CN101331229A - Immunostimulatory oligoribonucleotides - Google Patents

Immunostimulatory oligoribonucleotides Download PDF

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CN101331229A
CN101331229A CNA2006800440306A CN200680044030A CN101331229A CN 101331229 A CN101331229 A CN 101331229A CN A2006800440306 A CNA2006800440306 A CN A2006800440306A CN 200680044030 A CN200680044030 A CN 200680044030A CN 101331229 A CN101331229 A CN 101331229A
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cell
immunostimulating
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CN101331229B (en
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亚历山大·福斯巴赫
约尔格·沃尔梅尔
格雷森·B·利浦福德
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Pfizer Animal Health
Coley Pharmaceutical Group Inc
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Coley Pharmaceutical GmbH
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Abstract

The invention provides immunostimulatory compositions and use of those compounds in the preparation of medicaments for the treatment of disease as well as in vitro uses. In particular, the compositions of the invention include immunostimulatory oligoribonucleotides that incorporate a sequence-dependent immunostimulatory sequence motif. Specific modifications involving phosphate linkages, nucleotide analogs, adducts, and combinations thereof are provided. Compositions of the invention, which optionally can include an antigen, can be used alone or together with other treatments to stimulate or enhance an immune response. Also provided are compositions and methods useful for treating a subject having an infection, a cancer, an allergic condition, asthma, airway remodeling, or immunodeficiency. Immnostimulatory oligoribonucleotides of the invention are believed to stimulate Toll-like receptor 8 (TLR8).

Description

Immunostimulatory oligoribonucleotides
Technical field
Generally speaking, the present invention relates to field of immunology, relate more specifically to the immunostimulating molecule.More particularly, the present invention relates to have Yeast Nucleic Acid (RNA) molecule of immunostimulatory activity, comprise oligoribonucleotide.
Background technology
Toll sample acceptor (TLR) is pattern recognition acceptor (PRR) peptide family of high conservative, and its identification and pathogenic agent bonded molecular pattern (PAMP) play a crucial role in mammiferous congenital immunity (innate immunity).Identified at least ten family members at present, they are named as TLR1-TLR10.The endochylema structural domain of multiple TLR is characterised in that Toll-interleukin 1 receptor (TIR) structural domain.Medzhitov R et al.(1998)Mol Cell 2：253-8。The activation that TLR amplifies the identification priming signal cascade that microorganism is invaded, this guards in fruit bat (Drosophila) and Mammals.Report: the fit albumen MyD88 that contains the TIR structural domain combines with TLR, will raise to TLR with the factor 6 (TRAF6) of interleukin 1 receptor bonded kinases (IRAK) and tumour necrosis factor (TNF) receptors bind.It is believed that the dependent signal transduction pathway of MyD88-causes NF-κ B transcription factor and c-Jun NH 2The activation of terminal kinases (Jnk) mitogen (mitogen)-activated protein kinase (MAPK), this is in the immuno-stimulating and the committed step of inflammatory cytokine generation.Summary is consulted Aderem A et al. (2000) Nature 406:782-87 and Akira S et al. (2004) Nat Rev Immunol 4:499-511.
Identified a large amount of specificity T LR parts.The part of TLR2 comprises peptidoglycan and lipopeptid.Yoshimura A et al.(1999)J Immunol 163：1-5；Yoshimura A et al.(1999)J Immunol 163：1-5；Aliprantis AO et al.(1999)Science 285：736-9。Lipopolysaccharides (LPS) is the part of TLR4.Poltorak A et al.(1998)Science 282：2085-8；HoshinoK et al.(1999)J Immunol 162：3749-52。Bacterial flagellin is the part of TLR5.Hayashi F et al.(2001)Nature 410：1099-1103。Report: peptidoglycan is not only the part of TLR2, but also is the part of TLR6.Ozinsky A et al.(2000)Proc NatlAcad Sci USA 97：13766-71；Takeuchi O et al.(2001)Int Immunol 13：933-40。Nearest report: some lower molecular weight synthetic compound imidazole quinoline miaow quinoline not moral (imidazoquinolinesimiquimod, R-837) and resiquimod (resiquimod R-848) is the part of TLR7 and TLR8.Hemmi H et al.(2002)Nat Immunol 3：196-200；Jurk M et al.(2002)NatImmunol 3：499。
From unmethylated DNA of bacteria of recent discovery and synthetic analogue (CpG DNA) thereof is part (Hemmi H et al. (2000) the Nature 408:740-5 of TLR9; Bauer S et al. (2001) Proc Natl Acad Sci USA 98,9237-42) beginning, report has been arranged: the part of some TLR comprises some nucleic acid molecule.The RNA that has reported at present some type is an immunostimulating with sequence-independent manner or sequence dependent form.In addition, report: these panimmunity pungencys RNA can stimulate TLR3, TLR7 or TLR8.
Summary of the invention
Generally speaking, the present invention relates to contain the oligoribonucleotide (ORN) of the immunostimulating of some immunostimulating RNA motif, and the using method that contains related immune irritating compositions and this para-immunity pungency ORN and the composition of this para-immunity pungency ORN.Immunostimulating ORN of the present invention is applicable to and anyly is used for stimulating or device (setting) that enhancing immunity is replied or use.As hereinafter disclosed, immunostimulating ORN of the present invention is particularly useful for preparing the pharmaceutical composition that contains adjuvant, vaccine and other medicament, and described pharmaceutical composition is used for the treatment of various disease conditions, comprises infection, cancer, transformation reactions and asthma.Therefore, the present invention relates to the immunostimulating composition that contains immunostimulating ORN of the present invention in some aspects, and their using method.As hereinafter disclosed, immunostimulating ORN of the present invention and immunostimulating composition are particularly useful in the following method, described method is used for immune cell activated, the vaccine inoculation experimenter, treatment suffers from the experimenter of immune system defect, treatment suffers from the experimenter of infection, treatment suffers from the experimenter of autoimmune disease, treatment suffers from the experimenter of cancer, treatment suffers from the experimenter of allergic conditions, and treatment suffers from asthma, airway remodeling (airway remodeling), promotes the experimenter of epi-position expansion and antibody dependent cellular cytotoxicity (ADCC).
As hereinafter disclosed in more detail, immunostimulating ORN of the present invention is characterised in that they comprise the immunostimulating RNA motif of at least a sequence dependent.The RNA sequence that the immunostimulating RNA motif of sequence dependent is normally short, although this motif also can contain modification in certain embodiments, phosphoric acid connection, modified nuclear base (nucleobase), modified sugar, nucleotide analog or its any combination between for example modified Nucleotide.As described in greater detail below, in one embodiment, immunostimulating RNA motif is present in the environment of longer immunostimulating ORN of the present invention.Immunostimulating RNA motif also may reside in the environment of chimeric DNA:RNA nucleic acid molecule.
The immunostimulating RNA motif of sequence dependent and adding have had the immunostimulating ORN of these motifs to be disclosed as the agonist of TLR8.More specifically, at least some sequence dependent immunostimulating RNA motif, immunostimulating ORN and immunostimulating DNA:RNA nucleic acid molecule to be disclosed as be the agonist of TLR8 rather than the agonist of TLR7.
The immunostimulating RNA motif of some aspects is N-U-R according to the present invention 1-R 2
N is a ribonucleotide, and N does not comprise U.In some embodiments, N is adenosine or cytosine(Cyt) (C) or derivatives thereof.
U is the uridylic or derivatives thereof.
R is a ribonucleotide, wherein R 1And R 2One of at least be adenosine (A) or cytosine(Cyt) or derivatives thereof.R is not U, unless N-U-R 1-R 2Contain at least two A.
ORN of the present invention contains at least one and contains in some embodiments more than (promptly 2,3 or 4 a) immunostimulating motif N-U-R 1-R 2ORN does not contain the TLR7/8 motif.The ORN preferred length is 4-100, and randomly contains at least one place backbone modifications.
N in some embodiments-U-R 1-R 2Can contain at least 3 As or at least 2 Cs.Randomly, N-U-R 1-R 2Contain at least one G or C.
In some embodiments, ORN is not ACCCAUCUAUUAUAUAACUC (SEQID NO:89).
In some other embodiment, the ORN motif is separated by non-nucleotide joint and 5 ' ribonucleotide.Also in some other embodiment, the ORN motif is separated by non-nucleotide joint and 3 ' ribonucleotide.Randomly, the ORN motif is separated by non-nucleotide joint and 5 ' and 3 ' ribonucleotide.
ORN also comprises pharmaceutically useful vehicle, and it randomly is lipid vehicle such as N-[1-(2,3-two oleoyl oxygen) propyl group]-N, N, N trimethyl ammonium methylsulfuric acid ester (DOTAP).In some other embodiment, ORN is not compound with DOTAP.
ORN can be strand or two strands.
In some other embodiment, ORN contains at least one AU.Also in some other embodiment, ORN contains at least one CU.
In some embodiments, ORN is one of following: A*U*A*G*G*C*A*C (SEQ IDNO:4), G*C*C*A*C*C*G*A*G*C*C*G*A*A*U*A*U*A*C*C (SEQ IDNO:11), A*U*A*U*A*U*A*U*A*U*A*U*A*U*A*U*A*U*A*U (SEQ IDNO:12), U*U*A*U*U*A*U*U*A*U*U*A*U*U*A*U*U*A*U*U (SEQ IDNO:13), A*A*U*A*A*U*A*A*U*A*A*U*A*A*U*A*A*U*A*A (SEQ IDNO:16), A*A*A*U*A*A*A*U*A*A*A*U*A*A*A*U*A*A*A*U (SEQ IDNO:17), A*A*A*A*U*A*A*A*A*U*A*A*A*A*U*A*A*A*A*U (SEQ IDNO:18), C*U*A*C*U*A*C*U*A*C*U*A*C*U*A*C*U*A*C*U (SEQ IDNO:24), U*U*A*U*U*A*U (SEQ ID NO:30), U*A*U*A*U*A*U (SEQ IDNO:33), C*C*G*A*G*C*C*G*C*A*U*U*A*C*C*C (SEQ ID NO:48), C*C*G*A*G*C*C*G*A*U*U*G*A*A*C*C (SEQ ID NO:76), C*C*G*A*G*C*C*G*A*A*U*A*C*C*C*C (SEQ ID NO:42), C*C*G*A*G*C*C*A*U*A*U*A*U*A*U*C (SEQ ID NO:39), C*C*G*A*G*C*C*G*A*U*A*U*U*A*C*C (SEQ ID NO:65), C*C*G*A*G*C*C*G*A*A*U*C*C*C*C*C (SEQ ID NO:44), C*C*G*A*G*C*C*G*C*C*U*A*C*C*C*C (SEQ ID NO:47), C*C*G*A*G*C*C*A*U*A*U*A*U*C*C*C (SEQ ID NO:38), C*C*G*A*G*C*C*G*C*U*A*U*A*C*C*C (SEQ ID NO:37), C*C*G*A*G*C*C*G*A*A*U*A*A*C*C*C (SEQ ID NO:40), C*C*G*A*G*C*C*G*C*U*A*U*C*C*C*C (SEQ ID NO:55), C*C*G*A*G*C*C*G*A*A*G*G*U*A*C*C (SEQ ID NO:82), C*C*G*A*G*C*C*G*A*A*G*A*U*A*C*C (SEQ ID NO:85), C*C*G*A*G*C*C*G*A*A*U*G*U*A*C*C (SEQ ID NO:63), C*C*G*A*G*C*C*G*C*C*U*A*A*C*C*C (SEQ ID NO:43), C*C*G*A*G*C*C*G*C*A*U*A*U*C*C*C (SEQ ID NO:36), C*C*G*A*G*C*C*G*A*A*G*C*U*A*C*C (SEQ ID NO:87), C*C*G*A*G*C*C*G*C*A*U*A*C*C*C*C (SEQ ID NO:45), C*C*G*A*G*C*C*G*C*A*U*A*A*C*C*C (SEQ ID NO:41), C*C*G*A*G*C*C*G*A*A*G*G*U*G*C*C (SEQ ID NO:83), C*C*G*A*G*C*C*G*C*A*U*C*C*C*C*C (SEQ ID NO:46), C*C*G*A*G*C*C*G*A*A*G*C*U*G*C*C (SEQ ID NO:88), C*C*G*A*G*C*C*G*C*C*G*C*C*C*C*C (SEQ ID NO:35), C*C*G*A*G*C*C*G*A*A*G*C*U*C*C*C (SEQ ID NO:84), or C*C*G*A*G*C*C*G*A*A*G*G*C*A*C*C (SEQ ID NO:56).
ORN gets rid of the TLR7/8 motif especially.The TLR7/8 motif can contain and for example is selected from following ribonucleoside acid sequence:
(i)5′-C/U-U-G/U-U-3′，
(ii)5′-R-U-R-G-Y-3′，
(iii)5′-G-U-U-G-B-3′，
(iv) 5 '-G-U-G-U-G/U-3 ' and
(v)5′-G/C-U-A/C-G-G-C-A-C-3′，
Wherein C/U is cytosine(Cyt) (C) or uridylic (U), and G/U is guanine (G) or U, and R is a purine, and Y is a pyrimidine, and B is U, G or C, and G/C is G or C, and A/C is VITAMIN B4 (A) or C.
In multiple embodiments, 5 '-C/U-U-G/U-U-3 ' is CUGU, CUUU, UUGU or UUUU.
In multiple embodiments, 5 '-R-U-R-G-Y-3 ' is GUAGU, GUAGC, GUGGU, GUGGC, AUAGU, AUAGC, AUGGU or AUGGC.In one embodiment, base sequence is GUAGUGU.
In multiple embodiments, 5 '-G-U-U-G-B-3 ' is GUUGU, GUUGG or GUUGC.
In multiple embodiments, 5 '-G-U-G-U-G/U-3 ' is GUGUG or GUGUU.In one embodiment, base sequence is GUGUUUAC.
In multiple embodiments, 5 '-G/C-U-A/C-G-G-C-A-C-3 ' is GUAGGCAC, GUCGGCAC, CUAGGCAC or CUCGGCAC.
One aspect of the present invention provides the immunostimulating composition that comprises immunostimulating ORN of the present invention and adjuvant.In multiple embodiments, adjuvant is adjuvant, immunostimulating adjuvant that produces storage effect (depot effect) or the adjuvant that produces storage effect and stimulating immune system.In one embodiment, the immunostimulating composition of this aspect is the conjugate of immunostimulating ORN and adjuvant according to the present invention.In the embodiment aspect this according to the present invention, immunostimulating ORN and adjuvant are covalently bound.They are not puted together in some other embodiment.In one embodiment, adjuvant is the agonist of TLR9.In one embodiment, adjuvant is the CpG nucleic acid of immunostimulating.
Composition of the present invention can randomly contain antigen.Therefore, one aspect of the present invention provides following vaccine, and wherein said vaccine contains immunostimulating ORN of the present invention and antigen.One aspect of the present invention provides a kind of vaccine, and described vaccine contains immunostimulating ORN of the present invention and antigenic conjugate.In one embodiment, the conjugate of this aspect contains the immunostimulating ORN covalently bound with antigen according to the present invention.In some other embodiment, they are not puted together.In a plurality of embodiments, antigen can be antigen itself.Antigen can be any antigen, comprises cancer antigen, microbial antigen or allergen.
One aspect of the present invention provides the immunostimulating composition, and described composition contains the conjugate of immunostimulating ORN of the present invention and lipophilic portion.In one embodiment, the ORN of immunostimulating and lipophilic portion are covalently bound.In one embodiment, lipophilic portion is selected from cholesteryl, palmityl and fatty acyl group.In one embodiment, lipophilic portion is the derivative of cholesterol, for example cholesteryl.
In one embodiment, immunostimulating ORN contains at least one deoxyribonucleotide.Described at least one deoxyribonucleotide can appear at outside the immunostimulating RNA motif Anywhere usually.In a plurality of embodiments, described at least one deoxyribonucleotide is 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23 or 24 successive deoxyribonucleotide.The present invention has also considered to comprise the immunostimulating ORN of discrete deoxyribonucleotide.In a plurality of embodiments, at least one deoxyribonucleotide is 5 ' end, 3 ' end or 5 ' and the 3 ' two ends of immunostimulating ORN.Described at least one deoxyribonucleotide is also corresponding to the DNA part of chimeric DNA:RNA molecule.In one embodiment, the DNA component of chimeric DNA:RNA molecule comprises CpG nucleic acid, i.e. the TLR9 agonist.In one embodiment, the DNA of chimeric DNA:RNA molecule and RNA are partly covalently bound by phosphate bond between Nucleotide.In another embodiment, the DNA of chimeric DNA:RNA molecule and RNA are partly covalently bound by joint (for example non-nucleotide joint).
One aspect of the present invention provides the immunostimulating composition, and described composition comprises covalence closed, the nucleic acid molecule part strand, dumb-bell shape, and wherein at least one strand of this molecule partly comprises immunostimulating RNA motif of the present invention.
One aspect of the present invention provides the pharmaceutical composition of the composition that comprises the aforementioned any aspect of the present invention and optional pharmaceutically acceptable vehicle, described composition be selected from following delivery vehicle and combine: cation lipid, liposome, spiral helicine (cochleate), virion, immunostimulating mixture (ISCOM), particulate, microballoon, millimicro ball, unilamellar vesicle (LUV), multilamellar vesicle, oil-in-water emulsion, water-in-oil emulsion, newborn body (emulsome) and polycation peptide.In the embodiment aspect this according to the present invention, pharmaceutical composition comprises antigen.
Can in atomizer or sucker, prepare ORN, for example metered-dose inhaler (metered doseinhaler) or Diskus.In some embodiments, ORN also comprises extra composition, for example chemotherapeutic, antiviral agent or pharmaceutically acceptable vehicle.Pharmaceutically acceptable vehicle can be configured to and be used for injection or mucosal administration.
In addition, according to these aspects of the present invention and others, in multiple embodiments, immunostimulating ORN can choose wantonly comprise between at least one 5 '-5 ' Nucleotide connect, connect between at least one 3 '-3 ' Nucleotide, at least one comprises between 5 of shank '-5 ' Nucleotide and connects, at least one comprises between 3 of shank '-3 ' Nucleotide and connect, or its any combination.In one embodiment, shank is the non-nucleotide shank.
In addition, also according to these aspects of the present invention and others, in multiple embodiments, immunostimulating ORN can choose wantonly to comprise between at least one 2 '-2 ' Nucleotide and connect or its any combination between connection, at least 2 '-5 ' Nucleotide between connection, at least one 2 '-3 ' Nucleotide.In preferred embodiments, connect between described at least one 2 '-2 ' Nucleotide, connect between at least one 2 '-3 ' Nucleotide or at least 2 '-5 ' Nucleotide between connect and be present in outside the immunostimulating RNA motif.
Also according to these and other aspect of the present invention, in one embodiment, immunostimulating ORN comprises at least one multiplication units (multiplier unit).Therefore, in certain embodiments, immunostimulating ORN of the present invention can have branched structure.The ramose composition can comprise 3 of arbitrary combination '-5 ', 5 '-5 ', 3 '-3 ', 2 '-2 ', 2 '-3 ' or 2 '-5 ' Nucleotide between key.In one embodiment, immunostimulating ORN comprises at least two multiplication units, produces so-called dendrimer (dendrimer).In addition, in certain embodiments, immunostimulating ORN of the present invention can comprise two or more immunostimulating RNA motifs, and it is the linear ORN random alignment in the dissimilar arm upper edge of branched structure for example, or property ORN random alignment all along the line and being positioned on the dissimilar arm of branched structure.The optional CpG nucleic acid that comprises at least one immunostimulating of branched structure (comprising dendrimer) is for example as the independent arm of branched structure.
Also according to these and other aspect of the present invention, in one embodiment, immunostimulating ORN does not comprise CG DNA or RNA dinucleotides.
One aspect of the present invention provides and has been used to reduce the method that inhibitive ability of immunity CD4+ regulates (Treg) cell.The method of this aspect comprises the steps: the composition of CD4+Treg cell and containing of significant quantity TLR8-specific immunity stimulation of the present invention ORN is contacted according to the present invention, to reduce the effect of CD4+Treg cell inhibiting.In one embodiment, composition comprises TLR8-specificity ORN and immunostimulating CpG nucleic acid, and wherein TLR8-specificity ORN is not connected with immunostimulating CpG nucleic acid.In one embodiment, composition contains TLR8-specificity ORN and immunostimulating CpG nucleic acid, and wherein specific ORN of TLR8-and immunostimulating CpG nucleic acid exist as conjugate.
The present invention provides the method that is used for regulating and control experimenter's immunne response on the other hand.The method of this aspect comprises the step of the experimenter being used the present composition of significant quantity according to the present invention.In some embodiments, ORN can be delivered to the experimenter, with treatment autoimmune disease or airway remodeling in the experimenter.ORN can be applied to the experimenter separately or with antigen.Randomly, send ORN with mouth for example, nose, hypogloeeis, intravenously, subcutaneous, mucous membrane, breathing, direct injection with through the approach of skin.ORN can be delivered to the experimenter with the inducing cell factor expression with significant quantity, for example TNF α, IL-10, IL-6, IFN-γ, MCP1 and IL-12.
One aspect of the present invention provides the method to experimenter's vaccine inoculation.The method of this aspect comprises the step to experimenter's administration of antigens and immunostimulating ORN of the present invention according to the present invention.
One aspect of the present invention provides the method that is used for the treatment of the experimenter, and described experimenter suffers from communicable disease or has the risk of suffering from communicable disease.The method of this aspect comprises the step of the experimenter being used the present composition of significant quantity according to the present invention.In one embodiment, this method comprises the step of the experimenter being used the immunostimulating ORN of the present invention of significant quantity.In one embodiment, the experimenter suffers from virus infection.Virus infection can be for example hepatitis B or hepatitis C.Also can use antiviral agent to the experimenter.Randomly, antiviral agent is connected with ORN.
One aspect of the present invention provides the method that is used for the treatment of the experimenter, and described experimenter suffers from cancer or has cancered risk.The method of this aspect comprises the step of the experimenter being used the present composition of significant quantity according to the present invention.In one embodiment, this method comprises the step of the experimenter being used the immunostimulating ORN of the present invention of significant quantity.In one embodiment, also the experimenter is used chemotherapy or radiation.
One aspect of the present invention provides the method that is used for the treatment of the experimenter, and described experimenter suffers from cancer or has cancered risk.The method of this aspect comprises the step of the experimenter being used the composition of significant quantity according to the present invention, and described composition contains TLR8-specific immunity pungency ORN of the present invention to reduce the effect of CD4+Treg cell inhibiting.In one embodiment, composition comprises TLR8-specificity ORN and immunostimulating CpG nucleic acid, and wherein TLR8-specificity ORN is not connected with immunostimulating CpG.In one embodiment, composition comprises TLR8-specificity ORN and immunostimulating CpG nucleic acid, and wherein TLR8-specificity ORN and immunostimulating CpG nucleic acid exist as conjugate.
One aspect of the present invention provides the method that is used for the treatment of the experimenter, and described experimenter suffers from allergic conditions or has the risk of suffering from allergic conditions.The method of this aspect comprises the step of the experimenter being used the present composition of significant quantity according to the present invention.In one embodiment, this method comprises the step of the experimenter being used the immunostimulating ORN of the present invention of significant quantity.In one embodiment, the experimenter suffers from rhinallergosis.
One aspect of the present invention provides the method that is used for the treatment of the experimenter, and described experimenter suffers from asthma or has the risk of suffering from asthma.The method of this aspect comprises the step of the experimenter being used the present composition of significant quantity according to the present invention.In one embodiment, this method comprises the step of the experimenter being used the immunostimulating ORN of the present invention of significant quantity.In one embodiment, the asthma that increased the weight of by virus infection of asthma.ORN can be applied separately or with allergen.
Another aspect of the present invention provides the method that is used for the treatment of the experimenter who suffers from airway remodeling.The method of this aspect comprises the step of the experimenter being used the immunostimulating ORN of the present invention of significant quantity according to the present invention.
One aspect of the present invention provides the method that is used to improve antibody dependent cellular cytotoxicity (ADCC).The method of this aspect comprises the steps: that the experimenter of ADCC that needs are increased uses the immunostimulating ORN of the present invention and the antibody of significant quantity according to the present invention, to increase ADCC.In one embodiment, antibody is the antibody to other antigen-specific of cancer antigen or cancer cells expression.In one embodiment, antibody is IgG antibody.
One aspect of the present invention provides the method that is used to strengthen the epi-position expansion.The method of this aspect comprises following sequential steps according to the present invention: immune cell is contacted with antigen, subsequently this cell is contacted with the immunostimulating ORN of the present invention of two doses at least.In one embodiment, carry out in this method body.In one embodiment, this method comprises the steps: with the amount that can effectively induce multiple epi-position specific immune response the experimenter to be used the vaccine that comprises antigen and adjuvant, subsequently the experimenter is used the immunostimulating ORN of the present invention of at least two doses.This method relates to the application of treatment scheme in one embodiment, described scheme causes the immunity system antigen-exposed among the experimenter, with the amount that can effectively induce multiple epi-position specific immune response the experimenter is used the immunostimulating ORN of the present invention of at least two doses then.In multiple embodiments, treatment plan is operation, radiation, chemotherapy, other cancer medicament, vaccine or cancer vaccine.In one embodiment, the immunostimulating ORN of described at least two doses separates each other at least one day to being applied in a week.In one embodiment, the immunostimulating ORN of described at least two doses separates each other and was applied at least one thoughtful one month.In one embodiment, the immunostimulating ORN of described at least two doses separates each other and was applied by six months by at least one moon.
On the one hand, the present invention is the N-U-R that comprises of cell by will expressing TLR8 and following dosage 1-R 2RNA oligoribonucleotide (ORN) contact, wherein N is that ribonucleotide and N do not comprise U, U is the uridylic or derivatives thereof, R is a ribonucleotide, wherein R 1And R 2One of at least be adenosine (A) or cytosine(Cyt) or derivatives thereof, wherein R is not U, unless N-U-R 1-R 2Contain at least two A, wherein said ORN does not comprise the TLR7/8 motif, and wherein said ORN length is 4-100, and inflammatory cytokine production and IFN-α production of wherein responding to ORN are not significantly induced for background before the described consumption effective stimulus.In some embodiments, the IFN-α that responds to ORN produces and to be less than 300 pg/ml.In one embodiment, ORN is not ACCCAUCUAUUAUAUAACUC (SEQ ID NO:89).ORN can with N-[1-(2,3-two oleoyl oxygen) propyl group]-N, N, N trimethyl ammonium methylsulfuric acid ester (DOTAP) is compound or not compound.
In some embodiments, the cell of expression TLR8 is monocyte or mDC.Also in some other embodiment, the cell of expressing TLR8 is external or intravital.
These and other feature of the present invention will be described in more detail in the detailed Description Of The Invention part.
Summary of drawings
Fig. 1 is by ORN inductive cytokine production when being described in the PBMC stimulation.By measuring IFN-α and TNF-α cytokine production, observe the difference between TLR8 and the TLR7/8.On complete titration curve, use with DOTAP (25 μ g/ml, 1/3 extent of dilution) compound and specify ORN (2 μ M, 1/3 extent of dilution) or stimulate the human PBMC with R-848 (2 μ M, 1/3 extent of dilution).Collect supernatant liquor after 16 hours, and (Fig. 1 a) and TNF-α (Fig. 1 b) to measure IFN-α by ELISA.Data presentation the average of three blood donors of at least three parts of independent experiments.DOTAP does not show effect separately.ORN can be compound with DOTAP, and R-848 is not compound.DOTAP is separately contrast.In Fig. 1 c, using with DOTAP (2.2 μ g/ml) or with the specified ORN of R-848 (2 μ M) compound 0.2 μ M stimulates the human PBMC.Collect supernatant liquor after 16 hours and measure IFN-α (left figure) and TNF-α (right figure) by ELISA.Data presentation the average of 3 donors (± SEM).
Fig. 2 is one group of histogram, its described separated pDC (Fig. 2 a), when monocyte (Fig. 2 b) and mDC (Fig. 2 c) stimulate by ORN inductive cytokine production.With with 10 μ g/mlDOTAP, 0.5 μ M CpG ODN or DOTAP compound 0.5 μ M ORN or independent substratum irritation cell, and measure IFN-α (Fig. 2 a), TNF-α (Fig. 2 c) and IL-12p40 (Fig. 2 c).
Fig. 3 is one group of histogram, its set forth PBMC when stimulating by ORN inductive cytokine production.With specifying ORN (0.5 μ M ORN) to stimulate the human PBMC, and measure IFN-α (Fig. 3 A) and TNF-α (Fig. 3 B) and pass through Luminex commercial measurement cytokine production by elisa technique with 10 μ g/ml DOTAP compound.
Fig. 4 be set forth to specify ORN (Fig. 4 a) and TNF-α (Fig. 4 b) maximum activity histogram relatively.With stimulating the human PBMC with DOTAP (since 25 μ g/ml, extent of dilution 1/3) compound ORN (7 kinds of concentration are since 2 μ g/ml, 1/3 extent of dilution), and to two parts separately in the experiment the average maximum activity of the 0.6 μ M 3-6 of a place blood donor measure.
Fig. 5 be set forth IFN-α maximum activity (Fig. 5 a) with IFN-α EC50 (Fig. 5 b) histogram relatively.Being used for DOTAP compound ORN stimulates the human PBMC and measures IFN-α.
Fig. 6 is a set of diagrams, its compared have TLR8 (SEQ ID NO:13) or TLR7/8 (SEQID NO:21) ORN to the PBMC of 3 blood donors, separated pDC or separated monocytic titration curve.With with DOTAP (since 25 μ g/ml, 1/ 4Extent of dilution) compound ORN (4 kinds of concentration, since 1 μ g/ml, 1/ 4Extent of dilution) irritation cell.Collect supernatant liquor after 16 hours and pass through Luminex commercial measurement cytokine production.Figure shows SEQ ID NO:21 (0,3 μ M) cytokine production per-cent.
Fig. 7-1 has shown one group of column diagram to 7-4, and it is set forth in the average maximum activity of 3 blood donors of any concentration to PMBC, separated monocyte, separated pDC and CD14-CD123-PBMC.With with DOTAP (since 25 μ g/ml, 1/ 4Extent of dilution) compound ORN (4 kinds of concentration, since 1 μ g/ml, 1/ 4Extent of dilution) irritation cell.Collect supernatant liquor after 16 hours and pass through Luminex commercial measurement cytokine production.The positive reaction of red square indication on the background of DOTAP and substratum.
Fig. 8 is one group of column diagram, and it has shown the difference between TLR8 (SEQ ID NO:13) and TLR7/8 (SEQID NO:21) ORN.With with DOTAP (since 25 μ g/ml, 1/ 4Extent of dilution) compound ORN (4 kinds of concentration, since 1 μ g/ml, 1/ 4Extent of dilution) irritation cell.Collect supernatant liquor after 16 hours and pass through Luminex commercial measurement cytokine production.The average maximum cell factor under any concentration of pictorialization measurement is produced, and it is expressed as the per-cent of TLR8 ORN (SEQ ID NO:13) than TLR7/8 ORN (SEQ ID NO:21).Show at separated pDC, PBMC, separated monocyte and CD123-CD14-PBMC.
Fig. 9 is set of diagrams and curve, and it has shown in the HEK-293 of stable transfection cell the reaction by the TLR8 ORN (SEQ ID NO:13) and the TLR7/8 ORN (SEQ ID NO:21) of TLR8 effect.With HEK-293 cytositimulation 16 hours, described HEK-293 cell was read report and people TLR8 stable transfection with NF κ B-luciferase with specified ORN.Remove supernatant liquor after 16 hours, with lysis and measure luciferase activity or cytokine levels.Fig. 9 a and 9b have shown stimulates the multiple of back NF κ B-luciferase to induce.Fig. 9 c has shown stimulates the multiple of back NF κ B-luciferase to induce when having inhibitor.Fig. 9 d has shown that the post-stimulatory IP-10 that measures by luciferase assay stimulates.
Figure 10 is the set of diagrams table, and it has shown with the surface marker that is rich in AU or be rich on the people pDC that the ORN of GU stimulates expresses.With with 25 μ g/ml DOTAP compound 1 μ M ORN or use separately DOTAP (Figure 10 a), or specified amount with DOTAP compound ORN or use DOTAP (Figure 10 b-10c) to hatch purified pDC of CD123+ (Figure 10 a and 10b) or separated monocyte (Figure 10 c) separately.Collecting cell is also with CD123, CD11c and HLA-DR antibody (Figure 10 a and 10b) or CD14 and CD19 (Figure 10 c) dyeing after 16 hours.Expressing measurement cell surface marker thing by CD86 (Figure 10 a and 10b) or CD80 (Figure 10 c) activates.Figure 10 a shows facs analysis, and it has proved that ORN (SEQ ID NO:13) that is rich in AU and the ORN (SEQ ID NO:21) that the is rich in GU CD86 surface marker when pDC stimulates shows difference in expressing.Figure 10 b is that the CD86 surface marker expression when setting forth people pDC stimulation is a dose-dependently.Figure 10 c is the figure that shows following content: the ORN (SEQ ID NO:21) that is rich in the ORN (SEQ ID NO:13) of AU and is rich in GU shows indifference in the expression of CD80 surface marker when human PBMC's (data not shown) and the stimulation of CD14-positive cell.
Figure 11 is one group of column diagram, and it has shown the difference between TLR8 ORN (SEQ ID NO:13) and the TLR7/8ORN (SEQ ID NO:21).Use SEQ ID NO:5 ORN in contrast.Ox PBMC and 10 μ g/ml ORN (HD) or 2.5 μ g/ml ORN (LD) were hatched 48 hours.Collect supernatant liquor and use elisa assay.Figure 11 a-c has shown the level of IL-12, IFN-γ and TNF-α respectively.
Figure 12 is one group of chart that shows following content: the interior or external ORN SEQ ID NO:13 that is rich in AU that do not respond to of mouse cell paste.The cell that uses be mouse macrophage Raw264.7 cell (Figure 12 a), J774 cell (Figure 12 b), purified mouse CD11c+ cell (sv129 mouse) (Figure 12 c-12g) and the interior mouse cell of body.Estimate cytokine concentration by ELISA.
Figure 13 is a chart of showing following content: rat spleen cells does not respond to the ORNSEQ ID NO:13 that is rich in AU.To merge from the splenocyte of 3 Sprague-Dawley rats, and with SEQ ID NO:21, SEQ ID NO:13 (all compound, 1/5 extent of dilution), R-848 or the DOTAP of prescribed concentration with 62.5 μ g/ml DOTAP separately (62.5 μ g/ml-＞1/5 extent of dilution) stimulate.Collect supernatant liquor after 20 hours and measure the TNF-alpha levels by ELISA.
Detailed Description Of The Invention
A part of the present invention relates to the present inventor to the discovery of the immune excitant RNA motif of a large amount of sequence-specifics. Have been found that at present: the molecule (making up separately or with some other composition) that contains immune excitant RNA motif is important immune irritant compound, it is suffered from illness or has in experimenter's the large metering method of ill disease risk usefully being used for the treatment of, and induces, increases or change that immunity replys can be favourable in the described illness. In one embodiment, the employed immune irritating compositions of the present invention of this paper is immune excitant ORN of the present invention.
Have been found that some sequence-specific RNA motif is that immunity is irritating, it is opposite with other motif that acts on TLR7 and TLR8 (be rich in GU and be rich in CU) by the TLR8 effect. RNA oligonucleotides (RON) (preferably containing the sequence that is rich in AU) is replied by the TLR8 immune stimulatory. In different types of ORN (for example contain AU and contain the ORN that GU repeats), observed the difference between IFN-α, TNF-α, IFN-γ and the IL-12 production. What is interesting is, have been found that immune excitant ORN of the present invention produces strong front inflammatory cytokine and replys, and except the IFN-α molecule relevant with IFN-α. IFN-α production is reduced or shortage when stimulating with these novel ORN.
The immune excitant RNA motif of some aspects is N-U-R according to the present invention1-R 2。
N is ribonucleotide, and N does not comprise U. In some embodiments, N is adenosine or cytimidine (C) or derivatives thereof.
U is the uracil or derivatives thereof.
R is ribonucleotide, wherein R1And R2One of at least be adenosine (A) or cytimidine or derivatives thereof. R is not U, unless N-U-R1-R 2Contain at least two A.
ORN of the present invention contains at least one and contains more than (namely 2,3 or 4 a) immune excitant motif N-U-R in some embodiments1-R 2 ORN does not contain the TLR7/8 motif.
ORN is oligonucleotides. Optional ground, this oligonucleotides length is 4-100. ORN length also can be for example 8-40,15-25 or 20-30 nucleotides. ORN contains at least one backbone modifications optionally.
N in some embodiments-U-R1-R 2Can contain at least 3 As or at least 2 Cs. Optional ground, N-U-R1-R 2Contain at least one G or C.
In some embodiments, ORN is not ACCCAUCUAUUAUAUAACUC (SEQ ID NO:89).
ORN also can comprise pharmaceutically useful delivery body, and it is lipid delivery body such as N-[1-(2,3-, two oleoyl oxygen) propyl group optionally]-N, N, N trimethyl ammonium methylsulfuric acid ester (DOTAP). In some other embodiment, ORN is not compound with DOTAP. In some other embodiment, can medicinal delivery body can be peptide, such as the polycation peptide. The polycation peptide comprise for example multiple poly-D-lysine, poly arginine and polypeptide (its more than 5, particularly contain alkaline amino acid, particularly arginine or lysine residue more than 50% more than the scope of 8 amino acid residues) or its mixture, and can for example contain naturally occurring insect antiseptic protein.
In some other embodiment, ORN contains at least one AU.
Except sequence-specific, be effective as the immune excitant RNA motif of single stranded RNA, partially double stranded RNA or complete double-stranded RNA.
For ORN of the present invention and have the TLR7/8 motif ORN of (namely containing the repetition of GU), observe the clear difference between the production of IFN-α and IFN-α correlation molecule and other front inflammatory cytokine such as TNF-α, IFN-γ, IL-10, IL-6 and IL-12. Has N-U-R1-R 2The ORN of the present invention of motif, for example those ORN that contain AU or AUU repetition (SEQ ID NO:12, SEQ ID NO:13) show without IFN-α cytokine production when PBMC and pDC stimulation. On the contrary, the ORN with three or more U (SEQ ID NO:14, SEQ ID NO:15) in the row induces IFN-α production, although there is As. What is interesting is that when using identical, single chamber with the ORN group of A exchange G, the strong IFN-α when observing PBMC and stimulating produces. The existence that the data that this paper exists are pointed out two different ORN kinds strongly: a kind of cell of expressing TLR8 that acts on, such as monocyte and mDC (SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:16-SEQ ID NO:18), contain N-U-R of the present invention1-R 2The ORN of motif, another kind act on and express the two cell of TLR7/8, for example contain monocyte, mDC and the pDC (SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:19-SEQ ID NO:23) of CU, GU and GUU sequence.
Therefore, ORN of the present invention has induce immune response and does not induce ability with respect to significant quantity IFN-α or the IFN-α correlation molecule of background. Preferably change with respect to the IFN-α of background or IFN-α correlation molecule level with respect to the IFN-α of the significant quantity of background or IFN-α correlation molecule and to be less than 20 %. In some embodiments, it is less than 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1%. In some other embodiment, derivative IFN-α or correlation molecule equal background or are less than background level. Also in some other embodiment, the IFN-α that is induced by ORN of the present invention be less than or the IFN-α that equals to be induced by TLR7/8 ORN 20%. IFN-α optional 300pg/ml of being less than in testing in vitro by ORN of the present invention induces maybe can have the EC50 greater than 1.5 μ M.
IFN-α correlation molecule used herein is the cell factor or the factor relevant with the IFN-alpha expression. These molecules include but not limited to MIP1-β, IP-10 and MIP1-α.
Report CD4+Treg cell is expressed TLR8 at present, and the transduction of the TLR8 signal in these cells reduces or reversed their immune excitant function. Peng G et al. (2005) Science 309:1380-4. Observe the CD4+Treg cell population of increase in the patient who suffers from the polytype cancer, wherein immunosupress can help the growth of immunity " escape " and these cancers of downward modulation. Therefore, estimate the Treg-mediation inhibition to be reversed in the treatment cancer be favourable.
PRN gets rid of the TLR7/8 motif especially. Have been found that the TLR7/8 motif can produce dominant result, it has shielded the immunostimulatory properties of the uniqueness of ORN of the present invention. The TLR7/8 motif comprises for example ribonucleotide acid sequence, such as 5 '-C/U-U-G/U-U-3 ', 5 '-R-U-R-G-Y-3 ', 5 '-G-U-U-G-B-3 ', 5 '-G-U-G-U-G/U-3 ' or 5 '-G/C-U-A/C-G-G-C-A-C-3 '. C/U is cytimidine (C) or uracil (U), and G/U is guanine (G) or U, and R is purine, and Y is pyrimidine, and B is U, G or C, and G/C is G or C, and A/C is adenine (A) or C. 5 '-C/U-U-G/U-U-3 ' can be CUGU, CUUU, UUGU or UUUU. In a plurality of embodiments, 5 '-R-U-R-G-Y-3 ' is GUAGU, GUAGC, GUGGU, GUGGC, AUAGU, AUAGC, AUGGU or AUGGC. In one embodiment, the base sequence is GUAGUGU. In a plurality of embodiments, 5 '-G-U-U-G-B-3 ' is GUUGU, GUUGG or GUUGC. In a plurality of embodiments, 5 '-G-U-G-U-G/U-3 ' is GUGUG or GUGUU. In one embodiment, the base sequence is GUGUUUAC. In a plurality of embodiments, 5 '-G/C-U-A/C-G-G-C-A-C-3 ' is GUAGGCAC, GUCGGCAC, CUAGGCAC or CUCGGCAC.
Generally speaking, the present invention relates to immunostimulatory oligoribonucleotides, it comprises one or more immune excitant RNA motifs, contains the immune irritating compositions of one or more immune excitant ORN of the present invention, and the using method of immune excitant ORN of the present invention and immune irritating compositions.
Term used herein " RNA " and " the natural RNA " of being connected are intended to represent by the covalently bound two or more ribonucleotides (be each molecule comprise with the phosphoric acid base be connected the ribose that pyrimidine nuclear base (for example guanine, adenine, cytimidine or uracil) is connected with purine) together of 3 '-5 ' phosphoric acid diester linkage.
The irritating RNA motif of immunity may reside in the end (when immune excitant ORN has free-end) of immune excitant ORN. For example, the immune excitant RNA motif that has the immune excitant ORN of free-end and be positioned at immune excitant ORN end can be used as XaM or as MXbExist, wherein M represents immune excitant RNA motif, XaAnd XbRepresent independently of one another the one or more identical or different nucleotides of immune excitant ORN except immune excitant RNA motif.
Perhaps, immune excitant RNA motif can be connected with in its both-side ends at least one extra nucleotides of immune excitant ORN, no matter should whether have free-end by immunity excitant ORN. For example, the immune excitant ORN that has the nucleotides of free-end and immune excitant RNA motif flank can be used as XaMX bExist, wherein M represents immune excitant RNA motif, XaAnd XbRepresent independently of one another the one or more identical or different nucleotides of immune excitant ORN except immune excitant RNA motif.
In different embodiments, the immune excitant ORN that comprises immune excitant RNA motif can contain single motif or more than one immune excitant RNA motif. It may be favourable having among the single immune excitant ORN that two or more immune excitant RNA motifs are considered to, if for example motif is spaced so that immune excitant ORN can be in conjunction with two or more TLR the time. For example, immune excitant ORN can be in conjunction with two or more TLR8 acceptors, thus amplification or modify the immune spread effect that obtains.
When immune excitant ORN comprised more than an immune excitant RNA motif, immune excitant ORN can be used as M in one embodiment1XM 2Exist, wherein M1And M2Represent independently of one another immune excitant RNA motif, X represents the one or more identical or different nucleotides of immune excitant ORN except immune excitant RNA motif. In one embodiment, X comprises non-nucleotide joint as herein described. In one embodiment, X comprises branch units as herein described.
When existing more than an immune excitant RNA motif among the immune excitant ORN, this motif generally can occur in any position along immune excitant ORN. For example, when having two motifs, they can be present in the end of immune excitant ORN separately. Perhaps, a motif may reside in an end, and a motif can be enclosed in its both-side ends by at least one outer nucleotides of immune excitant ORN. Also in another embodiment, each motif can be enclosed in its both-side ends by at least one extra nucleotides of immune excitant ORN.
Immunity excitant ORN includes but not limited to following, shows from left to right to read 5 ' to 3 ':
In some embodiments, ORN is one of active ORN of showing of following table 1 and table 2, for example below: U*U*A*G*G*C*A*C (SEQ ID NO:2), A*U*A*G*G*C*A*C (SEQ ID NO:4), G*C*C*A*C*C*G*A*G*C*C*G*A*A*U*A*U*A*C*C (SEQ ID NO:11), A*U*A*U*A*U*A*U*A*U*A*U*A*U*A*U*A*U*A*U (SEQ ID NO:1 2), U*U*A*U*U*A*U*U*A*U*U*A*U*U*A*U*U*A*U*U (SEQ ID NO:1 3), A*A*U*A*A*U*A*A*U*A*A*U*A*A*U*A*A*U*A*A (SEQ ID NO:1 6), A*A*A*U*A*A*A*U*A*A*A*U*A*A*A*U*A*A*A*U (SEQ ID NO:1 7), A*A*A*A*U*A*A*A*A*U*A*A*A*A*U*A*A*A*A*U (SEQ ID NO:1 8), C*U*A*C*U*A*C*U*A*C*U*A*C*U*A*C*U*A*C*U (SEQ ID NO:24), U*U*A*U*U*A*U (SEQ ID NO:30), U*A*U*A*U*A*U (SEQ ID NO:33), C*C*G*A*G*C*C*G*C*A*U*U*A*C*C*C (SEQ ID NO:48), C*C*G*A*G*C*C*G*A*U*U*G*A*A*C*C (SEQ ID NO:76), C*C*G*A*G*C*C*G*A*A*U*A*C*C*C*C (SEQ ID NO:42), C*C*G*A*G*C*C*A*U*A*U*A*U*A*U*C (SEQ ID NO:39), C*C*G*A*G*C*C*G*A*U*A*U*U*A*C*C (SEQ ID NO:65), C*C*G*A*G*C*C*G*A*A*U*C*C*C*C*C (SEQ ID NO:44), C*C*G*A*G*C*C*G*C*C*U*A*C*C*C*C (SEQ ID NO:47), C*C*G*A*G*C*C*A*U*A*U*A*U*C*C*C (SEQ ID NO:3 8), C*C*G*A*G*C*C*G*C*U*A*U*A*C*C*C (SEQ ID NO:37), C*C*G*A*G*C*C*G*A*A*U*A*A*C*C*C (SEQ ID NO:40), C*C*G*A*G*C*C*G*C*U*A*U*C*C*C*C (SEQ ID NO:55), C*C*G*A*G*C*C*G*A*A*G*G*U*A*C*C (SEQ ID NO:82), C*C*G*A*G*C*C*G*A*A*G*A*U*A*C*C (SEQ ID NO:85), C*C*G*A*G*C*C*G*A*A*U*G*U*A*C*C (SEQ ID NO:63), C*C*G*A*G*C*C*G*C*C*U*A*A*C*C*C (SEQ ID NO:43), C*C*G*A*G*C*C*G*C*A*U*A*U*C*C*C (SEQ ID NO:36), C*C*G*A*G*C*C*G*A*A*G*C*U*A*C*C (SEQ ID NO:87), C*C*G*A*G*C*C*G*C*A*U*A*C*C*C*C (SEQ ID NO:45), C*C*G*A*G*C*C*G*C*A*U*A*A*C*C*C (SEQ ID NO:41), C*C*G*A*G*C*C*G*A*A*G*G*U*G*C*C (SEQ ID NO:83), C*C*G*A*G*C*C*G*C*A*U*C*C*C*C*C (SEQ ID NO:46), C*C*G*A*G*C*C*G*A*A*G*C*U*G*C*C (SEQ ID NO:88), C*C*G*A*G*C*C*G*C*C*G*C*C*C*C*C (SEQ ID NO:35), C*C*G*A*G*C*C*G*A*A*G*C*U*C*C*C (SEQ ID NO:84) or C*C*G*A*G*C*C*G*A*A*G*G*C*A*C*C (SEQ ID NO:56).
As mentioned above, RNA is the polymer by the ribonucleotide of 3 '-5 ' phosphoric acid diester linkage connection. In certain embodiments, immune excitant ORN of the present invention is RNA. Yet immune excitant ORN of the present invention is not limited to RNA, as will be described below.
In one embodiment, immune excitant ORN of the present invention can comprise two or more modified nuclear base, i.e. derivatives of A, C, G and U. The particular of the nuclear base that these are modified includes but not limited to the cytimidine that 5-replaces (5-methyl-cytimidine for example, 5-fluoro-cytimidine, 5-chloro-cytimidine, 5-bromo-cytimidine, 5-iodo-cytimidine, 5-hydroxyl-cytimidine, 5-methylol-cytimidine, 5-difluoromethyl-cytimidine, that do not replace or replace 5-alkynes base-cytimidine), the cytimidine that 6-replaces, the cytimidine (for example N4-ethyl-cytimidine) that N4-replaces, the 5-aza-cytosine, 2-sulfydryl-cytimidine, iso-cytosine, false iso-cytosine, with the similar thing of the cytimidine of carbocyclic fused ring system (N for example, N '-propylene cytimidine Huo phenoxazine), with uracil and derivative thereof (5-fluoro-uracil for example, 5-bromo-uracil, 5-bromo vinyl-uracil, 4-sulphur generation-uracil, 5-hydroxyl-uracil, 5-propinyl-uracil), thymine derivative (2-thio-thymine for example, the 4-thio-thymine, the thymidine that 6-replaces), guanosine derivative (7-deazaguanine, the guanine (for example 7-denitrogenation-7-(C2-C6) alkynes base guanine) that 7-denitrogenation-7-replaces, the guanine that 7-denitrogenation-8-replaces, hypoxanthine, the guanine (for example N2-methyl-guanine) that N2-replaces, guanine (for example 8-hydroxyl guanine and 8-bromine are for guanine) and 6-thioguanine that 8-replaces) or adenosine derivative (5-amino-3-methyl-3H, 6H-thiazolyl [4,5-d] pyrimidine-2, the 7-diketone, 2,6-diaminopurine, 2-aminopurine, purine, indoles, adenine, the adenine that replaces (N6-methyl-adenine for example, 8-oxygen-adenine)). Base also can be by universal base (for example 4-methyl-indoles, 5-nitro-indoles, 3-nitro-pyrrole, P-base and K-base), aromatic ring (for example benzimidazole or two chloro-benzimidazoles, 1-methyl isophthalic acid H-[1,2,4] triazole-3-carboxylic acid amine), aromatic ring system (for example fluorobenzene or difluoro phenylbenzene) or hydrogen atom (dSpacer) replace. Preferred base modification is uracil and 7-denitrogenation-guanine. The U nuclear base that these are modified and the ribonucleic acid of their correspondences can get from commercial supplier.
The particular of modified G nuclear base comprises N2Guanine, 8-hydroxyl guanine, 6-thioguanine and 8-oxo guanine that the guanine that-dimethylguanine, 7-deazaguanine, guanozola, 7-denitrogenation-7-replace, 7-denitrogenation-7-(C2-C6) alkynes base guanine, 7-denitrogenation-8-replace. In one embodiment, modified G nuclear base is 8-hydroxyl guanine. These modified G nuclear bases and their corresponding ribonucleotide can derive from commercial supplier.
In certain embodiments, at least one β-ribose unit can be by β-D-deoxyribose or modified sugar unit displacement, wherein modified sugar unit for example is selected from β-D-ribose, α-D-ribose, β-L-ribose (in ' Spiegelmers '), α-L-ribose, 2 '-amino-2 '-deoxyribose, 2 '-fluoro-2 '-deoxyribose, 2 '-O-(C1-C6) alkyl ribose, preferred 2 '-O-(C1-C6) alkyl ribose is 2 '-the O-methylribose, 2 '-O-(C2-C6) alkene base ribose, 2 '-[O-(C1-C6) alkyl-O-(C1-C6) alkyl] ribose, LNA and α-LNA (Nielsen P et al. (2002) Chemistry-A European Journal 8:712-22), β-D-wood-furans sugar, α-arabinofuranose, 2 '-fluoro arabinofuranose and carbocyclic ring and/or ring-opened saccharides are like thing (for example be described in Vandendriessche et al. (1993) Tetrahedron 49:7223 in) and/or two ring sugar analogue (for example be described in Tarkov M et al. (1993) Helv Chim Acta 76:481 in).
Perhaps, each ribonucleotide of immune excitant ORN of the present invention is connected with ribonucleotide by joint connection, especially dealkalize base joint (abasic linkers (dSpacers)), three ethylene glycol unit or six vinyl ethylene glycol (hexaethylene glycol) unit of non-nucleic acid. Other joint is amino (alkylamino) joint of alkane, the amino joint of C3, C6 and C12 for example, and alkyl mercaptan (alkylthiol) joint, for example C3 or C6 mercaptan joint. Perhaps, the individual nucleotides of immune excitant ORN of the present invention is connected with ribonucleotide and is connected by aromatic residue, and described aromatic residue also can be replaced by the alkyl of alkyl or replacement.
RNA is the polymer that connects the ribonucleotide that links up by 3 '-5 ' phosphoric acid diester. The nucleotides of immune excitant ORN of the present invention also can link up by the connection of 3 '-5 ' phosphoric acid diester. Yet the present invention also comprises having the ORN that connects between unusual nucleotides, comprise specifically 5 '-5 ', 3 '-3 ', 2 '-2 ', 2 '-3 ' and 2 '-5 ' nucleotides between key. In one embodiment, the unusual connection of this class is excluded outside immune excitant RNA motif, although one or more this kind connection can be present in the other places of immune excitant ORN. For the immune excitant ORN with free-end, comprise between 3 '-a 3 ' nucleotides to connect and to cause immune excitant ORN to have two 5 ' free ends. On the contrary, for the immune excitant ORN with free-end, comprise between 5 '-a 5 ' nucleotides to connect and to cause immune irritating ORN to have two 3 ' free ends.
Immune irritating compositions of the present invention can contain two or more immune excitant RNA motifs, and it can connect by the unit of branch. Connect between nucleotides can be 3 '-5 ', 5 '-5 ', 3 '-3 ', 2 '-2 ', 2 '-3 ' or 2 '-5 ' connect. Wherein, title 2 '-5 ' is selected according to the carbon atom of ribose. Connecting between unusual nucleotides can be that the phosphoric acid diester connects, but it also can be modified to sulphur substituted phosphate or any other modified connection as herein described. Following formula shows the general structure of the immune excitant ORN of branch of the present invention by the nucleotides branch units. Nu wherein1、 Nu 2And Nu3Can be by 3 '-5 ', 5 '-5 ', 3 '-3 ', 2 '-2 ', 2 '-3 ' or 2 '-5 '-connect and connect. The branch of immunity excitant ORN also can relate to the use at non-nucleotide joint and dealkalize base interval. In one embodiment, Nu1、Nu 2And Nu3Represent identical or different immune excitant RNA motifs. In another embodiment, Nu1、Nu 2And Nu3Comprise at least one immune excitant RNA motif and at least one immune excitant CpG DNA motif.
Figure A20068004403000301
Immunity excitant ORN can contain unit double or three times (Glen Research, Sterling, VA), especially has the immune excitant ORN of 3 '-3 ' key. In one embodiment, double unit can be two with 1,3--[5-(4,4 '-dimethoxy three or four fourth phenolic aldehyde) the amyl group amide groups] propyl group-2-[(2-cyanogen ethyl)-(N, N-diisopropyl)]-Ya phosphorus acid amides be basic. In one embodiment, three times of unit can be with Tris-2,2,2-[3-(4,4 '-dimethoxy three or four fourth phenolic aldehyde) propyl group oxygen methyl] ethyl-[(2-cyanogen ethyl)-(N, N-diisopropyl)]-Ya phosphorus acid amides be integrated into the basis. Multiple double, three times or other many times of unit cause dendrimer to the branch of immune excitant ORN, and it is another embodiment of the present invention. The immune excitant ORN of branch can cause acceptor (such as TLR3, TLR7 and TLR8) crosslinked of immune excitant RNA, has from the immune excitant ORN of branch form not to compare different immunizations. In addition, branch or other the immune excitant ORN of many bodies (multimeric) synthetic can be stablized RNA and be not degraded, maybe can make weak or part effectively the RNA sequence demonstrate the useful immune activity level for the treatment of. Immunity excitant ORN also can contain connector unit, and it derives from peptide and modifies reagent or oligonucleotides-modified reagent (Glen Research). In addition, immune excitant ORN can contain by peptide (acid amides) and connect the one or more natural or non-natural amino acid residue that is connected with polymer.
3 '-5 ', 5 '-5 ', 3 '-3 ', 2 '-2 ', 2 '-3 ' and 2 '-5 ' nucleotides between connect can be direct or non-directly. In this context, directly connection refers to that phosphoric acid disclosed herein connects or modified phosphoric acid connects, and does not contain the insertion blank area. Inserting blank area is to connect from phosphoric acid disclosed herein or the modified different organic moiety of phosphoric acid connection, and it can comprise for example polyethylene glycol, three ethylene glycol, six ethylene glycol, dSpacer (being dealkalize base deoxidation nucleotides), two times of unit or three times of unit.
In certain embodiments, immune excitant ORN and another entity are puted together generation and are puted together thing. The combination of any two or a plurality of entities of puting together that thing refers to be bonded to each other by any physical chemistry approach (comprising hydrophobic interaction and covalent coupling) used herein.
In another embodiment, immune excitant ORN can with puted together by the small-molecular weight part of immune excitant Receptor recognition. This receptor is the member of TLR family preferably, for example TLR2, TLR3, TLR4, TLR7, TLR8 or TLR9. The small-molecular weight part is the mimicry for the native ligand of these acceptors. Example includes but not limited to: stimulate the two R-848 (Resiquimod), R-837 (Imiquimod of TLR7 and TLR8; ALDARATM, 3M Pharmaceuticals), 7-denitrogenation-guanosine, 7-sulphur generation-8-oxygen-guanosine and 7-pi-allyl-8-oxygen-guanosine (Loxoribine). D-glucopyranose derivatives such as 3D-MPL (TLR4 part) also can put together with immune excitant ORN. Pam3-Cys is an example of the TLR2 part that can put together with immune excitant ORN. The oligodeoxynucleotide that contains the CpG motif is the TLR9 part, and these also can be puted together with immune excitant ORN of the present invention. In one embodiment, at least one comprises stimulating transduce oligodeoxynucleotide and the immune excitant ORN of the present invention of effective CpG motif of TLR9 signal to put together. Enter the multimerization that can cause acceptor of puting together of a molecule for the part of different TLR, the immunity of comparing enhancing that this causes from any single this class part causes stimulates or different immune stimulation spectrums.
One aspect of the present invention provides the thing of puting together of immune excitant ORN of the present invention and lipophilic portion. In certain embodiments, immune excitant ORN and lipophilicity part are covalently bound. The lipophilicity part can be present on the one or more ends of the immune excitant ORN with free-end usually; although in certain embodiments; the lipophilicity part can exist in the other places of immune excitant ORN, thereby and does not require that immune excitant ORN has free-end. In one embodiment, immune excitant ORN has 3 ' end, and the lipophilicity part is terminal covalently bound with described 3 '. The lipophilicity group generally can be cholesteryl, modified cholesteryl, cholesterol derivative, the cholesterol that is reduced, substituted cholesterol, cholestane, C16 alkyl chain, bile acid, cholic acid, taurocholate, deoxidation cholic acid, oleoyl foundation stone cholic acid (oleyl litocholic acid), oleoyl cholenic acid, glycolipid, phosphatide, sheath fat, class isoprene such as steroids, vitamin such as vitamin E, saturated fatty acid, unrighted acid, fatty acid esters such as triglycerides, pyrene, laver alkali, Texaphyrine, adamantane amine, acridine, biological element, cumarin, fluorescein, Luo Danming, Texas-Red, digoxin (digoxygenin), two pairs of methoxy trityls, t-butyl dimethyl silane, t-butyl phenylbenzene monosilane, cyanine dye (for example Cy3 or Cy5), Hoechst 33258 dyestuffs, psoralen or brufens. In certain embodiments, lipophilicity partly is selected from cholesteryl, palm base and fatty acyl group. In one embodiment, lipophilicity partly is cholesteryl. Think and comprise that one or more these class lipophilicitys parts give again their extra stability for nuclease degradation among the immune excitant ORN of the present invention. When having two or more lipophilicity part among the single immune excitant ORN of the present invention, this class lipophilicity part can be selected independently of one another.
In one embodiment, lipophilicity partly is attached on the 2 ' position of nucleotides of immune excitant ORN. Lipophilicity group or or be connected with the heterocycle nuclear base of the nucleotides of immune excitant ORN extraly. The lipophilicity part can be covalently bound by any suitable direct or indirect connection and immune excitant ORN. In one embodiment, this connection is directly and is ester or acid amides. In one embodiment, this connection is indirectly and comprises the interval part that for example the nucleotide residue of one or more dealkalize bases, oligomeric ethylene glycol are such as three ethylene glycol (interval 9) or six ethylene glycol (interval 18) or alkanediol such as butanediol.
In one embodiment, immune excitant ORN of the present invention advantageously makes up with cation lipid or cationic peptide. Cation lipid and cationic peptide are considered to help immune excitant ORN is transported in chamber, endosome district, and TLR8 is present in wherein. In one embodiment, cation lipid be DOTAP (N-[1-(2,3-, two oleoyl oxygen) propyl group]-N, N, N-trimethyl ammonium methylsulfuric acid ester). DOTAP is considered to the transhipment of RNA oligomer be entered cell and be transported to specifically in the chamber, endosome district, and it can discharge the RNA oligomer with the pattern of pH dependence in the there. In the time of in chamber, endosome district, RNA can interact with TLR in some cell, causes the signal transduction pathway that the TLR mediation of generation is replied in the design immunity. Other reagent with similar characteristic (comprise and be transported to chamber, endosome district) can replace DOTAP or use except DOTAP. Other liquid formulations comprises for example EFFECTENETM(the non-liposomal lipid matter with specific DNA enhanced squeezing) and SUPERFECTTM(novel effect dendrimeric technology). The lipid body can commerce derive from Gibco BRL, for example LIPOFECTINTMAnd LIPOFECTACETM, they are by cation lipid such as N-[1-(2,3 two oleoyl oxygen)-propyl group]-N, N, N-trimethyl ammonium chloride (DOTMA) and dimethyl bromination octacosane ammonium (DDAB) form. Method for the manufacture of the lipid body is known in the art and is described in many publications. The lipid body is also summarized by Gregoriadis G (1985) Trends Biotechnol 3:235-241.
In one embodiment, immune excitant ORN of the present invention is the form with covalence closed, dumb-bell shape molecule of a level structure and secondary structure. As disclosed herein, this class ring-type oligoribonucleotide comprises two single-stranded loops that connect by the double-stranded section that inserts in one embodiment. In one embodiment, at least one single-stranded loop comprises immune excitant RNA motif of the present invention. Other molecule covalence closed, dumb-bell shape of the present invention comprises chimeric DNA:RNA molecule, and wherein for example at least part of DNA of being of double-stranded section (for example homologous dimerization body dsDNA or the dimer DNA:RNA of hospital) and the ring of at least one strand comprise immune excitant RNA motif of the present invention. Perhaps, the double-stranded section of chimeric molecule is RNA.
In certain embodiments, immune irritating ORN is separated. Separated molecule is substantially pure and molecule that do not contain other material, described other material usually is present in the nature with described molecule or in the body in the system, exists degree to reach can put into practice and the suitable degree of described molecule purpose purposes. ORN is especially enough pure and enough not celliferous other biological components for the immunity excitant, thereby is applicable to for example produce the medicine preparation. Because separated excitant ORN of the present invention can with can in the medicine preparation, mix by medicinal delivery body, so immune excitant ORN can only comprise by weight the in a small amount preparation of percentage. But immune excitant ORN is substantially pure, because the separating substances that described ORN can be combined with it in the life system substantially.
For the purposes among the present invention, immune excitant ORN of the present invention can with or adapt to any of a large amount of steps well known in the art and come from new synthetic. The inferior phosphorus acid amides method (Beaucage SL et al. (1981) Tetrahedron Lett 22:1859) of β cyanogen ethyl for example; Nucleosides H-phosphoric acid ester method (Garegg P et al. (1986) Tetrahedron Lett 27:4051-4; Froehler BC et al. (1986) Nucl Acid Res 14:5399-407; Garegg P et al. (1986) Tetrahedron Lett 27:4055-8; Gaffney BL et al. (1988) Tetrahedron Lett 29:2619-22). These chemistry can be undertaken by commercially available a large amount of automatic nucleic acid synthesizers. Other synthetic method that is suitable for according to the present invention is disclosed among Uhlmann E et al. (1990) Chem Rev 90:544-84 and Goodchild J (1990) the Bioconjugate Chem 1:165.
Can in solution or at solid support, carry out the synthetic of oligoribonucleotide. In solution, preferably block coupling reaction (dimer, tripolymer, the tetramer etc.), and the synthetic preferred monomer members piece that uses of solid phase carries out in the process of substep. (Eckstein F (1991) Oligonucleotides and Analogues is described, A Practical Approach, IRL Press, Oxford) different chemistry, for example phosphoric acid three ester methods, H-phosphoric acid ester method and inferior phosphorus acid amides method. Although in phosphoric acid three ester methods, reactive phosphorus group according to inferior phosphorus acid amides and H-phosphoric acid ester approach, uses in the coupling reaction to have more reactive Phosphor+III derivative in oxidation speed+V. In rear two kinds of approach, P (V) derivative that phosphorus oxidized generation behind coupling step is stable. If oxidant is iodine/water/base, then after protecting, remove-insurance obtains the phosphoric acid diester. On the contrary, if oxidant is vulcanizing agent such as Beaucage ' s reagent, then after protecting, remove-insurance obtains the sulphur substituted phosphate.
Being used for synthetic effective ways of oligoribonucleotide is combinations of using the solid support synthetic method of (described for oligodeoxynucleotide at first such as Matteucci and Caruthers) inferior phosphorus acid amides chemistry. Matteucci MD et al. (1981) J Am Chem Soc 103:3185.
To the synthetic of oligoribonucleotide be similarly to the synthetic of oligodeoxynucleotide, difference is that the 2 ' hydroxyl that exists in the oligoribonucleotide must be by suitable hydroxy-protective group protection. Monomer can be for example by 2 '-O-t-butyl dimetylsilyl (TBDMS) radical protection in the RNA monomer members piece. Yet, use to contain 2 '-O-three isopropyl monosilane yloxymethyl (TOM) group (TOM-Protecting-GroupTM) the synthetic RNA of monomer produced higher coupling by report and render a service because the TOM blocking group shows the steric hindrance lower than TBDMS group. Although use fluoride to remove the TBDMS blocking group, the quick remove-insurance of using methylamine in the ethanol/water at room temperature to reach the TOM group is protected. In oligomerization (ribose) nucleotides is synthetic, preferably extend from 3 '-5 ' terminal chain, this ribonucleotide acid unit by will having 3 '-phosphorus (III) group or 5 ' the oh group coupling that dissociates of its derivative that is activated and another nucleotide units are reached.
Synthesizing to use automation DNA/RNA synthesizer to carry out expediently. The synthetic circulation that wherein can use synthesizer supplier to recommend. For the ribonucleotide phosphoramidite monomer, coupling time is compared longer (for example 400 seconds) with the deoxidation nucleoside monomers. As solid support, can use 500 to 1 000
Figure A20068004403000351
Controlled cellular glass (CPG) support thing or organic Support Polymer, for example primer is supported thing PS200 (Amersham). Solid support contains first nucleosides by its 3 '-terminal combination usually, for example two pairs of methoxy trityls of 5 '-O--N-6-benzoyl adenosine. After two pairs of methoxy trityl group of three monoxones cutting 5 '-O-, the chain extension is reached in the use for example inferior phosphorus acid amides of-2 '-O-t-butyldimethylsilyl-nucleosides-3 ' of two pairs of methoxy trityl-N-protecteds of 5 '-O--O-. After the continuous repetitive cycling, by with concentrated ammonia liquor/ethanol (3: 1, v: v) 30 ℃ process 24 hours with the oligoribonucleotide finished from supporting thing cutting and remove-insurance to protect. Use at last triethylamine/HF that the TBDMS blocking groups is excised. Can analyze by the oligoribonucleotide of ion exchange column high pressure liquid chromatography (HPLC), Ion paired RP HPLC or polyacrylamide gel electrophoresis (PAGE) purification of crude and by the mass spectrum method.
5 ' puts together behind thing synthetic directly in solid phase is synthetic the inferior phosphorus acid amides of molecule to be connected and 5 ' hydroxyl coupling of terminal nucleotide. Can the commercial multiple inferior phosphorus amide derivatives that obtains this class part, for example cholesterol, acridine, biological element, general element draw human relations (psoralene), ethylene glycol or aminoalkyl residue. Perhaps, can introduce the aminoalkyl function between synthesis phase in solid phase, deriving after its permission is synthesized by the conjugate molecules (for example active ester, different thiocarbamide or iodo-acetyl amine) that activates.
It is synthetic usually by using corresponding adorned solid support to finish that 3 ' end is puted together thing, the solid support that described support thing for example can the commercial cholesteryl that obtains be derived. Yet, put together also and can between nucleotides, key, nuclear base or ribose residue (for example 2 ' of ribose-position) carry out.
For the ring-type oligoribonucleotide, the extension of oligonucleotide chain can be carried out at nucleotides PS solid support (Glen Research) by the inferior phosphorus acid amides of Application standard chemistry. Then use phosphoric acid three ester coupling steps (Alazzouzi et al. (1997) Nucleosides Nucleotides 16:1513-14) to carry out cyclization at solid support. When protecting with the final remove-insurance of ammonium hydroxide, the unique product that in fact enters in the solution is the ring-type oligonucleotides of expectation.
Ring-type oligoribonucleotide of the present invention comprises the RNA of closed loop, and can comprise the single stranded RNA that has or do not have double-stranded RNA.For example, in one embodiment, described ring-type oligoribonucleotide comprises double-stranded RNA and presents the dumbbell conformation with two single standard rings that described two rings connect by the double-stranded section that inserts.CpG oligodeoxynucleotide covalence closed, dumb-bell shape has been described in U.S.Pat.No.6, in 849,725.In another embodiment, the ring-type oligoribonucleotide comprises double-stranded RNA and presents following configuration that described configuration has the three or more single-stranded loop that connects by the double-stranded section that inserts.In one embodiment, immunostimulating RNA is arranged in one or more strand sections.
Immunostimulating ORN of the present invention is applicable to separately or with other reagent such as adjuvant and is used in combination.Adjuvant used herein is represented: strengthen the material of replying antigenic activated immune cell (for example body fluid and/or cellullar immunologic response) except that antigen.Adjuvant promotes the gathering and/or the activation of accessory cell, with the special immunne response of enhancement antigen.Adjuvant is used to strengthen the efficient of vaccine, and described vaccine promptly is used to induce the antigen composition that contains at antigenic protective immunity.
Adjuvant generally includes adjuvant, immunostimulation adjuvant of calling in storage effect and the adjuvant of creating storage effect and stimulating immune system.The adjuvant of establishment storage effect used herein is meant and causes that antibody slowly discharges in vivo, thereby prolongs the adjuvant of immunocyte to antigen-exposed.This adjuvant includes but not limited to alum (for example aluminium hydroxide, aluminum phosphate); The prescription of emulsion-based comprises mineral oil, non-mineral oil, water-in-oil or water-in-oil bag oil-emulsion, oil-in-water emulsion for example the Seppic ISA series of Montanide adjuvant (for example Montanide ISA 720; AirLiquide, Paris, France); MF-59 (with the stable water bag squalene emulsion of Span 85 and Tween 80, Chiron Corporation, Emeryville, Calif.); (contain and stablize washing composition and the oil-in-water emulsion that becomes the micelle agent with PROVAX; IDEC Pharmaceuticals Corporation, San Diego, Calif.).
The immunostimulation adjuvant is to cause immune system cell activatory adjuvant.It can for example cause the production of immunocyte and the secretion of cytokine.This class adjuvant includes but not limited to the saponin(e of purifying from Q.saponaria tree bark, as the QS21 (glycolipid of wash-out in the 21st peak of HPLC fraction; Aquila Biopharmaceuticals, Inc., Worcester, Mass.); Poly [two (carboxylic acid phenoxy group (carboxylatophenoxy)) phosphonitrile] (PCPP polymer; Virus Research Institute, the U.S.); Lipopolysaccharide derivant such as monophosphoryl lipid A (MPL; Ribi ImmunoChem Research, Inc., Hamilton, Mont.), Muramyl dipeptide (MDP; Ribi), andthreonyl-Muramyl dipeptide (t-MDP; Ribi); OM-174 (the glycosamine disaccharides relevant with lipid A; OM Pharma SA, Meyrin is Switzerland) with Leishmania elongation factor (purified Leishmania albumen; Corixa Corporation, Seattle, Wash.).The adjuvant of this kind also comprises CpGDNA.
The adjuvant of creating storage effect and stimulating immune system is the compound with above-mentioned two kinds of functions.This class adjuvant include but not limited to ISCOMS (immunostimulating complex, its contain blended saponin(e, lipid and with can keep the particle that antigenic hole forms the virus size; CSL, Melbourne, Australia); SB-AS2 (SmithKline Beecham adjuvant system #2, it is the oil-in-water emulsion that contains MPL and QS21: SmithKline Beecham Biologicals[SBB], Rixensart, Belgium); SB-AS4 (SmithKline Beecham adjuvant system #4, it contains alum and MPL; SBB, Belgium); Non-ionic block copolymer, (these contain the hydrophobicity polyoxypropylene linear chain that flank is a polyoxyethylene chain for its formation micelle such as CRL 1005; Vaxcel, Inc., Norcross, Ga.); (SAF contains the oil-in-water emulsion of Tween 80 and the segmented copolymer of non-example with the Syntex adjuvant prescription; Syntex Chemicals, Inc., Boulder, Colo.).
One aspect of the present invention provides the adjuvant that self comprises immunostimulating ORN of the present invention.In another embodiment, the invention provides the adjuvant (combination adjuvant) that comprises immunostimulating ORN of the present invention and at least a other adjuvant.Described other adjuvant can comprise the adjuvant of creating the storage effect, immunostimulating adjuvant, create adjuvant and any combination thereof of storage effect and stimulating immune system.In one embodiment, immunostimulating ORN of the present invention and at least a other adjuvant are covalently bound each other.Combination adjuvant according to the present invention is compared with the effect sum that immunostimulation ORN is independent and at least a other adjuvant is independent, can show collaborative immunostimulation.In addition or or, can show that according to combination adjuvant of the present invention independent with immunostimulating ORN or at least a other adjuvant compares separately, the immunostimulation spectrum of change.For example, combination adjuvant can provide the more Th1/Th2 immunostimulation of branch's form in one embodiment, or it can provide the Th1/Th2 immunostimulation of (skewed) form that more tilts in another embodiment.Those skilled in the art are understood that how to select individual components to promote the immunostimulation of desired type, for example about Th1 and branch or the more inclination more of Th2 feature.Th1 and Th2 further describe hereinafter.
A kind of composition also is provided, and described composition comprises immunostimulating ORN of the present invention and adds another adjuvant, and wherein said other adjuvant is a cytokine.In one embodiment, said composition is the conjugate of immunostimulating ORN of the present invention and cytokine.
Cytokine is by the inducing inflammatory reaction of many kinds of cells produce and immunoreactive soluble protein and glycoprotein.Interchange between the cytokine mediated immune system cell, function and the propagation in order to raise cell and to regulate them is made on local and general ground.The category of cytokine comprises the medium and the setter of the medium of congenital immunity and setter, adaptive immunity, and the stimulator of hemopoietic.Be included in the cytokine is interleukin (IL-1 for example, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18 and interleukin-11 9-32 (IL-19-IL-32), inter alia), chemokine (IP-10 for example, RANTES, MIP-1 α, MIP-1 β, MIP-3 α, MCP-1, MCP-2, MCP-3, MCP-4, the eotaxin, I-TAC and BCA-1, inter alia) and other cytokine, comprise 1 type Interferon, rabbit (for example IFN-α and IFN-β), 2 type Interferon, rabbit (for example IFN-γ), tumor necrosis factor-alpha (TNF-α), (TGF-β and multiple G CFS (CSF) comprise GM-CSF to transforming growth factor-beta, G-CSF and M-CSF.
Also provide and comprised the composition that immunostimulating ORN of the present invention adds immunostimulating CpG nucleic acid.In one embodiment, this is combined as the conjugate of immunostimulating ORN of the present invention and CpG nucleic acid, for example RNA:DNA conjugate.In one embodiment, said composition is the mixture of immunostimulating ORN of the present invention and CpG nucleic acid, promptly is not the RNA:DNA conjugate.
This paper uses immunostimulating CpG nucleic acid to be meant natural or the synthetic dna sequence dna, and it contains the activation or the propagation of CpG motif and stimulating immune system cell.Immunostimulating CpG nucleic acid is described in a large amount of patent, disclosed patent application and other publications of publishing, and comprises U.S.Pat.Nos.6,194,388; 6,207,646; 6,214,806; 6,218,371; 6,239,116 and 6,339,068.In one embodiment, immunostimulating CpG nucleic acid is the long CpG oligodeoxynucleotide (CpG ODN) of 6-100 Nucleotide.In one embodiment, immunostimulating CpG nucleic acid is the long CpG oligodeoxynucleotide (CpG ODN) of 8-40 Nucleotide.
Immunostimulating CpG nucleic acid comprises different types of CpG nucleic acid.A kind of can activate the B cell but when inducing IFN-α with the NK cell activation relative a little less than; This kind has been named as category-B.Typically, category-B CpG nucleic acid is by complete stability, and it has unmethylated CpG dinucleotides in some preferred base environment.Consult for example U.S.Pat.Nos.6,194,388; 6,207,646; 6,214,806; 6,218,371; 6,239,116 and 6,339,068.Another kind can be induced IFN-α and NK cell activation, but when stimulating the B cell relatively a little less than, this kind has been known as category-A.Category-A CpG nucleic acid typically has the sequence of phosphoric acid diester CpG dinucleotides of at least 6 Nucleotide of the palindrome and stable poly G sequence at 5 ' and 3 ' two ends.Consult for example disclosed International Patent Application WO 01/22990.Another Class Activation B cell of CpG nucleic acid and NK cell are also induced IFN-α; This kind has been known as the C class.As at first analyzing, C class CpG nucleic acid comprises category-B type sequence and the palindromic sequence or the approximate palindromic sequence that are rich in GC typically by complete stability.This kind has been described in the disclosed U.S. patent application 2003/0148976, and its full content is incorporated this paper by reference into.
Immunostimulating CpG nucleic acid comprises that also the full content of described application is incorporated this paper by reference into as disclosed so-called soft and medium-soft CpG nucleic acid in the disclosed U.S. patent application 2003/0148976.Soft and the medium-soft immunostimulating CpG nucleic acid of this class is integrated the combination of key between nuclease resistance and nuclease sensitivity Nucleotide, and wherein dissimilar keys is according to some principle location.
Also provide to comprise the composition that immunostimulating ORN of the present invention fastens another adjuvant, wherein said another adjuvant is lipopeptid such as Pam3Cys, cationic polysaccharide such as chitosan, or cationic peptide such as protamine.In one embodiment, composition is the conjugate of immunostimulating ORN of the present invention and another adjuvant.
One aspect of the present invention provides and has comprised immunostimulating ORN of the present invention and antigenic vaccine." antigen " used herein expression: can be by any molecule of T cell antigen receptor or B cell antigen receptor identification.This term comprises the molecule that is identified as any kind of external source by host immune system widely.Antigen generally comprises but is not limited to peptide and non-peptide mimicry, small molecules, lipid, glycolipid, polysaccharide, carbohydrate, virus and viral extract and multicellular organism such as the parasite and the allergen of cell, cell extract, protein, polypeptide, peptide, polysaccharide, polysaccharide conjugates, polysaccharide and other molecule.For for the antigen of protein, polypeptide or peptide, this class antigen can comprise the antigenic nucleic acid molecule of this class of coding.Antigen includes but not limited to more specifically: cancer antigen, and it comprises cancer cells and in cancer cells or the molecule of expressing on the cancer cells; Microbial antigen, it comprises microorganism and in microorganism or the molecule of expressing on the microorganism; And allergen.Therefore, the present invention provides in certain embodiments and has been used for cancer, infectious agent and allergenic vaccine.
One aspect of the present invention provides immunostimulating ORN of the present invention to be used to prepare the purposes of medicament, and described medicament is used for the vaccine inoculation experimenter.
One aspect of the present invention provides the method that is used to prepare vaccine.This method comprises the steps: immunostimulating ORN of the present invention placed with antigen and optional the direct of pharmaceutically acceptable vehicle and combines.
In multiple embodiments, antigen is microbial antigen, cancer antigen or allergen." microbial antigen " used herein is meant the antigen of microorganism, and it includes but not limited to virus, bacterium, parasite and fungi.This class antigen comprises complete microorganism and natural conivium or fragment or derivative thereof, also comprises same or similar and induce synthetic compound to the special immunne response of this microorganism with natural microbial antigen.If compound is induced the antigenic immunne response of natural microbial (body fluid and/or cell), then itself and natural microbial Antigens are seemingly.This class antigen is that this area routine is used and is that this area routine techniques personnel are known.
Virus is little infectious agent, and it generally contains nucleic acid core and protein enclosure, but the biology that can not live on one's own life.Virus also can take to lack the form of proteinic INA.Do not exist virus can duplicate therein life cell the time, virus can not exist.Virus enters special life cell and breeding by endocytosis or direct injection DNA (phage), causes disease.Fan Zhi virus can be released or infect other cell then.Some viruses are the virus that contains DNA, and other is the virus that contains RNA.In some respects, the present invention also is intended to treat disease, infectious month first protein (prion) is replicated in progression of disease in the described disease, described disease for example mad cow disease (is a mad cow disease, BSE) or the scrapie in the animal infect or the Creutzfe1dt-Jakob disease of philtrum.
Virus includes but not limited to: enterovirus (including but not limited to picornaviridae section, for example poliovirus, Coxsackie virus (coxsackie virus), ECHO virus (echo virus)), rotavirus, adenovirus, hepatitis virus.The special example of the virus of having found in the people includes but not limited to: (for example human immunodeficiency virus such as HIV-1 (are also referred to as HTLV-III, LAV or HTLV-III/LAV or HIV-III to Retroviridae; With other conivium, for example HIV-LP)); Picornaviridae (for example poliovirus, hepatitis A virus; Enterovirus, human coxsackievirus, rhinovirus, ECHO virus); Calciviridae (for example causing the bacterial strain of gastro-enteritis); Togaviridae (for example equine encephalitis virus, rubella virus); Flaviviridae (for example dengue fever virus, encephalitis, yellow fever virus); Coronaviridae (for example coronavirus); Rhabdoviridae (for example stomatitis herpesvirus, rabies virus); Filoviridae (for example Ebola virus); Paramyxoviridae (for example parainfluenza virus, mumps virus, Measles virus, respiratory syncytial virus); Orthomyxoviridae (for example influenza virus); Bunyaviridae (for example Hantaan virus (Hantaan viruses), bunya virus, Phlebovirus (phleboviruses) and Nairo virus); Arenaviridae (hemorrhagic fever virus); Reoviridae (for example reovirus, orbiviurses and rotavirus); Birnaviridae; Hepadnaviridae (hepatitis B virus); Parvoviridae (parvovirus group); Papovaviridae (Papillomavirus, polyomavirus); Adenoviridae (most of adenovirus); Herpesviridae (hsv (HSV) 1 and 2, varicella zoster virus, cytomegalovirus (CMV)); Poxviridae (alastrim virus, vaccinia virus, poxvirus); Iridoviridae (for example African swine fever virus); With non-classified virus (cause of disease of spongiform encephalopathy (etiological agent) for example, the cause of disease of hepatitis D (the defective satellite that is considered to hepatitis B virus), cause of disease (the kind 1=internal transmission of non-first type, non-hepatitis B; The transmission of kind 2=parenteral) (being hepatitis C); Norwalk and correlated virus and Astrovirus).
Bacterium is by the binary vegetative unicellular organism of fissioning.They are classified and name according to its morphology, staining reaction, nutrition and metabolism needs, antigenic structure, chemical constitution and genetic homogeny.Bacterium can be divided into three classes according to its morphological form: spherical (coccus), directly bar-shaped (bacillus) and bending or spiral bar-shaped (vibrios, Campylobacter, spirillum and spirochete).Bacterium also more generally is divided into two class biology, Gram-positive and Gram-negatives according to their staining reaction.Gram is meant the dyeing process that carries out usually in microbiology laboratory.The Gram-positive biology keeps dyeing and shows intense violet color behind staining procedure.But gram-negative biological does not keep dyeing absorbs redying, thus and demonstration pink colour.
Communicable bacterium includes but not limited to Gram-negative and gram positive bacterium.Gram positive bacterium includes but not limited to: the kind of the kind of Pasteurella, the kind of Staphylococci and Streptococcus.Gram negative bacterium includes but not limited to: the kind of Escherichia coli, Pseudomonas and the kind of Salmonella.The special example of infective bacterial includes but not limited to: Helicobacter pyloris, Borrelia burgdorferi, Legionella pneumophilia, Mycobacteria sps (M.tuberculosis for example, M.avium, M.intracellulare, M.kansasii, M.gordonae), Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria meningitidis, Listeria monocytogenes, Streptococcus pyogenes (A organizes Streptococcus), Streptococcus agalactiae (B organizes Streptococcus), Streptococcus (viridans group), Streptococcus faecalis, Streptococcus bovis, Streptococcus (anaerobism kind), Streptococcus pneumoniae, pathogenicCampylobacter sp., Enterococcus sp., Haemophilus influenzae, Bacillusanthracis, Corynebacterium diphtheriae, Corynebacterium sp., Erysipelothrix rhusiopathiae, Clostridiumperfringens, Clostridium tetani, Enterobacter aerogenes, Klebsiella pneumoniae, Pasturella multocida, Bacteroides sp., Fusobacterium nucleatum, Streptobacillus moniliformis, Treponema pallidum, Treponema pertenue, Leptospira, Rickettsia and Actinomyces israelli.
Parasite is to depend on other biology and the biology of survival, so it must enter or infect other biology to continue their life cycle.Infected biology (being the host) provides nutrition and habitat to parasite.Although this term parasite with its most widely implication can comprise all infectious agents (being bacterium, virus, fungi, protozoon and worm), but generally speaking, this term is used for representing separately protozoon, worm and ectoparasite arthropods (for example tick, mite etc.).Protozoon is single celled biology, but its cell is interior or duplicate in the extracellular, especially in the extracellular matrix of blood, enteron aisle or tissue.Worm is almost always at extracellular multicellular organism (except that Trichinella spp.).Worm need withdraw from and shift from elementary host usually and enter among the secondary host, thereby duplicates.Opposite with these above-mentioned kinds, ectoparasite arthropods forms the parasitism with host's health outside surface.
Parasite comprises the cytozoon of cytozoon and obligate.Parasitic example includes but not limited to Plasmodium falciparum, Plasmodium ovale, Plasmodium malariae, Plasmdodium vivax, Plasmodium knowlesi, Babesia microti, Babesia divergens, Trypanosoma cruzi, Toxoplasma gondii, Trichinellaspiralis, Leishmania major, Leishmania donovani, Leishmania braziliensis, Leishmania tropica, Trypanosoma gambiense, Trypanosoma rhodesiense and Schistosoma mansoni.
Fungi is an eukaryote, and only some causes infection in the vertebrates Mammals.Because fungi is an eukaryote, so they are significantly different on size, structure establishment, life cycle and reproduction mechanisms with the bacterium of protokaryon.Fungi is classified with its morphological feature, reproduction pattern and cultural characteristic usually.Although fungi can cause dissimilar diseases in the experimenter, the mycetism that for example suck respiratory allergies behind the fungal antigen, (for example Amanitaphalloides toxin of being produced by poisonous mushroom and phallotoxin and the aflatoxin produced by the aspergillar kind) causes because the picked-up toxicant, but not every fungi produces communicable diseases.
The infectivity fungi can cause infection general or shallow.Elementary systemic infection can take place in the normal healthy experimenter, and opportunistic infection the most often are present among the immunocompromised experimenter.The most common fungi material that causes elementary systemic infection comprises Blastomyces, Coccidioides and Histoplasma.The common fungi that causes opportunistic infection in immunocompromised or immunosuppressant experimenter includes but not limited to the kind of Candida albicans, Cryptococcusneoformans and multiple Aspergillus.Systemic fungal infection is internal's a invasive infection.The biological lung of process usually, gi tract or intravenous catheter enter health.The infection of these types can be caused by elementary pathogenic fungi or opportunistic fungi.
The fungi infestation of shallow relates to the externally surperficial growth of fungi and does not invade interior tissue.Typical shallow fungi infestation comprises the epidermis fungi infestation that relates to skin, hair or nail.
The disease relevant with fungi infestation comprises aspergillosis, blastomycosis, moniliosis, chromoblastomycosis, coccidioidomycosis, torulosis, the fungi eye infections, fungi hair, nail and skin infections, histoplasmosis, keloidal blastomycosis, mycetoma, otomycosis, paracoccidioidomycosis, the Penicillium marneffei of distribution, phaeohyphomycosis, rhinosporidiosis, sporotrichosis and zygomycosis.
The microorganism that other medical science is relevant is extensively described in the literature, for example consults C.G.AThomas, Medical Microbiology, and Bailliere Tindall, Great Britain 1983, its whole content is incorporated this paper by reference into.Each content of previous list all is illustrative but not is intended to restriction.
Term used herein " cancer antigen " and " tumour antigen " make convertibly and are used for representing compound, as peptide, protein or glycoprotein, it is relevant with tumour or cancer cells, and can excite immunne response during expression on the antigen presenting cell surface in main histocompatibility complex (MHC) branchs subenvironment.Cancer antigen by the cancer cells differential expression can be utilized with target cancer cell.Cancer antigen is the antigen that possible stimulate the tomour specific immunne response significantly.In these antigens some are encoded by normal cell, although must do not expressed by normal cell.These antigens can be characterized as being some stage antigen (for example embryonal antigen and fetal antigen) expression and transient expression usually reticent (promptly not expressing), that only breaking up in normal cell.Other cancer antigen is by the cytogene coding of sudden change, for example oncogene (for example activated ras oncogene), suppressor gene (for example Tu Bian p53), derive from inner the disappearance or the fusion rotein of chromosome translocation.Also have other cancer antigen to encode, for example the virogene that is loaded with on RNA and the DNA tumour virus by virogene.
Cancer antigen can be by preparation cancer cells crude extract (for example described in Cohen PA et al. (1994) the Cancer Res 54:1055-8), by partial purification antigen, by recombinant hormone or preparing from cancer cells from newly synthetic by known antigens.Cancer antigen includes but not limited to by recombinant expressed antigen, its immunogenicity part or its complete tumour or cancer or cell.This class antigen can separate or preparation by reorganization ground or by any other means known in the art.
The example of tumour antigen comprises MAGE, MART-1/Melan-A, gp100, DPP IV (DPPIV), adenosine deaminase binding protein (ADAbp), cyclophilin b, colorectum related antigen (CRC)--C017-1A/GA733, carcinomebryonic antigen (CEA) and immunogenicity epi-position CAP-1 and CAP-2, etv6, aml1, prostate specific antigen (PSA) and immunogenicity epi-position PSA-1 thereof, PSA-2 and PSA-3, prostatic specific membrane antigen (PSMA), T-cell receptors/CD3-ζ chain, the tumour antigen of MAGE-family (MAGE-A1 for example, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, MAGE-A12, MAGE-Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-B3), MAGE-Xp4 (MAGE-B4), MAGE-C1, MAGE-C2, MAGE-C3, MAGE-C4, MAGE-C5), the tumour antigen of GAGE-family (GAGE-1 for example, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE-8, GAGE-9), BAGE, RAGE, LAGE-1, NAG, GnT-V, MUM-1, CDK4, tyrosine oxidase, p53, MUC family, HER2/neu, p21ras, RCAS 1, α-peptide protein, the E-cadherin, α-catenin, white and the γ catenin of beta-catenin, p120ctn, gp100 Pmel117, tumour antigen, lmp-1, the P1A of PRAME, NY-ESO-1, cdc27, adenomatous polyposis coli albumen (APC), fodrin, connection protein 37, Ig-idiotype, p15, gp75, GM2 and GD2 ganglioside, viral product such as human papillomavirus albumen, Smad family, nuclear antigen (EBNA)-1, brain glycogen phosphorylase, SSX-1, SSX-2 (HOM-MEL-40), SSX-1, SSX-4, SSX-5, SCP-1 and the CT-7 of EBV-coding, and c-erbB-2.Restriction is not represented in this tabulation.
" allergen " used herein is the molecule that can excite immunne response, and described immunne response is characterised in that produces IgE.Allergen also is the material that can induce allergic response or asthma to reply in the experimenter of susceptible.Therefore, in the context of the present invention, the term allergen is represented the antigen of special type, and it causes by the antibody-mediated allergic response of IgE.
Allergenic tabulation is huge and can comprises pollen, insect venom, animal scurf dust, fungal spore and medicine (for example penicillin).The allergenic example of natural animal and plant comprises with the special protein of subordinate: Canis (Canis familiaris); Dermatophagoides (for example Dermatophagoides farinae); Felis (Felis domesticus); Ambrosia (Ambrosiaartemisiifolia); Lolium (for example Lolium perenne and Lolium multiflorum); Cryptomeria (Cryptomeria japonica); Alternaria (Alternaria alternata); Alder; Alnus (Alnus gultinosa); Betula (Betula verrucosa); Quercus (Quercus alba); Olea (Olea europa); Artemisia (Artemisia vulgaris); Plantago (for example Plantagolanceolata); Parietaria (for example Parietaria officinalis and Parietaria judaica); Blattella (for example Blattella germanica); Apis (for example Apis multiflorum); Cupressus (for example Cupressus sempervirens, Cupressus arizonica and Cupressus macrocarpa); Juniperus (for example Juniperus sabinoides, Juniperus virginiana, Juniperus communis and Juniperus ashei); Thuya (for example Thuya orientalis); Chamaecyparis (for example Chamaecyparis obtusa); Periplaneta (for example Periplaneta americana); Agropyron (for example Agropyron repens); Secale (for example Secale cereale); Triticum (for example Triticum aestivum); Dactylis (for example Dactylis glomerata); Festuca (for example Festuca elatior); Poa (for example Poapratensis and Poa compressa); Avena (for example Avena sativa); Holcus (for example Holcus lanatus); Anthoxanthum (for example Anthoxanthum odoratum); Arrhenatherum (for example Arrhenatherum elatius); Agrostis (for example Agrostisalba); Phleum (for example Phleum pratense); Phalaris (for example Phalaris arundinacea); Paspalum (for example Paspalum notatum); Sorghum (for example Sorghum halepensis) and Bromus (for example Bromus inermis).
One aspect of the present invention provides immunostimulating ORN of the present invention and antigenic conjugate.In one embodiment, immunostimulating ORN of the present invention and antigen are covalently bound.It can be that the covalency of any suitable species connects that covalency between immunostimulating ORN and the antigen connects, if immunostimulating ORN and antigen so in conjunction with the time keep measurable functionally active of setting up separately.In one embodiment, the covalency connection is direct.In another embodiment, it is indirect that covalency connects, and for example passes through shank.Covalently bound immunostimulating ORN and antigen can be processed to discharge each other in cell.It was sent and compares and can be enhanced when in this way, arbitrary component was delivered to cell and uses as independent preparation or independent component.In one embodiment, antigen is antigen itself, and promptly it is preformed antigen.
One aspect of the present invention provides a kind of pharmaceutical composition, and it comprises and delivery medium bonded composition of the present invention.In multiple embodiments, delivery medium can be selected from cation lipid, liposome, spirochete, virion, immunostimulating complex (ISCOM), particulate, microballoon, millimicro ball, unilamellar vesicle (LUV), multilamellar vesicle, oil-in-water emulsion, water-in-oil emulsion, newborn body and polycation peptide and optional pharmaceutically useful vehicle.Pharmaceutically useful vehicle is discussed hereinafter.Pharmaceutical composition of the present invention randomly can further comprise antigen.Composition of the present invention is with the antigen of existence, uses suitable method to be admitted in the physical bond with delivery medium.The immunostimulating composition can be included in the delivery medium, or on the surface that exposes of its solvent that may reside in delivery medium or combination with it.In one embodiment, immunostimulating ORN is present on the surface that the solvent of delivery medium exposes or combination with it, and if have antigenic words, it is included in the delivery medium.In another embodiment, immunostimulating ORN and antigen the two all be present on the solvent exposed surface of delivery medium or combination with it.Also in another embodiment, antigen is present on the solvent exposed surface of delivery medium or combination with it, and immunostimulating ORN is included in the delivery medium.Also in another embodiment, immunostimulating ORN and antigen the two (if comprising antigenic words) all are included in the delivery medium.
The present invention also provides the using method of immunostimulating composition of the present invention.One aspect of the present invention provides the method for immune cell activated.The method of this aspect comprises the steps: immunocyte is contacted in or the body external with the present composition of significant quantity according to the present invention, with immune cell activated.Composition of the present invention can randomly contain antigen." immunocyte " used herein is meant the cell of any bone marrow derived, and it participates in immunne response inborn or that adapt to.Immune cell includes, but are not limited to dendritic cells (DC), NK cell (NK) cell, monocyte, scavenger cell, granulocyte, bone-marrow-derived lymphocyte, plasmocyte, T lymphocyte and precursor cell thereof.
Term used herein " significant quantity " is meant and causes the necessary of the biological effect expected or amount fully.Significant quantity can still must not be subject to the amount of using in the single administration.
Term used herein " immune cell activated " is meant that inducing immune cells enters the active state relevant with immunne response.Term " immune cell activated " be meant induce and increase immunne response the two.This paper uses term " immunne response " to be meant that any aspect of inborn or the immunne response that adapts to, described immunne response have reflected immune cell propagation, carried out the activation that effector immunologic function or production relate to the gene product of immunne response.The gene product that relates to immunne response can comprise the interior and cell surface molecule (for example some cluster of differentiation (CD), transcription factor and genetic transcription thing) of distinctive cell of excretory product (for example antibody, cytokine and chemokine) and immunologic function.Term " immunne response " can be applied to individual cells or cell population.
Can assess the production of cytokine by any method in the Several Methods well known in the art, described method comprises that fluorescence activated cell sorting (FACS) is analyzed and ThermoScript II/polymerase chain reaction (RT-PCR) in biological respinse mensuration, enzyme-linked immunosorbent assay (ELISA), the cell.
In one embodiment, the generation of inflammatory cytokine immunne response before immunne response relates to.Preceding inflammatory cytokine immunne response can comprise expression any in some cytokine and the chemokine, comprises IFN-γ, TNF-α, IL-12, IL-10, IL-6 and any combination thereof.It gets rid of IFN-α with regard to purpose of the present invention specifically.
In one embodiment, immunne response relates to the rise of immunocyte activated cell surface marker, for example CD25, CD80, CD86 and CD154.The method that is used to measure the cell surface expression of this class mark is well known in the art and comprises facs analysis.
In order to measure the immunne response in cell or the cell population, in one embodiment, cell or cell population are expressed TLR8.Cell can be expressed TLR natively, maybe can it be operating as by the suitable expression vector of introducing TLR in cell and express TLR.In one embodiment, the cell of acquisition or cell population are peripheral blood lymphocytes (PBMC).In one embodiment, the cell of acquisition or cell population are for expressing the clone of TLR.In one embodiment, the cell of acquisition or cell population are for expressing the transient transfection body of TLR.In one embodiment, the cell of acquisition or cell population are the stable transfection bodies of expressing TLR.
Also in order to be used for measuring the immunne response of cell or cell population, introducing the sub-construct of report in cell or cell population can be easily, and described construct responds to the intracellular signal transduction that TLR causes.In one embodiment, report is the gene that is positioned under the NF-κ B promoter regulation here.In one embodiment, the gene that is positioned under the described promoter regulation is a luciferase.Under suitable activation condition, luciferase reports that sub-construct expressed and send detectable optical signal, and described signal can use luminometer to measure quantitatively.This class reports that sub-construct and the sub-construct of other suitable report can commercially obtain.
The present invention has also considered the purposes of the acellular method of monitoring TLR activated.
The present invention relates to composition and the method that is used for the treatment of purposes in some aspects.Immunostimulating composition of the present invention can use separately or be used in combination with other therapeutical agent.Immunostimulating composition and other therapeutical agent can while or continuous administration.When immunostimulating composition of the present invention and other therapeutical agent were used simultaneously, they can be identical or independently use in the prescription, but use simultaneously.In addition, when immunostimulating composition of the present invention and other therapeutical agent are used simultaneously, they can by identical or independently route of administration be applied, but use simultaneously.When using of immunostimulating composition of the present invention temporarily separated with using of another therapeutical agent, immunostimulating composition of the present invention and another therapeutical agent were by continuous administration.Temporal separation can be a big approximate number minute or can be more of a specified duration between these compound administration.In one embodiment, immunostimulating composition of the present invention was applied before using another therapeutical agent.In one embodiment, immunostimulating composition of the present invention is applied after using another therapeutical agent.In addition, when immunostimulating composition of the present invention and another therapeutical agent during by continuous administration, they can by identical or independently route of administration be applied.Other therapeutical agent includes but not limited to be applicable to adjuvant, antigen, vaccine and the medicine of treatment infection, cancer, transformation reactions and asthma.
An aspect of of the present present invention provides the method to experimenter's vaccine inoculation.The method of this aspect comprises the step to the experimenter's administration of antigens and the present composition according to the present invention.In one embodiment, administration of antigens comprises the nucleic acid of using coding for antigens.
" experimenter " used herein is meant vertebrates.In a plurality of embodiments, the experimenter is people, non-human primates or other Mammals.In certain embodiments, the experimenter is mouse, rat, guinea pig, rabbit, cat, dog, pig, sheep, goat, ox or horse.
In order to use in vaccine inoculation experimenter's method, composition of the present invention comprises antigen in one embodiment.Antigen can separate from ORN of the present invention or is covalently bound with it.In one embodiment, composition of the present invention self does not comprise antigen.In this embodiment, this antigen can be applied to the experimenter discretely or with composition of the present invention with composition of the present invention.Independently use independent, the site that comprises the time or the independence of route of administration, or time and site or route of administration are all independent.When composition of the present invention and antigen during in time by individual application, antigen can be applied before or after composition of the present invention.In one embodiment, antigen after composition of the present invention is used 48 hours to being applied in 4 weeks.This method also considered after initial application antigen and composition, and the antigen of one or more booster immunization dosage is used separately, composition is used separately or the using of antigen and composition.
The present invention also comprises: can prepare the experimenter who meets with unknown antigen in the future by the experimenter being used composition of the present invention, wherein said composition does not contain antigen.According to this embodiment, experimenter's immunity system is prepared as has stronger the replying of antigen that the experimenter is met with after a while, and described experience is for example by environment or occupational exposure.These class methods can be used for for example may being exposed to traveller, medical worker and the soldier of microbiological materials.
An aspect of of the present present invention provides treatment to suffer from the experimenter's of immune system defect method.The method of this aspect comprises composition of the present invention that the experimenter is used significant quantity step with the treatment experimenter according to the present invention." immune system defect " used herein is meant the immunity system of being prevented the unusually ability at antigen generation immunne response.In one embodiment, immune system defect is a kind of disease or illness, and wherein experimenter's immunity system is with normal ability effect, or the immunne response of wherein strengthening the experimenter can be applicable to tumour or cancer or the infection of for example eliminating among the experimenter." experimenter who suffers from immune deficiency " used herein is meant following experimenter, and wherein experimenter's immunity system is prevented at the ability of antigen generation immunne response.The experimenter who suffers from immune deficiency comprises experimenter with acquired immunodeficiency and the experimenter with innate immune system defective.The experimenter who suffers from acquired immunodeficiency includes but not limited to: suffer from the chronic inflammatory illness the experimenter, suffer from chronic renal insufficiency or renal failure the experimenter, suffer from infection the experimenter, suffer from cancer the experimenter, accept immunosuppressive drug the experimenter, accept the experimenter of other immunosuppressant therapy and suffer from underfed experimenter.In one embodiment, the experimenter has repressed CD4+T cell population.In one embodiment, the experimenter has infected human immunodeficiency virus (HIV) or has suffered from acquired immune deficiency syndrome (AIDS) (AIDS).Thereby the method for this aspect provides the method that is used for replying or strengthening taking place at experimenter's booster immunization of the stronger immunne response of needs the ability of immunne response according to the present invention.
The compositions and methods of the invention can use separately or be used in combination with being applicable to other reagent and method that treatment is infected.One aspect of the present invention provides treatment to suffer from the experimenter's of infection method.The method of this aspect comprises the present composition from significant quantity to the experimenter who suffers from infection that the use step with the treatment experimenter according to the present invention.
An aspect of of the present present invention provides treatment to suffer from the experimenter's of infection method.The method of this aspect comprises to the experimenter who suffers from infection and uses the present composition of significant quantity and the infection medicine step with the treatment experimenter according to the present invention.
An aspect of of the present present invention provides immunostimulating ORN of the present invention to be used to prepare the purposes of medicine, and described medicine is used for the treatment of the infection among the experimenter.
An aspect of of the present present invention provides the composition that is applicable to that treatment is infected.Composition according to this aspect comprises immunostimulating ORN of the present invention and infection medicine.
The term " treatment " that this paper uses about the experimenter's that suffers from disease or illness aspect should be illustrated among the experimenter and prevent, improves or eliminate a disease or at least one sign or the symptom of illness.
" experimenter who suffers from infection " is following experimenter, and described experimenter suffers from the illness that the invasion of the shallow table of experimenter, partial or general is caused by infectious microorganism.Infectious microorganism can be aforesaid virus, bacterium, fungi or parasite.
Infection medicine includes but not limited to: antiseptic-germicide, antiviral agent, anti-mycotic agent and antiparasitic.For example " anti-infection agent ", " microbiotic ", " antiseptic-germicide ", " antiviral agent ", " anti-mycotic agent ", " antiparasitic ", " parasiticide " have definite implication to this area routine techniques personnel and define in the medical article of standard.In brief, antiseptic-germicide kills or suppresses bacterium, and comprises that microbiotic and other have the synthetic or natural compound of similar functions.Antiviral agent can separate from natural origin or be synthetic, and is applicable to and kills or suppress virus.Anti-mycotic agent is used to treat the fungi infestation and the opportunistic and elementary systemic fungal infection of shallow table.Antiparasitic kills or suppresses parasite.A lot of microbiotic are low-molecular-weight molecules, and its stimulation meta-bolites as cell such as microorganism is produced.Microbiotic nonspecific infection microorganism special, be not present in one or more functions or structure in the host cell.
One of problem of anti-infective therapy is the side effect that takes place among the host when treating with anti-infection agent.For example, many anti-infection agents can kill or suppress the microorganism of wide spectrum, and not special to the species of particular type.Anti-infection agent treatment with these types causes killing the normal microflora and the infective micro-organisms of living among the host.The forfeiture of described flora can cause the disease complication and the host is easy to by other pathogenic infection, because the effect to the barrier of infectious agent is competed and played to described flora and infectious agent.Other side effect can be caused host's the non-microorganism cell or the specificity or the nonspecific action of tissue by these chemical entities.
Another problem that is extensive use of anti-infection agent is the development of the microorganism strains of antibiotics resistance.Develop the S.aureus of the enterococci of vancomycin resistance, the pneumococci of penicillin resistance, multiple resistance and the tuberculosis bacterial strain of multiple resistance, and become the main clinical problem.The widely-used bacterial isolates that may produce many antibiotics resistances of anti-infection agent.Therefore, need new anti-infective strategy to resist these microorganisms.
Effectively killing or suppress on a large scale, the antibiotic microbiotic of bacterium is meant Broad spectrum antibiotics.The antibiotic microbiotic of other type is mainly effective to the bacterium of Gram-positive or Gram-negative kind.The microbiotic of these types is known as narrow-spectrum antibiotic.Single biology or disease effectively and not are known as limit spectrum microbiotic (limited-spectrum antibiotics) at other microbiotic of the bacterium of other type.
Antiseptic-germicide is classified according to their main binding mode sometimes.Antiseptic-germicide generally is cell walls synthetic inhibitor, cytolemma inhibitor, protein synthesis inhibitor, nucleic acid is synthetic or depressant of functions and competitive inhibitor.The cell walls synthetic inhibitor suppresses a step in the cell walls building-up process, usually in bacterial peptide glycan synthetic.The cell walls synthetic inhibitor comprises beta-lactam antibiotics, natural penicillin, semisynthetic penicillin, Ampicillin Trihydrate, clavulanic acid, cephalosporins and bacitracin.
Beta-lactam is the microbiotic that contains the quaternary beta-lactam nucleus, its inhibiting peptide glycan synthetic final step.Beta-lactam antibiotics can be a synthetic or natural.The beta-lactam antibiotics of being produced by penicillium is natural penicillin, for example penicillin G or penicillin v.These fermentations by Penicillium chrysogenum produce.Natural penicillin has the activity of narrow spectrum, and effective at Streptococcus, Gonococcus and Staphylococcus usually.The gram positive bacterium also natural penicillin of effective other type is comprised penicillin-f, X, K and O.
The modification of the 6-amino-penicillanic acid molecule that semisynthetic penicillin is normally produced by mould.6-amino-penicillanic acid can be modified by adding side chain, and this has produced to compare with natural penicillin has the more penicillin of active or multiple other favorable characteristics of wide spectrum.The semisynthetic penicillin of some types has the wide spectrum at Gram-positive and gram negative bacterium, but by the penicillinase deactivation.These semisynthetic penicillin comprise Ampicillin Trihydrate, Gepcillin, Oxazacillin, azlocillin, mezlocillin and piperacillin.The semisynthetic penicillin of other type has the narrower activity at gram positive bacterium, thereby but has flourishing characteristic not by the penicillinase deactivation.These comprise for example X-1497, dicloxacillin and nafcillin.Some wide spectrum semisynthetic penicillins can for example clavulanic acid and Sulbactam (sulbactam) be used in combination with beta-lactamase inhibitor.Beta-lactamase inhibitor does not have anti-microbial effect, but they act on the inhibition penicillinase, thereby protects semisynthetic penicillin not to be degraded.
The beta-lactam antibiotics of another kind of type is a cephalosporins.They are to the degraded sensitivity of bacterium β-Nei Xiananmei, thereby and usually not separately not effectively.Yet cephalosporins is the penicillinase resistance.They are effective to multiple Gram-positive and gram negative bacterium.Cephalosporins includes, but are not limited to cefoxitin, Cephapirin, Cephalexin Monohydrate Micro/Compacted, Cefamandole, cefaclor, Cephazolin, cephalofruxin, cefoxitin, cefotaxime, cefsulodin, cefetamet, Cefixime Micronized, ceftriaxone, cefoperazone, ceftazime and latamoxef.
Bacitracin is the microbiotic of another kind, and to suppress cell walls from the release of following molecule synthetic by suppressing muramyl peptide subunit or peptidoglycan for it, and described molecule is delivered to subunit in the outside of film.Although bacitracin is effectively to gram positive bacterium, because its high toxicity, its purposes is limited in the topical application usually.
Carbapenems is another kind of wide spectrum beta-lactam antibiotics, and it is synthetic that it can suppress cell walls.The example of carbapenem includes, but are not limited to imipenum (imipenem).Monocycle beta-lactam also is the wide spectrum beta-lactam antibiotics, and it comprises excellent Tener (euztreonam).By a kind of antibiotic vancomycin that Streptomyces produces, also be synthetic effective to gram-positive microorganism by suppressing cytolemma.
Another kind of antiseptic-germicide is the antiseptic-germicide that belongs to the cytolemma inhibitor.These compounds destroy the structure of bacterial film or suppress its function.A problem that belongs to the antiseptic-germicide of cytolemma inhibitor is: because the similarity of phosphatide in bacterium and the eukaryote film, they can generation effect in eukaryotic cell and in the bacterium.Therefore, these compounds are seldom enough special allowing these compounds to be used by general ground, and the topical application of prevention high dosage.
A kind of clinical useful cytolemma inhibitor is polymyxin (Polymyxin).Polymyxin is by combining the inteferometer coating function with membrane phospholipid.It is effective that polymyxin is primarily aimed at gram negative bacterium, and be normally used for less toxicity antibiosis is have in the serious Pseudomonas infection or Pseudomonas infection of resistance.Use the relevant serious side effects of this compound with general and comprise damage kidney and other organ.
Other cytolemma inhibitor comprises amphotericin B (Amphotericin B) and nystatin (Nystatin), and it is the anti-mycotic agent that is mainly used in treatment systemic fungal infection and the Candida yeast infection.Imidazoles is the another kind of microbiotic that belongs to the cytolemma inhibitor.Imidazoles is used as antiseptic-germicide and anti-mycotic agent, for example is used for the treatment of yeast infection, dermatozoon infection and systemic fungal infection.Imidazoles includes but not limited to clotrimazole (clotrimazole), miconazole (miconazole), KETOKONAZOL (ketoconazole), itraconazole (itraconazole) and fluconazole (fluconazole).
Many antiseptic-germicides are protein synthesis inhibitors.These compounds stop bacterium composite structure protein and enzyme, thereby cause the inhibition or the necrocytosis of bacterial cell growth or function.Usually, these compounds disturb the process of transcribing or translating.The antiseptic-germicide that obstruction is transcribed includes but not limited to Rifampin (Rifampins) and Tibutol (Ethambutol).The Rifampin that suppresses RNA polymerase has broad spectrum of activity, and effective to Gram-positive and gram negative bacterium and Mycobacterium tuberculosis.Tibutol is effective to Mycobacterium tuberculosis.
The antiseptic-germicide that sealing is transcribed disturbs bacterial ribosome, is translated into protein to stop mRNA.Usually, the compound of this kind includes but not limited to tetracyclines, paraxin, Macrolide (for example erythromycin) and aminoglycoside (for example Streptomycin sulphate).
Aminoglycoside is a kind of microbiotic of being produced by bacterium Streptomyces, for example Streptomycin sulphate, kantlex, tobramycin, amikacin (amikacin) and gentamicin.Aminoglycoside has been used for infecting at the various bacteria that is caused by Gram-positive and gram negative bacterium.Streptomycin sulphate has been widely used as the main medicine of treatment tuberculosis (tuberculosis).Gentamicin is used to (particularly with the tobramycin combination) comprises Pseudomonas at multiple Gram-positive and gram negative bacterium infection.Kantlex is used to comprise the Staphylococci of penicillin resistance at many gram positive bacteriums.A side effect of restriction aminoglycoside clinical application is: under the essential dosage of effect, life-time service shows the damage renal function and causes the injury of acoustic nerve that causes deafness.
The translational inhibitor antiseptic-germicide of another kind of type is a tetracyclines.Tetracyclines is a kind of microbiotic, and it is wide spectrum and effective at multiple Gram-positive and gram negative bacterium.The example of tetracyclines comprises tsiklomitsin, Minocycline HCl, Vibravenos and duomycin.They are important for the bacterium of the many types of treatment, but are even more important in the treatment of Lyme disease (Lyme disease).Because their hypotoxicity and minimum direct side effect, tetracyclines is used by the medical circle excess and abuses, and causes a plurality of problems.For example, their excess is used the extensive generation that has caused resistance.
Antiseptic-germicide such as Macrolide combine with the 50S ribosomal subunit is reversible, and the extension by peptidyl transferase arrestin matter or stop uncharged tRNA to discharge from bacterial ribosome, or the two has concurrently.These compounds comprise erythromycin, Roxithromycin, clarithromycin, romicil and Azythromycin.Erythromycin is active to most gram positive bacteriums, Neisseria, Legionella and Haemophilus, is not active to Enterobacteriaceae still.Closed protein matter peptide bond forms between synthesis phase lincomycin and clindamycin are used at gram positive bacterium.
The translational inhibitor of another kind of type is a paraxin.Paraxin suppresses the bacterial enzyme peptidyl transferase in conjunction with 70 S rrna, thus the growth of peptide chain during the prevention protein synthesis.A severe side effect relevant with paraxin is aplastic anemia.In small portion (1/50,000) patient, aplastic anemia takes place under the paraxin dosage of effectively treating bacterium.Because the death that anaemia causes, the microbiotic paraxin of once being prescribed are in a large number seldom used now.It still is used for life-threatening situation (for example typhoid) because of validity.
Some antiseptic-germicides destroy the synthetic or function of nucleic acid, thus for example combine with DNA or RNA they signal can not they include but not limited to by reading: quinolones and trimethoprim-sulfamethoxazole (co-trimoxazole) (the two is the synthetic chemical) and rifomycin (natural or semisynthetic chemical).Quinolone is by suppressing the dna replication dna of DNA gyrase (gyrase) sealing bacterium, and bacterium needs described gyrase to produce their cyclic DNA.They are wide spectrums, and example comprises norfloxicin, Ciprofloxacin, enoxacin, Nalidixic Acid and temafloxacin.Nalidixic Acid is the sterilant in conjunction with DNA gyrase (topoisomerase), and it is crucial for duplicating of DNA, and allows superhelix to be released and formation again, suppresses DNA gyrase activity.The main application of Nalidixic Acid is treatment lower urinary tract infection (UTI), because it is to the common cause of UTI---and the kind of some kinds of Gram-negative bacterias such as E.coli, Enterobacter aerogenes, K.pneumoniae and Proteus is effective.Trimethoprim-sulfamethoxazole is the combination of Sulfamethoxazole (sulfamethoxazole) and trimethoprim (trimethoprim), and its sealing is made the bacterium of the required folic acid of DNA Nucleotide and synthesized.Rifampin is the derivative to gram positive bacterium (comprising Mycobacterium tuberculosis and the meningitis that is caused by Neisseria meningitidis) and the activated rifomycin of some gram negative bacteriums.Rifampin and the β subunit of polysaccharase combine and seal the interpolation of first Nucleotide (its be activate polysaccharase necessary), thereby sealing mRNA is synthetic.
Another kind of antiseptic-germicide is the compound that plays the competitive inhibitor effect of bacterial enzyme.Almost all similar with the bacterial growth factor structure and the competition of competitive inhibitor combines, but does not carry out metabolic function in cell.These compounds comprise the sulfanilamide (SN) of sulfamido and chemically modified form, and it has higher or anti-microbial activity widely.Sulfamido (for example Sulfafurazole and the pyridine of methylamine benzyl) is applicable to treatment Streptococcus pneumoniae, beta hemolytic streptococcus and E.coli, and has been used for the treatment of uncomplicated UTI that is caused by E.coli and the treatment that is used for meningococcal meningitis.
Antiviral agent is to prevent that cell is by virus infection or prevent the compound of virus replication in the cell.Existence is than antimicrobial drug antiviral drug still less, because the dna replication dna in the process of virus replication and the host cell is closely related, makes nonspecific antiviral agent poisonous to host cell usually.Have some stages in the process of virus infection, it can be sealed by antiviral agent or suppress.These stages comprise that virus combine the synthetic of (immunoglobulin (Ig) or binding peptide), uncoating (for example amantadine), virus mRNA or translates the maturation (for example proteinase inhibitor) of the duplicating of (for example Interferon, rabbit), viral RNA or DNA (for example nucleoside analog), new virus protein and viral sprout and discharge with host cell.
Another category of antiviral agent is a nucleoside analog.Nucleoside analog is and the synthetic compound of nucleoside analogues, still has incomplete or unusual ribodesose or ribose groups.In a single day nucleoside analog is in the cell just by phosphorylation, produces the triphosphate form, and it is integrated among viral DNA or the RNA with normal Nucleotide competition.In case the triphosphate form of nucleoside analog is integrated in the nucleic acid chains of growth, it causes the irreversible chain termination that combines and cause thus with varial polymerases.Nucleoside analog includes, but are not limited to acyclovir (being used for the treatment of hsv and varicella one varicella zoster virus), ganciclovir (gancyclovir) (being used for the treatment of cytomegalovirus), iodoxuridine, ribavirin (being used for the treatment of respiratory syncytial virus (respiratory syncitial virus)), dideoxyinosine, zalcitabine and zidovudine (Zidovodine).
Another kind of antiviral agent comprises cytokine, for example Interferon, rabbit.Interferon, rabbit is by the cell of virus infection and immunocyte excretory cytokine.The following effect of Interferon, rabbit: the special receptors bind with on the infected cell adjacent cells, cause the change in the cell, described change protection cell is avoided virus infection.α and interferon-are also induced the expression of I class and II class MHC molecule on the infected cells surface, and the host immune cell recognition appears being used in the antigen that causes increasing.α and interferon-can be used as recombinant form and obtain, and have been used to treat chronic hepatitis B and hepatitis C infection.Under antagonism viral therapy effective dosage, Interferon, rabbit has severe side effect and for example has a fever, do not accommodate and lose weight.
Immunoglobulin therapy is used to prophylaxis of viral infections.The immunoglobulin therapy that is used for virus infection be used for the different of infectation of bacteria because immunoglobulin therapy is by in conjunction with the extracellular virus body and stop their to attack and enter cell to the virus infection susceptible, rather than antigen-specific.This therapy is applicable to prophylaxis of viral infections in antibody is present in one period among the host.Two types immunoglobulin therapy is arranged usually, normal immunoglobulin therapy and hyperimmune globulin therapy.Normal immunoglobulin therapy utilizes a kind of antibody product, and described antibody product prepares from the serum of normal blood donor and merges.The product of this merging has the low antibody titer to large-scale Human virus, and described virus is hepatitis A, parvovirus, enterovirus (particularly in the newborn infant) for example.The antibody that the utilization of hyperimmune globulin therapy prepares from the serum of following individuality, described individuality have the high antibody titer to concrete virus.Use these antibody at special virus then.The example of hyperimmune globulin comprises Z/G (be applicable in immunocompromised children and newborn infant and prevent varicella), Human Rabies Immunoglobulin's (being applicable to by in the experimenter's of mad animal bite the post-exposure prophylaxis), hepatitis B immune globulin (being applicable to the prevention hepatitis B virus, particularly in being exposed to this viral experimenter) and RSV immunoglobulin (Ig) (being applicable to the treatment respiratory syncytial virus infection).
Anti-mycotic agent is applicable to treatment and preventing infection fungi.Anti-mycotic agent is classified by their mechanism of action sometimes.Some anti-mycotic agents are by suppressing glucosylceramide synthase as the effect of cell walls inhibitor.These include but not limited to basiungin/ECB.The film integrality unstability is fixed to be acted on other anti-mycotic agent by making.These comprise, but be not limited to imidazoles, for example clotrimazole, sertaconzole, fluconazole, itraconazole, KETOKONAZOL, miconazole and voriconazole, and FK 463, amphotericin B, BAY 38-9502/MK 991, pradimicin, UK 292, butenafine and Terbinafine.Other anti-mycotic agent is by degraded chitin (for example chitinase) or immunosuppression (501 breast frost) effect.
Parasiticide is directly to kill the parasite material.This compounds is known in the art and can commercially obtains usually.The example that is applicable to the parasiticide that the people uses includes but not limited to albendazole, amphotericin B, Rochagan, bithionol, chloroquine HCl, chloroquini phosphas, clindamycin, dehydroemetine, diethylcarbamazine, diloxanide (diloxanide furoate), eflornithine, Nifurazolidone, glucocorticosteroid, Halfan (halofantrine), Iodoquinol (iodoquinol), ivermectin (ivermectin), mebendazole (mebendazole), Mefloquine hydrochloride (mefloquine), meglumine antimonate (meglumine antimoniate), melarsoprol (melarsoprol), Metrifonate (metrifonate), metronidazole (metronidazole), niclosamide (niclosamide), nifurtimox (nifurtimox), oxamniquine (oxamniquine), paromycin (paromomycin), Pentamidine Isethionate (pentamidine isethionate), piperazine (piperazine), praziquantel (praziquantel), primaquine (primaquine phosphate), chloroguanide (proguanil), pyrantel (pyrantel pamoate), Pyrimethamine hcl-sulfamido (pyrimethanmine-sulfonamides), Pyrimethamine-Sulfadoxine (pyrimethanmine-sulfadoxine), hydrochloric acid quinacrine (quinacrineHCl), Quinine Sulphate Di HC, quinaglute (quinidine gluconate), Spiramycin Base, sodium stibogluconate (sodium stibogluconate sodium antimony gluconate), Suramine, tsiklomitsin, Vibravenos, Thiabendazole, tinidazole, trimethroprim-sulfamethoxazole and Trypothane.
ORN also is applicable to treatment and prevention autoimmune disease.Autoimmune disease is a class disease, wherein experimenter's self antibody and host tissue reaction, or wherein effector T lymphocyte and endogenous self peptide react automatically and cause disorganization.Therefore, caused immunne response at experimenter's self antigen (being called self antigen).Autoimmune disease includes but not limited to rheumatoid arthritis, Crohn ' s disease, multiple sclerosis, systemic lupus erythematous (SLE), the autoimmunization encephalomyelitis, myasthenia gravis (MG), Hashimoto ' s thyroiditis, Goodpasture ' s syndrome, pemphigus (for example pemphigus vulgaris), Grave ' s disease, autoimmune hemolytic anemia, autoimmune thrombocytopenic purpura, the scleroderma that has the anticol original antibody, mixed connective tissue disease, polymyositis, pernicious anemia, primary Addison ' s disease, the autoimmunization dependency is sterile, glomerulonephritis (crescentic glomerulonephritis for example, proliferative glomerulonephritis), bullous pemphigoid;
Figure A20068004403000581
Syndrome, insulin resistant and autoimmune diabetes.
This paper uses " self antigen " to be meant the antigen of normal host tissue.Normal host tissue does not comprise cancer cells.Therefore, in the context of autoimmune disease, the immunne response that causes at self antigen is the immunne response of not expecting, and helps to destroy and the damage healthy tissues, and be the immunne response of expectation, and help the destruction of tumour or cancer at the immunne response that cancer antigen causes.Therefore, the present invention is directed to the treatment autoimmune disorder some aspect, do not recommend ORN is used with self antigen, especially belong to those self antigen of the target of autoimmune disorder.
In other cases, ORN can be sent with the self antigen of low dosage.A large amount of zooscopies have proved that the antigenic mucosal administration of low dosage can cause the state of immune low reactivity or " tolerance ".Actual mechanism shows it is cytokine mediated immune deviation, described deviation from Th1 to the deviation of replying that is mainly Th2 and Th3 (being the TGF-b domination).Effective inhibition of low dosage antigen delivery also can suppress incoherent immunne response (onlooker's inhibition), and it is subjected to significant concern in autoimmune disease (for example rheumatoid arthritis and SLE) treatment.The onlooker suppress to relate to Th1-anti--regulate, the secretion of inhibitor cytokine in local environment, the cytokine and the Th1 cytokine of short inflammation are released in mode antigen-specific or antigen non-specific in the described local environment.This paper uses " tolerance " to be meant this phenomenon.In fact, be that effectively described disease comprises in the treatment of oral tolerance a large amount of autoimmune diseases in animal: experimental autoimmune encephalomyelitis (EAE), experimental autoimmunization myasthenia gravis, collagen-induced sacroiliitis (CIA) and insulin-dependent diabetes.In these models, the prevention of autoimmune disease is relevant with the migration that inhibition and the body fluid and the cell response of antigen-specific are replied from Th1 to Th2/Th3.
The compositions and methods of the invention can use separately or be used in combination with other reagent and the method that are applicable to the treatment cancer.One aspect of the present invention provides treatment to suffer from the experimenter's of cancer method.The method of this aspect comprises the present composition that the experimenter who suffers from cancer the is used significant quantity step with the treatment experimenter according to the present invention.
One aspect of the present invention provides treatment to suffer from the experimenter's of cancer method.The method of this aspect comprises the experimenter who suffers from cancer is used the composition of the present invention of significant quantity and the anti-cancer therapies step with the treatment experimenter according to the present invention.
One aspect of the present invention provides immunostimulating ORN of the present invention to be used to prepare the purposes of medicine, and described medicine is used for the treatment of the cancer among the experimenter.
One aspect of the present invention provides the composition that is applicable to the treatment cancer.Composition according to this aspect comprises immunostimulating ORN of the present invention and cancer drug.
The experimenter who suffers from cancer be have can detected cancer cells the experimenter.Cancer can be pernicious or nonmalignant cancer.This paper uses " cancer " to be meant uncontrolled cell growth, and it disturbs the normal function of body member and system.The cancer of moving and entering vitals from their original position can finally cause experimenter's death by the deterioration of affected organ.Hematopoiesis cancer for example leukemia can be defeated normal hematopoiesis compartment in the experimenter, thereby causes hematopoiesis failure (with the form of anaemia, thrombopenia and neutrophil leucocyte minimizing), finally causes death.
Metastasis is the cancer cells in a zone, and its position with primary tumor is different, derives from cancer cells is disseminated to health from initial tumour other parts.During the diagnosing primary tumor mass, can monitor the existence of experimenter's metastasis.Except that the special symptom of monitoring, metastasis is surveyed by being used alone or in combination nuclear magnetic resonance (MRI) scanning, computer tomography (CT) scanning, blood and platelet count, Liver Function, chest X-ray examination and bone scanning usually.
Cancer includes but not limited to: rodent cancer, cholangiocarcinoma; Bladder cancer; Osteocarcinoma; Brain and central nervous system (CNS) cancer; Mammary cancer; Cervical cancer; Choriocarcinoma; The colon and the rectum cancer; The reticular tissue cancer; Digestive system cancer; Carcinoma of endometrium; The esophageal carcinoma; Cancer eye; The H﹠N cancer; Last intracutaneous true tumor; Kidney; Laryngocarcinoma; Leukemia; Liver cancer; Lung cancer (for example minicell and nonsmall-cell lung cancer); Lymphoma comprises Hodgkin ' s and Non-Hodgkin ' s lymphoma; Melanoma; Myelomatosis; Neuroblastoma; Oral carcinoma (for example lip, tongue, mouth and pharynx cancer); Ovarian cancer; Carcinoma of the pancreas; Prostate cancer; Retinoblastoma; Rhabdosarcoma; The rectum cancer; The respiratory system cancer; Sarcoma; Skin carcinoma; Cancer of the stomach; Carcinoma of testis; Thyroid carcinoma; Uterus carcinoma; Urinary system cancer and other cancer (carcinomas), gland cancer and sarcoma (sarcomas).
Immunostimulating composition of the present invention can also be applied with anti-cancer therapies.Anti-cancer therapies comprises cancer medicament, radiation and operation technique." cancer medicament " used herein is meant and is used the reagent that is to treat cancer to experimenter, purpose." treatment cancer " used herein comprise preventing cancer generation, reduce the symptom of cancer, and/or suppress the growth of the cancer determined.In others, the cancer medicament is applied the experimenter of taking place under the risk of cancer to being in, and purpose is to reduce the risk that cancer takes place.Herein disclosed is the polytype medicament that is used for the treatment of cancer.With regard to the purpose of this specification sheets, the cancer medicament is classified as chemotherapeutic, immunotherapeutic agent, cancer vaccine, hormonotherapy and biological response modifier.
Chemotherapeutic can be selected from: Rheumatrex, vincristine(VCR), Zorubicin, cis-platinum, not sacchariferous chloroethylnitrosoureas, 5 FU 5 fluorouracil, ametycin, bleomycin, Dx, Dacarbazine, safe plain, fragyline, Meglamine GLA, valrubicin, carmustine and poliferposan, MMI270, BAY 12-9566, the RAS farnesyl transferase inhibitor, farnesyl transferase inhibitor, MMP, MTA/LY231514, LY264618/Lometexol, Glamolec, CI-994, TNP-470, with U.S. new/Hycamtin (Hycamtin/Topotecan), PKC412, valspodar (Valspodar)/PSC833, Novantrone (Novantrone)/Mitroxantrone, Metaret/ Suramine (Suramin), Batimastat (Batimastat), E7070, BCH-4556, CS-682,9-AC, AG3340, AG3433, Incel/VX-710, VX-853, ZD0101, ISI641, ODN 698, TA2516/Marmistat, BB2516/Marmistat, CDP 845, D2163, PD183805, DX8951f, Lemonal DP 2202, FK 317, molten chain bacterium (Picibanil)/OK-432, AD 32/ valrubicin (Valrubicin), strontium chloride (Metastron)/strontium derivative, Temodal/ Temozolomide (Temozolomide), Evacet (Evacet)/Mycocet, Yewtaxan/ taxol (Paclitaxel), safe plain (Taxol)/taxol (Paclitaxel), Xi Luoda (Xeload)/capecitabine (Capecitabine), Furtulon (Furtulon)/doxifluridine (Doxifluridine), the oral taxol of Cyclopax/, oral Taxan, the SPU-077/ cis-platinum, HMR 1275/Flavopiridol, CP-358 (774)/EGFR, CP-609 (754)/RAS oncogene inhibitor, the oral platinum of BMS-182751/, UFT (Tegafur/uridylic), Ergamisol/ LEVAMISOLE HCL (Levamisole), eniluracil (Eniluracil)/776C8 5/5FU enhanser, Campto/ LEVAMISOLE HCL (Levamisole), Camptosar/ irinotecan (Irinotecan), Tumodex/Ralitrexed, Leustatin/ CldAdo (Cladribine), the Paxex/ taxol, the Doxil/ Mycocet, the Caelyx/ Mycocet, Fuda China (Fludara)/fludarabine (Fludarabine), Pharmarubicin/ epirubicin (Epirubicin), DepoCyt, ZD1839, LU 79553/Bis-Naphtalimide, LU 103793/ dolastatin (Dolastain), the Caetyx/ Mycocet, strong (the Gemzar)/gemcitabine (Gemcitabine) of selecting, ZD 0473/Anormed, YM 116, iodine particle (Iodine seeds), CDK4 and CDK2 inhibitor, the PARP inhibitor, D4809/Dexifosamide, Ifes/Mesnex/ ifosfamide (Ifosamide), brave and fierce (Vumon)/teniposide (Teniposide), Paraplatin (Paraplatin)/carboplatin (Carboplatin), cis-platinum (Plantinol)/cis-platinum, etoposide (Vepeside)/Etoposide (Etoposide), ZD 9331, taxotere (Taxotere)/Docetaxel (Docetaxel), the prodrug of guanine cytosine arabinoside, 10-deacetyltaxol, nitrosourea, alkylating agent such as melphelan and endoxan, aminoglutethimide (Aminoglutethimide), asparaginase, busulfan (Busulfan), carboplatin, Chlorambucil (Chlorombucil), Spongocytidine-hydrochloride (Cytarabine HCl), gengshengmeisu (Dactinomycin), daunorubicin hydrochloride (DaunorubicinHCl), estramustine phosphate sodium (Estramustine phosphate sodium), Etoposide (VP16-213), fluorodeoxyuridine (Floxuridine), Fluracil (5-FU), flutamide, hydroxyurea (hydroxycarbamide), ifosfamide (Ifosfamide), Intederon Alpha-2a, α-2b, leuprorelin acetate (Leuprolide acetate) (LHRH-releasing factor analogs), lomustine (Lomustine) (CCNU), hydrochloric acid chlormethine (Mechlorethamine HCl) (mustargen), mercaptopurine (Mercaptopurine), mesna (Mesna), mitotane (Mitotane) (o.p '-DDD), mitoxantrone hydrochloride (Mitoxantrone HCl), Sostatin (Octreotide), Plicamycin (Plicamycin), procarbazine hydrochloride (Procarbazine HCl), streptozocin (Streptozocin), Tamoxifen Citrate (Tamoxifen citrate), thioguanine, plug is for sending (Thiotepa), vinealeucoblastine(VLB) (Vinblastinesulfate), amsacrine (Amsacrine) (m-AMSA), azacitidine (Azacitidine), Erthropoietin, altretamine (Hexamethylmelamine) (HMM), interleukin-(Interleukin) 2, mitoguazone (Mitoguazone) (methyl-GAG; Methyl-prop keto-aldehyde two-methyl GAG; MGBG), pentostatin (Pentostatin) (2 ' deoxycoformycin), semustine (Semustine) (Semustine), teniposide (Teniposide) (VM-26) and vindesine sulfate, but be not limited only to this.
Immunotherapeutic agent can be selected from 3622W94,4B5, ANA Ab, anti--FLK-2, anti-VEGF, ATRAGEN, AVASTIN (rhuMAb-VEGF (bevacizumab); Genentech), BABS, BEC2, BEXXAR (tositumomab (tositumomab); GlaxoSmithKline), C225, CAMPATH (alemtuzumab (alemtuzumab); Genzyme Corp.), CEACIDE, CMA 676, EMD-72000, ERBITUX (Cetuximab (cetuximab); ImClone Systems, Inc.), Gliomab-H, GNI-250, HERCEPTIN (trastuzumab (trastuzumab); Genentech), IDEC-Y2B8, ImmuRAIT-CEA, ior c5, ior egf.r3, ior t6, LDP-03, LymphoCide, MDX-11, MDX-22, MDX-210, MDX-220, MDX-260, MDX-447, MELIMMUNE-1, MELIMMUNE-2, Monopharm-C, NovoMAb-G2, Oncolym, OV103, Ovarex, Panorex, Pretarget, Quadramet, Ributaxin, RITUXAN (Mabthera (rituximab); Genentech), SMART 1D10 Ab, SMART ABL 364 Ab,, SMART M195, TNT and ZENAPAX (daclizumab; But be not limited only to this Roche).
Cancer vaccine can be selected from cancer vaccine, Gp75 antigen, GMK melanoma vaccine, MGV Sphingolipids,sialo conjugate vaccine, HeR2/neu, Ovarex, M-Vax, O-Vax, L-Vax, STn-KHL theratope, BLP25 (MUC-1), liposome idiotype vaccine, Melacine (Melacine), peptide antigen vaccine, toxin/antigen vaccine, the vaccine based on MVA, PACIS, BCG vacine, TA-HPV, TA-CIN, the DISC-virus andImmuCyst/TheraCys of EGF, anti-idiotype, but is not limited only to this.
The compositions and methods of the invention can use separately, or are used in combination with being applicable to allergic other reagent of treatment and method.One aspect of the present invention provides treatment to suffer from the experimenter's of allergic conditions method.The method of this aspect comprises that the composition of the present invention that the experimenter who suffers from allergic conditions is used significant quantity is with the treatment experimenter according to the present invention.
One aspect of the present invention provides treatment to suffer from the experimenter's of allergic conditions method.The method of this aspect comprises the experimenter who suffers from allergic conditions is used the composition of the present invention of significant quantity and anti-allergic therapy with the treatment experimenter according to the present invention.
One aspect of the present invention provides immunostimulating ORN of the present invention to be used to prepare the purposes of medicine, and described medicine is used for the treatment of the allergic conditions among the experimenter.
One aspect of the present invention provides the composition that is applicable to the treatment allergic conditions.Composition according to this aspect comprises immunostimulating ORN of the present invention and transformation reactions medicament.
" experimenter who suffers from allergic conditions " represents experiencing at present or before once living through the experimenter who responds to allergenic atopic reaction.
" allergic conditions " or " transformation reactions " is meant the acquired supersensitivity to material (allergen).Allergic conditions includes but not limited to eczema, rhinallergosis or rhinitis (coryza), spring fever (hay fever), allergic conjunctivitis, bronchial asthma, urticaria (hives) and food allergy, other atopy illness comprises atopic dermatitis; Allergy (anaphylaxis); Drug allergy and angioedema.
Typically, transformation reactions be with at the relevant of short duration illness of allergenic antibody producing, described antibody is from concrete immunoglobulin (Ig) kind IgE.By the generation that general source of the gas allergen is replied of IgE mediation also is the factor that indication trends towards taking place the inducement of asthma.If allergen meets with on basophilic leucocyte (circulating in blood) or mastocyte (the being scattered in solid tissue everywhere) surface and the special IgE of IgE Fc acceptor (FceR) bonded, then cell is activated, cause the production and the release of medium, described medium is histamine, serotonin and lipid medium for example.
When tissue sensitivity's immunoglobulin (Ig) of IgE type and the reaction of external source allergen, atopic reaction takes place.IgE antibody combines with mastocyte and/or basophilic leucocyte, and when being subjected to allergen with the terminal bridge joint of antibody molecule and stimulating, these specialized cells discharge the chemical mediator (vasoactive amines) of atopic reactions then.Histamine, platelet activation factor, arachidonic acid metabolite and serotonin are the known best media of philtrum atopic reaction.Histamine and other vasoactive amines are stored in mastocyte and the basophil usually.Mastocyte is dispersed in animal tissues everywhere, and basophilic leucocyte circulates in vascular system.These cells are made histamine and it are stored in the cell, take place unless relate to the incident of IgE bonded specialization sequence, cause its release.
The symptom of atopic reaction is according to the position in the health and difference, and described position is IgE and antigen reactive position.Take place if be reflected on the respiratory epithelium, then symptom normally sneeze, cough and asthma reaction.Occur in (for example under the situation of food allergy) in the digestive tube, then common stomachache and diarrhoea if interact.The atopic reaction of general (after for example using penicillin after honeybee bites or to the experimenter) can be serious, and usually is life-threatening.
Transformation reactions is relevant with Th2 type immunne response, and it is converted to IgE to small part by Th2 cytokine IL-4 and IL-5 and antibody isotype and characterizes.Th1 and Th2 immunne response are mutual re, thereby the immunne response of Th2 type can be prevented or improve to immunne response towards the skew of Th1 type immunne response, comprises transformation reactions.Therefore, immunostimulating ORN of the present invention self is applicable to and treats the experimenter who suffers from allergic conditions, because immunostimulating ORN can make the immunne response of immunne response deflection Th1 type.Perhaps or in addition, immunostimulating ORN of the present invention can be used in combination the experimenter who suffers from allergic conditions with treatment with allergen.
Immunostimulating composition of the present invention also can with anti-allergic therapy combined administration.Be used for the treatment of or prevent allergic ordinary method to relate to the use of transformation reactions medicament or hyposensitization therapy.Be used for the treatment of or prevent during therapy comprises in more allergic development and the use of anti-IgE antibodies.The seriousness of allergic symptoms is regulated in the antihistamine of the chemical mediator of sealing atopic reaction and other medicines help, but can not react by prevention of allergic, and allergic response is not subsequently acted on.Preferred hyposensitization therapy, described hyposensitization therapy was induced originally at allergenic IgG type and was replied by giving (usually by subcutaneous injection) low dose of allergic effect.Think that the existence of IgG helps the production of neutralization medium, the production of described medium derives from inducing of IgE antibody.The initial usefulness very allergen of low dosage is treated the experimenter to avoid inducing serious reaction, and dosage is slowly increased.The therapy of the type is dangerous, because in fact the experimenter has been applied the compound that causes allergic response and can have caused serious atopic reaction.
The transformation reactions medicament includes but not limited to, antihistamine, cortin and prostaglandin(PG) inductor.Antihistamine is the compound of offsetting the histamine of mastocyte or basophil release.These compounds are well known in the art, and are generally used for treating transformation reactions.Antihistamine includes, but are not limited to acrivastine (acrivastine), astemizole (astemizole), azatadine (azatadine), azelastine (azelastine), betatastine, Parabromdylamine (brompheniramine), buclizine (buclizine), cetirizine (cetirizine), the cetirizine analogue, chlorphenamine (chlorpheniramine), clemastine (clemastine), CS 560, Cyproheptadine (cyproheptadine), Desloratadine (desloratadine), dexchlorpheniramine (dexchlorpheniramine), ebastine (ebastine), epinastine (epinastine), fexofenadine (fexofenadine), HSR 609, hydroxyzine (hydroxyzine), levocabastine (levocabastine), loratidine, epoxytropine tropate (methscopolamine), mizolastine (mizolastine), promise astemizole (norastemizole), phenindamine (phenindamine), promethazine (promethazine), Pyrilamine (pyrilamine), terfenadine (terfenadine) and tranilast (tranilast).
Cortin includes but not limited to: methylprednisolone (methylprednisolone), prednisolone (prednisolone), prednisone (prednisone), beclometasone (beclomethasone), budesonide (budesonide), dexamethasone (dexamethasone), flunisolide (flunisolide), fluticasone propionate (fluticasone propionate) and triamcinolone (triamcinolone).Although dexamethasone is the reflunomide with anti-inflammatory action, it is not used for the treatment of transformation reactions or asthma with the form that sucks usually, because it has secular inhibition side effect by high absorption and under effective dose.Yet dexamethasone can be used to treat transformation reactions or asthma according to the present invention, because when using with combination of compositions of the present invention, thereby it can use with low dosage and reduces side effect.Use some relevant side effects to comprise cough, mogiarthria, white mouth (moniliosis) and the systemic effect when the high dosage more, for example suprarenal gland inhibition, glucose intolerance, osteoporosis, aseptic necrosis of bone, cataract generation, growth-inhibiting, hypertension, myasthenia, thinning of skin (skin thinning) and easy the scratch with reflunomide.Barnes﹠amp; Peterson (1993) Am Rev Respir Dis148:S1-S26 and Kamada AK et al. (1996) Am J Respir Crit Care Med 153:1739-48.
The compositions and methods of the invention can use separately or be used in combination with other reagent and the method that are applicable to treatment asthma.One aspect of the present invention provides treatment to suffer from the experimenter's of asthma method.The method of this aspect comprises the present composition that the experimenter who suffers from asthma the is used significant quantity step with the treatment experimenter according to the present invention.
One aspect of the present invention provides treatment to suffer from the experimenter's of asthma method.The method of this aspect comprises the experimenter who suffers from asthma is used the composition of the present invention of significant quantity and the anti-asthma therapy step with the treatment experimenter according to the present invention.
One aspect of the present invention provides immunostimulating ORN of the present invention to be used to prepare the purposes of medicine, and described medicine is used for the treatment of the asthma among the experimenter.
One aspect of the present invention provides the composition that is applicable to treatment asthma.Composition according to this aspect comprises immunostimulating ORN of the present invention and asthma medicament.
" asthma " used herein is meant the illness of respiratory system, it is characterized in that the inflammation of air flue and narrow, and air flue is to the reactivity of the raising of inhalation.Asthma (but not exclusively) usually combines with atopy or allergic conditions.The symptom of asthma comprise by air-flow hinder stridulating of causing, asthma, chest is urgent and the periodical attack of cough.The airway inflammation relevant with asthma can by observe a large amount of physiology change monitored, for example the degrading of airway epithelia, basilar membrane collagen deposition, oedema, mastocyte activation, inflammatory cell (comprising neutrophil leucocyte, eosinophilic granulocyte and lymphocyte) infiltration down.As the result of airway inflammation, it is chronic that asthmatic patient often experiences airway hyperreactivity, airway limitation, respiratory symptom and disease.Airway limitation comprises that polarity bronchoconstriction, air flue oedema, cervical plug form and airway remodeling, and these features often cause bronchial obstruction.Half basilar membrane fibrosis (sub-basement membrane fibrosis) can take place under the certain situation of asthma, causes the pulmonary function permanent anomaly.
The asthma that studies show that in the past few years may be to be interacted by the complexity between other cell of inherent in inflammatory cell, medium and the air flue and tissue to cause.Mastocyte, eosinophilic granulocyte, epithelial cell, scavenger cell and activated T cells all play important effect in the inflammatory process relevant with asthma.Djukanovic R et al.(1990)Am Rev Respir Dis 142：434-457。Think these cells can be by secretion preformed and synthetic medium influence air flue function recently, described medium can act on local organization directly or indirectly.Thought that T lymphocyte (Th2) subgroup is by discharging cytokine optionally and set up that disease is chronic to play an important role in the alterative inflammation of regulating air flue.Robinson DS et al.(1992)N Engl J Med 326：298-304。
Asthma is a kind of illness of complexity, and it takes place in the different steps of growing, and can be classified as acute, subacute or chronic according to the degree of symptom.It is relevant that acute inflammatory response and cell enter raising in early days of air flue.Subacute inflammatory response relates to the activation of raising of cell and intrinsic cell, and this causes more persistent inflammation pattern.Chronic inflammatory diseases is replied cell injury and the ongoing repair process that is characterised in that lasting level, and it can cause the permanent anomaly in the air flue.
" experimenter who suffers from asthma " is meant that the experimenter who suffers from respiratory disorder, described illness are characterised in that the inflammation of air flue and narrow, and air flue is to the reactivity of the raising of inhalation.The factor relevant with asthma attack includes, but are not limited to allergen, cold temperature, motion, virus infection and SO 2
As mentioned above, asthma may be relevant with Th2 type immunne response, and it is converted to IgE to small part by Th2 cytokine IL-4 and IL-5 and antibody isotype and characterizes.Th1 and Th2 immunne response are mutual re, thereby the immunne response of Th2 type can be prevented or improve to immunne response towards the skew of Th1 type immunne response, comprises transformation reactions.Therefore, modified oligoribonucleotide analogue of the present invention is applicable to self treats the experimenter who suffers from asthma, because described analogue can make the immunne response of immunne response deflection Th1 type.Perhaps or in addition, modified oligoribonucleotide analogue of the present invention can be used in combination the experimenter who suffers from asthma with treatment with allergen.
Immunostimulating composition of the present invention also can with the asthma therapies combined administration.Be used for the treatment of or the ordinary method of prevention of asthma relates to the use of anti-allergic therapy (as mentioned above) and a large amount of other reagent (comprising inhalation).
The medicine that is used for the treatment of asthma is divided into two classes usually, rapid delivery of pharmaceuticals and long-term control medicine.Asthmatic patient adopts the long-term control medicine to reach and to keep the control of long-term asthma on the basis of every day.The long-term control medicine comprises anti-inflammatory agent, for example cortin, Gerhard Cromme (chromolyn) sodium and nedocromil (nedocromil); Long-acting bronchodilator, for example long-acting beta 2-agonist and methyl xanthine; With the leukotrienes modifier.Rapid delivery of pharmaceuticals comprises fugitive β 2-agonist, anticholinergic agents and general cortin.Exist with these medicines in every kind of relevant many side effects, do not have a kind of medicine or its combination can prevent or treat fully asthma.
Asthmatic medicament includes, but are not limited to the inhibitor and the proteinase inhibitor of PDE-4 inhibitor, bronchodilator/β-2 agonist, K+ channel opener, VLA-4 antagonist, neurokinin (neurokin) antagonist, thromboxane (thromboxane) A2 (TXA2) synthetic inhibitor, xanthine, arachidonic acid antagonist, 5 fats oxidn enzyme inhibitorss, TXA2 receptor antagonist, TXA2 antagonist, 5-lipox activation of protein.
Bronchodilator β 2-agonist is a compounds that causes bronchiectasis or smooth muscle loosening.Bronchodilator/β 2-agonist includes, but are not limited to Salmeterol (salmeterol), salbutamol (salbutamol), salbutamol (albuterol), terbutaline (terbutaline), D2522/ formoterol (formoterol), Partusisten (fenoterol), bitolterol (bitolterol), pirbuterol methyl xanthine (pirbuerol methylxanthines) and Orciprenaline (orciprenaline).Long-acting beta 2Agonist and bronchodilator are the compounds that is used for preventing for a long time symptom except that anti-inflammatory therapy.Long-acting beta 2Agonist includes, but are not limited to Salmeterol and salbutamol.These compounds usually and cortin be used in combination, and without any the inflammation therapy time, do not use.The side effect that with in the overdose for example tachycardia, skeletal muscle tremble for they, hypokalemia and QTc prolong at interval is relevant.
Methyl xanthine (comprising for example theophylline) has been used to long-term control and prevention symptom.These compounds cause the bronchiectasis by phosphodiesterase suppresses and possible adenosine antagonism causes.The acute toxicity that dosage is relevant is the particular problem of these type compounds.Therefore, thus must monitor conventional serum-concentration and calculate toxicity and limit the therapeutic scope (therapeutic range) that the metabolic clearance rate individual difference causes.Side effect comprises tachycardia, the rhythm of the heart market of overrunning, nausea and vomiting, central nervous system excitement, headache, epileptic seizures, spitting blood, hyperglycemia and hypokalemia.Fugitive β 2Agonist includes, but are not limited to salbutamol (albuterol), bitolterol (bitolterol), pirbuterol (pirbuterol) and terbutaline (terbutaline).With fugitive β 2Agonist uses that some relevant undesirable actions comprise that tachycardia, skeletal muscle tremble, hypokalemia, lactic acid increase, headache and hyperglycemia.
Gerhard Cromme (chromolyn) sodium and nedocromil are used as the long-term control medicine, and described medicine is used to prevent by kinetic primary symptoms of asthma or the allergic symptoms that caused by allergen.These compounds are considered to by disturbing muriate approach function to seal allergenic early stage and late phase response.They are also stablized mast cell membrane and suppress the activation and the release from inosineophils and epithelial cell thereof of medium.Usually need using of four to six weeks to reach maximum benefit.
Anticholinergics is normally used for alleviating the acute bronchus spasm.These compounds are considered to play a role by the competitive inhibition mAChR.Anticholinergics includes, but are not limited to ipratropium bromide.These compounds only can reverse the bronchospasm of cholinomimetic mode (cholinerigically) mediation, do not change antigenic any reaction.Side effect comprise dry and breathe secretion (respiratory secretions) if, in some individualities, increase stridulate and the visual field when spraying into eyes fuzzy.
Immunostimulating ORN of the present invention is also applicable to the treatment airway remodeling.Airway remodeling is thickened by smooth muscle cell proliferation in the air flue and/or submucosa and causes, and finally causes airway constriction, causes flow limitation.Immunostimulating ORN of the present invention can prevent further reconstruction, and even may reduce the tissue construction that process of reconstruction causes.
Immunostimulating ORN of the present invention also is applicable to survival, differentiation, activation and the maturation that promotes dendritic cells.The oligoribonucleotide of immunostimulating has cell survival, differentiation, activation and the sophisticated unique ability that promotes dendritic cells.
Immunostimulating ORN of the present invention also improves the cytotoxicity (ADCC) of natural killer cell lytic activity and antibody dependent.ADCC can be undertaken by being used in combination immunostimulating ORN with antibody, and described antibody pair cell target such as cancer cells are special.When immunostimulating ORN and antibody combined administration were given the experimenter, experimenter's immunity system was induced the kill tumor cell.Be applicable to that the antibody in the ADCC process comprises and the interactional in vivo antibody of cell.Special many these antibody-likes of pair cell target are described in this area, and many can commercially the acquisition.In one embodiment, described antibody is IgG antibody.
In some aspects, the invention provides the method that is used to strengthen the epi-position expansion.This paper uses " epi-position expansion " to be meant that epitope specificity is from the various accessory and/or hiding epi-position that turns on described protein (intramolecularly expansion) or other protein (intermolecular expansion) of initially concentrated, main epitope specificity immunne response (point to self or exogenous protein).The epi-position expansion causes the immunne response that multi-epitope is special.
Immunne response was made up of initial amplification phase (magnification phase) and downward modulation phase afterwards, the described amplification phase can be deleterious (as in autoimmune disease) or useful (as in vaccine inoculation), and the described downward modulation phase reverts to immunity system stable state and produces memory.The epi-position expansion can be the integral part in these two stages.The enhancing of epi-position expansion allows experimenter's immunity system to determine extra target epi-position (it is not responded to the immune system identification of original therapy scheme at first) in tumour takes place, reduce the possibility of escape variant in the tumour population simultaneously, thereby influence advancing of disease.
Oligoribonucleotide of the present invention is applicable to promoting treatment to go up the epi-position expansion in the useful indication (for example cancer, virus and infectation of bacteria, and transformation reactions).Described method comprises the steps: the experimenter is used the vaccine that comprises antigen and adjuvant in one embodiment, subsequently the experimenter is used the immunostimulating ORN of the present invention of at least two doses of certain consumption, described consumption is effectively induced the special immunne response of multi-epitope.This method comprises the steps: the experimenter is used the vaccine that comprises tumour antigen and adjuvant in one embodiment, subsequently the experimenter is used the immunostimulating ORN of the present invention of at least two doses of certain consumption, described consumption is effectively induced the special immunne response of multi-epitope.This method relates in one embodiment uses a kind of treatment plan, described treatment plan causes the immunity system antigen-exposed among the experimenter, use at least twice immunostimulatory oligoribonucleotides of the present invention subsequently,, promptly promote the epi-position expansion to induce the special immunne response of multi-epitope.In a plurality of embodiments, this treatment plan is surgical operation, radiation, chemotherapy, other cancer drug, vaccine or cancer vaccine.
Except immunostimulation therapy subsequently, treatment plan can carry out with the immunopotentiating agent combination.For example, when treatment plan was vaccine, it can combine with adjuvant and use.The combination of vaccine and adjuvant can be mixture or independently use, i.e. injection (being identical drainage district (drainage field)).Use must not be simultaneously.If use the injection of non-while, then arrangement of time can comprise first injection adjuvant, is vaccine formulation then.
After finishing treatment plan, beginning immunostimulation monotherapy.Target and other factors should be depended in best frequency of administration, time length and site, but can for example be every month or using per bimester the in six months to 2 years time period.Perhaps, using can be based on every day, weekly or carry out in per two weeks, or use can be in one day, a week or January repeatedly.In some cases, the time length of using can be depending on the length of therapy, and for example it can or stop after the several years after a week, after one month, after 1 year.In other cases, monotherapy can be a successive, as using intravenous drip.Immunostimulant can be applied to the common drainage district of target.
With regard to the purposes in the therapy, different dosage may be essential for the treatment experimenter, and this depends on the activity, route of administration of compound, purpose (being preventative or therapeutic), the character of illness and seriousness experimenter's the age and the body weight of immunity.Using of given dose can be finished by single administration with the form of individual dose unit or the form of several small dosage units.With the special interval of separately several weeks or several months repeatedly application dosage be applicable to the immunne response of strengthening antigen-specific.
In conjunction with instruction provided herein, by in various active compound and the weighing factor (for example potential, relative bioavailability, weight in patients, the seriousness of adverse side effect and the preference pattern of using), selecting, can plan effectively preventative or therapeutic treatment system, described treatment system does not cause remarkable toxicity and is in full force and effect for the concrete experimenter of treatment.The significant quantity that is used for any concrete application can be according to following factor and difference, and described factor is as the disease of treatment or illness, the concrete therapeutical agent of using, experimenter's build, or the seriousness of disease or illness.One of this area routine techniques personnel can rule of thumb determine the significant quantity of concrete nucleic acid and/or other therapeutical agent and must not carry out unsuitable experiment.
Experimenter's dosage of compound as herein described is typically from about 0.1mg to 10,000mg, more typically from about 1mg/ to 8000mg, the most typically in the scope from about 10mg to 100mg.With experimenter's body weight, typical dosage from about 0.1mg to 20mg/kg/ days, more typically from about 1 to 10mg/kg/ day, the most typically in about 1 to 5mg/kg/ day scope.
The pharmaceutical composition that contains nucleic acid and/or other compound can be applied by any suitable pathways that is used for drug administration.Can obtain multiple route of administration.Certainly, the concrete pattern of selection will depend on one or more concrete reagent of selection, the concrete illness and the required dosage of result of treatment of treatment.Generally speaking, method of the present invention can be used the acceptable any mode of administration of medical science and carries out, and promptly produces the efficient immune level and does not cause any pattern of unacceptable undesirable action clinically.Preferred mode of administration is in this paper discussion.For the purposes in the treatment, the nucleic acid of significant quantity and/or other therapeutical agent can be applied to the experimenter by any pattern, described pattern is delivered to the surface of expectation with described reagent, for example mucous membrane, general.
Using pharmaceutical composition of the present invention can finish by any approach known to the skilled.The approach of using includes but not limited in the mouth, parenteral, intravenously, intramuscular, intraperitoneal, nose, in the hypogloeeis, tracheae, suction, subcutaneous, eye, vagina and rectum.With regard to asthma or allergic treatment or prevention, this compounds preferably is inhaled into, takes in or is applied by the general approach.The general approach comprises oral and parenteral.The preferred in some embodiments medicine that sucks, because directly be delivered to lung, the inflammation site that asthmatic patient Central Plains is sent out.Usually use the equipment of some types to be used for using by suction.The equipment of these types comprises metered dose inhaler (MDI), breathe the MDI start, Diskus (DPI), with the interval/repository and the atomizer of MDI combination.
Therapeutical agent of the present invention can be delivered to concrete tissue, cell type by means of carrier, or is delivered to immunity system, or the two has concurrently." carrier " is meant any media that can be convenient to composition is transferred to target cell in the implication the most widely at it.Carrier usually with immunostimulatory nucleic acid, antibody, antigen and/or disease specific drug transport to target cell, have the degraded of minimizing for the degraded scope that obtains when not having carrier.
Usually, be applicable to that carrier of the present invention is divided into two classes: biological vehicle and chemical/physical carrier.Biological vehicle and chemical/physical carrier are applicable to sending and/or taking in of therapeutical agent of the present invention.
The various biological carrier is used to nucleic acid delivery, this belong to immunostimulatory nucleic acid or comprise in the therapeutic agent delivery of immunostimulatory nucleic acid the most suitable.
Except the biological vehicle that this paper discusses, the chemical/physical carrier can be used to send the therapeutical agent that comprises immunostimulatory nucleic acid, antibody, antigen and illness specificity medicament.This paper uses " chemical/physical carrier " to be meant that except from the natural or synthetic molecules bacterium or the viral source, they can nucleic acid delivery and/or other medicines.
The preferred chemical/physical carrier of the present invention is a colloidal dispersion system.Colloidal dispersion system comprises the system based on lipid, comprises oil-in-water emulsion, micelle, blended micelle and liposome.The preferred colloid system of the present invention is a liposome.Liposome is to be applicable in the body or the artificial rust vascular (vessel) of external delivery vector.Show: size range can be with big macromole encapsulate from the big unilamellar vesicle (LUV) of 0.2-4.0mm.RNA, DNA and complete virion can be delivered to cell by encapsulate in aqueous interior and with biologically active form.Fraley et al.(1981)Trends Biochem Sci6：77。
Can pass through liposome and special part (for example monoclonal antibody, sugar, glycolipid or protein) the coupling tissue that the liposome target is concrete.Can be used for the part of liposome target immunocyte is included but not limited to: with the special acceptor of immunocyte and interactional complete molecule of molecule (as antibody) or fragment, the cell surface marker thing of described acceptor and molecule and immunocyte interacts.This class part can easily be identified by well known to a person skilled in the art in conjunction with measuring.Also in some other embodiment, can be by making liposome by target on cancer the coupling of one of the immunotherapeutical antibody of liposome and preamble discussion.In addition, carrier can with the coupling of nuclear target peptide, described nuclear target peptide can point to carrier host cell nuclear.
The lipid prescription that is used for transfection can obtain with commercial sources from QIAGEN, for example EFFECTENE TM(non-liposome lipid) and SUPERFECT with special DNA compression enhanser TM(novel effect dendrimer technology).
Liposome can obtain with commercial sources from Gibco BRL, for example LIPOFECTIN TMAnd LIPOFECTACE TM, its by cation lipid such as N-[1-(2,3dioleyloxy)-propyl group]-N, N, N-trimethyl ammonium chloride (DOTMA) and dimethyl octacosyl brometo de amonio (DDAB) form.The method that is used to make liposome is well known in the art, and is described in many publications.Liposome is also summarized by Gregoriadis G (1985) Trends Biotechnol 3:235-241.
Some cation lipid (especially comprise N-[1-(2,3 two oleoyl oxygen)-propyl group]-N, N, N-trimethyl ammonium methylsulfuric acid ester (DOTAP)) particularly advantageous when demonstration is made up with modified oligoribonucleotide analogue of the present invention.
In one embodiment, media is to be applicable to biocompatible particulate or the implant of implanting or being applied to mammalian receptors.The exemplary biological erosion that is applicable to this method is separated implant and is described in PCT international application no.PCT/US/03307 (open No.WO95/24929 is entitled as " PolymericGene Delivery System ").Biocompatible, preferred biodegradable polymer matrix that PCT/US/0307 has described, described matrix is used to comprise the foreign gene that is under the suitable promoter regulation.Polymer matrix can be used to reach the lasting release of therapeutical agent in the experimenter.
The polymer matrix optimization is the form of particulate, for example microballoon (its amplifying nucleic acid and/or other therapeutical agent are scattered in solid polymer matrix everywhere) or microcapsule (its amplifying nucleic acid and/or other therapeutical agent are stored in the core of polymer shell).The polymer matrix that is used to comprise other form of therapeutical agent comprises film, dressing, gel, implants and support.Select the size and the composition of polymer matrix equipment, make the good release dynamics of generation in the tissue that described matrix is introduced into.This is according to the size of delivering method selection polymer matrix to be used, and described delivering method is injected into tissue typically or by aerosol suspension is used into nose and/or lung zone.Preferably, when using the aerosol approach, polymer matrix and nucleic acid and/or other therapeutical agent are included in the tensio-active agent media.Can select the polymer substrate composition, make it both have good degradation rate, the material by bioadhesion forms again, thereby further improves the validity that shifts when matrix is applied to nose that continues damage and/or lung surface.Also can select not degrade but substrate composition by discharging in the time period that is diffused in prolongation.In some preferred embodiments, by implant nucleic acid is administered to the experimenter during by acute administration when other therapeutical agent.Be applicable to that the biocompatible microballoon of sending (for example mouth or mucosal delivery) is disclosed among Chickering et al. (1996) Biotech Bioeng 52:96-101 and Mathiowitz E etal. (1997) Nature 386:410-414 and the PCT patent application WO97/03702.
Abiotic degradable and biodegradable polymer matrix all can be used for giving the experimenter with nucleic acid and/or other therapeutic agent delivery.Preferred biodegradable matrices.This class polymer can be natural or the synthetic polymer.Polymer according to desired time of releasing section select, usually by a few hours to one year or order more of a specified duration.Typically, the release of scope on the time period between a few hours and three to 12 months is expected most, especially for nucleic acid reagent.Polymer randomly is the form of hydrogel (its can be adsorbed to many its weight approximately 99%) in water, perhaps randomly crosslinked with polyvalent ion or other polymer.
The interested especially bioadhesion polymer of people comprises H.S.Sawhney, C.P.Pathak andJ.A.Hubell in Macromolecules, and hydrogel is separated in the biology erosion that (1993) 26:581-587 describes, and this paper is incorporated in the instruction of this reference into.These comprise the poly hyaluronic acid, casein, gelatin, glutin, polyanhydride, polyacrylic acid, alginate, chitosan, poly (methacryloxyethyl acid methyl esters), poly (Jia Jibingxisuanyizhi), poly (butyl methacrylate), poly (Propenoic acid, 2-methyl, isobutyl ester), poly (N-Hexyl methacrylate), poly (isodecyl methacrylate), poly (lauryl methacrylate), poly (phenyl methacrylate), poly (methyl acrylate), poly (isopropyl acrylate), poly (isobutyl acrylate) and poly (vinylformic acid stearyl).
If therapeutical agent is a nucleic acid, then also compression agent (compaction agent) is used in expectation.The compression agent also can be used separately or use with bio-carrier or chemical/physical carrier combinations.This paper use " compression agent " thus in being meant and the negative charge on the nucleic acid allow a kind of reagent of nucleic acid boil down to fine granular, for example histone.The compression of nucleic acid is convenient to nucleic acid and is taken in by target cell.The compression agent can be used separately, promptly with by the form nucleic acid delivery of the more effective absorption of cell, or more preferably its and one or more above-mentioned carrier combinations use.
Other exemplary composition that can be used for promoting nucleic acid to absorb comprises other chemical media, microinjection composition, electroporation and the homologous recombination composition (for example being used for nucleic acid is integrated in the previously selected site of target cell genome) that transports in calcium phosphate and the born of the same parents.
Compound can be used (for example in salt solution or damping fluid) separately or use any delivery medium known in the art to use.For example Yi Xia delivery medium is described: spirochete (Gould-Fogerite et al., 1994,1996); Emulsomes (Vancott et al., 1998, Lowell et al., 1997); ISCOMs (Mowat et al., 1993, Carlsson et al., 1991, Hu et., 1998, Morein et al., 1999); Liposome (Childers et al., 1999, Michalek et al., 1989,1992, de Haan 1995a, 1995b); Bacteria carrier (for example Salmonella, Escherichia coli, Bacillus Calmette-Gu é rin, Shigella, Lactobacillus) (Hone et al. lives, 1996, Pouwels et al., 1998, Chatfield et al., 1993, Stover et al., 1991, Nugent et al., 1998); Live vector (for example cowpox, adenovirus, herpes simplex) (Gallichan et al., 1993,1995, Moss et al., 1996, Nugent et al., 1998, Flexner et al., 1988, Morrow et al., 1999); Microballoon (Gupta et al., 1998, Jones et al., 1996, Maloy et al., 1994, Mooreet al., 1995, O ' Hagan et al., 1994, Eldridge et al., 1989); Nucleic acid vaccine (Fynan et al., 1993, Kuklin et al., 1997, Sasaki et al., 1998, Okada et al., 1997, Ishii et al., 1997); Polymer (for example carboxymethyl cellulose, chitosan) (Hamajima et al., 1998, Jabbal-Gill et al., 1998); Polymer ring (Wyatt et al., 1998); Proteoplast (Vancott etal., 1998, Lowell et al., 1988,1996,1997); Sodium Fluoride (Hashi et al., 1998); Transgenic plant (Tacket et al., 1998, Mason et al., 1998, Haq et al., 1995); Virosomes (Gluck et al., 1992, Mengiardi et al., 1995, Cryz et al., 1998) and virus-like particle (Jiang et al., 1999, Leibl et al., 1998).
Prescription of the present invention is applied in pharmaceutically useful solution, and described solution contains the salt, buffer reagent, sanitas of pharmaceutically acceptable concentration, suitable vehicle, adjuvant and other optional therapeutic component routinely.
The pharmaceutically acceptable vehicle of term is represented to be applicable to and is administered to people or other vertebrate solid or liquid filling agent, thinner or encapsulate materials that one or more are fit to.The term vehicle is represented natural or synthetic organic or inorganic composition, and activeconstituents makes up with it so that use.The composition of pharmaceutical composition also can be mixed with each other with compound of the present invention in the following manner: do not have the significantly interaction of the drug effectiveness of damage expectation.
For Orally administered, can be prepared compound (being nucleic acid, antigen, antibody and other therapeutical agent) by active compound and pharmaceutically acceptable vehicle well known in the art being mixed to come easily.This class vehicle makes compound of the present invention can be configured to tablet, alkyl, lozenge, capsule, liquid, gel, syrup, paste, suspension etc., is used for by experimenter's orally ingestible to be treated.The pharmaceutical preparation that is used for oral use can be used as solid excipient and obtains, and randomly grinds mixture and the process mixture particle that obtains if desired after adding proper supplementary material, obtains tablet or lozenge nuclear.Suitable vehicle is weighting agent such as sugar especially, comprises lactose, sucrose, N.F,USP MANNITOL or sorbyl alcohol; Cellulose preparation such as W-Gum, wheat starch, rice fecula, yam starch, gelatin, Tragacanth (gum tragacanth), methylcellulose gum, Vltra tears, Xylo-Mucine and/or Povidone (PVP).If desired, can add for example sodium alginate of for example crosslinked Povidone of disintegrating agent, agar or Lalgine or its salt.Randomly, oral preparations also can be formulated in and be used in salt solution or the damping fluid and the internal acid condition, or can not be applied with any vehicle.
Lozenge nuclear provides with suitable dressing.For this purpose can be used spissated sugar soln, its optional gum arabic, talcum, polyvinylpyrrolidone, carboxyvinyl polymer gel, polyoxyethylene glycol and/or titanium dioxide, lacquer solution (lacquer solution) and appropriate organic solvent or solvent mixture of containing.Dyestuff or pigment may be added to the various combination that is used to identify or characterize active compound doses in tablet or the lozenge dressing.
The pharmaceutical preparation that can orally use comprises the push-fit capsule of being made by gelatin, and the soft seal capsule of being made by gelatin and softening agent (for example glycerine or sorbyl alcohol).The push-fit capsule can contain and weighting agent such as lactose, tackiness agent such as starch and/or lubricant such as talcum or Magnesium Stearate and optional stablizer blended activeconstituents.In soft capsule, active compound dissolves in or is suspended in the suitable liquid, for example fatty oil, whiteruss, or in the liquid macrogol.In addition, can add stablizer.Also can use preparation to be used for the microballoon that mouth is used.This class microballoon has been that this area is determined.Being used for mouthful all prescriptions of using should be the dosage that is applicable to that this class is used.
For containing for clothes use, composition can be taked the tablet prepared in a usual manner or the form of lozenge.
For using by suction, the compound that is used for purposes of the present invention can be sent with suitable propelling agent (for example Refrigerant 12, trichlorofluoromethane, dichloro tetrafluoro ethane, carbonic acid gas or other suitable gas) expediently with the form from the sprays of supercharging packing or atomizer.Under the situation of pressurised aerosol, unitary dose can be determined by the value that is provided for sending measured quantity.The capsule and the cartridge case (for example gelatin) that are used for sucker or insufflator can be configured to the powdered mixture that contains compound and suitable powder basis (for example lactose or starch).
When expectation systemic delivery compound, it can be configured to and be used for by the injection parenteral administration, for example by injecting (bolus injection) or continuous infusion.The prescription that is used to inject can exist with unit dosage, for example ampoule or have in the multi-dose container of sanitas of interpolation.Composition can adopt the form of suspension, solution or emulsion in oiliness for example or the aqueous media, and can contain preparaton such as suspension agent, stablizer and/or dispersion agent.
The formula of medicine that is used for parenteral administration comprises the aqueous solution of the active compound of water-soluble form.In addition, the suspension of active compound can be prepared as suitable oily injection suspensions.Suitable lipophilic solvent or media comprise fatty oil (as sesame oil) or synthetic fatty acid ester (as ethyl oleate) or triglyceride level, or liposome.Water injection suspension liquid can contain the material that improves suspension viscosity, for example Xylo-Mucine, sorbyl alcohol or dextran.Choose wantonly, suspension also can contain suitable stabilizers or improve the reagent of compound dissolution degree, to allow the preparation highly concentrated solution.
Perhaps, active compound can be the powder type that is used for before use with suitable medium (for example sterile pyrogen-free water) combination.
Composition also can be formulated in rectum or vaginal compositions for example in suppository or the enema, and it for example contains conventional suppository base, as theobroma oil or other glyceryl ester.
Except aforementioned formula, compound also can be configured to the storage preparation.The long-acting prescription of this class can be with suitable polymer or hydrophobic material (emulsion in for example acceptable oil) or ion exchange resin preparation, or is configured to slightly soluble derivative (for example slightly soluble salt).
Pharmaceutical composition also can comprise suitable solid or gel phase vehicle or vehicle.The example of this class vehicle or vehicle includes but not limited to lime carbonate, calcium phosphate, multiple sugar, starch, derivatived cellulose, gelatin and polymer such as polyoxyethylene glycol.
Suitable liquid or solid pharmaceutical dosage forms for the water-based that for example is used for sucking or salt brine solution, by micro-capsule embedding, spiral embedding (encochleated), dressing at little gold grain, be included in liposome, by sprinkling, aerosol, be used for implanting the little group of skin or dryly will be rubbed into the sharp-pointed article of skin.Pharmaceutical composition also comprises tablet, (little) capsule, suppository, syrup, emulsion, suspension, frost, the drops of particle, powder, tablet, dressing or prolongs the preparation of release active compound, use vehicle and additive and/or auxiliary material, for example disintegrating agent, tackiness agent, Drug coating, swelling agent, lubricant, seasonings, sweeting agent or solubilizing agent in the described preparation as mentioned above routinely.Pharmaceutical composition is applicable to multiple drug delivery system.The summary summary that is used for the method that medicine sends is referring to Langer R (1990) Science 249:1527-1533, and it is by with reference to incorporating this paper into.
Nucleic acid and optional other therapeutical agent and/or antigen can self (pure) be applied or be applied with the form of pharmacologically acceptable salt.When being used for medicine, salt should be pharmaceutically useful, but can use non-pharmacologically acceptable salt to prepare its pharmacologically acceptable salt expediently.These salt include, but are not limited to from the salt of following acid preparation: hydrochloric acid, Hydrogen bromide, sulfuric acid, nitric acid, phosphoric acid, toxilic acid, acetate, Whitfield's ointment, p-toluenesulphonic acids, tartrate, citric acid, methylsulfonic acid, formic acid, propanedioic acid, succsinic acid, naphthalene-2-sulfonic acid and Phenylsulfonic acid.This class salt also can be prepared as basic metal or alkaline earth salt, for example the sodium salt of hydroxy-acid group, sylvite or calcium salt.
Suitable reducing comprises: acetate and salt (1-2%w/v); Citric acid and salt (1-3%w/v); Boric acid and salt (0.5-2.5%w/v); With phosphoric acid and salt (0.8-2%w/v).Suitable sanitas comprises Benzalkonii Chloridum (0.003-0.03%w/v); Trichloro-butyl alcohol (0.3-0.9%w/v); P-Hydroxybenzoate (0.01-0.25%w/v) and Sodium Mercurothiolate (0.004-0.02%w/v).
Composition can exist expediently with unit dosage form, and can be by the known any method preparation of pharmacy field.All methods all comprise the step that makes compound and vehicle combination, and described vehicle is formed one or more auxiliary agents (accessory ingredients).Unobstructed, by with compound equably and directly with solid vehicle or these two combination of liquid vehicle, fine dispersion, product is shaped prepares composition.Liquid dosage unit is pipe or ampoule.Solid dosage unit is tablet, capsule and suppository.
Other delivery system can comprise instant-free (time-release), postpone to discharge or prolong the release delivery system.This class system can be avoided the repetitive administration of compound, promotes the facility to experimenter and doctor.The delivery system of many types is obtainable, and is that this area routine techniques personnel are known.They comprise polymer basic system such as poly (rac-Lactide-glycollide), copolymerized oxalate, polycaprolactone, polyesteramide, many polyorthoesterses, poly hydroxybutyric acid and poly acid anhydrides.The medicine that contains aforementioned polymer microcapsule is disclosed in for example U.S.Pat.No.5, in 075,109.Delivery system also comprises following non-polymer system: lipid comprises sterol such as cholesterol, cholesteryl ester and lipid acid or neutral fat such as glycerine list, two and three esters; The hydrogel delivery systme; The silica gel system; System based on peptide; The wax dressing; Use the compressed tablets of conventional tackiness agent and vehicle; The implants of meromixis etc.Special example includes, but are not limited to: (a) erosion system (erosional system), and reagent wherein of the present invention is involved with the form in the matrix, U.S.Pat.Nos.4 for example, 452,775,4,675,189 and 5,736, disclosed those and (b) indiffusion system (diffusional system) in 152, wherein activeconstituents with controlled speed from for example U.S.Pat.Nos.3,854,480,5,133,974 and 5,407, the polymer infiltration described in 686.In addition, can use the hardware delivery system based on pump, some of them are applicable to implantation.
Following embodiment will further set forth the present invention, and described embodiment should not be considered to be used for further restriction by any way.
Embodiment
Embodiment 1
People PMBC is to containing N-U-R 1 -R 2 The responsiveness of oligoribonucleotide
Method: Luminex technology
The Luminex color code globule that is called microballoon is divided into 100 different groups.Each globule group can be used the actual packet quilt special to concrete biological assay, allows to catch from sample and detect special analyte.In the easy analyser of Luminex, the dye inside of each microsphere particle is identified in laser excitation, and the sub-dyestuff of any report also is hunted down between this test period.Each globule group is studied many readings, further confirm the result.In this way, the Luminex technology allows the quick also mensuration of 100 uniquenesses of accurately compound as many as in single sample.
Separation of human peripheral blood lymphocytes (PBMC) from the donor of health is coated with and stimulated 16 hours with multiple test and contrast immunostimulant it.Collect supernatant liquor after 16 hours, then by the ELISA determination and analysis.With the condition of the compound and complete titration curve of DOTAP (7 concentration) under test contain N-U-R 1-R 2Oligoribonucleotide, described curve is from beginning with 25 μ g/ml DOTAP compound, 2 μ M ORN and having an extent of dilution step of 1/3.Also comprise some negative control, comprise independent usefulness substratum and use DOTAP separately that (25mg/ml cultivates datum hole; " liposome ").The contrast immunostimulant comprise imidazole quinoline R-848 (2 μ M, 1/3 extent of dilution step and 7 concentration), the report TLR7 part, have the ORN of TLR7 motif such as AU and AUU sequence (SEQ IDNO:13-SEQ ID NO:15), ORN with TLR8 motif such as CU, GU and GUU sequence (SEQ ID NO:19-SEQ ID NO:23).The result is presented in Fig. 1 and 3.
Use separated pDC, monocyte and mDC to stimulate the similar mensuration that is used to test different ORN sequences.With with 10 μ g/ml DOTAP, 0.5 μ M CpG ODN compound, 0.5 μ MORN, or separately with DOTAP or substratum irritation cell.Collect supernatant liquor after 16 hours and measure IFN-α, TNF-α and IL-12p40 by ELISA.The result is presented among Fig. 2.
Fig. 1 has shown the clear difference between the TNF-α and IFN-α production when with the SEQ IDNO:21 that contains the SEQ ID NO:12 of AU sequence and contain the GU sequence PBMC being stimulated.Other sequential analysis shows that it is that another is induced TNF-α and does not produce the ORN of IFN-α that CUA repeats (SEQ ID NO:24).Contain AU and compare similar result with longer ORN (SEQ ID NO:12-SEQ ID NO:23), but efficient and ability reduce with the shorter ORN demonstration that GU repeats (SEQ ID NO:29-SEQ ID NO:34).
Fig. 2 and 6 has shown on separated monocyte, pDC and mDC the analysis to AU-ORN (SEQ ID NO:13) and GU-ORN (SEQ ID NO:21), reflected for the strong IFN-α production that reduces of AU-ORN (SEQ ID NO:13) with for two kinds of ORN TNF-α and IL-12p40 production clearly.IFN-α when stimulating from the ORN of pDC produces TLR7 mediation seemingly, and produces TLR8 mediation seemingly from the TNF-α of separated monocyte and mDC and IL-12p40.
Luminex result has been reflected the result suitable with the ELISA data, and has proved that difference main between GU-ORN and the AU-ORN is owing to the IFN-α production gene/cytokine (Fig. 3 and 8a) relevant with IFN-α.In addition, gene/cytokine that other Luminex data presentation is relevant with IFN-α with IFN-α is opposite, and other cytokine/chemokine is not subjected to from the influence of an outlier of CD123-CD14-cell (Fig. 7 and 8a-d).This IL-6 production may be owing to the B cell-stimulating of TLR7 mediation.
Embodiment 2
Comparison to the IFN-α and the TNF-α maximum activity of oligoribonucleotide
With stimulating the human PBMC with DOATP compound ORN.Collect supernatant liquor after 16 hours and measure TNF-α and the IFN-alpha levels.Measured 3-6 blood donor and two independent experiments average/maximum activity at 0.6 μ M place.The result is presented among Fig. 4.These data are clearly different between TLR8 and TLR7/8 motif: have motif N-U-R 1-R 2The IFN-α that is presented under the 300pg/ml of ORN produce, and TLR7/8ORN when being presented at PBMC and stimulating higher IFN-α production (Fig. 4 a).TLR8 and TLR7/8 separate with red line.On the contrary, the measurement of TNF-alpha levels point out to have the ORN of TLR8 and have the TLR7/8 motif ORN the two all stimulate TNF-α production.
Embodiment 3
Comparison to the IFN-α maximum activity and the IFN-α EC50 of oligoribonucleotide
With stimulating the human PBMC with DOATP compound ORN.Collect supernatant liquor after 16 hours and measure the INF-alpha levels.Measured 3-6 blood donor and two independent experiments 0.6 μ M place and the EC50 place of titration curve (scope 2 μ M are to 0.9nM) fully on average/maximum activity.The result is presented among Fig. 5.EC50 and maximum activity show the comparable result who relates to TLR8 and TLR7/8 motif.The low high maximum activity of EC50/ is represented TLR7/8ORN, and (Fig. 5 a) and high EC50 and low maximum activity are represented TLR8ORN (Fig. 5 b).
His-and-hers watches 1 listed ORN sequence is carried out IFN-α and TNF-α production test when the human PBMC stimulates.Stimulated the human PBMC 16 hours with specified ORN, collect supernatant liquor and measure cytokine production by ELISA.Table 2 has been summarized minimum/maximum activity and the EC50 of ORN at IFN-α and TNF-α production.
Table 1:
+: cytokine production
-: acellular factor production.
Table 2
Figure A20068004403000821
Figure A20068004403000841
Embodiment 4
When people PMBC stimulated, synthetic ORN was different between IFN-α and TNF-α release
With with 25 μ g/ml DOTAP compound 1 μ M ORN or use separately DOTAP (Figure 10 a), or specified amount with DOTAP compound ORN or use DOTAP (Figure 10 b-10c) to hatch purified pDC of CD123+ (Figure 10 a and 10b) or separated monocyte (Figure 10 c) separately.Collecting cell is also with CD123, CD11c and HLA-DR antibody (Figure 10 a and 10b) or CD14 and CD19 (Figure 10 c) dyeing after 16 hours.The facs analysis of CD86 is shown the ORN (SEQ ID NO:21) that is rich in the ORN (SEQ ID NO:13) of AU and is rich in GU shows difference when pDC stimulates in the expression of CD86 surface marker.Cause considerably less CD86 to activate with the ORN that is rich in AU (SEQ ID NO:13) stimulation, and cause significant CD86 to activate with the ORN that is rich in GU (SEQ ID NO:21) stimulation.It is cascade dependent (Fig. 1 Ob) that this activation is confirmed as.The ORN (SEQ ID NO:21) that is rich in the ORN (SEQ ID NO:13) of AU and is rich in GU shows indifference (Figure 10 c) in the CD80 surface marker is expressed when human PBMC's (data not shown) and CD14-positive cell stimulate.
Embodiment 5
The ORN (SEQ ID NO:21) that is rich in the ORN (SEQ ID NO:13) of AU and is rich in GU is with agent The mode that amount relies on stimulates special people TLR8 signal transduction
Personnel selection TLR3 or TLR8 expression plasmid and the unresponsive HEK-293 cell of NF κ B-luciferase reporter gene construct stable transfection.With specified ORN sequence (10 μ M, compound) or control stimulation (10 μ M R-848,50 μ g/ml polyIC, 3,3 μ M ODN10103 or 50 μ g/ml DOTAP) cell was hatched 16 hours with 50 μ M/mlDOATP.Activate by measuring luciferase activity measuring N F κ B-.The result induces as the multiple that is higher than background (substratum) and provides.(Fig. 9 a) to have shown representative experiment of 6 independent multiple.
With with the ORN of DOTAP (50 μ g/ml-＞1/3 extent of dilution) compound prescribed concentration or use DOTAP (50 μ g/ml-＞1/3 extent of dilution) separately with the HEK-293 cytositimulation of the stable transfection of expressing human TLR8 16 hours.Activate by measuring luciferase activity measuring N F κ B-.The result induces as the multiple that is higher than background (substratum) and provides.Representative experiments of 3 independent multiple (Fig. 9 b) have been shown.
Personnel selection TLR8 expression plasmid and the unresponsive HEK-293 cell of NF κ B-luciferase reporter gene construct stable transfection.With specified ORN sequence (15 μ M, compound with 75 μ M/ml DOATP) or control stimulation (15 μ M R-848 or 75 μ g/ml DOTAP) and with substratum (left side), 200nM Bafilomycin (Baf., in) or 1mM chloroquine (CQ, the right side) cell was hatched 16 hours.Activate by measuring luciferase activity measuring N F κ B-.The result induces as the multiple that is higher than background (substratum) and provides.Representative experiments of 4 independent multiple (Fig. 9 c) have been shown.
RPMI 8226 cells and 1000U/mlIntron A were hatched 3 hours, use the substratum washed twice, use stimulating of prescribed concentration then with DOTAP (50 μ g/ml-＞1/3 extent of dilution) compound ORN.Measure the release of cytokines of IP-10 by ELISA.The result provides with pg/ml.Representative experiments of 3 independent multiple (Fig. 9 d) have been shown.
These digital proofs SEQ ID NO:13 and SEQ ID NO:21 are to the specificity of TLR8.
TLR8ORN (SEQID NO:13), TLR 7/8 ORN (SEQ ID NO:21) and contrast ORN (SEQ ID NO:5) (table 3) with high dosage (H.D., 10 μ g/ml) and low dosage (L.D., 2.5 μ g/ml) repeat this mensuration.Only SEQ ID NO:21 handles and has caused significant IL-12 and TNF-α to produce (being respectively Figure 11 a and 11b).All ORN stimulate the production (Figure 11 c) of IFN-γ.
SEQID NO ORN
SEQID NO：5 C＊C＊G＊U＊C＊U＊G＊U＊U＊G＊U＊G＊U＊G＊A＊C＊U＊C
SEQID NO：13 U＊U＊A＊U＊U＊A＊U＊U＊A＊U＊U＊A＊U＊U＊A＊U＊U＊A＊U＊U
SEQID NO：21 U＊U＊G＊U＊U＊G＊U＊U＊G＊U＊U＊G＊U＊U＊G＊U＊U＊G＊U＊U
Embodiment 6
In the mouse macrophage body or externally the ORN (SEQ ID NO:13) that is rich in AU is not replied
(Figure 12 a), J774 cell (Figure 12 b) and purified CD11c+ cell (Milteny, magnetic bead mark) separate from sv129 mouse boosting cell (Figure 12 c-12e) and use with DOTAP (50 μ g/ml and dilute with the ORN) ORN of compound prescribed concentration, independent R-848 or DOTAP (50 μ g/ml) stimulates the Raw264.7 cell.16 hours (Figure 12 a and 12b) or 20 hours (Figure 12 c-12e) back is collected supernatant liquor and is used for the ELISA of TNF-α (Figure 12 a and 12b), IL-12p40 (Figure 12 c), IFN-α (Figure 12 d) and IP-10 (Figure 12 e).Data representation from the body one by one (Figure 12 a and 12b) of at least three experiments and the average (Figure 12 c-12e) of 3 mouse.In order to measure the ability of the ORN body internal stimulus mouse cell that is rich in AU, use ORN injection sv 129 mouse (n=5/ group) and the blood sampling after 3 hours of the specified amount of preparing with DOTAP (60,20 or 6 μ g/ml).In whole blood, measure IL-12p40 (Figure 12 f), IFN-α (Figure 12 g) and IP-10 (Figure 12 h) production with ELISA.
Embodiment 7
Purified rat spleen cells is not replied the ORN SEQ ID NO:13 that is rich in AU
To merge and with SEQID NO:21, SEQ ID NO:13 (all compound, 1/5 extent of dilution), R-848 or DOTAP independent (the 62.5 μ g/ml-＞1/5 extent of dilution) stimulation of prescribed concentration from the splenocytes of 3 Sprague-Dawley rats with 62.5 μ g/ml DOTAP.Collect supernatant liquor after 20 hours and measure the TNF-alpha levels by ELISA.As shown in Figure 13, cause TNF-α production with the ORNSEQ ID NO:21 stimulation of being rich in GU, and cause not producing TNF-α with the ORN SEQ ID NO:13 that is rich in AU production.
Embodiment 8
Rodent cells is replied the failure of the ORN SEQ ID NO:13 that is rich in AU can be by between species The TLR8 polymorphism causes
Stimulate people and Niu cell to cause cytokine production with the ORN that is rich in AU, stimulate mouse and rat cell then not to cause.Carry out TLR8 sequence alignment and analysis.The leucine that is rich in the protein sequence of TLR8 comparison display structure territory 1 repeats intensive difference among (LRR) 3 between different vertebratess (people, monkey, orangutan, dog, ox, pig, mouse and rat).Although people, orangutan and monkey are high conservatives, with the physiognomy ratio, rat, mouse and pig are proved to be in the position 106 (mouse), 103 (rats) or 102 (pigs) deletion 4AA, and ox is proved to be the have 2AA insertion of (105-106).What is interesting is that pig and ox demonstrate another 2AA deletion in same area (position 97).The deletion of being rich in the leucine repeat region of possible structural domain 1 can be disturbed the ORN combination of being rich in AU.
Equivalent
Aforementioned printed instructions is considered to enough make those skilled in the art realize the present invention.The embodiment that scope of the present invention is not provided limits, because embodiment only is intended to set forth separately one aspect of the present invention, the embodiment of other functional equivalent all within the scope of the present invention.Except shown and described herein, those skilled in the art describes obviously multiple modification of the present invention according to preamble, and these are all within the scope of additional claim.Advantage of the present invention is not necessarily comprised by each embodiment of the present invention.
All reference, patent and the patent publications of quoting among the application all together integral body incorporate this paper by reference into.
Sequence table
＜110〉Coley Pharmaceutical GmbH
Coley Pharmaceutical GmbH
＜120〉immunostimulating ribonucleotide
<130>C1041.70053WO00
<150>60/739,529
<151>2005-11-25
<150>60/778,989
<151>2006-03-03
<160>91
<170>PatentIn version 3.3
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<220>
＜223〉synthetic oligonucleotide
<220>
<221>misc_feature
<222>(3)..(3)
＜223〉wherein n is an inosine
<220>
<221>misc_feature
<222>(6)..(6)
＜223〉wherein n is an inosine
<220>
<221>misc_feature
<222>(9)..(9)
＜223〉wherein n is an inosine
<220>
<221>misc_feature
<222>(12)..(12)
＜223〉wherein n is an inosine
<220>
<221>misc_feature
<222>(15)..(15)
＜223〉wherein n is an inosine
<220>
<221>misc_feature
<222>(18)..(18)
＜223〉wherein n is an inosine
<400>28
uunuunuunu unuunuunuu 20
<210>29
<211>7
<212>RNA
＜213〉artificial sequence
<220>
＜223〉synthetic oligonucleotide
<400>29
uuguugu 7
<210>30
<211>7
<212>RNA
＜213〉artificial sequence
<220>
＜223〉synthetic oligonucleotide
<400>30
uuauuau 7
<210>31
<211>7
<212>RNA
＜213〉artificial sequence
<220>
＜223〉synthetic oligonucleotide
<400>31
ugugugu 7
<210>32
<211>7
<212>RNA
＜213〉artificial sequence
<220>
＜223〉synthetic oligonucleotide
<400>32
ucucucu 7
<210>33
<211>7
<212>RNA
＜213〉artificial sequence
<220>
＜223〉synthetic oligonucleotide
<400>33
uauauau 7
<210>34
<211>7
<212>RNA
＜213〉artificial sequence
<220>
＜223〉synthetic oligonucleotide
<400>34
guaguag 7
<210>35
<211>16
<212>RNA
＜213〉artificial sequence
<220>
＜223〉synthetic oligonucleotide
<400>35
ccgagccgcc gccccc 16
<210>36
<211>16
<212>RNA
＜213〉artificial sequence
<220>
＜223〉synthetic oligonucleotide
<400>36
ccgagccgca uauccc 16
<210>37
<211>16
<212>RNA
＜213〉artificial sequence
<220>
＜223〉synthetic oligonucleotide
<400>37
ccgagccgcu auaccc 16
<210>38
<211>16
<212>RNA
＜213〉artificial sequence
<220>
＜223〉synthetic oligonucleotide
<400>38
ccgagccaua uauccc 16
<210>39
<211>16
<212>RNA
＜213〉artificial sequence
<220>
＜223〉synthetic oligonucleotide
<400>39
ccgagccaua uauauc 16
<210>40
<211>16
<212>RNA
＜213〉artificial sequence
<220>
＜223〉synthetic oligonucleotide
<400>40
ccgagccgaa uaaccc 16
<210>41
<211>16
<212>RNA
＜213〉artificial sequence
<220>
＜223〉synthetic oligonucleotide
<400>41
ccgagccgca uaaccc 16
<210>42
<211>16
<212>RNA
＜213〉artificial sequence
<220>
＜223〉synthetic oligonucleotide
<400>42
ccgagccgaa uacccc 16
<210>43
<211>16
<212>RNA
＜213〉artificial sequence
<220>
＜223〉synthetic oligonucleotide
<400>43
ccgagccgcc uaaccc 16
<210>44
<211>16
<212>RNA
＜213〉artificial sequence
<220>
＜223〉synthetic oligonucleotide
<400>44
ccgagccgaa uccccc 16
<210>45
<211>16
<212>RNA
＜213〉artificial sequence
<220>
＜223〉synthetic oligonucleotide
<400>45
ccgagccgca uacccc 16
<210>46
<211>16
<212>RNA
＜213〉artificial sequence
<220>
＜223〉synthetic oligonucleotide
<400>46
ccgagccgca uccccc 16
<210>47
<211>16
<212>RNA
＜213〉artificial sequence
<220>
＜223〉synthetic oligonucleotide
<400>47
ccgagccgcc uacccc 16
<210>48
<211>16
<212>RNA
＜213〉artificial sequence
<220>
＜223〉synthetic oligonucleotide
<400>48
ccgagccgca uuaccc 16
<210>49
<211>16
<212>RNA
＜213〉artificial sequence
<220>
＜223〉synthetic oligonucleotide
<400>49
ccgagccgcu uaaccc 16
<210>50
<211>16
<212>RNA
＜213〉artificial sequence
<220>
＜223〉synthetic oligonucleotide
<400>50
ccgagccgca auuccc 16
<210>51
<211>16
<212>RNA
＜213〉artificial sequence
<220>
＜223〉synthetic oligonucleotide
<400>51
ccgagccgcu auuccc 16
<210>52
<211>16
<212>RNA
＜213〉artificial sequence
<220>
＜223〉synthetic oligonucleotide
<400>52
ccgagccgca auuccc 16
<210>53
<211>16
<212>RNA
＜213〉artificial sequence
<220>
＜223〉synthetic oligonucleotide
<400>53
ccgagccgcu uacccc 16
<210>54
<211>16
<212>RNA
＜213〉artificial sequence
<220>
＜223〉synthetic oligonucleotide
<400>54
ccgagccgca uucccc 16
<210>55
<211>16
<212>RNA
＜213〉artificial sequence
<220>
＜223〉synthetic oligonucleotide
<400>55
ccgagccgcu aucccc 16
<210>56
<211>16
<212>RNA
＜213〉artificial sequence
<220>
＜223〉synthetic oligonucleotide
<400>56
ccgagccgaa ggcacc 16
<210>57
<211>16
<212>RNA
＜213〉artificial sequence
<220>
＜223〉synthetic oligonucleotide
<400>57
ccgagccgac uuuacc 16
<210>58
<211>16
<212>RNA
＜213〉artificial sequence
<220>
＜223〉synthetic oligonucleotide
<400>58
ccgagccgag uuuacc 16
<210>59
<211>16
<212>RNA
＜213〉artificial sequence
<220>
＜223〉synthetic oligonucleotide
<220>
<221>misc_feature
<222>(10)..(10)
＜223〉wherein n is a thymidine
<400>59
ccgagccgan uuuacc 16
<210>60
<211>16
<212>RNA
＜213〉artificial sequence
<220>
＜223〉synthetic oligonucleotide
<400>60
ccgagccgaa uuuacc 16
<210>61
<211>16
<212>RNA
＜213〉artificial sequence
<220>
＜223〉synthetic oligonucleotide
<400>61
ccgagccgac uguacc 16
<210>62
<211>16
<212>RNA
＜213〉artificial sequence
<220>
＜223〉synthetic oligonucleotide
<220>
<221>misc_feature
<222>(10)..(10)
＜223〉wherein n is a thymidine
<400>62
ccgagccgan uguacc 16
<210>63
<211>16
<212>RNA
＜213〉artificial sequence
<220>
＜223〉synthetic oligonucleotide
<400>63
ccgagccgaa uguacc 16
<210>64
<211>16
<212>RNA
＜213〉artificial sequence
<220>
＜223〉synthetic oligonucleotide
<400>64
ccgagccgau cuuacc 16
<210>65
<211>16
<212>RNA
＜213〉artificial sequence
<220>
＜223〉synthetic oligonucleotide
<400>65
ccgagccgau auuacc 16
<210>66
<211>16
<212>RNA
＜213〉artificial sequence
<220>
＜223〉synthetic oligonucleotide
<400>66
ccgagccgau guuacc 16
<210>67
<211>16
<212>RNA
＜213〉artificial sequence
<220>
＜223〉synthetic oligonucleotide
<400>67
ccgagccgag uucacc 16
<210>68
<211>16
<212>RNA
＜213〉artificial sequence
<220>
＜223〉synthetic oligonucleotide
<220>
<221>misc_feature
<222>(10)..(10)
＜223〉wherein n is a thymidine
<400>68
ccgagccgan uucacc 16
<210>69
<211>16
<212>RNA
＜213〉artificial sequence
＜220〉synthetic oligonucleotide
<400>69
ccgagccgau cucacc 16
<210>70
<211>16
<212>RNA
＜213〉artificial sequence
<220>
＜223〉synthetic oligonucleotide
<220>
<221>misc_feature
<222>(11)..(11)
＜223〉wherein n is a thymidine
<220>
<221>misc_feature
<222>(13)..(13)
＜223〉wherein n is a thymidine
<400>70
ccgagccgau nunacc 16
<210>71
<211>16
<212>RNA
＜213〉artificial sequence
<220>
＜223〉synthetic oligonucleotide
<400>71
ccgagccgau uucacc 16
<210>72
<211>16
<212>RNA
＜213〉artificial sequence
<220>
＜223〉synthetic oligonucleotide
<220>
<221>misc_feature
<222>(13)..(13)
＜223〉wherein n is a thymidine
<400>72
ccgagccgau uunacc 16
<210>73
<211>16
<212>RNA
＜213〉artificial sequence
<220>
＜223〉synthetic oligonucleotide
<400>73
ccgagccgau uuaacc 16
<210>74
<211>16
<212>RNA
＜213〉artificial sequence
<220>
＜223〉synthetic oligonucleotide
<400>74
ccgagccgau ugcacc 16
<210>75
<211>16
<212>RNA
＜213〉artificial sequence
<220>
＜223〉synthetic oligonucleotide
<220>
<221>misc_feature
<222>(13)..(13)
＜223〉wherein n is a thymidine
<400>75
ccgagccgau ucnacc 16
<210>76
<211>16
<212>RNA
＜213〉artificial sequence
<220>
＜223〉synthetic oligonucleotide
<400>76
ccgagccgau ugaacc 16
<210>77
<211>16
<212>RNA
＜213〉artificial sequence
<220>
＜223〉synthetic oligonucleotide
<400>77
ccgagccgag uucacc 16
<210>78
<211>16
<212>RNA
＜213〉artificial sequence
<220>
＜223〉synthetic oligonucleotide
<220>
<221>misc_feature
<222>(10)..(10)
＜223〉wherein n is a thymidine
<400>78
ccgagccgan uucacc 16
<210>79
<211>16
<212>RNA
＜213〉artificial sequence
<220>
＜223〉synthetic oligonucleotide
<220>
<221>misc_feature
<222>(10)..(10)
＜223〉wherein n is a thymidine
<400>79
ccgagccgan uucacc 16
<210>80
<211>16
<212>RNA
＜213〉artificial sequence
<220>
＜223〉the synthetic Nucleotide of raising
<400>80
ccgagccgaa uucacc 16
<210>81
<211>16
<212>RNA
＜213〉artificial sequence
<220>
＜223〉synthetic oligonucleotide
<400>81
ccgagccgag cucacc 16
<210>82
<211>16
<212>RNA
＜213〉artificial sequence
＜223〉synthetic oligonucleotide
<400>82
ccgagccgaa gguacc 16
<210>83
<211>16
<212>RNA
＜213〉artificial sequence
<220>
＜223〉synthetic oligonucleotide
<400>83
ccgagccgaa ggugcc 16
<210>84
<211>16
<212>RNA
＜213〉artificial sequence
<220>
＜223〉synthetic oligonucleotide
<400>84
ccgagccgaa gcuccc 16
<210>85
<211>16
<212>RNA
＜213〉artificial sequence
<220>
＜223〉synthetic oligonucleotide
<400>85
ccgagccgaa gauacc 16
<210>86
<211>16
<212>RNA
＜213〉artificial sequence
<220>
＜223〉synthetic oligonucleotide
<400>86
ccgagccgaa gguccc 16
<210>87
<211>16
<212>RNA
＜213〉artificial sequence
<220>
＜223〉synthetic oligonucleotide
<400>87
ccgagccgaa gcuacc 16
<210>88
<211>16
<212>RNA
＜213〉artificial sequence
<220>
＜223〉the synthetic Nucleotide of raising
<400>88
ccgagccgaa gcugcc 16
<210>89
<211>20
<212>RNA
＜213〉artificial sequence
<220>
＜223〉synthetic oligonucleotide
<400>89
acccaucuau uauauaacuc 20
<210>90
<211>22
<212>DNA
＜213〉artificial sequence
<220>
＜223〉synthetic oligonucleotide
<400>90
tcgtcgtttt cggcggccgc cg 22
<210>91
<211>21
<212>DNA
＜213〉artificial sequence
<220>
＜223〉synthetic oligonucleotide
<400>91
tcgtcgtttt tcggtcgttt t 21

Claims (107)

  1. One kind be used to stimulate before the inflammatory cytokine method of producing, described method comprises:
    The N-U-R that comprises of the cell of TLR8 and following dosage will be expressed 1-R 2The external contact of RNA oligoribonucleotide (ORN), wherein N is that ribonucleotide and N do not comprise U, U is the uridylic or derivatives thereof, R is a ribonucleotide, wherein R 1And R 2One of at least be adenosine (A) or cytosine(Cyt) or derivatives thereof, and wherein R not U, unless N-U-R 1-R 2Contain at least two A, wherein said ORN is not ACCCAUCUAUUAUAUAACUC (SEQ ID NO:89) and does not comprise the TLR7/8 motif, and wherein said ORN length is 4-100, described consumption can produce by the preceding inflammatory cytokine of effective stimulus, and makes that the IFN-α production that wherein responds to ORN is not significantly induced for background.
  2. One kind be used to stimulate before the method for inflammatory cytokine, comprising:
    The N-U-R that comprises of the cell of TLR8 and following dosage will be expressed 1-R 2The external contact of RNA oligoribonucleotide (ORN), wherein N is that ribonucleotide and N do not comprise U, U is the uridylic or derivatives thereof, R is a ribonucleotide, wherein R 1And R 2One of at least be adenosine (A) or cytosine(Cyt) or derivatives thereof, wherein R is not U, unless N-U-R 1-R 2Contain at least two A, wherein said ORN do not comprise the TLR7/8 motif and not with N-[1-(2,3-two oleoyl oxygen) propyl group]-N, N, N trimethyl ammonium methylsulfuric acid ester (DOTAP) is compound, and wherein said ORN length is 4-100, described consumption can effective stimulus before inflammatory cytokine production and IFN-α production of wherein responding to ORN for background, significantly do not induced.
  3. 3. claim 1 or 2 method, the IFN-α production that wherein responds to described ORN is less than 300pg/ml.
  4. 4. the described method of claim 1, wherein said ORN and N-[1-(2,3-two oleoyl oxygen) propyl group]-N, N, N trimethyl ammonium methylsulfuric acid ester (DOTAP) or polycation peptide are compound.
  5. 5. claim 1 or 2 described methods, wherein N is adenosine or cytosine(Cyt) (C) or derivatives thereof.
  6. 6. claim 1 or 2 described methods, wherein N is adenosine or cytosine(Cyt).
  7. 7. claim 1 or 2 described methods, wherein U is a uridylic.
  8. 8. claim 1 or 2 described methods, wherein said ORN comprises at least one AU.
  9. 9. claim 1 or 2 described methods, wherein said ORN comprises at least one CU.
  10. 10. claim 1 or 2 described methods, the cell of wherein expressing TLR8 is the dendritic cell of monocyte or monocyte derived.
  11. 11. claim 1 or 2 described methods, the cell of wherein expressing TLR8 is mDC.
  12. 12. claim 1 or 2 described methods, wherein said ORN contains at least two AU.
  13. 13. claim 1 or 2 described methods, wherein said ORN contains at least three AU.
  14. 14. claim 1 or 2 described method, wherein N-U-R 1-R 2Contain at least 3 As.
  15. 15. claim 1 or 2 described method, wherein N-U-R 1-R 2Contain at least 2 Cs.
  16. 16. claim 1 or 2 described method, wherein N-U-R 1-R 2Contain at least one G or C.
  17. 17. claim 1 or 2 described methods, wherein said ORN contains at least one place backbone modifications.
  18. 18. claim 1 or 2 described methods, wherein said ORN is a strand.
  19. 19. comprise N-U-R 1-R 2RNA oligonucleotide (ORN), wherein N is that ribonucleotide and N do not comprise U, U is the uridylic or derivatives thereof, R is a ribonucleotide, wherein R 1And R 2One of at least be adenosine (A) or cytosine(Cyt) or derivatives thereof, wherein R is not U, unless N-U-R 1-R 2Contain at least two A, and wherein said ORN is not ACCCAUCUAUUAUAUAACUC (SEQ ID NO:89) and do not comprise the TLR7/8 motif, and wherein said ORN length is 4-100 and comprises at least one place backbone modifications.
  20. 20. the described ORN of claim 19, it also comprises pharmaceutically useful vehicle.
  21. 21. the described ORN of claim 19, wherein said ORN and N-[1-(2,3-two oleoyl oxygen) propyl group]-N, N, N trimethyl ammonium methylsulfuric acid ester (DOTAP) is compound.
  22. 22. the described ORN of claim 19, wherein said ORN is a strand.
  23. 23. comprise N-U-R 1-R 2RNA oligonucleotide (ORN), wherein N is that ribonucleotide and N do not comprise U, U is the uridylic or derivatives thereof, R is a ribonucleotide, wherein R 1And R 2One of at least be adenosine (A) or cytosine(Cyt) or derivatives thereof, wherein R is not U, unless N-U-R 1-R 2Contain at least two A, wherein said ORN do not comprise the TLR7/8 motif and not with N-[1-(2,3-two oleoyl oxygen) propyl group]-N, N, N trimethyl ammonium methylsulfuric acid ester (DOTAP) is compound, and wherein said ORN length is 4-100, and comprise at least one place backbone modifications, and be formulated in the pharmaceutically useful vehicle.
  24. 24. claim 19 or 23 described ORN, wherein N is adenosine or cytosine(Cyt) (C) or derivatives thereof.
  25. 25. claim 19 or 23 described ORN, wherein N is adenosine or cytosine(Cyt).
  26. 26. claim 19 or 23 described ORN, wherein U is a uridylic.
  27. 27. claim 19 or 23 described ORN, wherein ORN comprises at least two AU.
  28. 28. claim 19 or 23 described ORN, wherein ORN comprises at least two CU.
  29. 29. claim 19 or 23 described ORN, wherein N-U-R 1-R 2Comprise at least 3 As.
  30. 30. claim 19 or 23 described ORN, wherein N-U-R 1-R 2Comprise at least 2 Cs.
  31. 31. claim 19 or 23 described ORN, wherein N-U-R 1-R 2Comprise at least one G or C.
  32. 32. claim 19 or 23 described ORN, wherein said ORN is a strand.
  33. 33. claim 19 or 23 described ORN, wherein said ORN are one of following: U*U*A*G*G*C*A*C (SEQ ID NO:2), A*U*A*G*G*C*A*C (SEQ IDNO:4), G*C*C*A*C*C*G*A*G*C*C*G*A*A*U*A*U*A*C*C (SEQ IDNO:11), A*U*A*U*A*U*A*U*A*U*A*U*A*U*A*U*A*U*A*U (SEQ IDNO:12), U*U*A*U*U*A*U*U*A*U*U*A*U*U*A*U*U*A*U*U (SEQ IDNO:13), A*A*U*A*A*U*A*A*U*A*A*U*A*A*U*A*A*U*A*A (SEQ IDNO:16), A*A*A*U*A*A*A*U*A*A*A*U*A*A*A*U*A*A*A*U (SEQ IDNO:17), A*A*A*A*U*A*A*A*A*U*A*A*A*A*U*A*A*A*A*U (SEQ IDNO:18), C*U*A*C*U*A*C*U*A*C*U*A*C*U*A*C*U*A*C*U (SEQ IDNO:24), U*U*A*U*U*A*U (SEQ ID NO:30) or U*A*U*A*U*A*U (SEQ IDNO:33).
  34. 34. claim 19 or 23 described ORN, it also comprises atomizer.
  35. 35. claim 19 or 23 described ORN, it also comprises sucker.
  36. 36. the described ORN of claim 35, wherein said sucker is a metered dose inhaler.
  37. 37. the described ORN of claim 35, wherein said sucker is a powder inhalator.
  38. 38. claim 19 or 23 described ORN, it also comprises chemotherapeutic.
  39. 39. claim 19 or 23 described ORN, it also comprises antiviral agent.
  40. 40. claim 19 or 23 described ORN, it also comprises antigen.
  41. 41. claim 20 or 23 described ORN, wherein pharmaceutically useful vehicle is used for injection by preparation.
  42. 42. claim 20 or 23 described ORN, wherein pharmaceutically useful vehicle is used for mucosal administration by preparation.
  43. 43. one kind is used for the treatment of method for cancer, it comprises the ORN that the experimenter of needs is used among the claim 19-23 each with the amount of effective treatment cancer.
  44. 44. the described method of claim 43, it also comprises uses chemotherapy to the experimenter.
  45. 45. the described method of claim 43, it also comprises uses radiation to the experimenter.
  46. 46. a method that is used for the treatment of asthma, it comprises the ORN that the experimenter of needs is used among the claim 19-23 each with the amount of effective treatment asthma.
  47. 47. one kind is used for the treatment of allergic method, it comprises the ORN that the experimenter of needs is used among the claim 19-23 each with the amount of effective treatment asthma.
  48. 48. the described method of claim 47, wherein said experimenter suffers from rhinallergosis.
  49. 49. a method that is used in experimenter regulation and control immunne response, it comprises the ORN that the experimenter of needs is used among the claim 19-23 each with the amount of effective regulation and control immunne response.
  50. 50. the described method of claim 49, wherein said ORN is delivered to described experimenter to treat autoimmune disease in the experimenter.
  51. 51. the described method of claim 49, wherein said ORN is delivered to described experimenter to treat airway remodeling in the experimenter.
  52. 52. the described method of claim 49, wherein said ORN is delivered to described experimenter and non-delivery of antigens.
  53. 53. the described method of claim 49, wherein said ORN is delivered to described experimenter to be selected from following approach: mouth, nose, hypogloeeis, intravenously, subcutaneous, mucous membrane, breathing, direct injection and through skin.
  54. 54. the described method of claim 49, wherein said ORN is delivered to described experimenter with the amount of effective inducing cell factor expression.
  55. 55. the described method of claim 53, wherein said cytokine are selected from the group that TNF α, IL-10, IL-6, IFN-γ, MCP1 and IL-12 constitute.
  56. 56. a method that is used for the treatment of the asthma that is increased the weight of by virus infection comprises the experimenter of needs is used among the claim 19-23 each ORN with following dosage, described consumption is the asthma that increased the weight of by virus infection of treatment effectively.
  57. 57. a method that is used for transmissible disease, it comprises the ORN that the experimenter of needs is used among the claim 19-23 each with the amount of the described transmissible disease of effective treatment.
  58. 58. the described method of claim 57, wherein said experimenter suffers from virus infection.
  59. 59. the described method of claim 58, wherein said virus infection are hepatitis B.
  60. 60. the described method of claim 58, wherein said virus infection are hepatitis C.
  61. 61. the described method of claim 58, it also comprises uses antiviral agent to the experimenter.
  62. 62. the described method of claim 61, wherein said antiviral agent is connected with ORN.
  63. 63. the described method of claim 57, wherein said ORN is sent by being selected from following approach: mouth, nose, hypogloeeis, intravenously, subcutaneous, mucous membrane, breathing, direct injection and through skin.
  64. 64. claim 19 or 23 described ORN, wherein said ORN motif is separated by non-nucleotide joint and 5 ' ribonucleotide.
  65. 65. claim 19 or 23 described ORN, wherein said ORN motif is separated by non-nucleotide joint and 3 ' ribonucleotide.
  66. 66. claim 19 or 23 described ORN, wherein said ORN motif is separated by non-nucleotide joint and 5 ' and 3 ' ribonucleotide.
  67. 67. claim 19 or 23 described ORN, wherein said ORN is formulated in the polycation vehicle.
  68. 68. the described method of claim 43, it also comprises the CpG nucleic acid of the experimenter being used immunostimulating.
  69. 69. the described method of claim 68, wherein said ORN and described immunostimulating CpG nucleic acid are used independently.
  70. 70. the described method of claim 68, wherein said ORN and described immunostimulating CpG nucleic acid are configured to conjugate.
  71. 71. the described method of claim 68, wherein said ORN and described immunostimulating CpG nucleic acid are configured to mixture.
  72. 72. one kind is used to reduce the method that inhibitive ability of immunity CD4+ regulates (Treg) cell, described method comprises: the CD4+Treg cell is contacted with the following composition of following dosage, described consumption effectively reduces the effect of CD4+Treg cell inhibiting, and described composition comprises each described ORN among the claim 19-23.
  73. 73. the described method of claim 72, wherein said composition also contain immunostimulating CpG nucleic acid.
  74. 74. the described method of claim 73, wherein said ORN is connected with described immunostimulating CpG nucleic acid.
  75. 75. the described method of claim 73, wherein said ORN and described immunostimulating CpG nucleic acid exist as conjugate.
  76. 76. the purposes of each ORN composition in the medicament that is used for the treatment of cancer is made among the claim 19-42.
  77. 77. the described purposes of claim 76, wherein said medicament uses with chemotherapy.
  78. 78. the described purposes of claim 76, wherein said medicament uses with radiation.
  79. 79. each ORN composition is in the purposes that is used for making the medicament for the treatment of asthma among the claim 19-42.
  80. 80. each ORN composition is in the purposes that is used for making the allergic medicament of treatment among the claim 19-42.
  81. 81. the described purposes of claim 80, wherein said transformation reactions is rhinallergosis.
  82. 82. each ORN composition is in the purposes of the medicament that is used for making the regulation and control immunne response among the claim 19-42.
  83. 83. the described purposes of claim 82, wherein said medicament is used for the treatment of autoimmune disease.
  84. 84. the described purposes of claim 82, wherein said medicament is used for the treatment of airway remodeling.
  85. 85. the described purposes of claim 82, wherein said medicament is used for using with antigen.
  86. 86. the described purposes of claim 82, wherein said medicament are used for using by being selected from following approach: mouth, nose, hypogloeeis, intravenously, subcutaneous, mucous membrane, breathing, direct injection and through skin.
  87. 87. the described purposes of claim 82, wherein said immunne response comprises cytokine induction.
  88. 88. the described purposes of claim 87, wherein said cytokine are selected from TNF α, IL-10, IL-6, IFN-γ, MCP1 and IL-12.
  89. 89. the purposes of each ORN composition in the medicament that is used for the treatment of the asthma that is increased the weight of by virus infection is made among the claim 19-42.
  90. 90. the purposes of each ORN composition in the medicament that is used for the treatment of transmissible disease is made among the claim 19-42.
  91. 91. the described purposes of claim 90, wherein said medicament is used for the treatment of virus infection.
  92. 92. the described purposes of claim 91, wherein said virus infection are hepatitis B.
  93. 93. the described purposes of claim 91, wherein said virus infection are hepatitis C.
  94. 94. the described purposes of claim 91, wherein said medicament is used for using with antiviral agent.
  95. 95. the described purposes of claim 94, wherein said antiviral agent is connected with described ORN.
  96. 96. the described purposes of claim 90, wherein said medicament are used for sending by being selected from following approach: mouth, nose, hypogloeeis, intravenously, subcutaneous, mucous membrane, breathing, direct injection and through skin.
  97. 97. claim 19 or 23 described ORN, wherein said ORN motif is separated by non-nucleotide joint and 5 ' ribonucleotide.
  98. 98. claim 19 or 23 described ORN, wherein said ORN motif is separated by non-nucleotide joint and 3 ' ribonucleotide.
  99. 99. claim 19 or 23 described ORN, wherein said ORN motif is separated by non-nucleotide joint and 5 ' and 3 ' ribonucleotide.
  100. 100. the described purposes of claim 76, wherein said medicament are used for using together with immunostimulating CpG nucleic acid.
  101. 101. the described purposes of claim 100, wherein said ORN and described immunostimulating CpG nucleic acid are used for individual application.
  102. 102. the described purposes of claim 100, wherein said ORN and described immunostimulating CpG nucleic acid are configured to conjugate.
  103. 103. the described purposes of claim 100, wherein said ORN and described immunostimulating CpG nucleic acid are configured to mixture.
  104. 104. one kind is used to reduce the method that inhibitive ability of immunity CD4+ regulates (Treg) cell, described method comprises: the CD4+Treg cell is contacted with the following composition of following dosage, described consumption effectively reduces the effect of CD4+Treg cell inhibiting, and described composition comprises each described ORN among the claim 19-23.
  105. 105. the described method of claim 104, wherein said composition also contain immunostimulating CpG nucleic acid.
  106. 106. the described method of claim 105, wherein said ORN is connected with described immunostimulating CpG nucleic acid.
  107. 107. the described method of claim 105, wherein said ORN and described immunostimulating CpG nucleic acid exist as conjugate.
CN2006800440306A 2005-11-25 2006-11-22 Immunostimulatory oligoribonucleotides Active CN101331229B (en)

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