KR101458947B1 - Interleukin-8 aptamers and uses thereof - Google Patents

Abstract

The present invention relates to an RNA aptamer that binds to IL-8 and its use. More specifically, an aptamer that specifically binds to IL-8 in an RNA library is selected using SELEX, and the aptamer is selected from IL-8 It was confirmed that IL-8-induced neutrophil migration, intracellular signal transduction, and invasion of cancer cells were inhibited by binding with high binding force. In addition, a truncated form of the aptamer was prepared, and the truncated form of the aptamer binds to IL-8 with high binding force to induce IL-8-induced neutrophil migration and intracellular signaling, and Ca ^{2 +} mobilization. Therefore, it was confirmed that the aptamer binding to IL-8 of the present invention can be effectively used for the prevention and treatment of cancer or inflammatory diseases.

Description

Interleukin-8 aptamers and uses thereof < RTI ID = 0.0 >

The present invention relates to an RNA aptamer binding to IL-8 (interleukin-8) and its use, and more particularly to an aptamer which binds to IL-8 and inhibits IL-8 induced function, To a pharmaceutical composition for the prophylaxis and treatment of cancers and inflammatory diseases including tumors.

The aptamer is a unique single-stranded structure that folds into a unique 3D structure and binds specifically to the target with high binding force at low nanomolar to picomolar concentrations. -strand) refers to DNA or RNA oligonucleotides. The aptamer is generally identified in a library of randomized sequences (> 10 ¹⁵ ) in vitro by methods known as SELEX (systematic evolution of ligands by exponential enrichment). Since the SELEX method was developed in 1990, aptamers targeting proteins, phospholipids, sugars, nucleic acids, and even human plasma and whole cells, It was identified.

Aptamers are frequently compared to antibodies in that they bind specifically to the target and are broadly applicable. Compared to antibodies, aptamers are readily produced by chemical synthesis, are stable at high temperatures, are easy to access targets, are chemically easy to modify, are non-immunogenic, and nontoxic ) As a therapeutic agent. On the other hand, nuclease-mediated degradation causes aptamers to have a short half-life. These limitations may be overcome using various chemical transformations. For example, pyrimidine nucleotides can be modified with 2'-fluorin, 2'-F or 2'-amine (2'-amine, 2'-NH ₂ ) ) Nucleotides into 2'-OMe, or by chemical changes such as 3'-inverted dT, 3'-3'-dT, to increase the nuclease resistance of the aptamer.

Interleukin-8 (IL-8), also known as CXCL8, is the first chemokine identified. IL-8 is also known as a chemoattractant of neutrophil. IL-8 is a monocyte, macrophage, endothelial cell, and endothelial cell in response to proinflammatory stimuli, chemical and enviromental stresses, and steroid hormones. It is produced by various types of cells such as endothelial cells, epithelial cells, and tumor cells. IL-8 contains an ELR motif and Glu-Leu-Arg, a sequence essential for receptor binding and essential for chemotactic activity, and also promotes angiogenesis through CXCR1 and CXCR2. CXCR1 (IL-8RA) and CXCR2 (IL-8RB) are G protein-linked receptors that bind to IL-8 with high binding force and deliver signals that begin with IL-8 into cells. In addition, IL-8 has other biological activities important in the development of cancer. Tumor-derived IL-8 can promote the proliferation, survival, migration, and invasion of cancer cells and activate endothelial cells to promote angiogenesis. The contribution of IL-8 in tumor progression is crucial for the development of therapeutic agents for targets and for the treatment of cancer.

Accordingly, the inventors of the present invention selected an aptamer specifically binding to IL-8 in an RNA library using SELEX while studying an aptamer capable of treating cancer by inhibiting the activity of IL-8, Binding to IL-8 with high binding force to inhibit IL-8-induced neutrophil migration, intracellular signaling, and invasion of cancer cells. In addition, a truncated form of the aptamer was prepared and bound to IL-8 with high binding force to inhibit IL-8-induced neutrophil migration and intracellular signaling and Ca ²⁺ mobilization Thus, the present invention has been accomplished by confirming that the aptamer can be effectively used for the treatment and prevention of cancer or inflammatory diseases.

It is an object of the present invention to provide an aptamer which inhibits IL-8-induced function in association with IL-8 (interleukin-8) and uses thereof.

The present invention provides an aptamer that specifically binds to IL-8.

The present invention also provides a pharmaceutical composition for the prevention and treatment of cancer or inflammatory diseases comprising an aptamer that specifically binds to IL-8.

The aptamer that binds to IL-8 (interleukin-1) of the present invention binds to IL-8 with high binding force to inhibit the activity of IL-8, thereby inhibiting IL-8-induced cancer and inflammatory diseases And can be usefully used as a pharmaceutical composition for prevention and treatment.

Figure 1A is a graph showing the binding capacity of an RNA pool and an RNA pool obtained through SELEX to IL-8.
Fig. 1B is a diagram showing the RNA obtained from an RNA pool divided into several groups according to sequence similarity.
Figure 1C shows that the # 8 aptamer in the RNA pool specifically binds His-IL-8 and does not bind to other CXC-chemokines His-GRO-alpha and His-GRO-beta.
2A and 2B are diagrams for predicting the structure of an aptamer of the present invention.
Figure 3 is a graph showing that the aptamer of the present invention inhibits neutrophil migration.
Figure 4 shows that 8A-44 (SEQ ID NO: 23) strongly binds to IL-8.
Figure 5A shows that 8A-44 (SEQ ID NO: 23) inhibits the migration of neutrophils induced by IL-8.
Figure 5B shows that 8A-44 (SEQ ID NO: 23) inhibits the activity of ERK induced by IL-8.
Figures 6A and 6B show that 8A-44 (SEQ ID NO: 23) inhibits IL-8 induced intrusion in IL-8 induced oral cancer cell lines HSC-3 and 686-LN-e cell lines.
FIG. 7 shows the binding potency of 8A-35 (SEQ ID NO: 24) and 8A-30 (SEQ ID NO: 25) to IL-8 in the truncated form of 8A-44 (SEQ ID NO: 23).
Figure 8A is a diagram showing that 8A-44 (SEQ ID NO: 23), 8A-35 (SEQ ID NO: 24) and 8A-30 (SEQ ID NO: 25) inhibit the migration of neutrophils induced by IL-8.
Fig. 8B shows that 8A-44 (SEQ ID NO: 23), 8A-35 (SEQ ID NO: 24) and 8A-30 (SEQ ID NO: 25) inhibit the activity of ERK induced by IL-8.
9 is a concentration 8A-44 (SEQ ID NO: 23), 8A-35 (SEQ ID NO: 24) and 8A-30 (SEQ ID NO: 25) is a Ca ^{2 +} mobilization (mobilization) induced by IL-8 in the neutrophil cells Dependent inhibition.

Hereinafter, the present invention will be described in detail.

The present invention relates to an RNA aptamer comprising 30 RNAs specifically binding to IL-8 (interleukin-8), wherein the 4 th to 30 th bases from the 5 'end of the aptamer are those comprising a base of SEQ ID NO: 22 RNA < / RTI >

The present invention also provides an RNA aptamer comprising 35 RNAs specifically binding to IL-8, wherein the 7 th to 30 th bases from the 5 'end of the aptamer are composed of the bases of SEQ ID NO: 22 Provides RNA aptamer.

Also, the present invention provides an RNA aptamer comprising 44 RNAs specifically binding to IL-8, wherein the 7 th to 30 th bases from the 5 'end of the aptamer are composed of the bases of SEQ ID NO: 22, 27, and SEQ ID NO: 28, and a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 27 and SEQ ID NO: 28, and 80% or more thereof, respectively.

The above-mentioned "RNA aptamer" is a short single-stranded oligonucleotide capable of forming a three-dimensional structure capable of binding to a target substance with high affinity and specificity, and in most cases can specifically bind to a target protein But can be efficiently used to inhibit the function of the target protein. Repeated in vitro screening procedures allow selection of specific RNA molecules, aptamers, from any RNA library (AD Ellington and JW Szostak, Nature 346: 818-822, 1990; C. Tuerk and L. Gold, Since the size of the RNA library is large (1014-1015) and it is likely to produce RNA molecules by in vitro enzymatic action, the RNA library may contain other biological libraries or synthetic libraries for screening high affinity aptamers (Science 249: 505-510, 1990) Library (EN Brody and L. Gold, Rev. Mol. Biotechnol. 74: 5-13, 2000).

In a preferred embodiment of the present invention, the present inventors performed SELEX using an RNA library to select an RNA aptamer that binds to IL-8 and inhibits IL-8 induced function, and as a result, 15 sequences divided into five groups were selected (see FIG. 1B). 8A-W (SEQ ID NO: 16) specifically binds to IL-8, and the binding affinity to IL-8 is about 19 times higher than that of the RNA library But not to other CXC-chemokines GRO-alpha and GRO-beta (see Fig. 1C).

In addition, the present inventors produced a truncated aptamer using the 8A-W (SEQ ID NO: 16) as a template. (SEQ ID NO: 23) was prepared by PCR in the presence of 8A-1F1R, 8A-2F1R, 8A-2F2R and 8A-44 Truncated form RNA aptamer of SEQ ID NO: 24) and 8A-30 (SEQ ID NO: 25) was further prepared and its two-dimensional structure was predicted using an M fold program ).

Further, the present inventors have found that the migration of neutrophil by the treatment of 8A-W (SEQ ID NO: 16), 8A-1F1R and 8A-44 (SEQ ID NO: 23) among the obtained truncated forms of aptamers is concentration- , But it was not affected by 8A-2F2R or 8A-2F1R (see FIG. 3).

In addition, it was confirmed from the above results that 8A-44 (SEQ ID NO: 23) is sufficient for binding to IL-8 and inhibits the function of IL-8. 8A-44 (SEQ ID NO: 23) bound to IL-8 with high binding force compared to the control group, whereas it was found to be rapidly dissociated (see FIG. 4 and Table 1).

The present inventors also found that 8A-44 (SEQ ID NO: 23) suppressed IL-8 induced neutrophil migration in a concentration-dependent manner (see FIG. 5A) and inhibited IL-8 induced ERK activity at high concentrations (See FIG. 5B).

In addition, it was confirmed that intrusion of IL-8-induced oral cancer cell line was suppressed in a concentration-dependent manner of 8A-44 (SEQ ID NO: 23) (see FIG. 6). The results show that 8A-44 (SEQ ID NO: 23) can bind to purified IL-8 and naturally secreted cell-derived IL-8 and suppress its function.

8A-35 (SEQ ID NO: 24) and 8A-30 (SEQ ID NO: 25) binding activity to IL-8 showed 8A-35 (SEQ ID NO: 24) bound to IL-8 with stronger binding force but 8A-30 (SEQ ID NO: 25) showed slightly weaker binding (see FIG. 7 and Table 2).

In addition, it has been shown that 8A-44 (SEQ ID NO: 23) and its truncated forms 8A-35 (SEQ ID NO: 24) and 8A-30 (SEQ ID NO: 25) inhibit the function of IL- Akt signaling, and Ca < ^{2 +} & ^{gt ;} mobilization (Fig. 8A, 8B and Fig. 9).

Therefore, the aptamers of the present invention are useful for the prevention and treatment of IL-8 induced cancer and inflammatory diseases by significantly inhibiting intracellular signal transduction pathways induced by IL-8 bound to human IL-8 And thus it can be used as an effective composition.

The present invention provides a pharmaceutical composition for the treatment and prevention of cancer or inflammatory diseases including the aptamer.

The aptamer of the present invention is most preferably but not limited to inhibit IL-8-induced IL-8-induced function. In addition, the structure of the aptamer preferably comprises a main stem and a loop structure, but can be used if it contains the sequence of the aptamer of the present invention.

In addition, the aptamers can also be modified with 2'-fluoro-pyrimidine-modified 2'-F-dCTP and 2'-F-dUTP instead of CTP and UTP to have RNaseA resistance. pyrimidine-modified RNA library to SELEX, but not limited thereto. Any method known in the art can be used as long as it has a resistance to RNaseA.

In addition, the aptamer may include, but is not limited to, one or more modifications selected from the group consisting of:

-OH group in the 2 ' carbon position of the nucleotide in the nucleotide is selected from the group consisting of -Me (methyl), -OMe, -NH2, -F (fluorine), -O-2-methoxyethyl- A modification by substitution with ethyl (methylthioethyl), -O-3-aminopropyl, -O-3-dimethylaminopropyl, -ON-methylacetamido or -O-dimethylamidooxyethyl;

A modification in which the oxygen in the sugar structure in the nucleotide is replaced with sulfur; And

Nucleotide linkage is a modification of a phosphorothioate, borano phosphophate or methyl phosphonate bond

In addition, the present invention provides an aptamer-complex conjugated with a macromolecular substance having biocompatibility with the RNA aptamer of the present invention, but is not limited thereto. The macromolecular substance is preferably, but not limited to, polyethylene glycol (PEG), diacylglycerol (DAG), or monoclonal antibody.

The present invention provides a pharmaceutical composition for the prevention and treatment of cancer or inflammatory diseases comprising the RNA aptamer fmf of the present invention.

The cancer is preferably oral cancer and skin cancer induced by overexpression of IL-8. According to a preferred embodiment of the present invention, oral cancer is more preferably, but not limited thereto.

In addition, the inflammatory diseases include inflammatory diseases such as dermatitis, atopy, conjunctivitis, periodontitis, rhinitis, otitis, sore throat, tonsillitis, pneumonia, gastric ulcer, gastritis, Crohn's disease, colitis, hemorrhoids, gout, ankylosing spondylitis, rheumatic fever, lupus, fibromyalgia, It is preferable to be selected from the group consisting of psoriasis arthritis, osteoarthritis, rheumatoid arthritis, shoulder inflammation, tendinitis, hay fever, tendinitis, myositis, hepatitis, cystitis, nephritis, sjogren's syndrome, multiple sclerosis and acute and chronic inflammatory diseases But not limited to this.

The composition containing the RNA aptamer of the present invention may further contain one or more active ingredients showing the same or similar functions in addition to the above components.

The composition of the present invention may further comprise a pharmaceutically acceptable additive, wherein pharmaceutically acceptable additives include starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, calcium hydrogen phosphate, lactose Starch glycolate, sodium starch glycolate, carnauba wax, synthetic aluminum silicate, stearic acid, magnesium stearate, aluminum stearate, calcium stearate, calcium stearate, , White sugar, dextrose, sorbitol and talc. The pharmaceutically acceptable additives according to the present invention are preferably included in the composition in an amount of 0.1 to 90 parts by weight, but are not limited thereto.

That is, the composition of the present invention can be administered in various formulations of oral and parenteral administration at the time of actual clinical administration. In the case of formulation, a diluent such as a filler, an extender, a binder, a wetting agent, a disintegrant, . ≪ / RTI > Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose, Sucrose, lactose, gelatin, and the like. In addition to simple excipients, lubricants such as magnesium stearate talc may also be used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions and syrups, and various excipients such as wetting agents, sweetening agents, fragrances, preservatives and the like may be included in addition to water and liquid paraffin, which are simple diluents commonly used . Formulations for parenteral administration may include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used as the non-aqueous solvent and suspension agent. Examples of suppository bases include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin, and the like.

The composition of the present invention may be administered orally or parenterally in accordance with the intended method, and may be administered orally, parenterally or intraperitoneally, rectally, subcutaneously, intravenously, intramuscularly, And is administered by a suitable method, including intra-lesional administration if necessary for local immunosuppressive treatment. The dosage varies depending on the patient's body weight, age, sex, health condition, diet, administration time, administration method, excretion rate, and severity of disease.

The composition of the present invention can be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy, and biological response modifiers for neuroprotection.

Hereinafter, the present invention will be described in detail with reference to examples and production examples.

However, the following examples and preparation examples illustrate the present invention specifically, and the content of the present invention is not limited by the following examples and production examples.

< Example  1> In vitro IL For -8 RNA Of app tamer  Screening, cloning and sequencing

<1-1> SELEX Using RNA Aptamer  Selection

To obtain an RNaseA-resistant RNA aptamer for human IL-8, SELEX was performed using 2'-F-dCTP and 2'-F-dUTP, respectively, instead of CTP and UTP.

Specifically, 40 randomized single-stranded DNA libraries with a fixed linker region, a T7 promoter, and a restriction enzyme site located next to each other Were mixed with an equimolar dNTP mix and 0.25 uM of 5'- and 3'-primers were added, respectively, followed by 10 cycles of polymerase chain reaction (PCR) And converted to double-stranded DNA. The 5'- and 3'-primers were synthesized in bioneer (Daejeon, Korea) as the following sequence.

DNA library (N40, 40 random nucleotide sequence): 5'-GGGAGAGCGGAAGCGTGCTGGGCCN40CATAACCCAGAGGTCGATGGATCCCCCC-3 ',

5'-primer (SEQ ID NO: 1): 5'-GGTAATACGACTCACTATAGGGAGAGCGGAAGCGTGCTGGG-3 ', and

3'-primer (SEQ ID NO: 2): 5'-GGGGGGATCCATCGACCTCTGGGTTATG-3.

A 2'-fluoro-pyrimidine-modified RNA library containing the above 40 random nucleotides, the starting material for SELEX, was amplified using a Dura-Scribe T7 transcription kit The PCR products were prepared in vitro using a transcription procedure according to the manufacturer's instructions of the manufacturer of the DuraScribe T7 Transcription Kit (Epicenter Biotechnology, Madison, Wis., USA). The transcription reaction consisted of 0.2 ug of double-stranded DNA template with T7 promoter, 5 mM ATP, 5 mM GTP, 5 mM 2'-F-dCTP, 5 mM 2'-F-dUTP, 10 mM DTT, 20 [mu] l dura- scribe T7 reaction solution (DuraScribe T7 reaction buffer) containing a rib T7 enzyme mixture (DuraScribe T7 enzyme mix) was reacted overnight at 37 ° C. Then, the mixture was treated with DNase I (Epi Center Biotechnology) at 37 ° C for 15 minutes, and the RNA separated by electrophoresis in 8% acrylamide / 7 M urea gel Was extracted with phenol / chloroform extraction, ethanol precipitation and elution buffer [500 mM ammonium acetate, 1 mM EDTA, 0.2% (w / v) Sodium dodecyl sulfate (SDS), pH 0.7], and re-obtained. Library RNA pools or pools of RNA for each SELEX cycle were incubated with 200 μl of binding buffer to remove RNA binding His-GRO-α, His-GRO-β or magnetic beads A 200 nM recombinant human His-GRO-alpha or His-GRO-beta protein was added to a binding buffer [30 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1.5 mM MgCl2, 2 mM DTT, And 10 ul of MagneHis Ni particles (Promega, Madison, Wis., USA) at room temperature for 30 minutes. The pretreated RNA pool was reacted at room temperature for 30 minutes with stirring to perform binding assay with 100 nM His-IL-8 protein (feldan), and the resulting RNA-IL-8 complex was reacted with beads And a precipitate is formed. The precipitate formed was washed four times with binding buffer and treated with 200 ug / ml proteinase K at 37 캜 for 1 hour to elute RNA bound to IL-8 protein. The reassorted RNA was amplified by RT-PCR and then transcribed in vitro to generate an RNA pool for performing the next SELEX. The concentration of His-IL-8 was doubled every 5 times for more stringent conditions, and after 15 successive runs, the selected RNA was amplified by RT-PCR. The amplified PCR product was cloned into pcDNA3 (Invitrogen, Carlsbad, CA, USA) and then transformed into E. coli (DH5?).

As a result, 15 IL-8-specific RNAs (SEQ ID NOS: 11 to 21) were obtained as shown in Fig. 1, and as a result of IL-8-specific binding measurement of the obtained RNA pool, SELEX (FIG. 1A). As shown in FIG. 1B, the obtained RNA was divided into several groups according to the sequence similarity (FIG. 1B).

<1-2> Radiation Labeled RNA  Coupled measurement IL -8 and check binding

In order to confirm that the aptamer obtained in the above Example <1-1> had a specific binding force specifically to IL-8, the radiolabeled RNA binding assay was performed.

Specifically, 1 ug RNA was dephosphorylated by treatment with 20 U CIAP (TaKaRa, Tokyo, Japan) at 37 캜 for 30 minutes, and the dephosphorylated RNA was purified by phenol / chloroform extraction and ethanol elution. Purified RNA was treated with 10 U T4 polynucleotide kinase for 30 min at 37 ° C and phosphorylated with ³² P-labeled γ-ATP (PerkinElmer, Waltham, MA, USA) . Then, a binding assay was performed in the same manner as in Example <1-1>, except that 50 nM library and IL-8-specific RNA (unlabeled: labeled = 10: 1) mu] M binding buffer containing 200 [mu] M His-GRO- [alpha], His-GRO- [beta], or His-IL-8 protein and 10 ul magnetic Ni particles. The IL-8-binding RNA eluted with the proteinase K was assayed by measuring the counter per minute (cpm) using a scintillation counter (Beckman, Coulter, CA, USA).

As a result, the # 8 aptamer specifically binds to His-IL-8 and does not bind to other CXC-chemokines His-GRO-a and His-GRO- (Fig. 1C). Therefore, the # 8 aptamer of the present invention was named 8A-W (SEQ ID NO: 16).

<1-3> IL Lt; RTI ID = 0.0 &gt; Of app tamer  Shape ( truncated form Production

IL-8 8A-W (SEQ ID NO: 16) was serially truncated from the 5 'and 3' ends to identify minimal sequences that bind to IL-8 and inhibit its function. For this purpose, PCR was performed by a method well known in the art by combining primers of 1F (SEQ ID NO: 3), 1R (SEQ ID NO: 4), 2F (SEQ ID NO: 5) and 2R (8A-1F1R, 8A-2F2R, 8A-2F1R, 8A-44 (8A-1F2R, SEQ ID NO: 23)]. (8A-1F2R), SEQ ID NO: 23) using the primers 35F (SEQ ID NO: 7) / 35R (SEQ ID NO: 8) and 30F (SEQ ID NO: 9) / 30R -35 (SEQ ID NO: 24), and 8A-30 (SEQ ID NO: 25).

At this time, the following primers were used for PCR:

1F (SEQ ID NO: 3): 5'-GGTAATACGACTCACTATAGGGGGCTTATCATTCCATTTAGTG-3 '

1R (SEQ ID NO: 4): 5'-GGGTTATGTCTGATGGGAAG-3 '

2F (SEQ ID NO: 5): 5'-GGTAATACGACTCACTATAGGGTCCATTTAGTGTTATGATAACC-3 '

2R (SEQ ID NO: 6): 5'-TGATGGGAAGGTTATCATAAC-3 '

35F (SEQ ID NO: 7): 5'-GGTAATACGACTCACTATAGGGGGCTTATCATTCCATTTAGT-3 '

35R (SEQ ID NO: 8): 5'-GGTTATCATAACACTAAATGGAATGATAAGCCC-3 '

30F (SEQ ID NO: 9): 5'-GGTAATACGACTCACTATAGGGTTATCATTCCATTTAGTGTTA-3 ', and

30R (SEQ ID NO: 10): 5'-TTATCATAACACTAAATGGAATGATAACC-3 '.

As a result, as shown in Fig. 2, the secondary structure of the RNA of the entire sequence and its cleaved RNA was determined using the M fold program (http://mfold.rna.albany.edu/?=mfold ). 8A-W (SEQ ID NO: 16) consists of four stem / loops and 8A-1F1R and 8A-44 (8A-1F2R, The longest main stem / ring part of the. However, 8A-2F1R and 8A-2F2R showed a completely different form from the template 8A-W, and thus the longest major stem / loop structure of 8A-W (SEQ ID NO: 16) .

< Example  2 > 8A-44 (SEQ ID NO: 23) IL -8-induced neutrophil migration ( migration ) And ERK  Inhibit phosphorylation

<2-1> Isolation of neutrophils from human blood

Neutrophils were isolated from whole human blood by density gradient method using Histopaque (Sigma Aldrich, St. Louis, Mo., USA).

Specifically, the venous blood was carefully stacked on the same amount of histopaque solution and centrifuged at 1,500 rpm for 30 minutes at room temperature, and the neutrophil membrane (white membrane between red blood cells and histopaque) was collected. Hypotonic lysis was performed to remove erythrocytes from neutrophil membranes. First, 30 ml of distilled water was added and gently pipetted, and PBS was added to the neutrophil membrane so that the final amount was 10 ml. After 1 minute, 10 ml of 4% NaCl was added to the solution, mixed well, and centrifuged at 1,200 rpm for 7 minutes at room temperature. To obtain neutrophils, the cells were washed with RPMI1640 medium (Weljin, Daegu, Korea) and cultured on a plate using RPMI1640 medium supplemented with 10% FBS.

<2-2> Measurement of neutrophil migration migration assay )

A neutrophil migration assay was performed to determine if aptamers specifically binding to IL-8 produced by the method of Example 1 inhibited the function of IL-8.

Specifically, the various concentrations of RNA aptamer and 100 ng / ml IL-8 (R & D system) obtained in Example 1 were added to serum-free RPMI 1640 medium for 30 minutes at room temperature and pretreated with stirring, 2-1 > were collected and suspended in serum-free RPMI 1640 medium. The wells in the lower part of each of IL-8 into the RNA aptamer mixture, the 2 × 10 ⁶ cells were placed in the transwell insert part (transwell insert). After incubation at 37 ° C for 2 hours, the insert was removed from the chamber, WST-1 (Takara) was added to the wells below, and reacted at 37 ° C for 1 hour. Neutrophil migration was assessed by measuring the absorbance of formazan in the sample at 450 nm using a microplate reader.

As a result, the IL-8-induced neutrophil migration was detected as concentration-dependent by 8A-W (SEQ ID NO: 16), 8A-1F1R, or 8A-1F2R (8A-44, SEQ ID NO: 23) , But it was not affected by 8A-2F2R or 8A-2F1R. Based on the above results, it was confirmed that an aptamer specifically binding to IL-8 having the sequence of 21-66 nucleotides (8A-44, SEQ ID NO: 23) is sufficient to bind to IL-8 and inhibits IL-8 activity Respectively.

<2-3> IL 8 &lt; / RTI &gt; (SEQ ID NO: 23) Of app tamer  Confirm binding

In order to confirm the binding ability of 8A-44 (8A-1F2R, SEQ ID NO: 23) consisting of 44 members of the 21-66 nucleotide sequence sufficient to bind to IL-8 to IL-8 in the result of Example <2-2> , A complementary sequence of 8A-44 (SEQ ID NO: 23) (8A-44COM, SEQ ID NO: 26) as a negative control and a ProteOn XPR36 assay using surface plasmon resonance (SPR) Binding capacity to IL-8 was measured via a radiolabeled rat, Hurricles, CA, USA).

Specifically, the assay was used with a slight change in the conventional method. The His-tagged IL-8 protein was added to 10 mM sodium acetate buffer (pH 8.0) so as to have a concentration of 1 uM and the protein was immobilized on a GLM sensor chip by amine coupling. 30 < / RTI > ul / ml for 5 minutes. After the chip surface was inactivated with 1 M ethanolamine, the cells were washed with PBST running buffer (10 mM sodium phosphate, pH 7.4, 150 mM NaCl, and 0.005% Tween 20) Each diluted 8A-44 (SEQ ID NO: 23) aptamer was flowed vertically for 1 minute at a flow rate of 100 ul / ml at concentrations of 20, 10, 5, 2, 1, and 0 nM. The dissociation was observed for 10 minutes and 12.5 mM NaHO was flowed into the regenerator before the aptamer was drained. All interconnection sensorgrams were collected, processed and analyzed using ProteOn Manager software and channel reference [interspot] - and double reference [0 nM, blank (blank) Subtracted sensorgrams are simply fitted to the Langmuir model at 1: 1 and the association ( k _a ) and dissociation ( k _d ) (parameters). The equilibrium dissociation constants ( K _D ) were calculated by Equation (1) below. The R max (Rmax) of all sensorgrams is less than 200 resonance units (RUs), while the calculated χ ² value of each aptamer is about 5 RU.

As a result, as shown in FIG. 4 and Table 1, the K _D value of 8A-44 (SEQ ID NO: 23) against IL-8 showed a high binding force of 5.15 nM, but a high k _d value (2.73 × 10 ^-2 s- ¹ ), the bound 8A-44 (SEQ ID NO: 23) was cleaved at a rapid rate. The control, 8A-44COM (SEQ ID NO: 26) did not bind to IL-8 (results not shown).

Parameter k _a k _d K _D unit 1 / Ms 1 / s M 8A-44 5.30 × 10 ⁶ 2.73 x 10 ^-2 5.15 × 10 ^-9

&Lt; 2-4 > 8A-44 (SEQ ID NO: 23) Of app tamer , IL -8 Movement of induced neutrophils and ERK  Identification of inhibition of phosphorylation

8A-44 (SEQ ID NO: 23) aptamer was inhibited by IL-8 induced neutrophil migration using the same method as in Example <2-2>, and 8A-44 (SEQ ID NO: 23) Western blot was performed to confirm that it inhibited ERK phosphorylation.

Specifically, 8A-44 (SEQ ID NO: 23) of 1 to 1000 nM and 100 ng / ml of IL-8 are pretreated, and neutrophils are prepared according to the preparation method of the neutrophil preparation of Example <2-2>. An RNA-IL-8 mixture is added to 2 x 10 ⁶ cells / ml neutrophils and phosphorylation is performed at room temperature for 2 minutes and 30 seconds. The phosphorylation reaction was stopped by adding an SDS sample buffer. The mixture was incubated with anti-phospho-p44 / 42 (p-ERK1 / 2), p44 / 42 MAPK (Santa Cruz Biotechnology, Santa Cruz, Calif., USA), which is an immunosuppressive agent (ERK1 / 2), phosphoric acid-Akt, Akt (CellSignaling Technologies, Danver, MA, USA) Western blotting was performed in a manner well known in the art.

As a result, as shown in Fig. 5A, the migration of neutrophils induced by IL-8 was significantly decreased in a concentration-dependent manner by the treatment of 8A-44 (SEQ ID NO: 23), whereas the control group 8A-44COM No. 26) showed no effect. In addition, as shown in Fig. 5B, it was confirmed that 8A-44 (SEQ ID NO: 23) inhibited ERK activity induced by IL-8 at a high concentration as opposed to 8A-44COM (SEQ ID NO: 26). The results indicate that 8A-44 (SEQ ID NO: 23) specifically binds human IL-8 and inhibits IL-8 induced intracellular signaling.

< Example  3 > 8A-44 (SEQ ID NO: 23) Of app tamer  Cancer cell-derived IL -8-induced invasion of oral cell line ( invasion ) Suppression

It has been shown that levels of IL-8 are increased in patients with invasive oral cancer. Therefore, it was confirmed that 8A-44 (SEQ ID NO: 23) of the present invention inhibits the invasion of IL-8-induced oral cancer cells by inhibiting IL-8.

Specifically, cultured under the condition of a human mouth cancer cell line HSC-3, and 686-LN-e is added to DMEM 10% FBS (Thermo Fisher Scientific dipik, Waltham, USA) in the medium (weljin), 37 ℃, 5% CO 2 Respectively. In addition, intrusion verification was performed in a 6.5 mm transwell chamber with a 8.0 um pore filter (Costa). The inner filter was pre-processed with 1 mg / ml cold-growth factor-reduced Matrigel matrix (BD Biosciences, Franklin Lake, NJ, USA) Lt; / RTI &gt; 8A-44 (SEQ ID NO: 23) aptamer or a 1000 ng / ml IL-8 neutralizing antibody at concentrations of 0.1, 1 and 5 uM were cultured in a medium (HSC-3 and 686-LN- conditioned media and pretreated for 30 min at room temperature. The cells were trypsinized to obtain a cell line, which was resuspended in serum-free DMEM. 8A-44 (SEQ ID NO: 23) aptamers in the medium were placed in the lower part of each well, and 1 × 10 ⁵ cells were added to each of the transwell inserts. After reacting at 37 ° C for 24 hours, the insert was fixed with Diff-Quick solution (SISMAX, Kobe, Japan) and stained. The stained cells were photographed with a phase contrast microscope (Olympus, Tokyo, Japan) and counted in five microscope field cells.

As a result, as shown in Fig. 6, the culture-induced intrusion of the HSC-3 and 686-LN-e cell lines showed a significant decrease in the concentration of 8A-44 (SEQ ID NO: 23) aptamers, -44 COM (SEQ ID NO: 26) had no effect. The results show that 8A-44 (SEQ ID NO: 23) aptamer can bind to purified IL-8 and naturally secreted cell-derived IL-8 and its function is inhibited.

< Example  4> cleavage mutation ( truncated mutation ) 8A-30 (SEQ ID NO: 24) and 8A-35 (SEQ ID NO: 25) Of app tamer IL Confirmation of bond to -8

Based on the above results, it was confirmed that 8A-44 (SEQ ID NO: 23) contained a sequence sufficient to specifically bind IL-8 and inhibit its function. Thus, it was confirmed that a minimal Oligonucleotides were identified. For this, 8A-35 (35mer, SEQ ID NO: 24) and 8A-30 (30mer, SEQ ID NO: 25) were used in the truncated form of 8A-44 (SEQ ID NO: 23). 8A-35 (SEQ ID NO: 24) is a form in which 9 nucleotides are truncated from the 3'end of 8A-44 (SEQ ID NO: 23) Terminus from the 3 'and 3' ends, and the stem / loop structure of both of the two aptamers is conserved. Using the above two aptamers, it was confirmed by binding with high binding force to IL-8 using the method of Example <2-3>.

As a result, as shown in Figure 7 and Table 2, 8A-35 k _a value of (SEQ ID NO: 24) is similar to 8A-44 (SEQ ID NO: 23) Although ^{1.71 × 10 6 M -1 s -1} , k d (SEQ ID NO: 24) strongly binds to IL-8 with a K _D value of 46.3 pM, showing a 7.92 × 10 ^-5 s ^-1 decrease by about 300-fold compared to 8A-44 . The k _a and k _d values of 8A-30 (SEQ ID NO: 25) are 4.31 × 10 ⁶ M ^-1 s ^-1 and 5.79 × 10 ^-2 s ^-1 , respectively, and have a K _D value of 13.5 nM, This showed a slight change compared to 8A-44 (SEQ ID NO: 23).

Parameter k _a k _d K _D unit 1 / Ms 1 / s M 8A-35 1.71 × 10 ⁶ 7.92 × 10 ^-5 4.63 × 10 ^-9 8A-30 4.31 × 10 ⁶ 5.79 x 10 ^-2 1.35 x 10 ^-8

< Example  5 (SEQ ID NO: 23), 8A-35 (SEQ ID NO: 24) and 8A-30 ERK  And Akt  Signal active, and Ca ^{2 +}  Mobilization ( mobilization ) Suppression

In order to confirm whether the truncated forms of the aptamers maintained the IL-8 inhibitory effect, the migration of IL-8-induced neutrophils and the migration of neutrophils using the methods of Examples <2-2> and <2-4> ERK inhibition, and also confirmed the inhibition of Ca ^{2 +} mobilization in neutrophils.

Specifically, the fluorescent Ca ^{2 +} indicator measurement of Ca ^{2 +} mobilization in neutrophils incubated with (indicator) is a fluorescence spectral photometer; was used (spectrofluorophotometer, Shimadzu RF-5301PC, Kyoto, Japan). The isolated neutrophils were reacted with 4 μM fura-2 AM in serum-free RPMI 1640 medium for 40 minutes at 37 ° C, washed 3 times, and resuspended in serum-free RPMI 1640 medium. (SEQ ID NO: 23), 8A-35 (SEQ ID NO: 24) and 8A-30 (SEQ ID NO: 25) of the present invention were incubated at room temperature For 30 minutes. Furanyl -2 The AM is added to the cell line is 8 × 10 rokke solution with no Ca ^{2 +} at a density of ^{5 cells (Locke's solution) [} 154 mM NaCl, 5.6 mM KCl, 1.2 mM MgCl 2, 5 mM HEPES (pH 7.3), 10 mM glucose, and 0.2 mM EGTA] and pretreated with a mixture of IL-8 (20 ng / ml) and aptamer (2, 20 nM). The change in fluorescence ration (340/380 nm) was confirmed to be a dual excitation wavelength of 340 nm and 380 nm and an emission wavelength of 500 nm.

As a result, IL-8-induced neutrophil migration inhibition of 8A-35 (SEQ ID NO: 24) and 8A-30 (SEQ ID NO: 25) Dependent. In addition, as shown in Figure 8B, the ability to inhibit IL-8-induced ERK and Akt signaling was also inhibited by 8A-35 (SEQ ID NO: 25) and 8A-30 (SEQ ID NO: 26) in a concentration-dependent manner. In addition, as shown in Figure 9, when 8A-44 (SEQ ID NO: 24), 8A-35 (SEQ ID NO: 25) and 8A- in Ca ^{2 +} movable upset decreases, especially 8A-35 (SEQ ID NO: 26) cells were blocked completely Ca ^{2 +} mobilization in the 20 nM low concentrations. The results demonstrate that 8A-44 (SEQ ID NO: 24), 8A-35 (SEQ ID NO: 25) and 8A-30 (SEQ ID NO: 26) bind to human IL- ). &Lt; / RTI &gt;

< Manufacturing example  1> Preparation of pharmaceutical preparations

<1-1> Sanje  Produce

2 g of the RNA aptamer of the present invention

Lactose 1 g

The above components were mixed and packed in airtight bags to prepare powders.

<1-2> Preparation of tablets

100 mg of the RNA aptamer of the present invention

Corn starch 100 mg

100 mg of milk

2 mg of magnesium stearate

After mixing the above components, tablets were prepared by tableting according to a conventional method for producing tablets.

&Lt; 1-3 > Preparation of capsules

100 mg of the RNA aptamer of the present invention

Corn starch 100 mg

100 mg of milk

2 mg of magnesium stearate

After mixing the above components, the capsules were filled in gelatin capsules according to the conventional preparation method of capsules.

&Lt; 1-4 >

1 g of the RNA aptamer of the present invention

Lactose 1.5 g

Glycerin 1 g

0.5 g of xylitol

After mixing the above components, they were prepared so as to be 4 g per one ring according to a conventional method.

<1-5> Preparation of granules

150 mg of the RNA aptamer of the present invention

Soybean extract 50 mg

200 mg of glucose

600 mg of starch

After mixing the above components, 100 mg of 30% ethanol was added and the mixture was dried at 60 캜 to form granules, which were then filled in a capsule.

< Manufacturing example  2> Manufacturing Method of Injection Solution

An injection solution containing 100 nM of RNA aptamer of the present invention as an active ingredient was prepared by the following method.

1'-chloro-3,2'-dihydroxacycone or 5'-chloro-2,3'-dihydroxystearic acid hydrochloride, 0.6 g of sodium chloride and 0.1 g of ascorbic acid were dissolved in distilled water to give 100 Ml. This solution was placed in a bottle and sterilized by heating at 20 DEG C for 30 minutes.

The components of the injection solution are as follows.

5 [mu] g (100 nM) of the RNA aptamer of the present invention

5'-chloro-3,2'-dihydroxacycone, or

5'-chloro-2, 3'-dihydroxystearic acid hydrochloride 1 g

0.6 g of sodium chloride

0.1 g ascorbic acid

Determination of distilled water

<110> National Cancer Center <120> Interleukin-8 aptamers and uses thereof <130> 12P-12-07 <160> 31 <170> Kopatentin 1.71 <210> 1 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> 5'-primer <400> 1 ggtaatacga ctcactatag ggagagcgga agcgtgctgg g 41 <210> 2 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> 3'-primer <400> 2 ggggggatcc atcgacctct gggttatg 28 <210> 3 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> 1F <400> 3 ggtaatacga ctcactatag ggggcttatc attccattta gtg 43 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 1R <400> 4 gggttatgtc tgatgggaag 20 <210> 5 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> 2F <400> 5 ggtaatacga ctcactatag ggtccattta gtgttatgat aacc 44 <210> 6 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> 2R <400> 6 tgatgggaag gttatcataa c 21 <210> 7 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> 35F <400> 7 ggtaatacga ctcactatag ggggcttatc attccattta gt 42 <210> 8 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> 35R <400> 8 ggttatcata acactaaatg gaatgataag ccc 33 <210> 9 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> 30F <400> 9 ggtaatacga ctcactatag ggttatcatt ccatttagtg tta 43 <210> 10 <211> 29 <212> DNA <213> Artificial Sequence <220> <30> <400> 10 ttatcataac actaaatgga atgataacc 29 <210> 11 <211> 92 <212> RNA <213> Artificial Sequence <220> <223> # 2 <400> 11 gggagagcgg aagcgugcug ggccucccga cgnuuaauuu gguacguuuc aguuaguauu 60 cuaccauaac ccagaggucg auggaucccg gg 92 <210> 12 <211> 94 <212> RNA <213> Artificial Sequence <220> <223> # 4, 11, 12 <400> 12 gggagagcgg aagcgugcug ggccacggcu ugaagcucgc aauuauaaau guuccauuca 60 guguuacaua acccagaggu cgauggaucc cggg 94 <210> 13 <211> 94 <212> RNA <213> Artificial Sequence <220> <223> # 5, 17, 19 <400> 13 gggagagcgg aagcgugcug ggccacggcu ugaagcucgc aaucauaaau guuccauuca 60 guguuccaua acccagaggu cgauggaucc cggg 94 <210> 14 <211> 91 <212> RNA <213> Artificial Sequence <220> <223> # 6 <400> 14 gggagagcgg aagcgugcug ggccaucuug cgcuccungc auccauuuug uguuugcuuu 60 gcucauaacc cagaggucga uggaucccgg g 91 <210> 15 <211> 86 <212> RNA <213> Artificial Sequence <220> <223> # 7 <400> 15 gggagagcgg aagcgugcug ggccuuauca uuuccauuua guguuaugau aacuucucca 60 uaacccagag gucgauggau cccggg 86 <210> 16 <211> 91 <212> RNA <213> Artificial Sequence <220> &Lt; 223 ># 8 (8A-W) <400> 16 gggagagcgg aagcgugcug ggcuuaucau uccauuuagu guuaugauaa ccuucccauc 60 agacauaacc cagaggucga uggaucccgg g 91 <210> 17 <211> 92 <212> RNA <213> Artificial Sequence <220> <223> # 9 <400> 17 gggagagcgg aagcgugcug ggccuuauca uuccauuuau uguuaugaua acuuuuccau 60 cagacauaac ccagaggucg auggaucccg gg 92 <210> 18 <211> 92 <212> RNA <213> Artificial Sequence <220> <223> # 14 <400> 18 gggagagcgg aagcgugcug ggccuuauca uuccauuuag uguuaugaua acuucuccau 60 cagacauaac ccagaggucg auggaucccg gg 92 <210> 19 <211> 93 <212> RNA <213> Artificial Sequence <220> <223> # 15 <400> 19 gggagagcgg aagcgugcug ggccuuauca uuccauuuag ggguuaugau aacuucucca 60 ucagacauaa cccagagguc gauggauccc ggg 93 <210> 20 <211> 93 <212> RNA <213> Artificial Sequence <220> <223> # 20 <400> 20 gggagagcgg aagcgugcug ggcgcuuauc acuccauuua guguuaugau aacuucucca 60 ucagacauaa cccagagguc gauggauccc ggg 93 <210> 21 <211> 93 <212> RNA <213> Artificial Sequence <220> <223> # 16 <400> 21 gggagagcgg aagcgugcug ggccuugugu gcguuccgau auaucaaucg auguuccaca 60 uaauucauaa cccagagguc gauggauccc ggg 93 <210> 22 <211> 27 <212> RNA <213> Artificial Sequence <220> <223> core sequences <400> 22 uuaucauucc auuuaguguu augauaa 27 <210> 23 <211> 44 <212> RNA <213> Artificial Sequence <220> <223> 8A-44 <400> 23 gggggcuuau cauuccauuu aguguuauga uaaccuuccc auca 44 <210> 24 <211> 35 <212> RNA <213> Artificial Sequence <220> <223> 8A-35 <400> 24 gggggcuuau cauuccauuu aguguuauga uaacc 35 <210> 25 <211> 30 <212> RNA <213> Artificial Sequence <220> <223> 8A-30 <400> 25 ggguuaucau uccauuuagu guuaugauaa 30 <210> 26 <211> 44 <212> RNA <213> Artificial Sequence <220> <223> 8A-44COM <400> 26 gggccgaaua guaagguaaa ucacaauacu auuggaaggg uagu 44 <210> 27 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> 8A-44_N <400> 27 gggggc 6 <210> 28 <211> 11 <212> RNA <213> Artificial Sequence <220> <223> 8A-44_C <400> 28 ccuucccauc a 11 <210> 29 <211> 54 <212> RNA <213> Artificial Sequence <220> <223> 8A-1F1R <400> 29 gggggcuuau cauuccauuu aguguuauga uaaccuuccc aucagacaua accc 54 <210> 30 <211> 44 <212> RNA <213> Artificial Sequence <220> <223> 8A-2F1R <400> 30 ggguccauuu aguguuauga uaaccuuccc aucagacaua accc 44 <210> 31 <211> 34 <212> RNA <213> Artificial Sequence <220> <223> 8A-2F2R <400> 31 ggguccauuu aguguuauga uaaccuuccc auca 34

Claims (12)

An RNA aptamer consisting of 30 RNAs specifically binding to IL-8 (interleukin-8) consisting of the nucleotide sequence of SEQ ID NO: 25.
An RNA aptamer consisting of 35 RNAs specifically binding to IL-8 consisting of the nucleotide sequence of SEQ ID NO: 24.
An RNA aptamer consisting of 44 RNAs specifically binding to IL-8 consisting of the nucleotide sequence of SEQ ID NO: 23.
4. The RNA aptamer according to any one of claims 1 to 3, wherein the RNA aptamer binds to IL-8 and inhibits IL-8 induced function.
4. The RNA aptamer according to any one of claims 1 to 3, wherein the RNA aptamer has one main stem and one loop structure.
4. The RNA aptamer according to any one of claims 1 to 3, wherein the RNA aptamer has RNase A resistance.
4. The RNA aptamer according to any one of claims 1 to 3, wherein the RNA aptamer comprises one or more variants selected from the group consisting of:
-OH group in the 2 ' carbon position of the nucleotide in the nucleotide is selected from the group consisting of -Me (methyl), -OMe, -NH2, -F (fluorine), -O-2-methoxyethyl- A modification by substitution with ethyl (methylthioethyl), -O-3-aminopropyl, -O-3-dimethylaminopropyl, -ON-methylacetamido or -O-dimethylamidooxyethyl;
A modification in which the oxygen in the sugar structure in the nucleotide is replaced with sulfur; And
The bond between the nucleotides is transformed into a phosphorothioate, borano phosphophate or methyl phosphonate bond.
A macromolecular material selected from the group consisting of an RNA aptamer according to any one of claims 1 to 3 and one selected from the group consisting of polyethylene glycol (PEG), diacylglycerol (DAG) and monoclonal antibody Bonded aptamer - complex.
delete A pharmaceutical composition for preventing and treating oral cancer, which comprises an aptamer according to any one of claims 1 to 3.
A pharmaceutical composition for antiinflammation comprising an aptamer according to any one of claims 1 to 3. delete

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