RU2789087C2 - Aptamers for use in inhibition and/or suppression of tlr9 activation - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to the field of biotechnology, specifically to aptamers; it can be used in medicine for use in therapy of a subject for inhibition or suppression of TLR9 activation. The used aptamer consists of a nucleic acid sequence SEQ ID No. 1 (GGT TGG TGT GGT TGG).

EFFECT: invention provides inhibition of TLR9 activation in a subject’s cell by specific interaction of an aptamer with TLR9 receptor.

9 cl, 5 dwg, 3 ex

Description

FIELD OF TECHNOLOGY

The present invention relates to novel aptamer molecules for use in treating a subject by inhibiting or suppressing TLR9 activation in a cell, a method for inhibiting or suppressing TLR9 activation in a cell using such aptamer molecules, a pharmaceutical composition and a kit containing such aptamer molecules, and the use of the aptamer molecules. to inhibit or suppress TLR9 activation.

BACKGROUND OF THE INVENTION

Toll-like receptors (TLRs) are present on many cells of the immune system and have been shown to be involved in the innate immune response (Hornung, V. et al., (2002) J. Immunol. 168:4531-4537). In vertebrates or mammals, this family consists of proteins called TLR1-TLR10, which are known to recognize pathogen-associated molecular patterns (PAMPs) in bacteria, fungi, parasites, and viruses (Poltorak, a. et al. (1998) Science 282:2085 -2088, Underhill, D.M., et al (1999) Nature 401:811-815, Hayashi, F. et al (2001) Nature 410:1099-1103, Zhang, D. et al (2004) Science 303: 1522-1526; Meier, A. et al. (2003) Cell. Microbial 5:561 570; Campos, M. A. et al. (2001) J. Immunol. 167: 416-423; Hoebe, K. et al. ( 2003) Nature 424: 743-748; Lund, J. (2003) J. Exp. Med. 198:513-520; Heil, F. et al. (2004) Science 303:1526-1529; Diebold, S.S., et al (2004) Science 303:1529-1531 Hornung, V. et al (2004) J Immunol 173:5935-5943).

TLRs are the key means by which mammals recognize and elicit an immune response to foreign molecules, and also provide the means by which innate and adaptive immune responses are linked (Akira, S. et al. (2001) Nature Immunol. 2:675-680; Medzhitov, R. (2001) Nature Rev. Immunol. 1:135-145). TLRs have also been shown to play a role in the pathogenesis of many diseases, including autoimmunity, infectious disease, and inflammation (Cook, D.N. et al. (2004) Nature Immunol. 5:975-979), and regulation of TLR-mediated activation using suitable agents can provide a remedy for the disease. In addition, activation of TLR9 in plasmacytoid dendritic cell (PDC) progenitors and B cells by self nucleic acids plays an essential role in the pathogenesis of systemic lupus erythematosus (SLE).

As part of the defense against extra- and intracellular pathogens, TLRs are found on the cell surface, but also inside the cell. While TLR2, 4, 5, and 6 are cell surface receptors that protect the cell from extracellular pathogens, TLR3, 7, 8, and 9 are generally found inside the cell to maintain protection against intracellular pathogens (Dowling and Dellacasagrande, 2016) Dowling , J.K. and Dellacasagrande, J. (2016) Methods Mol. Biol. Clifton NJ 1390, 3-27), (Diebold, S.S. et al. (2004) Science 303:1529-1531; Liew, F. et al. (2005) Nature 5:446-458; Hemmi H et al. (2002 ) Nat Immunol 3:196-200; Jurk M et al., (2002) Nat Immunol 3:499; Lee Jet al. (2003) Proc. Natl. Acad. Sci. USA 100:6646-6651); (Alexopoulou, L. (2001) Nature 413:732-738).

Several unmethylated CpG motifs present in bacterial and synthetic DNA have been shown to activate the immune system and induce antitumor activity through TLR9 (Tokunaga T et al., J. Natl. Cancer Inst. (1984) 72:955-962; Shimada S, et al., Jpn. H cancer Res., 1986, 77, 808-816 amamoto S, et al., Jpn. J. Cancer Res., 1986, 79, 866-73). In contrast, mammalian DNA generally lacks immunostimulatory activity, apparently due to the low frequency of CG sequences and because most CG sequences have methylated cytosine. Thus, it appears that cells of the mammalian immune system distinguish between bacterial DNA and self-DNA via the TLR9 receptor.

Other studies using antisense oligonucleotides containing CpG dinucleotides show stimulation of immune responses (Zhao Q, et al. (1996) Biochem. Pharmacol. 26:173-182). Subsequent studies have demonstrated that TLR9 recognizes unmethylated CpG motifs present in bacterial and synthetic DNA (Hemmi, H. et al. (2000) Nature 408:740-745).

Other modifications of CpG-containing thiophosphate oligonucleotides can also affect their ability to act as immune response modulators via TLR9 (see, for example, Zhao et al., Biochem. Pharmacol. (1996) 51:173-182; Zhao et al. ( 1996) Biochem Pharmacol 52:1537-1544 Zhao et al (1997) Antisense Nucleic Acid Drug Dev 7:495-502 Zhao et al (1999) Bioorg Med Chem Lett 9:3453-3458; Zhao et al (2000) Bioorg Med Chem Lett 10:1051-1054 Yu, D et al (2000) Bioorg Med Chem Lett 10:2585-2588 Yu D et al (2001) Bioorg Med Chem Lett 11:2263-2267 and Kandimalla, E et al (2001) Bioorg Med Chem 9:807-813).

In addition, structure-activity association studies have provided the identification of synthetic motifs and novel DNA-based compounds that induce specific immune response profiles that differ from those derived from unmethylated CpG dinucleotides (Kandimalla, E. et al. (2005) Proc Natl Acad Sci US A 102:6925-6930 Kandimalla, E. et al (2003) Proc Nat Acad Sci US A 100:14303-14308 Cong, Y et al (2003 ) Biochem Biophys Res Commun 310: 1133-1139 Kandimalla, E et al (2003) Biochem Biophys Res Commun 306:948-953 Kandimalla E et al (2003) Nucleic Acids Res 31:2393-2400; Yu, D. et al. (2003) Bioorg. Med. Chem. 11:459-464; Bhagat, L. et al. (2003) Biochem. Biophys. Res. Commun. 300:853 -861 Yu, D. et al (2002) Nucleic Acids Res.30:4460-4469 Yu, D. et al (2002) J. Med Chem.45:4540-4548 Yu, D. et al (2002) Biochem Biophys Res Commun 297:83-90 Kandimalla E et al (2002) Bioconjug Chem 13:966-974 Yu D et al (2002) Nucleic Acids R es. 30:1613-1619; Yu, D. et al. (2001) Bioorg. Med. Chern. 9:2803-2808; Yu, D. et al. (2001) Bioorg. Med. Chem. Lett. 11:2263-2267; Kandimalla, E. et al. (2001) Bioorg. Med. Chem. 9:807-813; Yu, D. et al. (2000) Bioorg. Med. Chem. Lett. 10:2585-2588; Putta, M. et al. (2006) Nucleic Acids Res. 34:3231-3238).

While TLR activation is involved in the formation of the immune response, uncontrolled stimulation of the immune system through TLR can exacerbate certain diseases, for example, in immunocompromised subjects. Thus, in cases of generally or specifically increased immune response caused by TLR9 activation, the use of TLR9 antagonists may be desirable.

In recent years, several groups have shown the use of synthetic oligodeoxyoligonucleotides (ODNs) acting as inhibitors of inflammatory cytokines (Lenert, P. et al. (2003) DNA Cell Biol. 22(10):621-631).

Using some synthetic ODNs, Lenert et al. reported the ability to produce inhibitory ODNs (Lenert, P. et al. (2003) DNA Cell Biol. 22(10):621-631). These inhibitory ODNs require two triplet sequences, a proximal "CCT" triplet and a distal "GGG" triplet. In addition to these triplet-containing inhibitory ODNs, several groups have reported other specific DNA sequences that can inhibit TLR9-mediated activation by CpG-containing ODNs. These "inhibitory" or "suppressive" motifs are rich in poly "G" (e.g. "GGGG") or "CG" sequences, tend to be methylated, and are present in the DNA of mammals and some viruses (see, e.g., Chen, Y., et al., Gene Ther. 8: 1024-1032 (2001); Stunz, L. L., Eur. J. Immunol. 32: 1212-1222 (2002).

Duramad, O., et al., J. Immunol., 174: 5193-5200 (2005) and US Patent Application US 2005/0239733 describe the structure of inhibitory DNA oligonucleotides containing the GGGG motif within sequences. Patole et al. demonstrate that GGGG containing ODN will suppress systemic lupus (Patole, P. et al. (2005) J. Am. Soc. Nephrol. 16:3273-3280). Additionally, Gursel, I., et al., J. Immunol., 171: 1393-1400 (2003) describe repeating TTAGGG elements that are present at high frequency in mammalian telomeres and down-regulate CpG-induced immune activation. Shirota, H., et al., J. Immunol., 173: 5002-5007 (2004) demonstrate that synthetic oligonucleotides containing the TTAGGG element mimic this activity and may be effective in the prevention/treatment of certain Th1-dependent autoimmune diseases .

US Patent Application US 11/549,048 discloses a new class of TLR antagonists that do not require a poly-G sequence. Kandimalla et al. also describes the use of these new compositions for the treatment and prevention of various diseases and disorders (11/549,048; 11/743,876; 12/140,334; 12/140,338; 12/244,199).

However, the problem remains of developing additional TLR antagonists that do not require a poly-G sequence. The number of TLR9 antagonists is still quite limited, and their efficacy and safety for use in humans and animals still needs to be confirmed. Therefore, it is desirable to have additional TLR9 antagonists that show advantageous inhibition profiles.

Such new, specially designed compounds and compositions will find applications in many clinically relevant applications, including the treatment and prevention of, for example, diseases and disorders with an immune stimulating component, as well as pain and inflammation. Such compounds and compositions are currently being tested for efficacy in asthma and allergic rhinitis or other allergic conditions (Basith S, Manavalan B, Lee G, Kim SG, Choi S. "Toll-like receptor modulators: a patent review (2006-2010) ." Expert Opin Ther Pat. Jun 2011: 927-944.).

Many more diseases have been associated with unwanted or unnatural TLR9 activation in patients. It has also been known that cardiac fibroblasts carry the TLR9 receptor and that activation of cardiac fibroblast TLR9 receptors appears to be involved in pathological conditions of the heart such as myocarditis, maintaining the pathological condition (Ohm IK, Alfsnes K, Belland Olsen M, Ranheim T , Sandanger ∅, Dahl TB, Aukrust P, Finsen AV, Yndestad A, Vinge LE "Toll-like receptor 9 mediated responses in cardiac fibroblasts" PLoS One No date: e104398.).

Blockade, suppression or inhibition of TLR9 activation in such cases may show a therapeutic effect, as has already been observed with Dynavax IRS 954 in experimental lupus (Pawar RD1, Ramanjaneyulu A, Kulkarni OP, Lech M, Segerer S, Anders HJ. "Inhibition of Toll- like receptor-7 (TLR-7) or TLR-7 plus TLR-9 attenuates glomerulonephritis and lung injury in experimental lupus." J Am Soc Nephrol. Jun 2007: 1721-1731.).

Currently, only a very limited number of aptamers are available that can be used to regulate TLR9 activation, for example, in autoimmune diseases, and their efficacy and safety for use in human patients still needs to be confirmed. Therefore, it is desirable to have additional oligonucleotide sequences that inhibit or repress TLR9 activation.

Therefore, it is an object of the present invention to provide novel antagonists for use in treating a subject by inhibiting or suppressing TLR9 activation in a cell.

In addition, another object of the present invention is to provide a method for inhibiting or suppressing TLR9 activation using novel TLR9 antagonists.

It is also an object of the present invention to provide a pharmaceutical composition and kit containing novel TLR9 antagonists.

Yet another object of the present invention is to provide the use of novel TLR9 antagonists to inhibit or suppress TLR9 activation.

DISCLOSURE OF THE INVENTION

This problem is solved by aspects of the present invention, as disclosed later in this document.

According to a first aspect of the present invention, an aptamer is provided for use in therapy of a subject by inhibiting or suppressing TLR9 activation in a cell.

In a preferred embodiment of the first aspect of the invention, the subject is a mammal, preferably the subject is a human.

In another preferred embodiment of the first aspect of the invention, the cell to be contacted or the subject to be treated demonstrates overexpression of TLR9 and/or overactivity of TLR9-mediated signaling.

In yet another preferred embodiment of the first aspect of the invention, the cell and/or subject to be treated is tested for TLR9 overexpression and/or TLR9-mediated signaling overactivity prior to inhibition of TLR9 activation in the cell and/or subject.

In a preferred embodiment of the first aspect of the invention, the cell is a glial cell, microglial cell, astrocyte, macrophage, B cell and/or dendritic cell, preferably a plasmacytoid dendritic cell.

In another preferred embodiment of the first aspect of the invention, the subject suffers from a disorder selected from an autoimmune disorder, an inflammatory disorder, an autoimmune connective tissue disease (ACTD) and/or a neurodegenerative disorder, preferably a disorder selected from, but not limited to, psoriasis, rheumatoid arthritis, universal alopecia, acute disseminated encephalomyelitis, Addison's disease, allergies, ankylosing spondylitis, antiphospholipid antibody syndrome, arteriosclerosis, atherosclerosis, autoimmune hemolytic anemia, autoimmune hepatitis, bullous pemphigoid, Chagas disease, chronic obstructive pulmonary disease, celiac disease, cutaneous), lupus erythematosus (CLE) dermatomyositis, diabetes mellitus, dilated cardiomyopathy (DCM), endometriosis, Goodpasture's syndrome, Graves' disease, Guillain-Barré syndrome, Hashimoto's disease, hidradenitis suppurativa, idiopathic thrombocytopenic purpura, inflammatory disease bowel disease, interstitial cystitis, localized scleroderma, multiple sclerosis (MS), myasthenia gravis, myocarditis, narcolepsy, neuromyotonia, pemphigus, pernicious anemia, polymyositis, primary biliary cirrhosis, rheumatoid arthritis (RA), schizophrenia, Sjögren's syndrome, systemic lupus erythematosus (SLE) ), systemic sclerosis, temporal arteritis, vasculitis, vitiligo, vulvodynia, Wegener's granulomatosis, traumatic pain, neuropathic pain, and acetaminophen toxicity.

In a preferred embodiment of the first aspect of the invention, the subject is suffering from a tumor/cancer, preferably the tumor/cancer is selected from the group consisting of breast cancer, cervical squamous cell carcinoma, gastric carcinoma, glioma, hepatocellular carcinoma, lung cancer, melanoma, cancer prostate, recurrent glioblastoma, recurrent non-Hodgkin's lymphoma, colon and rectal cancer.

In one preferred embodiment of the first aspect of the invention, the aptamer comprises the nucleic acid sequence of SEQ ID No. 1 (GGT TGG TGT GGT TGG) and/or a nucleic acid sequence that is at least 80% identical to SEQ ID No. 1.

According to a second aspect of the present invention, there is provided a method for inhibiting or suppressing TLR9 activation in a cell, comprising contacting a cell expressing TLR9 with an aptamer.

In a preferred embodiment of the second aspect of the invention, the method is performed in vitro/ex vivo .

In another preferred embodiment of the second aspect of the invention, the method further comprises the upstream step of testing the cell for TLR9 overexpression and/or TLR9-mediated signaling overactivity.

In a preferred embodiment of the second aspect of the invention, the cell is a mammalian cell, preferably the cell is a human cell.

In one preferred embodiment of the second aspect of the invention, the aptamer comprises the nucleic acid sequence of SEQ ID No. 1 (GGT TGG TGT GGT TGG) and/or a nucleic acid sequence that is at least 80% identical to SEQ ID No. 1.

According to a third aspect of the present invention, there is provided a pharmaceutical composition comprising an aptamer for use in accordance with the first aspect of the present invention and at least one pharmaceutically acceptable carrier.

According to a fourth aspect of the present invention, there is provided a kit containing at least one aptamer for use in accordance with the first aspect of the present invention and a container.

According to a fifth aspect of the present invention, the use of an aptamer as defined herein for inhibiting or suppressing TLR9 activation is provided.

In a preferred embodiment of the fifth aspect of the invention, the aptamer is used in vitro/ex vivo .

DESCRIPTION OF FIGURES

Figure 1 shows inhibition of TLR9 activation in ODN 2006 (TLR9 agonist)-stimulated HEK-Blue™ hTLR9 cells by adding increasing concentrations (in μM) of SEQ ID No. aptamer. 1 (gray bars). Potential intrinsic effects from increasing concentrations of the aptamer SEQ ID No. 1 for TLR9 activation in the absence of stimulation with the TLR9 agonist ODN 2006 (SEQ ID No. 2) were examined and shown as black bars. Changes in optical density are caused by TLR9-activated expression of alkaline phosphatase.

Figure 2 shows the effects of increasing concentrations of SEQ ID No. aptamer. 1 (in μM) on HEK-Blue™ TNF-α cells stimulated with 100 ng TNF-α (grey bars) and without TNF-α stimulation (black bars). Optical density changes are caused by TNF-α-activated expression of alkaline phosphatase.

Figure 3 shows inhibition of TLR9 activation in ODN 2006 (TLR9 agonist)-stimulated HEK-Blue™ hTLR9 cells by adding increasing concentrations (in μM) of SEQ ID No. aptamer. 3 (gray bars). Potential intrinsic effects from increasing concentrations of the aptamer SEQ ID No. 3 for TLR9 activation in the absence of stimulation with the TLR9 agonist ODN 2006 (SEQ ID No. 2) were examined and shown as black bars. Changes in optical density are caused by TLR9-activated expression of alkaline phosphatase.

Figure 4 shows the effects of increasing concentrations of SEQ ID No. aptamer. 3 (in μM) on HEK-Blue™ TNF-α cells stimulated with 100 ng TNF-α (grey bars) and without TNF-α stimulation (black bars). Optical density changes are caused by TNF-α-activated expression of alkaline phosphatase.

Figure 5 shows inhibition of TLR9 activation in ODN 2006 (TLR9 agonist)-stimulated HEK-Blue™ hTLR9 cells by adding increasing concentrations (in ng) of the TLR9 CpG antagonist ODN TTAGGG (SEQ ID No. 4; depicted as gray bars) ). Potential intrinsic effects of increasing concentrations of CpG ODN TTAGGG (SEQ ID No. 4) on TLR9 activation in the absence of stimulation with the TLR9 agonist ODN 2006 (SEQ ID No. 2) were investigated and shown as black bars. Changes in optical density are caused by TLR9-activated expression of alkaline phosphatase.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the identification of novel oligonucleotides of unexpected structure and nucleotide composition that are capable of interacting with, as well as inhibiting and repressing TLR9 receptor activation in eukaryotic cells.

Until now, only a very limited number of aptamers have been known from the prior art that appear to antagonize the TLR9 receptor. Some common structural motifs underlie previously known antagonistic oligonucleotides. However, in some cases, these common structures may contribute to reduced bioavailability and/or other difficulties due to stringent requirements regarding the pharmaceutical use of aptamers in therapy, for example, human patients. For this purpose, it has been highly desirable to provide additional, structurally different aptamers for use in targeted modulation of TLR9 activation.

The inventors have now discovered for the first time the claimed aptamers as TLR9 antagonists, which differ from the structural motifs of oligonucleotides widely known for their TLR9 receptor antagonistic effect. Thus, it is now possible to provide structurally novel oligonucleotides as TLR9 antagonists.

In accordance with one preferred embodiment of the present invention, the subject in which TLR9 activation is to be inhibited or suppressed is a vertebrate, more preferably the subject is a mammal. Within the meaning of the present invention, the group of mammals includes, but is not limited to, rats, mice, rabbits, cats, dogs, horses, cattle, cows, pigs, sheep, non-human primates, and humans. More preferably, the subject is a human.

In the context of the present invention, it should be understood that any disclosure that refers only to "subject" may additionally be construed as the same disclosure referring to "cell", and vice versa, unless one skilled in the art appreciates what is disclosure does not carry any technical meaning.

In accordance with another preferred embodiment of the present invention, at least one cell to be contacted with the aptamers of the invention shows overexpression of TLR9. Thus, a subject to be treated with aptamers according to the invention exhibits TLR9 overexpression in at least some of that subject's cells. More preferably, the subject to be treated suffers from a disorder and/or symptoms caused by overexpression of TLR9 in at least some of said subject's cells.

According to another preferred embodiment of the present invention, at least one cell to be contacted with the aptamers of the invention exhibits TLR9-mediated signaling overactivity. This may mean that the subject to be treated with the aptamers of the invention demonstrates TLR9-mediated signaling overactivity in at least some of the subject's cells. More preferably, the subject to be treated suffers from a disorder and/or symptoms caused by an overactivity of TLR9-mediated signaling in at least some of said subject's cells.

According to one preferred embodiment of the present invention, the cell and/or subject to be treated is tested for TLR9 overexpression and/or TLR9-mediated signaling overactivity prior to inhibition of TLR9 activation in the cell and/or subject. Thus, in accordance with another preferred embodiment of the present invention, the cells and/or subjects to be treated have been characterized as demonstrating TLR9 overexpression and/or TLR9-mediated signaling overactivity prior to contact with, or treatment with, the aptamers of the invention.

According to another preferred embodiment of the present invention, the subject to be treated has a positive test result for TLR9 overexpression and/or TLR9-mediated signaling overactivity.

It is within the ability of one skilled in the art to reliably test and determine whether a cell or subject exhibits TLR9 overexpression and/or TLR9-mediated signaling overactivity. In this context, by way of example, reference is made to the disclosure of Deering and Orange, Clin Vaccine Immunol. 2006 Jan;13(1):68-76. It provides methods for assessing TLR9 expression and/or TLR9-mediated signaling activity. Additional evidence regarding the evaluation of TLR9 expression/activity, which forms part of the state of the art, can be taken from the development of "TLR9 Test Strip 2216" from Invivogen, San Diego, CA, United States of America.

In accordance with one preferred embodiment of the present invention, TLR9 overexpression and/or TLR9-mediated signaling overactivity is expected if TLR9-mediated TNFα production is increased above a reference value for healthy subjects. In accordance with a preferred implementation, the value for the subject to be treated is determined as follows:

Determination of TLR9 overexpression and/or TLR9- overactivity mediated signaling through the use of TLR9-mediated TNFα production as a surrogate parameter.

Selection TLR9-carrying mononuclear cells peripheral blood (PBMC)

To evaluate a patient's TLR9 status, PBMCs of patients who carry the TLR9 receptor are isolated from a heparinized blood sample using Ficoll-Paque Plus density gradient separation (Amersham Biosciences, Uppsala, Sweden). Isolated PBMCs are resuspended in RPMI (GIBCO/BRL, Grand Island, NY) containing 10% heat-inactivated fetal bovine serum (FBS; HyClone, Logan, UT), 0.1 µmol non-essential amino acids, 10 mM HEPES, 1 mM sodium pyruvate, 50 U/ml penicillin, 50 μg/ml streptomycin sulfate, and 2 mM L-glutamine (GIBCO/BRL), collectively referred to as R10-FBS.

All media are filtered through a 0.2 µm disposable filter (Millipore, Billerica, MA) after preparation. PBMCs can be cryopreserved by resuspending the cells in 0.2 µm filter-sterilized FBS containing 10% dimethyl sulfoxide (DMSO; Fisher Scientific, Fiery Lawn, NJ) in a 1-mL polypropylene vial containing a neoprene liner (Corning, Corning, New York). The temperature of the vials is gradually lowered by approximately 1°C/min using a methanol-containing non-mechanical cryopreservation apparatus (Nalgene, Rochester, NY) in a freezer at -80°C. Prior to rapid thawing for use in the experiment, the cells are stored in liquid nitrogen.

After thawing, cell viability is determined by trypan blue exclusion (GIBCO/BRL) and any sample with <95% viability is discarded.

provocation agonist(ligand)-mediated TLR9 response on PBMC

TLR9 activity can be assessed by incubating TLR9 receptor- ^containing PBMCs with the specific agonist/ligand ODN 2216. 2216/ml, 100 µl/well) or wells containing control medium and incubated at 37°C for 24 h with 5% CO2. Ligand-free medium is used to determine the baseline of TNFα production.

Grade quantity educated TNFα through enzyme immunoassay (ELISA)

The amount of TNFα formed by PBMCs provoked by ODN 2216 in the blood of patients or controls was assessed by TNFα-specific ELISA technology.

For this purpose, Immulon microtiter plates (ThermoLabSystems, Franklin, Massachusetts) were incubated overnight at 4°C with 100 μl of human monoclonal immobilized anti-human TNFα antibody (BD Biosciences) in coating buffer (0.1 M sodium carbonate, pH 9.5 ). After removing the supernatant, the plates were washed three times with phosphate buffered saline (PBS) with 0.05% Tween 20 (PBST), pH 7.4, and then blocked with PBS containing 10% bovine serum albumin, pH 7.0, for 1 h at room temperature.

Supernatants from individual wells of TLR9 ligand-stimulated PBMCs are transferred to individual antibody-coated wells.

Preferably, the supernatants are diluted twice directly in the antibody-coated wells so that the amount of TNFα measured is in any case within the range of the standard TNFα curve. A serially diluted TNFα standard preparation serves as a standard curve (R&D Systems, Minneapolis, Minnesota).

After loading the sample and standard curve, the plates are incubated at room temperature for 2 h followed by five washes with PBST. To detect captured TNFα, 100 µl of biotinylated anti-human TNFα sandwich monoclonal antibody (BD Biosciences) along with avidin-horseradish peroxidase conjugate (BD Biosciences) should be added to each well and the plates should be incubated for 1 h at room temperature.

After washing five times with PBST and incubation with 100 µl of tetramethylbenzidine plus hydrogen peroxide substrate solution for 30 min at room temperature, the colorimetric reaction is stopped by adding 50 µl of 1 M sulfuric acid. Absorbance is read at 450 nm using a microtiter plate spectrophotometer (Biotek, Winuski, Vermont).

In accordance with one specific embodiment of the present invention, the TNFα value determined in accordance with the method described herein above for the subject to be treated is at least 50 pg/ml, preferably at least 60 pg/ml, more preferably , at least 75 pg/ml, even more preferably at least 90 pg/ml, even more preferably at least 100 pg/ml, even more preferably at least 110 pg/ml, even more preferably at least 125 pg/ml, even more preferably at least 150 pg/ml, most preferably at least 175 pg/ml.

In a preferred embodiment of the present invention, the cell to be contacted with the aptamers of the invention is a glial cell, microglial cell, astrocyte, macrophage, B cell and/or dendritic cell, more preferably a plasmacytoid dendritic cell. According to another more preferred embodiment, the cell to be contacted is a glial cell in the central nervous system.

In one preferred embodiment of the present invention, the subject has a disorder selected from an autoimmune disorder, an inflammatory disorder, an autoimmune connective tissue disease (ACTD) and/or a neurodegenerative disorder, more preferably the disorder is selected from, but not limited to, psoriasis, rheumatoid arthritis, alopecia universalis , acute disseminated encephalomyelitis, Addison's disease, allergies, ankylosing spondylitis, antiphospholipid antibody syndrome, arteriosclerosis, atherosclerosis, autoimmune hemolytic anemia, autoimmune hepatitis, bullous pemphigoid, Chagas disease, chronic obstructive pulmonary disease, celiac disease, cutaneous lupus erythematosus (CLE), , diabetes mellitus, dilated cardiomyopathy (DCM), endometriosis, Goodpasture's syndrome, Graves' disease, Guillain-Barré syndrome, Hashimoto's disease, hidradenitis suppurativa, idiopathic thrombocytopenic purpura, inflammatory bowel disease interstitial cystitis, localized scleroderma, multiple sclerosis (MS), myasthenia gravis, myocarditis, narcolepsy, neuromyotonia, pemphigus, pernicious anemia, polymyositis, primary biliary cirrhosis, rheumatoid arthritis (RA), schizophrenia, Sjögren's syndrome, systemic lupus erythematosus (SLE) ), systemic sclerosis, temporal arteritis, vasculitis, vitiligo, vulvodynia, Wegener's granulomatosis, traumatic pain, neuropathic pain, and acetaminophen toxicity.

In a more preferred embodiment, the subject suffers from an autoimmune disorder selected from the group consisting of systemic lupus erythematosus, multiple sclerosis, arteriosclerosis, inflammatory bowel disease, diabetes mellitus, allergies, and cancer, in particular autoimmune diabetes.

In another more preferred embodiment, the subject suffers from an autoimmune disorder that is associated with the presence of GPCR autoantibodies, even more preferably the autoimmune disorder is selected from the group consisting of cardiomyopathy, dilated cardiomyopathy (DCM), ischemic cardiomyopathy (iCM), peripartum cardiomyopathy (PPCM) , idiopathic cardiomyopathy, Chagas cardiomyopathy, chemotherapy-induced cardiomyopathy, Chagas megacolon, Chagas megaesophagus, Chagas neuropathy, benign prostatic hyperplasia, scleroderma, Raynaud's syndrome, peripheral arterial occlusive disease (PAOD), pre-eclamsia, kidney allograft rejection, glaucomacarditis, glaucomacarditis , hypertension, pulmonary hypertension, malignant hypertension, metabolic syndrome, alopecia, alopecia areata, migraine, Parkinson's disease, epilepsy, cluster headache, multiple sclerosis, depression, regional pain syndrome, unstable angina, system lupus erythematosus (SLE), schizophrenia, Sjögren's syndrome, periodontitis, atrial fibrillation, vitiligo, hemolytic uremic syndrome, stiffness syndrome, congenital heart block, type I diabetes mellitus, psoriasis, Alzheimer's disease, pathological fatigue, neurodermatitis, renal kidney disease, amyotrophic lateral sclerosis (ALS), Leber's optic nerve atrophy (LHON syndrome), allergic asthma, arrhythmia, refractory hypertension, type II diabetes mellitus, vascular dementia, non-Chagas megacolon, and/or orthostatic hypertension.

In another more preferred embodiment, the subject is suffering from a cardiac condition, in particular dilated cardiomyopathy and/or myocarditis.

In another more preferred embodiment of the present invention, the subject suffers from neuropathic pain resulting from the effects of diabetes mellitus or chemotherapy drugs on the nerves, trauma, surgical procedures, arthritis, AIDS, burn injuries, cerebral or lumbar spine disease, fibromyalgia, postischemic pain, tumors, viral neuralgia, sympathetic reflex dystrophy syndrome, phantom pain and pain after amputation, postherpetic neuralgia, complex regional pain syndrome or central pain syndrome.

In another more preferred embodiment of the present invention, the subject is suffering from an inflammatory disorder such as rheumatoid arthritis, spondyloarthropathies, gouty arthritis, osteoarthritis, systemic lupus erythematosus, or juvenile arthritis. In another more preferred embodiment of the present invention, the subject suffers from inflammation associated with asthma, allergic rhinitis, sinus disease, bronchitis, tuberculosis, acute pancreatitis, sepsis, infectious diseases, menstrual cramps, preterm labor, tendinitis, bursitis, skin-related conditions. such as psoriasis, eczema, atopic dermatitis, urticaria, dermatitis, contact dermatitis and burns, or from postoperative inflammation, including ophthalmic surgery such as cataract surgery and refractive surgery, vascular disease, migraine headaches, periarteritis nodosa, thyroiditis, aplastic anemia, Hodgkin's disease, sclerodoma, rheumatic fever, type I diabetes mellitus, neuromuscular junction diseases, including myasthenia gravis, white matter diseases, including multiple sclerosis, sarcoidosis, nephritis syndrome, Behçet's syndrome, polymyositis, gingivitis, nephritis, hypersensitivity, posttraumatic swelling, myocardial ischemia, allergic rhinitis, respiratory distress syndrome, systemic inflammatory response syndrome (SIRS), cancer-related inflammation, decreased tumor-associated angiogenesis , endotoxic shock syndrome, atherosclerosis, an ophthalmic disease such as retinitis, retinopathy, uveitis, ocular photophobia or acute eye tissue injury, lung inflammation such as associated with viral infections or cystic fibrosis, chronic obstructive pulmonary disease or acute respiratory distress syndrome, tissue rejection, graft-versus-host disorders, delayed-type hypersensitivity, and immune-mediated and inflammatory elements of CNS diseases such as Alzheimer's disease, Parkinson's disease, multiple sclerosis.

In another preferred embodiment of the present invention, the subject is suffering from a tumor/cancer, preferably the tumor/cancer is selected from the group consisting of breast cancer, squamous cell carcinoma of the cervix, gastric carcinoma, glioma, hepatocellular carcinoma, lung cancer, melanoma, cancer prostate, recurrent glioblastoma, recurrent non-Hodgkin's lymphoma, colon and rectal cancer.

In another more preferred embodiment of the present invention, the subject suffers from acute lymphoblastic leukemia, acute myelocytic leukemia, adrenocortical carcinoma, AIDS-associated cancer, AIDS-associated lymphoma, anal cancer, appendix cancer, astrocytoma (pediatric cerebellar or cerebral), basal cell carcinomas, bile duct cancer, bladder cancer, bone cancer, brainstem glioma, brain tumors (cerebellar astrocytoma, cerebral astrocytoma/malignant glioma, ependymoma, medulloblastoma, supratentorial primitive neuroectodermal tumors, optic pathway glioma, and hypothalamic glioma), breast cancer gland, bronchial adenomas/carcinoids, Burkitt's lymphoma, carcinoid tumors (children's, gastrointestinal), carcinomas of unknown primary location, lymphomas of the central nervous system (primary), cerebellar astrocytoma, cerebral astrocytoma/malignant glioma, p aka cervical, childhood cancer, chronic lymphocytic leukemia, chronic myelogenous leukemia, chronic myeloproliferative disorders, colon cancer, cutaneous T-cell lymphoma, desmoplastic small round cell tumor, endometrial cancer, ependymoma, esophageal cancer, Ewing's sarcoma of the Ewing tumor family, extracranial germ cell tumor (children), extragonadal germ cell tumor, extrahepatic bile duct cancer, ocular cancer (intraocular melanoma, retinoblastoma), gallbladder cancer, gastric cancer (stomach), carcinoid tumor of the gastrointestinal tract, stromal tumor of the gastrointestinal tract, germ cell tumors (children extracranial, extragonadal, ovarian), gestational trophoblastic tumor, gliomas (adults, brainstem in children, infantile cerebral astrocytoma, optic tract and hypothalamic in children), gastric carcinoid, hairy cell leukemia, head and neck cancer, hepatic cell carcinoma (liver nor), Hodgkin's lymphoma, hypopharyngeal cancer, hypothalamic glioma and glioma of the optic tract (in children), intraocular melanoma, islet cell carcinoma, Kaposi's sarcoma, kidney cancer (renal cell carcinoma), laryngeal cancer, leukemias (acute lymphoblastic, acute myeloid, chronic lymphatic, chronic myelogenous, hair cell), lip and mouth cancer, liver cancer (primary), lung cancer (non-small cell, small cell), lymphomas (AIDS-related, Burkett, cutaneous T-cell, Hodgkin's, non-Hodgkin's, primary in the central nervous system), macroglobulinemia (Waldenström), malignant fibrous bone histiocytoma/osteosarcoma, medulloblastoma (children), melanoma, intraocular melanoma, Merkel cell carcinoma, mesothelioma (adult malignant, pediatric), metastatic squamous neck cancer with latent primary localization, oral cancer , syndrome of multiple endocrine neoplasia (children), multiple myel myelodysplastic syndromes, myelodysplastic/myeloproliferative disorders, myelogenous leukemia (chronic), myeloid leukemia (acute adult, acute childhood), multiple myeloma, myeloproliferative disorders (chronic), cancer of the nasal cavity and paranasal sinuses, nasopharyngeal carcinoma , neuroblastoma, non-Hodgkin's lymphoma, non-small cell lung cancer, oral cavity cancer, oropharyngeal cancer, osteosarcoma/malignant fibrous bone histiocytoma, ovarian cancer, epithelial ovarian cancer (superficial epithelial stromal tumor), embryonic cell tumor of the ovary, borderline ovarian tumor, pancreatic cancer pancreatic (islet cell) cancer, paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pineal astrocytoma, pineal germinoma, pineoblastoma and supratentorial primitive neuroectodermal tumors (childhood), pituitary adenoma, plasma cell neoplasia, pleuropulmonary blastoma, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell carcinoma (kidney cancer), renal pelvis and ureteral transitional cell cancer, retinoblastoma, rhabdomyosarcoma ( pediatric), salivary gland cancer, sarcoma (from the family of tumors of Ewing, Kaposi, soft tissue, uterus), Cesari syndrome, skin cancer (non-melanoma, melanoma), skin carcinoma (Merkel cells), small cell lung cancer, cancer of the small intestine, soft sarcoma tissue, squamous cell carcinoma, squamous neck cancer with a hidden primary site (metastatic), gastric cancer, supratentorial primitive neuroectodermal tumor (childhood), T-cell lymphoma (skin), testicular cancer, throat cancer, thymoma (childhood), thymoma and cancer thymus, thyroid cancer, thyroid cancer (childhood), transitional cell cancer of the renal pelvis and ureter, trophobl aseptic tumor (gestational), primary site unknown (adult, pediatric), transitional cell carcinoma of the ureter and renal pelvis, urethral cancer, uterine cancer (intrauterine), uterine sarcoma, vaginal cancer, optic pathway glioma and hypothalamic glioma (childhood), vulvar cancer , Waldenstrom's macroglobulinemia and Wilms' tumor (children's).

In one preferred embodiment of the present invention, the aptamer sequences defined herein that antagonize the TLR9 receptor do not contain a cytosine nucleotide. Obviously, sequences that contain a sequence having an antagonistic effect on TLR9 may contain additional nucleotide sequences, including cytosine nucleotides.

In a more preferred embodiment of the present invention, the aptamer of the present invention described and disclosed herein comprises the nucleic acid sequence of SEQ ID No. 1 (GGT TGG TGT GGT TGG) and/or a nucleic acid sequence that is at least 80% identical to SEQ ID No. 1. In another more preferred embodiment, the aptamer contains the nucleic acid sequence of SEQ ID No. 1 (GGT TGG TGT GGT TGG), in particular, the aptamer consists of the nucleic acid sequence SEQ ID No. 1 (GGT TGG TGT GGT TGG).

In one preferred embodiment, the aptamer of the present invention, as disclosed and described herein, contains the nucleic acid sequence of SEQ ID No. 5 (GGT TGG TGT GGT TG), preferably the aptamer consists of the nucleic acid sequence SEQ ID No. 5 (GGT TGG TGT GGT TG).

The determination of percent identity between two sequences is carried out, according to the present invention, by using the mathematical algorithm of Karlin-Altschull (Proc. Natl. Acad. Sci. USA (1993) 90:5873-5877). Such an algorithm is the basis of the BLASTN and BLASTP programs in Altschul et al. (J. Mol. Biol. (1990) 215: 403-410). Nucleotide BLAST searches are performed using the BLASTN program. To obtain gapped alignments for comparison purposes, gapped BLAST is used as described in Altschul et al. (Nucleic Acids Res. (1997) 25: 3389-3402). When using the BLAST and BLAST programs with spaces, the default settings of the respective programs are used.

In accordance with a preferred embodiment of the present invention, the aptamer sequences form a part of the invention that consists of or contains a nucleic acid sequence that is at least 85% identical to the individualized aptamer sequences that are disclosed herein, more preferably at least 90% identical, even more preferably at least 95% identical.

For the purpose of the present invention, the term "aptamer" refers to an oligonucleotide that binds specifically and with high affinity to a target molecule. Under certain conditions, aptamers can fold into a specific three-dimensional structure. In one preferred embodiment of the present invention, the claimed aptamers specifically and with high affinity interact with the target receptor, more preferably with the TLR9 receptor.

According to a preferred embodiment, the inventive aptamer does not form part of a nanostructure composed of at least two components, wherein the minimum two components are a plurality of oligonucleotides and a plurality of G-quadruplex-forming nucleic acids associated with a plurality of oligonucleotides, more preferably the inventive aptamer does not form part of a nanostructure composed of at least two components. In this preferred embodiment, the two components are preferably structurally different. In addition, in this preferred embodiment, the bond between the plurality of oligonucleotides and the plurality of G-quadruplex-forming nucleic acids associated with the plurality of oligonucleotides in the nanostructure not claimed in this embodiment is a covalent bond or a non-covalent bond, the non-covalent bond being more preferably based on one or more of electrostatic interactions, van der Waals forces, π effects, or hydrophobic effects.

According to another preferred embodiment, the inventive aptamer does not form part of a stable, self-assembled nucleic acid nanostructure comprising a plurality of oligonucleotides, each internucleotide bond of the oligonucleotide being not a phosphorothioate bond, a plurality of G-quadruplex-forming nucleic acids bound to a plurality of oligonucleotides, wherein the G- quadruplex nucleic acids are not TAGGGTT, and a plurality of G-quadruplex stabilizing domains associated with G-quadruplex-forming nucleic acids, wherein the oligonucleotides, G-quadruplex-forming nucleic acids and G-quadruplex stabilizing domains form a plurality of G-quad structures.

According to another preferred embodiment, the inventive aptamer does not form part of a stable, self-assembling nucleic acid nanostructure comprising a plurality of oligonucleotides, a plurality of G-quadruplex-forming nucleic acids bound to a plurality of oligonucleotides, wherein the G-quadruplex-forming nucleic acids are not TAGGGTT, and a plurality of stabilizing G-quadruplex of domains associated with G-quadruplex-forming nucleic acids, wherein when at least one of the G-quadruplex-forming nucleic acids contains GG, GGG, or GGGG, and the oligonucleotide is a CpG oligonucleotide, the lipid is not a diacyl lipid, and the oligonucleotides forming G-quadruplex nucleic acids and G-quadruplex stabilizing domains form a variety of G-quad structures.

According to yet another preferred embodiment, the inventive aptamer does not form part of a nucleic acid nanostructure containing G-quadruplex stabilizing domains associated with the inventive aptamer.

The aptamer of the invention contains or consists of a sequence of nucleic acid molecules, nucleotides. According to a preferred embodiment, the aptamer of the invention consists of a nucleotide sequence as defined herein.

The aptamer of the invention preferably contains unmodified and/or modified D- and/or L-nucleotides. According to the common one-letter code for nucleic acid bases, "C" means cytosine, "A" means adenine, "G" means guanine, and "T" means or thymine if the nucleotide sequence is a DNA sequence, or "T" means uracil nucleotide if the nucleotide sequence is an RNA sequence. Unless otherwise indicated below, the term "nucleotide" refers to ribonucleotides and deoxyribonucleotides.

An aptamer of the invention may comprise or consist of a DNA or RNA nucleotide sequence and may therefore be referred to as a DNA aptamer or RNA aptamer, respectively. It should be understood that if the aptamer of the invention contains the RNA nucleotide sequence in the sequence motifs specifically indicated in the present invention, "T" means uracil.

For the sake of brevity, in the present invention, reference is made only to explicit DNA nucleotide sequences. However, it should be understood that the corresponding RNA nucleotide sequences are also included in the scope of the present invention.

According to one embodiment, the use of DNA aptamers is preferred. DNA aptamers are generally more stable in plasma than RNA aptamers. However, according to an alternative implementation, RNA aptamers are preferred. According to another embodiment, single stranded nucleotide sequences are preferred. According to another alternative implementation, double-stranded nucleotide sequences are preferred.

Aptamers of the invention may contain a nucleotide sequence containing 2'-modified nucleotides, for example, 2'-fluoro-, 2'-methoxy-, 2'-methoxyethyl and/or 2'-amino-modified nucleotides. The aptamer of the invention may also contain a mixture of deoxyribonucleotides, modified deoxyribonucleotides, ribonucleotides and/or modified ribonucleotides. Accordingly, the terms "2'-fluoro-modified nucleotide", "2'-methoxy-modified nucleotide", "2'-methoxyethyl-modified nucleotide" and/or "2-amino-modified nucleotide" refers to modified ribonucleotides and modified deoxyribonucleotides. .

The aptamer of the invention may contain modifications. Such modifications include, for example, alkylation, i. methylation, arylation or acetylation of at least one nucleotide, incorporation of enantiomers and/or fusion of aptamers with one or more other nucleotides or nucleic acid sequences. Such modifications may contain, for example, 5' and/or 3' PEG or 5' and/or 3' CAP modifications. Alternatively or in addition, the aptamer of the invention may contain modified nucleotides, preferably selected from closed nucleic acids, 2'-fluoro, 2'-methoxy and/or 2'-amino modified nucleotides.

Locked nucleic acids (LNA) are analogues of the corresponding RNA nucleotides, and the conformation has been fixed.

Closed nucleic acid oligonucleotides contain one or more bicyclic ribonucleosides, wherein the 2'-OH group is connected to the C ₄ carbon atom via a methylene group. The closed nucleic acids exhibit improved stability against nucleases compared to the corresponding unmodified RNA aptamers. The hybridization properties are also improved, which allows for the enhancement of the affinity and specificity of the aptamer.

Another preferred modification is the attachment of a so-called 3'-CAP, 5'-CAP structure and/or a modified guanosine nucleotide (eg 7-methyl-guanosine) to the 3' and/or 5' end of the aptamer. This modification of the 3' and/or 5' end has the effect that the aptamer is protected from rapid degradation from nucleases.

Alternatively or in addition, the aptamer of the invention may exhibit a pegylated 3' or 5' end. The 3' or 5' PEG modification comprises the addition of at least one unit of polyethylene glycol (PEG), preferably the PEG group contains from 1 to 900 ethylene groups, more preferably from 1 to 450 ethylene groups. In a preferred embodiment, the aptamer contains linear PEG units with HO-(CH ₂ CH ₂ O) _n -H, where n is an integer from 1 to 900, preferably n is an integer from 1 to 450.

The aptamer of the invention may be fully or partially configured as a peptide nucleic acid (PNA). The aptamers of the present invention can be further modified as described in Keefe AD et al., Nat Rev Drug Discov. 2010 Jul;9(7):537-50 or Mayer G, Angew Chem Int Ed Engl. 2009;48(15):2672-89 or in Mayer, G. and Famulok M.,Pharmazie in unserer Zeit 2007; 36:432-436.

The term "oligonucleotide" generally refers to a polynucleoside containing a plurality of linked nucleoside units. Such oligonucleotides can be obtained from existing sources of nucleic acid, including genomic or cDNA, but are preferably obtained by synthetic methods. In preferred embodiments, each nucleoside unit may encompass various chemical modifications and substitutions compared to wild-type oligonucleotides, including, but not limited to, a modified nucleoside base and/or a modified sugar unit.

Examples of chemical modifications are known to the person skilled in the art and are described, for example, in Uhlmann, E. et al. (1990) Chem. Rev. 90:543; "Protocols for Oligonucleotides and Analogs" Synthesis and Properties & Synthesis and Analytical Techniques, S. Agrawal, Ed, Humana Press, Totowa, USA 1993; and Hunziker, J. et al. (1995) Mod. Syn. Methods 7:331-417; and Crooke, S. et al. (1996) Ann. Rev. Pharm. Tox. 36:107-129.

Nucleoside residues can be linked to each other by any of the many known internucleoside bonds, inter alia, to improve the stability of the oligonucleotides against enzymatic degradation, for example, by nucleases. Such internucleoside linkages include, without limitation, phosphodiester, phosphorothioate, phosphorodithioate, alkylphosphonate, alkylphosphorothioate, phosphotriester, phosphoramidate, siloxane, carbonate, carboalkoxy, acetamidate, carbamate, morpholino, borane, thioether, bridging phosphoramidate, bridging methylene phosphonate, bridging phosphorothioate, and sulfonic internucleoside connections.

The term "oligonucleotide" also encompasses polynucleosides having one or more stereospecific internucleoside linkages (eg, (Rr)- or (Sr)-phosphorothioate, alkylphosphonate or phosphotriester linkages). As used herein, the terms "oligonucleotide" and "dinucleotide" are intended to expressly include polynucleosides and dinucleosides having any such internucleoside bond, whether or not the bond contains a phosphate group. In some preferred embodiments, these internucleoside bonds may be phosphodiester, phosphorothioate, or phosphorodithioate bonds, or combinations thereof, more preferably, the internucleoside bonds are phosphorothioate.

In addition, aptamers can be encapsulated in suitable carriers to protect their structural integrity as well as to facilitate their intracellular delivery. Preferred carriers include liposomes, lipid vesicles, microparticles, and the like.

Lipid vesicles are similar to plasma membranes and can be made to fuse with cell membranes. Most liposomes and multilamellar vesicles are not clearly fusogenic, mainly because the stored energy of the radius of curvature of the vesicle is minimal. Preferred lipid vesicles include small monolamellar vesicles. The small monolamellar vesicles intended to encapsulate the aptamers of the present invention are very fusogenic because they have a very small radius of curvature. The average diameter of a small monolamellar vesicle ranges from 5 nm to 500 nm; preferably 10 nm to 100 nm, more preferably 20 nm to 60 nm, including 40 nm. This size allows the vesicles to pass through the spaces between endothelial cells, thereby allowing systemic delivery of aptamer-containing vesicles after intravenous administration. The size of the beneficial vesicles can vary greatly and can be selected according to the specific application with the aptamer.

Small monolamellar vesicles can be easily obtained in vitro using procedures available in the art (as, for example, disclosed in WO 2005/037323 A2). Vesicle-forming compositions contain a phospholipid that is a stable vesicle former, preferably together with another polar lipid, and optionally with one or more additional polar lipids and/or raft formers. Preferred phospholipids that are stable vesicle formers include 1-palmitoyl-2-docosahexanoyl-sn-glycero-3-phosphocholine and 1,2-dioleoyl-sn-glycero-3-phosphocholine. Preferred polar lipids include: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphate, 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, 1 ,2-dioleoyl-sn-glycero-3-[phospho-l-serine], typical sphingomyelin, 1,2-dimyristoyl-sn-glycerol and 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine.

Other preferred polar lipids include phosphatidylserine, phosphatidylglycerol, mixed chain phosphatidylcholine, phosphatidylethanol, and phospholipids containing decosahexaenoic acids. One example of a preferred raft former is cholesterol.

One advantage of modifying the aptamer of the invention in one of the ways mentioned above is that the aptamer can be stabilized against deleterious influences such as, for example, nucleases present in the environment where the aptamer is used. These modifications are also suitable for adapting the pharmacological properties of the aptamer. Preferably, the modifications do not change the affinity or specificity of the aptamer.

The aptamer of the invention may also be conjugated to a carrier molecule and/or a reporter molecule. The carrier molecules comprise those that, when conjugated to the aptamer, prolong the plasma half-life of the conjugated aptamer in human plasma, for example by enhancing stability and/or by influencing the rate of excretion. One example of a suitable carrier molecule is PEG.

Reporter molecules contain molecules that allow the detection of the conjugated aptamer. Examples of such reporter molecules are GFP, biotin, cholesterol, dyes such as, for example, fluorescent dyes, electrochemically active reporter molecules and/or compounds containing radioactive residues, in particular radionuclides, suitable for PET (positron emission tomography) detection. ), such as, for example, ¹⁸ F, ¹¹ C, ¹³ N, ¹⁵ O, ⁸² Rb or ⁶⁸ Ga. Suitable carrier and reporter molecules, as well as methods for conjugating them to the aptamer of the invention, are well known to those skilled in the art.

The aptamers of the present invention are useful in the treatment of certain diseases by inhibiting or suppressing the activation of TLR9 in a cell. In the context of the present invention, aptamers are expected to be useful in human subjects as well as animal subjects. According to one embodiment, the aptamers are intended for use in human subjects. According to another embodiment, the aptamers are intended for use in animal subjects.

Diseases in which inhibition or suppression of TLR9 activation may be beneficial are those in which TLR9 activation produces undesirable symptoms or consequences in a subject exhibiting such TLR9 activation. According to a preferred embodiment, the disorder and/or disease is caused by TLR9 activation in the subject being treated. According to a particularly preferred embodiment, the disorder and/or disease is caused by TLR9 activation in a subject being treated for autoantibodies directed to G protein-coupled receptors.

Several diseases associated with TLR9 activation have already been reported or known in the literature. The present inventors have done further research in view of this association and have found that more diseases may be associated with such TLR9 activation or TLR9 overactivation than previously reported (data not shown).

In view of the affinity of the claimed aptamers for TLR9 and their antagonistic effects on it, there is evidence that any disease that is associated with the activation or overactivation of TLR9 can be effectively treated with the aptamers presented and claimed herein. Thus, in principle, all diseases that have been found to be associated with (over)activation of TLR9 are promising target diseases to be treated with aptamers in accordance with the present invention.

The aptamers of the present invention are capable of affecting, preferably antagonizing, the TLR9 receptor. Thus, in a preferred embodiment, the claimed aptamers act as TLR9 antagonists. Preferably, the claimed aptamers are capable of inhibiting or suppressing TLR9 activation. According to one preferred embodiment, the aptamers of the present invention do not affect the TLR7 receptor, more preferably they do not affect any other TLR receptor molecule. According to another preferred embodiment, the aptamers of the present invention also act on the TLR7 receptor.

By inhibiting pathological or undesired activation or overactivation of the TLR9 receptor, the potentially negative effects of TLR9 activation are neutralized and attenuated, and permanent or temporary TLR9 activation can be eliminated or reduced to normal levels. As a consequence, the extent and severity of disease caused by or associated with TLR9 activation can be substantially reduced. Thus, the present invention provides aptamers that are suitable for use in the treatment of diseases associated with TLR9 activation or overactivation.

According to another embodiment of the present invention, there is provided a method for inhibiting or suppressing TLR9 activation in a cell, comprising contacting a cell expressing TLR9 with an aptamer.

In a preferred embodiment of the present invention, the method is performed in vitro/ex vivo . More preferably, the cell to be contacted with the aptamer according to the method of the present invention does not form part of a whole living organism. In one embodiment, the cell to be contacted may be cultured in a cell structure. Such cultures of individual or groups of cells can be carried out as is commonly done in the art.

In another preferred embodiment of the method of the present invention, the method can be performed in vivo and/or on cells that form part of a whole living organism. According to this embodiment, the method preferably includes the additional upstream step of testing the cell(s) to be contacted for TLR9 activation, more preferably the cell(s) are tested for TLR9 overexpression and/or TLR9-mediated signaling overactivity.

In a preferred embodiment of the second aspect of the invention, the cell is a mammalian cell, preferably the cell is a human cell.

The present invention is also directed to a pharmaceutical composition containing at least one aptamer of the invention and optionally at least one pharmaceutically acceptable excipient. The invention is also directed to a pharmaceutical composition comprising an aptamer of the invention or a mixture of various aptamers of the invention and a pharmaceutically acceptable excipient such as, for example, a suitable carrier or diluent.

Preferably, the aptamer of the invention constitutes the active ingredient of the pharmaceutical composition and/or is present in an effective amount. The term "effective amount" means an amount of an aptamer of the invention that has a prophylactically, diagnostically or therapeutically relevant effect on a disease or pathological condition. The preventive effect prevents the outbreak of the disease. A therapeutically relevant effect alleviates to some extent one or more symptoms of a disease or partially or completely normalizes one or more physiological or biochemical parameters associated with or causing a disease or pathological conditions.

The appropriate amount to administer the aptamer of the invention is high enough to achieve the desired prophylactic, diagnostic or therapeutic effect. One of skill in the art will appreciate that the particular dose level, frequency, and period of administration to any particular mammal will depend on a wide range of factors, including potency of the particular components used, age, body weight, general health, sex, nutrition, time administration, route of administration, drug combinations, and the severity of a particular treatment. Using well-known means and methods, one skilled in the art can determine the exact amount through routine experimentation.

According to one embodiment of the pharmaceutical composition of the invention, at least 20% of the total aptamer content is the aptamer of the invention, preferably at least 50%, more preferably at least 75%, most preferably at least 95%.

When used for treatment, the pharmaceutical composition will generally be formulated in association with one or more pharmaceutically acceptable excipients. The term "adjuvant" is used herein to describe any ingredient other than the aptamer of the invention. The choice of excipient will depend to a large extent on the particular mode of administration. The excipients may be suitable carriers and/or diluents.

The pharmaceutical composition of the invention may be administered orally. Oral administration may include swallowing, so that the composition enters the gastrointestinal tract, or buccal or sublingual administration may be used, due to which the composition enters the bloodstream directly from the oral cavity.

Formulations suitable for oral administration include: solid formulations such as tablets; coated tablets, capsules containing particles, liquids or powders; lozenges (including those filled with liquid); and gummies; multi- and nanoparticles; gels; solid solutions; liposomes; films, suppositories, sprays and liquid formulations.

Liquid formulations include suspensions, solutions, syrups and elixirs. Such formulations may be used as fillers in soft or hard capsules and typically contain a carrier such as water, ethanol, polyethylene glycol, propylene glycol, methylcellulose, or a suitable oil, and one or more emulsifying agents and/or suspending agents. Liquid formulations can also be prepared by reconstituting a solid, for example from a sachet.

For a dosage form in the form of a tablet, depending on the dose, the aptamer of the invention can be from 0.1 wt. % up to 80 wt. % dosage form, more specifically, from 5 wt. % up to 60 wt. % dosage form. In addition to the aptamer of the invention, the tablets generally contain a disintegrant.

Examples of disintegrants include sodium starch glycolate, sodium carboxymethylcellulose, calcium carboxymethylcellulose, sodium croscarmellose, crospovidone, polyvinylpyrrolidone, methylcellulose, microcrystalline cellulose, lower alkyl-substituted hydroxypropylcellulose, starch, pregelatinized starch, and sodium alginate.

In general, the baking powder will be from 1 wt. % up to 25 wt. %, preferably from 5 wt. % up to 20 wt. % dosage form.

Tablets may contain additional adjuvants such as, for example, binders, surfactants, lubricants and/or other possible ingredients such as, for example, antioxidants, colorants, flavoring agents, preservatives and/or taste masking agents.

Tablet blends can be compressed directly or by means of a tablet roller. Alternatively, tablet blends or batch blends can be prepared using wet, dry or melt granulation, melt solidification or extrusion prior to tabletting. The final composition may contain one or more layers and may or may not be coated; it can even be encapsulated.

Solid formulations for oral administration may be formulated for immediate and/or modified release. Modified release formulations include delayed, delayed, intermittent, controlled, targeted, and programmed release.

The pharmaceutical composition of the invention may also be administered directly into the bloodstream, into a muscle, or into an internal organ. Suitable means for parenteral administration include intravenous, intraarterial, intraperitoneal, intrathecal, intraventricular, intraurethral, intrasternal, intracranial, intramuscular and subcutaneous. Suitable devices for parenteral administration include needle (including microneedle) injectors, needleless injectors and infusion equipment.

Formulations for parenteral administration are generally aqueous solutions which may contain excipients such as salts, carbohydrates and buffering agents (preferably up to a pH value of 3 to 9), however, for some applications they may be more suitable. formulated as a sterile anhydrous solution or as a dry form for use in conjunction with a suitable vehicle such as sterilized, pyrogen-free water.

Preparation of formulations for parenteral administration under sterile conditions, for example by lyophilization, can easily be accomplished using standard pharmaceutical techniques well known to those skilled in the art.

The solubility of a pharmaceutical composition of the invention used in the preparation of parenteral formulations can be improved by the use of suitable formulation techniques, such as the addition of solubility enhancers.

Formulations for parenteral administration may be formulated to be immediate and/or modified release formulations. Modified release formulations include delayed, delayed, intermittent, controlled, targeted, and programmed release. Thus, the compounds of the invention may be formulated as a solid, semi-solid, or thixotropic liquid for administration as an implantable depot providing a modified release of the active compound. Examples of such formulations include drug-coated stents and PGLA poly(dl-lactic-coglycolic)acid (PGLA) microspheres.

The pharmaceutical composition of the invention may also be applied topically to the skin or mucous membrane, ie dermally or transdermally. Typical formulations for this purpose include gels, hydrogels, lotions, solutions, creams, ointments, powders, dressings, foams, films, skin patches, cachets, implants, sponges, fibers, dressings, and microemulsions.

Liposomes can also be used. Typical carriers include alcohol, water, mineral oil, liquid petrolatum, white petrolatum, glycerin, polyethylene glycol, and propylene glycol. Penetration enhancers may be added. Other topical agents include delivery by electroporation, iontophoresis, phonophoresis, sonophoresis, and microneedle or needleless (eg, Powderject(TM), Bioject(TM), etc.) injection. Topical formulations may be formulated to be immediate and/or modified release formulations. Modified release formulations include delayed, delayed, intermittent, controlled, targeted, and programmed release.

For administration to human patients, the total daily dose of the aptamer of the invention and/or the pharmaceutical composition of the invention is typically in the range of 0.001 mg to 5000 mg, depending of course on the mode of administration. For example, the necessary daily dose for intravenous administration may be only 0.001 mg to 40 mg. The total daily dose may be administered in single or divided doses and may, at the discretion of the physician, be outside the typical range defined herein.

These doses are based on an average human subject weighing approximately 75 kg to 80 kg. The physician will readily be able to determine doses for subjects whose weight falls outside this range, such as neonates or the elderly.

In the context of the present invention, the aptamer may preferably be administered in combination with one or more vaccines, antigens, antibodies, cytotoxic agents, allergens, antibiotics, antisense oligonucleotides, a TLR antagonist, peptides, proteins, gene therapy vectors, DNA vaccines, adjuvants or kinase inhibitors. .

The present invention also encompasses a kit containing an aptamer of the invention, a container, and optionally, written instructions for use and/or with means of administration.

For the treatment and/or diagnosis of a disease, regardless of the route of administration, the aptamer of the invention is administered at a daily dose per treatment cycle of not more than 20 mg/kg body weight, preferably not more than 10 mg/kg body weight, more preferably selected from the range of 1 μg/kg to 20 mg/kg of body weight, most preferably selected from the range of 0.01 to 10 mg/kg of body weight.

In a preferred embodiment of the present invention, the aptamer can be used in vitro/ex vivo . In an alternative implementation, the aptamer can be used in vivo .

The production or mass production of the aptamers of the invention is well known in the art and is only routine.

All embodiments of the present invention described herein are considered to be capable of being combined in any combination, unless a person skilled in the art considers that such a combination does not carry any technical meaning.

EXAMPLES

Functional analysis to assess the antagonistic effect of aptamers inventions on the TLR9 receptor

A functional assay capable of identifying the antagonistic effects of oligonucleotides at the TLR9 receptor was established to evaluate the ability to inhibit or suppress TLR9 activation.

This functional assay uses the HEK-293 recombinant cell line and established TLR9 agonist/antagonist assay (HEK-Blue™ hTLR9 from InvivoGen, San Diego, CA, USA). The cell line used carries a functionally overexpressed human TLR9 receptor associated with a reporter gene, which is an alkaline phosphatase secreted after signaling by an NFkB-inducible promoter. The TLR9 activation signal is an elevated optical density (OD) value.

Recombinant HEK-293 cells that carry the reporter gene but not the TLR9 receptor serve as controls (HEK-Blue™ TNF-α cells from InvivoGen, San Diego, CA, USA). Here, the reporter gene is activated via TNFα. Using this control cell, it is possible to distinguish whether the effects are specific to the TLR9 receptor or whether they are caused by interference in the signaling cascade.

Example 1:

In this example, the inhibitory effect of SEQ ID NO. 1 (GGT TGG TGT GGT TGG) for the TLR9 receptor. HEK-Blue™ hTLR9 cells were pre-incubated with various concentrations of SEQ ID No. aptamer. 1 (0, 0.37, 1.1, 3.3, 10, and 20 µM aptamer) for 60 minutes before adding 300 ng/mL (38.86 nM) of the well-known TLR9 agonist ODN 2006 (SEQ ID No. 2: TCG TCG TTT TGT CGT TTT GTC GTT) and the cells were incubated for an additional 18 hours. Next, the optical density was determined using a standard alkaline phosphatase assay. The results of this analysis are shown as gray bars in Figure 1. Potential intrinsic effect of SEQ ID No. 1 for signaling cascade involving TLR9 was also tested in the absence of the ODN 2006 agonist and depicted as black bars in figure 1.

The same experimental setup using HEK-Blue™ TNF-α cells as described above was used as a control. These TNF-α sensitive cells were pre-incubated with different concentrations of SEQ ID No. aptamer. 1 (0, 0.37, 1.1, 3.3, 10 and 20 µM aptamer) for 60 minutes before 100 ng/ml TNF-α (molecular weight 17484 daltons; TNF-α final concentration: 5.7 nM), and the cells were incubated for an additional 18 hours. Next, the optical density was determined. The results of this analysis are shown as gray bars in Figure 2. Potential intrinsic effect of SEQ ID No. 1 for a signaling cascade involving TNF-α was also tested in the absence of the TNF-α stimulatory molecule and is depicted as black bars in Figure 2.

From these experiments and the results shown in Figures 1 and 2, it can be seen that the aptamer SEQ ID No. 1 (GGT TGG TGT GGT TGG) does not have any inherent or non-specific effects on the agonist/antagonist assay or reporter expression, but instead shows a clear inhibition and suppression of TLR9 activation.

Example 2:

As additional control experiments, the assay described above and in Example 1 was used to determine the potential inhibitory effects of another oligonucleotide sequence. To this end, the same experiments were performed, including controls, as described in Example 1 using SEQ ID No. 3 (TGG AGG TGG A) instead of SEQ ID No. 1 for comparison.

The results obtained with SEQ ID No. 3 on ODN 2006-stimulated HEK-Blue™ hTLR9 cells are shown in Figure 3, and on TNF-α stimulated HEK-Blue™ cells, TNF-α are shown in Figure 4, respectively.

From this set of control experiments, it can be seen that the tested 10-mer SEQ ID No. 3 shows no inhibitory effects on TLR9, but instead shows an independent and additive stimulatory effect on TLR9 in the absence (see black bars in Figure 3) or presence of an ODN 2006 agonist (see gray bars in Figure 3). Again, with the aptamer SEQ ID No. 3 (see figure 4) no effect on the signaling cascade was observed.

Example 3:

As a control for the significance and validity of the assay used in Examples 1 and 2 above, a control assay was performed using the established TLR9 CpG antagonist ODN TTAGGG (SEQ ID No. 4: TTA GGG TTA GGG TTA GGG TTA GGG; molecular weight 7575 Da ) at increasing concentrations (0, 30, 100, 300, 1000 and 3000 ng of the TLR9 antagonist, corresponding to 0, 3.96, 13.2, 39.6, 132 and 396 nM, respectively) instead of the aptamers from example 1 or 2, as described above.

The results obtained with the control antagonist on ODN 2006-stimulated HEK-Blue™ hTLR9 cells are shown in Figure 5. As expected, the established TLR9 antagonist was capable of inhibiting and/or suppressing TLR9 activation (see gray bars in Figure 5) and did not produce a significant intrinsic effect in the absence of the TLR9 agonist ODN 2006 (see black bars in figure 5).

--->

SEQUENCE LIST

<110> BERLIN KURES GMBH

<120> Aptamers for use in inhibition and/or suppression

TLR9 activation

<130> B 30246

<160> 4

<170> BiSSAP 1.3

<210> 1

<211> 15

<212> DNA

<213> Artificial sequence

<220>

<223> SEQ ID No. 1

<400> 1

ggttggtgtg gttgg 15

<210> 2

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> SEQ ID No. 2

<400> 2

tcgtcgtttt gtcgttttgt cgtt 24

<210> 3

<211> 10

<212> DNA

<213> Artificial sequence

<220>

<223> SEQ ID No. 3

<400> 3

tggaggtgga 10

<210> 4

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> SEQ ID No. 4

<400> 4

ttagggttag ggttagggtt aggg 24

<---

Claims (9)

1. Use of a method comprising administering to a human subject an effective amount of an aptamer for treating the subject by inhibiting or suppressing TLR9 activation in a human cell, wherein the aptamer consists of the nucleic acid sequence of SEQ ID No. 1 (GGT TGG TGT GGT TGG) and wherein the aptamer specifically interacts with the TLR9 receptor to inhibit TLR9 activation in the subject's cell. 2. Use according to claim 1, wherein the subject to be treated demonstrates overexpression of TLR9 and/or overactivity of TLR9-mediated signaling. 3. Use according to claim 1, wherein the subject to be treated has a positive test result for TLR9 overexpression and/or TLR9-mediated signaling overactivity. 4. Application according to any one of paragraphs. 2 or 3, where TLR9 overexpression and/or TLR9-mediated signaling overactivity in a subject is established if TLR9-mediated TNFα production is increased above a reference value for healthy subjects. 5. Use according to claim 4, wherein the value for the subject to be treated is determined by a method using TLR9-mediated TNFα production as a surrogate parameter and comprising the following steps: isolation of TLR9-transferring peripheral blood mononuclear cells (PBMC), provoking an agonist (ligand )-mediated TLR9 response to PBMC and assessment of the amount of TNFα formed by enzyme immunoassay (ELISA), where the specified value is at least 50 pg/ml, preferably at least 75 pg/ml, more preferably at least 100 pg/ml , most preferably at least 125 pg/ml. 6. Application according to any one of paragraphs. 1-4 or 5, wherein the subject is suffering from a disorder selected from an autoimmune disorder, an inflammatory disorder, an autoimmune connective tissue disease (ACTD) and/or a neurodegenerative disorder, preferably the disorder is selected from psoriasis, rheumatoid arthritis, alopecia universalis, acute disseminated encephalomyelitis, disease Addison, allergies, ankylosing spondylitis, antiphospholipid antibody syndrome, arteriosclerosis, atherosclerosis, autoimmune hemolytic anemia, autoimmune hepatitis, bullous pemphigoid, Chagas disease, chronic obstructive pulmonary disease, celiac disease, cutaneous lupus erythematosus (CLE), dermatomyositis, diabetes mellitus, dilatation (DCM), endometriosis, Goodpasture's syndrome, Graves' disease, Guillain-Barré syndrome, Hashimoto's disease, hidradenitis suppurativa, idiopathic thrombocytopenic purpura, inflammatory bowel disease, interstitial cystitis, localized scleroderma, multiple o sclerosis (MS), myasthenia gravis, myocarditis, narcolepsy, neuromyotonia, pemphigus, pernicious anemia, polymyositis, primary biliary cirrhosis, rheumatoid arthritis (RA), schizophrenia, Sjögren's syndrome, systemic lupus erythematosus (SLE), systemic sclerosis, temporal arteritis, vasculitis, vitiligo, vulvodynia, Wegener's granulomatosis, traumatic pain, neuropathic pain, acetaminophen toxicity and tumor/cancer, where preferably the tumor/cancer is selected from the group consisting of breast cancer, cervical squamous cell carcinoma, gastric carcinoma, glioma, hepatocellular carcinoma, lung cancer, melanoma, prostate cancer, recurrent glioblastoma, recurrent non-Hodgkin's lymphoma, colon and rectal cancer. 7. A method for inhibiting or suppressing TLR9 activation in a human cell, comprising contacting a human cell expressing TLR9 with an aptamer, wherein the method is performed in vitro/ex vivo and wherein the aptamer consists of a nucleic acid sequence of SEQ ID No. 1 (GGT TGG TGT GGT TGG) and interacts specifically with the TLR9 receptor to inhibit TLR9 activation in a subject's cell. 8. The method of claim 7, wherein the method further comprises testing the cell for TLR9 overexpression and/or TLR9-mediated signaling overactivity. 9. Use of a method comprising contacting a human cell expressing TLR9 with an effective amount of an aptamer consisting of the nucleic acid sequence of SEQ ID No. 1 (GGT TGG TGT GGT TGG), to inhibit or suppress TLR9 activation in a human cell, where the aptamer is used in vitro/ex vivo and where the aptamer specifically interacts with the TLR9 receptor to inhibit TLR9 activation in a subject cell.

Images