WO2018079864A1 - TNF-α 결합 압타머 및 그것의 치료적 용도 - Google Patents
TNF-α 결합 압타머 및 그것의 치료적 용도 Download PDFInfo
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Definitions
- the present invention relates to aptamers that specifically bind to TNF- ⁇ and their therapeutic uses.
- TNF- ⁇ is an pro-inflammatory cytokine secreted by cells of the immune system, including macrophages, monocytes, and reacts with cells of the immune system, and its abnormal overexpression is inflammatory reactions, including sepsis, infectious diseases, and autoimmunity.
- Various diseases such as disease, transplant rejection (Annu. Rev. Immunol. 10: 411-452, 1992; Annu. Rev. Med. 45: 491-503, 1994).
- Etanercept TM Enbrel TM, Infliximab TM, Arimumab TM, Hutrizumab Pegol (Cimzia TM) and the like are representative antibody pharmaceuticals targeting TNF- ⁇ .
- etanercept inhibits the secretion of TNF- ⁇ in LPS treated THP-1 cells but may have side effects by overexpressing cell binding TNF- ⁇ by 6-fold (Int Immunopharmacol. 8 (5): 679-).
- infliximab is a chimeric antibody with murine variable regions and human IgG 1 constant domains, and has problems causing ADCC (antibody dependent cellularcy to toxicity) and CDC (complement-dependent cytotoxicity).
- Busab pegol is a PEGylated humanized antibody, and pegylated proteins accumulate in kidney cells and form vacuoles, as well as problems that cause in situ reactions (Bioconjug Chem. 19). 24 (6): 915-25, 2013).
- Aptamers like antibodies, are nucleic acid ligands that can specifically bind to target proteins, and Macugen, which targets vascular endothelial growth factor (VEGF) as a drug for macular degeneration in the US FDA in 2004, Since approval, therapeutic development using aptamers has been active worldwide (Gene Ther 14 (4): 283-291, 2007).
- VEGF vascular endothelial growth factor
- Aptamer therapeutics have several advantages over antibody therapeutics, which are smaller and simpler molecules than antibodies, enabling chemical synthesis and facilitating necessary chemical modifications, and SELEX technology (Systematic Evolution of Ligands by Exponential enrichment, Science 249 (4968)). : 505-510, 1990; Methods Enzymol 267: 275-301, 1996; Methods Enzymol 318: 193-214, 2000; US Patent No. 5,475,096, US Patent No. 5,270,163, WO 91/19813). It can increase selectivity and affinity in vitro, and unlike protein antibody, it is heat stable, so it can be stored for a long time at room temperature.
- aptamers can be made for toxins, which are difficult to make polyclonal antibodies, and unlike antibody therapeutics, hardly generate an immune response in vivo, while antibodies must be prepared at the in vivo or cellular level, while aptamers are produced at the in vitro level. It is possible to control the pharmacokinetic and pharmacodynamic properties through chemical formulas and so on.
- the present invention discloses aptamers that specifically bind to TNF- ⁇ and their therapeutic uses.
- Another object of the present invention is to provide a therapeutic use of the aptamer.
- RNA aptamer of SEQ ID NO: 1 (“ATK001") and RNA of SEQ ID NO: 2 (“ATK007") having high binding affinity for TNF- ⁇ from these RNAs through a plate-based binding assay
- SPR Surface Plasmon Resonance
- Gel retardation method while the binding affinity of ErbB2, ErbB3 and IGF-BP1 to TNF- ⁇ was confirmed by SPR analysis.
- TNF- ⁇ there is a binding specificity for TNF- ⁇ by comparing with the binding capacity for the TNF- ⁇ , the ATK001 and ATK002 aptamer to be treated together with the recombinant human TNF- ⁇ treatment to cause cell damage in SK-HEP1 cells, liver cancer cell line
- hepatoglobin, Fibrogen- ⁇ , Fibronectin, Transferrin, and other inflammatory cytokines (TNF- ⁇ , IL1- ⁇ and IL6) and angiogenic substances (Acute phase proteins) that rapidly increase in expression when cells are damaged VEGF) expression was inhibited.
- inflammatory cytokines when acute phase proteins such as Hepatoglobin or inflammatory cytokines are proteins that increase their expression in order to protect them when cells are damaged, when the aptamer is treated together with TNF- ⁇ treatment causing cell damage Inhibition of the expression of these acute phase proteins, inflammatory cytokines, can be said to show an effective inhibitory activity against TNF- ⁇ of ATK001 and ATK007 aptamer.
- the present invention relates to ATK001 aptamer of SEQ ID NO: 1 or ATK007 aptamer of SEQ ID NO: 2, which binds to TNF- ⁇ , and, in another aspect, these pressures.
- the present invention relates to a pharmaceutical composition for inhibiting TNF- ⁇ or a pharmaceutical composition for preventing or treating a disorder or disease caused by abnormal overexpression of TNF- ⁇ as a therapeutic use of a tammer.
- TNF- ⁇ means any TNF- ⁇ present in mammals such as humans and mice that can be seen as TNF- ⁇ in its function and sequence. Preferably human TNF- ⁇ .
- aptamer refers to a nucleic acid ligand that binds to a target molecule as it is known in the art, preferably a nucleic acid ligand that binds specifically to the target molecule.
- 'specifically' binding means binding to a target molecule with a higher affinity than other molecules in a sample or human body.
- active ingredient alone means a component that can exhibit the desired pharmaceutical activity or itself can exhibit the pharmaceutical activity with a carrier inactive.
- TNF- ⁇ inhibition means that the aptamer of the present invention binds to TNF- ⁇ to inhibit the inherent biological activity of TNF- ⁇ . Inhibition of such TNF- ⁇ inherent biological activity can lead to the prophylactic or therapeutic effect of a disorder or disease mediated by TNF- ⁇ (or abnormal overexpression of TNF- ⁇ is harmful) by abnormal overexpression of TNF- ⁇ .
- disorders or diseases caused by abnormal overexpression of TNF- ⁇ are variously known in the art and mean such various disorders and diseases as known in the art.
- disorders and disorders include respiratory disorders, asthma, allergic and non-allergic asthma, asthma due to infection, asthma due to infection with respiratory syncytial virus (RSV), chronic obstructive pulmonary disease (COPD), airway inflammatory conditions , Eosinophilia, fibrosis and mucus hyperplasia, cystic fibrosis, pulmonary fibrosis, atopic disorders, atopic dermatitis, urticaria, eczema, allergic rhinitis, allergic gastroenteritis, skin inflammation and / or skin autoimmune conditions, gastrointestinal tract Inflammation and / or autoimmune conditions, inflammatory bowel disease (IBD), ulcerative colitis, Crohn's disease, inflammation of the liver and / or autoimmune conditions, cirrhosis, liver fibrosis, hepatitis
- IBD inflammatory bowel disease
- IBD ulcer
- the disorders and diseases include rheumatoid arthritis, osteoarthritis, combustible chronic arthritis, septic arthritis, Lyme arthritis, psoriatic arthritis, reactive arthritis, spondyloarthropathy, systemic lupus erythematosus, Crohn's disease, ulcerative colitis, inflammatory bowel disease , Insulin dependent diabetes mellitus, thyroiditis, asthma, allergic diseases, psoriasis, scleroderma, graft-versus-host disease, organ transplant rejection, acute or chronic immune disease associated with organ transplantation, sarcoidosis, atherosclerosis, disseminated intravascular coagulation, Kawasaki disease, Grave's disease, nephrotic syndrome, chronic fatigue syndrome, Wegener's granulomatosis, Henoch-Schoene purpura, microscopic vasculitis of the kidneys, chronic active hepatitis, uveitis, septic shock, toxin shock syndrome, sep
- SELEX technology is used to select aptamers that specifically bind to specific target molecules.
- SELEX technology is named after the term "Systematic Evolution of Ligands by Exponential enrichment" and the technology is described in Science 249 (4968): 505-510, 1990, U.S. Patent No. 5,475,096, U.S. Patent No. 5,270,163, International Patent Publication No. WO 91/19813 discloses specific methods for the selection of aptamers and the use of suitable reagents, materials, etc., Methods Enzymol 267: 275-301, 1996, Methods Enzymol 318: 193. -214, 2000.
- This SELEX technique starts from a single strand of RNA or DAN oligonucleotide nucleic acid library, wherein the oligonucleotides of the nucleic acid library generally contain random sequences between the 5 'and 3' ends and the 5 'and 3' ends. At the end, it contains a known sequence that is common among all oligonucleotides of the library.
- This known sequence includes a sequence to which forward / reverse primers bind, a promoter sequence of RNA polymerase, a restriction enzyme recognition sequence for manipulation such as cloning, and the like.
- the total length of the oligonucleotide including the 5 'end and the 3' end is usually 30-80 nucleotides, preferably 40-60 nucleotides.
- Synthesis of such oligonucleotides is well known in the art and examples thereof include solution phase synthesis techniques such as solid phase oligonucleotide synthesis techniques and triester synthesis methods. Specifically, Nucl. Acid Res. 14: 5399-5467, 1986, Tet. Lett. 27: 5575-5578, 1986, Nucl. Acid Res. 4: 2557, 1977, Lett., 28: 2449, 1978, and the like.
- RNA library can be obtained by transferring the DNA library to RNA polymerases such as T3, T4, and T7.
- the SELEX process basically includes the steps of (a) contacting the nucleic acid library with the target molecule under conditions suitable for binding to the target molecule, (b) separating the nucleic acid (oligonucleotide) that specifically binds the target molecule from the non-binding nucleic acid to obtain a target molecule-nucleic acid complex, and (c) amplifying the nucleic acid from the target molecule-nucleic acid complex.
- Such contacting step, binding step (obtaining target molecule-nucleic acid complex) and amplification step may be repeated one or more cycles, preferably five or more cycles, to select nucleic acid ligands having higher specificity and binding ability with the target molecule.
- the nucleic acid library is an RNA library
- the amplification is performed through cDNA synthesis before the amplification of step (c), and the RNA nucleic acid ligand is obtained by amplification after amplification. Sequencing can also be accomplished by cloning each individual nucleic acid ligand after amplification.
- the RNA aptamer specific for TNF- ⁇ of SEQ ID NO: 1 and SEQ ID NO: 2 was selected using a SELEX technique together with a plate-based binding assay.
- SELEX technology is known in the art for some of its improvements. For example, Counter-SELEX technology (Science 263 (5152): 1425-1429, 1994) to increase specificity with target molecules, Toggle SELEX technology (Mol Ther 4 (6): 567, considering non-clinical animal experiments in the development of aptamer therapy) -573, 2001) and Spiegelmer technology (Chem Biol 9 (3): 351-359, 2002) using enantiomers of target molecules and aptamers.
- the aptamer may be single-stranded RNA itself as disclosed in SEQ ID NO: 1 or 2, but these RNA aptamers are present at the sugar position, the phosphate position, the base position, the 5 'end and / or the 3' end of the ribonucleotide. It may also be a chemically modified (modified) aptamer.
- RNA or DNA is easily degraded by enteric or exonucleases in vivo.
- These chemically modified aptamers can improve the stability in vivo, thereby increasing the half-life of the body, thereby improving the pharmacodynamic and pharmacokinetic properties, and providing resistance to chemical and physical degradation, thereby improving storage stability. Can improve.
- the modification at the sugar position is such that the sugar of one or more arbitrary ribonucleotides of the RNA aptamer of the present invention, in one or more hydroxyl groups (OH groups) of the sugar, is a halogen group, aliphatic group, ether group, amine group It is modified by such as.
- OH groups hydroxyl groups
- 2'F-CTP and 2'F-UTP are used to prepare the RNA aptamer of the present invention of SEQ ID NOs: 1 and 2, in the following examples. It is said that all C and U of the RNA aptamers of the invention are fC (C with 2'-F modified) and fU (U with 2'-F modified).
- US Pat. No. 5,580,737 discloses a method for screening nucleic acid ligands specific for target molecules, including nucleotides modified with 2'-NH2, 2'-F, 2'-OMe.
- Modifications at sugar sites may also be made using sugar analogs instead of ribose.
- Such analogues include ⁇ -anomeric sugars, epimeric sugars such as arabinose, xyloses or lyxoses, and pyranose sugars.
- abasic nucleoside analogs such as furanose sugars, sedoheptuloses, methyl riboside, and more specifically, the International Patent Publication. See WO 2011/130195 and the like.
- RNA aptamers can be synthesized using T3, T4, T7, SP6 RNA polymerase or mutant T7 polymerase, and RNA aptamers comprising 2 'modified nucleotides can be synthesized in particular using mutant polymerases.
- Mutant T7 polymerase Y639F, Mutant T7 polymerase Y639F / H784A, Mutant T7 polymerase H784A, and the like are known in the art as mutant polymerases capable of preparing RNA aptamers comprising 2 'modified nucleotides.
- T7 polymerase Y639F is a mutant polymerase wherein the tyrosine residue has been changed to phenylalanine at position 639, and the 2 'modified nucleotide is used as a substrate using 2'-deoxy, 2'-NH 2- , or 2'-F-NTP. It is known in the art to be able to synthesize RNA comprising (Science, 286: 2305-2308, 1999) and has been widely used in the art to synthesize RNA comprising actual 2 ′ modified nucleotides. However, this mutant T7 polymerase Y639F has limitations in using 2 'modified NTP (particularly 2' modified GTP) with 2'-0Me, 2'-N 3, etc.
- Mutant T7 polymerase Y639F / H784A is a double mutant polymerase in which histidine is replaced with an alanine residue at position 784 in addition to Y639F mutant RNA polymerase and is based on a 2 'modified NTP having a 2'-0Me, 2'-N 3, etc. substrate.
- a single mutant T7 polymerase H784A whose histidine is replaced with an alanine residue at position 784 can also be used as a substrate with a 2 'modified NTP with 2'-0Me, 2'-N 3 , and the like.
- mutant T7 polymerase Y639F / H784A mutant T7 polymerase H784A, or the like (Nucleic Acids Res. 30 (24): 138, 2002).
- modifications at the phosphate sites include phosphates in which P (O) S ("thioate”), P (S) S ("dithioate”), P (O) NR2 ("amidate"), P (O) R, P ( O) OR ', CO or CH2 ("formacetal”).
- R or R ′ is independently H or substituted or unsubstituted alkyl, wherein substituted or unsubstituted alkyl (particularly alkyl having 1 to 20 carbon atoms) is an ether (—O—) bond, aryl, alkenyl, cyclo Alkyl, cycloalkenyl or araldyl.
- the linking group is -O-, -N-, -S- or -C-, through which the adjacent nucleotides bind to each other.
- Modifications at the base position include 5-position modifications of pyrimidine, 8-position modifications of purine, 4-position or 5-position modifications of uracil, exoclyclic amine modifications of cytosine, and the like.
- examples of the 5-position modification of pyrimidine include benzylcarboxyamide, benzylaminocarbonyl, naphthylmethylcarboxyamide, naphthylmethylaminocarbonyl, tryptaminocarboxyamide ( tryptaminocarboxyamide), trypaminocarbonyl, and the like
- RNA aptamers of the invention may be modified at the 5 'end and / or the 3' end.
- the 5 'end may be modified with -NH 2 derived from the 5'-hexylamine linker phosphoramidite.
- the 3 'end may be modified by binding reversed thymidine to 3'-3' (3 'capping).
- RNA aptamers of the invention may be modified by linking polyalkylene glycols (“PAGs”) with or without linkers.
- the linker is not particularly limited as long as it can function as a linker.
- amines R-NH 2
- Such polyalkylene glycols are known in the art to prevent renal filtration to improve blood half-life of aptamers.
- PAGs of the present invention typically have a molecular weight in the range of 5 to 100 kDa, but are selected and used in any size in consideration of the indications, formulations, routes of administration, etc., to be applied to the aptamers of the present invention. Generally in the range from 10 to 80 kD, preferably in the range from 30 to 50 kDA will be used.
- PEG polyethylene glycol
- PAG PEG
- PEG PEG
- PAG PEG
- PEG PEG
- PEG PEG
- PEG PEG
- Such alternatives may also be used in the aptamer modifications of the present invention, and examples of such alternatives include polyoxazoline (POZ), polyPEG, hydroxyethyl starch (HES), albumin, cholesterol that is a lipophilic compound, and the like.
- POZ polyoxazoline
- PEG polyPEG
- HES hydroxyethyl starch
- albumin cholesterol that is a lipophilic compound, and the like.
- Such alternatives may also be bound to the aptamers of the invention without or through linkers.
- the aptamer synthesis of the present invention is well known in the art, and for example, solution phase synthesis techniques such as solid phase oligonucleotide synthesis techniques and triester synthesis methods, and the like can be used.
- solution phase synthesis techniques such as solid phase oligonucleotide synthesis techniques and triester synthesis methods, and the like can be used.
- the aptamer of the present invention binds to TNF- ⁇ and has TNF- ⁇ inhibitory activity
- the aptamer of the present invention can be prepared as a pharmaceutical composition for the prophylactic and therapeutic use of TNF- ⁇ -mediated disorders and diseases. .
- the aptamer of the present invention, the active ingredient in the pharmaceutical composition of the present invention as long as it can exhibit TNF- ⁇ inhibitory activity, prophylactic and therapeutic effectiveness against TNF- ⁇ -mediated disorders and diseases, etc. It may be included in any amount (effective amount) appropriate depending on the dosage form, the route of administration, etc. A conventional effective amount will be determined within the range of 0.001% to 20.0% by weight based on the total weight of the composition.
- the term "effective amount” refers to intended medical and pharmacology such as a disease-improving effect mediated by TNF- ⁇ when the composition of the present invention is administered to a mammal, preferably a human, during the administration period according to the suggestion of a medical professional or the like. It refers to the amount of the active ingredient included in the composition of the present invention, which can exhibit an effect. Such effective amounts can be determined experimentally within the range of ordinary skill in the art.
- compositions of the present invention may be prepared in oral or parenteral formulations according to the route of administration by conventional methods known in the art, including pharmaceutically acceptable carriers in addition to the active ingredient.
- the route of administration here can be any suitable route, including the topical route, the oral route, the intravenous route, the intramuscular route, and direct absorption through mucosal tissue, and may be used in combination of two or more routes.
- An example of a combination of two or more routes is where a drug of two or more formulations according to the route of administration is combined, for example when one drug is administered first by the intravenous route and the other by the local route.
- Pharmaceutically acceptable carriers are well known in the art depending on the route of administration or formulation, and specific reference may be made to the pharmacopoeia of each country, including "Korea Pharmacopoeia.”
- composition of the present invention is prepared in an oral dosage form, powders, granules, tablets, pills, dragees, capsules, solutions, gels, syrups, suspensions, wafers according to methods known in the art with suitable carriers It may be prepared in a formulation such as.
- suitable carriers include lactose, glucose, sucrose, dextrose, sorbitol, sugars such as mannitol, xylitol, starch such as corn starch, potato starch, wheat starch, cellulose, methyl cellulose, ethyl cellulose, sodium carboxymethyl cellulose, Celluloses such as hydroxypropylmethylcellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, magnesium stearate, mineral oil, malt, gelatin, talc, polyol, vegetable oil, ethanol, and Cerrol etc. are mentioned.
- binders include starch, magnesium aluminum silicate, starch ferrite, gelatin, methylcellulose, sodium carboxymethylcellulose, polyvinylpyrrolidone, glucose, corn sweeteners, sodium alginates, polyethylene glycols, waxes, and the like.
- lactose, dextrose, sucrose, mannitol, sorbitol, cellulose, glycine etc. are mentioned as a diluent.
- compositions of the present invention When the pharmaceutical compositions of the present invention are prepared in parenteral formulations, they may be formulated in the form of injections, transdermal administrations, nasal inhalants and suppositories with suitable carriers according to methods known in the art.
- a suitable carrier may be an aqueous isotonic solution or suspension.
- an isotonic solution such as phosphate buffered saline (PBS) containing triethanol amine, sterile water for injection, or 5% dextrose may be used.
- PBS phosphate buffered saline
- transdermal administration it may be formulated in the form of an ointment, cream, lotion, gel, external solution, pasta, linen, aerosol, and the like.
- Nasal inhalants can be formulated in the form of aerosol sprays using suitable propellants, such as dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, etc. witepsol), tween 61, polyethylene glycols, cacao butter, laurin paper, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene stearate, sorbitan fatty acid esters and the like.
- suitable propellants such as dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, etc. witepsol
- tween 61 polyethylene glycols, cacao butter, laurin paper, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene stearate, sorbitan fatty acid esters and the like.
- compositions of the present invention may also be administered in the form of liposome drug delivery systems such as small unilamellar vesicles, large unilamella vesicles and multilamella vesicles.
- Liposomes can be prepared from various phospholipids containing cholesterol, stearylamine or phosphatidylcholine. Liposomal drug shear systems are well known in the art and may be referred to, eg, US Pat. No. 5,262,564.
- Preferred dosages of the pharmaceutical compositions of the invention range from 0.001 mg / kg to 1,000 mg / kg per day, preferably 0.001 mg / kg to day, depending on the condition, body weight, sex, age, severity of the patient and route of administration. May range from 1 g / kg. Such as when administered orally, may range from about 0.05 to 7,500 mg / kg per day. Administration can be done once a day or divided into several times. Such dosages are given for purposes of illustration only and should not be construed as limiting the scope of the invention in any aspect.
- the present invention can provide prophylactic and therapeutic uses for aptamers that bind to TNF- ⁇ and TNF- ⁇ -mediated diseases.
- FIG. 1 is a schematic view of the secondary structure of TNF- ⁇ aptamer ATK001 (a) and ATK007 (b).
- FIG. 2 is a graph illustrating measurement of TNF- ⁇ aptamer ATK007 and human TNF- ⁇ binding by a plate-based binding assay.
- Figure 3 shows the sensorgrams and dissociation constants of TNF- ⁇ aptamers ATK001 (a) and ATK007 (b) and human TNF- ⁇ by the SPR method.
- Figure 4 is a result of analyzing the TNF- ⁇ aptamer ATK001 (a) and ATK007 (b) by the gel retardation method is a graph (c) showing the amount of aptamer bound to TNF- ⁇ in percentage.
- TNF- ⁇ aptamer ATK001 and human TNF- ⁇ , ErbB2 and ErbB3 by SPR method and sensorgrams (b) of TNF- ⁇ aptamer ATK007 and human TNF- ⁇ , ErbB2 and ErbB3.
- Sensorgrams (c) of TNF- ⁇ aptamer ATK007 and IGF-BP1 and their dissociation constants are shown.
- TNF- ⁇ aptamer is a result of the effect of TNF- ⁇ aptamer on its expression at the transcription level of acute phase proteins.
- TNF- ⁇ aptamer 7 is a result of the effect of TNF- ⁇ aptamer on its expression at the transcription level of pro-inflammatory cytokines and angiogenesis.
- Each of the 5 'and 3' underlined regions is a fixed region where the forward and reverse primers bind, respectively, and Nn is a sequence in which n bases such as A, G, T, and C are randomly present.
- n bases such as A, G, T, and C are randomly present.
- a T7 RNA polymerase promoter sequence for RNA synthesis is bound to the 5 'end.
- Two groups of DNA libraries were prepared by PCR of each of the two single-stranded DNA oligonucleotides as a template.
- 1,000 pM DNA single-stranded oligonucleotide, primers of 2,500 pM PCR, 50 mM KCl, 10 mM Tris-Cl, pH 8.3, 3 mM MgCl 2 , 0.5 mM dNTP (dATP, dCTP, dGTP, and dTTP) PCR was performed by reacting 32 cycles with 0.1 U Taq DNA Polymerase (Perkin-Elmer, Foster City Calif.), followeded by 30 cycles of 95 ° C, 30 seconds of 55 ° C, and 1 minute of 72 ° C. It was then purified by a QIAquick-spin PCR purification column (QIAGEN Inc., Chatsworth Calif.).
- RNA degrading enzyme Using the T7 RNA polymerase as a template for each of the prepared two groups of DNA libraries, two groups of RNA libraries were prepared through in vitro transcription as follows. At this time, 2'F-CTP, 2'F-UTP including ATP and GTP and 2'-F-substituted pyrimidine were used to impart resistance to RNA degrading enzyme.
- the RNA library 10 14 nucleotide sequence / L concentration of the synthesized single-stranded nucleic acid in a concentration of 200 pM / 200 ⁇ l SELEX buffer (50 mM TrisCl (pH 7.4), 5 mM KCl, 100 mM NaCl, 1 mM MgCl) 2 , 0.1% NaN 3 ) was heated at 80 ° C. for 10 minutes and then left on ice for 10 minutes.
- a reaction solution was prepared by adding yeast tRNA (yeast tRNA, Life Technologies) and 0.2% BSA (bovine serum albumin, Merck) at a concentration of 5 times the concentration of the single stranded nucleic acid.
- Biotinylation of human TNF- ⁇ was carried out according to the protocol of the Protein Labeling kit (Jena Bioscience, Germany). A 10 mg / ml biotin solution was prepared using 1 M sodium bicarbonate solution and DMF (dimethylformamide) with distilled water. Human TNF- ⁇ solution was prepared by adding 1 M sodium bicarbonate solution prepared above to recombinant human TNF- ⁇ (Sigma-Aldrich, Germany) at a concentration of 100 mM Sodium bicarbonate and 10 mg / ml of human TNF- ⁇ .
- the human TNF- ⁇ solution and the biotin solution were mixed in the same amount and reacted at room temperature for 1 hour, and then reacted for 10 minutes by adding 2.5 ml reaction solution to a Sephadex G-25 column (Sigma-Aldrich, Germany). Thereafter, 10,000xg, centrifuged for 10 minutes to remove the free biotin of the supernatant and harvested biotinylated human TNF- ⁇ .
- RNA single-stranded nucleic acid prepared above was added to the harvested biotinylated human TNF- ⁇ 10 ⁇ g / ml concentration and reacted for 30 minutes to form a complex of RNA single-stranded nucleic acid and TNF- ⁇ after Dynabeads
- Dynabeads was synthesized in a biotinylated human TNF- ⁇ -single-nucleic acid complex obtained by reacting biotinylated human TNF- ⁇ with a single-stranded nucleic acid.
- M-280 Streptavidin was added and reacted to form a biotinylated human TNF- ⁇ -RNA single-stranded nucleic acid-bead complex using the binding of the biotin of the complex with the avidin of the beads, and the SELEX buffer (1x PBS, 1 mM magnesium acetate, pH). 7.0) and the magnetic material was used to harvest biotinylated human TNF- ⁇ -RNA single stranded nucleic acid-bead complex.
- RT-PCR was performed using the harvested complex as it was to amplify the DNA pool from RNA single-stranded nucleic acid binding to human TNF- ⁇ .
- 500nM primer was added to the harvested complex, denatured at 65 ° C. for 5 minutes, and then allowed to bind RNA and primer for 10 minutes at room temperature.
- 1 mM dNTP, 5X RT buffer, 25U AMV RTase (promega) was added to the reaction for 30 minutes at 37 °C, then heated at 95 °C for 5 minutes to cool at 4 °C to inactivate the reverse transcriptase.
- the synthesized cDNA was mixed with 2,500 pM of PCR primers, 50 mM KCl, 10 mM Tris-Cl (pH 8.3), 3 mM MgCl 2 , 0.5 mM dNTP (dATP, dCTP, dGTP, and dTTP) and added 0.1 PCR was performed by adding 32 U Taq DNA Polymerase (Perkin-Elmer, Foster City Calif.) To react 32 cycles under conditions of 30 seconds at 95 ° C, 30 seconds at 55 ° C, and 1 minute at 72 ° C. 3% agarose gel was confirmed, and in vitro transcription was performed in the same process as that of the RNA library, and RNA was synthesized and the selection process was repeated three times as described above.
- RNA single-stranded nucleic acid sequence identified in ⁇ Example 2> with TNF- ⁇ 0.5 mg / mL of recombinant human TNF- ⁇ protein was diluted in DPBS (Dulbecco's Phosphate-buffered Saline) A concentration of 15 ⁇ g / mL was made and 100 ⁇ l was added to a 96 well Maxisorb plate and incubated overnight at 4 ° C. After removing the TNF- ⁇ solution, the plate was washed three times at room temperature with 200 ⁇ l of wash buffer (DPBS + 0.05% Tween 20).
- wash buffer DPBS + 0.05% Tween 20
- the plate was then blocked with 200 ⁇ l of 10 mg / mL bovine serum albumin (BSA) dissolved in DPBS for 30 minutes at room temperature.
- BSA bovine serum albumin
- the BSA blocking solution was then removed and washed three times with 200 ⁇ l wash solution again.
- RNA single-stranded nucleic acid serially diluted with 0.1% BSA in DPBS was added to the plate and incubated for 3 hours at room temperature.
- 100 ⁇ l of rabbit monoclonal TNF- ⁇ antibody (Eputomics, USA) was added to the plate and incubated at room temperature for 60 minutes.
- the TNF- ⁇ antibody solution was then removed and the plate washed three times with 200 ⁇ l wash buffer.
- ATK001 or ATK007 had the best binding affinity with recombinant human TNF- ⁇ among the RNA single-stranded nucleic acids sequenced in ⁇ Example 2>, and ATK001 and ATK007 were repeated through five replicate experiments on ATK001 or ATK007. It was confirmed that the average value of the binding affinity for the recombinant TNF- ⁇ was 43 nM and 30 nM, respectively. Data for ATK001 obtained from one of these experiments is shown in FIG. 2.
- RNA aptamers ATK001 and ATK007 were denatured at 80 ° C. for 5 minutes and then restored at room temperature for 15 minutes to prepare analytes (RNA).
- the flow rate was changed to 30 ⁇ l / min to obtain kinetics.
- the prepared analyte was dissolved in 1 ⁇ HBS, prepared at a concentration of between 6.25 nM and 500 nM, and then flowed into a sensor chip to determine the binding affinity between the selected RNA aptamer and TNF- ⁇ protein (equilibrium dissociation constant: Quantification using Kd).
- Equilibrium dissociation constant (Kd) is set to 1: 1 binding of the selected RNA aptamer and TNF- ⁇ , and then the dynamic Ka / is derived from a plot curve of Req values obtained from the sensogram.
- Kd simultaneous program kinetic simultaneous Ka / Kd model program
- TNF- ⁇ Single-stranded nucleic acids (50 pM) binding to TNF- ⁇ selected in ⁇ Example 2> labeled with radioisotopes were reacted with increasing amount of TNF- ⁇ protein (0-320nM).
- the aptamer and TNF- ⁇ protein, binding buffer (30mM Tris-HCl (PH 7.5), 150mM NaCl, 1.5mM MgCl2, 2mM DTT), tRNA 3g, etc. were adjusted to a total 40 ⁇ L and bound for 30 minutes at room temperature.
- 6X BPB was added and electrophoresed on 6% native gel (6% polyacrylamide, 1X TBE, 10 mM MgCl2, 2% glycerol) at 120 V at 4 ° C and developed by exposure to X-ray film.
- the cleavage constant (Kd) was determined by calculating the amount of RNA aptamer binding to TNF- ⁇ relative to the total RNA aptamer.
- FIG. 4A and 4B show 50 pM RNA aptamer ATK001 (a) and 50 pM RNA aptamer ATK007 (b) labeled with radioisotopes, increasing the amount of TNF- ⁇ (0-320 nM).
- the resulting RNA aptamer (F) that did not bind to the resulting TNF- ⁇ -RNA aptamer conjugate (C) was photographed on a 4% unmodified acrylamide gel, and FIG. The percentage of RNA is calculated and graphed, which is the average of the measurements from three replicates.
- the selected ATK001 shows that ATK007 can bind strongly to the target protein by 1.2 times, showing more efficient binding ability.
- RNA aptamers ATK001 and ATK007 are 10 ⁇ 9 M (nM) level for TNF- ⁇ , about 10 4 nM level for ErbB2 and ErbB3dp, and IGF-BP1 (Insulin-like growth). It hardly binds to factor-binding protein 1), indicating a high binding force to TNF- ⁇ .
- hepatocarcinoma SK-HEP1 cells Twenty four hours prior to the experiment, hepatocarcinoma SK-HEP1 cells (ATCC) were placed in 96-well flat-bottom microtiter plates with DMEM medium containing 10% FBS at a density of 1.0 ⁇ 10 4 . Inoculation.
- DMEM medium containing 10% FBS at a density of 1.0 ⁇ 10 4 . Inoculation.
- 2 ⁇ g / mL aptamer ATK001 or ATK007
- 20 ⁇ g / mL anti-TNF- ⁇ mAB R & D systems
- RNA and target acute phase protein were then washed with warm PBS and then incubated in complete medium for an additional 48 hours.
- angiogenic substances and pro-inflammatory cytokines RT-PCR was performed with the primers of Tables 1 and 2.
- PCR products were electrophoresed on agarose gels to evaluate the effects of TNF- ⁇ aptamers ATK001 and ATK007 on the transcription of acute phase proteins, angiogenic and proinflammatory cytokines.
- GAPDH F 5-TATCGGACGCCTGGTTAC-3R: 5-TGCTGACAATCTTGAGGGA-3 407
- Hepatoglobin F 5-CCTGAATGTGAAGCAGTATGT-3R: 5-TTCTGTTTGAGTTTGATGAGC-3 338
- Fibrogen-gamma F 5-CTACTTCGCTGGTGGGGATG-3 R: 5-GCTTTGCAAGTCCATTGTCCA-3 493
- Transferrin F TGGAGACAGATGCTCCCTCC-3R: TTTGTGCTCTGTGTATGTGGTAAGG-3 119
- Feritin-l F 5-GAGACCACAAGCGACCCGCA-3 R: 5-GAGGTGACGGAGGGCTGGCT-3 138
- GAPDH F 5-TATCGGACGCCTGGTTAC-3R: 5-TGCTGACAATCTTGAGGGA-3 407
- TNF- ⁇ F 5-TTCTGTCTACTGAACTTCGGGGTGATCGGTCC-3R: 5-GTATGAGATAGCAAATCGGCTGACGGTGTGGG-3 468 IL-1 ⁇ F; 5-TGCGAATCTCCGACCACCACTACA-3R: 5-TGGAGGTGGAGAGCTTTCAGTTCATAT-3 295
- IL6 F 5-ATGAACTCCTTCTCCACAAGCGC-3 R: 5-GAAGAGCC-CTCAGGCTGGACTG-3.
- VEGF F 5-GAGTATATCTTCAAGCCGTCCTGT-3R: 5-ATCTGCATAGTGACGTTGCTCTC-3 230
- the TNF- ⁇ aptamer used in the experiment as shown in FIG. 7 the proinflammatory cytokines (TNF- ⁇ , IL-1 ⁇ and IL6) and angiogenic substances (VEGF) whose expression levels are increased in the acute phase response are transcription levels.
- the expression level was suppressed at.
- RAW 264.7 cells were seeded in wells of 12-well plates with RPMI 1640 and 10% FBS at a density of 1.0 ⁇ 10 5 .
- Cells were pretreated with 2U / ml of IFN- ⁇ (Peprotech, NJ) for 1 hour, followed by RNA of final concentration of 2 ⁇ g / mL TNF- ⁇ aptamer (ATK001 or ATK007) or final concentration of 2 ⁇ g / mL control
Abstract
Description
Gene | Primer Sequence | Product Size (bp) |
GAPDH | F: 5-TATCGGACGCCTGGTTAC-3R: 5-TGCTGACAATCTTGAGGGA-3 | 407 |
Hepatoglobin | F: 5-CCTGAATGTGAAGCAGTATGT-3R: 5-TTCTGTTTGAGTTTGATGAGC-3 | 338 |
Fibrogen-gamma | F: 5-CTACTTCGCTGGTGGGGATG-3 R: 5-GCTTTGCAAGTCCATTGTCCA-3 | 493 |
Fibronectin | F: 5-CCGTGGGCAACTCTGTC-3R: 5-TGCGGCAGTTGTCACAG-3 | 438 |
Transferrin | F: TGGAGACAGATGCTCCCTCC-3R: TTTGTGCTCTGTGTATGTGGTAAGG-3 | 119 |
Feritin-L | F: 5-GAGACCACAAGCGACCCGCA-3 R: 5-GAGGTGACGGAGGGCTGGCT-3 | 138 |
Gene | Primer Sequence | Product Size (bp) |
GAPDH | F: 5-TATCGGACGCCTGGTTAC-3R: 5-TGCTGACAATCTTGAGGGA-3 | 407 |
TNF-α | F: 5-TTCTGTCTACTGAACTTCGGGGTGATCGGTCC-3R: 5-GTATGAGATAGCAAATCGGCTGACGGTGTGGG-3 | 468 |
IL-1β | F; 5-TGCGAATCTCCGACCACCACTACA-3R: 5-TGGAGGTGGAGAGCTTTCAGTTCATAT-3 | 295 |
IL6 | F: 5-ATGAACTCCTTCTCCACAAGCGC-3 R: 5-GAAGAGCC- CTCAGGCTGGACTG-3. | 628 |
VEGF | F: 5-GAGTATATCTTCAAGCCGTCCTGT-3R: 5-ATCTGCATAGTGACGTTGCTCTC-3 | 230 |
Claims (16)
- 서열번호 1 또는 서열번호 2를 가지고, TNF-α에 결합하는 RNA 압타머 또는 그 변형체.
- 제1항에 있어서,상기 TNF-α는 인간 TNF-α 또는 마우스 TNF-α인 것을 특징으로 하는 압타머.
- 제1항에 있어서,상기 RNA 압타머는 변형된 압타머이고, 그 변형된 압타머는 리보뉴클레오타이드의 당 위치, 포스페이트 위치, 염기 위치, 5' 말단 위치 및 3' 말단 위치 중 어느 하나 이상의 위치에서 화학적으로 변형된 압타머인 것을 특징으로 하는 압타머.
- 제3항에 있어서,상기 변형된 압타머는 당 위치에서 변형된 압타머이고, 그 압타머는 당의 하나 이상의 하이드록실 기가 할로겐 기, 지방족 기, 에테르 기 및 아민 기 중 어느 하나로 변형된 것을 특징으로 하는 압타머.
- 제3항에 있어서,상기 변형된 압타머는 당 위치에서 변형된 압타머이고, 그 압타머는 당의 2'-OH 기에서 OMe, O-알킬, O-알릴, S-알킬, S-알릴 및 할로겐 중 하나로 변형돤 것을 특징으로 하는 압타머.
- 제1항에 있어서,상기 RNA 압타머는 변형된 압타머이고, 그 변형된 압타머는 서열번호 1 또는 서열번호 2에 있는 모든 C 및 U가 각각 2'-F가 수식된 C 및 2'-F가 수식된 U인 것을 특징으로 하는 압타머.
- 제1항에 있어서,상기 RNA 압타머는 포스페이트 위치에서 변형된 압타머이고, 그 변형된 압타머는 포스페이트가 P(O)S("thioate"), P(S)S("dithioate"), P(O)NR2("amidate"), P(O)R, P(O)OR', CO 또는 CH2("formacetal")로 변형된 것을 특징으로 하는 압타머.
- 제1항에 있어서,상기 RNA 압타머는 염기 위치에서 변형된 압타머이고, 그 변형된 압타머는 피리미딘의 5-위치, 푸린의 8-위치, 우라실의 4-위치, 우라실의 5-위치 또는 시토신의 고리 밖(exoclyclic) 아민 위치에서 변형된 것을 특징으로 하는 압타머.
- 제1항에 있어서,상기 RNA 압타머는 5' 말단 위치에서 변형된 압타머이고, 그 변형된 압타머는 5' 말단에 -NH2이 결합되어 변형된 것을 특징으로 하는 압타머.
- 제1항에 있어서,상기 RNA 압타머는 3' 말단 위치에서 변형된 압타머이고, 그 변형된 압타머는 역상 티미딘이 3'-3'으로 결합하여 변형된 것을 특징으로 하는 압타머.
- 제1항에 있어서,상기 RNA 압타머는 폴리알킬렌 글리콜이 링커를 통하여 또는 링커 없이 연결되어 변형된 것을 특징으로 하는 압타머.
- 제1항에 있어서,상기 RNA 압타머는 폴리에틸렌 글리콜이 링커를 통하여 또는 링커 없이 연결되어 변형된 것을 특징으로 하는 압타머.
- 제12항에 있어서,상기 RNA 압타머는 폴리에틸렌 글리콜이 아민(R-NH2)을 링커로 하여 5' 말단 위치에서 도입되어 변형된 것을 특징으로 하는 압타머.
- 제1항 내지 제13항 중 어느 한 항 기재의 압타머를 포함하는 TNF-α 억제용 약제학적 조성물.
- 제1항 내지 제13항 중 어느 한 항 기재의 압타머를 포함하는, TNF-α가 매개하는 질환의 치료 또는 예방용 약제학적 조성물.
- 제15항에 있어서,상기 TNF-α가 매개하는 질환은 호흡기 장애, 천식, 알레르기성 및 비알레르기성 천식, 감염으로 인한 천식, 호흡기 세포 융합 바이러스(RSV)에 의한 감염으로 인한 천식, 만성 폐쇄성 폐 질환(COPD), 기도 염증성 병태, 호산구 증가증, 섬유증 및 점액 과생성증, 낭포성 섬유증, 폐 섬유증, 아토피성 장애, 아토피성 피부염, 두드러기, 습진, 알레르기성 비염, 알레르기성 위장염, 피부 염증 및/또는 피부 자가면역 병태, 위장 기관의 염증 및/또는 자가면역 병태, 염증성 장 질환(IBD), 궤양성 대장염, 크론병, 간의 염증 및/또는 자가면역 병태, 간경화, 간 섬유증, B형 및/또는 C형 간염 바이러스에 의해 유발된 간 섬유증, 강피증, 종양 또는 암, 간세포 암종, 교모세포종, 림프종, 호지킨 림프종, 바이러스 감염, 세균 감염, 기생충 감염, HTLV-1 감염 또는 백신접종에 의한 면역 반응인 것을 특징으로 하는 약제학적 조성물.
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- 2016-10-24 EP EP16920238.9A patent/EP3530738A4/en not_active Withdrawn
- 2016-10-24 CN CN201680090346.2A patent/CN110023499A/zh active Pending
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Cited By (1)
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CN110023499A (zh) | 2019-07-16 |
EP3530738A4 (en) | 2020-06-03 |
US11028395B2 (en) | 2021-06-08 |
US20190323012A1 (en) | 2019-10-24 |
JP2019535249A (ja) | 2019-12-12 |
CA3041451A1 (en) | 2018-05-03 |
EP3530738A1 (en) | 2019-08-28 |
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