and the likes. It has already been well-known that techniques of inserting DNA into such vectors. It is possible to insert additionally targeting moieties such as genes with regard to selectable markers for making easy certification or selection for the transduced cells and/or genes encoding ligands acting as a receptor to a particular target cell in the retrovirus vector. Targeting may be performed by known processes using specific antigens.
It is possible to use plural vectors that are commercially available and known to in the art for the purposes of the present invention. Selecting appropriate vectors will be mainly dependent upon the sizes of the nucleic acids to be inserted into the vectors and specific host cells transformed with the vectors. Each vector contains various components, depending upon its functions (amplification and/or expression of foreign polynucleotides) and compatibilities to the specific host cells having thereof. Vector components generally comprises, but are not limited to, replication origins (especially if the vector is inserted into prokaryotes), selection marker genes, promoters, ribosome binding sites (RBS), signal sequences, foreign nucleic acids insert, and a transcription termination sequence.
For example, the vector of the present invention may comprise expression control sequences, which may have an effect on the expression of the proteins, such as a initiation codon, a termination codon, a poly-adenylation signal, enhancers, signal sequences for membrane-targeting or section, and the likes. The poly-adenylation sequence increases the safety of the transcripts and facilitates cytoplasm transports. Enhancer sequences are nucleic acid sequences which are located at various sites with regard to the promoters and increase transcription activity compared to a transcription activity by the promoters without the enhancer sequences. Signal sequences comprise, but are not limited to, PhoA signal sequence, OmpA signal sequence, and the likes in case the host cell is bacteria in Escherichia spp., α-amylase signal sequence, subtilisin sequence and the likes in case the host cell is bacteria in Bacillus spp., MF-α signal sequence, SUC2 signal sequence and the likes in case the hose cell is yeast, and insulin signal sequence, α-interferon signal sequence, antibody molecule signal sequence and the likes in case the host cell is mammals.
A category of vector is a ‘plasmid’ which refers to a circular, double-stranded DNA loop into which additional DNA fragments may be ligated. Another category of vector is a phage vector. Further another category of vector is viral vectors which additional DNA fragments may be ligated into the viral genome. Specific vectors can replicate autonomously into the host cells having the transfected the vectors (for example, viral vectors and episome mammalian vectors having bacterial replication origins). Other vectors (for example, non-episome mammalian vectors) may be integrated into the genome of a host cell as they transfect to the host cell, and thereby, being replicated together with the genome of the host cell. Besides, specific vectors may direct the expression of genes operatively linked to the vectors. Such vectors are referred herein as a “recombinant expression vector (or, shortly, “recombinant vector”). Generally, the expression vectors, which may be useful for recombinant DNA technologies, exist as a shape of plasmid.
It is possible to construct vector systems of the present invention through various processes known to in the art, and such processes are discloses in Sambrook et al, Molecular Cloning, A Laboratory manual, Cold Spring Harbor Laboratory Press (2001), which is incorporated herein by reference. For example, the vectors, which may be used in the present invention, may be manufactured by manipulating the usually used vectors in the art, plasmid (e.g., pSC101, ColE1, pBR322, pUC8/9, pUC19, pET and the likes), phage (e.g., λgt-4λB, λ-Charon, λΔz1, λGEM.TM.-11, M13 and the likes), virus (e.g., SV40 and the likes).
In a concrete embodiment of the present invention, nucleic acid molecules are inserted into host cells using virus expression systems (e.g., vaccinia or other pox viruses, retroviruses, lentivirus, baculovirus, adenovirus, or adeno-associated virus). Exemplarily, virus vector comprises, but are not limited to, retrovirus vectors derived from HIV, SIV, murine retroviruses, gibbon ape leukemia virus, AAV (adeno-associated viruses), adenoviruses and the likes (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). Retrovirus vectors derived from murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), ecotropic retroviruses, SIV (simian immunodeficiency virus), HIV (human immunodeficiency virus) and the likes have been 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, 53: 2374-2378; Miller et al., 1991, J. Virol. 65: 2220-2224; Rosenberg and Fauci 1993 in Fundamental Immunology, Third Edition, W. E. 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).
Constitutively or inducible promoters can be used in the present invention as pacific occasions that can be certified by a person having ordinary skill in the art arise. Plural promoters that recognized by various possible host cells have been widely known in the art. Selected promoters may be linked operatively to the cistron DNA encoding the polypeptides described herein by removing the removing the promoters from suppliers DNA through restriction enzyme digestions and then inserting the isolated promoter sequences into the selection vectors. It is possible to direct amplification and/or expression of the target genes using both natural promoter sequences and a plurality of foreign promoters. But, foreign promoters are generally more preferable to the natural targeting polypeptide promoters because the foreign promoters allows much transcription and high yield of the expressed target genes compared to the natural targeting polypeptide promoters.
Meanwhile, when the vector of the present invention is an expression vector and a host cell is a eukaryote, promoters derived from the genome of the mammalian cells (e.g., metallothionein promoter) or promoters derived from mammalian viruses (e.g. adenovinis late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus promoter and tK promoter of HSV) may be used, and they generally have poly-adenylation signal as a transcription termination signal.
Besides, when the recombinant vector of the present invention is a replicable repression vector, it may comprise a replication origin, which is a specific nucleic acid sequence for initiating replication. In addition, the recombinant vectors may comprise sequences encoding selectable markers. The selectable markers are intended to screen transformed cells by the vectors and markers giving selectable phenotypes such as drug resistances, nutritional requirements, cytotoxic agent resistances, or expressions of surface proteins may be used. The vectors of the present invention may comprise antibiotics resistant genes which have been conventionally used in the art, for example, ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin, and tetracycline resistant genes as selectable markers. It is possible to screen the transformed cells because only cells expressing the selectable markers can survive in an environment of treating elective agents. Representative example of the selectable markers may comprise an auxotrophic marker, ura4, leu1, his3 and the likes, but the selectable markers can be used in the present invention is not limited to such an example.
Various in vitro amplification techniques that amplify sequences sub-cloned to expression vectors have been known. There are PCR (polymerase chain reaction), LCR (ligase chain reaction), QP-replicase amplification and techniques using other RNA polymerases in such techniques (Sambrook et al., 1989, Molecular Cloning-A Laboratory Manual (2nd Ed.) 1-3; U.S. Pat. No. 4,683,202; PCR protocols: A guide to Methods and Applications, Innis et al. eds. Academic Press Inc., San Diego, Calif. 1990. Improved methods of cloning in vitro amplified nucleic acids are described in U.S. Pat. No. 5,426,039).
The vector of the present invention may be fused with other sequences in order to facilitate the purification of the peptide therefrom. Fused sequences comprises glutathione S-transferase (Pharmacia, USA), maltose binding protein (NEB, USA), FLAG (IBI, USA), 6×His (hexahistidine, Quiagen, USA) and the likes, and 6×His is most preferable. Owing to the additional sequences for purification, the proteins expressed in the host cell are purified with promptness and ease through affinity chromatography assay. If necessary, a sequence encoding Fc fragments may be fused with the vector in order to facilitate extracellular secretion of those peptides.
In accordance with an exemplary embodiment of the present invention, the peptides expressed by the vectors which comprises nucleotide sequences encoding IK factor or partial fragments of IK factor are purified by affinity chromatography. For example, it is possible to purify the desired peptides with promptness and ease by using glutathione as a substrate of glutathione-S-transferase when glutathione-S-transferase is fused with the vector, and by using Ni-NTA His-binding resin column (Novagen, USA) when the vector comprises 6×his.
It is possible to any host cells known in the art as long as the host cells make the vectors stably and continuously clone and express. For example, host cells may comprise E. coli JM109, E. coli BL21 (DE3), E. coli RR1, E. coli LE392, E. coli B, E. coli X 1776, E. coli W3110, Bacillus strains such as Bacillus subtilis, Bacillus thuringiensis, and enterobacteriaceae strains such as Salmonella typhimurium, Serratia marcescens and various Pseudomonas species.
Besides, yeast (Saccharomyces cerevisiae), insect cells (e.g., SF9 cell), human cells (e.g., CHO (Chinese hamster ovary) cell, W138, BHK, COS-7, 293, HepG2, 3T3, RIN and MDCK cell lines) may be used as host cells in case of transforming eukaryotes with the vectors of the present invention.
The vectors of the present invention may be used in modifying genetically the host cells in in vivo or ex vivo or in in vitro. With regard to methods of modifying genetically cells, various methods comprising cell transfection or transduction with viral vectors, calcium phosphate precipitation, methods of fusing recipient cells with bacterial protoplast containing DNA, method of treating recipient cells with liposome or microspheres containing DNA, DEAE dextran, receptor mediated endocytosis, electroporation, micro-injection, and gene bombardment are well known in the art.
Pharmaceutical Composition and Administration
In accordance with another embodiment of the present invention, the present invention relates to a pharmaceutical composition for treating arthritis, comprises a therapeutically effective amount of the above-described IK factor and fragments of IK factor; and, if necessary, a pharmaceutically acceptable carrier. In this case, IK factor and peptides, fragments of IK factor may be administered directly to a subject.
In accordance with further another embodiment of the present invention, the present invention relates to pharmaceutical composition for treating arthritis, comprises a gene delivery vehicle having a nucleic acid molecule encoding IK factor or fragments thereof; and, if necessary, a pharmaceutically acceptable carrier. Using the gene delivery vehicle is intended for so called a gene therapy.
Accordingly, the present invention relates to a pharmaceutical composition containing as an active ingredient a peptide, IK factor or fragments thereof, and/or a gene delivery vehicle comprising a nucleic acid molecule encoding IK factor or fragments thereof, or a method for treating arthritis comprising administering such an active ingredient to a subject. In this case, the active ingredient may be contained in an amount of 1.0 ng/mL˜10 μg/mL in the pharmaceutical composition.
In one embodiment, the present invention provides a pharmaceutical composition or a medicine containing the compounds of the present invention, a therapeutically inactive carrier, a diluent or an excipient as well as a method of using the compounds of the present invention for manufacturing the composition or medicine. In one example, the compounds may be formulated together with a physiologically acceptable carrier, i.e., a non-toxic carrier to a subject in a dose and concentration used in a shape of a galenical administration at an ambient temperature and appropriate pH, by admixing them with a desired purity. While pH of the formulation is depending upon specific uses and concentrations of the compounds, preferably in the range of about 3 to about 8, In one example, the compounds are formulated in acetate buffer solution at pH 5. In another embodiment, the compounds are sterilized. The compounds may be stored as solid or amorphous composition, freeze-dried formulation and solution.
The composition is formulated, medicated and administered in a manner that is consistent with an excellent medical practice. With regard to it, considering factors includes a specific disorder to treat, a specific patient to treat, a clinical condition of each patient, a cause of the disorder, a transferring site of an agonist, an administration route, an administration schedule, and other factors known to medical practitioners.
If necessary, it is possible to prepare sustained release dosage forms. An appropriated example of the sustained release dosage form comprises semi-permeable matrix of solid hydrophobic polymers containing the compounds, and the matrix is a shaped article, for example a film or a microcapsule. For example, the sustained release matrix comprises polyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate) or poly(vinylalcohol), polylactides, copolymers of L-glutamic acid and gamma-ethyl-L-glutamate, non-degradable ethylene-vinyl-acetate, degradable copolymers of lactic acid-glycolic acid, and poly-D-(−)-3-hydroxybutyric acid.
In one example, the pharmaceutically effective amount of the compounds, which is administered parenterally per dose, will be about 0.01-100 mg/kg, alternatively about 0.1-20 mg/kg daily based upon patients' weight, typical initial dose of the used compounds will be 0.3 to 15 mg/kg/day. In another embodiment, oral unit dosage forms, for example, table or capsule contains preferably about 5-100 mg of the compounds of the present invention.
The compounds used as an active ingredient of the composition may be administered by any appropriate means, for example, oral, local (including buccal and subglossal), rectal, vaginal, percutaneous, parenteral, subcutaneous, intraperitoneal, intrapulmonary, intradermal, intradural, epidural and intranasal means, and, if necessary, means for local treatment and intralesional administering. Parenteral injection comprises intramuscular, intravenous, intra-arterial, intraperitoneal, or subcutaneous administration.
The active ingredient compounds of the present invention may be administered with any convenient dosage forms, for example, tablet, powder, capsule, solution, dispersion, suspension, syrup, aerosol, suppository, gel, emulsion, patch and the likes. Such a composition may comprise typical components to a pharmaceutical formulation, for example, diluents, carriers, pH adjusters, sweetening agents, bulking agents, and additional activators.
Typical formulations are manufactured by mixing the compounds of the present invention with a carrier or an excipient. Appropriate carriers and excipients are well-known to a person having ordinary skill in the art and described in detail in the documents [Ansel, Howard C., et al., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems. Philadelphia: Lippincott, Williams & Wiklins, 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 contained in the pharmaceutical composition of the present invention comprises, but are not limited to, conventionally used carriers in formulation such as lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum Arabia, calcium phosphate, alginate, water, syrup, methyl cellulose, methyl hydroxy benzoate, propyl hydroxy benzoate, talc, magnesium stearate, and mineral oil.
The formulation may comprise one or more buffering agents, stabilizers, surfactants, wetting agents, lubricants, emulsifiers, suspending agents, conservatives, anti-oxidants, opacifying agents, processing aids, coloring agents, sweetening agents, odoriferous substances, diluents, and other known additives which provides medication (i.e. the compounds of the present invention or its pharmaceutical composition) with a nice appearance or gives help to manufacture of the pharmaceutical product (i.e. medication).
The pharmaceutical composition of the present invention may be manufactured in the shape of unit dosage form by formulating the compounds using the pharmaceutically acceptable carrier and/or excipients or incorporating the compounds into multiple dosages vessel according to techniques which can be accomplished easily by a person having ordinary skill in the art. The formulation of the composition may be a solution, a suspension or an emulsion in oil or aqueous media, or an extract, a powder, or a granule, a tablet or a capsule, and may further comprise dispersions or stabilizers.
The produced composition is dried, granulated, mixed with magnesium stearate, and compressed as a tablet form using conventional apparatuses. For example, aerosol formulation may be prepared by dissolving 5-400 mg of the compounds of the present invention in appropriate buffer solutions such as phosphates buffers, and if necessary, adding isotonic agents, for example salts such as sodium chloride to the solution, It is possible to remove impurities and contaminants by filtering the solution, for example, using 0.2 micrometer micro-filter.
Accordingly, one embodiment of the present invention comprises a pharmaceutical composition comprising the compounds or stereoisomers thereof or pharmaceutically acceptable salts thereof. Additional embodiment of the present invention comprises a pharmaceutical composition comprising the compounds or stereoisomers thereof or pharmaceutically acceptable salts thereof, and a pharmaceutically acceptable carrier or recipient.
The above-described gene medicine comprises a gene delivery vehicle including a nucleic acid molecule of interest. The gene delivery vehicle is manufacture in order to transport and express the nucleic acid molecule of interest. The detailed description for the nucleic acid molecule as a transportation object will be omitted in order to avoid over-writings with the above-mentioned description.
In order to manufacture the gene delivery vehicle, it is preferable that the nucleic acid molecule of interest exists within appropriate expression constructs. Within the expression constructs, it is preferable that the nucleic acid molecule of interest is linked operatively to a promoter. As used herein, the term “operatively linked” means a functional relationship between nucleic acid expression control sequences (e.g., promoter, signal sequence, or array at transcription control element binding sties) and other nucleic acid sequences, the control sequence regulates the transcription and/or translation of the other nucleic acid sequences through operatively links. In the present invention, the promoters, which are linked to the nucleic acid molecule of interest, are operated preferably in animal cells, more preferably mammalian cells in order to control transcription of the nucleic acid molecule of interest. The promoters comprises, but are not limited to, promoters derived from mammalian viruses or from the genomes of the mammalian cells, for example, CMV (cytomegalo virus) promoter, adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, HSV tK promoter, RSV promoter, EFL alpha promoter, metallothionein promoter, beta-actin promoter, promoter for human IL-2 gene, promoter for human IFN genes, promoter for human IL-4 gene, promoter for human lymphotoxin gene, promoter for human GM-CSF gene, cancer cell specific promoters (e.g., TERT promoter, PSA promoter, PSMA promoter, CEA promoter, E2F promoter and AFP promoter), and tissue specific promoters (e.g., albumin promoter). Preferably, the expression construct used in the present invention comprises poly-adenylation signal sequence (e.g. bovine growth hormone terminator and SV40 derived poly-adenylation sequence).
Gene delivery vehicles may be manufactured in various shapes, for example, 1) naked (recombinant) DNA molecules, 2) plasmids, 3) viral vectors and 4) liposome or niosome nesting the naked (recombinant) DNA molecules or the plasmids. When DNA is in a naked state, DNA is coated with biodegradable beads in order to transfer DNA further efficiently into cells (Ulmer et al., 1993, Science 259: 1745-1749; Cohen, 1993, Science 259: 1691-1692).
The nucleic acid molecule of interest may be applied to any of gene delivery systems used in conventionally gene therapy, preferably applied to plasmid, adenovirus (Lockett L J. et al., Clin. Cancer Res. 3: 2075-2080, 1997), adeno-associated virus (AAV; Lashford L S. et al., Gene Therapy Technologies, Applications and Regulations Ed, A. Merger, 1999), retrovirus (Gunzburg W H, et al., Retroviral vectors. Gene Therapy Technologies, Applications and Regulations Ed. A. Merger, 1999), Lentivirus (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: 649-647, 1999), baculovirus (King L. A. And Possee R D., The Baculovirus Expression System, Springer, 1992), liposome (Methods in Molecular Biology, Vol. 199, S. C. Basu and M. Basu (Eds.), Human Press, 2002) or niosome.
Transducing the above-described gene delivery vehicle into cells may be performed through various processes known in the art. In case the gene delivery vehicle is prepared based upon viral vectors in the present invention, it is possible to adopt processes using viral infections known in the art so as to transduce the gene delivery vehicle. The infection of the host cells using viral vectors are described in the above-described documents.
In case the gene delivery vehicle is naked recombinant DNA or plasmids in the present invention, it is possible to transfer genes by a micro injection (Capecchi, M. R., Cell. 22: 479, 1980; and Harland & Weintraub, J. Cell Biol. 101: 1094-1099; 1985); calcium phosphate precipitation (Graham, F L. et al., Virology, 52: 456, 1973; Chen & 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, T. K. et al., Gene, 10: 87, 1980; Nicolau & Sene, Biochim. Biophys. Acta, 721: 185-190, 1982; and Nicolau et al., Methods Enzymol., 149: 157-176, 1987); DHEA-dextran treating method (Gopal, Mol. Cell Biol., 5: 1188-1190, 1985); and gene bombardment (Yang et al., Proc. Natl. Acad. Sci., 87: 9568-9572, 1990).
Hereinafter, while the present invention will be described in more detail through various examples, the present invention is not limited to inventions described in the following examples.
1) Screening Active Sites of IK Factor
We screened active sites of IK factor using computer program. We screened the active sites by inputting and setting an amino acid sequence of a truncated fragment IK factor (tIK) of SEQ ID NO: 3 at in silico program ‘scanlite’ which is provided at a website http://scansite.mit.edu/motifscan_seq.phtml. All motif analysis data are based upon SWISS-PORT database. The screening result for the active sites of IK factor is shown in Table 1 below. In table 1, ‘score’ is a numerical value of actual interaction of the sites of IK factor with kinase, and ‘percentile’ is a numerical value showing what percentage the kinase motif associated with tIK among kinase motifs in SWISS-PROT database in top high rank is. Both S382 site and Y482 site are motif candidates presented in the all screening conditions, high/medium/low stringency conditions, and Y489 site is a motif candidate present in the screening conditions, medium/low/stringency conditions.
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 Excised Some Nucleotides
As shown schematically in
A mutant gene fragment (S382A), which has substituted nucleotides ‘GCC’ encoding alanine for the nucleotide residues encoding the 382nd amino acid residue Serine of SEQ ID NO: 2 (the 1144-1146th nucleotide residues of SEQ ID NO: 1), was prepared. Also, both a mutant gene fragment (Y489F), which has substituted nucleotides ‘TTC’ encoding phenylalanine for the nucleotide residues encoding the 489th amino acid residue Tyrosine of SEQ ID NO: 2 (the 1465-1467th nucleotide residues of SEQ ID NO: 1), and a mutant gene fragment (Y492F), which has substituted nucleotides ‘TTC’ encoding phenylalanine for the nucleotide residues encoding the 492nd amino acid residue Tyrosine of SEQ ID NO: 2 (the 1474-1476th nucleotide residues of SEQ ID NO: 1), were prepared.
Simultaneously, a dual mutant gene fragment (Y489492F), which has substituted nucleotides encoding phenylalanine for the nucleotide residues encoding 489th and the 492nd amino acid residues Tyrosine of SEQ ID NO: 2, and a triple mutant gene fragment (S382AY489492F), which has respectively substituted nucleotides encoding alanine for the nucleotide residues encoding the 382nd amino acid Serine of SEQ ID NO: 2 and nucleotides encoding phenylalanine for the nucleotide residues encoding the 489th and the 492nd amino acid residues Tyrosine of SEQ ID NO: 2, were also prepared. A HA-tag sequence (haemagglutinin sequence; SEQ 1D NO: 54) was added at 5′-end of each gene fragment for detecting, isolating and purifying the expressed proteins. Nucleotide sequences for the tIK nucleic acid fragment and the mutant nucleic acid fragments are shown in Table 2 below.
The tIK fragment and the mutant nucleic acid fragments were prepared by inserting inserts, which were amplified using fusion PCR method, into pcDNA 3.1 vectors. With regard to a S382 mutant, both a forward primer complementary to HA tag to a 5′-region of a tIK nucleic acid and a reverse primer having a nucleotide sequence encoding alanine in order to substitute the 382th Serine for alanine were prepared and used in PCR. The first gene fragment consisting of the HA tag sequence to a nucleotide encoding the 382nd alanine (Ala) was amplified using such primer sets and tIK gene as a template. And then, a forward primer including a nucleotide encoding alanine in order to replace the 382nd serine with alanine, and a reverse primer complementary to the 3′-end region of tIK gene were prepared so that the second gene fragment consisting of the nucleotides encoding the 382nd alanine to 3′-end of till(nucleic acids. 1 μL of each of two gene fragments obtained by PCR was added to 500 μL micro tube, 1 μL of each of another forward primer complementary nucleic acids from HA tag sequence to 5′-end region of tIK gene and another reverse primer complementary to 3′-end of tIK gene was added to the micro tube, and then polymerase buffer, dNTPs, polymerase, and distilled water was added to perform fusion PCR. The ends of the amplified fragments obtained by fusion PCR was cut by restriction enzymes and the amplified fragments were inserted into pcDNA 3.1 for the transformation, and plasmids including the inserts with the substituted amino acids were screened, and finally certified whether the plasmids has the substituted amino acid through sequencing. Other mutants were prepared using the same processes as described above. Table 3 shows the primer sets which were used for the amplification of the tIK nucleic acid fragment and the mutant nucleic acid fragments according to the present example.
aincluding EcoRI recognition site;
bincluding XhoI recognition site;
cincluding HindIII recognition site.
We certified the tIK and mutant nucleic acids by analyzing mRNA expression levels of upstream regulatory elements each of which control an expression of class II coactivator, CIITA that facilitates the expression of MHC class II. We selected CNOT1 (CCR4-NOT transcription complex subunit 1), CDCA3 (Cell division cycle-associated protein 3) and MAPK1 (Mitogen-activated protein kinase 1) as an upstream regulatory factor regulating the expression of CIITA. It was certified that the expression level of the factors (CNOT1, CDCA3, and MAPK1) which suppress the expression of CIITAA was increased in case of transfecting plasmids having inserted tIK gene into human Raji B cells without intrinsic UK factor, compared to a negative control group transfecting a control vector into the cells. It was considered that the tIK nucleic acid fragment increases the expression of factors suppressing CIITA, and thereby, having an effect upon the inhibition of the MHC class II expression. And then, we certified if there exist the same phenomena in the tIK mutant nucleic acid fragments, which has substituted amino acids for the amino acid of kinase motifs, based upon such analyses.
Concretely, plasmids each of which have an inserted tIK mutant fragment, S382A, Y489F, Y489492F, and S382AY489492F, respectively, were transfected into Raji B cells using electroporation, and then RAN was isolated in order to prepare cDNA. A plasmid inserting a tIK nucleic acid fragment was also used, and cDNA obtained by transfecting pcDNA 3.1 plasmid without inserted any of the nucleic acid fragments into the cells were used as a negative control. Quantitative real-time PCR was performed using the prepared cDNA, and the measured values (Ct values) were calculated as relative values based upon the result in the negative control group transfected with pcDNA 3.1 plasmid, using 2(−ΔΔCt) method. We used cDNA of the factors (CDCA3, CNOT1, and MAPK1) regulating the expression of CIITA as templates, and the primers sets used in certifying the expression of such factors are shown in Table 4.
A novel animal model (tIK-IL1RaKO mice), which induces autoimmune arthritis, was manufactured by cross-breeding an animal model, IL-1 receptor antagonist knock-out mouse (IL1RaKO mice, derived from Balb/c), which exhibits high immunological sensitivity and occurs autonomous arthritis in order to increase sensitivity to arthritis-inducing molecules, and tIK-transgenic mice (tIK., derived from Balb/c), which can express a nucleic acid molecule which has a nucleotide sequence of SEQ ID NO: 3 and encodes the truncated IK factor (tIK fragment). In order to manufacture transgenic mice expressing truncated IK factor (tIK), tIK-pcDNA 3.1 clones into which a nucleic acid molecule of SEQ ID NO: 3 is inserted were micro-injected into fertilized ova extracted from Balb/c female mouse. The ova were transplanted into the uterus of Balb/c female mouse at false pregnancy state so as to implant the ova, and offsprings were obtained 3 weeks later. In order to obtain the mouse gene through cross-breeding, the offsprings' tail were cut by 0.3 mm, tail lysis buffer (pH 8.0, Tris buffer 50 mM, EDTA 50 mM, 0.5% SDS in distilled water, 20 mg/mL proteinase K) was added and incubated in 55° C. constant-temperature water bath for 18 hours so as to obtain tail lysate.
The tail lysate was transferred to vacutainer, phenol/chloroform/isoamylalcohol (25:24:1) was added into the vacutainer, the vacutainer was inverted 50 times, and then the vacutainer was centrifuged (2,000 rpm, 5 minutes, room temperature) so as to remove the rest debris except genomic DNA. Chloroform was added to the centrifuged vacutainer, and then the vacutainer was inverted again, and then centrifuged (2,000 rpm, 5 minutes, room temperature) to separate only a genomic DNA layer. Transparent supernatant obtained by the isolation was transferred to another micro tube, 100% ethanol was added to the micro tube, and the micro tube was inverted to obtain genomic DNA of white skein shapes. The obtained genomic DNA was picked up using a pipette tip, washed in 75% ethanol, air-dried for 10 minutes, and then dissolved in distilled water. The first amplicon was amplified by PCR using the genomic DNA, extracted from the transgenic mice into which tIK nucleic acid fragments are inserted, as a template and primer sets for tIK genotyping (Forward Primer: SEQ ID NO: 33; Reverse Primer: SEQ ID NO: 34). The second amplicon was amplified by PCR using primer sets for IL1RaKO genotyping (Forward Primer: SEQ ID NO: 35; Reverse Primers: SEQ ID NOs: 36/37). Two amplicons were obtained through electrophoresis assay in 1% agarose gel to certify the amplification levels. In case both amplicons were detected in the electrophoresis results, it was certified that the genomic DNA was extracted from tIK-IL1RaKO double positive transgenic mice obtained through the cross-breeding.
We monitored the inducing levels of arthritis in tIK-IL1RaKO mice, new autoimmune arthritis animal model obtained by cross-breeding in Example 2, and IL1RaKO mice. In order to compare the arthritis-inducing levels in a natural state, 10 4-week-old tIK-IL1RaKO mice and 10 4-week-old IL1RaKO mice were fed in cages in the same conditions. During the feeding time, we scored the arthritis levels by observing arthredema levels in ankle sites of the mice in every week. Detailed scoring method is as follows: Arthritis assessment tests were recorded based upon mean arthritis index by Rosoliniec et al. And monitoring was proceeded continuously three times in a week. We scored paws for arthritis levels on a scale of 0 to 4 and summed the paw scores to yield individual mouse scores. The joint arthritis scores were evaluated blindly as follows: grade 0, no swelling; grade 1, slight swelling and erythema; grade 2, pronounced swelling; grade 3, joint rigidity; grade 4, maximal swelling. Each limb was graded as a score of 0-4, with a maximum possible score of 16 for each mouse. Also, arthritis-occurring levels were monitored so as to show incidence rate by the feeding period.
In order to certify the pathogenic states of the arthritis induced mice, an autopsy for the mice of each group (tIK-1RaKO group; and IL1RaKO group) was done after 16-week monitoring for the spontaneously induced arthritis groups so as to obtain joint tissues. The joint tissue was fixed in 10% neutral formalin, dipped in decalcification solution for 7 hours. The decalcificated joint was washed in water for 16 hours, dehydrated, de-alcoholized, and infiltrated to prepare a paraffin block. The paraffin block was cut using a section apparatus so as to obtain 7 mm section, and the section was attached to a slide for staining using hematoxylin & eosin (H&E staining). Staining process is as follows: deparaffinization was performed from the paraffin section, an enhydrous procedure was performed, and then nucleus of the cells was stained with hematoxylin. After washing it, cytoplasm was stained with eosin. Such stained cells were again dehydrated, de-alcoholized, and included, so as to certify the structural state of tarsal bone around the ankle and infiltrations by various immune cells. The results are shown in
Also, we certified the cartilage erosions extracted from the mice group using safranin O staining. Safranin O staining was performed subsequently by deparaffinization of the tissue section, enhydrous process, hematoxylin staining, safranin O staining, fast green staining, dehydration, de-alcoholization, inclusion. The results are shown in
In order to analyze comparatively the bone damages owing to inflammation in the spontaneously induced arthritis mice between the IL1RaKO mice group and the tIK-IL1RaKO mice group, an autopsy for the mice of each group was done after 16-week monitoring for the spontaneously induced arthritis groups so as to obtain joint tissues. The tissue was fixed in 10% neutral formalin for 18 hours for the micro CT imaging, which was performed using ex vivo micro CT scanner (Skyscan 1172). The sectional images of the tissues were photographed using X-ray source of 50 kV voltages and 200 μA currents and 15 μm resolution, and 0.5 mm filter. We observed that there are much bone damages and structural modifications in the tissue extracted from IL1RaKO mice, while the tissue extracted from the tIK-IL1RaKO mice maintains an original structure having little bone damages (
(1) Certification of mRNA Expression
In order to certify expression levels of inflammatory cytokines at the joint in spontaneously induced arthritis mice, an autopsy for the mice of each group was done after 16-week monitoring for the spontaneously induced arthritis groups so as to obtain joint tissues. Joints were grinded in LN2 and added in 400 μl of Trizol reagent to proceed with RNA isolation. Chloroform was added to the tissue and performed vortex strongly, and then the solution was incubated at room temperature for 5 minutes. After centrifuging (12,500 rpm, 15 min., 4° C.) the solution, supernatant was transferred to another micro tube. After adding isopropanol and glycoblue to the supernatant, the supernatant was incubated for 5 minutes. After another centrifugation (12,500 rpm, 10 min., 4° C.), the supernatant was filtered out. Pellet out of the supernatant was washed in 70% ethanol, was centrifugated (9,500 rpm, 5 min., 4° C.), and then air dried and dissolved in DEPC-water. Separated DNA was prepared as cDNA using QuantiTech Reverse Transcription kit (Quiagen). Quantitative real-time PCR was performed using the prepared cDNA (cDNA 1 μL, each of primer set 1 μL, 2XSYBR green mix 12.5 μL, Distilled water 9.5 μL, 40 cycles).
The measured result value (Ct value) per group was calculated as a relative value based upon the result in the wild type mouse group using 2(−ΔΔCt) method. We measured the expressions of the inflammatory cytokines (IL-1β, IL-16, and IL-17A) each of which is known to increase in the case of arthritis development, using the cDNA as a template. Primers for used in amplifying the inflammatory cytokines (IL1β, IL-16, and IL-17A) using the cDNA as a template are shown in Table 2, and measuring results are shown in
We observed that the inflammatory cytokines (IL-1β, IL-6) that are involved in inducing joint inflammation were much expressed in the joint of the IL1RaKO mice than the joint of the tIK-IL1RaKO mice. Also, IL-17, which is a major factor suppressing substrate expressions within cartilage cells and osteoblasts, inducing joint damages, and making tissue regeneration deficient, were expressed remarkably lower in the joint of the tIK-IL1RaKO mice. We certified that the nucleic acid fragments derived from IK gene may regulate expressions of inflammatory cytokines expressed in inducing arthritis, and therefore, may lower arthritis values.
(2) Certification of Cytokine Expressions Using Tissue Staining
In order to certify pathogenic states of the joints in spontaneously induced arthritis mice, an autopsy for the mice of each group (tIK-IL1RaKO mice group; and IL1RaKO mice group) was done after 16-week monitoring for the spontaneously induced arthritis groups so as to obtain joint tissues. The joint was fixed in 10% neutral formalin, dipped in decalcification solution for 7 hours. The decalcificated joint was washed in water for 16 hours, dehydrated, de-alcoholized, and infiltrated to prepare a paraffin block. The paraffin block was cut using a section apparatus so as to obtain 7 mm section, and the section was attached to a slide for performing staining inflammatory cytokines (IL-17, IL-1β, and TNF-α). Deparaffinization was performed from the paraffin section, and enhydrous procedure was performed. After attaching a primary antibody for IL-1, IL-1β, and TNF-α for 16 hours, secondary antibody with labeled biotin was attached. After incubating in ABC reagent and developing colors was performed using peroxidase substrate solution, and washed in water. And then, we certified expressions of the inflammatory cytokines around the joint synovial sites and Observed with a microscope. The result are shown in
In order to certify expression levels of inflammatory cytokines within a blood in the spontaneously induced arthritis mice, an autopsy for the mice of each group was done after 16-week monitoring for the spontaneously induced arthritis groups so as to obtain a blood through heart blood collection. The obtained blood was centrifugated (4,000 rpm, 15 min., 4° C.) so as to separate a serum, and we certified expression levels of inflammatory cytokines in serum through ELISA assay using it.
Describing MCP-1 ELISA kit (R&D system) as an example, standard, which was provide with the kit, was added in serial to a plate where anti-MCP-1 primary antibody was pre-coated, and then, serum stock solution was added to a well and incubated for 2 hours. After completing the incubation, all samples added into the well was removed, and the serum was washed with 0.5% PI3ST (Phosphate Buffered Saline Tween-20) four times. Conjugate solution, which can bind to MCP-1 bonded to the anti-MCP-1 antibody in well, added to the well and incubated at room temperature for 2 hours. After incubation, substrate solution was added and monitored if the substrate solution was reacted with the conjugate to develop colors, when the coloring levels of serum samples for measuring were within the standard color developing ranges, stop solution (2N H2SO4) was added, and then, we measured the obserbance at 459 nm wave-length using ELISA reader. It was possible to MCP-1 concentrations indicated by the obserbance of the sample based upon the absorbance of the standard concentration. Using the same process, we measured the concentrations of other cytokines except of MCP-1. In order to amplify a MCP-1 cytokine gene, a forward primer of SEQ ID NO: 44 and a reverse primer of SEQ ID NO: 45 were used, and other cytokine genes were amplified using the same primer sets described in example 6.
Assay results are shown in
It has been well-known that T cells of immune cells are major cells inducing disturbances of immune system. Activated T cells are differentiated into effector T cells such as Th1, Th2, Th17, and regulatory T cells. The developments and the progresses of disorders depend upon which differentiation paths are facilitated. Each of Th1 cell and Th17 cell secretes, respectively, IFN-γ and IL-17, and has been known to form main pathologies in inflammatory disorders such as diabetics, rheumatoid arthritis, and Crohn's disease.
In this case, we certified whether or not expressions of the pathogenic T cells are involved when arthritis is suppressed by the nucleic acid fragments derived from IK factor. An autopsy for the mice of each group was done after 16-week monitoring for the spontaneously induced arthritis groups so as to obtain a spleen. 100 μm of strainer was added to 100 mm Petri dish containing serum free RPMI media, and the spleen was placed onto the strainer. The spleen was tore into pieces using a syringe needle so that immune cells of the spleen passes into the dish containing media through the strainer. After grinding all spleen, immune cells spreading on the media was collected into a tube, and centrifugated (1,500 rpm, 5 min, 4° C.) so as to obtain only pellet. Red blood cell lysis buffer was added to the pellet and incubated at 4° C. for 5 minutes. Again, the solution was centrifugated (1,500 rpm, 5 min, 4° C.) so as to remove red blood cells, and finally to obtain splenocyte. After suspending the splenocyte in RMPI media containing FBS, 1×106 cells were chosen, and then, staining process were proceeded in order to analyze an amount of pathogenic I cells by flow cytometry, PMA (100 ng/mL) and ionomycin (200 ng/mL) was added to splenocyte obtained by per group, and was incubated for 4 hours, and then, golgi stop agent for staining intracellular IL-7 were added. The stimulated cell by PMA and ionomycin was washed in cold PBS, an antibody (PE-Cy5 conjugated) to a cell surface molecule of Th17 cells, CD4, was added to react at 4° C. for 30 minutes, and then the mixture washed in cold PBS. After increasing cellular permeability for transferring antibody into the cells and staining intracellular IL-17, another antibody (PE conjugated) to IL-17 was added to react at room temperature for 30 minutes. After completing all reactions, the mixture was washed using fixative solution, and finally the stained cells were fixed in 1% paraformaldehyde so as to use the cell in flow cytometry assay.
Th1 cells secreting IFN-γ were detected in 6.09% in the IL1RaKO mice but they were detected relatively lower amount of 3.24% in the tIK-IL1RaKO mice (See,
Such assay results accord with the expression analysis results for the inflammatory cytokines, which are known to be involved in differentiation of Th17 cells, certified in joints and sera extracted from the mice in examples 6 and 7. Accordingly, we certified that the nucleic acid fragments derived from IK factor can suppress the expressions of Th17 cells and Th1 cells, which are mainly concerned with inflammatory responses in case of developing arthritis, so that the fragments can inhibit the inflammatory responses owning to arthritis.
Macrophages activated by rheumatoid arthritis secrete various inflammatory cytokines and interacts with various immune cells so as to initiate and maintain immune responses in joints. Macrophage induces an activation of T cell by presenting antigen with the T cell as well as an activation of osteoclasts by secreting various cytokines. Because macrophage plays an important role in maintaining inflammatory responses caused by arthritis, we certified if macrophage is concerned with arthritis suppression by IK factor in this example.
An autopsy for the mice of each group (IL1RaKO mice and tIK-IL1RaKO mice) was done after 16-week monitoring so as to obtain a spleen as in example 8. The obtained splenocyte were stained using macrophage factor CD11b antibody (APC conjugated), pathogenic macrophage factor F4/80 antibody (PE-cy7 conjugated) and macrophage activating factor CD86 antibody (FITC conjugated). The stained cells were fixed in 1% paraformaldehyde solution so as to use the cell in flow cytometry assay. As shown in
We prepared short-length active peptides, which can be expressed and purified with ease compared to tIK factor, as partial fragments of IK factor so as to certify whether the peptides exhibit physical functions in case of applying them to the arthritis animal models. We synthesized the peptides including two amino acids (S382 and Y489 of SEQ ID: NO 2) which were expected to play an important role in function of IK factor in example 1. A peptide (SEQ ID NO: 47, S peptide) consisting of 14 amino acids comprising S382 and another peptide (SEQ ID NO: 48, Y peptide) consisting of 13 amino acids comprising Y489 were synthesized. In this case, each of S382 and Y489 were phosphorylated. Each active peptide was synthesized using Fmoc solid phase synthetic method. During the synthetic process, we removed residual agents were removed by repeating coupling-washing-deprotection-washing steps and used COMDEL device as a multi-channel automated synthesizer. Each active peptide was synthesized in the direction from C-terminal to N-terminal by the synthetic process. After completing all reactions, the synthetic peptides were separated from resins using reagent K. Separated peptides were precipitated by adding cold diethyl ether, washed again using diethyl ether, and then dried in vacuum state. We measured molecular weights of the synthetic peptides using mass spectrometer, and performed purification in case only the measured molecular weights are identical as expected molecular weights. The purification was performed as follows: Reverse-HPLC; C18 column, 220 nm wavelength.
We certified the macrophage activation levels by placing the S peptide and Y peptide in conditions stimulated with lipopolysaccharide (LPS). After co-transfecting simultaneously 500 ng/mL of each of S peptide and Y peptide into J774a.1 macrophage cell-lines, the cell-lines were treated with LPS (O111:B4) for 24 hours in order to certify the expression of inflammatory cytokines. It was shown that mRNA expressions of the inflammatory cytokines IL-6) in macrophage, which was treated LPS for 24 hours, was reduced in the active peptides treating group (See
In order to certify whether or not the IK factor active peptides have an influence on the differentiation of pathogenic T cells, splenocytes were extracted from wild type mouse as in Example 8. Only naive CD4 T cells were selectively separated from the splenocytes, and then were pre-treated with S peptide and Y peptide (100 ng/mL, or 500 ng/mL) for 1 hour. Culture media comprising various cytokines, which can be differentiated into pathogenic T cells, and neutral antibodies were added, and the same concentration of S peptide and Y peptide (100 ng/mL or 500 ng/mL) was added again when condition culture media was added. Culture media containing anti-CD3, anti-CD28 and IL-2 were added to the splenocytes for inducing differentiating to Th0 cells. Culture media containing anti-CD3, anti-CD28, IL-2, IL-12, and anti-IL14 were added to the splenocytes for inducing differentiating to Th1 cells. Also, culture media containing anti-CD3, anti-CD28, IL-6, TGF-β, anti-IL-4, and anti-IFN-γ were added to the splenocytes for inducing differentiating to Th17 cells. After culturing 2 days, culture media containing various cytokines and neutral antibodies were added to the cells at the same conditions for differentiating to pathogenic T helper cells. After 2 days, the pathogenic T helper cells were stained for flow cytometer assay.
First, phrobol 12-myristate-13-acetate (PMA, 50 ng/mL) and ionomycin (200 ng/mL) was added to the cultured cells. After incubating at 37° C. for 4 hours, golgi stop (monensin) for staining intracellular IL-17 was added. After incubation, the cells were washed cold PBS. After increasing cellular permeability for transferring antibody into the cells for staining intracellular cytokines (IL-17 and INF-γ), antibodies to IL-17 and IFN-γ was added and reacted for 30 minutes on ice. After completing the reaction, the cells were washed in fixative solution, and finally the stained cells were fixed in 1% paraformaldehyde solution for using flow cytometer assay.
As shown in
We injected active peptides into the spontaneously induced arthritis model (IL1RaKO mice) in order to assess if the active peptides of IK factor, S peptide and Y peptides, suppress arthritis. 7 IL1RaKO mice were used in each group. As active peptides, total 10 mg/kg of peptides (S peptide: 5 mg/kg; Y peptide: 5 mg/kg) were injected by intraperitoneal injection. As a negative control, same volume of phosphate buffed saline (PBS) was injected by intraperitoneal injection. Peptides were injected by every 2-3 day, and we monitored mice' weights, arthritis development levels and indices in injecting the peptides for 7 weeks. We found that the arthritis index in the peptide-treated mice group was lower than that in the negative control group which was injected by PBS (See,
(1) Flow Cytometer Assay
We measured the distribution of Th17 cells, which is a major factor in initiating and maintaining inflammatory responses by arthritis, by the injection of active peptides of IK factor. After 8 weeks of injecting active peptides according to example 12, an autopsy was done in order to obtain a spleen of each group. After extracting splenocytes by grinding the obtained spleen, PMA (50 ng/mL) and ionomycin (200 ng/mL) was added to the splenocytes. When incubating the splenocytes at 37° C. for 4 hours, golgi stop (monensin) for staining intracellular IL-17 was added. After incubating, the cells were washed in cold PBS, antibody (APC conjugated to surface molecule of pathogenic Th17 cells, CD4, was wadded, and then was reacted 4° C. for 30 minutes. After completing the reaction, the cells were washed. After increasing cellular permeability for transferring antibody into the cells and staining intracellular IL-17, another antibody (PE conjugated) to IL-17 was added to react at room temperature for 30 minutes. After completing all reactions, the mixture was washed in fixative solution, and finally the stained cells were fixed in 1% paraformaldehyde for flow cytometry assay.
Comparing assay results for the pathogenic Th17 cells in the splenocytes extracted from the arthritis-induced mice between the peptide-injected group and the PBS-injected group, there were less Th17 cells in the splenocytes extracted from the IL1RaKO mice injected by active peptides (See,
(2) Certification of Cytokine Expression Using Tissue Staining
We certified the expressions of inflammatory cytokines in joint synovial sites after injecting IK active peptides. After 8 weeks of injecting active peptides according to example 12, an autopsy was done in order to obtain a spleen of each group. Joint were fixed in 10% neutral formalin, and was dipped in decalcification solution for 7 hours. The decalcificated joint was washed in water for 16 hours, dehydrated, de-alcoholized, and infiltrated to prepare a paraffin block. The paraffin block was cut using a section apparatus so as to obtain 7 mm section, and the section was attached to a slide, and then staining for inflammatory cytokines (IL-17, IL1-β and TNF-α) was performed. After de-paraffinizing the paraffin section, an enhydrous procedure was performed. Primary antibodies to IL-17, IL1-β and TNF-α were attached to the section for 16 hours, and then secondary antibodies labeled biotin was attached. After incubating in ABC reagent, color developing process was performed using peroxidase substrate solution. And then, the mixture was washed in water. After certifying the expression of the inflammatory cytokines around the joint synovial sites, we observed the synovial sites with a microscope. The results are shown in
In order to express IK fragment (tIK) in a large quantity, tIK plasmid which inserts tagged FC sequence was transfected into baculovirus, and then tIK was expressed from insect cell (SF9 cell) infected with a recombinant baculovirus. A nucleic acid sequence (SEQ ID NO: 50) encoding thrombin recognition site and Fc tagging sequence (SEQ ID NO: 51) were linked to 3′-end of the tIK nucleic acid. After preparing viral seeds, baculovirus expressing tIK-Fc was infected into the insect cells of which amount are subsequently increased in the 1st infection (100 mm dish scale), the 2nd infection (T75 flask scale), and the 3rd infection (1 L conical flask). Finally, after infecting the insect cells in the 3rd infection for three days, 1 L of cell culture secreting tIK-Fc was obtained. The obtained UK-Fc protein in the cell culture was purified using immuno-precipitation method, when rProtein A resin was added to Sepharose column and then cell culture was injected into the column, expressed Fc in tIK-Fc protein contained cell culture binds to the resin so that tIK-Fc protein were remained in the column. We certified the expression ( 55 kDa) of the tIK-Fc protein using Commassie blue staining method (result not shown). In order to obtain pure tIK-Fc protein, we intended to separate the tIK-Fc region conjugates from the resin by severing non-covalent bonds between the Fe: region and rProtein. In order to sever the non-covalent bonds, the protein was eluted using Elute Buffer (0.1M Glycine buffer, pH 2.8), and then neutralized by Neutralization buffer (1M Tris-HCl, pH 8.0) for preventing structural modifications of the protein. Column washing process was performed by using washing buffer (0.15M NaCl-20 mM Na2HPO4, pH 8.0). All separated solutions with 7 fractions were obtained by Affinity Chromatography assay. And then, we performed SDA-PAGE assay for the each solution of 7 fractions and certified the elution of the protein. We found that the protein analyzed in SDS-PAGE has 55 kDa as tIK-Fc conjugated from (See,
Unlikely to retrovirus, since adeno-associated virus (AAV) does not cause disorders and integrates selectively into the 19th chromosome in human cells, it has been used as a gene therapy vector for chronic disease. Using those characteristics of AAV, we constructed recombinant adeno-associated virus (rAAV)-tIK vector system by gene cloning step in which the tIK nucleic acid fragment of SEQ ID NO: 3 was enveloped by inverted terminal repeat (ITR) of AAV so as to transfer the tIK nucleic acid fragment into host cells. We used GFP-expression inducible rAAV viral vector, pSP72-XP7-scAAV2-CMV-GFP, as an expression vector. HA tag sequence (SEQ NO: 54) was linked to 5′-end of tIK nucleic acid fragment, and PCR was performed using primers (Forward Primer: SEQ ID NO: 55; and Reverse Primer: SEQ ID NO: 56) which are complementary to both ends of tIK nucleic acid fragment. The ends of the amplified fragment obtained by such processes were cut by restriction enzymes, and the amplified fragment was inserted into the vector so as to prepare tIK-HA-sp72-XP7-scAAV2 recombinant expression vector (AAV2-tIK). After transforming the prepared recombinant expression vector into E. coli, it was possible to screen a plasmid with having inserted tIK amplified fragment. The tIK gene conjugated to adeno-associated viral genome was transfected into 293T cells by a transfection process using polyethyleneimine (PEI). The virus, which was certified to express tIK fragment using PEI, was again infected into 293T cells. We certified that tIK was expressed in 293 T cells by Western-blotting assay (See,
We applied the RAAV-tIK prepared in example 15 to in vivo disorder model and certified arthritis treating effect by the expression of the UK nucleic acid. Each of rAAV2-tIK and rAAV2-GFP viruses (respectively, 1×1011 vg/100 μL concentration) was intravenously injected through the tail of spontaneously induced arthritis model IL1RaKO mice. The rAAV2-tIK and rAAV2-GFP viruses were injected every two week, and we monitored the joint states and arthritis incidences in every one week. It was shown that arthritis incidences and arthritis indices were relatively lower in the IL1RaKO mice transfected by rAAV2-tIK compared to the IL1RaKO mice transfected by rAAV2-GFP (See,
Since Chinese hamster ovary (CHO) cells shows high expression efficiencies in case of transfecting genes, they have been widely used as cell lines for preparing gene recombinant protein medicines. In order to certify the expression efficiency of tIK in CHO cells, tIK plasmids (tIK-pcDNA 3.1), which has a HA tag sequence (SEQ ID NO: 54) linked 5′-end of the tIK nucleic acid, were transfected into CHO cells for the amplification of the tIK gene, a forward primer of SEQ ID NO: 55 and a reverse primer of SEQ ID NO: 56 was used. We certified the expression of tIK in CHO cells by Western blotting assay (See,
| Number | Date | Country | Kind |
|---|---|---|---|
| 10-2013-0041771 | Apr 2013 | KR | national |
| 10-2014-0038809 | Apr 2014 | KR | national |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 14784614 | Oct 2015 | US |
| Child | 15481787 | US |
