MXPA99005880A - G-rich oligo aptamers and methods of modulating an immune response - Google Patents

Abstract

Aptamer oligonucleotides specifically bind to the DNA binding site of proteins such as Sp1 and Sp1-related proteins which regulate the genes which encode costimulatory molecules such as CD28 and cytokines such as IL-2 and GMCSF. The oligonucleotides compete with the DNA-binding sites of regulatory proteins which specifically regulate molecules to modulate T-cell activation. This serves to modulate gene expression by preventing transcription of the gene. Aptamers are administered to provide therapies for diseases which involve aberrant T-cell activation such as psoriasis, Type I (insulin-dependent) diabetes mellitus, multiple sclerosis, autoimmune uveitis, rheumatoid arthritis, systemic lupus erythematosus, inflammatory bowel disease (Crohn's and ulcerative colitis), and septic shock and to regulate normal T-cell activation such as in allograft rejection.

Description

OLIGOAPTAMERS RICH IN G AND METHODS TO MODULATE AN IMMUNE RESPONSE FIELD OF THE INVENTION The field of the invention is immunology.

BACKGROUND OF THE INVENTION The pathogenesis and exacerbation of many prevalent T-cell-mediated diseases results from an inappropriate immune response driven by the abnormal activation of T cells. Many other diseases are considered to be caused by aberrant activation of T cells and include type I diabetes mellitus (dependent on insulin), thyroiditis, sarcoidosis, multiple sclerosis, autoimmune uveitis, rheumatoid arthritis, systemic lupus erythematosus, inflammatory bowel disease (Crohn's and ulcerative colitis) and aplastic anemia. In addition, various syndromes that include septic shock and tumor-induced cachexia may involve T cell activation and increased production of potentially toxic levels of lymphokines. The normal activation of T cells also mediates the rejection of transplanted cells and organs by providing the signals necessary for the effective destruction of "foreign" donor tissue.

REF .: 30474 The activation of T lymphocytes leads to proliferation of T cells and gene expression and secretion of specific immunomodulatory cytokines that require two independent signals. The first signal involves the recognition, by the specific T cell / CD3 receptor complex, of antigen presented by the major histocompatibility complex molecules on the surface of antigen-presenting cells (APC). Nonspecific intercellular interactions with the antigen between T cells and APCs provides a second signal which serves to regulate T cell responses to antigen. These secondary or costimulatory signals determine the magnitude of the T cell response to the antigen. The costimulated cells react by increasing the transcription levels of the cytokine-specific gene and by stabilizing the selected mRNAs. Activation of T cells in the absence of costimulation results in an aborted or anergic T cell response. A key costimulatory signal is provided by interaction of the T cell surface CD28 receptor with B7 related molecules in APC (Linsley and Ledbetter (1993) Annu Rev Immunol 11: 191-212). CD28 is constitutively expressed in 95% of CD4 + T cells (which provides auxiliary functions for the production of antibodies by B cells) and in 50% of CD8 + T cells (which have cytotoxic functions) (Yamada et al (1985 ) Eur J. Immunol 15: 1164-1168). After antigenic or mitogenic stimulation in vi tro, there is an additional induction of the superficial levels of CD28, as well as the production of certain immunomodulatory cytokines. These include interleukin-2 (IL-2), necessary for the cell cycle progress of T cells, interferon-gamma (IFN-γ), which shows a wide variety of antiviral and antitumor effects, and interieucine 8 (IL-). 8) known as a potent chemotactic factor for neutrophils and lymphocytes. It has been shown that these cytokines are regulated by the CD28 pathway of T cell activation (Fraser et al (1991) Science 251: 313-316, Seder et al (1994) J. Exp Med 179: 299-304, Wechsler et al. al (1994) J. Iwmunol 153: 2515-2523). IL-2, IFN? IL-8 are essential for promoting a wide range of immune responses and have been shown to be overexpressed in many disease states mediated by T cells. In psoriasis, activated T cells of the lesion release predominantly Thl cytokines such as IL- 2 and IFN? (Schlaak et al (1994) J Invest Derm 102: 145-149). These secreted cytokines induce normal keratinocytes to express the same phenotype (HLA DR + / ICAM-l +>) as found in psoriasis lesions (Baadsgaard et al (1990) J Invest Derm 95: 275-282). In addition, IL-8, by virtue of its proinflammatory properties in vi tro and in vivo, and because it is secreted in large quantities by both activated T cells and keratinocytes of psoriatic lesions, is considered a major contributor to the pathological changes observed in psoriatic skin such as hyperproliferation of keratinocytes. In addition, a member of the B7 family of receptors (the natural ligands for CD28 found in activated APCs), BB1, has been shown to be expressed in psoriatic keratinocytes but not in unaffected cutaneous keratinocytes (Nickoloff et al (1993) Am J Pathology 142: 1029-1040) underlining the importance of T cell activation in the pathogenesis of the disease. In other skin disorders mediated by T cells such as allergic contact dermatitis and lichen planus, CD28 is expressed in high concentrations in most skin and epidermal CD3 + T cells, but in normal skin and basal cell carcinoma (a disease cutaneous that is not mediated by T cells) CD28 is expressed only in perivascular T cells. Similarly, in both contact allergic dermatitis and lichen planus, B7 expression is found in dermal dendritic cells, skin APCs and keratinocytes, but not in normal skin and basal carcinoma cells (Simón et al (1994). ) J Invest Derm 103: 539-543). Therefore, this suggests that the CD28 / B7 pathway is an important mediator of skin diseases mediated by T cells.

The aberrant activation of T cells associated with certain autoimmune diseases caused by the loss of self-tolerance is characterized predominantly by the presence of CD28 + T cells and the expression of their ligand, B7, on the activated professional APCs (monocytes, macrophages or dendritic cells). These include autoimmune Graves thyroiditis (Garcia-Cozar et al (1993) Immunology 12 32), sarcoidosis (Vandernverghe et al (1993) Jnt Immunol 5: 317-321), rheumatoid arthritis (Verwilghen et al (1994) J "Immun? 153: 1378-1385) and systemic lupus erythematosus (Sfikakis et al (1994) Clin Exp I munol 96: 8-14) In the normal activation of T cells, which mediates the rejection of transplanted cells and organs, the union of CD28 by its appropriate B7 ligand during T-cell receptor coupling is critical for an appropriate halogenic response to foreign antigens, eg, on donor tissue (Azuma et al (1992) J. Exp Med 175: 353-360, Turka et al (1992) Proc Nat Acad Sci USA 89: 1102-1105) Traditional therapies for autoimmune diseases do not prevent the activation of T cells, the effector stage in self-reactive immune responses to self antigens, medications such as autoinflammatory drugs steroids and not steroid denos (NSAIDS) are currently used to reduce symptoms, but they do not prevent the progression of the disease. In addition, steroids can have side effects such as induction of osteoporosis, organ toxicity and diabetes, and can accelerate the process of cartilage degeneration and cause what are called post-injection redness of up to 2 to 8 hours. NSAIDs may have gastrointestinal side effects and an increased risk of agranulocytosis and iatrogenic hepatitis. Immunosuppressive drugs have also been used as another form of therapy, especially in advanced stages of disease. However, these drugs suppress the entire immune system and often the treatments have serious side effects including hypertension and nephrotoxicity. It has also been established that immunosuppressants such as cyclosporin and FK506 can not inhibit the CD28-dependent T cell activation pathway (June et al (1987) Mol Cell Biol 7: 4472-4481). Current agents which affect the activation of T cells include synthetic peptides, monoclonal antibodies and soluble forms of T cell activation molecules. To date, no competitive synthetic peptides have been identified for T cell activation molecules such as CD28, CD40L and the CAM family of adhesion molecules. Monoclonal antibodies (mAb) have been shown to have a possible therapeutic effect in such T cell-mediated diseases such as psoriasis (against CD4 (Prinz et al (1994) Lancet 338: 320-321)) and immunosuppression of normal activation of T cells in allografts (against VCAM-1 and VLA-4 (Isobe et al (1994) J "Immunol 153: 5810-5818).) However, with chronic treatment, the host animal develops antibodies against the monoclonal antibodies so that Its usefulness is limited: "Humanized" monoclonal antibodies have been developed which apparently reduce the risk of an immune response induced to these mAbs, however, they are still under development and, in addition, these new mAbs remain as large proteins and therefore can present difficulties in reaching their target sites Soluble forms of T cell activation molecules such as CTLA-4Ig, which contains the extracellular domain of the CTLA-4 gene, have been developed human (which is sequentially related to CD28), fused to a C string? Human Ig CTLA-4Ig has been shown to specifically block the normal activation of T cells by preventing rejection of xenogeneic cardiac allografts (Lenschow et al (1992) Science 257: 789-792) and allogeneics (Turka et al (1992) Proc Nati Acad Sci USA 89: 1102-1105) in rats and has therapeutic effects on the aberrant activation of T cells such as those found in rat autoimmune glomerulonephritis (Nishikawa et al (1994) Eur J Im unol 24: 1249-1254). Nevertheless, Soluble CTLA-4Ig presents limitations similar to that of monoclonal antibodies in addition to the cost of its production. Furthermore, the true function of this molecule similar to CD28 is not known and therefore needs to be fully determined before its therapeutic benefit can be evaluated. Inhibition of cell surface expression of CD28 leads to a lack of prolonged response or suppression of activated T cells. Inactivation prevents the proliferation of T cells and suppresses the specific production of T cells by specific immunoregulatory cytokines such as interieucin 2, interferon gamma and interieucin 8. Regulation of CD28 gene expression can be obtained using antisense and triple-shaped oligonucleotides. hybridizing oligodeoxyribonucleotides or oligoribonucleotides to DNA or RNA sequences within the paque gene CC28 or the codiplic region (? PCT ÜB96 / 0.L507, filed August 30, 1996). Oligonucleotides avoid many of the deficiencies of the current agents used to block the effects of normal and abnormal activation of T cells. However, these oligos (oligonucleotides) designed for antisense strategies are susceptible to degradation by intracellular nucleases or nucleases present in the extracellular medium It has previously been shown that DNA binding (or RNA) to the protein is a fundamental way by which the transcription of a gene is controlled. These regulatory proteins or transcription factors recognize DNA sequences with specific secondary structure and the following interaction can lead to positive or negative control of gene expression. Aptamers are short sequences of oligonucleotides 1-which can specifically bind specific proteins. It has been shown that different aptameric sequences can bind specifically to different proteins, for example, the sequence GGNNGG where N = guanosine (G), cytosine (C), adenosine (A) or thymidine (T) bind specifically to thrombin (Bock). et al (1992) Nature 355; 565-566"and" patent # 5582981 (1996) Toóle et al). However, no aptameric sequences have been described which can function as competitive inhibitors of DNA binding sites on regulatory proteins known as transcription factors. Transcription factors are a class of proteins which regulate genes by binding mainly to specific regulatory sequences in the promoter region towards the 5 'end of these genes. This interaction leads to the start of transcription. Certain transcription factors such as Spl, AP2, AP-1, EGR-1 and NF B are critical in the activation of T and B lymphocytes (Skerka et al. J Biol Chem 270: 22500-22506, Jung et al (1995) Ann. N And Acad Sci 766: 245-252). In some cases these transcription factors are induced by signals initiated after co-stimulation (Jung et al (1995) Ann N and Acad Sci 766: 245-252). Therefore, there is still a need for the development of agents and methods to interfere with the interaction of proteins with DNA binding site which may lead to suppression of certain immune pathways, including the costimulatory pathway.

BRIEF DESCRIPTION OF THE INVENTION The present invention provides aptamers that have a length of between 12 and 22 nucleic acid units, inclusive, and a sequence that includes at least 2 G-rich regions selected from the group consisting of GGnG, GGGG, GnGG, nGGG and GGGn, where G is guanidine and n is any nucleotide. In preferred embodiments, the oligonucleotides are designed to bind to specific regulatory proteins such as Spl and Spl-related proteins and to act to compete with the binding of these transcription factors to the promoter region of the genes which are under their control.

This serves to modulate gene expression by preventing transcription of the gene. Therefore, the antisense oligonucleotides are capable of inhibiting the function of RNA or DNA, either its translation into a protein, its translocation to the cytoplasm or any other activity necessary for its general biological function. The inability of the RNA or DNA to perform all or part of its functions results in a failure of a portion of the genome that controls the activation of T cells, so that it is expressed properly and in this way the metabolism is modulated. It is preferred to direct the aptameric nucleic acid decoy to compete with the DNA binding sites of regulatory proteins which specifically regulate molecules which can modulate the activation of T cells. It has been found that the CD28 protein is particularly useful for this approach . Inhibition of CD28 and expression of the gene challenged with CD28 is expected to be useful for the treatment of psoriasis or other skin diseases, syndromes with aberrant T cell activation, autoimmune disorders and allograft rejection. Methods are provided for modulating the activation of T cells, comprising contacting a patient with an oligonucleotide which competes with the DNA binding site of a regulatory protein so that the expression of a regulatory protein known to be inhibited is inhibited. it is able to modulate the activation of T cells. Oligonucleotides are preferred which bind to proteins such as Spl and Spl related proteins which regulate the transcription of CD28 and genes related to CD28. In another aspect of the invention, aptamers are administered to provide therapies for diseases which involve aberrant activation of T cells such as psoriasis, psoriasis exacerbated by AIDS and other skin diseases, diabetes mellitus type I (insulin dependent) thyroiditis, sarcoidosis, multiple sclerosis, autoimmune uveitis, rheumatoid arthritis, systemic lupus erythematosus, inflammatory bowel disease (Crohn and ulcerative colitis), septic shock, tumor-induced cachexia and aplastic anemia, and to regulate the normal activation of T cells, for example in allograft rejection. This can be obtained by perturbation in the synthesis and expression of the T cell activation molecules including CD28 and molecules related to CD28. In still another aspect of the present invention, aptamers are provided which are capable of binding to specific regulatory proteins such as Spl and Spl-related proteins and thereby inhibit the transcription of genes such as CD28 and CD28-related proteins which: a) they are normally regulated by these proteins, and b) they can modulate the T cell responses.

BRIEF DESCRIPTION OF THE DRAWINGS Figures 1A and IB are graphical representations of the in vitro stability of 32P labeled phosphorothioate, ICN 16064 (Sec # 4), in extracellular fluid and in Jurkat cells, respectively. Figures IC and ID are graphical representations of the stability of labeled phosphorothioate 32P, ICN 16214 (Sec # 21) in extracellular fluid and in Jurkat cells, respectively. Figures 1E and 1F are graphical representations of the time-dependent degradation (0-96 h) of each oligonucleotide ICN 16064 (Sec # 4) and ICN 16214 (Seq # 21), (2000 cpm) determined by 20% polyacrylamide denaturing gel electrophoresis followed by visualization using a Phosphorlmager. In the eluates, the percentage of intact full length of 32P-RT03S (0) and 32P-RTC06S (•) remaining at each time point, in relation to t = 0, from 1000 cpm of extracellular (figure 1E) is determined. ) and cellular (figure 1F) applied through Nickspin columns (Pharmacia). The molecular weight (Std), 32P-dNTP (N) and the free 32P-orthophosphate (P) standards were analyzed simultaneously. Figure 2 is a graphical representation of a gel shift assay which shows that oligonucleotides containing a sequence motif of 12 G-rich units (lane 5 and 11) provides a distinct A band which differs in electrophoretic displacement respect to band B observed with other phosphorothioate oligonucleotides subsequent to incubation with HeLa nuclear extract. Band C is 32P-oligo alone.

Figure 3 is a graphic representation of the expression of chloramphenicol acetyltransferase (CAT) following transfection of Jurkat cells with plasmid vectors containing a 226 bp insert from the promoter region CD28 (residues -197 to +28) (28b) or a mutant with a substitution at residues -51 or -22 with Sec # 3 of Table 1 (28h-1) towards the 5 'end of the CAT reporter gene, and subsequent treatment with and without phosphorothioate oligonucleotides ICN 16064 and ICN 16481 La7 Figure 4 is a graphical representation of the superdisplacement gel test showing that the binding of Spl to 28b, the region towards the 5'192 to +28 end of the CD28 gene is specific. Figure 5 is a graphical representation of the binding of Spl to the double-stranded oligo 28b labeled with 32P (which is derived from parental or original 28b - Sec # 1, table 1) and the competitive binding of the double-stranded oligo 28b cold and the aptameric oligos of figure A and 16481.

DETAILED DESCRIPTION OF THE SPECIFIC MODALITIES Aptameric oligonucleotides that specifically bind to the DNA binding site of regulatory proteins such as Spl and Spl-related proteins will avoid binding of the regulatory protein to the DNA-specific double-stranded region in the promoter region of the gene of interest. Competitive binding by the aptamer can prevent transcription of the gene and thereby inhibit the flow of genetic information from the DNA to the protein. The properties of the oligonucleotides which make it specific for its purpose also become versatile. Because the oligonucleotides are long chains of four monomer units they can be easily synthesized for any target RNA sequence. Oligonucleotide-mediated inhibition of gene expression has been demonstrated in many models and in in vitro systems, and has therapeutic potential as a novel strategy for treating many human diseases (Uhlmann and Peynan (1990) Chem Rev 90: 544-584. and Stec (1991) Oligonucleotides and analogues - A Practical Approach: 87-108, Miller et al (1981) Biochem 20: 1874-1880, Orson et al (1991) Nucleic Acids Res 19: 3435-3441, Helene and Toulme (1990) ) Biochem Biophys Acta 1049: 99-125, Thierry and Dritschilo (1992) Nucleic Acid Res 20: 5691-5698). Due to recent advances in the synthesis of nuclease-resistant oligonucleotides, including phosphorothioates Zon and Stec (1991) Oligonucleotides and analogues - A Practical Approach; 87-108 and phosphorothioate-3 '-hydroxypropylamine (Tam et al (1994) Nucleic Acid Res 22: 977-986), which shows increased cellular uptake, it is now possible to consider the use of oligonucleotides as a novel form of therapeutics. Aptameric oligonucleotides that target regulatory protein binding sites represent an alternative class of nucleic acid-based compounds and provide an ideal solution to the problems encountered with prior art solutions. They are directly involved in the modulation of specific gene expression and thus inactivate the expression of target proteins and not the competitive inhibition of soluble receptors for the target protein, an interaction which requires a complete understanding of the binding and affinity mechanism of the receptor-ligand interaction. Oligonucleotides are small molecules and therefore do not encounter the same steric problems as large inhibitor molecules.DESCRIPTION OF THE OBJECTIVES The objectives contemplated herein include molecules which can be regulated by transcription factors which play an essential role in the initiation or maintenance of an immune response. These include costimulatory molecules such as CD28 and cytokines such as IL-2, GM-CSF and IFNα. .

For therapy, an animal suspected or having a disease which can be treated by decreasing the expression of costimulatory molecules such as CD28 or molecules related to CD28, can be treated by administering oligonucleotides according to this invention. Oligonucleotides can be formulated in a pharmaceutical composition, which can include carriers, thickeners, diluents, buffers, preservatives, surfactants, liposomes or lipid formulations and the like in addition to the oligonucleotide. The pharmaceutical compositions may also include one or more active ingredients such as antimicrobial agents or anti-inflammatory agents, anesthetics and the like, in addition to the oligonucleotide. The pharmaceutical composition can be administered in many ways depending on whether a local or systemic treatment is desired, and the area to be treated. The administration can be topically (including ophthalmic, vaginal, rectal and intranasal), orally or by inhalation, or parenterally, for example by intravenous drops, or subcutaneous, intraperitoneal or intramuscular injection. Formulations for topical administration may include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable. Condoms or coated gloves may also be useful. Compositions for oral administration and in powders or granules, suspensions or solutions in water or in non-aqueous medium, capsules, sachets or tablets. Thickeners, flavorings, diluents, emulsifiers, dispersion aids or binders may be desirable. Formulations for parenteral administration may include sterile aqueous solutions which may contain buffers, liposomes, diluents and other suitable additives. The dosage depends on the severity and responsiveness of the condition to be treated, but will usually be one or more doses per day, with a course of treatment lasting from several days to several months or until the cure or when a decrease in the morbid state is obtained. People who are usually familiar with the technique can easily determine optimal dosages, dosing methodologies, and repetition rates. In a preferred systemic application, the aptamers to be administered intravenously in a dose of mg / kg once a day. In a preferred topical application, the aptamers will be administered in a 1-5% solution once per day. In a preferred lung application, the aptamers will be administered in an aerosol dose of 5 mg, once per day. The present invention utilizes aptameric oligonucleotides for use in the inhibition of RNA and DNA function which corresponds to proteins capable of modulating the activation of T cells. In the context of this invention, the term "oligonucleotide" refers to an oligomer or polymer of ribonucleic acid or deoxyribonucleic acid. This term includes oligomers consisting of naturally occurring bases, sugars and interazcar linkages (main structure) as well as oligomers that have portions that do not occur naturally but which function in a similar manner. Such modified or substituted oligonucleotides are often preferred over native forms due to their properties such as, for example, improved cellular uptake and increased stability in the presence of nucleases. The oligonucleotides according to this invention preferably comprise from about 3 to about 50 nucleic acid base units. It is further preferred that such oligonucleotides are constituted from about 8 to 30 units of nucleic acid bases, and it is further preferred that they have from about 12 to 22 nucleic acid base units. As will be appreciated, a nucleic acid base unit is a base-sugar combination suitably linked to an adjacent nucleic acid base unit via phosphodiester or other linkers. Oligonucleotides used in accordance with this invention conveniently and usually can be manufactured by the well-known technique of solid phase synthesis. Several suppliers that include Applied Biosystems sell equipment for such synthesis. Any other means can also be used for such a synthesis, however, the actual synthesis of oligonucleotides is within the capabilities of a person familiar with the art. It is also well known to use similar techniques to prepare other oligonucleotides such as phosphorothioates and 3'-amino-phosphorothioates. In accordance with this invention, persons usually familiar with the art will understand that the messenger RNA identified by the open reading frames (ORF) of the DNA from which they are transcribed include not only the DNA ORF information, but also associated ribonucleotides. which form regions known to such persons as the 5 'untranslated or 3' untranslated region and the ribonucleotides in an intervening sequence. Therefore, the oligonucleotides can be formulated according to this invention which have as their target all or a part of these associated ribonucleotides as well as with respect to the informational ribonucleotides. In preferred embodiments, the aptamer oligonucleotide interacts with the DNA binding site of a regulatory protein such as Spl proteins and Spl-related proteins, and thereby disrupts the expression of a gene encoding a protein involved in cell activation. T. In the preferred embodiments, the proteins to be regulated are CD28 and all homologs of the CD28 molecule. Oligonucleotides comprising sequences containing at least two G-rich regions defined as a four nucleotide region containing at least three guanosine residues (G) such as GGGG, GNGG, GGNG, where N = A, C are preferred. , G, U or T. In the invention, two such G-rich regions separated by a maximum of 6 residues, and preferably 4, are useful. or ones. little waste. Preferred sequence segments that may be useful in whole or in part are: 5 '3' SEQUENCE IDENTIFICATION TTG GAG GGG GTG GTG GGG FIGURE 1A GGG GAG GG GGG CTG GAA INC 16481 GGG GTG_GTG GGG ICN 16525 TTG GAG GGG GAG GGG GGG INC 16475 TTG GAG GGG GAG GTG GGG INC 16479 GGG TTG GAG GGG GTG GTG GGG INC 16065 Although it is considered that the illustrated sequences are accurate, the present invention is directed to correct sequences, although errors can be found. Oligonucleotides useful in the invention comprise one of these sequences, or portions thereof. Therefore, it is preferred to use any of these oligonucleotides as set forth above, or any of the similar oligonucleotides which can be prepared by persons usually familiar with the art from knowledge of preferred oligonucleotide targets for the modulation of the synthesis of T cell activation molecules that include CD28 and molecules related to CD28. The inhibition or modulation of CD28 production and / or CD28 homologues is expected to have significant therapeutic benefits in the treatment of diseases. In order to determine the effectiveness of the compositions, an assay or a series of tests is required.

EXAMPLES Oligonucleotides The oligodeoxynucleotides are synthesized in an automated DNA synthesizer (Applied Biosystems model 394) using the standard chemistry of phosphoramidite. The β-cyanoethylphosphoramidites, the synthesis reagents and the CPG polystyrene columns are purchased from Applied Biosystems (ABI, Foster City, CA). Columns 3-amino-modifier C3 CPG are purchased from Glen Research (Sterling, VA). For the phosphorothioate oligonucleotides, the standard oxidation flask is replaced with tetraethylthiuram / acetonitrile disulfide, and the standard ABI phosphorothioate program is used for stepwise addition of phosphorothioate linkages. After separation of the controlled pore glass column, the protecting groups are removed by treating the oligonucleotides with concentrated ammonium hydroxide at 55 ° C for 8 hours. The oligonucleotides are purified by CLAP using a reverse phase semiprep C8 column (ABI). After removal of the protective group DMT, treatment with 80% acetic acid and precipitation with ethanol, the purity of the product was determined by CLAP using an analytical C18 column (Beckman, Fullerton, CA). All oligonucleotides of > 90% purity was lyophilized to dryness. The oligonucleotides are reconstituted in sterile deionized water (ICN, Costa Mesa), adjusted to 400 μM after evaluation of D026 or nm aliquots are formed and stored at -20 ° C before their examination. In all cases, at least three batches of each oligonucleotide are used, which are included in Table 1.

In vitro studies of oligonucleotide stability Temporal stability analysis of oligonucleotides is performed as previously described (Tam et al (1994) Nucleic Acid Res 22: 977-986). Oligonucleotide degradation profiles are determined by means of electrophoresis and quantified using Nickspin columns.

Cell lines and T cell purification Mononuclear cells are isolated from peripheral blood (PBMC) of the yellow layer after centrifugation with Ficoll-Hypaque density gradient to 60 ml of blood from healthy donors. Subsequently, the T cells are purified from the PBMC using lymphokwik lymphocyte isolation reagent specific for T cells (LK-25T, One Lambda, Canoga Park CA). Subsequently incubated - an average yield of 40-60 x 106 T cells overnight at 37 ° C in 20-30 ml of RPMI-AP5 (RPMI-1640 medium (ICN, Costa Mesa, CA) containing HEPES 20 buffer mM, pH 7.4, autologous plasma 5%, L-glutamine 1%, penicillin / streptomycin 1% and 2-mercaptoethanol 0.05%) to remove any contaminating adherent cells. In all experiments the T cells were washed with RPMI-AP5 and then plated onto 96-well microtiter plates at a cell concentration of 2-3 x 10 6 cells / ml. The T-cell lymphoma cell line, Jurkat E6-1 cells (CD28 + / CD4 +) (152-TIB) are maintained in RPMI-10 (RPMI-1640 medium containing 20 mM HEPES buffer, pH 7.4, fetal bovine serum 10 % (FCS) (Hyclone, Logan, UT), L-glutamine 1% and penicillin / streptomycin 1%).

T cell activation induced by mitogen and treatment with oligonucleotide Prior to the addition of human peripheral T cells or T-cell lymphoma cell lines (0.2-0.3 x 106), 96-well microtiter plates were previously covered, in duplicate, with monoclonal antibody (mAB) against CD3 (6.25 -200 ng / well) (clone HIT 3a, Pharmingen, San Diego, CA) and washed twice with cold phosphate buffered saline, pH 7.4 (PBS). T cells treated with mAB against CD3 are subsequently activated by the addition of 2 ng of 12-myristate 13-phorbol acetate (PMA) (Calbiochem, La Jolla, CA), and incubated for 48 h at 37 ° C. T cells activated with anti CD3 / PMA are treated with 1-20 μM of oligonucleotides specific for CD28 and control oligonucleotides immediately after activation, and are re-treated 24 h later. The cells of a duplicate plate are used for immunofluorescence analysis and the AIA is used for cytokine studies, and the second plate is used for T cell proliferation analysis.

Immunofluorescence Studies After activation, 150 μl of cell supernatant from the first duplicate plate is transferred to another microplate for analysis of cell-derived cytokine production. The remaining cells are washed twice with isotonic saline, pH 7.4 (Becton Dickinson, Mansfield, MA) and resuspended in 50 μl in isotonic saline and divided into two samples. An aliquot of sample is stained concomitantly with either mAB PE-CD28 / FITC-CD4 and non-specific fluorescence assays are performed by staining the second aliquot with control monoclonal antibody varied as to isotype labeled with PE / FITC. All monoclonal antibodies labeled for fluorescence are obtained from Becton Dickinson (San José, CA). Incubations were performed in the dark at 4 ° C for 45 min using saturating concentrations of mAB. The unincorporated label is removed by washing with PBS before analysis with a FACScan flow cytometer (Becton Dickinson). The antigenic density was indirectly determined in the living cells that passed and is expressed as the medium fluorescence channel (MCF). The expression and surface of the CD4 + subset of cells stained with mAB CD28 is determined by subtracting the MCF from CD28 + CD4 + from the MCF of cells CD28"CD4". The viability of cells treated with control and treated with oligonucleotide, of all oligonucleotides in multiple donors by staining with a vital dye, propidium iodide, was determined in each batch. (final concentration, 5 μg / ml). The percentage of live cells which exclude propidium iodide by flow cytometry was determined and was > 90% (range 90-99%) after treatment with all batches of all oligonucleotides at a dose range of 1-20 μM.

Cytokine Analysis The concentrations of human cytokine derived from cells in cell supernatants were determined from the first duplicate plate. The mitogen-induced changes in interieucin-2 (IL-2) levels were determined using commercially available ELISA equipment (R &D systems Quantikine kit, Minneapolis, MN). All ELISA results are expressed as pg / ml.

Electrophoretic Mobility Displacement Analysis (EMSA). oligonucleotides were labeled at the 5 'end with [? -32P] -ATP (ICN, Costa Mesa, CA) using T4 polynucleotide kinase following the protocol of the manufacturers (Gibco BRL, Gaithersburg, MD). 10 μg of HeLa cell extract (Promega) is incubated with approximately 80,000 cpm of labeled oligonucleotide for 20 min at room temperature. Binding reaction mixtures contained 10 mM Tris-HCl (pH 7.5), 50 mM NaCl, 0.5 mM DTT, 0.5 mM EDTA, 1 mM MgCl 2, 4% glycerol, and 0.5 μg poly (d? .dC). The DNA-protein complexes are separated by electrophoresis through 4% polyacrylamide gel containing 0.5 x TBA buffer (50 mM Tris, 45 mM boric acid, 0.5 mM EDTA) for approximately 3 h at 100 V. The gel is dried and undergoes autoradiography using Phosphorlmager (Biorad, Richmond, CA).

Preparation of cDNA for the DNase Dactidal Fingerprint Assay and Gel Displacement Test The cDNA (approximately 300 base pairs) used in the DNA-protein binding in DNase fingerprint assays and the gel shift assays were isolated for plasmids pCAT3e 28b, pCAT3e 28h or pCAT3e 28h-1. 60 μg of each plasmid is digested with BglII, some are placed first on agarose gel to verify the linearity, the rest is then extracted with phenol / sevag, precipitated with ethanol and then resuspended in water and digested with Sacl. Again, a small portion is placed on the gel to verify if it has been cut. (Now two bands appear: a band of 4 kb and a band of 300 p.b.). It is extracted with phenol / sevag and precipitated with ethanol. For the next dephosphorylation, the pelleted DNA is resuspended in a small volume of water (62 μl), 1 μl of 20 U alkaline phosphatase / μl (Boehringer Mannheim, Indianapolis, IN) and 7 μl of 10X reaction buffer is added. After incubating the reaction mixture at 37 ° C for 1 h, add 7 μl of 0.2 M EGTA, pH 8.0, and the whole tube is heated at 65 ° C for 10 min. All 77 μl of the dephosphorylated DNA are placed on a 1% agarose gel to purify the 300 p.b. using a Qiaquick gel extraction equipment from Qiagen (Santa Clarita, CA). The final volume of the purified band of 300 p.b. is 70 μl and concentration is calculated as follows: 60 μg x (300 p.b./4300 p.b.) = 4.2 μg, assuming 50% recovery after all these manipulations: 2.1 μg / 70 μl = 30 ng / μl. For each labeling reaction at the end with 32P (cination), treatment with kinases) ^. 5μl to 7μl of 300 p.b. DNA are used. purified.

Polyacrylamide Gel Preparation for the Gel Displacement Test 4% non-denaturing polyacrylamide gel solution is prepared in 0.5X TBE according to the technical bulletin of Promega Gel Shift Assay Systems (acrylamide 4%, bisacrylamide 0.05%, glycerol 2.5%, 0.5X TBE). A 250 ml concentrate of the above gel solution is prepared, filtered and maintained at 4 ° C. For each use, 12.5 μl of TEMED and 187.5 μl of 10% ammonium persulfate are added to each 25 ml of the 4% gel solution concentrate, and poured into 16.5 cm x 16.5 cm x 0.75 mm glass plates. It is always allowed that the gel polymerizes during the night for optimal results. The gel is pre-cured in TBE 0.5X buffer for 30 min at 100 V, before loading the samples.

Formation and Purification of Double Chain Oligonucleotides The method used here is that of "Antiparallel polypurine phosphorothioate oligonucleotides form stable triplexes with the rat to (I) collagen gene promoter and inhibit transcription in cultured mouse fibroblast" by Jacob Joseph et al., In Nucleic Acids Research, 1997, vol. 25, No. 11, 2182-2188. Equal amounts of complementary single strands are heated at 80 ° C for 5 min in 0.25 M NaCl, followed by slow cooling to room temperature. The alignment of the double-stranded oligonucleotides is purified by electrophoresis in a 6% non-denaturing polyacrylamide gel (29: 1), subsequently trimmed, "crushed and rinsed", ethanol precipitated using the methods described in "Molecular cloning" , a laboratory manual "by Sambrook, Fritsch & Maniatis Approximately 20 ng of oligo d.s. it is used in each marking reaction.

Tagged at the End with 32P of DNA 150 to 200 ng of the 300 p.b. cDNA, or 20 ng of d.s. oligo with 10 μCi of [? -32P] ATP (4500 Ci / mmol, ICN, Irvine, CA) and 10 U of kinase and kinase IX buffer (both from Promega) in a volume of 10 μl at 37 ° C for 1 h, and purified on a Centri Spin-10 column (Princenton Separation, Adelphia, NJ). Approximately 80,000- 100,000 cmp of kinase-treated DNA is used in each gel displacement reaction.

Gel Displacement Test Proteins (nuclear extract or purified transcriptional factor) are incubated with gel displacement buffer IX (Promega, Madison, Wl) at room temperature for 5-10 min before the kinase-treated DNA is added and incubated for another 20-30 min at room temperature. The samples are then loaded in a previous run of 4% non-denaturing gel. After approximately 3-4 h of running at 100 V in TBE 0.5X, the gel is dried on two pieces of Whatman paper and exposed to an imager with phosphorus overnight.

Superdisplacement Test of Antibody Gel Antibody is pre-incubated for Spl (clone 1C6, Santa Cruz Bioteshnologies, Santa Cruz, CA) with purified SP1 (Promega) for 1 h before 32 P-labeled cDNA or oligo is added.

Completed the Gel Displacement Test Approximately 70-100 molar excess of unlabeled oligonucleotides (either single chain or double chain) are preincubated with protein at room temperature for approximately 30 min before the addition of labeled DNA with 32P.

Construction of pCAT3e 28b, pCAT3e 28h, pCAT3e 28h-l CDNA is produced towards the 5 'end of CD28 (-197 to +28) by RT-PCR using Jurkat total RNA as template. This piece of cDNA is first cloned into a cloning vector TA PCR 2.1 (Invitrogen, Carlsbad, CA). The same cDNA was subsequently subcloned into pCAT3e (Promega) by inserting it into the Xhol-Sacl site. pCAT3e 28h and pCAT3e 28h-l are mutants of pCAT3e 28b in which the -51 to -22 sequences are deleted and replaced by another 15 nucleotides.

Transfection (Transient Expression) One day before transfection, Jurkat cells are prepared in 2 or 3 T150s at 1: 4 or 1: 5 dilutions from 80-90% confluent cells. Just before transfection, all the cells are accumulated in a flask and counted (the concentration should be approximately 40 x 104 per ml) of 11 x 4 x 106 cells for 10 transaction reactions that are pelleted by centrifugation in 50 ml conical tubes. The cells are washed IX with half the original volume of PBS, then resuspended in 44 ml of previously heated fresh Jurkat medium (PRMI 1640 90%, FBS 10%, L-glutamate 1%, penicillin / streptomycin 1%), so that the final concentration is 1 x 106 / ml. 4 ml of cells are pipetted into each of the wells in 6-well plates. Pipette 2.5 μl of 2 mg / ml plasmid (pCAT3e series) into a 1.5 ml tube, add 147.5 μl of RPMI 1640 medium (without serum or antibiotics), and then add 20 μl of Qiagen Superfact reagent. to the plasmid / medium solution, they are mixed by pipetting 5 times several times, and allowed to settle at room temperature for 5-10 min. The transfection complex is added dropwise to the cells in each well, the plate is gently rotated to mix. Cells are incubated in an incubator with 5% C02 at 37 ° C, and collected for CAT assay after 24 h. If oligos are to be added after transfection, 50 μl of 400 μM concentrated oligo is added to the cells at the designated time (1 hour after transfection) and the cells are returned to the incubator.

CAT test After 24 h of incubation, the cells are harvested by pipetting the cells from each well into 15 ml conical tubes, making sure to rinse well with cell medium so that no cells are left. They are then centrifuged at 2000 rpm for 5 min at room temperature. The medium is separated with a pipette. Each cell pellet is washed 3X with 2 ml of PBS (PBS is added, vortexed, centrifuged and the medium is pipetted). Both final PBS wash is removed as possible with the tip of a pipette. 400 μl of indicator lysis buffer IX (Promega CAT Enzyme Assay System) is added to each cell pipette and transferred to a 1.5 ml tube. The cell pellet is incubated in lysis buffer at room temperature for 30 min, and vortexed occasionally. These tubes are heated at 60 ° C for 10 min at the end of the 30 min incubation, and then centrifuged at room temperature, 12,000 rpm, 2 min, the supernatant is pipetted. (lysate) to a new 1.5 ml tube. For each CAT assay reaction, 100 μl of lysate is used, and the rest is frozen at -80 ° C. Each CAT assay is adjusted as follows: 18.5 μ of water are combined with 100 μl of lysate, 5 μl of N-butyryl CoA 5 mg / ml (Promega) and 1.5 μl of chloramphenicol-1C 0.1 μCi / μl (ICN) in tube of 1.5 ml (total volume, 125 μl) and incubated at 37 ° C for 1 h. At the end of 1 h, 300 μl of xylene (ICN) are added to each tube, vortexed vigorously for 5 sec, centrifuged at full speed for 30 min at room temperature, 280 μl of the upper phase is pipetted. (xylene) _ and placed in a new tube. 100 μl of Tris 0.25 M, pH 8.0 to the 280 μl xylene phase, it is vortexed and centrifuged as in the previous one. Pipette 200 μl of the upper phase into a scintillation flask, add 5 ml of scintillation fluid, mix by inversion and count the samples in a scintillation counter.

The Stability of the In Vitro Oligonucleotide Widens the Biological Activity of the Phosphorothioate Oligoncleotides Modification of oligos with phosphorothioate internucleotide linkages can impart nuclease resistance and thus extend bioactivity from 1-2 h to 24 h (Stein, (1993) Science 261: 1004-1012). Here, we show that oligo rich in G, figure IA (sec. # 4) has greater stability in vi tro compared to phosphorothioate that is not rich in G, figure IB (Sec. # 21). In Figure IA, the electropherograms clearly show that, for both extracellular IA (S) and cellular IB (L), a larger intact amount marked with 32P Figure 1A (Sec # 4) remains considerably compared to the one remaining in the figure IB (Sec # 21) after 96 h of incubation with Jurkat cells. Consistent with this observation are the data from the Nickspin column (Figure IB). Here, the percentage of intact oligo recovered from Figure IA (Sec. # 4) after 96 h is 54% (S) and 59% (L) and from Figure IB (Sec # 21) is 10% (S) and 34% (L). These data suggest that greater nuclease resistance is imparted solely by the presence of G-rich regions in Figure IA (Sec # 4) and this is likely to be associated with the ability of this particular oligo to form folded secondary structures.

Inhibition of Functional Expression CD28 and Production of IL-2 Specific for CD28 in Human T Cells Activated by Aptameric Oligonucleotides Depends on a Specific Rich-G Motif Table 2 shows the relative inhibition of CD28 expression and production of CD28-specific IL-2 by sequence # 4 to 21 of the phosphorothioate oligonucleotide sequence (5 μM) of Table 1. Here, the Sequential requirements are precise for the bioactivity of these aptameric oligonucleotides. Table 2 shows that the inhibitory activity is sequence dependent, in particular, based on the presence of the motif that contains 2 G quartets separated by 4 bases (Sec # 5-8). These data suggest that the interaction of an oligo such as that of Figure IA (Sec # 4) and its putative objective, depend on the precise conformational requirement as that observed in an oligo-protein interaction instead of an acid hybridization requirement. nucleic: nucleic acid (as found with antisense and antigen models).

Oligonucleotides that Contain a Sequence Motive of 12 Specific Units Form a Specific Oligo Protein Complex Figure 2 shows the electrophoretic mobility shift analysis of 32 P-labeled oligonucleotides preincubated with HeLa cell extract. This list of oligos in Table 3 includes two [Figure IA (Sec # 4) and ICN 16481 (Sec. # 5)] which contains a 12-unit motif that presents two sets of tetradas G separated by 4 nucleotides. The motif presented by the oligos (lanes 5 and 11 where only the test oligos provide such oligo-protein displacement (band A) different from other phosphorothioate oligos. These data suggest that a specific protein-oligo interaction occurs with oligos containing a 12-unit motif.

Inhibition of Functional CD28 Expression in Human T Cells Activated by Aptameric Oligonucleotides Correlates with the Presence of the Specific Oligo-Protein Complex Table 4 compares the inhibitory effect of both the mitogen-induced CD28 expression and the production of IL-2 by certain phosphorothioate oligonucleotides at 5 μM, with its aptameric ability to form a specific oligo-protein complex when incubated with nuclear extract HeLa, an enriched source of transcription factors. These data clearly indicate a correlation between the inhibitory activity of the oligos that exhibit motif in the expression of CD28 and the secretion of IL-2 as well as the formation of a specific gel displacement band. Substitution within the two G-tetrads results in the loss of function and results in a disappearance of the oligo-protein complex.

The Promoter Region towards the 5 'end of CD28 -197 to +28 (28b) joins Spl The promoter region of CD28 labeled with 32P -197 +28 otherwise known as 28b is incubated with the Spl protein and serial triple dilutions of Spl antibody are made starting with 0.5 μg. A superdisplacement gel test is carried out and the DNA-protein-antibody complexes are separated after electrophoresis and the data are shown in the figure. The data indicate that Spl does not bind to region 28b of the CD28 promoter. This interaction is specific by following the serial dilution of the specific Spl antibody at 0.00617 μg the band of antibody Spl / 32P-28b / Spl (band B) disappears, leaving the band 28b / Spl (band A). This shows that 28b binds specifically to Spl. 28b marked with 32P free is the C band.

An Oligo -51 to -22 Derived from the Promotora Region Towards the 5 'End CD28 -197 to +28 (28b) and which contains the 12-unit Rich G Motive, can also be joined to Spl In an effort to restrict the precise binding region Spl ^ in 28b to the G-rich motif in the promoter region of CD28 -197 +28, an oligo of 30 double-stranded units (ds) called oligo 28b was synthesized (Sec # 1 , table 1) which contains 12 units GGGGAGGAGGGG within its sequence. It is hypothesized that, this is a Spl junction site in the CD28 promoter region. After labeling with 32P, oligo 28b is incubated with Spl extract and it is intended that they bind to each other (figure 5, band A, lane 2 and 3). The competition with oligo 28b cold ds causes the band to disappear, showing that the union is specific for Spl (lane 4) Surprisingly, the G-rich oligos of single chain phosphorothioate, Figure IA (lane 5) and 16481 (lane £) (both of which contain a rich motif in G) but not the oligo control ICN 16476 (lane 7) compete for the union to Spl. These data show that in fact the G-rich oligos of phosphorothioate, Figure IA and ICN 16481 can act as aptamers in binding to the DNA binding site of Spl. The consequence of this interaction is to prevent Spl from binding to the Spl site at -51 to -22 in the promoter region and thereby inhibiting the Spl-mediated transcription of the CD28 gene and a decrease in the expression of the mature CD28 protein. . Therefore, aptamers and methods for modulating an immune response using such aptamers have been described.

Although specific embodiments have been described herein, the scope of the invention is not limited except by the interpretation of the appended claims.

LIST OF SEQUENCES (1) GENERAL INFORMATION: (i) APPLICANT: Robert Tam (ii) TITLE OF THE INVENTION: OLIGOAPTAMERS RICH IN G AND METHODS TO MODULATE AN IMMUNE RESPONSE (iii) SEQUENCE NUMBER: (iv) CORRESPONDENCE ADDRESS: (A) RECIPIENT: Crockett & Fish (B) STREET: 1440 N. Harbor Blvd., Suite 706 (C) CITY: Fullerton (D) STATE: California (E) COUNTRY: United States of America (F) ZIP: 92835 (v) READABLE FORM OF THE COMPUTER: (A) TYPE OF MEDIA: Flexible disk (B) COMPUTER: IBM PC Compatible (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) SOFTWARE OR PROGRAM: WordPerfect 6.1 (vi) CURRENT REQUEST DATA: (A) APPLICATION NUMBER: Not yet assigned (B) SUBMISSION DATE: November 21, 1995 (C) CLASSIFICATION: Not yet assigned (viii) ATTORNEY / AGENT INFORMATION (A) NAME: Fish, Robert D. (B) REGISTRATION NUMBER: 33,880 (C) REFERENCE NUMBER / FILE: 213/015 (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: 714-525-3433 (B) TELEFAX: 714-525-3303 (2) INFORMATION FOR SEC. FROM IDENT. NO: 1 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 base pairs • (B) TYPE: nucleic acid (C) TYPE OF HEBRA: Double (D) TOPOLOGY: unknown (ii) TYPE OF MOLECULE: DNA (genomic) (xi) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: l: GGGTTCCTCG GGGAGGAGGG GCTGGAACCC (3) INFORMATION FOR SEC. FROM IDENT. NO: 2: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 15 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: Double (D) TOPOLOGY: unknown (ii) TYPE OF MOLECULE: DNA (genomic) (xi) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 2: GGAGCACAGG GTGCT (4) INFORMATION FOR SEC. FROM IDENT. NO: 3: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 15 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: Double (D) TOPOLOGY: unknown (ii) TYPE OF MOLECULE: DNA (genomic) (xi) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 3 TCATCACAGG_GTGCT (5) INFORMATION FOR SEC. FROM IDENT. NO: 4: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 18 base pairs (B) TYPE: nucleic acid (C) TYPE OF FLEECE: Double (D) TOPOLOGY: unknown (ii) TYPE OF MOLECULE: DNA (genomic) (xi) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 4: TTGGAGGGGG TGGTGGGG (6) INFORMATION FOR SEC. FROM IDENT. NO: 5: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 18 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: Double (D) TOPOLOGY: unknown (ii) TYPE OF MOLECULE: DNA (genomic) (xi) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 5 GGGGAGGAGG GGCTGGAA (7) INFORMATION FOR SEC. FROM IDENT. NO: 6: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: Double (D) TOPOLOGY: unknown (ii) TYPE OF MOLECULE: DNA (genomic) (xi) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 6: GGGTTGGAGG GGGTGGTGGG G (8) INFORMATION FOR SEC. FROM IDENT. NO: 7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 base pairs (B) TYPE: nucleic acid (C) TYPE OF FLEECE: Double (D) TOPOLOGY: unknown (ii) TYPE OF MOLECULE: DNA (genomic) (xi) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 7: TTGGAGGGGG AGGAGGGG (9) INFORMATION FOR SEC. FROM IDENT. NO: 8: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 18 pairs of bases (B) TYPE: nucleic acid (C) TYPE OF HEBRA: Double (D) TOPOLOGY: unknown (ii) TYPE OF MOLECULE: DNA (genomic) (xi) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 8 TTGGAGGGGG AGGTGGGG (10) INFORMATION FOR SEC. FROM IDENT. NO: 9 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: Double (D) TOPOLOGY: unknown (ii) TYPE OF MOLECULE: DNA (genomic) (xi) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 9; TTGGAGGCGG TGGTGGCG (11) INFORMATION FOR SEC. FROM IDENT. NO: 10: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 18 base pairs (B) TYPE: nucleic acid (C) TYPE OF FLEECE: Double (D) TOPOLOGY: unknown (ii) TYPE OF MOLECULE: DNA (genomic) (xi) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 10: TTGGAGCCGG TGGTGGCC (12) INFORMATION FOR SEC. FROM IDENT. NO: 11: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 18 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: Double (D) TOPOLOGY: unknown (ii) TYPE OF MOLECULE: DNA (genomic) (xi) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 11 TTGGAGGGGC TCCTCGGG (13) INFORMATION FOR SEC. FROM IDENT. NO: 12: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 16 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: Double (D) TOPOLOGY: unknown (ii) TYPE OF MOLECULE: DNA (genomic) (xi) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 12: TTGGAGCCGG TGGTGG (14) INFORMATION FOR SEC. FROM IDENT. NO: 13: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 12 base pairs _ (B) TYPE: nucleic acid (C) TYPE OF HEBRA: Double (D) TOPOLOGY: unknown (ii) TYPE OF MOLECULE: DNA (genomic) (xi) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 13 GGGGTGGTGG GG (15) INFORMATION FOR SEC. FROM IDENT. NO: 14: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 10 base pairs (B) TYPE: nucleic acid (C) TYPE OF FLEECE: Double (D) TOPOLOGY: unknown (ii) TYPE OF MOLECULE: DNA (genomic) (xi) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 14: GGGGTTGGGG (16) INFORMATION FOR SEC. FROM IDENT. NO: 15: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 5 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: Double (D) TOPOLOGY: unknown (ii) TYPE OF MOLECULE: DNA (genomic) (xi) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 15: TGGGG (17) INFORMATION FOR SEC. FROM IDENT. NO: 16: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 4 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: Double (D) TOPOLOGY: unknown (ii) TYPE OF MOLECULE: DNA (genomic) (xi) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 16: GGGG (18) INFORMATION FOR SEC. FROM IDENT. NO: 17: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: Double (D) TOPOLOGY: unknown (ii) TYPE OF MOLECULE: DNA (genomic) (xi) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 17: CACTGCGGGG AGGGCTGGGG (19) INFORMATION FOR SEC. FROM IDENT. NO: 18: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: Double (D) TOPOLOGY: unknown (ii) TYPE OF MOLECULE: DNA (genomic) (xi) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 18 ATGGGGTGCA CAAACTGGGG (20) INFORMATION FOR SEC. FROM IDENT. NO: 19: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 15 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: Double (D) TOPOLOGY: unknown (ii) TYPE OF MOLECULE: DNA (genomic) (xi) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 19: AACGTTGAGG GGCAT (21) INFORMATION FOR SEC. FROM IDENT. NO: 20: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 18 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: Double (D) TOPOLOGY: unknown . (ii) TYPE OF MOLECULE: DNA (genomic) (xi) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 20 TTCCAGCCCC TCCTCCCC (22) INFORMATION FOR SEC. FROM IDENT. NO: 21: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 18 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: Double (D) TOPOLOGY: unknown (ii) TYPE OF MOLECULE: DNA (genomic) (xi) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 21: AACCTCCCCC ACCACCCC (23) INFORMATION FOR SEC. FROM IDENT. NO: 22: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 22 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: Double (D) TOPOLOGY: unknown (ii) TYPE OF MOLECULE: DNA (genomic) (xi) DESCRIPTION OF THE SEQUENCE: 'SEC. FROM IDENT. NO: 22 ATTCGATCGG GGCGGGGCGA GC (24) INFORMATION FOR SEC. FROM IDENT. NO: 23: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: Double (D) TOPOLOGY: unknown (ii) TYPE OF MOLECULE: DNA (genomic) (xi) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 23: CGCTTGATGA GTCAGCCGGA A (25) INFORMATION FOR SEC. FROM IDENT. NO: 24: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 27 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: Double (D) TOPOLOGY: unknownF MOLECULE: DNA (genomic) (xi) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 24 GATCGAACTG ACCGCCCGCG GCCCCT (26) INFORMATION FOR SEC. FROM IDENT. NO: 25: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 22 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: Double (D) TOPOLOGY: unknown (ii) TYPE OF MOLECULE: DNA (genomic) (xi) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 25: AGTTGAGGGG ACTTTCCCAG GC (27) INFORMATION FOR SEC. FROM IDENT. NO: 26: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 22 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: Double (D) TOPOLOGY: unknown (ii) TYPE OF MOLECULE: DNA (genomic) (xi) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 26 TGTCGAATGC AAATCACTAG AA (28) INFORMATION FOR SEC. FROM IDENT. NO: 27: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 27 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: Double (D) TOPOLOGY: unknown (ii) TYPE OF MOLECULE: DNA (genomic) (xi) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 27: AGAGATTGCC TGACGTCAGA GAGCTAG (29) INFORMATION FOR SEC. FROM IDENT. NO: 28: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 25 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: Double (D) TOPOLOGY: unknown (ii) TYPE OF MOLECULE: DNA (genomic) (xi) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 28 GCAGAGCATA TAAGGTGAGG TAGGA Table 1.

No. of ID oligo Sequence Sec. 28b 51 GGG TTC CTC GGG GAG GG GGG CTG GAA CCC 3 'CCC AAG GAG CCC CTC CTC CCC GAC CTT GGG 28h 51 GGA GCA CAG GGT GCT - 3 'CCT CGT GTC CCA CGA 28h - 1 5' TCA TCA CAG GGT GCT 3 'AGT AGT GTC CCA CGA 4 ICN 16064 5' TTG GAG GGG GTG GTG GGG 5 ICN 16481 5 'GGG GAG GAG GGGCTG GAA 6 ICN 16065 5 'GGG TTG GAG GGG GTG GTG GGG 7 ICN 16475 5' TTG GAG GGG GAG GAG GGG 8 ICN 16479 5 'TTG GAG GGG GAG GTG GGG 9 ICN 16480 5' TTG GAG GCG GTG GTG GCG 10 ICN 16538 5 'TTG GAG CCG GTG GTG GCC 11 ICN 16539 5' TTG GAG GGG CTC CTC GGG 12 ICN 16523 51 TTG GAG CCG GTG GTG G 13 ICN 16525 5 'GGG GTG GTG GGG 14 ICN 16526 5' G GGG TTG GGG 15 ICN 16483 5 'TG GGG 16 ICN 16482 5' G GGG 17 ICN 16527 5 'CAC TGC GGG GAG GGC TGG GG 18 ICN 16528 5' ATG GGG TGC ACA AAC TGG GG 19 ICN 16487 5 * AAC GTT GAG GGG CAT 20 ICN 16476 5 'TTC CAG CCC CTC CTC CCC 21 ICN 16214 5' AAC CTC CCC CAC CAC CCC 22 SP1 5 'ATT CGA TCG GGG CGG GGC GAG C 3' TAA GCT AGC CCC GCC CCG CTC G 23 API (c-jun) 5 * CGC TTG ATG AGT CAG CCG GAA 3 'GCG AAC TAC TCA GTC GGC CTT 24 AP2 51 GAT CGA ACT GAC CGC CCG CGG CCC CT 3' CTA GCT TGA CTG GCG GGC GCC GGG GA NF-KB 5 'AGT TGA GGG GAC TTT CCC AGG C 31 TCA ACT CCC CTG AAA GGG TCC G 26 OCT 1 5 'TGT CGA ATG CAA ATC ACT AGA A 3"ACA GCT TAC GTT TAG TGA TCT T 27 CREB 5' AGA GAT TGC CTG ACG TCA GAG AGC TAG_3_• TCT CTA ACG GAC TGC AGT CTC TCG ATC 28 TFIID 5 'GCA GAG CAT ATA AGG TGA GGT AGG A 3' CGT CTC GTA TAT TCC ACT CCA TCC T Table 2 expression oligo ID Sequence CD28 IL-2 ICN 16064 TTG GAG GGGGTG GTG GGG 100 100 ICN 16481 GGGGAG GAG GGGCTG GAA 100 100 ICN 16065 GGG TTG GAG GGG GTG GTG GGG 100 100 ICN 16475 TTG GAG GGG GAG GAG GGG 100 Ios ICN 16479 TTG GAG GGG GAG GTG GGG 100 100 ICN 16480 TTG GAG GC GGTG GTG GC G 31 38 ICN 16538 TTG GAG C CG GTG GTG GC C 40 57 ICN 16539 TTG GAG GGG CTC CTC GGG 44 25 ICN 16523 TTG GAG CCG GTG GTG G 38 57 ICN 16525 GGG GTG GTG GGG 100 120 ICN 16526 _ G GGG TTG GGG 30 39 ICN 16483 TG GGG 2 2 ICN 16482 G GGG 2 2 ICN 16527 CAC TGC GCG GAG GGC TGG GG 58 76 ICN 16528 A TG GGG TGC ACA AAC TGG GG 51 63 ICN 16487 AAC GTT GAG GGG CAT 26 52 ICN 16476 TTC CAG CCC CTC CTC CCC 29 22 ICN 16214 AAC CTC CCC CAC CAC CCC 4 2 A sequence of 12 units containing two quartets G separated by four bases confers oligo activity. The minimum sequence required for invitro activity of ICN 16064 is determined by the ability of sequence changes (in bold) to affect ICN 16064-mediated inhibition of CD28 expression in human T cells activated with PMA against CD3, and its effect on the production of activated IL-2 in Jurkat T cells. * The results are expressed in relation to the activity of ICN 16064 5 μm (100%) whose inhibition interval in seven experiments is 52-79% of CD28 expression and 76-89% of IL-2 production.

Table 3: Oligo Sequence Line No.

ICN 16064 TTG GAG GGG GTG GTG GGG 11, 12 ICN 16481 GGG GAG GAG GGG CTG GAA 5,6 ICN 16480 TTG GAG GCG GTG GTG GCG "7,8 ICN 16538 TTG GAG CCG GTG GTG GCC 1,2 ICN 16485 GTT GGA GAC CGG GGT TGG 3,4 ICN 16476 TTC CAG CCC CTC CTC CCC 9,10 I Oligo Sequence CD28 IL-2 ICN 16064 TTG GAG GGG GTG GTG GGG 100 100 If ICN 16481 GGG GAG GAG GGG CTG GAA 100 If ICN 16480 TTG GAG GCG GTG GTG GCG 31 38 No ICN 16538 TTG GAG CCG GTG GTG GCC 40 57 No ICN 16485 GTT GGA GAC CGG GGT TGG 11 15 No ICN 16476 TTC CAG CCC CTC CTC CCC 29 22 No ICN 16214 AAC CTC CCC CAC CAC CCC 4 2 No It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is the conventional one for the manufacture of the objects or products to which it refers.

Claims (25)

1. CLAIMS Having described the invention as above, the content of the following claims is claimed as property. An aptamer having a length of between about 12 and 22 nucleic acid units, inclusive, and a sequence which includes at least two rich regions in G which are selected from the group of GGnG, GGGG, GnGG, nGGG and GGGn, where G is guanidine and n is any nucleotide.
2. 2. The aptamer according to claim 1, characterized in that at least two of at least two regions are separated by less than 2 to 7 nucleotides, inclusive.
3. 3. The aptamer according to claim 1, characterized in that at least two of at least two regions are separated by 3 to 6 nucleotides, inclusive.
4. 4. The aptamer according to claim 1, characterized in that at least 2 of at least 2 regions are separated by 4 nucleotides.
5. The aptamer according to claim 1, characterized in that it competes for a nucleic acid binding site of an immune regulatory protein.
6. 6. The aptamer according to claim 2, characterized in that the immune regulatory protein is selected from the group of SP1, NFKB, EGR1 and AP2.
7. 7. The aptamer according to claim 1, characterized in that it competes for a nucleic acid binding site of an immune regulatory protein, wherein at least 1 of at least 2 G-rich regions comprises GGnG, and at least 2 of At least 2 regions are separated by 2 to 7 nucleotides.
8. 8. The aptamer according to claim 1, characterized in that it competes for a nucleic acid binding site of an immune regulatory protein, wherein at least 1 of at least 2 G-rich regions comprises GGGG, and at least 2 of at least 2 regions are separated by 2 to 7 nucleotides, inclusive.
9. The aptamer according to claim 1, characterized in that it competes for a nucleic acid binding site of an immune regulatory protein, wherein at least 1 one of at least 2 G-rich regions comprises GnGG, and therefore less 2 of at least 2 regions are covered by 2 to 7 nucleotides, inclusive.
10. 10. The aptamer according to claim 1, characterized in that it competes for a "nucleic acid binding site" of an immune regulatory protein, wherein at least 1 of at least 2 G-rich regions comprises nGGG or GGGn, and at least 2 of at least 2 regions are covered by 2 to 7 nucleotides, inclusive.
11. 11. The aptamer according to claim 1, characterized in that it comprises the sequence 5 'TTG GAG GGG GTG GTG GGG31 (Seq. Ident. No. 4).
12. 12. The aptamer according to claim 1, characterized in that it comprises the 5 'sequence GGG GAG GAG GGG CGT GAA31 (Ident. Sec. No. 5). The aptamer according to claim 1, characterized in that it comprises the 5 'sequence GGG GTG GTG GGG3' (Ident. Sec. No.
13. 13).
14. 14. The aptamer according to claim 1, characterized in that it comprises the sequence 5 'TTG GAG GGG GAG GAG GGG3' (Ident. Sec. No. 7). x
15. 15. The aptamer according to claim 1, characterized in that it comprises the sequence 5 'TTG GAG GGG GAG GTG GGG3' (Ident. Sec. No. 8).
16. 16. The aptamer according to claim 1, characterized in that it comprises the sequence 5 'GGG TTG GAG GGG GTG GGG3' (Ident. Sec. No. 6).
17. 17. A method to modulate the response of the system. Immune in a patient, characterized in that it comprises administering to the patient an aptamer according to any of claims 1 to 16.
18. 18. A method for treating a patient having a condition characterized by an inappropriate response of the immune system, characterized in that it comprises administering to the patient an aptamer according to any one of claims 1 to 16.
19. The method according to claim 18, characterized in that the condition comprises the graft-versus-host response.
20. 20. The method according to claim 18, characterized in that the condition comprises an autoimmune disease.
21. 21. The method according to claim 20, characterized in that the condition comprises rheumatoid arthritis.
22. 22. The method according to claim 20, characterized in that the condition comprises multiple sclerosis.
23. 23. The method according to claim 20, characterized in that the condition comprises lupus erythomatous.
24. 24. The method according to claim 20, characterized in that the condition comprises insulin-dependent diabetes mellitus.
25. 25. The method according to claim 20, characterized in that the condition comprises psoriasis.