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CN101340931A - Modulation of tlr-mediated immune responses using adaptor oligonucleotides - Google Patents

Modulation of tlr-mediated immune responses using adaptor oligonucleotides Download PDF

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Publication number
CN101340931A
CN101340931A CNA200680044386XA CN200680044386A CN101340931A CN 101340931 A CN101340931 A CN 101340931A CN A200680044386X A CNA200680044386X A CN A200680044386XA CN 200680044386 A CN200680044386 A CN 200680044386A CN 101340931 A CN101340931 A CN 101340931A
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oligonucleotide
tlr7
cell
tlr8
tlr
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M·朱尔克
J·沃尔莫
A·M·克莱格
E·尤尔曼
B·O·诺尔
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Coley Pharmaceutical GmbH
Coley Pharmaceutical Group Inc
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Coley Pharmaceutical GmbH
Coley Pharmaceutical Group Inc
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Abstract

The invention relates to the ability of certain oligonucleotides to modify the profile of TLR ligands such as TLR7, TLR8 and TLR9 ligands.

Description

Use and connect the immunne response that oligonucleotide is regulated the TLR mediation
Related application
The application requires JIUYUE in 2005 to submit in 27th according to 35U.S.C. ξ 119, denomination of invention is the U.S. Provisional Application serial number 60/720 of " use and connect the immunne response that oligonucleotide is regulated the TLR mediation ", 981 priority is incorporated herein by reference the full content of the document.
Invention field
The present invention relates to use specific nucleic acid to regulate the immunne response of TLR mediation.
Background of invention
With the reaction of some motif in the DNA of bacteria be autarcetic critical function.Known for a long time DNA of bacteria is mitogenetic for mammal bone-marrow-derived lymphocyte (B cell), and mammalian DNA generally is not like this.This immunity identification is oriented to the discovery that is positioned at the specific DNA sequences of center on the motif that comprises unmethylated CpG dinucleotide and has opened the field of leading to the molecular immunology means.(1995) Nature 374:546-9 such as Krieg AM.Can use the CpG dinucleotide that comprises in some preferred flanking sequence scope, promptly the synthetic oligodeoxynucleotidecombination of CpG motif (ODN) reproduces the immunostimulation of so-called CpG DNA.Reported that the ODN (CpG-ODN) that contains CpG brings into play many effects to immune various types of cells, comprise and prevent elementary B cell generation apoptosis, promoting cell cycle to enter and make immunne response be partial to the Th1-para-immunity replys, for example, induce interleukin-6 (IL-6), interleukin 12 (IL-12), IFN-(IFN-γ), the IgG2a of active antigen specificity cytolytic t lymphocyte (CTL) and inducing mouse.
Recently the immunoregulation effect of having reported CpG DNA relates to by Toll sample receptor 9 (TLR9) and carries out the signal conduction.Think that CpG DNA takes in cell and is delivered to the endosome compartment through the non-specific approach of sequence, wherein it and TLR9 interact in the sequence-specific mode.The TLR9 signal transduction path causes a large amount of immune function related genes to be induced, and comprises noticeable NF-κ B.
TLR is present in the big receptor family (pathogen associated molecular pattern or PAMP) of the specific molecular structure in the pathogen and is also referred to as pattern recognition receptor (PRR) for identification.The immunocyte of expression PRR is activated when identification PAMP and causes that the optimal adaptation immunne response produces.The PRR that is made up of 10 kinds of different TLR hypotype TLR1-TLR10 has been described.Described this class TLR and participated in identification double-stranded RNA (TLR3), lipopolysaccharide (LPS) (TLR4), bacterial flagellin (TLR5), little antiviral compound (TLR7 and TLR8) and DNA of bacteria or CpGODN (TLR9).(referring to summary (2003) the Curr Opin Drug DiscovDevel 6:204-17 of Uhlmann etc.).In addition, identified think with and by TLR7 and TLR8 and signal interactional RNA molecule takes place (referring to International Patent Application PCT/US03/10406).Think that this para-immunity stimulates the RNA molecule to have the base sequence that comprises at least one guanine and at least one uracil.Be rich in G, the immunostimulation RNA of U need not as to the described CpG motif of TLR9.Think that the corresponding RNA of actual discovery divides subclass to be present in ribosomal RNA (rRNA), transfer RNA (tRNA) is in messenger RNA (mRNA) and the viral RNA (vRNA).
After finding immunostimulation CpG DNA, the report that a large amount of descriptions have the short dna sequence of immunosuppressive action has appearred.Known gathering-G sequence is an inhibitive ability of immunity.Described among the PCT patent application WO 00/14217 that announces to comprise and suppressed motif N 1N 2GN 3The ODN of G, wherein N 1, N 2And N 3In at least two kinds be G (guanosine).Krieg and colleague have described one group of inhibition 15-mer ODN with 3 or 4 continuous G, and inductive apoptosis protection of they zest ODN capable of blocking and cell cycle enter.(2001) Antisense Nucleic AcidDrug Dev 11:247-56 such as Lenert P; (2002) Eur J Immunol 32:1212-22 such as Stunz LL; (2003) Antisense Nucleic Acid Drug Dev 13:143-50 such as Lenert P.It is reported that the immunosuppressive action of these ODN has specificity to CpG-ODN and relates to the mechanism of non-atomistic competition cellular uptake.(2002) Eur J Immunol 32:1212-22 such as Stunz LL.Klinman and colleague have reported single inhibitive ability of immunity ODN independently.(2002) Arthritis Rheum 46:2219-24 such as Zeuner RA; (2002) J Immunol169:5590-4 such as Yamada H.
Summary of the invention
The invention provides the method and composition of the immunne response of regulating the TLR mediation, comprise that suppressing some replys, and strengthens other and replys or its some combination.The present invention partly relates to following discovery: some oligonucleotide obviously can change the TLR signal transport properties of TLR part.Therefore, according to an embodiment, these " connection " oligonucleotide can mainly change into the TLR8 part with the TLR7 part, and according to another embodiment, they mainly change into the TLR8 part with the TLR7/8 part.
Therefore, provide the method for the immunne response that stimulates the TLR8 mediation among the present invention in one aspect, comprise to the experimenter that these needs are arranged give the immunne response that effective stimulus TLR8 mediates consumption the TLR7/8 part be connected oligonucleotide.
The present invention provides the immunne response that makes TLR7 mediation to be redirected method for the immunne response of TLR8 mediation in one aspect of the method, comprises that experimenter to the immunne response of experience TLR7 mediation gives effectively to make the immunne response of TLR7 mediation to be redirected the connection oligonucleotide of the consumption of the immunne response that mediates for TLR8.
The present invention provides in one aspect of the method and has comprised TLR7/8 part and the compositions that is connected oligonucleotide.
In one embodiment, the TLR7/8 part is the TLR7 part, such as the TLR7 ligands specific.The TLR7 part can be guanosine analogue, such as, but be not limited to the guanosine that C8-replaces.The TLR7 part can be 3M-001.The TLR7 part can be the chemical compound based on adenosine, such as, but be not limited to 6-amino-9-benzyl-2-(3-hydroxyl-propoxyl group)-9H-purine-8-alcohol or 6-amino-9-benzyl-2-butoxy-9H-purine-8-alcohol.The TLR7 part can-guanosine assorted for the 7-denitrogenation.
The example of the guanosine that C8-replaces comprises 7-pi-allyl-7,8-dihydro-8-oxo-guanosine (loxoribine), 7-thia-8-oxo guanosine (immunosine, isatoribine, ANA245,7-thia-8-oxo-7,8-dihydro guanosine, 5-amino-3-(β-D-ribofuranosyl)-3H, 6H-thiazole [4,5-d] pyrimidine-2, the 7-diketone), 8-TGR, 8-bromine guanosine, the 8-methylguanosine, 8-oxo-7,8-dihydro guanosine, C8-virtue amino-2 '-deoxyguanosine, C8-propinyl-guanosine, the guanosint riboside that C8-and N7-replace, 7-methyl-8-oxo guanosine, the amino guanosine of 8-, 8-hydroxyl-2 '-deoxyguanosine, the 7-denitrogenation is assorted-guanosine and 8-hydroxyl guanosine that 8-replaces.In certain embodiments, the guanosine that C8-replaces is a loxoribine, immunosine or 7-cadeguomycin.
In another embodiment, the TLR7/8 part is the TLR8 part, comprises the TLR8 ligands specific.The TLR8 part can be 3M-002.At part is in the embodiment of TLR8 part, and the immunne response that TLR8 mediates can strengthen 2-times at least than the immunne response level that does not connect acquisition under the oligonucleotide existence, 3-times, and 4-times, 5-times, 10-times, 15-times, more than 20-times or 20-times.
In another embodiment, the TLR7/8 part is a TLR7 part TLR8 part.The example of this class TLR 7/8 part is an imidazoquinolie.The example of imidazoquinolines comprises immidazoquinolinaminas, imidazopyridine amine, 6, the condensed cycloalkyl imidazopyridine of 7-amine, 1, Bridge 2 connects immidazoquinolinaminas, R-848 (S-28463 or resiquimod), 4-amino-2 ethoxyl methyls-α, alpha-alpha-dimethyl-1 H-imidazo [4,5-c] quinoline-1-ethanol, 1-(2-methyl-propyl)-1H-imidazo [4,5-c] quinoline-4-amine (R-837 or imiquimod) or S-27609.In some important embodiment, imidazoquinolie is R848.
The TLR7/8 part can be 3M-003.At part is in the embodiment of TLR7 and TLR8 part, and the immunne response that TLR8 mediates can strengthen 2-times at least than the immunne response level that does not connect the TLR8 mediation that obtains under the oligonucleotide existence, 3-times, 4-times, 5-times, 10-doubly, 15-times, more than 20-times or 20-times.
In one embodiment, connect oligonucleotide and comprise formula 5 ' X-N 5-X 3 ' or formula 5 ' X-N 43 ', wherein X can and can exist or not exist and N for any nucleotide 4And N 5Explain 4 or 5 successive T (thymidine), U (uracil) or A (adenine) make N 4Or N 5In each N identical.According to the difference of embodiment, oligonucleotide is 7,8,9,10,11,12,13,14,15,16,17 or 17 above length of nucleotides.In important embodiment, it comprise key between at least one thiophosphate nucleotide (up to and comprise complete D2EHDTPA main chain).
In a relevant embodiment, connect oligonucleotide and comprise formula 5 ' X a-TTTTT-X b3 ', X wherein aAnd X bCan be independently for any nucleotide and can exist or not exist.X aAnd X bCan be in one or more nucleotide (for example, 1-100 nucleotide).In one embodiment, oligonucleotide comprises successive T more than 6,7 or 7.In an important embodiment, connecting oligonucleotide is (dT) homopolymer, and it is optional to be 17 length of nucleotides.
In another embodiment, connect oligonucleotide and comprise formula 5 ' X a-UUUUU-X b3 ', X wherein aAnd X bCan be independently for any nucleotide and can exist or not exist.X aAnd X bCan be one or more nucleotide (for example, 1-100 nucleotide) that in one embodiment, oligonucleotide can comprise successive U more than 6,7 or 7.In an important embodiment, oligonucleotide is uracil (dU) homopolymer, and it is optional to be 17 length of nucleotides.
In another embodiment, connect oligonucleotide and comprise formula 5 ' X a-AAAAA-X b3 ', X wherein aAnd X bCan be independently for any nucleotide and can exist or not exist.X aAnd X bCan be in one or more nucleotide (for example, 1-100 nucleotide).In one embodiment, oligonucleotide can comprise successive A more than 6,7 or 7.In an important embodiment, oligonucleotide is adenine (dA) homopolymer, and it is optional to be 17 length of nucleotides.
In another embodiment, connect oligonucleotide and comprise formula 5 ' C n-T m-C p3 ', wherein n is the integer of 0-100, p is that integer and the m of 0-100 is the integer of 0-100.In one embodiment, n and p sum are the value that is equal to or less than m, make that the C content of whole oligonucleotide is 50%, are lower than 50%, are lower than 45%, be lower than 40%, be lower than 35%, be lower than 30%, be lower than 25%, be lower than 20%, be lower than 15%, be lower than 10%, be lower than 5% or lower.In certain embodiments, n is at 3-7, m at 2-10 and p at 4-8, as long as satisfy above-mentioned percentage ratio.Example comprises 5 ' C 3T 10C 43 ' (SEQ ID NO:1) and 5 ' C 4T 8C 53 ' (SEQ ID NO:2).
The connection oligonucleotide comprises the CpG motif or it can lack such motif.This motif can be unmethylated CpG motif.
In one embodiment, the experimenter is given the TLR7/8 part respectively and is connected oligonucleotide.In these and other embodiment, part and oligonucleotide can be given each other simultaneously.In another embodiment, oligonucleotide and TLR7/8 part are puted together each other.In one embodiment, make connection oligonucleotide and TLR7/8 part covalent bond.
In one embodiment, the connection oligonucleotide is DNA, and in another embodiment, it is RNA.In one embodiment, when independent use, connect oligonucleotide and be not immunostimulating.In one embodiment, independent connection oligonucleotide does not stimulate the immunne response of TLR7-or TLR8 mediation.
In one embodiment, described compositions further comprises antigen and/or described method and further comprises and give antigen to the experimenter.
In one embodiment, the experimenter has infection.In another embodiment, the experimenter has cancer.In another embodiment, the experimenter has anaphylaxis or asthma.
Described compositions can be for comprising the pharmaceutical preparation of pharmaceutically acceptable carrier.
Aforesaid TLR part is provided among the present invention in one aspect and has been connected oligonucleotide and is combined in preparation and is used for application the premunitive preparation of experimenter.
The method for preparing vaccine is provided among the present invention in one aspect.This method comprise with aforesaid TLR part be connected the step that oligonucleotide combination and antigen and optional pharmaceutically acceptable carrier are combined closely.
Aforesaid TLR part is provided among the present invention in one aspect and has been connected oligonucleotide and is combined in application in the preparation that preparation treatment experimenter infects.
The compositions that is used for the treatment of infection is provided among the present invention in one aspect, and it comprises aforesaid TLR part and is connected oligonucleotide combination and antiviral agents or medicament.
Application in aforesaid TLR part being provided among the present invention in one aspect and being connected the medicament that oligonucleotide is combined in preparation treatment experimenter cancer.
The compositions that is used for the treatment of cancer is provided among the present invention in one aspect, and it comprises aforesaid TLR part and is connected oligonucleotide combination and cancer with medicament.
Application in aforesaid TLR part being provided among the present invention in one aspect and being connected the medicament that oligonucleotide is combined in preparation treatment experimenter anaphylactic disease.
The compositions that is used for the treatment of anaphylactic disease is provided among the present invention in one aspect, and it comprises aforesaid TLR part and is connected oligonucleotide and is combined in preparation treatment experimenter anaphylaxis with medicament.
Application in aforesaid TLR part being provided among the present invention in one aspect and being connected the medicament that oligonucleotide is combined in preparation treatment experimenter asthma.
The compositions that is used for the treatment of asthma is provided among the present invention in one aspect, and it comprises aforesaid TLR part and is connected oligonucleotide and is combined in preparation treatment experimenter asthma with medicament.
The present invention provides TLR7/8 part and the method that is connected oligonucleotide identified in others.Therefore, the method of identifying the TLR8 part is provided among the present invention in one aspect, comprise making the cell of expressing TLR8 have and not be connected engaged test part in the presence of the oligonucleotide, and be determined at and be not connected the stimulation of the cell of the expression TLR8 of this test ligand of response under the oligonucleotide existence.According to there being the increase that connects stimulation in the presence of the oligonucleotide to identify the TLR8 part.The increase that stimulates preferably surpasses and does not connect the oligonucleotide existence irritation level of non-zero down.
The present invention provides the method for TLR7 part in one aspect of the method, comprise making cell of expressing TLR7 and the cell of expressing TLR8 have and not be connected engaged test part in the presence of the oligonucleotide, and be determined at and be not connected oligonucleotide and have the cell of the expression TLR7 of this test ligand of response and the stimulation of the cell of expression TLR8 down.Identify the TLR7 part according to the stimulation increase in stimulation minimizing that connects in the cell of expressing TLR7 in the presence of the oligonucleotide and the cell of expressing TLR8 are arranged.In aspect this, the increase of stimulation preferably surpasses the zero irritation level that does not connect under the oligonucleotide existence.
The present invention provides the method that connects oligonucleotide of identifying in one aspect of the method, comprise making the cell of expressing TLR8 be connected contact TLR7 part in the presence of the oligonucleotide having, and be determined at and stimulation that test is not connected the cell of the expression TLR8 that responds this TLR7 part under the oligonucleotide existence with test not.Can there be the level in the presence of the TLR7 part to test the level in the presence of the oligonucleotide and/or having the TLR7 part to compare with irritation level and in the level of not testing in the presence of the oligonucleotide with the known level that is connected in the presence of the oligonucleotide with having.In one embodiment, according to connecting oligonucleotide having the cytositimulation to expressing TLR8 that connects in the presence of the oligonucleotide when not having than it to increase to identify, condition is the immunne response (as by above-mentioned controlled trial mensuration) that this oligonucleotide self does not mediate TLR8 and mediates.
TLR7 and TLR8 part be connected oligonucleotide and can be any in those described in aforementioned aspect and the embodiment.The cell of expressing TLR7 or TLR8 can be for through transforming to express the cell of (for example dystopy ground) TLR7 or TLR8 for the cell of natural expression TLR7 or TLR8 or they.Can measure stimulation by signal conduction and downstream effect thereof through TLR7 or TLR8, comprise, but being not limited to gene expression (for example increases, manifest as the bonded report construct of promoter element of use and TLR7 or TLR8 downstream target), somatomedin (cytokine) is expressed, generation or secretion increase etc.
These and other aspect of the present invention is described hereinafter in more detail.
The accompanying drawing summary
Figure 1A-1B represents the ODN selective reinforcement and suppresses the activity to people TLR8 and TLR7 of R-848 mediation.Figure 1A represents that ODN 6056 (SEQ ID NO:3) and 1982 (SEQ ID NO:4) (with regard to sequence, referring to table 1 and 2) suppress the activity of R-848 to people TLR7.The widow (dT) that the HEK293 cell of stably express TLR7 and NF-κ B luciferase report construct and 2 μ M R-848 are measured shown in be with or without 17Under existing, homopolymer ODN 6056 or heteropolymer ODN 1982 hatch.The activity of independent R-848 is set at 100%.Triplicate all data points of mensuration and displaying meansigma methods (+/-SD).Shown in the result be one of twice above independent experiment.Figure 1B represents widow (dT) 17ODN sequence-selective reinforcement R-848 is to the activity of people TLR8.With the R-848 of the HEK293 cell of stably express hTLR8 and NF-κ B-luciferase report construct and recruitment not or 0.1,1 and 5 μ M widows (dT) are arranged 17Under existing, homopolymer ODN 6056 or heteropolymer ODN 1982 hatch.Reference culture medium background calculates the NF-kB activation to stimulate.Triplicate all data points of mensuration and displaying meansigma methods (+/-SD).Shown in the result be one of four independent experiments.
Fig. 2 A-C represents and few (dT) 17TLR7 part during the ODN coupling, loxoribine and 7-denitrogenation be assorted-and target that guanosine changes is special.In Fig. 2 A, with stably express hTLR7, the HEK293 cell of hTLR8 or hTLR9 and the loxoribine of recruitment are hatched.Measure the NF-kB activation and be shown as the multiplication that is higher than the culture medium background and stimulate by measuring uciferase activity after 16 hours.In Fig. 2 B, in the presence of the homopolymer of concentration shown in being with or without ODN 6056 or heteropolymer ODN 1982, hatch expressing the HEK293 cell of hTLR8 and the loxoribine of recruitment.The NF-kB activation is appointed as the multiplication that is higher than the culture medium background induces.In Fig. 2 C, do not have (solid circles) or have in the presence of (empty circles) 5 μ M ODN 6056 to the 7-denitrogenation of the HEK293 raji cell assay Raji recruitment of expressing hTLR8 assorted-guanosine (group on the left side) and inosine (group on the right side) and calculate the NF-κ B stimulation that is higher than the culture medium background.Triplicate all data points of measuring.Shown in data from three times the experiment one of.
Fig. 3 A-3B represents that ODN 6056 suppresses the NF-kB activation of TLR7 mediation in the HEK293 cell.The HEK293 cell of stably express hTLR7 and NF-κ B-luciferase report construct was hatched 16 hours with 2.5mM loxoribine (Fig. 3 A) or 2 μ M R-848 (Fig. 3 B) in the presence of the ODN 6056 of concentration shown in being with or without.Reference culture medium background calculates the NF-kB activation to stimulate.Triplicate all data points of mensuration and displaying meansigma methods (+/-SD).Shown in the result from one of twice independent experiment.
Fig. 4 A-4D represents that specificity ODN produces the inductive cytokine of loxoribine and is redirected.With human PBMC and 1mM loxoribine not or have in the presence of the ODN 6056 of recruitment and the ODN 1982 and hatch.Collect supernatant and supernatant after 24 hours and measure IFN-α (Fig. 4 A), IL-12p40 (Fig. 4 B), the amount of IFN-γ (Fig. 4 C) and TNF-α (Fig. 4 D) by ELISA.Numerical value represent 3 donors meansigma methods (+/-SEM).Data are from the once representational experiment in four experiments.
Fig. 5 A-5F is illustrated in and loxoribine and widow (dT) 17When ODN cultivates altogether the person monocytic cell is produced the stimulation of IL-12p40 and TNF-α.With the human PBMC with shown in the amount loxoribine and ODN hatch.Use anti-IL-12p40/p70 (Fig. 5 A) and anti-TNF-α (Fig. 5 B) antibody to carry out dyeing in the born of the same parents.With anti-CD14 and anti-CD 19 antibodies while staining cell.Pair cell gate CD14-positive cell and calculating IL-12p40/p70-or TNF-α-positive cell percentage.The result represent three donors meansigma methods (+/-SEM).Fig. 5 C-5F represents representational fluidic cell metering Dot blot.What show is one of twice independent experiment.
Fig. 6 A-6L is illustrated in and loxoribine and widow (dT) 17When ODN 6056 cultivates altogether NK cells of human beings is produced the stimulation of I FN-γ.With the human PBMC with shown in the amount loxoribine and ODN hatch.Use anti-IFN-gamma antibodies to carry out dyeing in the born of the same parents.With anti-CD56 and anti-cd 3 antibodies while staining cell.The pair cell gate CD56-positive/CD3 negative cells (NK cell) and calculate IFN-γ-positive cell percentage and in fluidic cell metering Dot blot, show.What show is two representational donors in five.
Fig. 7 A and 7B represent that loxoribine and ODN hatch the influence that the pair cell factor produces altogether from the isolated cell group.In Fig. 7 A, use CD14mAb from the human PBMC, divide mononuclear cell (96% purity) and with shown in the loxoribine of amount hatch being with or without in the presence of the ODN.Measure the amount of IL-12p40 in the supernatant by ELISA.Data display the result of two independent donors (Lycoperdon polymorphum Vitt and black bar).Be expressed as 3 similarly one of experiments.In Fig. 7 B, use BDCA-4mAb from the human PBMC of 2 donors (Lycoperdon polymorphum Vitt and black bar) enrichment PDC (85% purity) and with shown in loxoribine and ODN combination hatched 24 hours.By the IFN-α in the ELISA detection supernatant.What show is from 3 similar data of one of experiment.
Fig. 8 A represents hatch the effect of 16 hours hTLR8-LUC-293 cell with 50 μ M R-848 in the presence of the ODN of amount shown in having.Measure NF-κ B stimulation by measuring uciferase activity.To be set at 100%. Fig. 8 B by the independent inductive NF-kB activity of 50 μ M R-848 and be illustrated in the hTLR8-LUC-293 cell that the existence of ODN shown in the 1 μ M was hatched with the R-848 that increases concentration down 16 hours.Measure NF-κ B stimulation by measuring uciferase activity.
Fig. 9 A and 9B represent to use TLR7 ligands specific 6-amino-9-benzyl-2-butoxy-9H-purine-8-alcohol and the TLR7 and the TLR8 signal of the combination of ODN 6056 to conduct.Fig. 9 A shows the effect of this combination to the conduction of TLR7 signal.Fig. 9 B represents the effect of this combination to the conduction of TLR8 signal.
Figure 10 represents the effect to the TLR8 signal conduction of two kinds of different RNA s.Poly-(rU) 18(SEQID NO:5) do not having to stimulate TLR8 in the presence of the TLR7/8 part.Blended RNA widow (SEQ IDNO:16) can not stimulate TLR8 separately.
Figure 11 is illustrated in the TLR7/8 part and has the effect of following two kinds of RNAs to the conduction of R-848 TLR8 signal.
Figure 12 represents 6056 pairs of TLR7 parts of ODN (loxoribine of TLR8 mediation, immunosine) and the effect of chemical compound, described part both can be TLR7, can be TLR8 part (R-848) again, and described chemical compound was not a TLR7/8 part (ribavirin).
Figure 13 A-D represents immunosine (A), can for 3 ' terminal or at inner (B) or in the 5 ' oligonucleotide of terminal (C) bonded two kinds of immunosine variant/monomers (B and C) and the structure of 6-amino-9-benzyl-2-butoxy-9H-purine-8-alcohol (D).
Figure 14 represents to comprise the structure of the immunosine-oligonucleotide conjugate that connects base.
Figure 15 A-C represents R-848 (A), the structure of CL-029 (B) and immunosine (C) with can be in conjunction with the point that is connected base and/or oligonucleotide.
Detailed Description Of The Invention
The present invention partly relates to the discovery that some oligonucleotides can be regulated the immune response of TLR mediation. The immune response of adjusting TLR used herein mediation means to handle the ability of one or more TLR signal conduction. For example, the present invention can change the TLR characteristic of specific T LR part, so that having in the presence of some oligonucleotides, these parts are by different TLR conducted signals. As another example, having in the presence of some oligonucleotides, known TLR part by more than one TLR conducted signals only shows and carries out the signal conduction by a kind of TLR, and in many cases, such signal conduction is enhanced. In another example, having in the presence of some oligonucleotides, usefulness and the effect of the conduction of TLR signal are significantly increased. Therefore, the present invention can pass through TLR7, among TLR8 and the TLR9 any or suppress and/or strengthen the signal conduction by any combination wherein is selective.
The present invention partly relates to following observed result: by Imidazoquinoline derivatives resiquimod (R-848) and few (dT) of autoactivation TLR7 and TLR817Homopolymers oligonucleotides (ODN 6056) is hatched the remarkable R-848 that increased altogether to the activity of HEK 293 cells of expressing TLR8, and the signal conduction of TLR7 mediation is eliminated. Similarly, the guanosine analogue Loxoribine by autoactivation TLR7 and few (dT)17The combination of ODN 6056 is induced the signal conduction of TLR8 mediation in the sequence selective mode and is eliminated the signal conduction of TLR7 mediation.
By with few (dT)17Hatch altogether and also changed the cell factor distribution that Loxoribine is induced in people's immunocyte kind. IL-12, TNF-α or IFN-γ secrete in a large number, and IFN-α generation is eliminated. Although do not estimate to be subject to any specific mechanism constraint, be transferred to due to the monocyte that activates by itself and ODN combination but infer the change of in the inducing cell factor, observing and be because independent Loxoribine makes from the cell type of Plasmacytoid DC activation, and show not expressive function TLR7 of monocyte.
The present invention is used for regulating the immune response of certain limit thus. In a specific embodiment, the present invention be used for to suppress TLR7 signal conduction (with the immune response of relevant TLR7 mediation), optionally induces simultaneously TLR8 signal conduction (immune response that mediates with relevant TLR8). In another specific embodiment, the invention provides inducing (comprising enhancing) of TLR8 signal conduction (immune response that mediates with relevant TLR8). According to the difference that connects oligonucleotides character, can induce simultaneously TLR9 signal conduction (immune response that mediates with relevant TLR9). For example, comprise connection oligonucleotides and the TLR7 part of CpG motif, can induce TLR8 and TLR9 signal conduction (and relevant immune response) such as the combination of Loxoribine.
The present invention also can be used for treating a series of diseases, and they can have benefited from choosing wantonly not or inducing (comprising enhancing) of the inhibition of the immune response of TLR7 mediation in the presence of the immune response of TLR9 mediation and/or immune response that TLR8 mediates arranged.
The disease of inhibition that can have benefited from the immune response of TLR7 mediation comprises autoimmune disease, such as, but be not limited to lupus and rheumatoid arthritis, particularly those diseases relevant with dna virus infection. Can use the inhibition of the immune response of TLR7 mediation, follow the inducing of immune response of TLR8 mediation, wherein need to strengthen by T and regulate the t cell response that cell suppresses. This class disease includes, but are not limited to cancer and the HCV of some form, and HBV and HIV infect. The TLR7 inhibition of associating and TLR8 induce also and may be of value to the treatment autoimmune disease, or wherein need induce immune response, but avoid TLR7 xicity related. Do not rely on TLR7 mediation immune response regulation the TLR8 mediation immune response induce (comprising enhancings) especially to be used in to treat cancer and infection in vaccine or the non-vaccine environment.
The present invention also can be used for treating the disease that can have benefited from the cell factor generation, or wherein the experimenter is difficult to control or becomes it (for example is difficult to control to the effect of some cell factor, IFN-α is as what sometimes observe in the treatment virus infections).
TLR
Toll sample acceptor (TLR) is the polypeptide class family of the high conservative that plays a decisive role in the mammal natural immunity. 10 family members have been identified at present, called after TLR1-TLR10. The cytoplasm domain of various TLR is characterised in that Toll-interleukin 1 (IL-1) acceptor (TIR) domain. (1998) Mol cell 2:253-8 such as Medzhitov R. TLR identification microorganism is invaded the signal transduction cascade activation that causes evolution conservative in Drosophila (Drosophila) and the mammal. Reported that the connection albumen MyD88 that comprises the TIR domain is combined with TLR and IL-1 receptor-associated kinase (IRAK) and TNF (TNF) receptor associated factor 6 (TRAF6) have been replenished to TLR. Think that the conduction of MyD88 dependent signals is by way of causing NF-κ B transcription factor and c-Jun NH2Terminal kinases (Jnk) mitogen-activated protein kinase (MAPKs) activation, the committed step in the immune activation and inflammatory cytokine produce. With regard to summary, referring to (2000) Nature 406:782-87 such as Aderem A.
Think that TLR is differentially expressed in different tissues and various types of immunocyte. For example, it is reported people TLR7 at placenta, lung, spleen, lymph node is expressed in the tonsillotome and at Plasmacytoid precursor dendritic cells (pDCs). (2000) Eur Cytokine Netw 11:372-8 such as Chuang T-H); (2001) J Exp Med 194:863-9 such as Kadowaki N. It is reported people TLR8 at lung, PBL (PBL), placenta, spleen is expressed in the lymph node and at monocyte. (2001) J Exp Med 194:863-9 such as Kadowaki N; (2000) Eur Cytokine Netw 11:372-8 such as Chuang T-H. It is reported people TLR9 at spleen, lymph node, marrow is expressed among the PBL and at pDC and B cell. (2001) J Exp Med 194:863-9 such as Kadowaki N; (2001) Proc Natl Acad Sci U.S. 98:9237-42 such as Bauer S; (2000) Eur Cytokine Netw 11:372-8 such as Chuang T-H.
The nucleotides of known person and mouse TLR7 and amino acid sequence. For example, referring to GenBank registration number AF240467, AF245702, NM_016562, AF334942, NM_133211; And AAF60188, AAF78035, NP_057646, AAL73191 and AAL73192 are incorporated herein by reference used these content. It is reported that people TLR7 is 1049 amino acid lengths. It is reported that mouse TLR7 is 1050 amino acid lengths. TLR7 polypeptide class comprises having the extracellular domain that is rich in leucic recurring unit, membrane spaning domain and the born of the same parents' internal area that comprises the TIR domain.
The nucleotides of known person and mouse TLR8 and amino acid sequence. For example, referring to GenBank registration number AF246971, AF245703, NM_016610, XM_045706, AY035890, NM_133212; And AAF64061, AAF78036, NP_057694, XP_045706, AAK62677 and NP_573475 are incorporated herein by reference used these content. It is reported people TLR8 for to exist with at least two kinds of isotypes, and a kind of is 1041 amino acid lengths, and another kind is 1059 amino acid lengths. Mouse TLR8 is 1032 amino acid lengths. TLR8 polypeptide class comprises having the extracellular domain that is rich in leucic recurring unit, membrane spaning domain and the born of the same parents' internal area that comprises the TIR domain.
The nucleotides of known person and mouse TLR9 and amino acid sequence. For example, referring to GenBank registration number NM_017442, AF259262, AB045180, AF245704, AB045181, AF348140, AF314224, NM_031178; And NP_059138, AAF72189, BAB19259, AAF78037, BAB19260, AAK29625, AAK28488 and NP_112455 are incorporated herein by reference used these content. It is reported people TLR9 for to exist with at least two kinds of isotypes, and a kind of is 1032 amino acid lengths, and another kind is 1055 amino acid lengths. Mouse TLR9 is 1032 amino acid lengths. TLR9 polypeptide class comprises having the extracellular domain that is rich in leucic recurring unit, membrane spaning domain and the born of the same parents' internal area that comprises the TIR domain.
The immune response of TLR mediation
Term used herein " conduction of TLR signal " means and any aspect of conducting by the relevant intracellular signal of the signal conduction of TLR. Term used herein " immune response of TLR mediation " means the immune response with TLR signal conduction relevant (for example, as its result).
The immune response of TLR7 mediation is for conducting relevant replying with the TLR7 signal. The feature of the immune response of TLR7 mediation generally is IFN-α and IFN induction type cell factor, induces such as IP-10 and I-TAC. The cell factor IL-1 α/β that in the immune response of TLR7 mediation, induces, IL-6, IL-8, MIP-1 α/β and MIP-3 α/β level are lower than those levels of inducing in the immune response of TLR8 mediation.
The immune response of TLR8 mediation is for conducting relevant replying with the TLR8 signal. This is replied and is characterised in that proinflammatory cytokine, such as IFN-γ, and IL-12p40/70, TNF-α, IL-1 α/β, IL-6, IL-8, MIP-1 α/β and MIP-3 α/β induce.
The immune response of TLR9 mediation is for conducting relevant replying with the TLR9 signal. This feature of replying is that at least IFN-γ and IL-12 produce and/or secretion, but is lower than the level of the immune response acquisition that mediates by TLR8 in level.
TLR 7/8 part
" R-848 " used herein means to increase any activating agent (being TLR7 and/or TLR8 activator) of TLR7 and/or the conduction of TLR8 signal jointly. Therefore, when not having connection oligonucleotides of the present invention to have lower the use, some R-848 (is for example induced independent TLR7 signal conduction, the TLR7 ligands specific), some (for example induces independent TLR8 signal conduction, the TLR8 ligands specific), and other lures and both induces TLR7, induces again the conduction of TLR8 signal. The present invention have been found that when with this class part when being connected the oligonucleotides coupling, the TLR signal transport properties of these parts changes. In some instances, TLR7 signal conduction is suppressed and the conduction of TLR8 signal is induced. In other example, the conduction of TLR8 signal is induced (namely increasing from background level) or is strengthened (namely increasing from non-background level), and in other embodiments, TLR8 and the conduction of TLR9 signal are induced or strengthened.
Term used herein " TLR7 part " means to increase any activating agent (being the activator of TLR7) of TLR7 signal conduction. In this respect, TLR7 signal level of conduction can strengthen than the signal level of conduction that is pre-existing in, or it induces the background level that can surpass the signal conduction. The TLR7 part comprises, but be not limited to guanosine analogue, guanosine such as the C8-replacement, the mixture of the ribonucleotide that is mainly formed by G and U, guanosine nucleotides and RNA or RNA-quasi-molecule (PCT/US03/10406) and based on the compound of adenosine (6-amino-9-benzyl-2-(3-hydroxyl-propoxyl group)-9H-purine-8-alcohol (CL-029 for example, Sumitomo), 6-amino-9-benzyl-2-butoxy-9H-purine-8-alcohol (as shown in Figure 13 D) and other related compound, those described in US 6310070 B1). The TLR7 part also is disclosed in the J. Immunol.2005 such as Gorden, 174:1259-1268 (for example, 3M-001, N-[4-(4-amino-2-ethyl-1H-imidazo [4,5-c] quinoline-1-yl) butyl-] Methanesulfomide; C17H 23N 5O 2S；mw 361)。
Term used herein " guanosine analogue " means to have the guanine base of relating to, the guanosine of the chemical modification of guanosine nucleosides sugar or guanine base and guanosine nucleosides sugar-class nucleotides (not comprising guanosine). Guanosine analogue particularly including, but be not limited to the 7-denitrogenation assorted-guanosine.
Guanosine analogue further comprises the guanosine that C8-replaces, such as 7-thia-8-oxo guanosine (immunosine), the 8-TGR, 8-bromine guanosine, the 8-methylguanosine, 8-oxo-7,8-dihydro guanosine, C8-virtue amino-2 '-deoxyguanosine, C8-propinyl-guanosine, the guanosint riboside that C8-and N7-replace, such as 7-pi-allyl-8-oxo guanosine (Loxoribine) and 7-methyl-8-oxo guanosine, the amino guanosine of 8-, 8-hydroxyl-2 '-deoxyguanosine, the guanosine that the assorted 8-of 8-hydroxyl guanosine and 7-denitrogenation replaces.
Term used herein " TLR8 part " means to increase any activating agent (being the activator of TLR8) of TLR8 signal conduction. In this respect, TLR8 signal level of conduction can strengthen than the signal level of conduction that is pre-existing in, or it induces the background level that can surpass the signal conduction. The TLR8 part comprises the ribonucleotide mixture that mainly is comprised of G and U, guanosine nucleotides and RNA or RNA-quasi-molecule (PCT/US03/10406). Extra TLR8 part also is disclosed in the J.Immunol.2005 such as Gorden, (for example, 3M-002,2-propyl group thiazole [4,5-c] quinolin-4-amines also among the 174:1259-1268; C13H 13N 3S；mw 243)。
Some R-848 is TLR7 and TLR8 part. They comprise imidazoquinolines, the ribonucleotide mixture that mainly is comprised of G and U, guanosine nucleotides and RNA or RNA-quasi-molecule (PCT/US03/10406). Extra R-848 also is disclosed in the J. Immunol.2005 such as Gorden, (for example, 3M-003 among the 174:1259-1268,4-amino-2-(ethoxyl methyl)-α, alpha-alpha-dimethyl-6,7,8,9-tetrahydrochysene-1H-imidazo [4,5-c] quinoline-1-alcohol hydrate; C17H 26N 4O 2；mw 318)。
The imidazoquinolines immune response modifier thinks that they induce the expression of several cell factors, comprises interferon (for example, IFN-α), TNF-α and some interleukins (for example, IL-1, IL-6 and IL-12). Imidazoquinolie can stimulate the Th1 immune response, confirms as the ability of inducing the IgG2a level to increase according to it. It is reported that the imidazoquinolie activating agent can also suppress the Th2 cell factor, such as IL-4, IL-5 and IL-13 produce. Some cell factor that imidazoquinolines is induced is produced by macrophage or dendritic cells. It is reported that some kind of imidazoquinolie increases NK lysis active and stimulation B cell proliferation and differentiation, induces antibody to produce and secretion thus.
Imidazoquinolines used herein comprises the imidazoquinolie amine, the cycloalkyl imidazopyridine amine and 1 that imidazopyridine amine, 6,7-condense, and Bridge 2 connects the imidazoquinolie amine. These compounds are described in US Patent No. 4689338,4929624, in 5238944,5266575,5268376,5346905,5352784,5389640,5395937,5494916,5482936,5525612,6039969 and 6110929. The concrete kind of imidazoquinolines comprises R-848 (S-28463); 4-amino-2 ethoxyl methyls-α, alpha-alpha-dimethyl-1H-imidazo [4,5-c] quinoline-1-ethanol; 1-(2-methyl-propyl)-1H-imidazo [4,5-c] quinolin-4-amines (R-837 or imiquimod) and S-27609. Imiquimod is used for the topical therapeutic wart at present, such as genitals and anus wart, and tests in the topical therapeutic of basal-cell carcinoma.
Term used herein " TLR9 part " means to increase any activating agent (being the activator of TLR9) of TLR9 signal conduction. The TLR9 part particularly including, but be not limited to immunostimulatory nucleic acids and particularly CpG immunostimulatory nucleic acids.
Term used herein " immunostimulation CpG nucleic acid " mean can activating immune cell any nucleic acid that contains CpG. At least the C in the CpG dinucleotides is generally, but does not necessarily methylate. Immunostimulation CpG nucleic acid is described in the patent application of many granted patent and announcement, comprises US Patent No. 6,194,388; 6,207,646; 6,218,371; 6,239,116; 6,339,068; 6,406,705; With 6,429,199.
In certain embodiments, the TLR part is ligands specific. TLR7 ligands specific used herein is not for passing through TLR7 when having the present invention to connect in the presence of the oligonucleotides to use, but is not the part of TLR8 or TLR9 conducted signal. Similarly, the TLR8 ligands specific is not for passing through TLR8 when having the present invention to connect in the presence of the oligonucleotides to use, but is not the part of TLR7 or TLR9 conducted signal. Pass through TLR7 rather than TLR conducted signal when preferred TLR7 ligands specific uses in the presence of not connecting few nucleosides, and the TLR8 ligands specific passes through TLR8 rather than TLR conducted signal when using in the presence of not connecting oligonucleotides.
In certain embodiments, the TLR part is not RNA.
R-848 can stimulate TLR8 and TLR9 with the application that is connected oligonucleotides, produces simultaneously the downstream effect from two kinds of acceptors. For example, oligonucleotides stimulates TLR9 to cause, and for example IFN-α produces, and oligonucleotides stimulates TLR8 to cause, for example IL-12, TNF-α and IFN-γ generation. Part and oligonucleotides can be puted together each other or can be separated from one another with physics mode.
Connect oligonucleotides
The present invention with R-848 be connected the oligonucleotides coupling. Connection oligonucleotides used herein by suppressing through the conduction of the TLR7 of TLR7 part signal, is induced through the conduction of the TLR8 of TLR7 part signal and/or is strengthened through add the oligonucleotides of this ligand activity for the TLR8 signal conduction of the part of TLR7 and TLR8 part when using with R-848.
Term oligonucleotides used herein and term nucleic acid can Alternates. Preferred this term does not comprise plasmid, carrier and/or antisensenucleic acids. In certain embodiments, what oligonucleotides can be for immunostimulating, and in other embodiments, it can be for nonimmune irritating when using separately. Oligonucleotides can be any length, preferred 7 or 8 to 100 length of nucleotides.
One class connects oligonucleotide widely and comprises formula 5 ' X-N 4-X 3 ' or 5 ' X-N 5-X 3 ', wherein X can and can exist or not exist and N for any nucleotide 4And N 5Represent 4 and 5 successive T (thymidine), U (uracil) or A (adenine) make N 4Or N 5In each N all identical.Oligonucleotide can be 7,8,9,10,11,12,13,14,15,16,17 or 17 above length of nucleotides.It preferably comprise key between at least one thiophosphate nucleotide (up to and comprise complete D2EHDTPA main chain).
Therefore, a class connects oligonucleotide and comprises formula 5 ' X a-TTTTT-X b3 ', X wherein aAnd X bCan be independently for any nucleotide and can exist or not exist.X aAnd X bCan represent one or more nucleotide (for example, 1-100 nucleotide).Oligonucleotide can be 7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22, and 23 or 23 above length of nucleotides.Oligonucleotide can be saturated successive T more than 6,7 or 7.Preferred connection oligonucleotide is dT homopolymer (being the few dT of length described herein).Even more preferably to connect oligonucleotide be 17 length of nucleotides of thymidine (dT) homopolymer.Most preferably it comprise key between at least one D2EHDTPA nucleotide (up to and comprise complete D2EHDTPA main chain).
According to the difference of embodiment, connecting oligonucleotide can be by 100%T, 99%T, 98%T, 97%T, 96%T, 95%T, 94%T, 93%T, 92%T, 91%T, 90%T, 85%T, 80%T, 75%T, 70%T, 65%T, 60%T, 55%T, 50%T, 45%T or 45% following T form.
Another kind of connection oligonucleotide comprises formula 5 ' X a-UUUUU-X b3 ', X wherein aAnd X bCan be independently for any nucleotide and can exist or not exist.X aAnd X bCan represent one or more nucleotide (for example, 1-100 nucleotide).Oligonucleotide can be 7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22, and 23 or 23 above length of nucleotides.Oligonucleotide can be saturated successive U more than 6,7 or 7.In an important embodiment, oligonucleotide is the dU homopolymer, its be preferably 17 length of nucleotides and have key between at least one D2EHDTPA nucleotide (up to and comprise complete D2EHDTPA main chain).
According to the difference of embodiment, connecting oligonucleotide can be by 100%U, 99%U, 98%U, 97%U, 96%U, 95%U, 94%U, 93%U, 92%U, 91%U, 90%U, 85%U, 80%U, 75%U, 70%U, 65%U, 60%U, 55%U, 50%U, 45%U or 45% following U form.
Another kind of connection oligonucleotide contains formula 5 ' X a-AAAAA-X b3 ' wherein X aAnd X bCan be independently for any nucleotide and can exist or not exist.X aAnd X bCan represent one or more nucleotide (for example, 1-100 nucleotide).Oligonucleotide can be 7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22, and 23 or 23 above length of nucleotides.Oligonucleotide can be saturated successive A more than 6,7 or 7.In an important embodiment, oligonucleotide is the dA homopolymer, its be preferably 17 length of nucleotides and have key between at least one D2EHDTPA nucleotide (up to and comprise complete D2EHDTPA main chain.
According to the difference of embodiment, connecting oligonucleotide can be by 100%A, 99%A, 98%A, 97%A, 96%A, 95%A, 94%A, 93%A, 92%A, 91%A, 90%A, 85%A, 80%A, 75%A, 70%A, 65%A, 60%A, 55%A, 50%A, 45%A or 45% following A form.
Another kind of connection oligonucleotide comprises formula 5 ' C n-T m-C p3 ', wherein n be 0-100 integer (for example, 3-7), p be 0-100 integer (for example, 4-8) and m be 0-100 integer (for example, 2-10).Preferred n and p sum are equal to or less than the value of m, make that C content is 50%, are lower than 50%, are lower than 45%, are lower than 40%, are lower than 35%, are lower than 30%, are lower than 25%, are lower than 20%, are lower than 15%, are lower than 10%, are lower than 5% or lower.In certain embodiments, n is at 3-7, m at 2-10 and p at 4-8, as long as satisfy above-mentioned percentage ratio.Some kind of this formula as shown in Figure 8.Example 5 ' C 3T 10C 43 ' (SEQ ID NO:1), 5 ' C 4T 8C 53 ' (SEQID NO:2), 5 ' C 5T 6C 63 ' (SEQ ID NO:6), 5 ' C 6T 4C 73 ' (SEQ ID NO:7) and 5 ' C 7T 2C 83 ' (SEQ ID NO:8).
In certain embodiments, connect the saturated immunostimulation CpG of oligonucleotide motif.This motif can be for methylating or unmethylated.In the later case, complete oligonucleotide can be unmethylated or part can be for unmethylated, but the C of at least 5 ' CG 3 ' must not methylate.Motif and immunoregulatory action range is described in the following document does not methylate: United States Patent (USP), and such as US 6,194,388 B1; US 6,207,646 B1; US 6,239,116B1; With US 6,218,371 B1; With the patent application of announcing, such as PCT/US98/03678, PCT/US98/10408, PCT/US98/04703 and PCT/US99/09863.The full content of these patent rights and patent application is incorporated herein by reference.
Term " oligonucleotide " and " nucleic acid " can exchange that to use so that refer to be connected a plurality of nucleotide of phosphate group and tradable organic base (be sacchariferous molecule (for example ribose or deoxyribose) as mentioned above, described base be pyrimidine (for example, cytosine (C), thymidine (T) or uracil (U)) or purine (for example, adenine (A) or guanine (G)).Therefore, this term comprises DNA and RNA oligonucleotide.This term should comprise that also multinuclear glycosides (promptly deducting the polynucleotide of phosphoric acid) and any other contain the base of polymer.Oligonucleotide can be originated (for example, genome or cDNA) available from existing nucleic acid, but preferably synthetic (for example, producing with the nucleic acid synthesizer).
Oligonucleotide can be two strands or strand.In certain embodiments, when oligonucleotide was strand, it can form secondary and tertiary structure (for example selecting to hybridize with himself on the fragment from start to finish or along its length at its body by turning back or passing through) on himself.Therefore, although the primary structure of this class oligonucleotide can be for strand, what its higher structure can be for double-stranded or three chains.
Oligonucleotide can have the certain-length scope, but they preferably are equal to or less than 100 nucleotide (or base) length.Connecting oligonucleotide preferably is not plasmid or carrier.It is at least active not relevant with any antisense that they also preferably can not have an effect that antisense activity or the present invention show.
Oligonucleotide can have the main chain of modification.For example, they can comprise at least one and not be the internucleotide linkage of phosphodiester bond.This generic key can be phosphorothioate bond.In certain embodiments, oligonucleotide can have chimeric main chain (promptly by confirming two kinds of main chains that dissimilar nucleotide bond is set up jointly).
Term used herein " thiophosphate main chain " means the stabilizing sugar phosphate backbone of oligonucleotide, and wherein non-bridged phosphoric acid oxygen is replaced by the sulfur on the key between at least one nucleotide.Non-bridged in one embodiment phosphoric acid oxygen is replaced by the sulfur on the key between each nucleotide.
The source of oligonucleotide and preparation
Oligonucleotide of the present invention can be compared with DNA with natural RNA and comprise various chemical modifications and replacement, comprises bridge between the di-phosphate ester nucleoside, β-D-ribose unit and/or natural nucleus glycoside base (adenine, guanine, cytosine, cytosine, uracil).The example that those skilled in the art's known chemical is modified and being described in the following document, for example: (1990) Chem Rev 90:543 such as Uhlmann E; " Protocols for Oligonucleotides and Analogs " Synthesis and Properties﹠amp; Synthesis and Analytical Techniques, S.Agrawal, Ed, Humana Press, Totowa, the U.S. 1993; (1996) Annu Rev Pharmacol Toxicol 36:107-29 such as Crooke ST; With (1995) Mod Synth Methods 7:331-417 such as Hunziker J.Oligonucleotide of the present invention can have a kind of or many modification, and wherein the oligonucleotide of every kind of modification and the identical sequence be made up of n DNA or RNA is positioned on bridge between specific di-phosphate ester nucleoside and/or the specific β-D-ribose unit and/or specific natural nucleoside base position.
For example, oligonucleotide can comprise one or more modifications, and wherein every kind of modification is independently selected from:
A) be positioned at 3 of nucleoside ' and/or 5 ' end on the di-phosphate ester nucleoside between the adorned nucleoside bridge of bridge replace;
B) be positioned at 3 of nucleoside ' and/or 5 ' end on the di-phosphate ester nucleoside between bridge replaced by the dephosphorylation bridge;
C) the sugared phosphoric acid unit from sugared phosphate backbone is replaced by another kind of unit;
D) the adorned sugar unit in β-D-ribose unit replaces; With
E) the adorned nucleoside base of natural nucleus glycoside base replaces.
The example more specifically that is used for the oligonucleotide chemical modification is as follows.
Oligonucleotide can comprise the internucleotide linkage of modification, those such as above-mentioned (a) or (b).The key of these modifications can partly tolerate degraded (for example stable)." stable oligonucleotide " should refer to tolerate relatively this class of body endogenous cause of ill modify the degraded that causes (for example by outer-or Nei-Qie nuclease) oligonucleotide.In certain embodiments, the oligonucleotide with phosphorothioate bond maximum activity can be provided and prevent oligonucleotide take place because of inside and outside the born of the same parents-or degraded that Nei-the Qie nuclease causes.
Be positioned at 3 of nucleoside ' and/or 5 ' end on the di-phosphate ester nucleoside between bridge can adorned nucleoside between bridge replace, wherein bridge for example is selected from D2EHDTPA, phosphorodithioate, NR between the nucleoside of Xiu Shiing 1R 2-phosphoramidate, borine phosphate ester (boranophosphate), Alpha-hydroxy benzylphosphonic acid ester, phosphoric acid-(C 1-C 21)-O-Arrcostab, phosphoric acid-[(C 6-C 12) aryl-(C 1-C 21)-O-alkyl] ester, (C 1-C 8) phosphonate ester and/or (C 6-C 12) the arylphosphonic acid ester bond, (C 7-C 12)-alpha-hydroxymethyl-aryl (for example, being disclosed among the WO 95/01363), wherein (C 6-C 12) aryl, (C 6-C 20) aryl and (C 6-C 14) aryl is optional by halogen, alkyl, alkoxyl, nitro, cyano group replaces, and R wherein 1And R 2Independently of one another is hydrogen, (C 1-C 18)-alkyl, (C 6-C 20)-aryl, (C 6-C 14)-aryl-(C 1-C 8)-alkyl, preferred hydrogen, (C 1-C 8)-alkyl, preferred (C 1-C 4)-alkyl and/or methoxy ethyl, or R 1And R 2Constitute with the nitrogen that carries them and to comprise another and be selected from O, the heteroatomic 5-6-unit heterocycle of S and N.
Be positioned at 3 of nucleoside ' and/or 5 ' end on the di-phosphate ester nucleoside between bridge can be replaced that (the dephosphorylation bridge is disclosed in by the dephosphorylation bridge, " the Methods inMolecular Biology " of Uhlmann E and Peyman A for example, Vol.20, " Protocols for Oligonucleotidesand Analogs ", S.Agrawal, Ed., Humana Press, Totowa 1993, Chapter16, among the pp.355 ff), wherein the dephosphorylation bridge for example is selected from dephosphorylation bridge formal (formacetal), 3 '-sulfo-formal, the methyl hydroxylamine, oxime, methylene dimethyl-hydrazono-, dimethylene sulfone and/or silicyl.
Can be replaced by another kind of unit from sugared phosphate backbone (promptly by the first sugared phosphate backbone of forming of sugared mono phosphoric acid ester) sugared phosphoric acid unit (promptly constituting bridge between the unitary β of sugared phosphoric acid-D-ribose and di-phosphate ester nucleoside each other), wherein another kind of unit for example is suitable for (for example constituting " morpholino-derivant " oligomer, described in (1989) Nucleic Acids Res 17:6129-41 such as Stirchak EP), promptly, for example, by morpholino-derivant unit replacement or formation polyamide nucleic acid (" PNA "; For example, described in (1994) Bioconjug Chem 5:3-7 such as Nielsen PE), that is, for example,, for example replaced by 2-amino-ethyl glycine by the PNA backbone units.Oligonucleotide can have other carbohydrate backbone modifications and replacement, and such as the peptide nucleic acid(PNA) with phosphate (PHONA), locked nucleic acid (LNA) has the oligonucleotide that alkyl is connected the main chain part of base or amino connection base with having.Alkyl connects that base can be for side chain or non-side chain, replaces or unsubstituted and chiral purity or racemic mixture.
β-ribose unit or β-D-2 '-deoxyribose unit can replace by adorned sugar unit, and wherein the sugar unit of Xiu Shiing for example is selected from β-D-ribose, α-D-2 '-deoxyribose, L-2 '-deoxyribose, 2 '-F-2 '-deoxyribose, 2 '-the F-arabinose, 2 '-O-(C 1-C 6) alkyl-ribose, 2 '-the O-methylribose, 2 '-O-(C 2-C 6) alkenyl-ribose, 2 '-[O-(C 1-C 6) alkyl-O-(C 1-C 6) alkyl]-ribose, 2 '-NH 2-2 '-deoxyribose, β-D-furyl xylose, α-arabinofuranosyl, 2, two deoxidation-the β of 4--D-erythro-pyranohexose and carboxylic acid are (for example, be described in Froehler (1992) J Am Chem Soc 114:8320) and/or open chain sugar analogue (for example, being described in (1993) Tetrahedron 49:7223 such as Vandendriessche) and/or dicyclo sugar analogue (for example, being described in (1993) Helv Chim Acta 76:481 such as Tarkov M).
In certain embodiments, the sugar of modification is the ribose of 2 ' modification.In certain embodiments, sugar is 2 '-the O-methylribose, particularly 1 or 2 is the nucleotide that connects by key between di-phosphate ester or di-phosphate ester-class nucleoside.
Nucleic acid also comprises the purine class and the miazines of replacement, such as C-5 propine pyrimidine and 7-denitrogenation assorted-base that purine radicals that 7-replaces is modified.(1996) Nat Biotechnol14:840-4 such as Wagner RW.Purine class and miazines include, but are not limited to adenine, cytosine, and guanine and thymus pyrimidine and other nuclear base natural and non-natural exists replace and unsubstituted aromatics part.
The base of modifying is the base of generally finding in DNA and RNA chemically being different from, such as T, and C, G, A and U, but have basic chemical structure with naturally occurring base.For example, the nucleoside base of modification can be selected from hypoxanthine, uracil, dihydrouracil, pseudouracil, 2-thiouracil, 4-thiouracil, 5-amino-uracil, 5-(C 1-C 6)-alkyl urea pyrimidine, 5-(C 2-C 6)-alkenyl uracil, 5-(C 2-C 6)-alkynyl uracil, 5-(hydroxymethyl) uracil, 5-chlorouracil, 5-fluorouracil, 5-bromouracil, 5-hydroxyl cytosine, 5-(C 1-C 6)-alkyl cytosine, 5-(C 2-C 6)-alkenyl cytosine, 5-(C 2-C 6)-alkynyl cytosine, 5-chlorine cytosine, 5-flurocytosine, 5-bromine cytosine, N 2-dimethylguanine, 2,4-diaminourea-purine, the 8-azapurine, the 7-deazapurine of replacement, preferred 7-denitrogenation is assorted-7-replace and/or the 7-denitrogenation assorted-purine that 8-replaces, 5-hydroxymethyl cytosine, N4-alkyl cytosine, for example, N4-ethyl cytosine, 5-hydroxyl deoxycytidine, 5-hydroxymethyl deoxycytidine, N4-alkyl deoxycytidine, for example, N4-ethyl deoxycytidine, the dezyribonucleoside of 6-sulfo-deoxyguanosine and nitro-pyrrole, C5-propinyl pyrimidine and diaminopurine, for example, 2, the 6-diaminopurine, inosine, 5-methylcytosine, 2-aminopurine, 2-amino-6-chloropurine, other trim of hypoxanthine or natural nucleus glycoside base.The implication of this catalogue is typical, but and has been not interpreted as the qualification effect.
In concrete formula as herein described, can mix the base of modification.For example, cytosine can replace by adorned cytosine.The cytosine of modification used herein is the pyrimidine bases analog that the naturally occurring or non-natural of cytosine exists, and it can replace this base, but can not damage the immunostimulatory activity of oligonucleotide.The cytosine of modifying includes, but are not limited to cytosine (for example, 5-methyl-cytosine that 5-replaces, 5-fluoro-cytosine, 5-chloro-cytosine, 5-bromo-cytosine, 5-iodo-cytosine, 5-hydroxyl-cytosine, 5-methylol-cytosine, 5-difluoromethyl-cytosine and 5-alkynyl-cytosine unsubstituted or that replace), the cytosine that 6-replaces, the cytosine that N4-replaces (for example, N4-ethyl-cytosine), 5-aza-cytosine, 2-sulfydryl-cytosine, iso-cytosine, vacation-iso-cytosine, cytosine analog (for example, the N with the ring system of condensing, N '-propylene cytosine Huo phenoxazine) and uracil and derivant thereof (5-fluoro-uracil for example, 5-bromo-uracil, 5-bromo vinyl-uracil, 4-sulfur-uracil, 5-hydroxyl-uracil, 5-propinyl-uracil).In the preferred cytosine some comprises 5-methyl-cytosine, 5-fluoro-cytosine, 5-hydroxyl-cytosine, 5-methylol-cytosine and N4-ethyl-cytosine.In another embodiment of the invention, the cytosine base is by universal base (for example, 3-nitro-pyrrole, P-base), and aromatics ring system (for example, fluorobenzene or two fluorobenzene) or hydrogen atom (dSpacer) replace.
As another example, guanine can replace by adorned guanine base.The guanine of modification used herein is the natural existence of guanine or the purine bases analog that non-natural exists, and it can replace this base, but can not damage the immunostimulatory activity of oligonucleotide.The guanine of modifying includes, but are not limited to the assorted guanine of 7-denitrogenation, the 7-denitrogenation is assorted-guanine that 7-replaces (such as the 7-denitrogenation assorted-7-(C 2-C 6) the alkynyl guanine), the 7-denitrogenation is assorted-guanine that 8-replaces, and hypoxanthine, the guanine that N2-replaces is (for example, N2-methyl-guanine), 5-amino-3-methyl-3H, 6H-thiazole [4,5-d] pyrimidine-2 also, the 7-diketone, 2, the 6-diaminopurine, 2-aminopurine, purine, indole, adenine, the adenine of replacement (for example, N6-methyl-adenine, 8-oxo-adenine), the guanine (for example, 8-hydroxyl guanine and 8-bromine guanine) and the 6-thioguanine of 8-replacement.In another embodiment of the invention, guanine base is by universal base (for example, 4-methyl-indole, 5-nitro-indole and K-base), aromatics ring system (for example benzimidazole or dichloro benzimidazole, 1-methyl isophthalic acid H-[1,2,4] triazole-3-Methanamide) or hydrogen atom (dSpacer) replace.
With regard to application of the present invention, can use any de novo synthesis oligonucleotide of the present invention in many methods well-known in the art, comprise, for example, beta-cyano ethyl phosphoramidite method ((1981) Tetrahedron Lett 22:1859 such as Beaucage SL) or nucleoside H-phosphonate ester method (Garegg etc. (1986) Tetrahedron Lett 27:4051-4; FroehlerBC etc. (1986) Nucleic Acid Res 14:5399-407; Garegg etc. (1986) Tetrahedron Lett 27:4055-8; Gaffhey etc. (1988) Tetrahedron Lett29:2619-22).These chemical methodes can be undertaken by being purchased the automatic nucleic acid synthesizer.These oligonucleotide are called synthetic oligonucleotide.Isolating oligonucleotide generally means isolating oligonucleotide from the bonded composition of common reality.As an example, isolating oligonucleotide can be for from cell, nucleus, isolating oligonucleotide in mitochondrion or the chromatin.
Can use the main chain of the automatic technology synthetic modification of using phosphoramidate or H-phosphonate ester chemistry, such as D2EHDTPA.For example, can be as U.S. Pat 4,469, prepare aryl-and alkyl-phosphonic acid ester described in 863; And can pass through automatization's solid phase synthesis, use be purchased reagent preparation alkyl phosphotriester class (wherein charged oxygen part is as U.S. Pat 5,023,243 and European patent EP 092,574 described in alkylation).The method for preparing other dna backbone and modify and replace (for example, (1990) Chem Rev 90:544 such as Uhlmann E has been described; Goodchild J (1990) Bioconjugate Chem 1:165).
Isolating
In certain embodiments, TLR7/8 part and/or connection oligonucleotide have been separated.Isolating part or oligonucleotide are for pure or do not contain other and generally find to reach realistic scale and be suitable for part or the oligonucleotide that it specifies the material of using in the system in natural or in vivo basically.Especially, part or oligonucleotide are for enough pure and be enough to not celliferous other biotic component, so that for example be used for production pharmaceutical preparation.Because isolating part or oligonucleotide can be blended in the pharmaceutical preparation with pharmaceutically acceptable carrier, so part or oligonucleotide can only account for the little percentage ratio of weight of formulation.However, but isolating ligands or oligonucleotide, make with its basically from it biofacies with separate in the bonded material.
Conjugate
Term used herein " conjugate " means by any plysiochemical directly or indirectly any combination of two or more ingredients connected to one another that interacts.In one embodiment, conjugate is the combination by direct or indirect two or more ingredients connected to one another of covalent bonding.
Provide among the present invention in one aspect and comprised TLR part and the compositions that is connected oligonucleotide.In one embodiment, form conjugate by covalent bond.In one embodiment, conjugate comprises and connects base.
Conjugate can comprise that of the present invention one or more connect oligonucleotide and one or more TLR7/8 parts.Selectively or in addition.Conjugate can comprise one or more other molecules, includes, but are not limited to antigen or other medicines.
Figure 13-15 illustration many features of this class conjugate, the junction point that comprises part and oligonucleotide be connected base promote this class in conjunction with in optional application.
In one embodiment, conjugate is for connecting oligonucleotide and some TLR7 ligands specific, such as, but not limited to the conjugate of immunosine or loxoribine.Connect oligonucleotide can for, but be not limited to length at 8-20, preferred 10-20 and more preferably 14-18 length of nucleotides gather (dT) oligonucleotide.In certain embodiments, if particularly connect oligonucleotide from as immunostimulating or by the TLR9 conducted signal, do not comprise so connecting oligonucleotide and some TLR7/8 part, such as the conjugate of the guanosine of imidazoquinolines and C8-replacement.
Figure 13 A-C illustration can be used for structure and two kinds of monomer variants thereof of the immunosine of associated methods.Variant shown in Figure 13 B can combine on interior location or 3 ' end with oligonucleotide.Therefore, can exist these with variant that oligonucleotide is connected in one or more.Variant shown in Figure 13 C can combine on 5 ' end with oligonucleotide.Figure 14 illustration use to connect base immunosine combined with oligonucleotide.Figure 15 illustration connection base and/or oligonucleotide can be combined into R-848, the junction point on CL-029 and the immunosine.Conjugate of the present invention can comprise 1,2,3,4, connects base more than 5 or 5, or selectively, they can not contain and connect base.The example of conjugate is poly-(dT) 14-L 2-IM (wherein L represents to connect base and IM and represents that immunosine and subscript represent each unitary quantity in the conjugate) (SRQ ID NO:9).Another example is IM-L 2-(dT) 14(SEQ ID NO:10).Difference between these examples is to dispose immunosine and is connected base on 3 ' end or 5 ' end.
Connecting base can connect for any reactive moieties on the oligonucleotide, includes, but are not limited to main chain phosphate group or sugared hydroxyl.For example, can be with them by di-phosphate ester, D2EHDTPA, methyl phosphonate and/or amido link are introduced.
Connect base and in fact can be non-nucleotide.They comprise, for example, alkaline residue (dSpacer), few ethylene glycol is such as 2,2'-ethylenedioxybis(ethanol). (base 9 at interval) or six ethylene glycol (base 18 at interval) or alkane-glycol, such as butanediol.Connecting base can be bonded to each other by di-phosphate ester or phosphorothioate bond.Other connects base and connects base for alkylamino, such as C3, and C6, the amino base that connects of C12, and also have alkyl thiol to connect base, connect base such as C3 or C6 sulfydryl.Can also in a kind of conjugate, use dissimilar connection bases.
Oligonucleotide can be connected by aromatic moieties, and these residues can further be replaced by the alkyl of alkyl or replacement.Oligonucleotide can also comprise two-fold or triple unit, and this makes a kind of or dissimilar multiple parts combine with oligonucleotide.Oligonucleotide can also comprise the connection base of being modified reagent or the generation of oligonucleotides-modified reagent by peptide.In addition, they can comprise one or more natural or alpha-non-natural amino acid residues that connects by peptide (amide) key.
By the synthetic different oligonucleotide of the method set up and they can be in the solid phase synthesis process online being connected to each other.Selectively, can behind synthetic individual unit, they be connected to each other.
Immunne response
In one embodiment, the immunne response of TLR mediation is replied for the Th1-para-immunity.Term used herein " Th1-class " means the property feature with Th1 immunne response.The Th1 immunne response can comprise on feature induces some cytokine, such as IFN-γ, and secretion of IgG2a immunoglobulin (in the mice) and macrophage activation.Term " Th1-class " is opposite with term " Th2-class ", and " Th2-class " means the property feature with Th2 immunne response as described below.
The Th1-para-immunity is replied and can be comprised that some is characterised in that cytokine relevant with the Th1 immunne response and any the expression in the chemotactic factor, comprises IFN-α, IFN-β, IFN-γ, TNF-α, IL-12, IL-18, IP-10 and any combination thereof.Think that Th1 immunne response and Th2 immunne response are counter regulation mutually.In certain embodiments, the Th1-para-immunity is replied and can be comprised some Th2 relevant cell factor, comprises IL-4, IL-5, the inhibition of IL-10 and IL-13.The Th1-para-immunity is replied the expression that can comprise some antibody isotype, comprises (in mice) IgG2a, with or not with the inhibition of some Th2 associated antibodies isotype, comprise IgE and (in mice) IgG1.In one embodiment, the Th1-para-immunity is replied to Th1 and is replied.
The Th2-para-immunity is replied and can be comprised that some is characterised in that cytokine relevant with the Th2 immunne response and any the expression in the chemotactic factor, comprises IL-4, IL-5, IL-10, IL-13 and any combination thereof.In certain embodiments, the Th2-para-immunity is replied the inhibition that can comprise some Th1 relevant cell factor.The Th2-para-immunity is replied the expression that can comprise some antibody isotype, comprise IgE and (in mice) IgG1, with or not with the inhibition of some Th1 associated antibodies isotype, comprise (in mice) IgG2a, in one embodiment, the Th2-para-immunity is replied to Th2 and is replied.
Therefore, the present invention provides the method for inducing experimenter Th1-para-immunity to reply in one embodiment.Induce the Th1-para-immunity to reply and comprise that increasing or strengthen the Th1-para-immunity replys.This method comprises the TLR7/8 part of the present invention that gives effective dose to the experimenter and is connected oligonucleotide so that the step of inducing experimenter Th1-para-immunity to reply.
In one embodiment, the invention provides the method that inhibition experimenter Th2-para-immunity is replied.This method comprises the TLR7/8 part of the present invention that gives effective dose to the experimenter and is connected oligonucleotide so that suppress the step that experimenter Th2-para-immunity is replied.These class methods can be applied to especially treat have and be characterised in that with the disease of the dominant immunne response of Th2 characteristic or be in experimenter in its risk.This class disease includes, but are not limited to anaphylaxis and asthma.
In one embodiment, immunne response comprises the cell surface marker of activated immune cell, such as CD25, and CD80, the up regulation of CD86 and CD154.The method that is used to measure the cell surface expression of this class labelling is well-known in the art and comprises facs analysis.
In one embodiment, in order to measure the immunne response in cell or the cell mass, cell or cell mass are expressed at least a, and in some cases, only preferred TLR7, a kind of among TLR8 or the TLR9.The natural expression TLR of cell maybe can handle it so that by suitable TLR expression vector transfered cell is expressed TLR.In one embodiment, cell or cell mass obtain as peripheral blood lymphocytes (PBMC).In one embodiment, cell or cell mass obtain as the cell line of expressing TLR.In one embodiment, cell or cell mass obtain as the cell line of using the TLR transient transfection.In one embodiment, cell or cell mass are as the cell line of using the TLR stable transfection.
In addition, in order to measure the immunne response in cell or the cell mass, can expediently the intracellular signal conduction to TLR there be (promptly being subjected to its adjusting) the cell report construct transfered cell or the cell mass of replying.In one embodiment, this class reporter gene is the gene of configuration under the control of NF-κ B promoter.In one embodiment, the gene of configuration is a luciferase under this promoter control, but, can use other reporter gene, comprises green fluorescent protein (GFP), beta galactosidase, alkali phosphatase etc.Under suitable activatory condition, as an example, but expressing luciferase report construct and its emission can be used the sensed light signal of luminometer quantitative assay.These and other report construct is what be purchased.In other embodiments, can be according to cytokine, such as IFN-α, TNF-α, IL-12, the immunne response of TLR mediation is measured in the generation (mRNA or protein) of IFN-γ etc. or secretion.Described example has been represented these types.The present invention also pays close attention to the application that detects the activatory not celliferous method of TLR.
The present invention also comprises the activation of inducing antigen-specific immunne response (as described in more detail) and non-specific natural immunity and the method for wide spectrum toleration that infectivity is attacked.Term antigen non-specific natural immunity used herein activation means the activated immune cell of non-B cell, comprises the NK cell, and T cell or other can be not rely on immunocyte that antigenic mode reacts or some combination of these cells.Induce the wide spectrum toleration that infectivity is attacked, because immunocyte is activity form and causes reaction to any intrusion chemical compound or microorganism.These cells not necessarily trigger the specific antigen specificity.This is used in particular for biological war and other above-mentioned environment, such as the traveller.
The experimenter
Experimenter used herein means people or inhuman vertebrates.Non-human vertebrate comprises livestock animals, companion animals and laboratory animal.Inhuman experimenter is also particularly including inhuman primates and rodent.Inhuman experimenter also particularly including, but be not limited to chicken, horse, cattle, pig, goat, Canis familiaris L., cat, Cavia porcellus, hamster, ermine and rabbit.
The experimenter who is in the generation disease risks used herein means known or suspects the experimenter who contacts pathogen, known described pathogen causes disease or associated, or known or suspection susceptible generation disease (for example, the genetic marker of disease family history or disease family history).
Provide treatment to have the experimenter's of immune system defect method among the present invention in one aspect.The method of this aspect of the present invention comprises and gives the present composition of effective dose so that treat this experimenter to the experimenter.Immune system defect used herein means the immune system support ability of antigenic immunne response is descended unusually.In one embodiment, immune system defect is disease or disorder, and wherein experimenter's immune system can't work in the normal capacity scope, or wherein it can be used to strengthen experimenter's immunne response, for example eliminates experimenter's tumor or cancer or infection.Experimenter with immune system defect used herein means and exists the support of experimenter's immune system for example to the experimenter of the ability drop of antigenic immunne response.Experimenter with immune system defect comprises the experimenter of acquired immunodeficiency and the experimenter with innate immunity defective.Experimenter with acquired immunodeficiency comprises, but be not limited to have the experimenter of chronic inflammatory disease, experimenter with chronic renal insufficiency or renal failure, experimenter with infection, experimenter with cancer, accept the experimenter of immunosuppressant, accept the experimenter of other immunosuppressant therapy and have underfed experimenter.In one embodiment, the experimenter has the CD4+T-cell mass of inhibition.In one embodiment, the experimenter has human immunodeficiency virus (HIV) and infects or have an acquired immune deficiency syndrome (AIDS) (AIDS).The method of the ability of immunne response is replied or strengthened supporting to the method for this aspect of the present invention for the experimenter that more violent immune response demands is arranged provides booster immunization thus
Inhibition used herein should refer to descend than result under the normal condition or effect.
The treatment that relates to disease or illness used herein should refer to get involved this class disease or illness so that prevention or slow down disease or illness takes place, and prevents or has slowed down progress, organizes its progress or eliminates them.For example, the treatment cancer comprises that (for example, prevention state precancer), the growth of cancers that alleviates the symptom of cancer and/or suppress to set up take place prophylaxis of cancer.
Illness
The present invention relates to treat the disease that has benefited from suppressing and/or inducing the immunne response of some TLR mediation.The immunostimulation of used herein and TLR7 mediation or the relevant disease of immunne response mean any disease or other illness that exists the immune activation relevant with the conduction of TLR7 signal and this class to activate deleterious experimenter.This class illness relates generally to TLR7 signal conduction activation as replying contact TLR7 part.
The immunostimulation of used herein and TLR8 mediation or the relevant illness of immunne response mean any disease or other illness that exists the immune activation relevant with the conduction of TLR8 signal and this class to activate deleterious experimenter.This class illness relates generally to TLR8 signal conduction activation as replying contact TLR8 part.
The immunostimulation of used herein and TLR9 mediation or the relevant illness of immunne response mean any disease or other illness that exists the immune activation relevant with the conduction of TLR9 signal and this class to activate deleterious experimenter.This class illness relates generally to TLR9 signal conduction activation as replying contact TLR9 part.
Infect
The present invention can be used for directly treating such as the treatment of infection illness.Infection means the experimenter and exists in the active born of the same parents or any illness of outer microbiological anomaly set of born of the same parents or colony.This quasi-microorganism can be for endogenic for the experimenter, or they can be for ectogenic for the experimenter.Experimenter with infection is shallow because of showing for having the experimenter, and part or whole body are subjected to the disease that infective micro-organisms is invaded or the growth of the naturally occurring endogenous microbiological anomaly of experimenter up regulation produces.Infective micro-organisms can be aforesaid virus, antibacterial, fungus or parasite.Various types of microorganisms can cause infecting, and are included as the microorganism of antibacterial, are the microorganism of virus, for the microorganism of fungus be parasitic microorganism.
Antibacterial is for passing through the vegetative unicellular organism of binary fission.They are based on its form, staining reaction, nutrition and metabolism requirement, antigenic structure, chemical composition and genetic homogeny classification and name.Antibacterial can be categorized as three races based on its morphologic species: spherical (coccus), directly-rod (bacillus) and bending or spiral rod (vibrio (vibrio), campylobacter (Campylobacter), spiral Pseudomonas (spirillum) and spirillum (spirochaete)).Antibacterial also more generally is categorized as two class biologies, i.e. gram positive bacteria and gram negative bacteria based on its staining reaction.Gram means the colouring method that carries out usually in Microbiological Lab.The Gram-positive biology keeps dyeing and demonstrates darkviolet behind dying operation.Gram-negative biological does not keep dyeing, but absorbs after stain and demonstrate pink thus.
Infectious bacteria includes, but are not limited to gram negative bacteria and gram positive bacteria.Gram positive bacteria includes, but are not limited to pasteurella (Pasteurella) kind, staphylococcus (Staphylococci) kind and Streptococcus (Streptococcus) kind.Gram negative bacteria includes, but are not limited to bacillus coli (Escherichia coli), Rhodopseudomonas (Pseudomonas) kind and Salmonella (Salmonella) kind.The instantiation of infectious bacteria comprises, but be not limited to: helicobacter pylori (Helicobacterpyloris), B. burgdorferi (Borrelia burgdorferi), invade lung legionella (Legionella pneumophilia), mycobacteria (Mycobacteria) kind (mycobacterium tuberculosis (M.tuberculosis) for example, Mycobacterium avium (M.avium), Mycobacterium intracellulare (M.intracellulare), mycobacterium kansasii (M.kansasii), mycobacterium gordonae (M.gordonae)), staphylococcus aureus (Staphylococcusaureus), Diplococcus gonorrhoeae (Neisseria gonorrhoeae), Neisseria meningitidis (Neisseria meningitidis), Listeria monocytogenes (Listeria monocytogenes), wine brewing Streptococcus pyrogenes (Streptococcuspyogenes) (A family Streptococcus), streptococcus agalactiae (Streptococcus agalactiae) (B family Streptococcus), Streptococcus (viridans streptococci family (viridans group)), enterococcus faecalis (Streptococcus faecalis), bargen's streptococcus (Streptococcusbovis), Streptococcus (anaerobism kind), streptococcus pneumoniae (Streptococcuspneumoniae), the kind of pathogenic campylobacter (Campylobacter sp.), the kind of Enterococcus (Enterococcus sp.), Haemophilus influenzae (Haemophilusinfluenzae), Bacillus anthracis (Bacillus anthracis), corynebacterium diphtheriae (Corynebacterium diphtheriae), the kind of Corynebacterium (Corynebacterium sp.), erysipelothrix rhusiopathiae (Erysipelothrixrhusiopathiae), aerogenesis folder film clostridium (Clostridium perfringens), clostridium tetani (Clostridium tetani), clostridium perfringen (Enterobacteraerogenes), Klebsiella pneumonia (Klebsiella pneumoniae), multocida (Pasturella multocida), the kind of Bacteroides (Bacteroidessp.), tool shuttle nuclear bacillus (Fusobacterium nucleatum), Streptobacillus moniliformis (Streptobacillus moniliformis), thorough white spirillum (Treponemapallidum), superfine treponema (Treponema pertenue), Leptospira (Leptospira), rickettsiae (Rickettsia) and actinomyces israelii (Actinomyces israelli).
Antibacterial includes, but are not limited to the pasteurella kind, staphylococcus kind, Streptococcus kind, bacillus coli, Rhodopseudomonas kind and Salmonella kind.The instantiation of infectious bacteria comprises, but be not limited to helicobacter pylori, B. burgdorferi is invaded the lung legionella, mycobacteria kind (mycobacterium tuberculosis for example, Mycobacterium avium, Mycobacterium intracellulare, mycobacterium kansasii, mycobacterium gordonae), staphylococcus aureus, Diplococcus gonorrhoeae, Neisseria meningitidis, Listeria monocytogenes, wine brewing Streptococcus pyrogenes (A family Streptococcus), streptococcus agalactiae (B family Streptococcus), Streptococcus (viridans streptococci family), enterococcus faecalis, bargen's streptococcus, Streptococcus (anaerobism kind), streptococcus pneumoniae, the kind of pathogenic campylobacter, the kind of Enterococcus, Haemophilus influenzae, Bacillus anthracis, corynebacterium diphtheriae, the kind of Corynebacterium, erysipelothrix rhusiopathiae, aerogenesis folder film clostridium, clostridium tetani, clostridium perfringen, Klebsiella pneumonia, multocida, the kind of Bacteroides, tool shuttle nuclear bacillus, Streptobacillus moniliformis, thorough white spirillum (Treponema pallidum), superfine treponema, Leptospira, rickettsiae and actinomyces israelii.
Virus is for generally comprising nucleic acid core and protein coat, but the little pathogen infection of the organism that can't independently survive.Virus can also take to lack the form of proteinic infectious nucleic acid.Virus can not be survived in the presence of the living cells that does not have it to duplicate therein.Virus enters specific living cells by endocytosis or DNA (phage) direct injection and breeding and causes disease.The virus of breeding can discharge and infect extra cell subsequently.Some virus is the virus that contains DNA, and other virus is for containing the virus of RNA.In certain aspects, the invention still further relates to the treatment disease, wherein in disease process, relate to protein virus, such as, for example mad cow disease (is a bovine spongiform encephalopathy, BSE) or the animal scrapie infects or people's creutzfeldt-jakob disease.
Virus includes, but are not limited to enterovirus (include, but are not limited to Pironavirus section (picornaviridae) virus, such as poliovirus, Coxsackie virus, ECHO virus), rotavirus, adenovirus, hepatitis virus.The instantiation of having found in the people includes, but are not limited to: (for example, the human immunodeficiency virus (is also referred to as HTLV-III, LAV or HTLV-III/LAV or HIV-III such as HIV-1 to Retroviridae (Retroviridae); With other separator, such as HIV-LP; Pironavirus section (for example, Pironavirus section, hepatitis A virus; Enterovirus, human coxsackievirus, rhinovirus, ECHO virus); Calciviridae (bacterial strain that for example, causes gastroenteritis); Togaviridae (Togaviridae) (for example, equine encephalitis virus, rubella virus); Flaviviridae (Flaviviridae) (for example, dengue virus, encephalitis, yellow fever virus); Coronaviridae (Coronaviridae) (for example, coronavirus); Rhabdoviridae (Rhabdoviridae) (for example, herpes stomatitis virus, rabies virus); Filoviridae (Filoviridae) (for example, Ebola virus); Paramyxoviridae (Paramyxoviridae) (for example, parainfluenza virus, mumps virus, Measles virus, respiratory syncytial virus); Orthomyxoviridae family (Orthomyxoviridae) (for example, influenza virus); Bunyan Viraceae (Bunyaviridae) (for example, Hantaan virus, cloth Buddhist nun virus, sand fly virus and Nairobi sheep virus (Nairo viruses)); Arenaviridae (Arenaviridae) (hemorrhagic fever virus); Reoviridae (Reoviridae) (for example, reovirus, Orbivirus (orbiviurses) and rotavirus); Birnavirus section (Birnaviridae); Have a liking for hepatovirus section (Hepadnaviridae) (hepatitis B virus); Parvoviridae (Parvoviridae) (parvovirus belongs to (parvoviruses)); Papovaviridae (Papovaviridae) (human papillomavirus, polyoma virus); Adenoviridae (Adenoviridae) (most of adenovirus); Herpetoviridae (Herpesviridae) (herpes simplex virus (HSV) 1 and 2, varicella zoster virus, cytomegalovirus (CMV)); Poxviridae (Poxviridae) (smallpox virus, vaccinia virus, poxvirus); Iris Viraceae (Iridoviridae) (for example, African swine fever virus); With unfiled virus (for example, the spongiform encephalopathy cause of disease, δ hepatitis cause of disease (think hepatitis B virus comitative aspect), the non-a non-b hepatitis substance (is propagated in 1 class=body; 2 classes=non-intestinal transmitted (being hepatitis C); Norwalk virus and correlated virus and Astrovirus).
The example of having found in the people includes, but are not limited to: (for example, (for example, the human immunodeficiency virus (is also referred to as HTLV-III, LAV or HTLV-III/LAV or HIV-III such as HIV-1 to Retroviridae; With other separator, such as HIV-LP; Pironavirus section (for example, Pironavirus section, hepatitis A virus; Enterovirus, human coxsackievirus, rhinovirus, ECHO virus); Calciviridae (bacterial strain that for example, causes gastroenteritis); Togaviridae (for example, equine encephalitis virus, rubella virus); Flaviviridae (for example, dengue virus, encephalitis, yellow fever virus); Coronaviridae (for example, coronavirus); Rhabdoviridae (for example, herpes stomatitis virus, rabies virus); Filoviridae (for example, Ebola virus); Paramyxoviridae (for example, parainfluenza virus, mumps virus, Measles virus, respiratory syncytial virus); Orthomyxoviridae family (for example, influenza virus); Bunyan Viraceae (for example, Hantaan virus, cloth Buddhist nun virus, sand fly virus and Nairobi sheep virus); Arenaviridae (hemorrhagic fever virus); Reoviridae (for example, reovirus, Orbivirus and rotavirus); Birnavirus section; Have a liking for hepatovirus section (hepatitis B virus); Parvoviridae (parvovirus genus); Papovaviridae (human papillomavirus, polyoma virus); Adenoviridae (most of adenovirus); Herpetoviridae (herpes simplex virus (HSV) 1 and 2, varicella zoster virus, cytomegalovirus (CMV)); Poxviridae (smallpox virus, vaccinia virus, poxvirus); Iris Viraceae (for example, African swine fever virus); With unfiled virus (for example, spongiform encephalopathy cause of disease, δ hepatitis cause of disease (think hepatitis B virus comitative aspect), i.e. hepatitis C; Norwalk virus and correlated virus and Astrovirus).
Fungus is a most eukaryotes, and wherein only minority causes vertebrate mammals to infect.Because fungus is a most eukaryotes, so they are in size, structure organization, life cycle and breeding substrate aspect obviously are different from the protokaryon antibacterial.Generally based on morphological characteristic, modes of reproduction and cultural characteristic classification fungus.Although fungus can cause the dissimilar disease of experimenter, such as the respiratory system anaphylaxis behind the suction fungal antigen, because of taking in toxicant, the mycetism that the aflatoxin class that phallotoxin that produces such as amanita phalloides (Amanitaphalloides) toxin and poisonous mushroom and Eurotium (aspergillus) kind produce causes, but be not that all funguses all cause infectious disease.
Infectious fungus can cause whole body or show shallow infection.The constitutional systemic infection can occur among the NHS, and opportunistic infection is most commonly in the immunocompromised host experimenter.Cause the modal fungal pathogens of constitutional systemic infection to comprise Blastomyces (Blastomyces), ball spore Pseudomonas (Coccidioides) and Histoplasma (Histoplasma).In immunocompromised host or immunosuppressant experimenter, cause the common fungus of opportunistic infection to include, but are not limited to white candida mycoderma (Candida albicans), the kind of Cryptococcus histolyticus (Cryptococcus neoformans) and various Eurotiums.The whole body fungal infection is the invasive infection of internal organs.Usually by non-, gastrointestinal tract or intravenous catheter enter in the body organism.The infection of these types can cause because of constitutional pathomycete or opportunistic fungus.
Show shallow fungal infection and comprise that fungus grows on the outer surface, but intrusive body inner tissue not.The shallow fungal infection of typical table comprises and relates to skin, the cutaneous fungal infection of hair or fingernail.
The disease relevant with fungal infection comprises aspergillosis, blastomycosis, candidiasis, chromomycosis, coccidioidomycosis, cryptococcosis, the fungoid eye infections, fungoid hair, fingernail and skin infection, histoplasmosis, Lobomycosis, Madura mycomycete disease, otomycosis, paracoccidioidomycosis, dispersivity Ma Nifushi penicillium sp (Penicilliummarneffei), paeohyphomycosis, rhinosporidiosis, sporotrichosis and zygomycosis.
Fungus comprises yeast and mycete.The example of fungus comprises, but be not limited to the kind of Eurotium, comprise Aspergillus fumigatus (Aspergillus fumigatus), Blastomyces dermatitidis (Blastomycesdermatitidis), the kind of mycocandida (Candida spp.), comprise white candida mycoderma, thick ball spore bacterium (Coccidioides immitis), novel Cryptococcus (Cryptococcus neoformans), histoplasma capsulatum (Histoplasmacapsulatum), Pneumocystis carinii (Pneumocystis carinii), the kind (Rhizopus spp.) of kind of root Mucor (Rhizomucor spp.) and Rhizopus.
Parasite is to depend on other organism so that the organism of survival, and must enter or infect another kind of organism thus to continue its life cycle.The organism that infects, promptly the host provides nutrition and habitat for parasite.Although at it the most widely on the implication, the term parasite can comprise all pathogen (be antibacterial, virus, fungus, protozoacide and anthelmintic), but generally speaking, this term is used for only referring to protozoacide, anthelmintic and ectoparasitic arthropod (for example, Ticks, Ticks is stepped on).Protozoacide be can be in born of the same parents and born of the same parents outside, particularly at blood, intestinal to or the extracellular matrix of tissue in the unicellular organism body that duplicates.Anthelmintic is the multicellular organisms outside the born of the same parents kind (Trichinella spp.) of Trichinella (except) all the time almost.Anthelmintic generally need and propagate into Secondary host so that duplicate from the Primary host disengaging.With above-mentioned these type opposite, ectoparasite arthropod and host's health outer surface form parasitism.
Parasite comprises born of the same parents entozoa and special born of the same parents entozoa.Parasitic example comprises, but be not limited to Plasmodium falciparum (Plasmodium falciparum), Plasmodium ovale (Plasmodium ovale), malariae (Plasmodium malariae), Plasmodium vivax (Plasmdodium vivax), Plasmodium knowlesi, babesia microti (Babesia microti), Babesia divergens, Oswaldocruzia (Trypanosomacruzi), toxoplasma gondii (Toxoplasma gondii), Trichinella spiralis, Leishmania major, Leishmania donovani (Leishmania donovani), leishmania brasiliensis (Leishmania braziliensis), crithidia cunninghami (Leishmania tropica), castellanella gambiense (Trypanosoma gambiense), trypanosoma rhodesiense (Trypanosoma rhodesiense) and schistosoma mansoni (Schistosoma mansoni).
Other infectious organisms (being protista) comprises the kind (Plasmodium spp.) of Plasmodium, such as Plasmodium falciparum, and malariae, Plasmodium ovale and Plasmodium vivax; And toxoplasma gondii.Blood-borne and/or blood-borne comprise the kind of Plasmodium, babesia microti (Babesia microti), Babesia divergens, Chlamydiatrachomatis, crithidia cunninghami, the kind of leishmaniasis (Leishmaniaspp.), leishmania brasiliensis, Leishmania donovani, castellanella gambiense and trypanosoma rhodesiense (lethargus), Oswaldocruzia (american trypanosomiasis) and Mus toxoplasma.
Other medically relevant microorganism has extensively been described in the document, for example, referring to C.G.A Thomas, Medical Microbiology, Bailliere Tindall, Great Britain1983 is incorporated herein by reference the full content of the document.
Cancer
Provide treatment to have the experimenter's of cancer method among the present invention in one aspect.
Experimenter with cancer be have can detected cancerous cell the experimenter.Cancer can be pernicious or non-malignant cancer." cancer " used herein means the uncontrolled growth of cell of the normal function effect of disturbing organ and system.Finally can be encroached on organ dysfunction and degenerate and cause experimenter's death from the migration of its home position and the cancer that is planted in vitals by making.Leukemia, such as leukemia can the excessive competition experimenter the normal hematopoiesis chamber, cause hemopoietic depletion (anemia, the form of thrombocytopenia and neutropenia) thus and finally cause death.
Be different from the primary tumor position, shift to diffusing to the cancerous cell district that the health other parts produce from primary tumor because of cancerous cell.When the diagnosing primary tumor mass, can monitor the existence of experimenter's metastasis.Usually can pass through separately or coupling NMR (Nuclear Magnetic Resonance)-imaging (MRI) scanning, computed tomography (CT) scanning, blood and platelet count, Liver Function, Chest X-rays is with scanning and monitoring specific symptoms detect metastasis admittedly.
Cancer includes, but are not limited to basal cell carcinoma, cancer of bile ducts; Bladder cancer; Osteocarcinoma; Brain and central nervous system (CNS) cancer; Breast carcinoma; Breast carcinoma; Choriocarcinoma; Colon and rectal cancer; The connective tissue cancer; Digestive system cancer; Carcinoma of endometrium; The esophageal carcinoma; Cancer eye; Head and neck cancer; Last Intradermal tumor; Renal carcinoma; Laryngeal carcinoma; Leukemia; Hepatocarcinoma; Pulmonary carcinoma (for example, minicell and non-small cell); Lymphoma comprises He Jiejin and non Hodgkin lymphoma; Melanoma; Myeloma; Neuroblastoma; Oral cancer (for example, lip, tongue, oral cavity and pharynx); Ovarian cancer; Cancer of pancreas; Cancer of pancreas; Retinoblastoma; Rhabdomyosarcoma; Rectal cancer; Respiratory system carcinoma; Sarcoma; Skin carcinoma; Gastric cancer; Carcinoma of testis; Thyroid carcinoma; Uterus carcinoma; Urinary system cancer and other cancer, adenocarcinoma and sarcoma.
The autoimmune illness
Relate to the illness that innate immunity is replied or the Th1-para-immunity is replied and comprise inflammation, acute and chronic allograft rejection, graft versus host disease (GvHD), some autoimmune disease and sepsis.Consider that the selectivity to the conduction of TLR signal that the present invention can realize suppresses, the present invention can be used for the treatment of this class illness.
Autoimmune disease generally can be categorized as antibody-mediated, the cell-mediated or antibody-mediated combination cell-mediated with T-of T-.Think that the combination of connection ODN of the present invention and TLR part is used for the treatment of the various types of autoimmunity that relate to immunity antibody-mediated or that T-is cell-mediated, comprise insulin-dependent (I type) diabetes, rheumatoid arthritis, multiple sclerosis, systemic lupus erythematosus (sle) (SLE) and inflammatory bowel (being Crohn disease and ulcerative colitis).Can utilize the animal model of these autoimmune diseases and they are used for estimating the effect that the present invention is combined in these diseases.Other autoimmune disease includes, but are not limited to alopecia areata, acquired hemophilia, ankylosing spondylitis, antiphospholipid syndrome, autoimmune hepatitis, autoimmune hemolytic anemia, behcet's syndrome, cardiomyopathy, sprue dermatitis, confirmed fatigue immune dysfunction syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy, this syndrome of mound, cicatricial pemphigoid, the CREST syndrome, cold agglutinin disease, discoid lupus, essential mixed cryoglobulinemia, fibromyalgia, fibromyositis, Ge-Ba syndrome, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura, IgA nephropathy, adolescent arthritis, lichen planus, myasthenia gravis, polyarteritis, polychondritis, polyglandular syndrome, dermatomyositis, the constitutional gamma globulin lacks mass formed by blood stasis, constitutional bile liver cirrhosis, psoriasis, Raynaud phenomenon, Reiter syndrome, sarcoidosis, stiff man syndrome, high peace arthritis (Takayasu arthritis), temple tremulous pulse/giant cell arteritis, uveitis, vasculitis and vitiligo.
In several autoimmune diseases, observe antibody usually at autoantigen.For example, with regard to systemic lupus erythematosus (sle), the autoantibody of strand and double-stranded DNA or RNA has been described.(1999) J Immunol 163:6306-13 such as Vallin H; (1999) JImmunol 163:3304-12 such as Hoet RM; Ven Venrooij (1990) J Clin Invest86:2154-60.The autoantibody level of finding in the autoimmune patients serum is relevant with severity of disease.For example, the complete macromolecular particle of the mode annunciations of the autoantibody that produces in people SLE can be for immunogenic such as the complex that contains RNA-or DNA self, and anti-nucleic acid antibody can produce thus.(1992) Mol Biol Rep 16:127 such as Lotz M; MohanC etc. (1993) J Exp Med 177:1367-81.For example, this class is from the cell inflammation that DNA that king's cell or be present in discharges in the microorganism that contains DNA or RNA the autoimmune patients serum or RNA can cause facilitating autoimmune disease of withering.Fatenejad S (1994) J Immunol152:5523-31; (2002) Isr Med Assoc J 4:706-12 such as Malmegrim KC; (2001) Arthritis Res 3:253-8 such as Newkirk MM.In fact, can be from inducing by thinking that facilitating group to examine the SLE serum of effective immunne response of the IFN-α secretion control that immunological diseases take place identifies the sequence that contains CpG.(2001) Scand J Immunol54:543-50 such as Magnusson M;
Figure A20068004438600431
L etc. (2001) J Exp Med 194:F59-63.In addition, the anti-RNA antibody epitope that can identify and its are by being rich in G, and the sequence of U is formed.(1992) Proc Natl Acad Sci U.S. 89:8864-8 such as Tsai DE; (1993) JImmunol 150:1137-45 such as Tsai DE.
Anaphylaxis
" anaphylaxis illness " or " anaphylaxis " mean the acquired allergy to material (anaphylactogen).The anaphylactoid experimenter that conduct is replied anaphylactogen takes place or has taken place in advance before answering feeling the pulse with the finger-tip in " experimenter with anaphylaxis illness ".The anaphylaxis illness includes, but are not limited to eczema, allergic rhinitis or rhinitis, and pollinosis, anaphylaxis conjunctivitis, bronchial asthma, urticaria (urticaria) (urticaria (hives)) and food anaphylaxis, other atopy illness comprises atopic dermatitis; Anaphylaxis; Drug allergy; And angioedema.
Anaphylaxis is generally from the certain kinds Immunoglobulin IgE to anaphylactogen and produces the relevant ictal illness of antibody.IgE mediation Aeroallergens is replied also for showing the factor of susceptible generation asthma.If anaphylactogen runs into and basophilic leukocyte (circulation in the blood) or mastocyte (disperse omnipresence in solid tissue) the bonded specific IgE of lip-deep IgE Fc receptor (Fc ε R), cell is activated so, cause amboceptor such as histamine, 5-hydroxy tryptamine and lipid mediators produce and discharge.
Anaphylaxis takes place when the IgE class is organized sensitization immunoglobulin and exogenous allergen reaction.IgE antibody discharges anaphylactoid chemical mediator (vasoactive amine) with the cell that mastocyte and/or basophilic leukocyte combine and these are special when the anaphylactogen that is subjected to bridging antibody molecule end so stimulates.Histamine, platelet activating factor, arachidonic acid metabolite and 5-hydroxy tryptamine are wherein known people's best anaphylaxis amboceptor.Histamine and other vasoactive amine generally are stored in mastocyte and the basophil.These mastocytes disperse omnipresence in animal tissue, and basophilic leukocyte circulates in vascular system.These cells are produced in cell and are stored histamine, occur otherwise relate to the bonded consequence dedicated sequences of the IgE that causes its release.
Anaphylactoid symptom changes according to the difference of position in the body, wherein IgE and antigen-reactive.If reaction takes place along respiratory epithelium, symptom is generally sneeze so, cough and asthma reaction.With regard to food anaphylaxis, if in digestive tract, interact, so common unusual pain and diarrhoea.For example, give penicillin at bee sting or to the anaphylaxis experimenter after, the systemic anaphylaxis system may serious and common life-threatening.
It is relevant that anaphylaxis and Th2-para-immunity are replied, and its feature is at least partially in Th2 cytokine IL-4 and IL-5 and to the antibody allotype conversion of IgE.Th1 and Th2 immunne response are oppositely to regulate each other, and the feasible skew that immunne response is replied the Th1-para-immunity can prevent or improve the Th2-para-immunity and reply, and comprises anaphylaxis.
Asthma
" asthma " used herein means respiratory disorder, it is characterized in that airway inflammation and narrow and air flue increase the reactivity that sucks pathogen.Asthma is relevant with atopy or anaphylaxis illness usually, but is not unique.The symptom of asthma comprises the wheezing that causes because of airway obstruction, draws last breath feeling of chest tightness and cough outbreak repeatedly.Can detect the airway inflammation relevant by observing many physiological changes with asthma, such as, airway epithelia strips off, collagen deposition under the basement membrane, and edema, the mastocyte activation, inflammatory cell infiltration comprises neutrophilic leukocyte, eosinophilic granulocyte and lymphocyte.As the result of airway inflammation, airway hyperreactivity takes place in asthmatic patient usually, flow limitation, respiratory symptom and disease chronicity.Flow limitation comprises acute bronchoconstriction, the air flue edema, and slime plug forms and Airway Remodeling, and these features cause bronchial obstruction usually.In some situation of asthma, fibrosis under the basement membrane may take place, cause the pulmonary function persistent anomaly.
Research has in the past few years disclosed asthma may be because of the inflammatory cell of air flue residence, and the complexity interaction institute in amboceptor and other cell and the tissue causes.Mastocyte, acidophil, epithelial cell, macrophage all can play an important role in the inflammatory process relevant with asthma with activating T cell.(1990) Am Rev Respir Dis142:434-457 such as Djukanovic R.Think that these cells can form and the synthetic recently amboceptor of may be directly or indirectly effect being organized in the part influences the air flue function by secretion.Recognize that also t lymphocyte subset group (Th2) is playing an important role aspect the allergic inflammation in the disease chronicity adjusting air flue by discharging selective cytokine and setting up.(1992) N Engl J Med326:298-304 such as Robinson DS.
Asthma is that the complicated disease that different phase produces is taking place, and can be categorized as acute, subacute or chronic based on the symptom degree.Acute inflammatory reaction is raised into air flue relevant with cell in early days.The subacute inflammation reaction relates to the residential cell activation that cell is raised and caused persistency inflammation pattern.Chronic inflammatory reaction is characterised in that primary cellular defect and the carrying out property repair process that continues level, may cause the persistency in the air flue unusual.
" experimenter with asthma " for having the experimenter of respiratory disorder, it is characterized in that airway inflammation and narrow and the airway reactivity that sucks pathogen increased.With asthma relevant factor takes place and include, but are not limited to anaphylactogen, cold temperature is tempered viral infection and SO 2
Asthma as herein described can be replied relevantly with the Th2-para-immunity, and its feature is at least partially in Th2 cytokine IL-4 and IL-5 and to the antibody allotype conversion of IgE.Th1 and Th2 immunne response are oppositely to regulate each other, and the feasible skew that immunne response is replied the Th1-para-immunity can prevent or improve the Th2-para-immunity and reply, and comprises anaphylaxis.
TLR7/8 part of the present invention also is used to improve the dendritic cell survival with the combination that is connected oligonucleotide, differentiation and ripe.
Provide the method that strengthens the epi-position diffusion of the present invention in aspect some." epi-position diffusion " used herein means the epitope specificity variation of going up subdominant and/or hiding epi-position to this albumen (intramolecularly diffusion) or other albumen (intermolecular diffusion) from the initially concentrated advantage epitope specificity immunne response that is oriented to self or extrinsic protein.The epi-position diffusion produces multiple epitope specificity immunne response.
Antimicrobial
The TLR7/8 part be connected oligonucleotide combination and can use jointly with one or more antimicrobials.Antimicrobial or medicine include, but are not limited to antibacterial, antiviral agent, antifungal and antiparasitic.Term, such as " anti-infective ", " antibiotic ", " antibacterial agent ", " antiviral agent ", the implication that " antifungal ", " antiparasitic " and " parasiticide medicament " are fully established based on those skilled in the art and being defined in the standard medical textbook.In brief, antibacterial agent kills and wounds antibacterial and comprises antibiotic and other has the synthetic or native compound of similar functions.Antiviral agent can separate from natural origin or synthesize and be used to kill and wound or suppress virus.Antifungal is used for the treatment of the shallow fungal infection of table and chance and constitutional whole body fungal infection.Antiparasitic kills and wounds or suppresses parasite.Many antibiotic are by cell, such as the low-molecular-weight molecule of microorganisms as secondary metabolite.Generally speaking, antibiotic disturbs microorganism is had specificity and is not present in one or more functions or structure in the host cell.
One of problem relevant with the infection therapy is the side effect that takes place in the host with anti-infective treatment.For example, many anti-infectives can kill and wound or suppress broad-spectrum micro-organisms and the kind of particular type is not had specificity.Use the anti-infective treatment of these types to cause killing and wounding normal flora and the infective micro-organisms of living among the host.Described flora disappearance can cause the disease complication and make other pathogen of host's susceptible, because this flora and infectious pathogen are competed and worked as barrier.Other side effect may produce as specificity or the non-specific result that be of these chemical entity to host's non-microorganism cell or tissue.
Be to take place the antibiotic resistance strain of microorganism with another problem of widely-used anti-infective.Vancomycin resistance enterococcus has appearred, the penicillin resistance streptococcus pneumoniae, and multidrug resistant staphylococcus aureus and multidrug resistant pulmonary tuberculosis bacterial strain, and become clinical subject matter.Widely-used anti-infective can produce the strains of many antibacterials.As a result of, need new infection strategy to resist these microorganisms.
The antibacterium antibiotic that effectively kills and wounds or suppress extensive antibacterial is called broad ectrum antibiotic.The antibacterium antibiotic of other type is mainly effective to Gram-positive or Gram-positive bacterioid.The antibiotic of these types is called narrow-spectrum antibiotic.Effectively resist single creature or disease, but other invalid antibiotic of other type antibacterial is called limited spectrum antibiotic.
Antibacterial agent is sometimes based on its main model of action classification.Generally speaking, antibacterial agent is the cell wall synthetic inhibitor, cell membrane inhibitor, protein synthesis inhibitor, nucleic acid synthetic or depressant of functions and competitive inhibitor.The cell wall synthetic inhibitor suppresses the cell wall building-up process and is generally the step of bacterial peptide polysaccharide in synthetic.The cell wall synthetic inhibitor comprises beta-Lactam antibiotic, natural penicillin, semisynthetic penicillin, ampicillin, clavulanic acid, cephalosporins and bacitracin.
Beta-Lactam antibiotic is the antibiotic that comprises the synthetic final step of peptide for inhibiting polysaccharide of 4-unit beta-lactam nucleus, and beta-Lactam antibiotic can be for synthetic or natural.The beta-Lactam antibiotic that is produced by Penicillium (penicillium) is a natural antibiotics, such as benzylpenicillin or penicillin V.They produce by fermentation Penicllium chrysogenum (Penicillium chrysogenum).Natural penicillin has narrow spectrum activity and generally to Streptococcus (Streptococcus), gonococcus (Gonococcus) and staphylococcus (Staphylococcus) are effective.Also the natural penicillin to effective other type of gram positive bacteria comprises penicillin F, X, K and O.
Semi-synthetic penicillins is generally the trim of the molecule 6-amino-penicillanic acid of mycete generation.Can modify 6-amino-penicillanic acid by adding side chain, described side chain produces to have than the natural penicillin penicillin of broad spectrum of activity or various other advantageous feature more.The semisynthetic penicillin of some type has the wide spectrum to gram positive bacteria and gram negative bacteria, but by the penicillinase inactivation.These semisynthetic penicillins comprise the ampicillin, carbenicillin, oxazacillin, azlocillin, mezlocillin and piperacillin.The semisynthetic penicillin of other type has narrower activity to gram positive bacteria, but has occurred not by the characteristic of penicillinase inactivation, and they comprise, for example methicillin, dicloxacillin and nafcillin.Some wide spectrum semisynthetic penicillin can with beta-lactamase inhibitor, all clavulanic acids and Sulbactam coupling.Beta-lactamase inhibitor does not have anti-microbial effect, but they play the inhibition penicillinase, prevents the semisynthetic penicillin degraded thus.
The beta-Lactam antibiotic of another kind of type is a cephalosporins.They are to the degraded sensitivity of antibacterial beta-lactamase, and therefore use is always invalid separately.Yet, cephalosporins tolerance penicillinase.They are effective to various gram positive bacterias and gram negative bacteria.Cephalosporins includes, but are not limited to cefalotin, cefapirin, cefalexin, cefamandole, cefaclor, cefazolin, cefuroxime, cefoxitin, cefotaxime, cefsulodin, cefetamet, cefixime, ceftriaxone, cefoperazone, cephalo pyridine and latamoxef.
Bacitracin is for being released into the outside synthetic another kind of antibiotic of cell wall that suppresses of film by suppressing muramyl peptide subunit or Peptidoglycan from the molecule of sending this Asia animal.Although bacitracin is effective to gram positive bacteria, its application is generally limited to topical because of its high toxicity.
Carbapenem is that another kind can suppress the synthetic wide spectrum beta-Lactam antibiotic of cell wall.The example of carbapenem includes, but are not limited to imipenum.Single bacterium amine is the wide spectrum beta-Lactam antibiotic also, and comprises euztreonam.The antibiotic vancomycin that streptomyces (Streptomyces) produces is synthetic also effective to gram positive bacteria by suppressing cell membrane.
Another kind of antibacterial agent is the antibacterial agent of cell membrane inhibitor.These chemical compounds destroy the bacterial membrane structure or suppress its function.Use a problem to be that they can generation effect in prokaryotic cell and antibacterial because of the similarity in the phospholipid in antibacterial and eukaryotic cell membrane as the antibacterial agent of cell membrane inhibitor.Therefore, these chemical compounds almost are not enough to allow these chemical compound whole bodies to use specificity and prevent to use the high dose topical.
A kind of clinical useful cell membrane inhibitor is a polymyxin.Polymyxin disturbs the film function by the binding film phospholipid.Polymyxin mainly effectively and generally is used for serious pseudomonal infection or the antibiotic pseudomonal infection of tolerance low toxicity to gram negative bacteria.These give the relevant side effect of this chemical compound with whole body and comprise infringement to kidney and other organ.
Other cell membrane inhibitor comprises amphotericin B and nystatin, and they are the antifungal that is mainly used in treatment whole body fungal infection and mycocandida yeast infection.Imidazoles is the another kind of antibiotic of cell membrane inhibitor that is.Imidazoles for example, is used for the treatment of yeast infection as antibacterial agent and antifungal, and dermatozoon infects and the whole body fungal infection.Imidazoles includes, but are not limited to clotrimazole, miconazole, ketoconazole, itraconazole and itraconazole.
Many antibacterial agents are protein synthesis inhibitor.These chemical compounds prevent antibacterial composite structure albumen and enzyme and cause bacterial cell growth thus or function suppresses or cell death.Generally speaking, these chemical compounds disturb and transcribe or translation process.The antibacterial agent that blocking-up is transcribed includes, but are not limited to rifampicin and ethambutol.The rifampicin of inhibitory enzyme RNA polymerase has broad spectrum of activity and effective to gram positive bacteria and gram negative bacteria and mycobacterium tuberculosis (Mycobacteriumtuberculosis).Ethambutol is effective to mycobacterium tuberculosis.
The antibacterial agent of blocking-up translation disturbs bacterial ribosome to be translated into protein to prevent mRNA.Generally speaking, such chemical compound includes, but are not limited to Tetracyclines, chloromycetin, Macrolide (for example erythromycin) and aminoglycoside (for example streptomycin).
Aminoglycoside is by antibacterial streptomyces (Streptomyces), such as, streptomycin for example, kanamycin, kanamycin, amikacin and gentamycin produce a class antibiotic.Aminoglycoside is useful to the bacterial infection that various gram positive bacterias and gram negative bacteria cause.Streptomycin is widely used as the phthisical main medicine of treatment.Gentamycin is useful to many gram positive bacterias and Gram-negative bacteria strain, comprises pseudomonal infection, especially with the tobramycin coupling.Kanamycin is useful to many gram positive bacterias, comprises the penicillin resistance staphylococcus.A kind of side effect that limits its aminoglycoside of using clinically is verified under the requisite dosage for effect, prolongs to use the infringement renal function and cause the auditory nerve infringement, thereby causes deafness.
The translational inhibitor antibacterial agent of another kind of type is a Tetracyclines.Tetracyclines is a class antibiotic, and they are broad-spectrum and effective to various gram positive bacterias and gram negative bacteria.The example of Tetracyclines comprises tetracycline, minocycline, doxycycline and chlortetracycline.They are important to the antibacterial for the treatment of many types, but the treatment Lyme disease in particular importance.As the result of its hypotoxicity and minimum direct side effect, the medical community excess is used and is misapplied Tetracyclines and has problems.For example, excess is used drug resistance is extensively taken place.
Antibacterial agent prolongs or prevents that not charged tRNA from discharging or they both from bacterial ribosome in conjunction with 50S ribosomal subunit and by peptidyl transferase Profilin matter such as Macrolide is reversible.These chemical compounds comprise erythromycin, Roxithromycin, clarithromycin, oleandomycin and azithromycin.Erythromycin has activity to most of gram positive bacteria, neisseria (Neisseria), and Legionnella (Legionella) and Haemophilus spp (Haemophilus), but invalid to enterobacteriaceae (Enterobacteriaceae).Lincomycin and clindamycin that peptide bond in the blocking protein building-up process forms are useful to gram positive bacteria.
The translational inhibitor of another kind of type is a chloromycetin.Chloromycetin prevents the polypeptide chain growth in the protein building-up process thus in conjunction with the 70S ribosome that suppresses the bacterial enzyme peptidyl transferase.A kind of serious side effects relevant with chloromycetin is aplastic anemia.Aplastic anemia is the effectively streptomycin dosage of treatment antibacterial generation down in small scale (1/50,000) patient.In case open antibiotic, use chloromycetin hardly because of the consequence of dying from anemia so according to there be limited evidence currently of higher dosage prescription.It still is applied to life-threatening situation (for example, typhoid fever) because of its effectiveness.
Some antibacterial agent destroys the synthetic or function of nucleic acid, for example in conjunction with DNA or RNA, makes its information not read.They include, but are not limited to quinolones and bactrim, have both comprised synthetic chemicals, comprise rifomycins again, promptly natural or semi-synthetic chemicals.Quinolones is blocked bacterium DNA replication by suppressing dna gyrase, and antibacterial needs this kind of enzyme to produce its circulation DNA.They comprise norfloxacin for broad-spectrum and example, ciprofloxacin, enoxacin, nalidixan and Temafloxacin.Nalidixan is the antibacterial in conjunction with dna gyrase (topoisomerase), described enzyme be dna replication dna essential and allow superhelix lax and form again, thereby suppress the dna gyrase activity.Being mainly used in of nalidixan in treatment lower urinary tract infection (UTI), because it is to the gram negative bacteria of several types, such as escherichia coli (E.coli), clostridium perfringen (Enterobacter aerogenes), the kind of Klebsiella pneumonia (K.pneumoniae) and Proteus (Proteus) is effective, and these bacterium are the common cause of UTI.Bactrim is the drug combination of Sulfamethoxazole and trimethoprim, and its blocking-up constitutes the antibacterial of the required folic acid of DNA nucleotide and closes.Rifampicin is a Ryfamycin derivative, and it has activity to gram positive bacteria (comprising the meningitis that mycobacterium tuberculosis and Neisseria meningitidis (Neisseriameningitidis) cause) and some gram negative bacteria.Rifampicin adds the requisite primary nucleotide of activated polymerization enzyme in conjunction with the beta subunit and the blocking-up of polymerase, and it is synthetic to block mRNA thus.
Another kind of antibacterial agent is the chemical compound as the bacterial enzyme competitive inhibitor.Competitive inhibitor structurally almost all with the bacterial growth factor type like and competitive the combination, but can't finish metabolic function in the cell.These chemical compounds comprise sulfonamides and even have a sulfanilamide chemical modification form higher and more broad spectrum antibiotic activity.Sulfonamides (for example sulfafurazole and trimethoprim) is used for the treatment of streptococcus pneumoniae (Streptococcus pneumoniae), uncomplicated UTI that beta hemolytic streptococcus and escherichia coli and being used for the treatment of cause because of escherichia coli and treatment meningococcal meningitis.
Antiviral agent is the chemical compound that prevents that virus infected cell or intracellular virus from duplicating.The antiviral agents that exists because virus replication is very relevant with the interior dna replication dna of host cell, makes non-specific antiviral agent toxic to the host usually far fewer than antimicrobial drug.In virus infection, there are the several stages that can be blocked or suppress by antiviral agent.These stages comprise that virus (immunoglobulin or binding peptide class) and host cell adhere to, virus uncoating (for example, shelling), virus mRNA is synthesized or (is for example translated, interferon), viral RNA or dna replication dna (for example, nucleoside analog), new virus albumen maturation (for example, protease inhibitor) and virus are sprouted and are discharged.
Another kind of antiviral agent is a nucleoside analog.Nucleoside analog is and nucleoside analogues, but has the synthetic compound of incomplete or unusual deoxyribose or ribose groups.In case nucleoside analog is in cell, they compete the triphosphoric acid form of mixing viral DNA or RNA just by phosphorylation thereby produce with normal oligodeoxynucleotide.In case the triphosphoric acid form of nucleoside analog is impregnated in the nucleic acid chains of growth, it just causes irreversibly combining with varial polymerases and taking place thus chain termination so.Nucleoside analog comprises, but be not limited to acyclovir (being used for the treatment of herpes simplex virus and varicella zoster virus), ganciclovir (being used for cytomegalovirus), idoxuridine, ribavirin (being used for the treatment of respiratory syncytial virus), didanosine, dideoxycytidine and zidovudine (Azidothymidine).
Another kind of antiviral agent comprises cytokine, such as interferon.Interferon is the cell and the excretory cytokine of immunocyte of viral infection.Interferon is by working in conjunction with the specificity health on the cell adjacent with infection cell, cause preventing that it is by the change in the cell of viral infection, α and beta-interferon be I class and the II class MHC developed by molecule on the inductive infection cell also, the antigen presentation that causes increasing is so that the host immune cell recognition can utilize α and beta-interferon as recombinant forms will be used for the treatment of chronic hepatitis B and hepatitis C.Be effective under the dosage of antiviral therapy, interferon has serious side effects, such as heating, and sense of discomfort and losing weight.
Immunoglobulin therapy is used for prophylaxis of viral infections.The immunoglobulin therapy that is used for viral infection is different from bacterial infection, because it is not an antigenic specificity, immunoglobulin therapy is by in conjunction with the extracellular virus body and prevent that it from adhering to and the cell that enters the viral infection sensitivity works.This therapy is used for the time bar that prophylaxis of viral infections antibody is present in the host, generally speaking, has two types immunoglobulin therapy, promptly general immunoglobulin therapy and hyperimmune globulin therapy.General immune albumin therapy is used the antibody product by preparation of normal blood donor's serum and collection.The product of this collection comprises extensive Human virus, such as hepatitis A, and parvovirus, enterovirus (especially in ewborn infant) hangs down titer antibody.The hyperimmune globulin therapy is used the antibody by the individual serum preparation of the titre that specific virus is had high antibody.Those antibody are useful to specific virus subsequently.The example of hyperimmune globulin comprises Z/G (chickenpox that is used for impaired child of epidemic prevention and ewborn infant), Human Rabies Immunoglobulin's (being used for) by the experimenter's of mad animal bite post-exposure prophylaxis, hepatitis B immune globulin (being used to prevent hepatitis B, the experimenter of especially contact virus) and RSV immunoglobulin (being used for the treatment of respiratory syncytial virus infection).
Antifungal is used for the treatment of and the prevention infection fungus.Antifungal is sometimes according to its mechanism of action classification.Some antifungal works by suppressing glucosylceramide synthase as the cell wall inhibitor.They include, but are not limited to basiungin/ECB.Other antifungal works by making the film integrality unstability.They include, but are not limited to imidazoles, such as clotrimazole, and Demlofix, fluconazol, itraconazole, ketoconazole, miconazole and excellent upright health azoles and FK 463, amphotericin B, BAY 38-9502, MK 991, pradimicin, UK 292, Butenafine and terbinafine.Other antifungal works by decomposing chitin (for example, chitinase) or immunosuppressant (501 frost).
Parasiticide is the parasitic activating agent of direct killing.This compounds is as known in the art and generally commercially available.The example that is used for the parasiticide of human body administration includes, but are not limited to albendazole, amphotericin B, benznidazole, bithionol, chloroquine HCl, Arechin (Polfa), clindamycin, dehydroemetine, diethylcarbamazine, diloxanide furoate, eflornithine, furazolidone, glucocorticoids, halofantrine, diiodohydroxyquinoline (Iodoquinol), ivermectin, mebendazole, mefloquine, Glucantime, melarsoprol, metrifonate, metronidazole, niclosamide, nifurtimox, oxamniquine, paromomycin, hydroxyethylsulfonic acid. pentamidine, piperazine, praziquantel, primaquine phosphate, proguanil, Pyrantel Pamoate, pyrimethamine-sulfonamides, Pyrimethamine-Sulfadoxine, quinacrine HCl, quinine sulfate, the gluconic acid quinidine, spiramycin, sodium stibogluconate (sodium antimonyl gluconate), suramin, tetracycline, doxycycline, thiabendazole, tinidazole, trimethoprim-sulfamethoxazole and tryparsamide.
Anti-cancer therapies
TLR7/8 part of the present invention be connected oligonucleotide combination can with the anti-cancer therapies coupling.
Anti-cancer therapies comprises the cancer medication, radiation and surgical operation." cancer medication " used herein means experimenter's administration so that the activating agent of treatment cancer.This paper has described the various types of medicines that are used for the treatment of cancer.With regard to the purpose of this description, the cancer medication is categorized as chemotherapeutics, immunotherapeutic agent, cancer vaccine, hormonotherapy and biological response modifier.
Chemotherapeutics can be selected from methotrexate (methotrexate), vincristine, amycin, cisplatin, not sacchariferous chloroethylnitrosoureas, 5-fluorouracil, ametycin, bleomycin, doxorubicin, dacarbazine, taxol, fragyline, Meglamine GLA, valrubicin, carmustine (carmustaine) and poliferposan, MMI270, BAY12-9566, RAS farnesyl transferase inhibitor, farnesyl transferase inhibitor, MMP, MTA/LY231514, Alimta, LY264618/lometexol, Glamolec, CI-994, TNP-470 and newly beautiful/hycamtin, PKC412, valspodar/PSC833, Nuo Xiaolin/mitoxantrone (Mitroxantrone), Metaret/ suramin, batimastat, E7070, BCH-4556, CS-682,9-AC, AG3340, AG3433, Incel/VX-710, VX-853, ZD0101, ISI641, ODN698, TA 2516/Marmistat, BB2516/Marmistat, CDP 845, D2163, PD183805, DX8951f, Lemonal DP 2202, FK 317, Picibanil/OK-432, AD 32/ valrubicin, strontium chloride/strontium derivant, Temodal/ temozolomide, Evacet/Mycocet, the Yewtaxan/ paclitaxel, taxol/paclitaxel, Xeload/ capecitabine, fortulon/doxifluridine, the oral paclitaxel of Cyclopax/, oral taxane, SPU-077/ cisplatin, HMR 1275/Flavopiridol, CP-358 (774)/EGFR, CP-609 (754)/RAS oncogene inhibitor, the oral platinum of BMS-182751/, UFT (ftorafur/uracil), levamisole (Ergamisol)/levamisole (Levamisole), eniluracil/776C85/5FU reinforcing agent is opened general opening up/levamisole, Camptosar/irinotecan, Tumodex/Ralitrexed, cladibrine/cladribine, Paxex/ paclitaxel, Doxil/Mycocet, pattern Lay/Mycocet, Fuda China/fludarabine, Pharmarubicin/ epirubicin, DepoCyt, ZD1839, LU 79553/Bis-Naphtalimide, LU103793/Dolastain, the Caetyx/ Mycocet, strong selecting/gemcitabine, ZD0473/Anormed, YM 116, propiodal, CDK4 and CDK2 inhibitor, PARP inhibitor, D4809/Dexifosamide, Ifes/ Mesnex/Ifosamide, brave and fierce/teniposide, Paraplatin/carboplatin, cisplatin (Plantinol)/cisplatin (cisplatin), etoposide (Vepeside)/etoposide (Etoposide), ZD 9331, docetaxel/docetaxel, guanine cytosine arabinoside prodrug, 10-deacetyltaxol, nitroso ureas, alkylating agent is such as melphalan (melphelan) and cyclophosphamide, aminoglutethimide, asparaginase, busulfan, carboplatin, chlorambucil, cytosine arabinoside HCl, actinomycin D, daunorubicin HCl, phosphoric acid estramustine sodium, etoposide (VP16-213), floxuridine, fluorouracil (5-FU), flutamide, hydroxyurea (Hydroxyurea) (hydroxyurea (hydroxycarbamide)), ifosfamide, Intederon Alpha-2a, α-2b, leuprorelin acetate (LHRH-releasing factor analogs), lomustine (CCNU), chlormethine HCl (chlormethine), purinethol, mesna, mitotane (o.p '-DDD), mitoxantrone HCl, octreotide, plicamycin, procarbazine HCl, streptozocin, Tamoxifen Citrate, thioguanine, plug is for group, vinblastine sulfate, amsacrine (m-AMSA), azacitidine, erythropoietin (Erthropoietin), altretamine (HMM), interleukin II, mitoguazone (methyl-GAG; Methyl-glyoxal is two-methyl GAG; MGBG), pentostatin (2 ' pentostatin), semustine (Semustine), teniposide (VM-26) and vindesine sulfate, but be not limited thereto.
Immunotherapeutic agent can be selected from 3622W94,4B5, and ANA Ab, anti-FLK-2, anti-VEGF, ATRAGEN, (shellfish is cut down the pearl monoclonal antibody to AVASTIN; Genentech), BABS, BEC2, BEXXAR (tositumomab; GlaxoSmithKline), C225, CAMPATH (A Lun pearl monoclonal antibody; GenzymeCorp.), CEACIDE, CMA 676, EMD-72000, ERBITUX (Cetuximab; The ImClone system, Inc.), Gliomab-H, GNI-250, HERCEPTIN (Herceptin; Genentech), IDEC-Y2B8, ImmuRAIT-CEA, ior c5, ior egf.r3, iort6, 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 (Rituximab; Genentech), SMART 1D10 Ab, SMART ABL 364Ab, SMART M195, TNT and ZENAPAX (daclizumab; But be not limited thereto Roche).
Cancer vaccine can be selected from EGF antiidiotype cancer vaccine, Gp75 antigen, GMK Melacine, MGV ganglioside combined vaccine, Her2/neu, Ovarex, M-Vax, O-Vax, L-Vax, STn-KHL Theratope, BLP25 (MUC-1), liposome idiotypic vaccine, Melacine, the peptide antigen vaccine, toxin/antigen vaccine is based on the vaccine of MVA, PACIS, BCG vaccine, TA-HPV, TA-CIN, DISC-virus and ImmuCyst/TheraCys, but be not limited thereto.
The antianaphylaxis medicine
TLR7/8 part of the present invention be connected oligonucleotide can with the coupling of antianaphylaxis medicine.
Be used for the treatment of or the common method of Polyglucan reaction comprises and uses anaphylaxis medication or desensitization therapy.Be used for the treatment of or some evolution therapy of Polyglucan reaction comprises in the use and anti-IgE antibodies.The seriousness that medicine of antihistaminic and other blocking-up anaphylaxis chemistry amboceptor effect helps to regulate allergic symptom, but can't the Polyglucan reaction and subsequently anaphylactic response do not had effect.Desensitization therapy is by specifying low dose of anaphylactogen, usually by at subcutaneous injection, carries out so that induce the IgG-class of this anaphylactogen replied.Think exist IgG antibody help in and predisposition lead the amboceptor that IgE antibody produces and produce.The initial anaphylactogen treatment experimenter who uses very low dose induces severe reaction and slowly increases this dosage avoiding.Such therapy is dangerous, because to the actual chemical compound that has given to cause producing anaphylactic response and severe allergic reaction of experimenter.
The anaphylaxis medication includes, but are not limited to antihistaminic, corticosteroid and prostaglandin inducer.Antihistaminic is the chemical compound of the histamine of counteracting mastocyte or basophilic leukocyte release.These chemical compounds are well-known in the art and are usually used in treating anaphylaxis.Antihistaminic includes, but are not limited to acrivastine, astemizole, azatadine, azelastine, betatastine, brompheniramine, buclizine, cetirizine, the cetirizine analog, chlorphenamine, clemastine, CS 560, Cyproheptadine, Desloratadine, dexchlorpheniramine, ebastine, epinastine, fexofenadine, HSR 609, hydroxyzine, levocabastine, loratadine, Methscopolamine, mizolastine, norastemizole, phenindamine, promethazine, pyrilamine, terfenadine and tranilast.
Corticosteroid includes, but are not limited to methylprednisolone, prednisolone, prednisone, beclometasone, budesonide, dexamethasone, flunisolide, fluticasone propionate and triamcinolone.Although dexamethasone is the corticosteroid with antiinflammatory action, it also irregularly is used for the treatment of anaphylaxis or asthma with the suction form, because it absorbs remarkable and have the long term inhibition side effect under effective dose.Yet, can use dexamethasone treatment anaphylaxis or asthma according to the present invention because when the time with itself and compositions coupling of the present invention, can with its with the low dosage administration so that alleviate side effect.Use some relevant side effect to comprise cough, dysphonia, thrush (candidiasis) with corticosteroid, and under higher dosage, be general action, suppress such as the adrenal gland, glucose intolerance, osteoporosis, aseptic necrosis of bone, cataract forms, growth inhibited, hypertension, muscle weakness, skin graph thinning and easily scratch.Barnes﹠amp; Peterson (1993) Am Rev Respir Dis 148:S1-S26; With (1996) Am J Respir Crit Care Med 153:1739-48 such as KamadaAK.
Antasthmatic
TLR7/8 part of the present invention be connected oligonucleotide combination can with the antasthmatic coupling.
The medicine (being antasthmatic) that is used for the treatment of asthma generally is divided into two classes, i.e. rapid release medicament and long-acting control medicament.Asthmatic patient is that long-acting control medicament is taken on the basis with the every day, so that realize and keep control to persistency asthma.Long-acting control medicament comprises antiinflammatory, such as corticosteroid, and chromolyn sodium and nedocromil; Long-acting bronchodilator is such as long-acting beta 2-agonist and methylxanthine class; With the leukotriene regulator.Immediate release drug comprises fugitive β 2Agonist, anticholinergic and whole body corticosteroid.Exist and each the autocorrelative many side effect of these medicines, and do not have a kind of medicine can prevent or treat fully asthma separately or with compound mode in the chemicals.
The asthma medication includes, but are not limited to the PDE-4 inhibitor, bronchodilator/β-2 agonist, the K+ channel opener, the VLA-4 antagonist, neurokin antagonist, TXA2. (TXA2) synthetic inhibitor, xanthine, the arachidonic acid antagonist, 5 lipoxidase inhibitors, TXA2 receptor antagonist, the TXA2 antagonist, 5-lipox activated protein inhibitor and protease inhibitor.
Bronchodilator/β 2Agonist is the chemical compound that a class causes bronchiectasis or smooth muscle loosening.Bronchodilator/β 2Agonist includes, but are not limited to salmaterol, salbutamol, albuterol, terbutaline, D2522/ formoterol, fenoterol, bitolterol, pirbuterol, methylxanthine class and orciprenaline.Long-acting beta 2Agonist and bronchodilator are for also being used for preventing for a long time the chemical compound of symptom except that being used for anti-inflammatory therapy.Long-acting beta 2Agonist includes, but are not limited to salmaterol and albuterol.If these chemical compounds are normal with the corticosteroid coupling and do not use any anti-inflammatory therapy, generally just do not use them.They are relevant with side effect under overdose, such as tachycardia, and the skeletal muscle vibration, hypokalemia and QTc prolong at interval.
The methylxanthine class comprises, for example theophylline has been used for long-term control and prevention symptom.These chemical compounds suppress because of phosphodiesterase and possible adenosine antagonism causes bronchiectasis.The relevant acute toxicity of dosage is for using the particular problem of these type compounds.As a result of, must the conventional serum-concentration of monitoring so that explanation toxicity and the narrow therapeutic domain that produces because of the individual variation on the metabolite clearance.Side effect comprises tachycardia, tachyarrhythmia, and nausea and vomiting, the central nervous system stimulates, headache, epilepsy, hematemesis, hyperglycemia and hypokalemia.Fugitive β 2Agonist includes, but are not limited to albuterol, bitolterol, pirbuterol and terbutaline.With give fugitive β 2Some ill effect that agonist is relevant comprises tachycardia, the skeletal muscle vibration, and hypokalemia, lactic acid increases, pain and hyperglycemia.
The long-acting control medicine of symptoms of asthma that Chromolyn sodium and nedocromil cause because of exercise as main prevention or the allergic symptom that produces because of anaphylactogen.Think that these chemical compounds can block because of infecting the early stage and late phase response of chloride channel function to anaphylactogen.They are also stablized mast cell membrane and suppress activation and amboceptor activates and discharges from inosineophil and epithelial cells.Generally need the administration 4-6 time limit in week so that obtain maximum helpfulness.
Anticholinergic generally is used to alleviate the acute bronchus spasm.Think that these chemical compounds work by the competitive inhibition muscarinic cholinergic receptor.Anticholinergic includes, but are not limited to ipratropium bromide.These chemical compounds only reverse the bronchospasm of cholinergic mediation, but do not change any to antigenic anti-.Side effect comprises xerostomia and respiratory secretions, and if the increase of stridulating in some individuality is spray eye then cause blurred vision.
Compositions of the present invention can also be used for the treatment of Airway Remodeling.Airway Remodeling thickens the airway constriction that causes and finally cause causing flow limitation because of tela submucosa in smooth muscle cell proliferation and/or the air flue.Compositions of the present invention can further prevent to reinvent and even the further tissue stack that reduces because of the remodeling process generation.
Adjuvant
TLR7/8 part of the present invention be connected oligonucleotide can with other reagent coupling, such as adjuvant.Adjuvant used herein means non-antigen, and the TLR7/8 part is replied antigen with the enhancing that is connected oligonucleotide, for example, and the material of the activated immune cell of body fluid and/or cellullar immunologic response.Adjuvant promotes accessory cell accumulation and/or activation so that the enhancement antigen specific immune response.Adjuvant is used to strengthen efficacy of vaccines, promptly contains the antigenic compositions that is used to reply to antigenic protective immunity.
Adjuvant generally comprises the adjuvant that produces long-acting, the adjuvant of immunostimulation adjuvant and generation long-acting and stimulating immune system.The adjuvant of generation long-acting used herein is to cause the adjuvant that can slowly discharge in vivo, prolongs immunocyte thus and contacts with antigenic.Such adjuvant includes, but are not limited to aluminum (for example, aluminium hydroxide, aluminum phosphate); The preparation of emulsion-based comprises mineral oil, non-mineral oil, and water-in-oil type or oil-in-water emulsion, the oil-in-water type solvent, (for example, Montanide ISA 720 such as Seppic ISA series Montanide adjuvant; AirLiquide, Paris, France); MF-59 is (with the stable water cornerite zamene Emulsion of Span 85 and Tween 80; Chiron Corporation, Emeryville, Calif.); And PROVAX (comprises the oil-in-water type solvent of stablizing detergent and micelle plasticizer; IDECPharmaceuticals Corporation, San Diego, Calif.).
Immunity-stimulant is for causing the activatory adjuvant of immune system cell.For example, it can cause immunocyte to produce and secrete cytokines.Such adjuvant includes, but are not limited to the saponins of purification from the bark of Q.saponaria tree, (uses the glycolipid of HPLC fractionated eluting on the 21st peak such as QS21; Aquila Biopharmaceuticals, Inc., Worcester, Mass.); Poly-[two (carboxylic acid phenoxy group) phosphonitrile (PCPP polymer; VirusResearch Institute, the U.S.); The derivant of liopopolysaccharides is such as a phosphoryl lipid A (MPL; Ribi ImmunoChem Research, Inc., Hamilton, Mont.), muramyldipeptide (MDP; Ribi) and threonyl-muramyldipeptide (t-MDP; Ribi); OM-174 (the glycosamine disaccharide relevant with lipid A; OM Pharma SA, Meyrin, Switzerland); With the leishmaniasis elongation factor (the leishmaniasis albumen of purification; CorixaCorporation, Seattle, Wash.).Such adjuvant also comprises CpG DNA.
The adjuvant that produces long-acting and stimulating immune system is those based on both chemical compound of above-mentioned definite function.Such adjuvant includes, but are not limited to ISCOMS, and (comprise blended saponins, lipid and formation have the granule immunostimulating complex of the hole virus size that can keep passable; CSL, Melbourne, Australia); SB-AS2 (SmithKlineBeecham adjuvant system #2, it is the oil-in-water emulsion that comprises MPL and QS21: SmithKline Beecham Biologicals[SBB], Rixensart, Belgium); SB-AS4 (the SmithKline Beecham adjuvant system #4 that comprises aluminum and MPL; SBB, Belgium); Form micellar nonionic block copolymer, (they comprise the hydrophobicity polyoxypropylene straight chain that is positioned at the polyoxyethylene chain flank such as CRL 1005; Vaxcel, Inc., Norcross, Ga.); (SAF comprises the oil-in-water emulsion of Tween 80 and nonionic block copolymer with Syntex adjuvant goods; Syntex Chemicals, Inc., Boulder, Colo.).
Adjuvant also with can be lipopeptid, such as Pam3Cys; Cationic polysaccharide is such as chitosan; Or cationic peptide, as protamine.
Cytokine
TLR7/8 part of the present invention be connected oligonucleotide combination can also with the cytokine coupling.Cytokine is soluble protein and the glycoprotein that is produced by transmitting inflammation and immunoreactive many cell types.Communication between the cytokine mediated immune system cell is worked so that raise cell and regulate its function and propagation at local and whole body.The cytokine classification comprises the amboceptor and the regulator of natural immunity, the amboceptor of adaptive immunity and regulator and hemopoiesis stimulant.Comprise interleukin (for example, IL-1, IL-2, IL-3 in the cytokine, 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 1 9-32 (IL-19-IL-32) etc.), chemotactic factor (for example, IP-10, RANTES, MIP-1 α, MIP-1 β, MIP-3 α, MCP-1, MCP-2, MCP-3, MCP-4, eotaxin, I-TAC and BCA-1 etc.) and other cytokine, comprise 1 type interferon (for example, IFN-α and IFN-β), 2 type interferon (for example, IFN-γ), tumor necrosis factor-alpha (TNF-α), transforming growth factor-beta (TGF-β) and various colony stimulating factor (CSFs) comprise GM-CSF, G-CSF and M-CSF.
Antigen
TLR7/8 part of the present invention be connected oligonucleotide combination and can choose wantonly in vaccine product and the antigen coupling." antigen " used herein means can be by any molecule of T-cell antigen receptor or the identification of B-cell antigen receptor.This term extensively comprises the molecule that is used as any kind that foreign body discerned by host immune system.Antigen generally comprises, but is not limited to cell, cell extract, protein, polypeptide class, peptide class, polysaccharide, polysaccharide conjugates, the peptide and the non-peptide mimics of polysaccharide and other molecule, micromolecule, lipid, lipoprotein, the glycolipid class, polysaccharide, carbohydrate, viral and viral extract and multicellular organism are such as parasite and anaphylactogen.Be protein just, the antigen of polypeptide class or peptide class, this class antigen can comprise the antigenic nucleic acid molecules of this class of coding.More particularly, antigen includes, but are not limited to cancer antigen, comprises cancerous cell and in cancerous cell or the molecule of expressing on it; Microbial antigen comprises microorganism and in microorganism or the molecule of expressing on it; And anaphylactogen.Therefore, the present invention provides in certain embodiments and has been used for cancer, the vaccine of pathogen and anaphylactogen.
In various embodiments, antigen is microbial antigen, cancer antigen or anaphylactogen." microbial antigen " used herein is the antigen of microorganism, and includes, but are not limited to virus, antibacterial, parasite and fungus.This class antigen comprises complete microorganism and natural separator thereof and fragment or derivant and identical with natural microbial antigen or similar and induce the synthetic compound that this microorganism is had antigen-specific immune responses.If induce (body fluid and/or cell) to the antigenic immunne response of natural microbial, chemical compound and natural microbial Antigens are seemingly.This class antigen is used always in the art and is that those skilled in the art are well-known.The present invention specifies and to comprise any the antigen that derives from the pathogen as herein described.
Term used herein " cancer antigen " and " tumor antigen " can exchange use, it refers to chemical compound, such as peptide, protein, lipoprotein or glycoprotein, it is combining with tumor or cancerous cell during expression on the delivery cell and can cause immunne response at antigen-be under main histocompatibility complex (MHC) the branchs subenvironment.Cancer antigen is differentially expressed and can utilize target cancer cell thus by cancerous cell.Cancer antigen is for stimulating the antigen of tangible tumour-specific immune response.In these antigens some encoded by normal cell, but not necessarily by its expression.These are antigenic to be characterised in that (promptly not expressed) that is generally tranquillization in normal cell, is only expressed and temporary transient the expression in some stage of differentiation, such as embryonal antigen and fetal antigen.Other cancer antigen is by the mutant cell gene code, and such as oncogene (for example, the active ras oncogene), suppressor gene (for example, mutant p53) is because of the fusion rotein of inherence disappearance or chromosome translocation generation.Other cancer antigen can be by viral gene, such as those gene codes that carry on RNA and the DNA oncovirus.
For example, can by partial purification antigen, prepare cancer antigen by cancerous cell by the crude extract of preparation cancerous cell described in (1994) Cancer Res 54:1055-8 such as Cohen PA by recombinant technique or by the de novo synthesis known antigens.Cancer antigen includes, but are not limited to recombinant expressed antigen, the immunogenicity part of complete tumor or cancer or its cell.This class antigen can or well known to a person skilled in the art any alternate manner classification or preparation by recombination form.
The example of tumor antigen comprises MAGE, MART-1/Melan-A, gp100, DPP IV (DPPIV), ABP (ADAbp), cyclophilin b, colorectum related antigen (CRC)-C017-1A/GA733, carcinoembryonic antigen (CEA) and immunogenicity epi-position CAP-1 and CAP-2, etv6, amll, prostate specific antigen (PSA) and immunogenicity epi-position PSA-1 thereof, PSA-2 and PSA-3, prostate specific membrane antigen (PSMA), T-cell receptors/CD3-ζ chain, MAGE family tumor antigen is (for example, MAGE-A1, 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), GAGE family tumor antigen is (for example, GAGE-1, 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, tryrosinase, p53, MUC family, HER2/neu, p21ras, RCAS1, α-Jia Taidanbai, E-cadherin, α-catenin, white and the γ-catenin of beta-catenin, p120ctn, gp100pmel117, PRAME, NY-ESO-1, cdc27, adenomatous polyposis coli albumen (APC), fodrin, connect protein 37, the Ig-idiotype, p15, gp75, GM2 and GD2 gangliosides, viral product, such as the human papilloma virus toxalbumin, Smad family tumor antigen, 1mp-1, P1A, the antigen (EBNA)-1 of EBV-coding, brain glycogen phosphorylase, SSX-1, SSX-2 (HOM-MEL-40), SSX-1, SSX-4, SSX-5, SCP-1 and CT-7 and c-erbB-2.This catalogue is not to have meaned the qualification effect.
" anaphylactogen " used herein is for causing the molecule of the immunne response that is characterised in that IgE produces.Anaphylactogen also be can be in the susceptible experimenter material of induced hypersensitivity or asthma reaction.Therefore, in the context of the present invention, the term anaphylactogen means the antigen that can cause by the antibody-mediated anaphylactoid particular type of IgE.
The catalogue of anaphylactogen is a large amount of and can comprises pollen, insecticide venom class, animal wool scurf bits dust, fungal spore and medicine (for example, penicillin).The example of natural animal and phytosensitinogen comprises has specific protein to following accessory: Canis (Canis) (domesticated dog (Canis familiaris)); Dermatophagoides (Dermatophagoides) (for example, dust demodicid mite (Dermatophagoides farinae)); Felis (Felis) (Felisdomesticus); Ambrosia (Ambrosia) (artemisiifolia (Ambrosia artemisiifolia)); Lolium (Lolium) (for example, rye grass (Lolium perenne) and Itanlian rye (Lolium multiflorum)); Cryptomeria (Cryptomeria) (Japanese cedar (Cryptomeria japonica)); Alternaria (Alternaria) (lattice chain lattice spore (Alternaria alternata) mutually); Alder; Alder (Alnus) (European alder (Alnusgultinosa)); Betula (Betula) (wart skin birch (Betula verrucosa)); Oak belongs to (Quercus) (white oak (Quercus alba)); Olive belongs to (Olea) (Fructus oleae europaeae (Olea europa)); Artemisia (Artemisia) (Radix Artemisia ordosicae (Artemisia vulgaris)); Plantago (Plantago) (for example, buckhorn plantain (Plantago lanceolata)); The rose grass belongs to (Parietaria) (for example, Parietaria officinalis and Parietariajudaica); Cockroach (Blattella) (for example, Groton bug (Blattellagermanica)); Apis (Apis) (for example, Apis multiflorum); CypressCypressus (Cupressus) (for example, cupressus sempervirens (Cupressus sempervirens), green dried cypress (Cupressus arizonica) and cupressus macrocarpa (Cupressus macrocarpa)); Juniperus Linn. (Juniperus) (for example, Juniperus sabinoides, Juniperusvirginiana, Juniperus communis L. (Juniperus communis) and A Xi Juniperus oxycedrus (Juniperusashei)); Thuya (for example, Thuya orientalis); Chamaecyparis Space (Chamaecyparis) (for example, Japanese cypress (the Chamaecyparis obt U.S.)); Periplaneta (Periplaneta) (for example, periplaneta americana (Periplaneta americana)); Agropyron (Agropyron) (for example, couchgrass (Agropyron repons)); Secale (Secale) (for example, rye (Secale cereale L.) (Secale cereale)); Triticum (Triticum) (for example, Semen Tritici aestivi (Triticumaestivum)); Orchardgrass (Dactylis) (for example, orchardgrass (Dactylis glomerata)); Festuca (Festuca) (for example, Isodon ternifolius (Festuca elatior)); Annual bluegrass belongs to (Poa) (for example, English grass (Poa pratensis) and Canada blue grass (Poacompressa)); Avena (Avena) (for example, Herba bromi japonici (Avena sativa)); Holcus (Holcus) (for example, yorkshire fog grass (Holcus lanatus)); Anthoxanthum (Anthoxanthum) (for example, Hemerocallis citrina Baroni thatch (Anthoxanthum odoratum)); Oatgrass (Arrhenatherum) (for example, Herba avenae fatuae (Arrhenatherum elatius)); Cut a strand grass and belong to (Agrostis) (for example, white bent (Agrostis alba)); Ladder forage spp (Phleum) (for example, timothy grass (Phleum pratense));
Figure A20068004438600631
Grass (for example, belongs to (Phalaris)
Figure A20068004438600632
Grass (Phalaris arundinacea)); Paspalum (Paspalum) (for example, Paspalum notatum); Sorghum (Sorghum) (for example, Sorghum halopensis); And Brome (Bromus) (for example, awnless brome (Bromus inermis)).
Antibody and ADCC
TLR7/8 part of the present invention be connected oligonucleotide combination and also increase natural killer cell lytic activity and antibody dependent cellular cytotoxicity (ADCC).Can use the pair cell target, ADCC is carried out in the combination that has specific antibody such as cancerous cell, makes experimenter's immune system killing tumor cell.The antibody that is used for ADCC operation comprises with cells in vivo interactional antibody takes place.Described many these class pair cell targets in this area and had specific antibody and many for being purchased.In one embodiment, antibody is IgG antibody.
Being used for treatment antibody of the present invention can be to antigen (for example, thin mattress, virus, parasite or fungal antigen), and cancer or tumor associated antigen and autoantigen have specificity.Preferred antibody for those identifications and in conjunction be present on the cell or wherein antigenic those.The example of suitable antibody includes, but are not limited to Rituxan TM(Rituximab, anti-CD 20 antibodies), Trastuzumab (Herceptin), Quadramet, Panorex, IDEC-Y2B8, BEC2, C225, Oncolym, SMART M195, ATRAGEN, Ovarex, Bexxar, LDP-03, ior t6, MDX-210, MDX-11, MDX-22, OV103,3622W94, anti-VEGF, Zenapax, MDX-220, MDX-447, MELMMUNE-2, MELIMMUNE-1, CEACIDE, Pretarget, NovoMAb-G2, TNT, Gliomab-H, GNI-250, EMD-72000, LymphoCide, CMA 676, Monopharm-C, 4B5, ior egf.r3, ior c5, BABS, anti-FLK-2, MDX-260, ANA Ab, SMART1D10 Ab, SMART ABL 364 Ab, CC49 (mAb B72.3), ImmuRAIT-CEA, anti-IL-4 antibody, anti-IL-5 antibody, anti-IL-9 antibody, anti-Ig antibody, anti-IgE antibodies, serum-deutero-hepatitis B antibody, reorganization hepatitis B antibody etc.
Can be used for other antibody of the present invention similarly and comprise A Lun pearl monoclonal antibody (B cell chronic lymphocytic leukemia), lucky trastuzumab ozogamicin (CD33+ acute myeloid leukaemia), hP67.6 (CD33+ acute myeloid leukaemia), the Ying Fuli Xidan resists (inflammatory bowel and rheumatoid arthritis), Embrel (rheumatoid arthritis), Embrel, MDX-210, oregovomab, anti-EGF receptor mAb, MDX-447, anti-tissue factor albumen (TF), (Sunol); Ior-c5, c5, edrecolomab, ibritumomab tiuxetan, the antiidiotype mAb analogies of Ganglioside, GD3 epi-position, anti-HLA-Dr10mAb, anti-CD 33 humanization mAb, anti-CD52 humAb, anti-CD1mAb (ior t6), MDX-22, Celogovab, anti-17-1AmAb, shellfish is cut down the pearl monoclonal antibody, daclizumab, anti-TAG-72 (MDX-220), high-molecular-weight protein polysaccharide antiidiotype mAb analogies (I-Mel-1), high-molecular-weight protein polysaccharide antiidiotype mAb analogies (I-Mel-2), anti-CEA Ab, hmAbH11, anti-DNA or DNA associated protein (histone) mAb, Gliomab-H mAb, GNI-250mAb, anti-CD22, CMA 676), the antiidiotype people mAb of GD2 ganglioside, ior egf/r3, anti-ior c2 glycoprotein mAb, ior c5, anti-FLK-2/FLT-3mAb, anti-GD-2 bispecific mAb, antinuclear autoantibody, anti-HLA-DR Ab, anti-CEA mAb, palivizumab, shellfish is cut down the pearl monoclonal antibody, A Lun pearl monoclonal antibody, BLyS-mAb, anti-VEGF2, anti-Trail receptor; B3 mAb, mAb BR96, breast carcinoma; With Abx-Cb1 mAb.
Immunoglobulin therapy
Activating agent of the present invention can also with standard and the coupling of hyperimmune globulin therapy.Standard immunoassay globulin therapy is used the antibody product by preparation of normal blood donor's serum and collection.The product of this collection comprises extensive antigen, such as the low titer antibody of those (for example, antibacterial, virus, such as hepatitis A, parvovirus, enterovirus, fungus and parasites) in the infectious pathogen.The hyperimmune globulin therapy is used the antibody by the individual serum preparation of the titre that specific virus is had high antibody.The example of hyperimmune globulin comprises Z/G (chickenpox that is used for impaired child of epidemic prevention and ewborn infant), Human Rabies Immunoglobulin's (being used for) by the experimenter's of mad animal bite post-exposure prophylaxis, hepatitis B immune globulin (being used to prevent hepatitis B, the experimenter of especially contact virus) and RSV immunoglobulin (being used for the treatment of respiratory syncytial virus infection).
Dosage and administration
Part and oligonucleotide can be formulated in the independent compositions that common use realizes required effect.For example, can will connect oligonucleotide and the TLR7/8 part is mixed with each other and to experimenter's administration or keep as combination while exposing cell basically.As another example, can with connect oligonucleotide and TLR7/8 part at different time to experimenter's administration or maintenance and cells contacting.As another example, can connect oligonucleotide and TLR7/8 part to experimenter's different parts.
As mentioned above, term " effective dose " generally refers to the consumption that must or be enough to realize required biological agent.By at various reactive compounds and weighted factor, such as effect, relative bioavailability, weight in patients, select in the seriousness of adverse side effect and the preferred administering mode, can effectively prevent or therapeutic scheme in conjunction with instruction plan provided herein, it can not produce tangible toxicity and treat particular subject completely effectively.The effective dose that is used for any application-specific is according to such as disease of being treated or illness, the specific oligonucleotides that gives, experimenter's size or the disease for the treatment of or illness this class factor of seriousness difference and change.Those skilled in the art can rule of thumb needn't overuse and test the effective dose of determining specific connection oligonucleotide and TLR7/8 part and/or antigen and/or other therapeutic agent.
Generally at each about 10ng-10mg of administration, this depends on it is every day to the experimenter's dosage that is used for whole body or local delivery chemical compound described herein, and weekly, or every month and any between them use At All Other Times, otherwise just carry out as required.Be more typically, whole body or local dose scope be at each about 1 μ g-1mg of administration, and the most general be at about 10 μ g-100 μ g, wherein several days or several all administrations 2-4 time at interval.High dose may be that parenterai administration is required.Yet, in certain embodiments, can use to be higher than above-mentioned typical doses 5-10 the non-intestinal dosage that is used for these purposes of 000 times of scope.
With regard to any chemical compound as herein described, can measure the treatment effective dose according to animal model at first.Can and give the dosage that the effect adjustment of chemical compound is used based on relative bioavailability.Dose titration is fully belonged to those skilled in the art's limit of power as method well-known in the art to maximum effect based on said method and other.
Route of administration
With regard to clinical practice, can with TLR7/8 part of the present invention be connected oligonucleotide combination and give separately or it is mixed with the delivery complexes of realizing the route of administration of required therapeutic effect by any suitable effectiveness.Route of administration comprises intestinal and parenterai administration approach.The example of intestinal canal administration approach comprises the oral cavity, stomach, intestinal and rectum.The limitative examples of parenterai administration approach comprises intravenous, and intramuscular is subcutaneous, intraperitoneal, and in the sheath, local injection, part, nose, mucosa and lung.
Delivery vector
(for example, in saline or buffer) or use any delivery vector as known in the art to give described oligonucleotide and TLR7/8 part and/or antigen and/or other therapeutic agent of connecing separately.
For example, can with TLR7/8 part of the present invention be connected oligonucleotide combination and directly give the experimenter or give with the delivery of nucleic acids complex.The delivery of nucleic acids complex should refer to use the targeting mode in conjunction with (for example, ion or covalent bond; Or be encapsulated in wherein) nucleic acid molecules (for example, cause the affine combination higher to target cell.The example of delivery of nucleic acids carrier comprises and sterin (for example, cholesterol), the bonded nucleic acid of lipid (for example, cation lipid, virion or liposome) or target cell specific-binding agent (for example, the part of target cell specificity health identification).It is fully stable to prevent the remarkable uncoupling before target cell is taken in that preferred complex can keep in vivo.Yet this complex can cracking under intracellular felicity condition, so that discharge oligonucleotide with functional form.
The present invention has described and operable other delivery vector comprises helix (Gould-Fogerite etc., 1994,1996); Emulsomes (Vancott etc., 1998, Lowell etc., 1997); ISCOMs (Mowat etc., 1993, Carlsson etc., 1991, Hu etc., 1998, Morein etc., 1999); Liposome (Childers etc., 1999, Michalek etc., 1989,1992, de Haan 1995a, 1995b); Bacteria carrier (for example, Salmonella, bacillus coli, bacillus calmette-guerin vaccine, escherich's bacillus (Shigella) alive, Lactobacillus (Lactobacillus)) (Hone etc., 1996, Pouwels etc., 1998, Chatfield etc., 1993, Stover etc., 1991, Nugent etc., 1998); Hepatovirus carrier (for example, cowpox, adenovirus, herpes simplex) (Gallichan etc., 1993,1995, Moss etc., 1996, Nugent etc., 1998, Flexner etc., 1988, Morrow etc., 1999); Microsphere (Gupta etc., 1998, Jones etc., 1996, Maloy etc., 1994, Moore etc., 1995, O ' Hagan etc., 1994, Eldridge etc., 1989); Nucleic acid vaccine (Fynan etc., 1993, Kuklin etc., 1997, Sasaki etc., 1998, Okada etc., 1997, Ishii etc., 1997); Polymer (for example, carboxymethyl cellulose, chitosan) (Hamajima etc., 1998, Jabbal-Gill etc., 1998); Polymer ring (Wyatt etc., 1998); Albuminous body (Vancott etc., 1998, Lowell etc., 1988,1996,1997); Sodium fluoride (Hashi etc., 1998); Transgenic plant (Tacket etc., 1998, Mason etc., 1998, Haq etc., 1995); Virion (Gluck etc., 1992, Mengiardi etc., 1995, Cryz etc., 1998); Virus-like particle (Jiang etc., 1999, Leibl etc., 1998).Other delivery vector is as known in the art.
Pharmaceutical composition
Can with TLR7/8 part of the present invention be connected the pharmaceutical composition that the oligonucleotide formulated in combination becomes also to comprise pharmaceutically acceptable carrier.Pharmaceutically acceptable carrier means one or more compatibility solids or liquid filling agent, diluent or encapsulation material, and they are suitable for people or other vertebrates are given.Carrier is natural or synthetic organic or inorganic component, and they and active component are associated with the required effect that is beneficial to.The composition of mixed together pharmaceutical composition in one way makes the interaction that does not have the required efficacy of drugs of obvious damage.
With regard to oral administration, be easy to by reactive compound is merged preparation chemical compound (promptly being connected oligonucleotide and TLR7/8 part, antigen and/or other therapeutic agent) with pharmaceutically acceptable carrier well-known in the art.This class carrier can be mixed with chemical compound of the present invention tablet by experimenter's orally ingestible of being treated, pill, lozenge, capsule, liquid, gel, syrup, unguentum, suspension etc.Can obtain the pharmaceutical preparation as solid excipient, the optional gained mixture and if desired of grinding obtains label or ingot core adding proper auxiliary agent post-treatment granulate mixture.Suitable excipient is in particular filler, such as saccharide, comprises lactose, sucrose, mannitol or sorbitol; Cellulosics, such as, for example, corn starch, wheaten starch, rice starch, potato starch, gelatin, gum tragacanth, methylcellulose, hydroxypropyl emthylcellulose, sodium carboxymethyl cellulose and/or polyvinylpyrrolidone (PVP).If desired, can add disintegrating agent, such as crospolyvinylpyrrolidone, agar or alginic acid or its salt are such as sodium alginate.Choose wantonly and can also in saline or buffer, prepare oral formulations, so that neutralize inner acid condition, or can administration under the situation of not using any carrier.
Give ingot core enclose suitable coatings.For this purpose, can use priming, it can be chosen wantonly and comprise Radix Acaciae senegalis, Pulvis Talci, polyvinylpyrrolidone, carbopol gel, Polyethylene Glycol and/or titanium dioxide, lacquer solution and appropriate organic solvent or solvent mixture.Dyestuff or pigment can be joined in tablet or the lozenge coatings so that identify or characterize the combination of different activities chemical compound dosage.
The pharmaceutical preparation that can orally use comprises by pushing of making of gelatin-formula capsule and by gelatin and plasticizer, the soft seal capsule of making such as glycerol or sorbitol.Push-the formula capsule can comprise active component and filler, such as lactose, and binding agent, such as starch and/or lubricant, such as Pulvis Talci or stearic acid, the mixture of magnesium and optional stabilizing agent.In soft capsule, reactive compound can be dissolved in or be suspended in suitable liquid, such as fatty acid, in liquid paraffin or the liquid macrogol.In addition, can add stabilizing agent.Can also use microsphere as the oral administration preparation.This class microsphere is fully to define in this area.All oral medicinal preparations all should be suitable for this class administration on dosage.
With regard to sucking administration, compositions can adopt the tablet of preparation in a usual manner or the form of lozenge.
Can suck lung road, especially bronchus by use standard suction apparatus, and the alveolar that sucks the deep lung of more specifically saying so gives described chemical compound.Can come the aerosol form of self-pressurization medicated bag or aerosol apparatus, send chemical compound by means of the suitable propellant of use, described propellant, for example, dichlorodifluoromethane, Arcton 11, dichlorotetra-fluoroethane, carbon dioxide or other suitable gas.With regard to pressurized aerosol, can send through the valve of metered amount by outfit and measure dosage unit.Suction apparatus used herein is the administration aerosol, such as any device of the chemical compound of dry powder form.Such equipment is well-known in the art and such as Remington:The Science and Practice of Pharmacy, 19 ThEdition, 1995, Mac Publishing Company, Easton, Pennsylvania describes in detail in the description of finding in the 1676-1692 page or leaf.Suction apparatus has also been described, such as U.S. Pat 6,116,237 in many United States Patent (USP)s.
" powder " used herein means the compositions of being made up of finely divided solid particle.Preferred compound relatively free-flow and can be scattered in suction apparatus neutralization subsequently times experimenter suck, make chemical compound reach lung so that can penetrate alveolar." dry powder " means the powder composition with water capacity, makes granule be easy to be scattered in the suction apparatus and forms aerosol.Water capacity generally is lower than about 10% weight (%w) water, and in certain embodiments for being lower than about 5%w and preferably being lower than about 3%w.Can use the polymer formulation powder maybe can choose wantonly and use other material, such as liposome, albumin and/or the preparation of other carrier.
Those skilled in the art can be concrete treatment application choice aerosol dose and delivery system, such as described in the following document, for example: Gonda, I. " Aerosols for deliveryof therapeutic and diagnostic agent to the respiratory tract; " Critical Reviews in Therapeutic Drug Carrier Systems, 6:273-313 (1990); And Moren, " Aerosol dosage forms andformulations, " Aerosols in Medicine.Principles, Diagnosis andTherapy, Moren etc., Eds., Elsevier, Amsterdam, 1985.
When the needs systemic delivery, can for example carry out parenterai administration preparation chemical compound for by injection by bolus injection or continuous infusion.Injection preparation can be made unit dosage forms, for example, add the ampoule or the multi-dose container form of antiseptic.These compositionss can adopt such as the suspension in oil or aqueous carrier, solution or Emulsion form and can comprise formulation aid, and such as suspending agent, stabilizing agent and/or dispersant.
The pharmaceutical preparation that is used for parenterai administration is included in the aqueous solution of the reactive compound of water-soluble form.In addition, the suspension of reactive compound can be prepared into suitable oily injection suspension.Suitable wherein solvent or carrier comprise fatty oil, such as Oleum sesami or synthetic fatty acid esters, such as ethyl oleate or triglyceride or liposome.Moisture injection suspension can comprise the material that increases suspension viscosity, such as sodium carboxymethyl cellulose, and sorbitol or glucosan.Optional this suspension can also comprise suitable stabilizers or increase the reagent of compound dissolution degree so that prepare highly enriched solution.
Selectively, reactive compound can be for using suitable carriers before use, for example the powder type of aseptic aqueous solution.
Chemical compound can also be mixed with rectum or compositions for vaginal use, such as suppository or enema,retention, for example, it comprises suppository base commonly used, such as cocoa butter or other glyceride type.
Except that above-mentioned preparation, chemical compound can also be mixed with durative action preparation.Can use suitable polymers or hydrophobic material (for example being mixed with) or ion exchange resin or awkward soluble derivatives, for example, prepare this class durative action preparation as indissoluble salt at the Emulsion that can accept in the oil.
Pharmaceutical composition can also comprise suitable solid or gel phase carrier or excipient.The example of this class carrier or excipient includes, but are not limited to calcium carbonate, calcium phosphate, and various saccharides, starch, cellulose derivative, gelatin and polymer are such as polyethylene glycols.
Suitable liquid or solid pharmaceutical dosage form is: for example, sucks with moisture or saline solution, and with its microencapsulation, internal coilingization, coating is included on the liposome on the microcosmic gold grain, atomizing; Aerosol; Be used for implanting the granule of skin or make it dry to put skin under on sharp object.Pharmaceutical composition also comprises granule, powder, tablet, coated tablet, (little) capsule, suppository, syrup, Emulsion, suspension, cream, drip or have the preparation of prolongation release of active compounds, in said preparation, use preparation excipient and additive and/or auxiliary agent usually as mentioned above, such as disintegrating agent, binding agent, coating materials, sweller, lubricant, correctives, sweeting agent or solubilizing agent.Pharmaceutical composition is applicable to various delivery systems.Just pass the simple summary of prescription method,, the document is incorporated herein by reference referring to Langer R (1990) Science 249:1527-33.
Can give with activating agent of the present invention and optional other therapeutic agent and/or antigen itself (only) or with the form of pharmaceutically acceptable salt.When being used for medicine, these salt should be pharmaceutically acceptable, but non-pharmaceutically acceptable salt also can be used to prepare its pharmaceutically acceptable salt in convenience ground.Those that this class salt includes, but are not limited to be equipped with by following processed with acid: hydrochloric acid, hydrobromic acid, sulphuric acid, nitric acid, phosphoric acid, maleic acid, acetic acid, salicylic acid, right-toluenesulfonic acid, tartaric acid, citric acid, methanesulfonic acid, formic acid, malonic acid, succinic acid, naphthalene-2-sulfonic acid and benzenesulfonic acid.In addition, this class salt can be prepared into alkali metal or alkali salt, such as the sodium of hydroxy-acid group, first or calcium salt.
Suitable reducing comprises: acetic acid 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 antiseptic comprises benzalkonium chloride (0.003-0.03%w/v); Chlorobutanol (0.3-0.9%w/v); Parabens (0.01-0.25%w/v) and thimerosal (0.004-0.02%w/v).
Pharmaceutical composition of the present invention comprises activating agent of the present invention and optional antigen and/or other therapeutic agent of choosing the effective dose that comprises wantonly in pharmaceutically acceptable carrier.The pharmaceutically acceptable carrier of term means one or more compatibility solids or liquid filling agent, diluent or encapsulation material, and they are suitable for people or other vertebrates administration.The term carrier is represented natural or synthetic organic or inorganic component, and they and active component merging are used so that help.In one way with the composition of pharmaceutical composition and chemical compound fusion of the present invention and be mixed with each other, make the interaction that does not have the required efficacy of drugs of obvious damage.
Screening technique
The present invention provides the method for identifying the TLR7/8 part and/or connecting oligonucleotide in other powder.The step of this method is according to different change of the activating agent of identifying (being part and oligonucleotide), and no matter part is TLR7, and TLR8 or TLR7/8 part still are used for the cell type of this method etc.
Based on instruction as herein described, the TLR7 part can stimulate the conduction of independent TLR7 signal according to it, stimulates TL8 signal conduction (from background level) in the presence of the oligonucleotide of connection and/or is having in the presence of the oligonucleotide of connection TLR7 stimulation inhibition identify having.The TLR8 part can be according to identifying conducting with TLR8 signal for the time enhancing (from being higher than background level) that is connected the oligonucleotide coupling.The stimulation of part TLR7 and 8 (when using separately) can be identified according to these active any combinations.The connection oligonucleotide can suppress TLR7 by the TLR7 part according to it, induces TLR8 stimulation and/or enhancing to identify from the ability of the TLR8 stimulation of TLR8 part by the TLR7 part.
These methods can be in vivo or external carrying out.The external test record in an embodiment.The suitable result that reads includes, but are not limited to be used for the IL-12 that TLR8 stimulates, TNF-α and/or IFN-γ and the IFN-α that is used for the TLR7 stimulation.These algoscopys are selectively used the report construct with reporter gene, and described reporter gene is with conduction has the transcription regulatory element (for example, NF-κ B response element) of replying to connect to TLR7 and/or TLR8 signal.These constructs are described in an embodiment.These algoscopys can be measured and transcribe increment or decrement adjusting, and translation increment or decrement are regulated, protein expression and/or secretion etc.
As an example, TLR8 part algoscopy can comprise that the cell (or cell mass) that makes expression TLR8 is being with or without engaged test part in the presence of the connection oligonucleotide.Cell is preferably expresses TLR8, but does not express the cell of TLR7 or TLR9.According to cell to test ligand have or immune oligonucleotide in the presence of reply and measure the conduction of TLR8 signal.To not have then with there being oligonucleotide exists following comparing at the test ligand that enhanced TLR8 signal transport properties in the presence of the oligonucleotide is arranged to be accredited as the TLR8 part.This algoscopy comprises that also use do not express the cell analysis TLR7 signal conduction of the expression TLR7 of TLR8 or TLR9.It is in the probability that the TLR7 part that converts the conduction of TLR8 signal in the presence of the oligonucleotide to is arranged that a kind of analysis in back can be used to get rid of part.
From the similar measurement result of using known ligand as shown in Figure 12.Loxoribine and immunosine (TLR7 ligands specific) are having stimulation TLR8 signal conduction in the presence of the ODN 6056.Can estimate that the TLR7 ligands specific of inferring has similar characteristics qualitatively.The conduction of the TLR8 signal of R-848 (TLR7 and TLR8 part) is enhanced having in the presence of the ODN 6056.Can estimate that the TLR8 part of inferring has similar characteristics qualitatively.Neither TLR7, the ribavirin of non-again TLR8 part all can not stimulate the conduction of TLR8 signal being with or without in the presence of the ODN 6056, represent the specificity of described algoscopy to the TLR7/8 part.
Connection oligonucleotide algoscopy comprises to comprise makes the cell (or cell mass) of expressing TLR8 be with or without TLR7, and there are engaged test oligonucleotide down in TLR8 or TLR7/8 part.If part is TLR7 part (when using separately), this algoscopy can be measured TLR7 signal conduction inhibition and/or inductive TLR8 signal conduction when oligonucleotide and part coupling so.If part is TLR8 part (when using separately), enhanced TLR8 signal conduction is compared in the effect when this algoscopy can be determined at oligonucleotide with the part coupling so during with independent use part.If ligand stimulation TLR7 and TLR8 (when using separately), this algoscopy is measured above-mentioned among the result one or more read so.This measurement result and positive control can be measured comparison (for example using the known algoscopy that is connected oligonucleotide) etc.This method can also comprise whether be used to measure oligonucleotide self is the algoscopy of TLR7 and/or TLR8 part.
These methods can be used to identify part, and it is weak irritating when using separately, but has new and/or enhanced signal transport properties with the oligonucleotide coupling time.
Further explain the present invention by the following example, but these embodiment never should be regarded as further qualification.
Embodiment
Material and method
Oligonucleotide and reagent
All oligonucleotide are all available from Biospring (Frankfurt, German) or by ColeyPharmaceutical GmbH (Langenfeld, Germany) provide, identify and purity by ColeyPharmaceutical GmbH control and have by limulus test mensuration can not detected level of endotoxin (＜0.1EU/ml) (BioWhittaker, Verviers, Belgium).Be suspended in oligonucleotide among the aseptic no endotoxic Tris-EDTA (Sigma, Deisenhofen, Germany) and storage and operation under aseptic condition, to prevent microorganism and contaminated with endotoxins.Use does not contain endotoxin Tris-EDTA and carries out all dilutions.Oligonucleotide sequence is listed in table 1 and 2.At first loxoribine (7-pi-allyl-7,8-dihydro-8-oxo-guanosine) (Sigma) is dissolved in 1NNaOH, dilution and use 1N HCl are adjusted to 7.4 (19) with pH in the RPMI-culture medium.R-848 (1-(2-hydroxyl 2-methyl-propyl)-2-methyl isophthalic acid H-imidazo [4,5-c] quinoline-4-amine) is commercial synthetic and be dissolved in 10%DMSO by GLSynthesis (Worcester, MA, the U.S.).With the 7-denitrogenation assorted-guanosine (ChemGenes, Wilmington, MA, the U.S.) dissolves with 1M in 1N NaOH.(Sigma) is dissolved in H with inosine 2O.In not containing endotoxic Tris-EDTA, carry out all dilutions.
TLR measures
Expressing human TLR9, the HEK293 cell of the stable transfection of TLR8 or TLR7 is (16,20) as mentioned above.In brief, by using the carrier electroporation transfection HEK293 cell of expressing corresponding human TLR and 6xNF-κ B-luciferase report plasmid.With stable conversion body (3x10 4Individual cells/well) with loxoribine or R-848 not or have in the presence of the ODN in 37 ℃ times and humidification incubator and hatched 16 hours.Each data point is by carrying out in triplicate.Cell lysis and mensuration luciferase gene activity (using from Perkin-Elmer Zaventem, the BriteLite test kit of Belgium).The reporter gene activity that reference does not add the culture medium of oligonucleotide calculates stimulation index.
Cell proliferation
From the peripheral blood buffy coat goods of healthy people's donor available from Blood Bank of theUniversity of D ü sseldorf (Germany) and by with Ficoll-Hypaque (Sigma) centrifugal purification PBMC.With cultured cell in RPMI 1640 culture medium of cell under 37 ℃, 5% (v/v) heat-killed people AB serum (BioWhittaker) or the heat-killed FCS of 10% (v/v) have been replenished in this culture medium, 2mM L-glutaminate, 100U/ml penicillin and 100 μ g/ml streptomycins (all from Sigma).
Cytokines measurement and fluidic cell determination and analysis
With 5x10 6The concentration suspension PBMC of individual cell/ml and join (250 μ l/ hole) in the 96 hole circle base plates.PBMC hatched together with different ODN and/or loxoribine concentration and shown in time point gather culture supernatants (SN).If do not use at once, so SN be stored under-20 ℃, until needed so far.Use is purchased the ELISA test kit (with regard to IL-12p40, from BD Biosciences, Heidelberg, Germany), and IFN-γ and TNF-α (from Diaclone, Besancon, France) or use and to be purchased antibody (PBL, New Brunswick, NJ, the U.S.) the inside ELISA of IFN-γ of research and development estimates the amount of the cytokine among the SN.
With regard to dyeing in the born of the same parents, in the 96 hole circle base plates (250 μ l/ hole) of oligonucleotide and/or the loxoribine concentration of amount shown in containing with 5x10 6Individual cell/ml is hatched PBMC and is added brefeldin A solution (BD Biosciences).PBMC was hatched 6 hours.With regard to IFN-γ dyeing in the born of the same parents, cell was hatched 16 hours with oligonucleotide and/or loxoribine, after this add brefeldin A solution.Gather cell and use Intraprep reagent, carry out dyeing in the born of the same parents according to manufacturer's scheme (Beckman-Coulter, Neuss, Germany).With identifying mononuclear cell (CD14 +), B cell (CD19 +) and NK cell (CD56 +, CD3 -) the suitable antibodies staining cell.Use FACSCalibur obtains the flow cytometry logarithmic data and the program CellQuest that uses a computer analyzes (all from BD Biosciences).All monoclonal antibodies (mAb) that are used for flow cytometric analysis are all available from BD Biosciences, except from the CD11c of Diaclone, from Immunotech (Marseille, CD14 France) and from the CD123 of Miltenyi (Bergisch Gladbach, Germany).Use CD14 cell separation test kit as described in the manufacturer (Miltenyi) from complete PBMC the separation of human mononuclear cell.In order to measure purity, use and identify to the mAb staining cell of CD11c and CD14 and by flow cytometry.In all experiments, mononuclear cell purity surpasses 95%.Mononuclear cell (4x10 with purification 6Individual cell/ml) hatched 24 hours with the ODN that increases concentration.Use BDCA-4 pDC separating kit as enrichment PDC as described in the manufacturer (Miltenyi).By using to CD123 (from Miltenyi), the mAb dyeing of HLA-DR and CD11c (from BD Biosciences) confirms the PDC purification.Purity is about 85%.With cell (5x10 5Individual cell/ml, 250 μ l/ holes) with or do not cultivate 24 hours with oligonucleotide and loxoribine.Measure IFN-α or IL-12p40 among the SN as mentioned above.
The result
By hatching altogether the sequence selective of the signal conduction of TLR8 mediation is strengthened with the homopolymer oligonucleotide
The HEK293 cell of stably express hTLR8 and NF-κ B-luciferase report construct and R-848 (1-(2-hydroxy-2-methyl propyl group)-2-methyl isophthalic acid H-imidazo [4,5-c] quinoline-4 amine) are hatched being with or without in the presence of the oligonucleotide.Analyze the influence of hatching the NF-kB activation that TLR8 is mediated with different oligonucleotide altogether then.
Identified the widow (dT) of containing the D2EHDTPA main chain 17Homopolymer ODN, promptly ODN 6056, and it significantly increases the NF-kB activation level (Figure 1B) that R-848 stimulates.Independent ODN 6056 is not in that reach under the 25 μ M concentration can be at the tangible NF-kB activation of cell moderate stimulation (being no more than 2.5 times of backgrounds) (unpub data) of expressing TLR8.It is dependent that effect shows as oligonucleotide, because be not all same fully enhancing TLR8 signal conduction (for example, incoherent heteropolymer (ODN 1982) can not influence the NF-kB activation of R-848 strongly) of oligonucleotide of all tests.
R-848 and ODN 6056 are hatched altogether and have not only been increased the TLR8 effect, and have significantly improved TLR8 activation usefulness.The EC50 of R-848 have 0.1 μ M ODN 6056 (0.1 μ M ODN6056:EC50 (R-848)=4.9 μ M, promptly from three times the experiment meansigma methods; Independent EC50 (R-848)＞30 μ M) there is down significantly decline.Yet the ODN 6056 of high concentration has reduced EC50 (1 μ M ODN 6056:EC50 (R-848)=1.4 μ M more; 5 μ M ODN:EC50 (R-848)=0.4 μ M).This effect is specific to ODN 6056, because descend quite lowly (5 μ M 1982:EC50 (R-848)=19.0 μ M) for ODN 1982 EC50.Opposite with the stimulation to TLR8, ODN 6056 (or ODN 1982) is hatched the NF-κ B that can not influence the CpG mediation altogether with the TLR9 part CpG ODN 2006 on the HEK293 cell of expressing hTLR9 stimulates (unpub data).
Study the possible sequence that is used for observed potentiation and required and tested several different D2EHDTPA ester homopolymers (table 1).Few (dT) 17(ODN 6056) have the strongest promotion characteristic, are few (dU) subsequently 17(SEQ ID NO:11) and few (dA) 17(SEQ ID NO:12).To few (dC) 17(SEQ ID NO:13) and randomization widow (dN) 15The enhancing that has detected significance descends.Heteropolymer ODN 1982 only shows minimum increased activity, and few (dG) 24(SEQ IDNO:14) do not have enhanced activity, but suppresses the NF-κ B stimulation of R-848 mediation.Sequence with ODN 6056 of phosphodiester backbone does not influence the stimulation of TLR8 mediation.The length of oligonucleotide also shows has influence: few (dA) 24(SEQ ID NO:15) is lower than widow (dA) with the synergism that R-848 shows 17(SEQ ID NO:12).
In the stimulation that has TLR7 ligands specific in the presence of the homopolymer T oligonucleotide to the cell of expressing TLR8
R-848 is the part (16) of hTLR7 and hTLR8, and loxoribine (7-pi-allyl-7,8-dihydro-8 oxos-guanosine) is described as TLR7 ligands specific (18).The loxoribine that concentration reaches 10mM can not activate with the conduction of the NF-κ B signal in the HEK293 cell of hTLR8 or hTLR9 transfection, but the signal conduction (Fig. 2 A) of activation hTLR7 mediation.Although be the TLR7 part, will express the HEK293 cell of hTLR8 and loxoribine and oligonucleotide and hatch altogether and activated NF-κ B signal conduction (Fig. 2 B).This concentration dependent effect is only having ODN 6056, but not ODN 1982 observes (Fig. 2 B) under existing.Use another kind of TLR7 part, the 7-denitrogenation is assorted-and guanosine similarly observes (18).Independent 7-denitrogenation is assorted-and guanosine reaching 5mM concentration and do not having in the presence of the ODN 6056 expressing the cell non-activity of hTLR8, but the NF-kB activation that stimulation TLR8 mediates in the presence of the ODN 6056 is being arranged.The ODN 6056 that also will show on the HEK293 cell of chemical compound inosine (unpub data) that activates the NF-κ B signal conduction in the HEK293 cell in non-specific mode and the hTLR8 that expresses is hatched (Fig. 2 C) altogether.Inosine changes having in the presence of the ODN6056 activation of NF-κ B do not compared with the non-specific activation of the inosine that reaches 10mM concentration.
Use has been observed similar effect based on the chemical compound 6-amino-9-benzyl-2-butoxy-9H-purine-8-alcohol of adenosine.When using separately, this chemical compound is the TLR7 part, but is not TLR8 part (Fig. 9 A and 9B).Poly-(dT) arranged 17Connect oligonucleotide and exist when using down, its concentration stimulates TLR8 signal conduction (Fig. 9 B).
The sequence dependent of TLR7 signal conduction suppresses
These data representation micromolecule TLR7 part is induced TLR8 dependency NF-κ B signal conduction having in the presence of some oligonucleotide.However, still in the HEK293 cell of expressing TLR7, effect is opposite.Oligonucleotide is in fact as Figure 1A, 3 and 9A shown in suppress NF-kB activation by the TLR7 mediation of TLR7 ligands specific and TLR7/8 part.This effect shows as sequence-non-specific, because suppress hTLR7 activation (unpub data) in the phosphorothioate oligonucleotide of all tests up to now.
The TLR8 part signal conduction of using RNA to connect oligonucleotide strengthens
Use R-848 similarly to test with the oligonucleotide that is connected based on RNA as the TLR7/8 part.Figure 10 represents blended sequence oligonucleotides and poly-(rU) 18The effect that oligonucleotide stimulates NF-κ B in the hTLR8-LUC-293 cell.Poly-(rU) 18Oligonucleotide (rU *RU *RU *RU *RU *RU *RU *RU *RU *RU *RU *RU *RU *RU *RU *RU *RU *RU; SEQ ID NO:5) when using separately, passes through the TLR8 conducted signal, as (Figure 10) of using this oligonucleotide that the slight activation of NF-κ B is confirmed.Blended sequence RNA oligonucleotide (rG *RC *RC *RA *RC *RC *RG *RA *RG *RC *RC *RG *RA *RA *RG *RG *RC *RA *RC *RC; SEQ ID NO:16) when using separately, can not pass through the TLR8 conducted signal.
In Figure 11, the R-848 of hTLR8-LUC-HEK293 cell and recruitment is hatched having in the presence of the following material: (a) TE and 50 μ g/ml DOTAP, or (b) ORN shown in the 5 μ M are having in the presence of the 50 μ g/ml DOTAP.After 16 hours, determine the stimulation of NF-κ B by measuring uciferase activity.Reference only has the background under the culture medium existence to calculate stimulation index.The figure shows poly-(rU) 18Oligonucleotide to TLR8 signal conduction act on this oligonucleotide and R-848 coupling the time significantly strengthened (Figure 11).Neither TLR7, the blended sequence RNA oligonucleotide of non-again TLR8 part also strengthens the conduction of TLR8 signal by R-848 as shown in Figure 11.Tables of data Benq also can be used as junctional complex in the oligonucleotide of RNA and works.
Loxoribine and few (dT) 17ODN is hatched altogether and is changed the cytokine characteristic that the human PBMC produces
Studied loxoribine and ODN 6056 and 1982 and hatched effect altogether people's immunocyte.Hatching with independent loxoribine has stimulated the human PBMC to produce IFN-α (Fig. 4 A).Yet, having in the presence of the ODN that increases concentration, the inductive IFN-α of loxoribine reduces to background level.Although use ODN all to observe this effect, ODN 6056 seems based on stronger inhibitory action.
Also studied other production of cytokines.Independent loxoribine only induces minimum IL-12p40 to secrete (Fig. 4 B) from the human PBMC.On the contrary, having in the presence of the ODN 6056, IL-12p40 produces in a large number, and the ODN 1982 of same concentrations can not induce the IL-12p40 that is higher than the background that uses the single culture base with loxoribine.IL-12p70 (unpub data) and IFN-γ have been obtained similar result (Fig. 4 C).When being hatched altogether, loxoribine and ODN 6056 also stimulated TNF-α to produce.Yet, when ODN 1982 is hatched altogether with loxoribine, observed TNF-α and produced, but, be starkly lower than the result of hatching acquisition with ODN 6056 altogether.There is not a kind of TNF-α secretion (Fig. 4 D) of inducing significant level in independent ODN and the loxoribine.
Using loxoribine and widow (dT) 17Mononuclear cell produced IL-12p40 and TNF-α when ODN stimulated altogether
The combination of loxoribine and ODN has activated and has expressed the HEK293 cell of TLR8, but has suppressed the signal conduction of TLR7 mediation.According to inferring main source and the positive pDCs of TLR7-that people TLR8-positive immunocyte is IL-12 and TNF-α is the main source of IFN-α.FACS dyeing does not disclose most of CD 14+ cell generation TNF-α (Fig. 5 A) when the human PBMC is hatched altogether with loxoribine and ODN 6056 in the born of the same parents.Independent loxoribine or only produce the positive mononuclear cell of a small amount of TNF-with ODN 1982 combination.The positive monocytic percentage ratio of IL-12-is lower than the percentage ratio of TNF-α.Yet the synergism of loxoribine and ODN 6056 combinations can obviously detect (Fig. 5 B) to IL-12 in the born of the same parents.When using ODN and loxoribine to stimulate, once can in CD 19+B cell, not detect IL-12 or TNF-α (unpub data) in the born of the same parents in three experiments.Also studied the cell source of IFN-γ.Loxoribine and ODN combination of stimulation high IL-12 level, and studies confirm that formerly IL-12 induces the IFN-γ secretion (21-23) in the NK cell.Therefore, in NK cells of human beings, estimate IFN-γ and produce (Fig. 6).In fact, IFN-γ in the NK cell produces and can observe when human PBMC and loxoribine and ODN 6056 are hatched altogether, but, independent stimulation can not induced as producing by the obvious IFN-γ that is confirmed from the IFN-γ protein ELISA of human PBMC's supernatant.
In order to confirm the stimulation as the monocytic direct TLR mediation that loxoribine and ODN 6056 are replied, purification is hatched being with or without in the presence of ODN 6056 or 1982 from human PBMC's mononuclear cell and with loxoribine.Isolating mononuclear cell only produces IL-12p40 (Fig. 7 A) or TNF-α (unpub data) conduct replying two kinds of stimulus object loxoribines and ODN 6056 combinations.
Human PBMC and little immunomodulatory compounds class R-848 or Luo Suoli are hatched the IFN-α that induces pDCs and are produced (Fig. 7 B and (24,25)).Therefore, ODN 6056 has been become the direct result of the signal conduction inhibition of TLR7 mediation among the pDCs by the inhibitory action to IFN-α generation of loxoribine stimulation.In order to study this probability, separation of human pDCs and use loxoribine are having widow (dT) 17Homopolymer or heteropolymer ODN (Fig. 7 B) exist down to be stimulated.The people pDC of enrichment with but loxoribine produce a large amount of IFN-α when hatching.On the contrary, having eliminated IFN-α when hatching altogether with ODN 6056 or ODN 1982 produces.In addition, the inhibitory action of ODN 6056 shows as the inhibitory action that is better than ODN 1982, because it is lower to eliminate the required ODN concentration of IFN-α generation.
Fig. 8 represents to use the data of TLR8-LUC-293 cell generation.Use the R-848 (50 micromole) of constant density and the appointment ODN of recruitment.The luciferase of independent R-848 is read the result be set at standardized 100%.The result is shown in left group.Fig. 8 right group is represented the effect in the poly-C oligonucleotide of the 1 micromole 17mer of T content in the presence of the R-848 of recruitment is arranged that increases.Even use two kinds of thymidines all to strengthen active and use that thymidine is active more than 6 kinds or 6 kinds significantly increases.
Discuss
Data acknowledgement of the present invention can have the ability of regulating hTLR8 in the presence of some D2EHDTPA ODN.HTLR8 is the most approaching relevant with hTLR7 (26), as (16) that ability reflected of little immune-stimulating compound R-848 activation hTLR7 and hTLR8.Yet hTLR7 is more more responsive than hTLR8 to the stimulation of R-848, because the R-848 concentration that the TLR7 activation needs is lower.R848 and D2EHDTPA widow (dT) 17Hatch altogether and make TLR8 more responsive and suppress the TLR7 activation, but this independent T homopolymer is to any not significantly effect among these TLR the stimulation of R848.
Whether can influence the TLR activation of other stimulus object in order to measure the T homopolymer, study it two kinds of TLR7 parts, i.e. loxoribine and 7-denitrogenation be assorted-effect of guanosine, they do not stimulate TLR8 (18).Surprisingly, with regard to two kinds of parts, few (dT) 17Existence cause converting TLR8 dependency NF-kB activation fully to wherein few (dT) from TLR7- 17Suppress the signal conduction of TLR7 mediation.Possible situation is, the weak affine combination that people TLR8 can the gene pairs loxoribine, and its possibly can't detect in available bioassary method.The existence of some oligonucleotide can strengthen the affinity of loxoribine to TLR8 by unknown mechanism, makes the TLR8 that can detect the loxoribine mediation in the stable transfected cells stimulate.
Can not get rid of oligonucleotide to the conduction of general signal by way of or the probability of picked-up mechanism influence.Yet, few (dT) 17To using hTLR7 (conduction of NF-κ B signal suppresses), the HEK293 cell of hTLR9 (no effect) or hTLR8 (the strong enhancing of signal conduction) transfection has this fact of different model of action and has come down hard upon this probability.Observed synergism is not limited to the cell of unconventionality expression TLR8.People TLR7 mainly expresses in pDCs and B cell.These cells are not expressed TLR8, and TLR8 expresses (27-30) in medullary cell.In fact, measure people's immunocyte, such as the synergism of loxoribine in the mononuclear cell and the ODN pair cell factor (IL-12 and TNF-α) generation with endogenous TLR8 expression.On the contrary, the inductive IFN-α of most of loxoribine and R-848 produces (our data and (4,24,25)) by the activation of the TLR7 among the people pDC.IFN-α among these TLR7 stimulus object activation pDC produces and is subjected to suppressing fully because of hatching with D2EHDTPA ODN, and this is consistent with observed inhibitory action in the TLR7-cells transfected.D2EHDTPA ODN suppresses not have the obvious sequence dependency in the signal conduction of TLR7 mediation in transfectional cell or pDC, this with to the observed sequence of TLR8-the selective stimulating effect is opposite.The combination of ODN and loxoribine not only causes converting mononuclear cell-deutero-IL-12 and TNF-α to from the deutero-IFN-α of pDC-, and causes IFN-γ to secrete from the NK cell.NK cells of human beings lacks TLR7 and TLR8 expresses (31), and making that the stimulation of NK cell-deutero-IFN-γ shows as may be by the indirect action (32) of IL-12 mediation.When simply adding some oligonucleotide, observed the change fully of the immunization characteristic of loxoribine mediation jointly.In addition, these data have been pointed out to use and have been certain sequence dependent effect of the ODN that is rich in D2EHDTPA T of the most effective ODN, and described ODN can be with the coupling of TLR7 ligands specific so that activation TLR8.
Some reports show TLR7 and the TLR8 (27-29) on bone marrow dendritic cell and the monocytes mRNA level.There are not the available data that are illustrated in actual TLR protein expression in these cells.In these experiments, do not detect loxoribine to monocytic direct stimulation.On the contrary, only the combination of loxoribine and oligonucleotide induces mononuclear cell-deutero-cytokine to produce.These results have opposed that the functional expression of TLR7 in mononuclear cell will point out that TLR8 is as by the receptor of oligonucleotide and loxoribine targeting.In addition, independent loxoribine can not induced a large amount of IL-12, but has reported and produce IL-12 (29) among the inductive human PBMC of R-848, the ability consistent (16) that this stimulates TLR with R-848.It is very difficult to notable difference to use the chemical compound that stimulates two kinds of healths to activate TLR in the cell that can express TLR7 and TLR8.Ito etc. (33) have reported that mDC produces IL-12, but does not produce IFN-α when using R-848 to stimulate.On the contrary, when using R-848 or other TLR ligand stimulation, pDC does not produce IL-12, but does not produce IFN-α (25), has shown that induce the IFN-α among pDCss that with TLR7 mediate of IL-12 in mDC of TLR8 mediation induces.Even R-848 or loxoribine and widow (dT) 17ODN is hatched altogether and also can not caused people pDC to produce IL-12, this prompting sort signal be not enough to activate among the pDC suitably by way of.
What cause concern is to infer model, wherein some TLR can have (may for allosteric) oligonucleotide can in conjunction with and the regulatory site that works of action effect molecule.With regard to TLR7, phosphorothioate oligonucleotide shows as antagonist and works.Therefore the TLR7 of oligonucleotide binding can suppress the TLR7 part combine with it capsule in conjunction with or for example, conducted by intravital correct conformational change correction downstream signal by aggravation.On the other hand, the oligonucleotide that is rich in T combine with TLR8 can be used as can increase R-848 or other micromolecule part class loxoribine combine with active TLR8 capsule in conjunction with or (allosteric) activator of increasing the downstream signal conduction work.Can not determine it is to have relate to two kinds at present, combination of micromolecule part and effect ODN combination still on same loci, occur in conjunction with the territory.The allosteric enhancer of known receptor from several different families.For example, Knoflach etc. (34) have reported as metabotropic glutamate receptors, i.e. the evaluation of two micromoleculars that work of the allosteric promoter of a large amount of G albumen coupling health families.The labor of receptor structure is positioned promoter binding site in the membrane spaning domain.Other allosteric promoter that studies show that micromolecule can be used as muscarinic acetylcholine recep-tor work (35).Some 2-aminothiophene class as adenosine receptor A1 allosteric promoter by stablizing ligand-receptor-G-albumen ternary complex and increasing empty receptor and G-protein binding (38) work (36,37).Interesting is that Gao etc. (39) find that a series of imidazoquinolies are derived so that work as the allosteric promoter that is combined in the agonist on the people A3 adenosine receptor.(40) such as recent Rutz use the surface plasmon resonance biosensor analysis to confirm directly to block with micromolecule the TLR9 protein binding of CpG ODN and purification.Their discovery shows that these molecules directly combine with the TLR9 extracellular domain, thereby the antagonist that conducts as the signal that TLR9 mediates works.Therefore, described oligonucleotide and micromolecule can be directly in conjunction with TLR7 and 8.That uses isolating protein and corresponding receptors ligand can further have insight into the definite mechanism of action of the combination of micromolecule TLR part and oligonucleotide to TLR7 or TLR8 in conjunction with research.These find the molecular signal transmission mechanism of understanding TLR and drug research and medicament research and development significant.The present invention has confirmed that the signaling activity of two member TLR8 of TLR family and TLR7 can be by existing or non-existent some oligonucleotide is handled.These results have pointed out the new mode of use loxoribine (or other micromolecule) with the combination change immunne response of some oligonucleotide jointly.Combination by these molecules can be oriented to the cytokine characteristic that different TLR change TLR micromolecule part again by making its signal conduction.The immunne response that changes or strengthen may be useful to the treatment various diseases.
Table 1
?SEQ?ID?NO：?(ODN) Sequence The %R-848 activity
?3?6056 T *T *T *T *T *T *T *T *T *T *T *T *T *T *T *T *T 271±12
?11 U *U *U *U *U *U *U *U *U *U *U *U *U *U *U *U *U 236±14
?12 A *A *A *A *A *A *A *A *A *A *A *A *A *A *A *A *A 228±37
?13 C *C *C *C *C *C *C *C *C *C *C *C *C *C *C *C *C 183±5
?15 A *A *A *A *A *A *A *A *A *A *A *A *A *A *A *A *A *A *A *A *A *A *A *A 160±16
N *N *N *N *N *N *N *N *N *N *N *N *N *N *N 156±19
?4?1982 T *C *C *A *G *G *A *C *T *T *C *T *C *T *C *A *G *G *T *T 121±21
?16 T_T_T_T_T_T_T_T_T_T_T_T_T_T_T_T_T 98±7
?14 G *G *G *G *G *G *G *G *G *G *G *G *G *G *G *G *G *G *G *G *G *G *G *G 76±12
Table 1: different ODN are to the active synergistic comparison of R-848
With the R-848 that expresses the HEK293 cell of hTLR8 and NF-κ B report construct and 50 μ M not or have shown in the 5 μ M and hatch in the presence of the ODN.Will be in that not have R-848 causes in the presence of the ODN NF-κ B stimulus settings be 100% and calculate the active effect to R-848 thus.Numerical value represent 2-4 time the experiment meansigma methods (± SD).The A of " N " expression random order, C, G or T, " *" expression thiophosphate main chain, " _ " expression phosphodiester backbone.
Table 2
T2 T ＊T
T4 T ＊T ＊T ＊T
T6 T ＊T ＊T ＊T ＊T ＊T
T8 T ＊T ＊T ＊T ＊T ＊T ＊T ＊T
T10 T ＊T ＊T ＊T ＊T ＊T ＊T ＊T ＊T ＊T
T12 T ＊T ＊T ＊T ＊T ＊T ＊T ＊T ＊T ＊T ＊T ＊T
T14 T ＊T ＊T ＊T ＊T ＊T ＊T ＊T ＊T ＊T ＊T ＊T ＊T ＊T
T17 T ＊T ＊T ＊T ＊T ＊T ＊T ＊T ＊T ＊T ＊T ＊T ＊T ＊T ＊T ＊T ＊T
C17 C ＊C ＊C ＊C ＊C ＊C ＊C ＊C ＊C ＊C ＊C ＊C ＊C ＊C ＊C ＊C ＊C
C7T2C8 C ＊C ＊C ＊C ＊C ＊C ＊C ＊T ＊T ＊C ＊C ＊C ＊C ＊C ＊C ＊C ＊C
C6T4C7 C ＊C ＊C ＊C ＊C ＊C ＊T ＊T ＊T ＊T ＊C ＊C ＊C ＊C ＊C ＊C ＊C
C5T6C6 C ＊C ＊C ＊C ＊C ＊T ＊T ＊T ＊T ＊T ＊T ＊C ＊C ＊C ＊C ＊C ＊C
C4T8C5 C ＊C ＊C ＊C ＊T ＊T ＊T ＊T ＊T ＊T ＊T ＊T ＊C ＊C ＊C ＊C ＊C
C3T10C4 C ＊C ＊C ＊T ＊T ＊T ＊T ＊T ＊T ＊T ＊T ＊T ＊T ＊C ＊C ＊C ＊C
T17 T ＊T ＊T ＊T ＊T ＊T ＊T ＊T ＊T ＊T ＊T ＊T ＊T ＊T ＊T ＊T ＊T
The expression phosphorothioate bond
List of references
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2.Krieg，A.M.2003.CpGmotifs：the?active?ingredient?in?bacterial?extracts？Nat.Med.9：831-835.
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20.Bauer，S.，C.J.Kirschning，H.Hacker，V.Redecke，S.Hausmann，S.Akira，H.Wagner，and?G.B.Lipford.2001.Human?TLR9?confers?responsiveness?to?bacterial?DNA?via?species-specific?CpGmotif?recognition.Proc.Natl.Acad.Sci.U.S.A.98：9237-9242.
21.Magram，J.，S.E.Conmaughton，R.R.Warrier，D.M.Carvajal，C.Y.Wu，J.Ferrante，C.Stewart，U.Sarmiento，D.A.Faherty，and?M.K.Gately.1996.IL-12-deficient?mice?are?defective?in?IFNgamma?production?and?type?1?cytokine?responses.Immunity.4：471481.
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23.Trinchieri，G.2003.Interleukin-12?and?the?regulation?of?innate?resistance?and?adaptive?immunity.Nat.Rev.Immunol.?3：133-146.
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25.Gibson，S.J.，J.M.Lindh，T.R.Riter，R.M.Gleason，L.M.Rogers，A.E.Fuller，J.L.Oesterich，K.B.Gorden，X.Qiu，S.W.McKane，R.J.Noelle，R.L.Miller，R.M.Kedl，P.Fitzgerald-Bocarsly，M.A.Tomai，and?J.P.Vasilakos.2002.Plasmacytoid?dendritic?cells?producecytokines?and?mature?in?response?to?the?TLR7?agonists，imiquimod?and?resiquimod.Cell?Immunol.218：74-86.
26.Du，X.，A.Poltorak，Y.Wei，and?B.Beutler.2000.Three?novel?mammalian?toll-like?receptors：gene?structure，expression，and?evolution.Eur.Cytokine?Netw.11：362-371
27.Jarrossay，D.，G.Napolitani，M.Colonna，F.Sallusto，and?A.Lanzavecchia.2001.Specialization?and?complementarity?in?microbial?molecule?recognition?by?human?myeloidand?plasmacytoid?dendritic?cells.Eur.J.Immunol.31：3388-3393.
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30.Iwasaki，A.and?R.Medzbitov.2004.Toll-like?receptor?control?of?the?adaptive?immune?responses.Nat.Immunol.?5：987-995.
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34.Knoflach，F.，V.Mutel，S.Jolidon，J.N.Kew，P.Malherbe，E.Vieira，J.Wichmann，and?J.A.Kemp.2001.Positive?allosteric?modulators?of?metabotropic?glutamate?1?receptor：characterization，mecbanism?of?action，and?binding?site.Proc.Natl.Acad.Sci.U.S.A.98：13402-13407.
35.Waelbroeck，M.2003.Allosteric?drugs?acting?at?muscarinic?acetylcholine?receptors.Neurochem.Res.28：419-422.
36.Bruns，R.F.and?J.H.Fergus.1990.Allosteric?enhancement?of?adenosine?A1?receptor?bindingand?function?by?2-amino-3-benzoylthiophenes.Mol.Pharmacol.38：939-949.
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Equivalents
Think that the specification of writing above is enough to make those skilled in the art to implement the present invention. The embodiment that scope of the present invention is not limited to provide, because these embodiment are intended to as the independent explanation to one aspect of the invention, and the embodiment of equivalence also belongs to scope of the present invention on other function. Except shown and described herein those, those skilled in the art obviously can also carry out various modification to the present invention and belong to the scope of appended claim according to top description. Advantage of the present invention is not necessarily contained by each embodiment of the present invention.
With all lists of references of quoting among the application, patent and patent application intactly are incorporated herein by reference.
Sequence table
<110>Coley?Pharmaceutical?Group，Inc.
Coley?Pharmaceutical?GmbH
Jurk，Marion
Vollmer，Jorg
Krieg，Arthur?M
Uhlmann，Eugen
Noll，Bernhard?O
＜120〉use the immunne response that connects oligonucleotide adjusting TLR mediation
<130>C1041.70051WO00
＜140〉do not specify
＜141〉herewith
<150>US?60/720,981
<151>2005-09-27
<160>20
<170>PatentIn?version?3.3
<210>1
<211>17
<212>DNA
＜213〉artificial sequence
<220>
＜223〉synthetic oligonucleotide
<400>1
cccttttttt?tttcccc
<210>2
<211>17
<212>DNA
＜213〉artificial sequence
<220>
＜223〉synthetic oligonucleotide
<400>2
cccctttttt?ttccccc 17
<210>3
<211>17
<212>DNA
＜213〉artificial sequence
<220>
＜223〉synthetic oligonucleotide
<400>3
tttttttttt?ttttttt 17
<210>4
<211>20
<212>DNA
＜213〉artificial sequence
<220>
＜223〉synthetic oligonucleotide
<400>4
tccaggactt?ctctcaggtt 20
<210>5
<211>18
<212>RNA
＜213〉artificial sequence
<220>
＜223〉synthetic oligonucleotide
<400>5
uuuuuuuuuu?uuuuuuuu 18
<210>6
<211>17
<212>DNA
＜213〉artificial sequence
<220>
＜223〉synthetic oligonucleotide
<400>6
cccccttttt?tcccccc 17
<210>7
<211>17
<212>DNA
＜213〉artificial sequence
<220>
＜223〉synthetic oligonucleotide
<400>7
cccccctttt?ccccccc 17
<210>8
<211>17
<212>DNA
＜213〉artificial sequence
<220>
＜223〉synthetic oligonucleotide
<400>8
cccccccttc?ccccccc 17
<210>9
<211>15
<212>DNA
＜213〉artificial sequence
<220>
＜223〉synthetic oligonucleotide
<220>
＜221〉misc_ feature
<222>(15)..(15)
＜223〉n connects base-connection base-immunosine
<400>9
tttttttttt?ttttn 15
<210>10
<211>15
<212>DNA
＜213〉artificial sequence
<220>
＜223〉synthetic oligonucleotide
<220>
＜221〉misc_ feature
<222>(1)..(1)
＜223〉n is that immunosine-connects base-connection base
<400>10
nttttttttt?ttttt 15
<210>11
<211>17
<212>RNA
＜213〉artificial sequence
<220>
＜223〉synthetic oligonucleotide
<400>11
uuuuuuuuuu?uuuuuuu 17
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<211>17
<212>DNA
＜213〉artificial sequence
<220>
＜223〉synthetic oligonucleotide
<400>12
aaaaaaaaaa?aaaaaaa 17
<210>13
<211>17
<212>DNA
＜213〉artificial sequence
<220>
＜223〉synthetic oligonucleotide
<400>13
cccccccccc?ccccccc 17
<210>14
<211>24
<212>DNA
＜213〉artificial sequence
<220>
＜223〉synthetic oligonucleotide
<400>14
gggggggggg?gggggggggg?gggg 24
<210>15
<211>24
<212>DNA
＜213〉artificial sequence
<220>
＜223〉synthetic oligonucleotide
<400>15
aaaaaaaaaa?aaaaaaaaaa?aaaa 24
<210>16
<211>20
<212>DNA
＜213〉artificial sequence
<220>
＜223〉synthetic oligonucleotide
<400>16
gccaccgagc?cgaaggcacc 20
<210>17
<211>17
<212>DNA
＜213〉artificial sequence
<220>
＜223〉synthetic oligonucleotide
<400>17
tttttttttt?ttttttt 17
<210>18
<211>10
<212>DNA
＜213〉artificial sequence
<220>
＜223〉synthetic oligonucleotide
<400>18
tttttttttt 10
<210>19
<211>12
<212>DNA
＜213〉artificial sequence
<220>
＜223〉synthetic oligonucleotide
<400>19
tttttttttt?tt 12
<210>20
<211>14
<212>DNA
＜213〉artificial sequence
<220>
＜223〉synthetic oligonucleotide
<400>20
tttttttttt?tttt 14

Claims (42)

1. stimulate the method for the immunne response of TLR 8 mediations, comprise to the experimenter that these needs are arranged give the immunne response that effective stimulus TLR 8 mediates consumption TLR 7/8 part be connected oligonucleotide.
2. make the immunne response of TLR7 mediation be redirected method, comprise that experimenter to the immunne response of experience TLR7 mediation gives effectively to make the immunne response of TLR7 mediation to be redirected the connection oligonucleotide of the consumption of the immunne response that mediates for TLR8 for the immunne response of TLR 8 mediations.
3. claim 1 or 2 described methods, wherein TLR 7/8 part is the TLR7 ligands specific.
4. claim 1 or 2 described methods, wherein the TLR7 ligands specific is the guanosine that C8-replaces.
5. the described method of claim 4, wherein the guanosine of C8-replacement is a 7-pi-allyl-7,8-dihydro-8-oxo-guanosine (loxoribine), 7-thia-8-oxo guanosine (immunosine), the 8-TGR, 8-bromine guanosine, 8-methylguanosine, 8-oxo-7,8-dihydro guanosine, C8-virtue amino-2 '-deoxyguanosine, C8-propinyl-guanosine, the guanosint riboside that C8-and N7-replace, 7-methyl-8-oxo guanosine, the amino guanosine of 8-, 8-hydroxyl-2 '-deoxyguanosine, the 7-denitrogenation is assorted-guanosine and 8-hydroxyl guanosine that 8-replaces.
6. the described method of claim 4, wherein the guanosine that replaces of C8-is a loxoribine.
7. the described method of claim 3, wherein the TLR7 ligands specific for the 7-denitrogenation assorted-guanosine.
8. the described method of claim 3, wherein the TLR7 ligands specific is 6-amino-9-benzyl-2-(3-hydroxyl-propoxyl group)-9H-purine-8-alcohol or 6-amino-9-benzyl-2-butoxy-9H-purine-8-alcohol.
9. claim 1 or 2 described methods, wherein TLR 7/8 part is TLR 8 ligands specifics.
10. claim 1 or 2 described methods, wherein TLR 7/8 part is TLR 7 parts and TLR 8 parts.
11. the described method of claim 10, wherein TLR 7/8 part is an imidazoquinolie.
12. the described method of claim 11, wherein imidazoquinolie is an immidazoquinolinaminas, imidazopyridine amine, 6, the condensed cycloalkyl imidazopyridine of 7-amine, 1, the immidazoquinolinaminas that Bridge 2 connects, R-848 (S-28463 or resiquimod), 4-amino-2-ethoxyl methyl-α, alpha-alpha-dimethyl-1H-imidazo [4,5-c] quinoline-1-ethanol, 1-(2-methyl-propyl)-1H-imidazo [4,5-c] quinoline-4-amine (R-837 or rice quinoline are not special) or S-27609.
13. the described method of claim 11, wherein imidazoquinolie is R848.
14. claim 1 or 2 described methods wherein connect oligonucleotide and comprise unmethylated CpG motif.
15. claim 1 or 2 described methods wherein connect oligonucleotide and do not contain unmethylated CpG.
16. claim 1 or 2 described methods wherein connect oligonucleotide and comprise 5 ' N-TTTTT-N 3 ', wherein N is any nucleotide.
17. claim 1 or 2 described methods wherein connect oligonucleotide and comprise 5 ' N-TTTTTT-N 3 ', wherein N is any nucleotide.
18. claim 1 or 2 described methods, wherein connecting oligonucleotide is the dT homopolymer.
19. claim 1 or 2 described methods wherein connect oligonucleotide and comprise the thiophosphate backbone modifications.
20. claim 1 or 2 described methods wherein give TLR 7/8 part respectively and are connected oligonucleotide.
21. claim 1 or 2 described methods, wherein the TLR7/8 part be connected oligonucleotide and be bonded to each other.
22. claim 1 or 2 described methods, wherein connecting oligonucleotide is DNA.
23. claim 1 or 2 described methods, wherein connecting oligonucleotide is RNA.
24. claim 1 or 2 described methods further comprise giving antigen to the experimenter.
25. claim 1 or 2 described methods, wherein the experimenter has infection.
26. claim 1 or 2 described methods, wherein the experimenter has cancer.
27. claim 1 or 2 described methods, wherein the experimenter has anaphylaxis or asthma.
28. compositions comprises TLR 7 ligands specifics that are selected from the pure and mild 6-amino of 6-amino-9-benzyl-2-(3-hydroxyl-propoxyl group)-9H-purine-8--9-benzyl-2-butoxy-9H-purine-8-alcohol and is connected oligonucleotide.
29. compositions comprises TLR 8 ligands specifics and is connected oligonucleotide.
30. claim 28 or 29 described compositionss wherein connect oligonucleotide and comprise unmethylated CpG motif.
31. claim 28 or 29 described compositionss wherein connect oligonucleotide and do not contain unmethylated CpG.
32. claim 28 or 29 described compositionss wherein connect oligonucleotide and comprise 5 ' N-TTTTT-N 3 ', wherein N is any nucleotide.
33. claim 28 or 29 described compositionss wherein connect oligonucleotide and comprise 5 ' N-TTTTTT-N 3 ', wherein N is any nucleotide.
34. claim 28 or 29 described compositionss, wherein connecting oligonucleotide is the dT homopolymer.
35. claim 28 or 29 described compositionss wherein connect oligonucleotide and comprise key between at least one thiophosphate nucleotide.
36. claim 28 or 29 described compositionss, wherein connecting oligonucleotide is DNA.
37. claim 28 or 29 described compositionss, wherein connecting oligonucleotide is RNA.
38. claim 28 or 29 described compositionss wherein connect oligonucleotide and TLR 7 ligands specifics or TLR 8 ligands specifics and are bonded to each other.
39. claim 28 or 29 described compositionss further comprise antigen.
40. claim 28 or 29 described compositionss, wherein said composition is pharmaceutical preparation.
41. identify the method for TLR8 part, comprise and make the cell of expressing TLR8 have and not be connected engaged test part in the presence of the oligonucleotide, with the stimulation that is determined at and is not connected the cell of the expression TLR8 of this test ligand of response under the oligonucleotide existence, wherein according to the increase evaluation TLR8 part that connects stimulation in the presence of the oligonucleotide is being arranged.
42. identify the method that connects oligonucleotide, comprise and make the cell of expressing TLR8 contact the TLR7 part in the presence of the oligonucleotide having to be connected with test not, with be determined at and test is connected oligonucleotide and has the stimulation of the cell of the expression TLR8 of this TLR7 part of response down, wherein according to the increase evaluation connection oligonucleotide that connects in the presence of the oligonucleotide the cytositimulation of expressing TLR8 is being arranged.
CNA200680044386XA 2005-09-27 2006-09-27 Modulation of tlr-mediated immune responses using adaptor oligonucleotides Pending CN101340931A (en)

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WO2007038720A3 (en) 2007-06-21
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