CN1181021A - Method and compositions for regulation of CD28 expression - Google Patents

Abstract

Oligomers are provided which are capable of reducing CD28 gene in a T cell. In addition to specific sequences, the invention includes a class of oligomers having at least two sequences of GGGG separated by 3 to 5 bases. Targets include CD28 gene, 5' untranslated regions and initiation codon, and their respective transcripts. Methods include use of the oligomers to moderate the pathogenic effects on the immune system in immune system-mediated diseases including graft versus host disease, septic shock syndrome, viral diseases, psoriasis, Type I (insulin-dependent) diabetes mellitus, thyroiditis, sarcoidosis, multiple sclerosis, autoimmune uveitis, rheumatoid arthritis, systemic lupus erythematosus and inflammatory bowel disease.

Description

Regulate the method and composition that CD28 expresses

I. invention field

The invention belongs to by using oligomer, particularly the effective oligomer of disease to the treatment immune-mediated comes the field of regulator gene expression.II. background of invention

Though immune system plays conclusive effect in the infection that protection higher organism body is avoided being in peril of one's life, immune system also is a key component in the pathogeny of multiple disease.The disease that immune system works comprises the autoimmune disease of immune system and autoantigen reaction, as systemic lupus erythematosus (sle), or with the immunomodulating relevant disease that causes by the exotic antigen reaction, as the graft versus host disease seen at transplant rejection.

The morbidity of many common T-cell mediated diseases and deterioration are to be caused by the inappropriate immunoreation that undesired T-cell-stimulating drives.The existence of activated T cell in the cell-mediated dermatosis of multiple T the report (Simon.et al., (1994) J.Invest Derm., 103:539-543).As torment 2% west population and comprise that 4,000,000 American psoriasiss are exactly a kind of cutaneous disorder, the unusual corium and the epidermis that are characterized as keratinocyte hyper-proliferative and activated T cell soak into, many reports point out these activated T cells at psoriasis (Baadsgaard et al., (1990) J.Invest Derm., 95:275-282, Chang et al., (1992) Arch.Derm, 128:1479-1485, Schlaak etal., (1994) J.Invest Derm., 102:145-149) and the AIDS psoriasis (Duvic (1990) J.Invest Derm., 90:38S-40S) the main effect in that increase the weight of.In psoriasis, activated damage T cell mainly discharge Thl cytokine (IL-2, IFN-) (Schlaak etc., (1994) J.Invest Derm., 102:145-149).The normal keratinocyte of these excretory cytokine inductions express with psoriasis damage the identical phenotype (HLA DR+/ICAM-1+) found (Baadsgaard etc., (1990) J.Invest Derm., 95:275-282).And because its body is interior and the former character of external inflammation, and secretion in a large number from the keratinocyte of activated T cells and psoriasis damage, IL-8 is considered to the main cause of finding pathological change of psoriasis skin such as keratinocyte hyper-proliferative.Moreover the BB1 of one of receptor B7 family (activate APC and go up the natural aglucon of finding of CD28) has been presented in the psoriasis and has expressed, do not influence the Keratoderma cell (Nickoloff etc., (1993) Am.J.Pathology, 142:1029-1040).

Other several diseases are considered to be caused by the abnormal T cell-stimulating, comprise I type (insulin dependency) diabetes, thyroiditis, sarcoidosis, multiple sclerosis, autoimmune uveitis, rheumatic arthritis, systemic lupus erythematosus (sle), inflammatory bowel (Crohn ' s and ulcerative colitis) and autoimmune hepatitis.In addition, multiple condensation disease comprises that the cachexia of septic shock and tumor inducing may be relevant with the generation of the horizontal lymphokine of genotoxic potential of t cell activation and increase.Normal t cell activation is also by providing the rejection to effectively destructive essential signal mediation transplanted cells of " external " donor tissue and organ.

The lymphocytic activation of T-that causes T cell proliferation, gene expression and specific immune to regulate cytokine secretion needs two independently signals.First signal comprises the antigen by special TXi Baoshouti/the main histocompatibility complex in CD3 complex identification antigen presenting cell (APCs) surface molecule is offered.The interphase interaction of antigen non-specific sexual cell is provided for regulating the T cell to antigen reactive second signal between T cell and the APCs.These secondarys or costimulatory signal decision T cell are to antigenic response amplitude.Irritation cell reacts by the mRNAs that increases specific cell factor gene transcriptional level and stable selection altogether.There is not under the stimulation situation altogether the t cell responses of that t cell activation leads to the failure or anergy.The costimulatory signal of a key by the interaction of the B7 correlation molecule on T cell surface receptor CD28 and the APC provide (Linsley and Ledbetter (1993) Ann.Rev.Immunol., 11:191-212).CD28 goes up constitutive expression (Yamada etc., (1985) Eur.J.Immunol.15:1164-1168) at the CD8+T cell (having cytotoxicity) of 95% CD4+T cell (B cell antibody has been produced miscellaneous function) and 50%.After antigen or external mitogenesis stimulated, the generation with some immunomodulating cytokines of inducing of CD28 surface level further took place.They comprise interleukin-22 (IL-2), for the T cell cycle carries out needing; IFN-is brought into play antiviral and antitumor action and interleukin-22 (IL-8) widely, is neutrophilic granulocyte and the effective chemotactic factor of lymphocyte.These cytokines have shown by the CD28 path of t cell activation regulates (Fraser etc., (1991) Science, 251F 313-316, Seder etc., (1994) J.Exp.Med., 179:299-304, Wechsler etc., (1994) J.Immunol., 153:2515-2523).IL-2, IFN-and IL-8 are to promoting that immunne response is essential widely, the different cell-mediated morbid state of many T that is presented at descended expression.

In some T cell-mediated cutaneous disorder such as anaphylaxis contact dermatitis and lichen planus, high level expression in most of skins and epidermis CD3+T cell, but in normal skin and basal cell carcinoma (a kind of dermatosis of non-T cell mediation), CD28 is only at blood vessel week T cellular expression.Similarly, under anaphylaxis contact dermatitis and lichen planus situation, find that B7 is at the skin arborescent cell, skin APCs and keratinocyte are expressed, but do not express at normal skin and basal cell carcinoma (Simon etc., (1994) J.Invest Derm., 103:539-543).Therefore this prompting CD28/B7 path is the cell-mediated dermopathic important amboceptor of T.

By the relevant abnormal T cell-stimulating of some autoimmune disease of causing of forfeiture self tolerance mainly with CD28 ⁺The existence of T cell and at activated professional APC _s(mononuclear cell, macrophage becomes arborescent cell) gone up and expressed its aglucon B7 is feature.These comprise autoimmune Graves thyroiditis (Garcia-Cozar etc., (1993) Immunol., 12:32).Sarcoidosis (Vandenbergh etc., (1993) Int.Immunol., 5:317-321), rheumatic arthritis (Verwilgher etc., (1994) J.Immunol., 153:1378-1385) and system lupus erythematosus (Sfikakis etc. (1994) Clin.Exp.Immunol., 96:8-14).In the normal t cell activation of mediation transplanted cells and organ rejection response, it is extremely crucial (Azuma etc. that CD28 is reacted in conjunction with the suitable allogeneic for relative exotic antigen such as donor tissue in the TXi Baoshouti engaging process by its suitable B7 aglucon, (1992) J.Exp.Med., 175:353-360, Turka etc., (1992) Proc.Nat.Acad.Sci.USA, 89:11102-11105).

The traditional remedies of autoimmune disease does not stop t cell activation; To the effector step in the autoreactivity immunne response of autoantigen.Be used to improve symptom at present as steroid and nonsteroidal antiinflammatory drug (NSAIDS), but they do not stop the evolution of disease.In addition, steroid has side effect as inducing osteoporosis, organ toxicity and diabetes, and quicken the cartilage degradation process and cause 2-8 hour be called injection back flushing.NSAIDS has gastrointestinal side-effect and increases agranulocytosis and the danger of iatrogenic hepatitis.Immunosuppressive drug also is used as another kind of form of therapy, disease stage especially late.Yet these medicines suppress whole immune system and treatment often has serious adverse, comprise hypertension and nephrotoxicity.Immunosuppressant of also have setting up such as cyclosporin and FK506 can not suppress the t cell activation path that CD28 relies on (June etc., (1987), Mol.Cell.Biol., 7:4472-4481).

Provided the shortcoming of treatment present available medicine of immune-mediated disease and method, just interesting new method and the compositions that these diseases of treatment are provided.III. summary of the invention

The invention provides the method and composition of the disease of treatment immune-mediated.Compositions of the present invention has the ability that reduction CD28 expresses in interested cell, it alleviates immune pathogenic effects in the disease of immune-mediated successively.The title method that reduces the CD28 expression is also as reducing CD28 ⁺The method of the antigenic stimulation of T cell, thus CD28 reduced ⁺The activation level of T cell and the release of cytokines relevant with t cell activation comprise interleukin-2, interferon-and interleukin-8.Inventive compositions comprises many different oligomers that can reduce the CD28 expression.

One aspect of the present invention provides and can reduce the oligomer that CD28 expresses by disturbing CD28 to express.Oligomer of the present invention has and CD28 gene or CD28 gene transcripts, or its homeologous nucleic acid base sequence, and the homology here is enough to allow form under the condition nucleic acid double chain spiral or triple helix in born of the same parents.Oligomer of the present invention can be DNA, RNA, or various its synthetic analogues.In specific embodiment, oligomer has 11 to 50 bases, and it comprises at least two GGGG sequences of being separated by 3 to 5 bases.

Another aspect of the present invention provides genetically engineered plasmid, is used for expressing in the born of the same parents of oligomer of the present invention in cells of interest, preferred natural expression CD28 cell.

Another aspect of the present invention provides the pharmaceutical formulation that comprises the different oligomers of one or more the present invention.This pharmaceutical formulation can be fit to various forms of human body medications maybe will import the intravital cell administration of body.

Another aspect of the present invention provides the method for the disease of treatment immune-mediated.Method of the present invention comprises by giving effective dose oligomer of the present invention regulates the CD28 expression.Method of the present invention comprises the method for the treatment of autoimmune disease, the method for reduce inflammation, replying, the method that the cytokine that minimizing is selected produces, the method for deactivation T cell, and the method for immunosuppressant transplant patient.IV. accompanying drawing summary

Fig. 1 be 5 ' untranslated region sequence (1A) of CD28 gene and the mRNA sequence of people CD28 (1B, 1C).Figure 1B represents the neighbouring part that polynucleotide sequence is different with 1C.

Fig. 2 is with behind different the CD28 special 5 contrast thiophosphates and the processing of thiophosphate 3 ' hydroxyl propylamine oligonucleotide, the diagram of survival (living) T cell percentage.

Fig. 3 is anti--diagram that the inductive CD28 of CD3 monoclonal antibody/PMA expresses in the human T-cell from two donors (GV010 and JC011), (A) and the special opposition of CD28 according to substituted phosphate (B, in batches 1 and 2) and thiophosphate-3 '-influence diagram that hydroxyl propylamine (C) oligonucleotide is expressed in the periphery blood T cell from identical two donors the inductive CD28 of CD 3-resisting monoclonal antibody/PMA.

Fig. 4 illustrates A) mitogen is to the inducing T cell proliferation function from the human T-cell of donor KS006, and B) CD28 special with the influence of contrast thiophosphate oligonucleotide to the inductive human T-cell's propagation of CD 3-resisting monoclonal antibody/PMA.

Fig. 5 is shown in inducing action and the CD28 influence special and that contrast thiophosphate (B) thiophosphate-3 ' hydroxyl propylamine (C) oligonucleotide generates the inductive IL-2 of CD 3-resisting monoclonal antibody/PMA in people's periphery T cell that CD 3-resisting monoclonal antibody and PMA generate interleukin-22 (IL-2) among the human T-cell.

Fig. 6 illustrates CD 3-resisting monoclonal antibody and PMA and induces (A) to what IFN-among the human T-cell (IFN γ) produced, and CD28 special with influence that contrast thiophosphate (B) thiophosphate-3 ' hydroxyl propylamine (C) oligonucleotide generates the inductive IFN-of CD 3-resisting monoclonal antibody/PMA in people's periphery T cell.

Fig. 7 illustrate inducing action (A) that CD 3-resisting monoclonal antibody and PMA generate interleukin 8 among the human T-cell (IL-8) and CD28 special with influence that contrast thiophosphate (B) thiophosphate-3 ' hydroxyl propylamine (C) oligonucleotide generates the inductive IL-8 of CD 3-resisting monoclonal antibody/PMA in people's periphery T cell.

Fig. 8 be shown in and people's periphery T cell of and processing contrast thiophosphate 3 ' hydroxyl propylamine oligonucleotide special without CD28 in, CD 3-resisting monoclonal antibody and PMA dialogue Jie plain-2 acceptor (IL-2R, or be called CD25) (A) and inducing of (B) expressing of intracellular adhesion molecule 1 (ICAM-1, or be called CD54).

Fig. 9 illustrates CD 3-resisting monoclonal antibody and PMA handles front and back, the expression of CD28 in HUT 78 (A) and Jurkat (B) the human T-cell system, and the effect of the Jurket cell (C) handled at CD 3-resisting monoclonal antibody and PMA of the special thiophosphate oligonucleotide of CD28.

Figure 10 illustrates the influence of interleukin-22 generation in HUT 78 (A) that the special thiophosphate oligonucleotide of CD28 handles CD 3-resisting monoclonal antibody and PMA and Jurkat (B) the human T-cell system.

Figure 11 illustrates the influence of thiophosphate oligonucleotide to the surface expression and the cytokine secretion of accessory molecule in the activated T cell.

Figure 12 illustrates the influence of thiophosphate oligonucleotide to CD28 and CD25 mRNA level.

Figure 13 illustrates the relevant specificity to CD28 expression inhibiting effect that function is arranged with RT04S (SEQ IDNO:45) of oligonucleotide RT035 (SEQ ID NO:44).

Figure 14 diagram is by external tolerance-induced of the special oligonucleotide RT 03S of CD28 (SEQ ID NO:44) and RT04S (SEQ ID NO:45).

Figure 15 diagram ³²Thiophosphate RT03S of P labelling (SEQ ID NO:44) and RTC06S (SEQ ID NO:48) be the vitro stability in (following hurdle) after extracellular supernatant (going up the hurdle) and Jurkat lysis.V. the detailed description of particular embodiment

Described here is the method and composition that is used for the treatment of the immune-mediated disease, and wherein Qi Wang curative effect passes through to reduce the expression of CD28, thereby cancels activated CD28 ⁺T cell function and the activation that reduces other immune system cell obtain.The inventor finds that the t cell activation that antigen relies on can be by reducing CD28 at CD28 ⁺Expression in the T cell and being suppressed.The invention provides many chemical compounds that CD28 expresses that can be used for reducing in the T cell.

Invention described here comprises that find reducing can the conflict antigenic specificity of T cell of the expression of CD28 in the T cell activates.This discovery can be used to provide many non-oligomers of expressing with oligomer and the reduction CD28 of targeting CD28 to treat the method for immune-mediated disease.By adopting the reduction CD28 described in this application to express the discovery of biological effect, the several different methods of treatment immune-mediated disease is provided, these methods can be used the non-oligomer chemical compound of also not synthetic or purification.

One aspect of the present invention is to provide the technology that is considered to a kind of utilization of having set up " antisense " oligonucleotide or " antisense therapy " usually for the oligomer that can be used for suppressing some gene expression.Existing many about making up and use the article of antisense.Be typically: Stein et al., Science, 261: 1004-1012 (1993); Milligan, et al., The pharmaceutical chemistry magazine, 36: 1923-1937 (1993); Helene, et al., J.Biochim.Biophys.Acta, 1049: 99-125 (1990); Wagner, Nature., 372: 333-335 (1994); And Crooke and Lebleu, Antisense research And use, CRC Press, Boca Raton (1993).At a little terms " antisense " that use, except as otherwise noted, refer to and to form the oligomer (comprising oligonucleotide) of two strands or triple helix with conflict gene expression with polynucleotide.Antisense design of describing in these and other similar article and the principle of using can be used to design, make and use the various embodiments of the special oligomer of CD28 of the present invention by those of ordinary skills.

Oligomer of the present invention can be regulated the CD28 expression of gene.Oligomer of the present invention comprises that those have can be by the oligomer that forms double-stranded polynucleotide spiral or form the characteristic of double-stranded polynucleotide spiral by a part or the hybridization of a plurality of part with the CD28 gene with CD28 transcript (or its part) hybridization, and wherein spiralization can occur in the born of the same parents under the condition.Oligomer of the present invention comprises that also those can influence the oligomer of gene expression regulation, as interacting by the protein-nucleic acid between the regulating element that stops transcription factor and CD28 gene untranslated region as molecule bait (moleculardecoy).In addition, oligomer of the present invention comprises that those can form the oligomer of three chain polynucleotide spirals with one or more parts of CD28 gene, and wherein this spiralization can take place under the condition in cell.The nucleic acid base pair relationhip (as adenine-thymus pyrimidine, cytosine-guanine, uracil-thymus pyrimidine) of double-stranded spiral and triple helix is that those of ordinary skills are known, and can be used to design oligomer of the present invention.Can with the present invention form two strands or triple helix to oligomer CD28 gene or CD28 genetic transcription one's respective area be called by this oligomer " targeting ".

People CD28 is the 90 kDa homologous dimerization transmembrane glycoproteins on a kind of T of being present in cell subsets surface.CD28 is present in most of CD4 ⁺T cell and about 50%CD8 ⁺On the T cell.The DNA sequence of coding people CD28 is measured, is found in other place, at Lee etc., and Journal ofImmunology, 145:344-352 (1990) and obtainable gene database of the public such as GenBank.People CD28 gene comprises four exons, the proteic functional domain of each decision expection.Observed the transcription product that people CD28 gene produces different sizes.Oligomer of the present invention can design with reference to CD28 gene nucleotide series that separates or the deutero-cDNAs sequence of CD28 gene.The compositions and methods of the invention can easily be changed to be used for inhuman mammal from the CD28 gene order of non-human mammal by reference.The sequence of inhuman CD28 gene can obtain as gene bank hybridization probe and/or PCR (polymerase chain reaction) amplimer by using the CD28 gene order from people (or other mammal) that will before determine.Though disclosed CD28 gene nucleotide series is considered to accurately, even disclosed CD28 nucleotide base sequence contains sequence errors, the invention of this theme can be carried out by one of ordinary skill in the art.The method of the CD28 gene region (or CD28 gene transcripts) that suitable nucleotide base sequence mistake can be by the preface of resurveying oligomer targeting of the present invention in the open sequence records.The available conventional dna sequencing technology preface of resurveying.

Oligomer of the present invention preferably contains an about 11-50 nucleic acid base unit.Those skilled in the art appreciates the oligomer of invention easily and obviously is longer than 50 nucleic acid base units.At one more preferably in the embodiment of the present invention, oligomer comprises an about 8-25 nucleic acid base unit; A 14-22 nucleic acid base unit more preferably from about.The restriction of the preferred size of oligomer of the present invention only is suitable for the oligomer that born of the same parents give cell outward, is unsuitable for the special oligomer of CD28 that produces in the born of the same parents, as is used for Controlled Target host cell gene carrier and produces.

Oligomer of the present invention can have multiple different nucleic acid base sequence.Oligomer of the present invention can be selected come by with any area hybridization (by nucleic acid-nucleic acid interaction) of the CD28 transcript of CD28 gene in fact reducing the expression of CD28, or reduce the expression of CD28 by non-making nucleic acid molecular hybridization (by nucleic acid-protein-interacting) with identification CD28 gene non-translated sequence.For example, oligomer of the present invention can selectedly do can with translation district, the CD28 transcript untranslated region of CD28 transcript, the non-shear zone of CD28 transcript, the CD28 gene intron, the CD28 promoter sequence, CD28 regulates sequence, 5 ' medicated cap district of CD28 transcript, hybridization such as CD28 gene coding region (comprising the combination in different characteristic zone).At the preferred embodiment of the CD28 gene of oligomer of the present invention and CD28 gene transcripts translation and/or transcription initiation region at CD28 gene (and transcript).By selecting to change the position that spiralization takes place by oligomer nucleic acid base matched sequence on CD28 or CD28 gene transcripts, the effectiveness of oligomer promptly produces the required amount of expectation biological effect and will change.The preferred embodiment of oligomer of the present invention has the highest may rendeing a service.The effectiveness of the different oligomers of the present invention can be measured by the known various in vitro testses of persons skilled in the art.The embodiment of these methods can find at the experimental section of the application's book.One of ordinary skill in the art advocates not expect to produce the oligomer that targeting also is present in the polynucleotide sequence of the gene locus outside the CD28 gene.For example, do not expect to produce the oligomer (the Alu region description of CD28 is in Lee etc., Journal of Immunology, 145:352 (1990)) of Ala sequence in 5 ' untranslated region of targeting CD28 transcript.The use of the oligomer that forms two strands or triple helix with gene outside the CD28 gene or gene transcripts can be searched for and minimizes by carry out oligomer nucleotide base sequence homology at the polynucleotide sequence information that exists among obtainable data base of the public such as the GenBank.

In a preferred embodiment of the invention, oligomer of the present invention and selected target sequence show perfect nucleic acid base complementarity, and promptly each nucleic acid base can carry out base pairing with second (or the 3rd) nucleic acid base on another chain of Double helix (or triple helical) in the oligomer.Yet, one of ordinary skill in the art understand various to CD28 gene target special and/or can suppress the oligomer that CD28 expresses and may have and CD28 gene (arbitrary chain) nucleotide base sequence of CD28 gene transcripts or the complete crossability of CD28 specific regulating hypoproteinosis.

Oligomer has following nucleotide base sequence in the oligomer preferred embodiment of the present invention;

5′TTGTCCTGACGATGGGCTA3′?????(SEQ?ID?NO：1)??RT01

5′AGCAGCCTGAGCATCTTTGT3′????(SEQ?ID?NO：2)??RT02

5′TTGGAGGGGGTGGTGGGG3′??????(SEQ?ID?NO：3)??RT03

5 ' GGGTTGGAGGGGGTGGTGGGG3 ' (SEQ ID NO:4) RT04 has RT01 in particularly preferred embodiment of the present invention, RT02, and the oligomer of nucleotide base sequence shown in RT03 and the RT04 is a thiophosphate.Particularly preferred oligomer is Tam etc., the described thiophosphate of Nucl.Acid.Res.22:977-986 (1994)-3 ' hydroxyl propylamine.

Oligomer of the present invention can be designed to reduce CD28 and give expression in the T cell of oligomer of the present invention in the extracellular of internalization.Oligomer of the present invention reduces the expression of CD28 when in addition, can be designed to produce oligomer in passing through utilization expression vector cell.The inhibition that CD28 expresses can be finished by following manner: (I) disturb the CD28 genetic transcription, (ii) disturb transcribing of CD28 gene transcripts, (iii) disturb the processing of CD28 gene transcripts, or (i), (ii) and combination in any (iii).Accuracy and the mechanism of disturbing CD28 to express will depend on following factors, as the structure of specific oligomer, and the nucleotide base sequence of oligomer, the dosage of oligomer gives mode of this oligomer or the like.

Refer to naturally occurring polynucleotide such as DNA at a little terms " oligomer " that use, RNA and have the natural various artificial analog that has nucleic acid that forms two strands or triple helix ability with DNA or RNA.Many have the character that is better than DNA or RNA when using in the method for the invention for the natural oligomer of the artificial analog of polynucleotide that exists.These character comprise the high affinity to DNA/RNA, the Cobra venom endonuclease resistance, and the exonuclease resistance, fat-soluble, RNase H activity etc.For example, substitute phosphate ester oxygen forming alkyl phosphate oligonucleotide or alkyl phosphotriester oligonucleotide with alkyl or alkoxyl, to increase fat-soluble and/or to the resistance of nuclease digestion by phosphodiester bond between nucleotide.Such a nonionic oligomer is a feature with resistance and/or the increase cellular uptake that increases the nuclease hydrolysis, has kept the ability that forms stable compound with the complementary nucleic acid sequence simultaneously.

Though manyly clearly described at this as the natural oligomer of nucleic acid analog that exists, and/or for it be known to those skilled in the art that the many oligomers as nucleic acid analog that should see following exploitation are expected to be used to suppress the CD28 expression of gene by those skilled in the art easily.Provided below and passed through to select suitable nucleic acid base sequence, thereby be used as the different brief overview that can obtain the DNA/RNA analog at present of oligomer of the present invention with targeting CD28 gene (and transcript).The various oligomers of describing in those articles are possible be applicable in the method and composition of the present invention to suppress example that oligomer that CD28 expresses not of the same race may embodiment and unrestricted.

Methyl phosphorodithioate (and other alkyl phosphate) oligomer can be on solution and insoluble polymerization holder with the several different methods preparation (Agrawal and Fiftina, Nucleic acids research., 6: 3009-3024 (1979); Miller et al., Biochemistry, 18: 5134-5142 (1979); Miller et al., Biological The Chemicals., 255: 9659-9665 (1980); Miller et al., Nucleic acids research., 11: 5189-5204 (1983); Miller et al., Nucleic acids research., 11: 6225-6242 (1983); Miller et al., Biological Chemistry, 25: 5092-5097 (1986); Sinha et al., The tetrahedron communication. 24: 877-880 (1983); Dorman et al., The tetrahedron communication. 40: 95-102 (1984); Jager and Engels, Tetrahedron Communication., 25: 1437-1440 (1984); Noble et al., Nucleic acids research., 12: 3387-3404 (1984) Callahan et al., Institute of NAS newspaper, 83: 1617-1621 (1986); KoziolkiewiczEt al., Chemica Scripta, 26: 251-260 (1986); Agrawal and Goodchild, On four sides The body communication., 38: 3539-3542 (1987); Lesnikowski et al., The tetrahedron communication., 28: 5535-5538 (1987); Sarin et al., Institute of NAS newspaper, 85: 7448-7451 (1988).

Other oligoribonucleotide analog as oligomer is described in Inova etc., nucleic acids research, 15:6131 (1987) (2 '-the O-methyl ribonucleotides), Inova etc., FEBS Lett., 215:327 (1987).

About the narration of how making and use thiophosphate and phosphorodithioate referring to U.S. Patent No. 5,292,875, U.S. Patent No. 5,286,717, U.S. Patent No. 5,276,019, U.S. Patent No. 5,264,423, U.S. Patent No. 5,218,103, U.S. Patent No. 5,194,428, U.S. Patent No. 5,183,885, U.S. Patent No. 5,166,387, U.S. Patent No. 5,151,510, U.S. Patent No. 5,120,846, U.S. Patent No. 4,814,448, U.S. Patent No. 4,814,451, U.S. Patent No. 4,096,210, U.S. Patent No. 4,094,873, U.S. Patent No. 4,092,312, U.S. Patent No. 4,016,225, U.S. Patent No. 4,007,197, U.S. Patent No. 3,972,887, U.S. Patent No. 3,917,621, and U.S. Patent No. 3,907,815, Dagle et al. Nucleic acids research 18: 4751-4757 (1990), Loke et al., The U.S. Institute of national academy of sciences newspaper, 86: 3474-3478 (1989), LaPlanche, et al., Nucleic acids research., 14: 9081 (1986) and by Stec, et al., JACS. 106: 6077 (1984), and Steinet al., Nucleic acids research. 16: 3209-3221 (1988).

About the narration of how to make and using phosphoramidite referring to Agrawal et al., U.S. state Institute of academy of science of family newspaper., 85: 7079-7083 (1988), Dagle et al., Nucleic acids research, 18(6): 4751-4757 (1990), Dagle et al., Nucleic acids research. 19(8): 1805-1810 (1991),

Other interested polynucleotide analog comprises the chemical compound that contains acetal or thioaldehydes in the framing structure.About how making and use these chemical compounds referring to Gao et al., Biochemistry 31: 6228-6236 (1992), Quaedfileg et al., The tetrahedron communication. 33(21): 3081-3084 (1992), Jones et al., Organic chemistry. 58: 2983-2991 (1993).

Other interested polynucleotide analog comprises the chemical compound that contains silicyl and silica bridge in the framing structure.About how making and use these chemical compounds referring to Ogilvie and Cormier, The tetrahedron communication., 26(35): 4159-4162 (1985), Cormier and Ogilvie, Nucleic acids research. 16(10): 4583-4594 (1988), PCT publication WO94/06811.

Other interested polynucleotide analog comprises the chemical compound that contains silicyl and acetonyl ester bridge in the framing structure.About how making and use these chemical compounds referring to Gait et al., J.Chem, Soc., Perkin Trans, 1: 1684 (1974), Mungall and Kaiser, The organic chemistry magazine. 42(4): 703-706 (1977), and Coull et al., and The tetrahedron communication. 28(7): 745-748 (1987).

Polynucleotide analog with morpholino base skeleton key also is described.How to make and use the data of these nucleotide analogs to see United States Patent(USP) Nos. 5,034,506,5,235,033,5,034,506,5,185,444.

Have different amine, peptide and other achirality and/or the neutral polynucleotide analog that connects are described in: Caulfield et al., Biological organic and pharmaceutical chemistry communication., 3(12): 2771-2776 (1993), Mesmaeker et al., Biological organic and pharmaceutical chemistry communication., 4(3): 395-398 (1994); Angew, Chem, Int, Ed, Engl., 33(2): 226-229 (1994), U.S. Patent No. 5,166,315, and U.S. Patent No. 5,142,047.

The polynucleotide that have thioether and other sulfide linkage between subunit are described in: Schneider andBrenner, The tetrahedron communication., 31(3): 335-338 (1990), Huang et al., Organic chemistry is assorted Will., 56: 3869-3882 (1991); Musicki and Widlanski, The tetrahedron communication., 32(10): 1267-1270 (1991); Huang and Widlanski, The tetrahedron communication., 33(19): 2657-2660 (1992); And Reynolds et al., The organic chemistry magazine. 57: 2983-2985 (1992), and PCTpublication WO91/15500.

Other polynucleotide analog interested comprises peptide nucleic acid(PNA) (PNAs) and related polynucleotides analog.The narration of how to make and using peptide nucleic acid(PNA) is referring to Buchardt etc., Trends inBiotech., 11 (1993) and PCT publication WO93/12129.

Other oligomer that is used for Antisense Suppression is described in Thuong et al., The American National science Institute of institute newspaper., 84: 5129-5133 (1987), U.S. Patent No. 5,217,866, Lamond, Biochem. Soc.Transactions, 21: 1-8 (1993) (2 '-O-alkyloligoribonucleotides), Ono etal., The bioconjugates chemistry, 4: 499-508 (1993) (on 1 of its deoxyribose, be loaded with 2 of amino key '-Brdurd), Kawasai et al., The pharmaceutical chemistry magazine., 36: 831-841 (1993) (2 '-deoxidation-2 '-fluorine thiophosphate oligonucleotide).PCT publication WO93/23570, Augustyns et al., Nucleic acids research, 21(20): 4670-4676 (1993).

In addition, oligomer can further be modified stability and/or the increase cellular uptake to increase duplex.The embodiment of these modifications sees that title is the open WO93/24507 Nielsen of PCT of " the conformation restriction oligomer that is used for bonded amide containing of sequence-specific or amino-formate bond " etc., Science, 254:1497-1500 (1991), title is the open WO92/05186 of the PCT of " key between improved nucleotide ", title is the United States Patent (USP) 5 of open WO91/06629 of the PCT that submits to " oligonucleotide analogs that has new key " 24 days October nineteen ninety and submission on April 24,264,562, title is the open WO91/13080 of the PCT of " pseudonucleus glycosides and pseudonucleus thuja acid and polymer thereof ", title is the open WO91/06556 of PCT of " 2 '-oligonucleotide modified ", title is the open WO90/15065 of PCT that " exonuclease resistance oligonucleotide and preparation method " submitted to June 5 nineteen ninety, with U.S. Patent No. 5,256,775.

Oligomer of the present invention comprises multiple nucleic acid base.That finds in DNA or RNA is naturally occurring as cytosine, adenine, guanine, thymus pyrimidine, uracil and the hypoxanthic nucleic acid base, and oligomer of the present invention can contain the natural one or more nucleic acid bases that have the nucleic acid base synthetic analogues.These non-naturals exist heterocyclic base to include but not limited to azepine and the assorted pyrimidine analogue of denitrogenation, azepine and deazapurine analog and other heterocyclic base analog, wherein carbon on purine and the pyrimidine ring and nitrogen-atoms have and are one or morely replaced by following hetero atom, as oxygen, sulfur, selenium, phosphorus etc.Preferred base composition can participate in the base of a chain in the double-stranded polynucleotide for those, so that keep the base pairing structural relation with the natural base that exists on the complementary strand in the double-stranded polynucleotide.

The invention provides the method for the various immune disorders of many treatments.Term used herein " treatment " or " processing " refer to disease is prevented and improve the symptom that has occurred in individuality.One of ordinary skill in the art can approve of the treatment needn't be in full force and effect to the symptom of prevent disease generation or minimizing and disease association.Concerning patient, any alleviation to symptom seriousness, the progress of the delay of paresthesia epilepsy or postponement serious symptom degree expects that all the medicable immune disorder of the inventive method comprises that the T that expresses CD28 is cell-mediated or causes the special disease of sending out effect.The inhibition that CD28 expresses causes activating CD28 ⁺The normal cytokine-expressing that produces of T cell reduces, and these cytokines comprise interleukin-2, IFN-and interleukin 8.Therefore, method of the present invention includes but not limited to by interleukin-2, IFN-, interleukin-8.Or its combination and cause the treatment of diseases method, the cell-mediated immunoreation of T is interrupted or weakens whereby.The immune disorder example that this oligomer treats that doses a patient with comprises the organ-graft refection, septic shock, the cachexia of tumor inducing and various autoimmune disease.Can comprise following disease by the autoimmune disease of this method treatment: I type (insulin dependency) diabetes by the mediation of abnormal T cell-stimulating, thyroiditis, sarcoidosis, multiple sclerosis, the autoimmune uveitis, ulcerative colitis, aplastic anemia, system lupus erythematosus, rheumatic arthritis, inductive inflammation of parasite and granuloma, CrohnShi disease, psoriasis, polymyositis, dermatomyositis, scleroderma, vasculitis, psoriatic arthritis, the Graves disease, gravis mycelium, autoimmune liver and gall diseases etc.In addition, method and composition of the present invention provides the treatment of multiple syndrome, comprises the cachexia that septic shock and tumor cause, the effect of wherein causing a disease to small part is passed through activated CD28 ⁺The lymphokine mediation of T emiocytosis.

Methods for the treatment of diseases of the present invention comprises each step that gives this oligomer of patient's effective dose.Dosage accurately, the effective dose that promptly gives patient's the special oligomer of CD28 will be with the disease specific of multiple factor as being treated, the accurate oligomer in the therapeutic combination, patient's age and situation etc. and change.The step of determining suitable drug dosage is that persons skilled in the art are known, also can be at Remington ' s Pharmaceutical Science (latest edition), and Mark PublishingCompany, Easton finds among the Pa. etc.

With directly using outside the CD28 targeting oligomer to patient, the present invention's imagination obtains CD28 from patient ⁺The cell that cell maybe can be expressed CD28 can have or not have other cells.And the Therapeutic Method that transforms with one or more oligomers of the present invention.Conversion can be by several different methods well known by persons skilled in the art, as electroporation, and cation lipid such as DOTMA or DOSPA etc.Cell transformed imports in the body more again.

Another aspect of the present invention provides by giving the method for the special oligomer treatment of CD28 immune disorder, and wherein this oligomer produces by the recombination of polynucleotide expression vector in cell.The special oligomer of CD28 that produces in the cell must be RNA or dna molecular.Biology field technical staff knows the recombinant nucleotide vector that is used for interested polynucleotide expression.Be used for recombinant vector that interested polynucleotide express describe in detail and see " Somatic cell gene therapy", ed.P.L.Chang, CRC Press, Boca Raton (1995), R.C.Muligan, Science, 260: 926-932 (1993), F.W.Anderson, Science, 256: 808-873 (1992), Culver et al., Hum. Gene Ther., 2:107-109 (1991) etc.Can be incorporated in the T cellular genome or in the T cell cytosol by used suitable recombinant vector in this method of genetic engineering treatment immune disorder and to duplicate.The special oligomer of CD28 that is used for administration in the cell is preferably obviously long than the CD28 oligomer of extracellular administration.In this method preferred embodiment of CD28 administration in cell, the special oligomer of CD28 and one or more complete CD28 transcript or complete CD28 gene complementation; But the special oligomer of CD28 that produces in the suitable cell is shorter on length.Unlike the special oligomer of CD28 that the extracellular gives, the special oligomer of CD28 does not have the problem of interior picked-up of cell or exoenzyme hydrolysis.The recombinant vector that can comprise the special oligomer coding of the CD28 that directly doses a patient with this method of the special thing of CD28 in the born of the same parents.Perhaps, the carrier that the special oligomer of CD28 is produced directly gives to separate the cell (being stem cell, T cell, whole blood, bone marrow etc.) that obtains from patient, produces transformant thus.Transformant can be followed and import patient again.

The present invention also provides the expression vector that can express one or more oligomers of the present invention especially.But common these expression vectors comprise a promoter with operative combination and a coding can suppress the polynucleotide sequence of CD28 at the oligomer of T cellular expression.Though many different promoteres can be used for carrier of the present invention, preferred promoter can drive high level expression in the T cell.Expression vector of the present invention also comprises various adjusting sequences.Existing expression vector, particularly those carriers that clearly design for gene therapy easily are suitable for the expression of CD28 targeting oligomer of the present invention.With conventional technique for gene engineering such as Sambrook etc., Molecular Cloning, 2nd Ed., Clod SpringHarbor Press, Cold Spring Harbor, N Y (1989) is described, and these carriers can be suitable for the expression of CD28 targeting oligomer.

Another aspect of the present invention is to provide pharmaceutical preparation with treatment immune-mediated disease for giving oligomer of the present invention.The technical staff of pharmaceutical field can easily make these pharmaceutical preparation.These preparations comprise one or more oligomers of the present invention; And comprising more than a kind of dissimilar oligomer in embodiments of the invention, these oligomers quilts preferably can not the phase mutual cross.Pharmaceutical preparation of the present invention is fit to give individuality with multiple to selected medication suitable manner, comprise oral, intravenous, intramuscular, intraperitoneal and part etc.This pharmaceutical preparation also can contain one or more abiotic reactive compounds except that containing one or more different oligomers of the present invention, i.e. excipient is as stabilizing agent (promotion long term store), emulsifying agent, binding agent, thickening agent, salt, antiseptic etc.

The preparation of parenteral can comprise aseptic aqueous solution, also can contain buffer, diluent and other appropriate addns.Can design pharmaceutical preparation of the present invention to quicken the oligomer in the cellular uptake compositions, as oligomer being encapsulated into suitable liposome.

The pharmaceutical preparation of topical is particularly useful to topical therapeutic.The preparation of topical therapeutic comprises ointment, spray, gel, suspension, emulsion, emulsifiable paste etc.Local application's preparation also can comprise carrier material such as isopropyl alcohol, glycerol, paraffin, Solsperse 2000, Polyethylene Glycol etc. except that this oligomer.Pharmaceutical carrier also can comprise known chemical absorbing promoter.The example of short absorbent is as dimethyl acetylamide (U.S. Patent No. 3,472,931), ethapon or trifluoroethanol (U.S. Patent No. 3,891,757), some alcohol and composition thereof (British patent No.1,001,949) and british patent specification No.1,464,975.

This oligomer is except that the treatment purposes, and they also can be used as assay laboratory's instrument and study t cell activation.The T cell also has several surface receptors except that CD28 and antigen-specific TXi Baoshouti.Because possible and interaction reality between multiple receptor-mediated path are studied selected receptor biological nature separately and can be met difficulty.By giving to reduce the mechanism that CD28 expresses in the T cell, oligomer of the present invention and method also provide useful laboratory method for the T cell behavior that research does not rely on the CD28 activation pathway.

Can understand the present invention better by reference the following example.The following example is provided to explain the present invention, and not as restriction of the present invention.VI. embodiment-series 1

Oligonucleotide

Oligodeoxynucleotide is gone up with standard phosphoramidite method synthetic at automatic dna synthesizer (Applied Biosystems394 type).β-cyanoethyl phosphoramidite, (ABI, Foster City C.A) buys from Applied Biosystems for synthetic agent and CPG polystyrene columns.3 '-amido modified C3 CPG post available from Glen Research (Sterling, VA).To the thiophosphate oligonucleotide, the oxygen cylinder of standard is replaced by curing tetraethyl thion/acetonitrile, and carries out the progressively increase that thiophosphate connects with standard A BI thiophosphate program.After the cutting-out of controlled pore glass post, handle oligonucleotide at 55 ℃ with strong aqua ammonia and removed blocking group in 8 hours.Oligonucleotide by HPLC with anti-phase C8 post (ABI) purification that partly prepares.With 80% acetic acid treatment excision DMT and with behind the ethanol precipitation.Through (Beckman, Fullerton CA) have reached the purity of sample by HPLC with analyzing the C18 post.The oligonucleotide of all＞90% purity is frozen drying.Oligonucleotide is dissolved in the asepsis ionized water (1CN, Costa Mesa) again, and the assistant value by OD260nm transfers to 400 μ M to concentration, five equilibrium and before experiment in-20 ℃ of storages.Under all situations, use three batches to list in table 1 oligonucleotide at least. The purification of cell strain and T cell

The blood of 60ml healthy donors after Ficoll-Hypaque density gradient centrifugation, separating periphery blood monocytic cell from yellowish chromatograph (PBMCs).Use for the single-minded Lymphokwik lymphocyte separation agent of T cell (LK-25T, One Lambda, Canoga ParkC A) purification T cell from PBMCs then.Average then 40-60 * 10 ⁶The product of T cell is containing 20 μ M HEPES buffer, pH7.4,5% self blood plasma, the 1%L-glutamine, 20-30ml RPMI-APS (RPMI-1640) culture medium (ICN of 1% penicillin/streptomycin and 0.05%2-mercaptoethanol, Costa Mesa is incubated overnight in 37 ℃ in CA), removes the adhesive cell of any contamination.In all experiments, the T cell is washed with RPMI-APS, is layered on then on 96 orifice plates, every porocyte concentration 2-3 * 10 ⁶Cell/ml.

Table 1

AS: antisense TF: triple helix

SS: just RO: oligomer at random

Oligonucleotide	Sequence	??SEQ?ID ????NO：	Type	Target spot	Residue number N O.
						????RT01	??TTG?TCC?TGA?CGA?TGG?GCT?A	????1	????AS	??5′untranslated?region	??89-107
????RT02	??AGC?AGC?CTG?AGC?ATC?TTT?GT	????2	????AS	??AUG?codon	??94-113
						????RT03	??TTG?GAG?GGG?GTG?GTG?GGG	????3	????TF	??5′untranslated?region	??58-75
????RT04	??GGG?TTG?GAG?GGG?GTG?GTG?GGG	????4	????TF	??5′untranslated?region	??58-78
						????RTC01	??TAG?CCC?ATC?GTC?AGG?ACA?A	????5	????SS	??sense?strand?control?for?RT01
????RTC02	??ACA?AAG?ATG?CTC?AGG?CTG?CT	????6	????SS	??sense?strand?control?for?RT02
						????RTC03	??CTC?CAG?CCA?ATC?GGA?AGG?CTC?TTT?AA	????7	????RO	??random?oligo
????RTC04	??TTT?TGG?TTG?GTG?TGG?TTT?GTG	????8	????GT	??GT?control?for?RT03，RT04
						????RTC05	??GTG?TGT?GTG?TGT?GTG?TGT?GTG	????9	????GT	??GT?control?for?RT03，RT04
????RTC06	??AAC?CTC?CCC?CAC?CAC?CCC	????10	????SS	??sense?strand?control?for ??RT03，RT04

Table 2

AS: antisense TF: triple helix

Oligonucleotide	Sequence	??SEQ?ID ????NO：	Type	Target spot	The residue number
						????RT05	AGT?TGA?GAG?CCA?AGA?GCA?GC	????11	????AS	?AUG?codon	108-127

Oligonucleotide	Sequence	??SEQ?ID ????NO：	Type	Target spot	The residue number
						????RT06	GCT?AAG?GTT?GAC?CGC?ATT?GT	????12	????AS	The AUG codon	197-216
????RT07	AGT?CCT?TTG?TGA?AGG?GAT?GC	????13	????AS	The coding region	256-275
						????RT08	ACC?TGA?AGC?TGC?TGG?GAG?TA	????14	????AS	The coding region	313-332
????RT09	CCC?AAT?TTC?CCA?TCA?CAG?TT	????15	????AS	The coding region	352-371
						????RT10	GCA?AGC?TAT?AGC?AAG?CCA?GG	????16	????AS	The coding region	585-604
????RT11	CAG?GAG?CCT?GCT?CCT?CTT?AC	????17	????AS	The coding region	641-660
						????RT12	GTG?TCA?GGA?GCG?ATA?GGC?TG	????18	????AS	The coding region	746-765
????RT13	GGC?CTG?TCA?CAG?GAA?ATC?TC	????19	????AS	The coding region	949-968
						????RT14	AGC?CGG?CTG?GCT?TCT?G	????20	????AS	Termination codon	777-792
????RT15	AAA?TTG?GCA?TTG?GTG?GGC?C	????21	????AS	3 ' untranslated region	873-890
						????RT16	TAA?GTT?GGA?ATG?TGG?GCC?AT	????22	????AS	3 ' untranslated region	984-1003
????RT17	CTC?CCA?GAA?TCC?ACT?CCC?TT	????23	????AS	3 ' untranslated region	1049-1068
						????RT18	GCT?TGA?CTG?AGA?TGT?GCA?GG	????24	????AS	3 ' untranslated region	1089-1108
????RT19	TCC?TAG?CCT?TTC?TTC?TGC?AA	????25	????AS	3 ' untranslated region	1136-1155
						????RT20	CGT?ACG?CTA?CAA?GCA?TGG?G	????26	????AS	coding?region	178-196
????RT21	TGA?GAA?AGG?GAA?GAG?GCT?CC	????27	????AS	3 ' untranslated region	1065-1088
						????RT22	GAA?GTC?GCG?TGG?TGG?G	????28	????AS	coding?region	729-744
????RT23	AAA?TTA?GCC?AGG?CAT?CAT?GG	????29	????AS	5 ' untranslated region	-325?to-306
						????RT24	AGT?GGG?TGG?ATC?ATT?TGA?GG	????30	????AS	5 ' untranslated region	-244?to-225

Oligonucleotide	Sequence	??SEQ?ID ????NO：	Type	Target spot	The residue number
						????RT25	TGC?TTG?AAA?TCC?AGC?AGA?GA	????31	????AS	?5′untranslated?region	-139?to-120
????RT26	CAT?GAT?GGG?CTT?ATG?GGA?AT	????32	????AS	5 ' AP-1 sample element	-60?to-79
						????RT27	CAG?TGG?CTC?ACG?CCT?GTA	????33	????AS	?5′untranslated?region	-201?to-184
????RT28	GGG?GTT?GGT?TGG?TTG?TTT?GG	????34	????TF	5 ' AP-1 sample element	-518?to-499
						????RT29	GTG?TTT?GTG?TGG?GGT?TT	????35	????TF	5 ' AP-1 sample element	-158?to-142
????RT30	GGG?GTT?TTT?TGT?GTG?GT	????36	????TF	5 ' AP-1 sample element	-148?to-132

The t cell lymphoma cell strain, Jurkat E6-1 (CD28 ⁺CD24+) cell (152-T1B and) HUT78 (CD28-/CD4+) cell (T1B-161) (ATCC, Rockville, MD) remain on RPMI-10 and (contain 20 μ M HEPFS buffer, pH7.4,10% hyclone (FCS) (Hyclone, Logan, UT), the RPML-1640 of 1%L-glutamine and 1% penicillin/streptomycin) in. Inductive t cell activation of mitogen and oligonucleotide are handled

Adding people's periphery T cell or t cell lymphoma cell strain (0.2-0.3 * 10 ⁶) before, 96 duplicate orifice plates be coated with in advance purification anti--CD3 monoclonal antibody (mA6) (6.25-200ng/ hole) (clone HIT 3a, Pharmingen, San Diego, CA) and with the normal saline of cold phosphate-buffered, pH7.4 (PBS) washes secondary.Further (Calbiochem, La Jolla CA) activate and 37 ℃ of incubations 48 hours the T cell that anti--CD3 mA6-handles by adding 2ng phorbol 12-myristic acid 13-ethyl ester (PMA).Anti--CD3/PMA activated T cells is used special the handling with control oligonucleotide of 1-20 μ M CD28 immediately after activating, and handles after 24 hours again.Be used for immunofluorescence analysis to the T of one of paired flat board cell, supernatant is used for cytokine research, and second flat board is used for the T analysis of cell proliferation. Immunofluorescence research

After the activation, go to the cytokine that is used for the analysis of cells source in another piece plate from the 150ml cell conditioned medium liquid of first block of parallel-plate and produce.(Becton Dickinson, Mansfield MA) wash two times to remaining cell, are resuspended in the 50ml isotonic saline solution and are divided into two duplicate samples with isotonic saline solution pH7.4.One duplicate samples dyes altogether with PE-CD28/FITC-CD4 or PE-CD54/FITC-CD25, and assesses non-specific fluorescence by another duplicate samples that dyes of the special-shaped matched control monoclonal antibody with the PE/FITC labelling.(San Jose CA) obtains all fluorescent labeling monoclonal antibodies by BectonDickinson.With saturated mAb concentration in the dark, 4 ℃ of incubations 45 minutes.Before analyzing with FACScan flow cytometer (Becton Dickinson), the label that does not participate in the PBS flush away.Antigen density records in gate living cells indirect, and represents with fluorescence average channel (MCF).Specific antigen (CD54, CD25) surface expression is expressed as clean passage and changes (MCS), and promptly the MCF that deducts abnormal shape pairing (IgG1) the contrast mAb staining cell of FITC-or PE-labelling with the MCF of the painted cell of antigenic specificity mAb of FITC-or PE-labelling obtains.In other words, use the CD4 of CD28 mAb staining cell ⁺The surface expression of-subgroup is by CD28-CD4 ⁺The MCF of cell deducts CD28 ⁺CD4 ⁺MCF record.The survival rate that contrasts untreated and the acid-treated cell of oligonucleoside is by recording with every capable oligonucleotide in a plurality of donors of vital stain iodate third ingot (final concentration 5 μ g/ml) dyeing.The living cells percent that repels iodate third ingot records with flow cytometry, and in acid-treated all row of the oligonucleoside of dosage range 1-20 μ M＞90% (90-99%) Fig. 2. Cytokine analysis

Be determined in the cell conditioned medium liquid of first block of parallel-plate human cell factor concentration from cell.The variation of the interleukin-22 that mitogen causes (IL-2) level can get ELISA test kit (R ﹠amp by commodity in use; D systems Quantikine kit, Minneapolis, MN) or with the cell line CTLL-2 that relies on IL-2 (ATCC, Rockville MD) record with biological method.(Cambridge is MA) with R ﹠amp by using Endogen respectively in the variation of IFN-that mitogen causes and interleukin 8 (IL-8) level; D system (Quantikire kit, Minneapolis, MN) kit measurement.All ELISA results are expressed as pg/ml, and the CTLL-2 biological test method is right by the CTLL-2 cell with representative ³The H-thymus pyrimidine (ICN, Costa, Mesa, the count per minute that the cell that depends on IL-2 CA) participates in is represented. The T cell proliferation test

Second block of parallel-plate in all experiments is used for analyzing the variation of the T cell proliferation that mitogen causes.Anti-CD3/PMA activates 72h also to be had or not to have in the presence of the oligonucleotide, and cell is with 1 μ Ci ³H-thymus pyrimidine (ICN, Costa, Mesa, CA) pulse and 37 ℃ of incubated overnight.(Wallac, Gaithersburg MD) go up liquid flashing counting and record by collecting cell and at Wallac Betaplate calculating instrument to participate in the cell growth that the mitogen estimated causes by the radioactivity labelling. The inhibitory action that the special oligonucleotide of CD28 is expressed CD28 among the activation human T-cell

The human T-cell that anti-CD3/PMA handles, the surface expression of its CD28 (using immunofluorescence analysis) increases to MCS150 ± 9.27 (n=9) from 122 ± 7.74MCS of tranquillization T-cell.The CD28 expression difference is defined as the CD28 expression (Fig. 3 A) that mitogen causes in tranquillization and the activated T cell.Fig. 3 B and 3C represent (to represent with the S-oligomer with thiophosphate, Fig. 3 B) and thiophosphate-3 ' amine (represent with the A-oligomer, Fig. 3 C) the special opposition of the CD28 of form is according to the anti-CD3/PMA activated T cells of 2 donors of oligonucleotide processing, and every kind of oligonucleotide two is gone respectively.In four kinds of candidate's oligonucleotide RT01-RT04 (table 1) of dosage range 2-10 μ M, the thiophosphate of RT03 and RT04 and thiophosphate-3 '-the amine form is the active best inhibitor that the inductive CD28 of mitogen expresses, and all suppresses to induce the CD28 expression greater than 50% (IC50) at 5 μ M or still less the time.These two kinds of different oligonucleotide of length are designed one section hybridization with 5 ' upstream double-stranded DNA of CD28 genetic transcription initiation site.Do not see the inhibitory action that similar dose dependent is expressed the inductive CD28 of mitogen with control oligonucleotide RTC01 (SEQ ID NO:5)-RTC06 (SEQ ID NO:10) (table 1).All experiments are all used T cell from 7 donors, are carried out with every kind of oligonucleotide at least 3 row.Provable oligonucleotide is important to this fact of adjusting that CD28 expresses in the human T-cell, because periphery, epidermis and corium T-lymphocyte will be the target spots of the special oligonucleotide of CD28. The CD28 specific oligonucleotide is to the inhibitory action of the inductive T cell proliferation of mitogen

The mitogen effect that anti-CD3/PMA handles activates the observed propagation in back by resting cell to be increased provable. ³Participating in count per minute of H-thymus pyrimidine represented, is 301641 ± 47856 (n=9) in activated T cell, is 650 ± 566 (n=9) in tranquillization T cell.Anti-CD3 and PMA work in coordination with the effect of T cell proliferation, shown in Fig. 4 A.Fig. 4 B represent CD28 special with representativeness experiment contrast thiophosphate oligonucleotide antagonism CD3/PMA activated T cells proliferation function.In at least seven are independently tested, in all experiments of dosage range 2-10 μ M, the thiophosphate of RT03 and RT04 (data not shown) and thiophosphate-3 ' amine (Fig. 4 B) form is the active inhibitor of tool of the inductive T cell proliferation of mitogen, and suppressor T cell propagation reaches 45%.Do not see similar dose-dependent inhibitory action with control oligonucleotide RTC01 (SEQ ID NO:6)-RTC06 (SEQ ID NO:10) to the inductive T cell proliferation of mitogen.Use special oligonucleotide RT03 of CD28 and RT04 processing can reverse the hyper-proliferative of activated T cell here, prove that therefore adjusting CD28 path plays an important role to the active bio function and the T cell proliferation of t cell activation. The CD28 specific oligonucleotide is to the activated inhibitory action that produces from the T cell cytokine

Activated T cells produces panimmunity and regulates cytokine, comprises IL-2, IFN-and IL-8.The inductive limiting element to IL-2 and IL-8 of CD28 has been proved to be the promoter sequence that is positioned at two genes, thereby show by CD28 path adjusting (Fraser et al., (1991) Science251:313-316, Seder etc. (1994) J.Exp179:299-304).IFN-has also shown by the CD28 path regulates (Wechsler etc., J.Immunol., 153:2515-2523 (1994)).AntiCD3/PMA handle tranquillization T cell and significantly increase the level of whole three kinds of cytokines in T cell source (Fig. 5 A, 6A, 7A).Fig. 5,6 and 7 describe the thiophosphate (B) and the thiophosphate-3 ' amine of the special and control oligonucleotide of CD28 respectively Variant is to the influence from the activated T cell of identical representative donor.CD28 specific oligonucleotide RT03 (SEQ ID NO:3) and RT04 (SEQ ID NO:4) but not control oligonucleotide RT01 (SEQ ID NO:5)-RT06 (SEQ ID NO:10) (data not shown) suppresses the inductive IL-2 of mitogen in the activated T cell, the generation of IFN-and IL-8 in dose-dependent mode.Thiophosphate (the IC of CD28 specific oligonucleotide ₅₀5 μ M) and thiophosphate-3 ' amine (IC ₅₀10 μ M) form has activity equally in 2-10 μ M scope.Analog result with the visible whole three kinds of cytokines of 4 or how different donor.These observe a plurality of effector molecules that proof CD28 specific oligonucleotide also can be regulated the CD28 path of t cell activation. Oligonucleotide is to the specificity of CD28 expression inhibiting effect

The specificity of CD28 specific oligonucleotide RT03 and RT04 is by 3 kinds of method evaluations.

(1) do not rely on the t cell activation mark of CD28

First method is to determine whether the CD28 specific oligonucleotide can suppress not rely on that CD28 stimulates path altogether and the expression of other human T-cell's activation marker thing of working.The activation of tranquillization T cell significantly increases the expression of IL-2 receptor (CD25) and intracellular adhesion molecule ICAM-1 (CD54).Yet these accessory molecules are not subjected to the CD28 path to regulate (June etc., Mol.Cell Biol.7:4472-4481 (1987), Damle etc., J.Immunol., 149:2541 (1992)).Fig. 8 represents the influence of CD25 in the mitogen activated T cells (Fig. 8 A) and CD54 (Fig. 8 B) being expressed with control oligonucleotide that CD28 is special.With 2-10 μ M CD28 special handle with control oligonucleotide after, the expression of activated T cell CD25 and CD54 does not all have obvious decline.This proves that clearly the CD28 specific oligonucleotide is to suppressing the specificity that its target protein is expressed.

(2) cloudy T cell strain, the HUT78 of giving birth to of CD28

Second method is that proof CD28 path is the target spot of real CD28 specific oligonucleotide, and it is by CD28 relatively ⁺T chronic myeloid leukemia cell line Jurkat E6-1 and CD28-T cell lymphoma cell are that the generation of the inductive IL-2 of mitogen among the HUT78 proves.Fig. 9 A confirms at tranquillization and activates and all do not have the CD28 expression in the HUT78 cell, and the basic horizontal of CD28 rising (Fig. 9 B) behind the Jurkat E6-1 cell-stimulating.At CD28 ⁺In the Jurkat E6-1 cell, the special and control oligonucleotide of CD28 can suppress the inductive CD28 of mitogen and express the generation (Figure 10 B) of (Fig. 9 C) and IL-2.On the contrary in the CD28-HUT78 cell, the inductive IL-2 of mitogen produces the influence (Figure 10 A) that is not subjected to the special and control oligonucleotide of CD28.This proves that clearly these oligonucleotide only suppress the specificity of the IL-2 generation of CD28 adjusting.

(3) the single-minded activation of CD28 path

The third method is that anti-CD28 monoclonal antibody and mitogen (anti-CD3/PMA) are united use specifically by CD28 path activation tranquillization T cell, uses the same procedure with the employing of only using the mitogen activated T cell.The front has showed that anti-CD28 mAb and PMA or anti-CD3mAb unite use and stimulus signal be provided for tranquillization T cell, and only promote that CD28 relies on but not the T cell proliferation of TCR dependence and the increase (June etc., (1987) Mol.Cell Biol.7:4472-4481) that cytokine produces.CD28 special but not the thiophosphate of control oligonucleotide and the IL-2 that thiophosphate-3 ' amine variant can suppress to depend on CD28, the activation that IL-8 and IFN-produce, and the T cell proliferation (data not shown) of the activated tranquillization T of anti-CD28/ mitogen cell.This proves that clearly CD28 specific oligonucleotide only only acts on the CD28 path of t cell activation.VII. Embodiment-series 2

Oligonucleotide

With Applied Biosystems 394 dna synthesizer synthetic oligonucleotides.The standard oxidation bottle imports phosphorothioate bond after replacing with curing tetraethyl thion/acetonitrile.The purity of oligonucleotide is determined with analyzing HPLC.The oligonucleotide of all purity＞90% is by lyophilizing lay equal stress on (400 μ M) soluble in water.Every kind of oligomer of listing in table 3 and 5 is at least with 3 row.With the T4 polynucleotide kinase and ³²P-γ ATP obtains the oligonucleotide of 5 ' labelling. T cell activation research

From healthy donors by density gradient centrifugation and Lymphokwik (One Lambda) T cell enrichment separating periphery blood monocytic cell (PBMCs).The mononuclear cell that pollutes is removed by adhering on the plastics.T cell CD2+＞99% of purification, HLA-DR ⁺＜1%, CD25 ⁺＜5%.Obtain Jurkat E6-1 (CD28 from ATCC ⁺/ CD4 ⁺) T cell and HUT78 (CD28 ^-/ CD4 ⁺) T cell and monocytic series THP.Cell is from 0.2-0.3 * 10 ⁶/ hole concentration is cultivated, and (Pharmingen and 2ng phorbol-12-myristoyl 13-ethyl ester (PMA) (Calbiochem) activates with fixing CD 3-resisting monoclonal antibody (mAb) (HIT3A0.25 μ g/ml) onboard. Immunofluorescence research

Cell dyes altogether with the special-shaped matched control (Becton Dickinson) of PE-CD28/FITC-CD4 or PE-CD54/FITC-CD25 mAb or PE/FITC labelling.(CD28, CD54 CD25) determine by flow cytometry (FACScan, Becton Dickinson) cell surface antigen density.Survival rate is surveyed the CD4 from the contrast of a plurality of donors is untreated and oligomer is handled ⁺Cell is determined the repulsion of iodate third ingot, representative location＞90% (90-99%) behind the every capable 1-10 μ M usefulness incubation 48h of all oligonucleotide. Propagation and cytokine test

Breeder reaction is by surveying among the last 16h 3 ^HParticipating in of-thymus pyrimidine (1 μ Ci ICN) is next definite, and cell is collected on the filter paper, and it is synthetic to measure DNA behind liquid flashing counting on the Wallac Betaplate enumerator.Cytokine concentrations IL-2 in the culture supernatant, IL-8 (R ﹠amp; D system) and the ELISA test kit of IFN-γ (Endogen) test or by obtaining with cell line CTLL-2 (ATCC) biological test that depends on IL-2. RT-PCR and Southern analyze

With the total cell RNA of Trizol reagent (GIBCO/BRL) extracting.Carry out cDNA synthetic reaction (Promega) with oligomer (dT) 15 primers and AMV reverse transcriptase (H.C.).PCR reaction (Gene Amp PCR kit, Perkin-Elmer Cetus) is by containing 3 μ l cDNA, dVTPs (every kind 200 μ M), and 50 μ l mixture of every kind of primer 0.5 μ M and 1 Taq of unit polymerase are formed.The primer that uses is as follows: CD28,5 '-CTGCTCTTGGCTCTCAACTT-3 ' (justice) and 5 ' AAGCTATAGCAAGCCAGGAC-3 ' (antisense) interleukin-2 receptor p55 α strand primer (Stratagene) and pHE7 ribosomal gene.Kao，H.T.，Nevins，J.R.(1983)Mol.Cell?Biol.3，2058-2065。Amplification condition 94 ℃ 45 seconds, 57 ℃ 1 minute, 72 ℃ were carried out 35 circulations in 2 minutes, outer back 72 ℃ 8 minutes.The PCR product separates with 2% agarose, goes to Hybond N+ film (Amersham) and excessively also fix with 0.4M NaOH in 20 * SSC.Trace is used ³²The oligonucleotide probe hybridization of P-γ ATP labelling.Flushing trace reuse Phosphor Inager analyzes. The T test cell line that MLR and isoantigen are special

For the MLR reaction, PBMCs cultivates (1: 1) together with the PBMCs from the different individuality of HLA that ametycin (50 μ g/ml) is handled.In the special T test cell line of isoantigen, the PBMCs that handles from the isolating T cell of PBMCs and self ametycin of the healthy donors of tetanus toxin sensitization in the presence of tetanus toxin (2 μ g/ml List Biologicals) with 1: 1 ratio co-cultivation.In two experiments, 2 * 10 ⁵Cells/well was cultivated 6 days in 37 ℃ before further analyzing. External oligonucleotide stability study

(Tam, R.C., Li, Y., Noonberg, S., Hwang, D.G., Lui, G., Hunt, C.A., Garovoy, M.R. (1994) are carried out in the analysis of oligonucleotide time stability as mentioned previously Nuclear Acid research.22,977-986).By electrophoresis and Nickspin post quantitative assessment oligonucleotide degraded profile. The thiophosphate oligonucleotide is single-minded and influences activated T the inhibitory action that CD28 expresses Cell function

Figure 11 has concluded in the table 3 the thiophosphate oligonucleotide to the influence of the surface expression and the cytokine secretion of accessory molecule in the activated T cell.The oligomer that uses is designed 5 ' untranslated region (UT) hybridization with the DC28 gene, is antisense (AS) or rich G sequence.The contrast oligomer is positive-sense strand (SS) or complementary strand (CS) sequence.Handle tranquillization T cell (R) with anti-cd 3 antibodies and PMA and increased accessory molecule CD28 (A) in 48 hours, CD25 (B) and CD54 And cytokine IL-2 (D), the data that IFM γ (E) and IL-8 (F) express are represented with the mean standard deviation of three times sample.Twice adding (0 and 24h), 2 μ M ( ), 5 μ M (

) and 10 μ M ( ) the cumulative function of table 3 thiophosphate T cell function that activation is caused (IFN γ, IL-8) mensuration secretory cell molecular level is monitored by immunofluorescence analysis (accessory molecule) with CTLL-2 biological method (IL-2) and ELISA.The surface antigen density (MCS) of gate CD4+ cell alive is compared with the IgG1 contrast, measures with the increase of mean fluorecence passage.Depend on IL-2's ³H-thymus pyrimidine cell participates in count per minute (CPM) and measures, and immunoreation IFN γ and IL-8 measure with pg/ml.Shown in data all get from the experiment that the T cell from a donor carries out, be the representativeness experiment of 9 different donors.

Table 3 thiophosphate oligonucleotide

Oligomer sequence (5 ' to 3 ') is described SEQ ID

NO

RT01S?????TTG?TGG?TGA?CGA?TGG?GCT?A?????AS?5′UT?79-97???????42

RT02S?????AGC?AGC?CTG?AGC?ATC?TTT?GT????AS?5′UT?94-113??????43

RT03S?????TTG?GAG?GGG?GTG?GTG?GGG???????G-rich?5′UT?58-75???44

RT04S?????GGG?TTG?GAG?GGG?GTG?GTG?GGG???G-rich?5′UT?58-78???45

RTC01S????TAG?CCC?ATC?GTC?AGG?ACA?A?????SS?to?RT01???????????46

RTC02S????ACA?AAG?ATG?CTC?AGG?CTG?CT????SS?to?RT02???????????47

RTC06S????AAC?CTC?CCC?CAC?CAC?CCC???????CS?to?RT03???????????48

The selected D2EHDTPA oligomer ester of digital proof (table 3) can be blocked CD4 specifically ⁺Activating the CD28 that causes in the T cell expresses.In a representative donor (Figure 11 A), D2EHDTPA oligomer ester RT03S (SEQ ID NO:44) and RT04S (SEQ ID NO:45) (IC ₅₀≤ 5 μ M) CD28 with dosage correlation form inhibition activation-inducing expresses but not the expression of IL-2 receptor (CD25) or intracellular adhesion molecule-1 (ICAM-1 or CD54).With antisense scant polymer RT01S (SEQ ID NO:42) or RT02S (SEQ ID NO:43) or contrast oligomer RTC01S (SEQ ID NO:46), RTC02S (SEQ ID NO:47) does not see similar inhibitory action with RTC06S (SEQ ID NO:48).And we provide the active oligomeric thing to activate human T-cell's the evidence of transcribing the CD28 that regulates activation-inducing by blocking-up.At 10 μ M, RT03S (SEQ ID NO:44) and RT04S (SEQ ID NO:45) but not representational contrast oligomer RTC06S (SEQ ID NO:48) reduce the CD28 expression of activation-inducing but not the expression (Figure 12) of IL-2 receptor mrna.

Figure 12 shows that the thiophosphate oligonucleotide is in the influence of 10 μ M to CD28 and CD25mRNA level.After total cell RNA RT-PCR also analyzes with special radiolabeled probe Southern, do not detected and had (swimming lane 2) or have oligonucleotide RT03S (SEQ ID NO:44) (swimming lane 4), the level of the tranquillization (swimming lane 1) of CD28 and CD25mRNA and anti-CD3/PMA activation (6h) when RTC06S (SEQ ID NO:64) (swimming lane 5) and RT03D (SEQID NO:49) (swimming lane 6).The CD28 probe is from exon 2 (5 '-ACGGGGTTC AACTGTGATGGGAAATTGGGCAA-3 '), and the probe of IL-2 receptor originates from the initial primers mixed liquor.With originating from pHE7 the equal applied sample amount of probe hybridization post-evaluation of adopted primer is arranged.The RNA of HUT of CD28 defective (7) and THP (8) cell strain is with comparing.The data of showing are 3 representatives of experiment respectively.

Therefore, the biological activity thiophosphate parallels with its effect to the CD28 surface protein the special inhibition of CD28mRNA level.And, the active oligomeric thing has been cancelled the T cell function of activation-inducing, as RT03S (SEQ ID NO:44) and RT04S (SEQ ID NO:45) but not RT01S (SEQ ID NO:42) or RT02S (SEQ ID NO:43) or contrast oligomer RTC01S (SEQ ID NO:46), RTC02S (SEQ ID NO:47) and RTC06S (SEQ ID NO:48), obviously suppress the inductive cytokine IL-2 of anti-CD3/PMA in the activated T cell, the synthetic (IC of IFNr and IL-8 ₅₀≤ 5 μ M, scope is at 2-10 μ M) (Figure 11 B).

Because other stimulates path also can induce the synthetic (Damle of lymphokine in the activated T cell altogether, N.K., Klussman, K., Linsley, P.S., Aruffo, A. (1992) .J.Immunol, 148,1985-1992), whether be important to the biological activity of determining RT03S (SEQ ID NO:44) and RT04S (SEQID NO:45) if expressing special to the CD28 that function is arranged.So we have compared thiophosphate at CD28 ⁺T chronic myeloid leukemia cell line Jurkat E6-1 and CD28-T cell lymphoma cell are the influence that the inductive IL-2 of anti-CD3/PMA produces among the HUT 78.Sum up as Figure 13, handled resting cell (R) 48 hours with anti-cd 3 antibodies and PMA, make CD28 at Jurkat (A, the right side) but not the expression of LUT78 (A, a left side) cell increase.Yet, activate and make IL-2 generation increase in Jurkat (C, a left side) and HUT78 (D, a left side) cell.Active oligonucleotide RT03S (SEQ ID NO:44) and RT04S (SEQ IDNO:45) be at 1 μ M (), 2 μ M ( ), 5 μ M (

) and 10 μ M ( ) suppress to activate CD28 expression (B) and IL-2 level (C, the right side) in the Jurkat cell, but to activating the not influence of IL-2 secretion that depends on CD28 in the HUT78 cell (D, the right side).The data of showing are three representatives of experiment separately.

In Figure 13 A, the confirmation of immunocyte fluorescence analysis is at tranquillization and activate no CD28 expression in HUT 78 cells, and Jurkat E6-1 cell is through activating the raising of CD28 composition level.And in Jurkat E6-1 cell, the CD28 that RT03S (SEQ ID NO:44) and RT04S (SEQ IDNO:45) (1-10 μ M scope) significantly suppress activation-inducing expresses (Figure 13 B) and IL-2 generation (Figure 13 C).On the contrary, though activated HUT78 cell produces the IL-2 of similar level, do not observe similarly the lymphokine oligomer is relied on inhibitory action (Figure 13 D).

We have proved that also special anti-CD28 mAb and anti-CD3 unite the t cell activation of sensing (expressing CD28, IL-2, IL-8 and IFN γ) and blocked by biological activity D2EHDTPA oligomer ester (data not shown).Direct crosslinked the promotion of the previous CD28 of demonstration molecule relies on CD28 but not relies on the T cell proliferation of TCR and the increase (Jure that cytokine produces, C, H., Ledbetter, J.A., Gillespie, M.M., Lindsen, T., Thompson, C.B. (1987) Mol.Cell Biol.7,4472-4481).In a word, the data biological activity of effectively pointing out the active oligomeric thing is special to the CD28 path of t cell activation. The thiophosphate oligonucleotide mixes at T test cell line and the elementary allogeneic that isoantigen relies on The inhibitory action of T cell proliferative response in the lymphocyte reaction

We next step compared CD28 specific oligonucleotide RT03S (SEQ ID NO:44) and RT04S (SEQ ID NO:45) in the elementary immunoreactive usefulness of extracorporeal blocking antigen-specific.In Figure 14, and the tranquillization of CD28 (A, B) and activate that (E, F) level is represented in special T cell experiment of tetanus toxin (top series, A and B) and mixed lymphocyte reaction (end series, E and T).CD4 ⁺, the percentage ratio of CD28-hiT cell shows in the mark of right hand A, B, E and F.Oligomer is active in twice possible adding (0 and 96h), 1 μ M (), 2 μ M ( ), 5 μ M ( ) and 10 μ M ( ) table 1 the thiophosphate oligonucleotide with reduce in each experiment CD28-hi express the T cell (C, G) and activated T cell propagation (D, percentage rate H) is estimated.T cell activation was induced the reaction of tetanus toxin (top series) time, or induced behind stimulating factor PBMCs (Y) (end series) the stimuli responsive T cell of being handled by ametycin (X).These data are three representatives of experiment respectively.

In Figure 14, with tetanus toxin special T cell experiment (Figure 14 B) and elementary mixed lymphocyte reaction (Figure 14 F), after we have observed 6 days experimental stages, the appearance of the T cell subsets of the CD28-hi of expression activation-inducing.Add corresponding minimizing and CD28-hi expression (Figure 14 C, the relevant reduction of dosage 14G) that RT03S (SEQ ID NO:44) and RT04S (SEQ ID NO:45) (2-10 μ M) cause tetanus toxin special (Figure 14 D) and respond special (Figure 14 H) the T cell proliferation of cellular antigens.With RT01S (SEQ ID NO:42) or contrast oligomer RT01S (SEQ ID NO:46), RTC02S (SEQ ID NO:47) or RT06S (SEQ ID NO:48) do not observe similar action. External oligonucleotide stability has been expanded the biological activity of thiophosphate oligonucleotide

Known with the oligomer that connects between thiophosphate nucleotide modify can give with the nuclease resistance and thereby expand the external biological activity from 1-2 hour to 24 hours (Stein, C.A., Cheng, Y.C. (1993) Science 261,1004-1012).Table 4 shows that thiophosphate RT03S (SEQID NO:44) and RT06S (SEQ ID NO:48) are to continued presence Or the time effect of after the 2nd day (D) removes oligonucleotide from extracellular environment, surperficial CD28 being expressed.Monitor with the immunofluorescence cell art.The result is with activated T cell (MCF _A) and the acid-treated activated T cell (MCF of oligonucleoside _X) the surface antigen expression difference represents.Tranquillization T cell is expressed in the 119-121 scope at 2-4 days CD28." NO " representative can not be distinguished difference.

Table 4. thiophosphate is continuous or discontinuous

Time activity after oligonucleotide is handled

CD28 differential expression (MCF _A-MCF _X)

RT03S(SEQ?ID???????????RTC06S(SEQ?ID

NO：44)????????????????NO：47)

It MCF _A05 10 05 10

2???????????144.8??????18.4??????8.9??????9.3?????1.9????1.7????ND

3?????C?????135.6??????21.9??????15.9?????5.1?????ND?????ND?????3.2

3?????D?????136.8??????18.3??????11.8?????7.1?????1.7????3.8????1

4?????C?????128.9??????18.2??????9.7??????6.1?????ND?????ND?????ND

4?????D?????130.1??????2.3???????ND???????ND??????ND?????ND?????ND

As shown in table 4, the lasting of RT03S (SEQ ID NO:44) effect surpasses 24 hours, and continues 2,3,4 days corresponding to oligomer dosage.But removed RT03S (SEQ ID NO:44) at the 2nd day from extracellular environment, suppress to activate maintenance and eliminated fully in 24 hours in the back then in 24 hours.In identical time course, do not observe the oligomer activity with representativeness contrast oligomer RT06S (SEQ ID NO:48).Similar phenomena oligomer mediation to the activated T cell inhibited proliferation in observe (data not shown).Only thiophosphate is provided by the bioavailability increase provide RT03S (SEQ ID NO:44) the bioactive reason of significant prolongation of can not saying so.So it is relevant that we prove that the vitro stability that provides with RT03S (SEQ ID NO:44) secondary structure is provided also in the expansion of effect.

Figure 15 sums up ³²The result of the test of P labelling thiophosphate RT03S (SEQ ID NO:44) and RT06S (SEQ ID NO:48) vitro stability in extracellular supernatant (top series) and Jurkat cell lysate (end series).(A) every kind of oligonucleotide degraded of (0-96h) in time (2000cpm) is observed by electrophoresis reuse PhosphorImager on 20% polyacrylamide degeneration glue and is estimated.(B) the remaining complete total length of each time point ³²P-RT03S (SEQID NO:44) (o) and ³²P-RT06S (SEQ ID NO:48) () measures in the eluent by Nickspin post (Pharmacia) at 10000cpm extracellular supernatant and cell pyrolysis liquid with respect to the percentage ratio of t=0.While analyzing molecules amount standard (Std), ³²P-NTP (N) and free ³²P-orthophosphate (P).

In Figure 15 A, outer supernatant (S) of the clear demonstration pair cell of electrophoretogram and cell lysate (L), with Jurkat cell incubation 96h after remaining complete ³²P labelling RT03S (SEQID NO:44) is more than RT06S (SEQ ID NO:48).Nickspin post data are observed unanimity (Figure 15 B) therewith.Here, complete from RT03S (SEQ ID NO:44) behind the 96h, the complete oligomer ratio of recovery is 54% (S) and 59% (L), and RT06S (SEQ ID NO:48) is 10% (S) and 34% (L).In addition, only secondary structure is not enough to illustrate that enhanced nuclease resistance and RT03S (SEQ ID NO:44) are bioactive continues, because its di-phosphate ester counter pair RT03D inanimate object activity (table 5) almost in vitro stability research, half life only is 24 hours (data not shown).

Table 5 is responsible for suppressing CD28 and is expressed and CD28 dependency IL-2

The evaluation of the oligonucleotide sequence that produces

^*The relative inhibition oligomer sequence C D28 IL-2 SEQ ID that expresses

NO：RT03S?(D)????TTG?GAG? GGG?GTG?GT G?GGG??????100(3)???100(44)??44(49)RT11S???????? GGG?GAG?GA G?GGG?CTG?GAA??????100??????100??????50RT04S????????GGG?TTG?GAG? GGG?GTG?GT G??????123??????100??????45

GGGRT05S????????TTG?GAG? GGG?GAG?GA G?GGG??????136??????100??????51RT09S????????TTG?GAG? GGG?GAG?GT G?GGG??????126??????100??????52RT10S????????TTG?GAG? GCG?GTG?GT G?GCG??????31???????38???????53RT24S????????TTG?GAG? CCG?GTG?GT G?GCC??????40???????57???????54RT25S????????TTG?GAG? GGG?CTC?CT C?GGG??????44???????25???????55RT23S????????TTG?GAG? CCG?GTG?GT G?G????????38???????57???????56RT18S????????GGG?GTG?GT G?GGG????????????????103??????120??????57RT19S???????? G?GGG?TT G?GGG????????????????30???????89???????58RTC07S???????T G?GGG?????????????????????????2????????2????????59RTC08S??????? G?GGG??????????????????????????2????????2????????60RT20S????????CAC?TGC? GGG?GAG?GGC?T GG??????58???????76???????61

GGRT21S????????AT G?GGG?TGC?ACA?AAC?T GG??????51???????63???????62

GGRT15S????????AAC?GTT?GA G?GGG?CAT????????????26???????52???????63RT06S????????TTC?CAG?CCC?CTC?CTC?CCC??????????29???????22???????64RTC06S???????AAC?CTC?CCC?CAC?CAC?CCC??????????4????????2????????48

In preparing table 5 during data, the external activity of thiophosphate oligonucleotide suppresses CD28 expresses among the activated periphery human T-cell of anti-CD3/PMA ability by it and determines activating being used for that IL-2 produces in the Jurkat T cell.The result represents that with respect to the activity (100%) of 5 μ M RT03S (SEQ ID NO:44) for CD28 expresses 52-79%, IL-2 is produced as 76-89% to its inhibition scope in 7 experiments.The value of the di-phosphate ester form of RT03D (SEQ ID NO:49) is in round parentheses. Give the evaluation of the active minmal sequence of external biological

Active sulfur substituted phosphate RT03S (SEQ ID NO:44) is one 18 monomer, former the design and the hybridization of people CD28 gene 5 ' untranslated region, and contains the contiguous four G sequences of two covers.For identify to activation-inducing CD28 among the human T-cell express and the JurkatT cell in the sequence correlation factor of inhibitory action key of the IL-2 generation that relies on of CD-28, add, remove or replace base from RT03S (SEQ ID NO:44) selectivity, and survey activity (table 5) with respect to the parent oligomer.5 ' end (RT04S) add 3 G or between two four G sequences the zone add one or more variations (RT05 (SEQ ID NO:51), RT09S (SEQ ID NO:52) do not reduce the inhibitory action with respect to RT03S (SEQ ID NO:44).What is interesting is with respect to RT03S (SEQ ID NO:44) also not show activity variation of adopted sequence (RT11S (SEQ ID NO:50)) is arranged.And it is opposite, in two covers, four G, replace or remove one or more G (RT10S (SEQ ID NO:53) with cytosine, RT24S (SEQ ID NO:54), RT25S (SEQ ID NO:55), RT23S (SEQ ID NO:56) (SEQ ID NO:56)) cause significantly losing with respect to the active of RT03S (SEQ ID NO:44).Remove among RT03S (SEQ ID NO:44) 5 ' first four G 6 residues to the active not influence of the inhibition of oligonucleotide (RT18S (SEQ ID NO:57).On the contrary, (RT20S (SEQ ID NO:61), RT21S (SEQ ID NO:62) residue number then significantly reduces the inhibition activity with respect to RT03S (SEQ IDNO:44) to reduce (RT19S (SEQ ID NO:58)) or increase between two four G sequences.TGGGG, GGGG or contain the sequence of 4 continuous G such as RT15S (SEQ ID NO:63) almost suppresses active with respect to RT03S (SEQ ID NO:44).The biological activity of these digital proofs RT03S (SEQ ID NO:44) depends on the special sequence primitive that contains 4 the adjacent G of 2 covers that separated by 3-5 residue.

According to toleration (Boussiotis by hindering the CD28 function to be given, V.A., Freeman, G.J., Gray, G., Gribben, J., Nadler, L.M. (1993) J.Exp.Med.178,1753-1763), whether the CD28 expression inhibiting that detects the oligomer mediation can be important for external evoked T cell anergy and the special tolerance of isoantigen provide more effective strategy.We point out that D2EHDTPA oligomer ester RT03S (SEQ ID NO:44) and TR04S reduce the level inhibition people CD4 of mRNA and maturation protein by the dosage of corresponding oligomer ⁺The inductive CD28 of anti-CD3/PMA expresses in the T cell.And in order to prove target-specific, we have detected the influence that IL-2 receptor and ICAM-1 are expressed of oligomer mediation: two kinds known do not rely on the accessory molecule that the CD28 path regulates (Damle, N.K., etc., (1992) J.Immunol.148,1985-1992; June, C.H., etc., (1987) Mol.Cell Biol.7,4472-4481; Stein, C.A., etc., Y.-C. (1993) Science261,1004-1012; Boussiotis, V.A., etc., (1993) J.Exp.Med.178,1753-1763).Therefore, the surface expression of the protein level of active information, CD25 and CD54 opposing oligomer effect.

Regulate cytokine such as IL-2 by the direct induction of immunity of the common stimulation of CD28 path, expression in activated T cell of IFN γ and IL-8 (Fraser, J.D., etc., (1991) Science251,313-316 Jenkins, M.K., etc., (1991) J.Immunol.147,2461-2466; Seder, R.A. waits (1994) J.Eep.; Med.179,299-304; Wechsler, A.S., etc., (1994) J. Immunol.153,2515-2523) want successful toleration, the special oligomer of active CD28 must be cancelled this function.Give the IL-2 that causes activation-inducing with the active oligomeric thing, IFN γ and IL-8 produce thereupon and regulate.In order to emphasize that the active oligomeric thing suppresses to rely on the sharp specificity of CD28 function, we point out that they can not prevent that the IL-2 of the activation-inducing in CD28 deficient cells strain HUT78 from producing.And the maximum that IL-8 is produced that oligomer mediates in the activated T cell suppresses to surpass 50% the other adjusting path that the IL-8 that points out driving of preservation to depend on CD28 produces.The oligomer activity is not limited to the polyclone activated T cells, and the T cell proliferation significantly reduces in MLR and the special T test cell line of tetanus toxin because the CD28 level of inhibition activation-inducing causes.Thereby our active oligomeric object mediates the special tolerance of isoantigen outward, and is provided for that inducing T cell is low replys, as CTCA 4 Ig, a kind of high being seen aglucon of B7 conjugate of making a concerted effort catch strategy another may select.(Tan，P.，Anasetti，C.，Hansen，J.A.，Melrose，J.，Brunvand，M.，Bradshaw，J.，Ledbetter，J.A.，Linsley，P.S(1993) J.Exp.Med.177，165-173.)。

When measuring active pharmacophoric group effect and continue, we observe, and RT03S (SEQ ID NO:44) expresses activated CD28 and the T cell proliferation that relies on CD28 shows astonishing lasting inhibition, even handles back 96h at oligomer.Biological activity is not relevant with toxicity, reverses the inhibition activity fully because remove oligomer in 24h.Moreover, compare thiophosphate RT03S (SEQID NO:44) and RT06S (SEQ ID NO:48), our vitro stability studies show that the secondary structure in the rich G sequence mediation of RT03S (SEQ ID NO:44) increases doubly (Stein of the main nuclease resistance 2-4 relevant with thiophosphate, C.A., Cheng, Y, C. (1993) Seience 261,1004-1012). ³²The half life (96h) of P-RT03S (SEQ ID NO:44) expansion, is with its biological activity and continue relevant.In addition, di-phosphate ester coordination compound RT03D display body external stability and the biological activity of RT03S (SEQ ID NO:44) all reduce, with previous report (Maltese, J.Y., Sharma, H.W., Vassiler, L., Narayanan, R. (1995) Nucleic Acids Res.23 1146-1151) observes consistently, is responsible for so the single biological activity to RT03S (SEQ ID NO:44) of the stability that secondary structure gives increases.Thereby the nuclease stability that thiophosphate is modified and secondary structure is given may be to prolong RT03S (SEQ IDNO:44) to suppress active reason.

In antisense that depends on hybridization and anti-genetic model (antigene), in fact single base pair is replaced can elimination activity (Maltese, J.Y., Sharma, H.W., Vassiler, L., Narayanan, R. (1995) Nucleic Acids Res.23,1146-1151).On the contrary, the activity of the special oligomer of CD28 only just significantly reduces when the sequence replacement occurs in two covers, four G that the oligomer functional structure is needed.In addition, after secondary structure cationic stabilized effect (100mM KCl and NaCl) was present in RT03S (SEQ ID NO:44), the oligomer melting curve showed a kind of conversion character (data not shown), the tetrameric formation (Hardin of prompting G-, CC, Watson, T, Corregan, M., Bailey, C. (1992) Biochemistry 31,833-841).In a word, our data are produced evidence, and promptly the special oligomer of this class CD28 is not by relying on the mechanism of hybridization, and the secondary structure of sequence works, and may form by the G-tetramer and delimit the oligomer activity.Similarly, Bennett, C.F., Chiang, M.Y., Wilson-Lingardo, L., Wyatt, J.R. (1994) Nucleic Acids Res.22,3202-3209, the activity of D2EHDTPA oligomer ester that proves them is based on the G-tetramer that forms in the sequence that contains two covers 3 or more heterogeneous adjacent G, and this proposes the human phosphodiester enzyme A of oligomer mediation ₂Adjusting be by special nucleic acid-protein-interacting.

Discern at telomere (Blackburn, E.H. (1990) in the centriole by the differential protein of some G-tetramer structures J.Biol.Chem.265,5919-5921), immunoglobulin switch region (Shimizu, A., Honjo, T. (1984) Cell36,801-803) and a class be called proof (Bock.L.C., Griffin, L.C., Latham, J.A., Vermaas, E.H., Toole, J.J. (1992) in the adjusting oligomer of aptamers Nature355,564-566; Huizenga, D.E., Szostak, J.W. (1995) Biochemistry34,656-665; Bergan, R., Connell, Y., Fahmy, B., Kyle, E., Neckers, L. (1994) Nucleic Acids Res.22,2150-2154.In our research, can form the oligomer of intermolecular four chain G-tetramer structures from 4 successive G of a cover, as telomere (Smith, F.W., Feigon, J. (1992) Nature 356 164-167) suppresses CD28 more weakly and expresses.One of them example is RT15S (SEQ ID NO:63), and its rich G sequence is before confirmed to suppress C-myc by other people and expresses (sequence 14 in Burgess, T.L., Fisher, E.F., Ross, S.L., Bready, J.V., Qian, Y.-X., Bayewitch, L.A., Cohen, A.M., Herrera, C.J., Hu, S.S.-F., Kramer, T.B., Lott, F.D., Martin, F.H., Pierce, G.F., Simonet, L., Farrell, C.L. (1995) Proc.Natl.Acad, Sci. USA92,4051-4055).Another rich G structure, the intramolecularly G-tetramer have shown thrombin inhibitory action (Wang, K.Y., McCurdy, S., Shea, R.G., Swaminanthan, S., Bolton, the P.H. (1993) that mediates aptameric Biochemistry32,1989-1904; Macaya, R.F., Schultze, P., Smith, F.W., Roe, J.A., Feigon, J. (1993) Proc.Natl.Acad.Sci.USA90,3745-3749).The sequence analysis indication residue 3-4 of RT03S (SEQ ID NO:44), 7-8, the pairing G of 12-13 and 16-17 can form this G-tetramer structure potentially.Yet it is invalid to the IL-2 generation that blocking-up suppresses CD28 expression and CD28 dependence to remove the intermolecular tetrameric residue 1-6 of obstruction (RT18S (SEQ ID NO:57)).The activity of these Notes of Key Datas RT03S (SEQ ID NO:44) is from another G-tetramer structure.

RT03S (SEQ ID NO:44) have really one be similar to others predict primitive 12 sequence monomers (Smith, F.W., Feigon, J. (1993) Biochemistry 32,8682-8692), tetrameric formation is essential to dimerization G-.The dimerization G-tetramer can be from dna double chain type or parallel and antiparallel chain.Adjacent chain helps four G to form four accumulation G-tetramers here.By have 4 bases separately every chain forming of 12 residues of adjacent four G of two covers be a primitive, and form and stable relevant.We displaing core 12 unit sequences (RT18S (SEQ ID NO:57)) have similar activity to RT03S (SEQ ID NO:44).Replacement (the RT10S (SEQ ID NO:53) of G → C in two four G zones, RT23S (SEQ ID NO:56), RT24S (SEQ ID NO:54), RT25S (SEQID NO:55)) also cause relative RT03S (SEQ ID NO:44) to suppress loss of activity 56-69%, similarly, the base that a few cover G are separated is inserted (RT20S (SEQ ID NO:61), RT21S (SEQ ID NO:62)) or is eliminated that (RT19S (SEQ ID NO:58) reduces relative biological activity 52-70%.In a word, to have only the special sequence primitive that forms dimerization G-tetramer ability that the inhibit feature CD28 of D2EHDTPA oligomer ester mediation is expressed be important to these Notes of Key Datas.

This class dimerization G-tetramer is brought into play the mechanism of its biological effect and is not also known.But several trail of evidence confirm the hypothesis that this primitive can make our active oligomeric thing work as bait, the general interaction that stops dimerization G-tetramer promoter sequence and specific transcription factor by competition.1) corresponding to the oligomer in CD28 upstream region of gene district (RT11S (SEQ ID NO:50) performance and the identical biologic activity of RT03S (SEQ ID NO:44).2) our active oligomeric thing works by non-antisense mechanism.3) these oligomers are regulated CD28 mRNA expression; Thereby its biological activity is with directly the target protein interaction is irrelevant.4) generally to increase the probability of the promoter sequence that the G-tetramer forms be a kind of phenomenon (Evans, T., Schon, E., Grazyna, G.M., Patterson, J., Efstratiadis, A. (1984) of generally regulating to class G promoter region Nucleic acids research,12,8043-805; Kilpatrick, M.W., Torri, A., Kang, D.S., Engler, J.A., Wells, R.D. (1986) Journal of biological chemistry, 261,11350-11354; Clark, S.P., Lewis, C.D., Felsenfeld, G., (1990) Nucleic acids research,18,5119-5126.).5) double-stranded oligomer can be used as bait (Morishita, R., Gibbons, G.H., Horuchi, M., Ellison, K.E., Nakajima, M., Zhang, L., Kaneda, Y., Ogihara, T., Dzau, the V.J. (1995) of transcription factor E2F Proc, Natl.Acad.Sci.USA92,5855-5859).6) class G oligomer has shown (Perez, J.R., Li, Y., Stein, C.A., Majumder, S., vanOorschot, A., Narayanan, the R. (1994) of inducing of mediation Spl transcription factor Institute of NAS newspaper91,5957-5961).List of references is incorporated into own forces

All patents, application for patent and the article of quoting are included as a reference at this.Equivalent

It is enough that description that the front is write is considered to making those skilled in the art implement the present invention.In fact, be conspicuous to the various modifications of foregoing to organic chemistry or those skilled in the relevant art for finishing the present invention, also will be in the scope of following claim.

Claims (26)

1. can reduce the oligomer of CD28 gene expression in the T cell.

2. the oligomer of claim 1, wherein said oligomer can be hybridized with the CD28 gene transcripts.

3. the oligomer of claim 2, the start codon hybridization of wherein said oligomer and CD28.

4. the oligomer of claim 1, wherein said oligomer can with the CD28 gene recombination.

5. the oligomer of claim 4, the transcript hybridization of wherein said oligomer and CD28 start codon.

5. the oligomer of claim 1, wherein this oligomer comprises at least 11 nucleic acid bases and and is less than 50 nucleic acid bases.

6. the oligomer of claim 1, wherein said oligomer is DNA or RNA molecule.

7. the oligomer of claim 1, it has and is less than 22 bases and comprises sequence 5 ' TTGTCCTGACGATGGGCTA 3 ' (SEQ ID NO:1).

8. the oligomer of claim 1, it has and is less than 22 bases and comprises sequence 5 ' AGCAGCCTGAGCATCTTTGT 3 ' (SEQ ID NO:2).

9. the oligomer of claim 1, it has and is less than 22 bases and comprises sequence 5 ' TTGGAGGGGGTGGTGGGG 3 ' (SEQ ID NO:3).

10. the oligomer of claim 1, it has and is less than 22 bases and comprises sequence 5 ' GGGTTGGAGGGGGTGGTGGGG 3 ' (SEQ ID NO:4).

11. the oligomer of claim 1, it has and comprises at least two GGGG sequences of being separated by 3-5 base, the phosphorothioate backbone that 11-50 base arranged.

12. treat to the method for small part by the disease of CD28 mediation, described method comprises step: claim 1 oligomer from effective dose to patient that use.

13. treat to the method for small part by the disease of CD28 mediation, described method comprises step: use the oligomer of effective dose to patient, this oligomer comprises at least two GGGG sequences of being separated by 3-5 base and 11-50 base is arranged.

14. the method for claim 13, wherein this oligomer comprises sequence 5 ' TTGGAGGGGGTGGTGGGG 3 ' (SEQ ID NO:3).

15. the method for claim 13, wherein this oligomer comprises sequence 5 ' GGGTTGGAGGGGGTGGTGGGG 3 ' (SEQ ID NO:4).

16. the method for claim 12-15, wherein said dosing step further comprises the following step:

Take out expression CD28 cell from patient; Thereby said oligomer is imported described cell make the oligomer cell transformed, and give patient said oligomer cell transformed.

17. the method for claim 12-15, wherein said dosing step also comprises the following step:

Take out expression CD28 cell from donor; Thereby said oligomer is imported described cell make the oligomer cell transformed, and said oligomer cell transformed is imported patient.

16. the method for claim 12-15, wherein said oligomer is by the generation of transcribing of expression vector.

17. one group of recombinant expression carrier, but described carrier comprises promoter with operative combination and encodes and can suppress the polynucleotide sequence of the CD28 oligomer that inducibility is expressed in the T cell.

17. comprise the pharmaceutical preparation of claim 1 oligomer.

18. the medicine of claim 17, wherein 11-50 base of this oligomer is long and comprise at least two GGGG sequences of being separated by 3-5 base.

19. the medicine of claim 17, the wherein long 18-25 of this an oligomer base and comprise 5 ' TTGGAGGGGGTGGTGGGG 3 ' (SEQ ID NO:3) sequence.

20. the medicine of claim 17, the wherein long 18-50 of this an oligomer base and comprise 5 ' GGGTTGGAGGGGGTGGTGGGG3 ' (SEQ ID NO:4) sequence.

21. comprise the pharmaceutical preparation of at least two kinds of different oligomers of claim 17-20.

22. the pharmaceutical preparation of claim 17-21, wherein said preparation is suitable for parenteral.

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