CN101378776A - Novel synthetic agonists of TOll-like receptors containing CG dinucleotide modifications - Google Patents
Novel synthetic agonists of TOll-like receptors containing CG dinucleotide modifications Download PDFInfo
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- CN101378776A CN101378776A CNA2006800530586A CN200680053058A CN101378776A CN 101378776 A CN101378776 A CN 101378776A CN A2006800530586 A CNA2006800530586 A CN A2006800530586A CN 200680053058 A CN200680053058 A CN 200680053058A CN 101378776 A CN101378776 A CN 101378776A
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Abstract
The invention relates to the therapeutic use of oligonucleotides as immune modulatory agents in immunotherapy applications. More particularly, the invention provides immune modulatory oligonucleotide compositions for use in methods for generating an immune response or for treating a patient in need of immune modulation. The immune modulatory oligonucleotides of the invention preferably comprise novel pyrimidines and purines.
Description
Related application
The application requires the rights and interests of U.S. Provisional Application serial number 60/752,335 that December in 2005 submitted on the 20th and the U.S. Provisional Application serial number of submitting on August 4th, 2,006 60/821,458.Incorporate the full content of above-mentioned application instruction into this paper to put forward the mode of stating.
Background of invention
Invention field
The present invention relates generally to use immunity and the immunization therapy application of oligonucleotide as immunomodulator.More specifically, the present invention relates to new Chemical composition that and their method of use.These chemical compounds can produce unique cytokine/chemotactic factor spectrum (cytokine/chemokine profile) effectively by the immunne response of TLR9 mediation.
The association area general introduction
According to the subgroup difference of the cell that relates in replying, immunne response comprises congenital and adaptability is replied.For example, classical cell-mediated function, auxiliary (Th) cell of T that for example relates in delayed hypersensitivity and cytotoxic T lymphocyte (CTL) activation is the Th1 cell; And the Th cell that serves as B cell activation accessory cell is the Th2 cell.The type of immunne response is subjected to the influence of the cytokine of generation in response to the antigen contact.Difference by the Th1 and the cytokine of Th2 emiocytosis may be that these two kinds of different biological functions of subgroup are caused.
The Th1 cell participates in congenital reply of health to antigen (for example viral infection, intracellular pathogen and tumor cell).The result causes the secretion of IL-2 and IFN-γ and is accompanied by the activation of CTL.Known Th2 cellular response activates in antibacterial and parasite, can mediate the adaptive immune response (for example generation of IgE and eosinophilic granulocyte's activation) of health by secretion IL-4 and IL-5.
For example, in mammal, can induce the Th1 immunne response by the synthetic DNA of introducing antibacterial or containing non-methylated CpG dinucleotide, this immunne response is that specific oligonucleotide sequence (for example non-methylated CpG) is the result who passs the receptor on the certain immune cells, and described receptor is called pattern recognition receptor (PRRs).Some are Toll-sample receptors (TLRs) among these PRR certain.
Toll-sample receptor (TLRs) has been replied related closely with innate immunity.In vertebrates, known family's identification pathogen associated molecule that comprises the ten kinds of proteic Toll-of being called sample receptors (TLRs) is graphic.Among these ten kinds of albumen, known TLR3,7,8 and 9 is positioned in the intracellular endosome, and identification nucleic acid (DNA and RNA) and micromolecule are as nucleoside and nucleic acid metabolism thing.Known TLR3 and TLR9 discern nucleic acid such as dsRNA and the non-methylated CpG dinucleotide that is present in viral DNA and DNA of bacteria and the synthetic DNA respectively.DNA of bacteria activated immune system and anti-tumor activity (Tokunaga T et al., J.Natl.Cancer Inst. (1984) 72:955-962 have been shown; Shimada S, et al., Jpn.H cancer Res, 1986,77,808-816; Yamamoto S, et al., Jpn.J.Cancer Res., 1986,79,866-73).Use contain the CpG dinucleotide antisense oligonucleotide other research shown stimulated immunne response (Zhao Q, et al., Biochem.Pharmacol.1996,26,173-82).The follow-up TLR9 identification that studies show that is present in non-methylated CpG motif (the Hemmi H in DNA of bacteria and the synthetic DNA, Takeuchi O, Kawai T, Kaisho T, Sato S, Sanjo H, Matsumoto M, Hoshino K, Wagner H, Takeda K, Akira S.A Toll-like receptor recognizesbacterial DNA.Nature. (2000); 408:740-5).To other of the thiophosphate oligonucleotide that contains CpG modify also can influence they by TLR9 as the ability of immune response modifier performance function (referring to, Zhao et al. for example, Biochem.Pharmacol. (1996) 51:173-182; Zhao et al., Biochem Pharmacol. (1996) 52:1537-1544; Zhao et al., Antisense Nucleic AcidDrug Dev. (1997) 7:495-502; Zhao et al., Bioorg.Med.Chem.Lett. (1999) 9:3453-3458; Zhao et al., Bioorg.Med.Chem.Lett. (2000) 10:1051-1054; Yu etal., Bioorg.Med.Chem.Lett. (2000) 10:2585-2588; Yu et al., Bioorg.Med.Chem.Lett. (2001) 11:2263-2267; With Kandimalla et al., Bioorg.Med.Chem. (2001) 9:807-813).In addition, multiple synthetic motif and new structure have been identified by structure-activity relationship research, completely different [Kandimalla ER, Bhagat L that their inductive specific immune response spectrums (profile) and non-methylated CpG dinucleotide produce based on DNA, Li Y, Yu D, Wang D, Cong YP, Song SS, Tang JX, Sullivan T, Agrawal S.Proc Natl Acad Sci U S is A.2005; 102:6925-30.Kandimalla ER, Bhagat L, Zhu FG, Yu D, Cong YP, Wang D, Tang JX, Tang JY, Knetter CF, Lien E, Agrawal S.Proc Natl Acad Sci U S is A.2003; 100:14303-8.Cong YP, Song SS, Bhagat L, Pandey RK, Yu D, KandimallaER, Agrawal S.Biochem Biophys Res Commun.2003; 310:1133-9.Kandimalla ER, Bhagat L, Cong YP, Pandey RK, Yu D, Zhao Q, Agrawal S.Biochem Biophys ResCommun.2003; 306:948-53.Kandimalla ER, Bhagat L, Wang D, Yu D, Zhu FG, Tang J, Wang H, Huang P, Zhang R, Agrawal S.Nucleic Acids Res.2003; 31:2393-400.Yu D, Kandimalla ER, Zhao Q, Bhagat L, Cong Y, Agrawal S.Bioorg Med Chem.2003; 11:459-64.Bhagat L, Zhu FG, Yu D, Tang J, Wang H, Kandimalla ER, Zhang R, Agrawal S.Biochem Biophys Res Commun.2003; 300:853-61.Yu D, Kandimalla ER, Bhagat L, Tang JY, Cong Y, Tang J, Agrawal S.Nucleic Acids Res.2002; 30:4460-9.Yu D, Kandimalla ER, Cong Y, Tang J, Tang JY, Zhao Q, Agrawal S.J Med Chem.2002; 45:4540-8.Yu D, Zhu FG, Bhagat L, Wang H, Kandimalla ER, Zhang R, Agrawal S.Biochem Biophys ResCommun.2002; 297:83-90.Kandimalla ER, Bhagat L, Yu D, Cong Y, Tang J, Agrawal S.Bioconjug Chem.2002; 13:966-74.Yu D, Kandimalla ER, Zhao Q, Cong Y, Agrawal S.Nucleic Acids Res.2002; 30:1613-9.Yu D, Kandimalla ER, Zhao Q, Cong Y, Agrawal S.Bioorg Med Chem.2001; 9:2803-8.Yu D, KandimallaER, Zhao Q, Cong Y, Agrawal S.Bioorg Med Chem Lett.2001; 11:2263-7.Kandimalla ER, Yu D, Zhao Q, Agrawal S.Bioorg Med Chem.2001; 9:807-13.YuD, Zhao Q, Kandimalla ER, Agrawal S.Bioorg Med Chem Lett.2000; 10:2585-8, Putta MR, Zhu F, Li Y, Bhagat L, Cong Y, Kandimalla ER, Agrawal S.NucleicAcids Res.2006,34:3231-8].In addition, other modification to the thiophosphate oligonucleotide that contains CpG also can influence the ability that they play a role as immune response modifier.Referring to, Zhaoet al. for example, Biochem.Pharmacol. (1996) 51:173-182; Zhao et al., Biochem Pharmacol. (1996) 52:1537-1544; Zhao et al., Antisense Nucleic Acid Drug Dev. (1997) 7:495-502; Zhao et al., Bioorg.Med.Chem.Lett. (1999) 9:3453-3458; Zhao et al., Bioorg.Med.Chem.Lett. (2000) 10:1051-1054; Yu et al., Bioorg.Med.Chem.Lett. (2000) 10:2585-2588; Yu et al., Bioorg.Med.Chem.Lett. (2001) 11:2263-2267; With Kandimalla et al., Bioorg.Med.Chem. (2001) 9:807-813.
Oligonucleotide and oligodeoxynucleotide use in multiple field widely, include but not limited to that diagnosis and detection, PCR cause, the Antisense Suppression of gene expression, siRNA, fit, ribozyme and based on the immunotherapeutic agent of Toll-sample receptor (TLR ' s).Recently, many open files have been proved the purposes of oligodeoxynucleotide as immunomodulator, and in the immunization therapy of multiple disease such as allergy, asthma, autoimmune, cancer and infectious disease is used, use them separately or with they purposes as adjuvant.
These reports show still need create the new chemical entities that can produce unique immunne response.Yet the new chemical entities that creation can produce unique cytokine/chemokine mediated property immunne response and be identified as the TLR9 part is still a difficult problem.Consider that from ideal angle this difficult problem can be tackled by such means: the unique chemical group is introduced new chemical entities, thereby produce new immunotherapeutic agent, and after using, produce unique cytokine/the chemotactic factor spectrum.
The invention summary
The invention provides new chemical entities and be used to produce the purposes of unique cytokine/chemokine mediated property immunne response with them.New chemical entities can be used for regulating the immunne response that the oligonucleotide chemical compound causes.The method according to this invention can be modified cytokine/chemotactic factor spectrum of the immune regulative oligonucleotide generation that is used for the immunization therapy application.The inventor finds unexpectedly, makes the immunne response spectrum of generation have motility to the modification of immunomodulating dinucleotide.
Aspect first, the invention provides and comprise the immune regulative oligonucleotide that general formula is the immunostimulation dinucleotide of CG, wherein C is cytosine, 2 '-deoxidation cytosine, N
3-methyl-dC, dF or Ψ-different-dC, G are guanosine, 2 '-deoxyguanosine or N
1-methyl-dG; Condition is that G is N when C is cytosine or 2 '-deoxidation cytosine
1-methyl-dG; Further condition is that C is N when G is guanosine or 2 '-deoxyguanosine
3-methyl-dC, dF or Ψ-different-dC.
Aspect second, the invention provides pharmaceutical composition.These compositionss comprise disclosed component and pharmaceutically suitable carrier in first aspect of the present invention.
Aspect the 3rd, the invention provides the method that is used for producing immunne response vertebrates, described method comprises uses immune regulative oligonucleotide according to first or second aspect of the present invention to vertebrates.
Aspect the 4th, the invention provides be used for the treatment of suffer from cancer, the vertebrate method of disease that autoimmune disorder, airway inflammation, inflammatory disease, skin disorder, allergy, asthma or pathogen cause, this method comprises uses immune regulative oligonucleotide according to first or second aspect of the present invention to the patient.
Aspect the 5th, the invention provides the method for the disease that prophylaxis of cancer, autoimmune disorder, airway inflammation, inflammatory disease, skin disorder, allergy, asthma or pathogen cause in vertebrates, this method comprises uses immune regulative oligonucleotide according to first or second aspect of the present invention to vertebrates.
The accompanying drawing summary
Fig. 1 has described the linear synthetic one group of typical micromolecule joint that is suitable for immune regulative oligonucleotide of the present invention.
Fig. 2 has described the parallel synthetic one group of typical micromolecule joint that is suitable for immune regulative oligonucleotide of the present invention.
Fig. 3 is used for the linear synthetic synthetic schemes of immune regulative oligonucleotide of the present invention.DMTr=4,4 '-dimethoxytrityl; The CE=cyanoethyl.
Fig. 4 is used for the parallel synthetic synthetic schemes of immune regulative oligonucleotide of the present invention.DMTr=4,4 '-dimethoxytrityl; The CE=cyanoethyl.
Fig. 5 A-5D is presented at and uses after the immune regulative oligonucleotide of the present invention IL-12 in C57BL/6 mouse boosting cell culture and IL-6 level.More briefly, Fig. 5 A-5D shows that using the immune regulative oligonucleotide that contains new base can produce unique IL-12 and IL-6 spectrum.
Fig. 6 A and 6B are presented at and use after the immune regulative oligonucleotide of the present invention IL-6 in human PBMC's culture and IL-10 level.More briefly, Fig. 6 A-6B shows that using the immune regulative oligonucleotide that contains new base can produce unique IL-6 and IL-10 spectrum.
Fig. 7 is presented at and uses the immune regulative oligonucleotide of the present invention activation of TLR9 in the HEK293 cell afterwards, and activation is measured with the NF-kB activity of these cells.More briefly, Fig. 7 shows that using the immune regulative oligonucleotide that contains new base can produce unique TLR9 activation spectrum.
Fig. 8 after subcutaneous (s.c.) uses immune regulative oligonucleotide of the present invention, the IL-12 level in the C57BL/6 mice.More briefly, Fig. 8 shows that using the immune regulative oligonucleotide that contains new base in the body can produce unique IL-12 spectrum.
Fig. 9 is presented at and uses after the immune regulative oligonucleotide of the present invention the weight of C57BL/6 mice spleen.More briefly, Fig. 9 shows that using the immune regulative oligonucleotide that contains new base in the body can produce unique immunne response spectrum.
Figure 10 A-10D is presented at and uses after the immune regulative oligonucleotide of the present invention, the IL-5 in the mouse boosting cell of OVA sensitization, IL-12, IL-13 and IFN-γ level.More briefly, even Figure 10 A-10D shows in the presence of immune system activator (for example ovalbumin), use the immune regulative oligonucleotide that contains new base and still produce unique cytokine/chemotactic factor spectrum, its amount and base composition (base composition) according to the oligonucleotide of using changes.
Figure 11 shows that working concentration is the immune regulative oligonucleotide of 10 μ g/ml and the HEK293 cell that control compound activates the expression mouse TLR 9.Figure 11 shows that more briefly using the immune regulative oligonucleotide that contains new base can produce unique TLR9 activation spectrum.
Figure 12 A-12B shows in C57BL/6 mouse boosting cell culture, excretory the inducing of the immune regulative oligonucleotide pair cell factor of the present invention.Figure 12 A-12B shows that more briefly using the immune regulative oligonucleotide that contains new base can produce unique IL-6 and IL-12 scattergram, and its amount and base composition according to the oligonucleotide of using changes.
Figure 13 A and 13B show 72 hour the splenomegaly (Figure 13 A) of animal after accepting immune regulative oligonucleotide, control compound or the PBS of subcutaneous administration, and secrete (Figure 13 B) by the inductive IL-12 of immune regulative oligonucleotide after subcutaneous administration.More briefly, Figure 13 A and 13B show that using the immune regulative oligonucleotide that contains new base in the body can produce unique immunne response spectrum.
Figure 14 shows by the inductive human B cell propagation of immune regulative oligonucleotide.More briefly, Figure 14 shows that using the immune regulative oligonucleotide that contains new base can produce unique cell proliferation spectrum, and its amount and base composition according to the oligonucleotide of using changes.
Numerous embodiments describes in detail
The present invention relates to oligonucleotide and be used for the therapeutic use that immunization therapy is used as immunomodulator.This paper incorporates them into this paper by carrying stating by carrying the full content of stating granted patent, patent application and the list of references incorporating this paper into and quote as writing exactly particularly and individually.Under the inconsistent situation of any instruction of any document of quoting herein and this description, description should preferentially be used for purpose of the present invention.
The invention provides the method for the immunne response that the enhance immunity irritant compound causes, described immune-stimulating compound is used for immunization therapy and uses, for example, but be not limited to, in the mankind of adult and germling and the treatment of cancer, autoimmune disorder, asthma, respiratory system allergy, food anaphylaxis and antibacterial, parasite and viral infection in veterinary's application.Therefore, the present invention also provides the chemical compound that immunization therapy is had best immunostimulation level, and the method for preparing and use these chemical compounds.In addition, chemical compound of the present invention can be used as adjuvant and dna vaccination, antibody and allergen coupling; And and chemotherapeutics and/or antisense oligonucleotide coupling.
Aspect first, the invention provides the immune regulative oligonucleotide, it comprises the immunomodulating dinucleotide that at least one general formula is CG, and wherein C is cytosine, 2 '-deoxidation cytosine, N
3-methyl-dC, dF or Ψ-different-dC, G are guanosine, 2 '-deoxyguanosine, 2 '-deoxidation-7-denitrogenation guanosine, arabinose guanosine (arabinoguanosine) or N
1-methyl-dG; Condition is that G is N when C is cytosine or 2 '-deoxidation cytosine
1-methyl-dG; Further condition is that C is N when G is guanosine or 2 '-deoxyguanosine
3-methyl-dC, dF or Ψ-different-dC.
In an embodiment aspect this, the invention provides immune regulative oligonucleotide independent or that comprise at least two oligonucleotide, described at least two oligonucleotide they 3 ' end place or nucleoside between connect (internucleoside linkage) and locate or functionalized nuclear base place or sugar place is connected with the non-nucleotide joint, wherein at least one oligonucleotide be the immune regulative oligonucleotide and have accessible 5 ' hold.Can have identical nucleotide sequence or can have different nucleotide sequences by non-nucleotide joint oligonucleotide connected to one another, condition is that wherein at least one oligonucleotide contains at least one immunomodulating dinucleotide of the present invention.
Term used herein " accessible 5 ' end " is meant that 5 of oligonucleotide ' end is fully available, and the factor of identification and oligonucleotide binding and stimulating immune system can be near it.In oligonucleotide with accessible 5 ' end, 5 ' OH position of terminal sugar not with surpass two nucleoside residues or other disturbs with the interactional module of 5 ' end covalently bound arbitrarily.5 ' OH can randomly be connected in phosphate ester, thiophosphate, and perhaps phosphorodithioate module, aromatic series or aliphatic joint, cholesterol, perhaps other does not disturb the entity of accessibility.
For the present invention, the meaning of term " immunostimulatory oligonucleotide " or " immune regulative oligonucleotide " is the chemical compound that comprises at least one immunomodulating dinucleotide, and described chemical compound does not then have the immunomodulating effect if do not contain this immunomodulating dinucleotide." immunomodulating dinucleotide " be have general formula 5 '-dinucleotide of CpG-3 ', wherein ＂ C ＂ is naturally occurring pyrimidine nucleoside or its synthesis of derivatives in the mammal, ＂ G ＂ is naturally occurring purine nucleosides or its synthesis of derivatives in the mammal.Immune regulative oligonucleotide of the present invention can have an immunomodulating dinucleotide or several immunomodulating dinucleotides.For example, each immune regulative oligonucleotide can have 2,3,4 or more a plurality of immunomodulating dinucleotide, and these immunomodulating dinucleotides are identical, perhaps can accept modification as described herein independently.
Term ＂ CpG ＂ and ＂ CpG dinucleotide ＂ refer to dinucleotide 5 '-deoxycytidine-deoxyguanosine-3 ', wherein p connects between nucleoside, includes but not limited to that di-phosphate ester, thiophosphate and phosphorodithioate connect.
For the present invention, term " oligonucleotide " is meant the polymerized nucleoside (polynucleoside) that the nucleoside unit by a large amount of connections forms.These oligonucleotide can comprise that genome or cDNA source obtains, but preferably produce by synthetic method from existing nucleic acid source.In some embodiments, each nucleoside unit comprise heterocyclic base and furan pentose base (pentofuranosyl), trehalose, arabinose, 2 '-deoxidation-2 '-replace arabinose, 2 '-O-replaces arabinose or hexose glycosyl group.The nucleoside residue can be by connecting coupling each other between a lot of any known nucleoside.Connect between these nucleoside and include but not limited to di-phosphate ester, thiophosphate, phosphorodithioate, alkyl phosphate, alkyl thio-phosphonate (alkylphosphonothioate), phosphotriester, phosphoramidate, siloxanes, carbonic ester, alkoxy carbonyl group (carboalkoxy), acetyl aminate (acetamidate), carbamate, morpholino, borano, thioether, bridging phosphoramidate, the bridging methene phosphonate ester, the bridging thiophosphate, and connect between the sulfone nucleoside.Term " oligonucleotide " also comprises having connection (for example, (R between one or more three-dimensional special nucleoside
P)-or (S
P)-thiophosphate, alkyl phosphate or phosphotriester connect) polymerized nucleoside.Term used herein " oligonucleotide " and " dinucleotide " clearly are intended to comprise to have polymerized nucleoside and the dinucleotide that connects between this class nucleoside arbitrarily, and no matter whether this connection comprises phosphate.In specific embodiment, connecting between these nucleoside can be di-phosphate ester, and thiophosphate or phosphorodithioate connect, perhaps their combination.
In some embodiments, each oligonucleotide have from about 3 to about 35 nucleoside residues, perhaps from about 4 to about 30 nucleoside residues, perhaps from about 4 to about 18 nucleoside residues.In some embodiments, the immune regulative oligonucleotide comprises and has from about 1 to about 18, perhaps from about 1 to about 15, perhaps from about 5 oligonucleotide to about 14 nucleoside residues.Term used herein " approximately " expression exact number is not crucial.Therefore, the number of nucleoside residue is not crucial in the oligonucleotide, the oligonucleotide of few 1 or 2 nucleoside residue, and perhaps many 1 oligonucleotide to several nucleoside residues are considered the equivalent of each above-mentioned embodiment.In some embodiments, one or more in the oligonucleotide have 11 nucleotide or 18 nucleotide.In the linguistic context of immune regulative oligonucleotide, specific implementations have from about 13 to about 35 nucleotide, perhaps from about 13 to about 26 nucleotide, perhaps from about 11 to about 22 nucleotide.
Term " oligonucleotide " also comprises having other substituent polymerized nucleoside, and described other substituent group includes but not limited to, albumen base, lipophilic groups, intercalating agent, diamidogen, folic acid, cholesterol and diamantane (obsolete).Term " oligonucleotide " comprises that also other comprises the polymer of examining base arbitrarily, the oligonucleotide that includes but not limited to peptide nucleic acid(PNA) (PNA), has peptide nucleic acid(PNA) (PHONA), the morpholino-skeleton oligonucleotide of phosphate group and have the skeleton part that comprises alkyl joint or amino joint.
Oligonucleotide of the present invention can comprise naturally occurring nucleoside, the nucleoside of modification or its mixture.Term used herein " nucleoside of modification " is meant the heterocyclic base that comprises modification, the sugared module of modification or the nucleoside of their combination.In some embodiments, the nucleoside of modification is non-natural pyrimidine or a purine nucleosides as described herein.In some embodiments, the nucleoside of modification be 2 '-replace ribonucleotide, arabinose nucleoside or 2 '-deoxidation-2 '-replacement-galactoside.
For the present invention, term " 2 '-replace ribonucleotide " or " 2 '-replace galactoside " comprise such ribonucleotide or arabinose nucleoside, wherein the hydroxyl of 2 of the pentose module ' position be substituted and produce 2 '-replace or 2 '-ribonucleotide that O-replaces.These replacements are with the low alkyl group that comprises the saturated or undersaturated carbon atom of 1-6; perhaps replace with aryl with 6-10 carbon atom; wherein this alkyl or aryl can be unsubstituted; perhaps can replace, for example with halogen, hydroxyl, trifluoromethyl, cyano group, nitro, acyl group, acyloxy, alkoxyl, carboxyl, alkoxy carbonyl group or amino the replacement.2 '-O-replace ribonucleotide or 2 '-example of O-replacement-galactoside includes but not limited to 2 '-O-methylribonucleotide or 2 '-O-methyl galactoside and 2 '-O-methoxyethyl ribonucleotide or 2 '-O-methoxyethyl galactoside.
Term " 2 '-replace ribonucleotide " or " 2 '-replace galactoside " also comprise such ribonucleotide or arabinose nucleoside, wherein 2 '-hydroxyl is replaced by the low alkyl group that comprises 1-6 saturated or unsaturated carbon atom, perhaps amino or halogen group.The example of this class 2 '-replacement ribonucleotide or 2 '-replacement galactoside includes but not limited to, 2 '-amino, 2 '-fluoro, 2 '-pi-allyl and 2 '-propargyl ribonucleotide or galactoside.
Term " oligonucleotide " comprises hybridization oligonucleotide and chimeric oligonucleotide." chimeric oligonucleotide " is meant to have and surpasses the oligonucleotide that connects between one type nucleoside.An example of this chimeric oligonucleotide is to comprise thiophosphate, di-phosphate ester or phosphorodithioate zone and nonionic to connect the chimeric oligonucleotide that alkyl phosphate for example or alkyl thio-phosphonate (alkylphosphonothioate) connect (referring to for example, people's United States Patent (USP)s 5 such as Pederson, 635,377 and 5,366,878).
" hybridization oligonucleotide " is the oligonucleotide with the nucleoside that surpasses a type.An example of this hybridization oligonucleotide comprise ribonucleotide or 2 '-replace the ribonucleotide acid region, and the deoxyribonucleotide zone (referring to, for example, Metelev and Agrawal, United States Patent (USP) 5,652,355,6,346,614 and 6,143,881).
For the present invention, term " immunostimulatory oligonucleotide " or " immune regulative oligonucleotide " are meant the aforesaid oligonucleotide of such class, when it being applied to vertebrates for example when fish, poultry or mammal, its adjusting (for example inducing) immunne response.Term used herein " mammal " includes but not limited to rat, mice, cat, Canis familiaris L., horse, cattle (cattle), milch cow (cows), pig, rabbit, non-human primate and people.
For the present invention, " natural " nucleoside is meant the nucleoside that comprises one of five kinds of bases (for example, adenosine, guanosine, thymidine, cytosine and uridnine) of being prevalent among DNA or the RNA, and it has deoxyribose or ribose.For the present invention, " modification " or " non-natural " nucleoside is meant and comprises the modified natural nucleoside that has base and/or modified natural existence sugar module.The modified natural example of base that exists includes but not limited to those compositions that general formula I or general formula I I represent.For the present invention, " the similar thing of dinucleotide " is aforesaid immunostimulation dinucleotide, and wherein pyrimidine and/or purine nucleosides are non-natural nucleosides.Term ＂ C
*PG ＂ and ＂ CpG
*＂ refers to comprise the similar thing of immunostimulating dinucleotide of cytidine analog (non-natural pyrimidine nucleoside) or guanosine analogue (non-natural purine nucleosides) respectively.
Under different situations, dinucleotide is expressed as R ' pG, C
*PG or YZ, in these cases, R ', C
*Or Y represents synthetic or non-natural pyrimidine respectively, for example, but is not limited to N
3(that is, Ψ-different-dC) and deoxidation furyl glycosyl are (that is, dF) for-methyl-dC, vacation-different-deoxycytidine (pseudo-iso-deoxycytodine).In other cases, dinucleotide is expressed as CpR, CpG
*Or YZ, in these cases, R, G
*Or Z represents synthetic purine respectively, for example, but is not limited to N
1-methyl-dG or 7-denitrogenation-dG.Term used herein " pyrimidine nucleoside " refers to that the base composition in the nucleoside is the nucleoside of monocycle nuclear base.Similarly, term " purine nucleosides " refers to that the base composition in the nucleoside is the nucleoside of two nucleolus bases.For the present invention, " synthesize " pyrimidine or purine bases, the sugared module of non-natural existence or their combination that pyrimidine or purine nucleosides comprise that non-natural exists.
Pyrimidine nucleoside of the present invention has structure (I):
Wherein:
D is a hydrogen bond donor;
D ' is selected from hydrogen, hydrogen bond donor, hydrogen bond receptor, hydrophilic group, hydrophobic group, electron withdraw group and electron donating group;
D and D ' can be the parts of 5 yuan of rings or 6 yuan of rings;
A is nitrogen or hetero atom, replaces or unsubstituted hetero atom;
A ' is selected from hydrogen bond receptor, hydrophilic group, hydrophobic group, electron withdraw group and electron donating group;
A ＂ is carbon or nitrogen
X is carbon or nitrogen; With
S ' is pentose or hexose sugar ring, or the sugar of non-natural existence.
In some embodiments, sugar ring is by the phosphate ester module of phosphate ester module, modification or be suitable for pyrimidine nucleoside is connected to other joint module institute derivatization of another nucleoside or nucleoside analog.
Hydrogen bond donor includes but not limited to ,-NH-,-NH
2,-SH and-OH.Hydrogen bond receptor includes but not limited to, the theheterocyclic nitrogen atom of C=O, C=S and aromatic heterocycle, for example, the N3 of cytosine.
In some embodiments, the naturally occurring pyrimidine bases of base module right and wrong in (I).The example of the pyrimidine bases that non-natural exists includes, but not limited to 5-hydroxyl cytosine, 5-hydroxymethyl cytosine, N
3-methyl-dC, vacation-different-deoxycytidine (that is Ψ-different-dC); Deoxidation furyl glycosyl (that is, dF), 4-thiouracil and N4-alkyl cytosine, for example N4-ethyl cytosine.But in some embodiments, 5-bromine cytosine is left out especially.
In some embodiments, the sugared module S ' in (I) is modified naturally occurring sugared module.For the present invention, " naturally occurring sugared module " is the natural sugared module that exists as a nucleic acid part, for example, ribose and 2 '-deoxyribose, and " modified naturally occurring sugared module " is meant that not being natural exists as a nucleic acid part, but any sugar that can be used for the skeleton of oligonucleotide, for example, hexose.Arabinose and arabinose derivant are the examples of sugared module.
Purine nucleoside analogs of the present invention has structure (II):
Wherein:
D is nitrogen or hetero atom, replaces or unsubstituted hetero atom;
D ' is selected from hydrogen, hydrogen bond donor and hydrophilic group;
A is hydrogen bond receptor or hydrophilic group;
X is carbon or nitrogen;
Each L is the atom that independently is selected from C, O, N and S; With
S ' is pentose or hexose sugar ring, the perhaps sugar of non-natural existence.
In some embodiments, sugar ring is by the phosphate ester module, and the phosphate ester module of modification perhaps is suitable for pyrimidine nucleoside is connected to other joint module institute derivatizations of another nucleoside or nucleoside analog.
Hydrogen bond donor includes but not limited to ,-NH-,-NH
2,-SH and-OH.Hydrogen bond receptor include, but not limited to C=O, C=S ,-NO
2With the theheterocyclic nitrogen atom of aromatic heterocycle, for example, the N1 of guanine.
In some embodiments, the naturally occurring purine bases of base module right and wrong in (II).The example of the purine bases that non-natural exists includes, but not limited to 2-amino-6-thio-purine, 7-denitrogenation guanosine, N
1-methyl-dG and 2-amino-6-oxo-7-deazapurine.In some embodiments, (II) the sugared module S ' in is being naturally occurring sugared module or modified naturally occurring sugared module, as above faces the description of structure (I).
In some embodiments, the immunostimulation dinucleotide is selected from C
*PG, CpG
*And C
*PG
*, wherein the base of C is a cytosine, C
*Base be thymus pyrimidine, 5-hydroxyl cytosine, N
3-methyl-dC, N4-alkyl cytosine, vacation-different-deoxycytidine; Deoxidation furyl glycosyl, 4-thiouracil or other non-natural pyrimidine, or 2-oxo-7 denitrogenations-8-methyl purine, wherein when base is 2-oxo-7-denitrogenation-8-methyl purine, it preferably 1 by base and 1 of pentose '-covalent bond; The base of G is a guanosine, G
*Base be 2-amino-6-oxo-7-deazapurine, 2-oxo-7-denitrogenation-8-methyl purine, 6-thioguanine, 7-denitrogenation guanosine, inosine, N
1-methyl-dG, 6-oxo purine or other non-natural purine nucleosides, p are to be selected between the nucleoside of di-phosphate ester, thiophosphate and phosphorodithioate to connect, and condition is that at least one C or G are not respectively cytosine or guanosine.
The immune regulative oligonucleotide can comprise the immunostimulation module in the one or both sides of immunostimulation dinucleotide.Therefore, in some embodiments, immunostimulatory oligonucleotide comprises the immunostimulation territory of structure (III):
5’-Nn-N1-Y-Z-N1-Nn-3’(III)
Wherein:
The base of Y is cytosine, thymus pyrimidine, 5-hydroxyl cytosine, N4-alkyl-cytosine, N
3-methyl-cytosine, Ψ-different-dC, dF, 4-thiouracil or other non-natural pyrimidine nucleoside, or 2-oxo-7-denitrogenation-8-methyl-purine, wherein when base is 2-oxo-7-denitrogenation-8-methyl-purine, it is 1 by base and 1 of pentose ' position covalent bond preferably;
The base of Z is guanine, 2-amino-6-oxo-7-deazapurine, 2-oxo-7-denitrogenation-8-methyl purine, 2-amino-6-sulfo--purine, 7-denitrogenation guanosine, N
1-methyl-dG, 6-oxo purine or other non-natural purine nucleosides;
N1 and Nn independently, are preferably the non-natural or synthetic nucleoside of natural existence or the immunostimulation module that are selected from down group: no base (abasic) nucleoside, N when occurring at every turn
3-methyl-dC, N
1-methyl-dG, arabinose nucleoside, 2 '-BrdU, α-dezyribonucleoside, β-L-dezyribonucleoside and by connecting the nucleoside that is connected with the adjacent nucleoside of 3 ' side between the nucleoside of di-phosphate ester or modification, connect between the nucleotide of modifying and be selected from, but be not limited to, the joint of length from about 2 dusts to about 200 dusts, C2-C18 alkyl joint, poly-(ethylene glycol) joint, the amino butyl-1 of 2-, the ammediol joint, the glyceryl joint connects between 2 '-5 ' nucleoside, and thiophosphate, phosphorodithioate, or connect between the methyl phosphonate nucleoside;
Condition is that at least one N1 or Nn randomly are the immunostimulation modules;
Further condition is that at least one Y or Z are not respectively cytosine or guanosine;
Wherein n is from 0 to 30 numeral; With
Wherein the nuclear base of joint or derivatization or sugar directly are connected with another oligonucleotide or connect by the non-nucleotide joint between 3 ' end, nucleoside, and this oligonucleotide can be can not be immunostimulating also.
In some embodiments, YZ is cytosine, Ψ-different-dC, dF or N
3-methyl-dC and guanosine or N
1-methyl-dG.The immunostimulation module comprises natural phosphodiester skeleton and the modification in the phosphate ester skeleton, comprise, but be not limited to, methyl phosphonate, the methyl Thiophosphonate, phosphotriester, phosphoric acid sulfo-three esters (phosphothiotriesters), thiophosphate, phosphorodithioate, three ester prodrugs, sulfone, sulfonamides, sulfamate, dimethoxym ethane (formacetal), the N-methyl hydroxylamine, carbonic ester, carbamate, morpholino, boranophosphonate, phosphoramidate, primary amino radical-phosphoramidate particularly, N3 phosphoramidate and N5 phosphoramidate, with stereospecific connection (for example, (R
P)-or (S
P)-thiophosphate, phosphonate ester or phosphotriester connect).
In some embodiments, immunostimulatory oligonucleotide of the present invention also comprises having sugar-modified nucleoside, comprise, but be not limited to, 2 '-pentose that replaces, include but not limited to, 2 '-O-methylribose, 2 '-O-methoxyethyl ribose, 2 '-O-propargyl ribose and 2 '-deoxidation-2 '-fluoro ribose; 3 '-pentose that replaces, include, but not limited to 3 '-the O-methylribose; 1 ', 2 '-bi-deoxyribose; Arabinose; The arabinose that replaces includes, but not limited to the arabinose of 1 '-methyl arabinose, 3 '-methylol arabinose, 4 '-methylol arabinose, 3 '-hydroxyl arabinose and 2 '-replacement; Hexose includes, but not limited to 1, the 5-dewatering hexitol; And α-anomer (anomers).Modifying sugar and be 3 '-dezyribonucleoside or 3 '-O-replaces in the embodiment of ribonucleotide, and the immunostimulation module is connected to adjacent nucleoside by connection between 2 '-5 ' nucleoside.
In some embodiments, immunostimulatory oligonucleotide of the present invention also comprises the oligonucleotide with other saccharide backbone modification and replacement, the oligonucleotide that comprises peptide nucleic acid(PNA) (PNA), morpholino skeleton oligonucleotide and have the skeleton joint part of length from about 2 dusts to about 200 dusts, described joint includes but not limited to, alkyl joint or amino joint.The alkyl joint can be a branch branching or branchiess, replaces or unsubstituted and chiral purity or racemic mixture.In some embodiments, this class alkyl joint have from about 2 to about 18 carbon atoms.This class alkyl joint has from about 3 to 9 carbon atoms in some embodiments.Some alkyl joints comprise one or more functional groups that are selected from hydroxyl, amino, mercaptan, thioether, ether, amide, thioamides, ester, urea and thioether.Some these class functionalised alkyl joints are formula-O-(CH
2-CH
2-O-)
n(n=1-9) poly-(ethylene glycol) joint or the glycerol shown in.Some other functionalized alkyl joints are peptide or aminoacid.
In some embodiments, immunostimulatory oligonucleotide of the present invention also comprises the DNA hypotype, includes, but not limited to β-L-dezyribonucleoside and α-dezyribonucleoside.In some embodiments, immunostimulatory oligonucleotide of the present invention comprises that 3 ' modifies, and comprise and have coupled position between non-natural nucleoside (include but not limited to, 2 '-5 ', 2 '-2 ', 3 '-3 ' and 5 '-5 ' connection) nucleoside.
In some embodiments, immunostimulatory oligonucleotide of the present invention also comprises having the nucleoside of modifying heterocyclic base, comprise, but be not limited to 5-hydroxyl cytosine, 5-hydroxymethyl cytosine, 4-thiouracil, 6-thioguanine, 7-deazaguanine, inosine, nitro-pyrrole, C5-propinyl pyrimidine, N4-alkyl cytosine such as N4-ethyl cytosine, and diaminopurine, comprise, but be not limited to, 2, the 6-diaminopurine.
As specifying rather than limiting, for example, in immunostimulation territory with structure (III), connecting between the methyl phosphonate nucleoside of position N1 or Nn is the immunostimulation module, having the joint of about 2 dusts to about 200 angstrom lengths---the C2-C18 alkyl joint of position X1 is the immunostimulation module, and the β of position X1-L-dezyribonucleoside is the immunostimulation module.The exemplary position of immunostimulation module and structure are referring to following table 1.Be understood that, claim that a certain joint is the immunostimulation module of a certain ad-hoc location, the nucleoside residue that is meant this position its 3 '-the hydroxyl place is replaced by the joint of indication, thereby connects between the nucleoside that produces modification between the adjacent nucleoside of this nucleoside residue and its 3 ' side.Similarly, claim that connection is the immunostimulation module of a certain specific location between a certain modified nucleoside, be meant that the nucleoside residue of this position is connected with the adjacent nucleoside of 3 ' side by described connection.
Table 1
| The position | Typical immunostimulation module |
| N1 | Naturally occurring nucleoside, alkali-free yl nucleosides, N 3-methyl-dC, N 1-methyl-dG, arabinose nucleoside, 2 '-BrdU, β-L-dezyribonucleoside, C2-C18 alkyl joint, poly-(ethylene glycol) connect, the amino butyl-1 of 2-, and connect between ammediol joint (amino joint), 2 '-5 ' nucleoside, connect between the methyl phosphonate nucleoside |
| Nn | Naturally occurring nucleoside, alkali-free yl nucleosides, N 3-methyl-dC, N 1-methyl-dG, arabinose nucleoside, 2 '-BrdU, 2 '-O-replaces between ribonucleotide, 2 '-5 ' nucleoside and connects, methylphosphonic acid |
| Connect between the ester nucleoside, condition is that N1 and N2 can not be that no base connects |
Table 2 shows to have exemplary position and the structure that the upstream strengthens the interior immunostimulation module of immune regulative oligonucleotide in territory.Term used herein " spacer groups 9 " is meant suc as formula-O-(CH
2CH
2-O)
n-shown in poly-(ethylene glycol) joint, wherein n is 3.Term " spacer groups 18 " is meant suc as formula-O-(CH
2CH
2-O)
n-shown in poly-(ethylene glycol) joint, wherein n is 6.Term used herein " C2-C18 alkyl joint " is meant suc as formula-O-(CH
2)
qJoint shown in the-O-, wherein q is from 2 to 18 integer.Therefore, term " C3-joint " and " C3-alkyl joint " are meant that chemical formula is-O-(CH
2)
3The joint of-O-, it can be to replace or unsubstituted, divides branching or branchiess (for example 1,2,3, glycerol).For in spacer groups 9, spacer groups 18 and the C2-C18 alkyl joint each, joint connects with adjacent nucleoside by di-phosphate ester, thiophosphate or phosphorodithioate and is connected.
Table 2
| The position | Typical case's immunostimulation module |
| 5’N2 | Naturally occurring nucleoside, the amino butyl-1 of 2-, the ammediol joint |
| 5’N1 | Naturally occurring nucleoside, β-L-dezyribonucleoside, C2-C18 alkyl joint, poly-(ethylene glycol), no base joint, the amino butyl-1 of 2-, ammediol joint |
| 3’N1 | Naturally occurring nucleoside, 1 ', 2 '-bi-deoxyribose, 2 '-O-methyl-ribonucleotide, C2-C18 alkyl joint, spacer groups 9, spacer groups 18 |
| 3’N2 | Naturally occurring nucleoside, 1 ', 2 '-bi-deoxyribose, 3 '-connect between dezyribonucleoside, β-L-dezyribonucleoside, 2 '-O-propargyl-ribonucleotide, C2-C18 alkyl joint, spacer groups 9, spacer groups 18, methyl phosphonate nucleoside |
| 3’N3 | Naturally occurring nucleoside, 1 ', 2 '-connect between bi-deoxyribose, C2-C18 alkyl joint, spacer groups 9, spacer groups 18, methyl phosphonate nucleoside, connections between 2 '-5 ' nucleoside, d (G) n, poly-I-gather C |
| 3’N2+3’N3 | 1 ', 2 '-bi-deoxyribose, β-L-dezyribonucleoside, C2-C18 alkyl joint, d (G) n, the poly-C of poly-I- |
| 3’N3+3’N4 | 2 '-O-methoxyethyl-ribonucleotide, methyl phosphonate nucleoside between connection, d (G) n, the poly-C of poly-I- |
| 3’N5+3’N6 | 1 ', 2 '-bi-deoxyribose, C2-C18 alkyl joint, d (G) n, the poly-C of poly-I- |
| 5’N1+3’N3 | 1 ', 2 '-bi-deoxyribose, d (G) n, the poly-C of poly-I- |
Table 3 shows to have exemplary position and the structure that the downstream strengthens the interior immunostimulation module of immune regulative oligonucleotide in territory.
Table 3
| The position | Typical case's immunostimulation module |
| 5’N2 | Connect between the methyl phosphonate nucleoside |
| 5’N1 | Connect between the methyl phosphonate nucleoside |
| 3’N1 | Connection, 2 '-O-methyl between 1 ', 2 '-bi-deoxyribose, methyl phosphonate nucleoside |
| 3’N2 | 1 ', 2 '-bi-deoxyribose, β-L-dezyribonucleoside, C2-C18 alkyl joint, |
| 3’N3 | 3 '-dezyribonucleoside, 3 '-O-replacement ribonucleotide, 2 '-O-propargyl-ribonucleotide |
| 3’N2+3’N3 | 1 ', 2 '-bi-deoxyribose, β-L-dezyribonucleoside |
Immune regulative oligonucleotide of the present invention comprises at least two oligonucleotide, and they are junction or functionalized nuclear base place or sugared locating by the connection of non-nucleotide joint between its 3 ' end or nucleoside.For the present invention, " non-nucleotide joint " is meant the operational blocks which partition system that can be connected with oligonucleotide by key covalently or non-covalently.This class joint length from about 2 dusts to about 200 dusts.Hereinafter listed the example of several joints.Non-covalent connection includes, but not limited to electrostatic interaction, hydrophobic interaction, pi accumulation interaction and hydrogen bonding.Term " non-nucleotide joint " is not to be used to refer between the nucleoside of 3 ' hydroxyl of two nucleoside of aforesaid direct connection to connect, for example di-phosphate ester, thiophosphate or phosphorodithioate functional group.For the present invention, this direct 3 '-3 ' connection (not having joint to participate in) is considered to " nucleotide connection ".
In some embodiments, the non-nucleotide joint is a metal, includes but not limited to gold grain.In some other embodiments, the non-nucleotide joint is solubility or insoluble Biodegradable polymeric pearl.
In other embodiments, the non-nucleotide joint is the organic module with the functional group that allows to attach to oligonucleotide.Described attached be by any stable covalent bond.As non-limiting instance, joint can attach to any suitable location on the nucleoside.In some embodiments, joint attach to 3 '-hydroxyl.In this class embodiment, joint comprises hydroxy functional group, this hydroxy functional group by attach to 3 based on di-phosphate ester, thiophosphate, phosphorodithioate or non-connection (non-phosphate-basedlinkage) based on phosphate ester '-hydroxyl.
In some embodiments, the non-nucleotide joint is a biomolecule, includes, but not limited to polypeptide, antibody, lipoid, antigen, allergen and oligosaccharide.In some other embodiments, the non-nucleotide joint is a micromolecule.For the present invention, micromolecule be molecular weight less than 1, organic module of 000Da.In some embodiments, micromolecular molecular weight is less than 750Da.
In some embodiments, micromolecule is aliphatic hydrocarbon or aromatic hydrocarbons, described aliphatic hydrocarbon or aromatic hydrocarbons can randomly comprise one or more functional groups that are selected from hydroxyl, amino, mercaptan, thioether, ether, amide, thioamides, ester, urea and thiourea, described functional group or be arranged in the straight chain that connects oligonucleotide, perhaps thereon attached.Micromolecule can be a ring-type or acyclic.The example of micromolecule joint includes, but not limited to aminoacid, sugar, cyclodextrin, diamantane (obsolete), cholesterol, hapten and antibiotic.But for describing the purpose of non-nucleotide joint, term " micromolecule " is intended to not comprise nucleoside.
In some embodiments, the micromolecule joint is suc as formula HO-(CH
2)
o-CH (OH)-(CH
2)
pGlycerol shown in the-OH or glycerol homologue, wherein o and p are the integer from 1 to about 6, from 1 to about 4 or from 1 to about 3 independently.In some other embodiments, the micromolecule joint is 1, the derivant of 3-diaminourea-2-hydroxy propane.In this analog derivative some have chemical formula HO-(CH
2)
m-C (O) NH-CH
2-CH (OH)-CH
2-NHC (O)-(CH
2)
m-OH, wherein m is the integer between 0 to about 10,0 to about 6,2 to about 6 or 2 to about 4.
Non-nucleotide joints more of the present invention are allowed and are surpassed the attached of two oligonucleotide.For example, micromolecule joint glycerol has 3 and can supply the attached hydroxyl of oligonucleotide covalency.Therefore, immune regulative oligonucleotide more of the present invention comprise and surpass two oligonucleotide, and they are connected with the non-nucleotide joint at its 3 ' end.
Utilize automatization's synthesizer and phosphoramidite method (shown in the sketch map of Fig. 3 and 4, further describing in an embodiment), can synthesize immune regulative oligonucleotide of the present invention easily.In some embodiments, by the synthetic immune regulative oligonucleotide (referring to Fig. 3) of linear synthetic method.Term used herein " linear synthetic " is meant the end from the immune regulative oligonucleotide, and linear advancement is synthetic to the other end.Linear synthetic the permission at the monomeric unit that mixes identical or different (with regard to length, base composition and/or the chemical modification of mixing) in the immune regulative oligonucleotide.
Another kind of selective synthesis mode is " parallel synthetic ", wherein synthesizes and outwards carries out (referring to Fig. 4) from central joint module.The joint that is attached on the solid support can be used for parallel synthesizing, and as United States Patent (USP) 5,912,332 is described.In addition, can use general solid support (for example attached controllable bore diameter glass of phosphate ester).
The parallel synthetic of immune regulative oligonucleotide has several advantages with respect to linearity is synthetic: (1) parallel synthetic allow mix identical monomeric unit; (2) with linear synthetic different, all monomeric units all are synthetic simultaneously, thus the number of synthesis step and the synthetic time that needs with synthesize the identical of a monomeric unit; (3) minimizing of synthesis step increases the purity of final immune regulative oligonucleotide product and output.
When or end of synthesis that parallel synthetic rules carry out synthetic,, can utilize dense ammonia solution easily or the immune regulative oligonucleotide be carried out deprotection according to phosphoramidite supplier's recommendation if mixed modified nucleoside according to linearity.Can utilize the reversed-phase HPLC purification to product immune regulative oligonucleotide, detritylation, desalination and dialysis.
Table 4 shows the typical immune regulative oligonucleotide of the present invention.
The example of table 4A. immune regulative oligonucleotide sequence
| SEQ?ID?NO. | Sequence and |
| 1 | 5’-CTATCTGAC 1GTTCTCTGT-3’ |
| 2 | 5’-CTATCTGACG 1TTCTCTGT-3’ |
| 3 | 5’-CTATCTGTC 1GTTCTCTGT-3’ |
| 4 | 5’-CTATCTGTCG 1TTCTCTGT-3’ |
| 5 | 5’-CTATCTGAGC 1TTCTCTGT-3’ |
| 6 | 5’-CTATCTGAG 1CTTCTCTGT-3’ |
| 7 | 5’-TCTGAC 1GTTCT-X-TCTTGC 1AGTCT-5’ |
| 8 | 5’-TCTGACG 1TTCT-X-TCTTG 1CAGTCT-5’ |
| 9 | 5’-TCTGTC 1GTTCT-X-TCTTGC 1TGTCT-5’ |
| 10 | 5’-TCTGTCG 1TTCT-X-TCTTG 1CTGTCT-5’ |
| 11 | 5’-TCTGAGC 1TTCT-X-TCTTC 1GAGTCT-5’ |
| 12 | 5’-TCTGAG 1CTTCT-X-TCTTCG 1AGTCT-5’ |
| 13 | 5’-CTATCTGACGTTCTCTGT-3’ |
| 14 | 5’-CTATCTGTCGTTCTCTGT-3’ |
| 15 | 5 '-CTATCTCACCTTCTCTG-3 ' (contrast) |
| 16 | 5’-TCTGACGTTCT-X-TCTTGCAGTCT-5’ |
| 17 | 5’-TCTGACG 2TTCT-X-TCTTG 2CAGTCT-5’ |
| 18 | 5 '-TCTCACCTTCT-X-TCTTCCACTCT-5 ' (contrast) |
| 19 | 5 '-ACACACCAACT-X-TCAACCACACA-5 ' (contrast) |
| 20 | 5’-TCTGTCG 2TTCT-X-TCTTG 2CTGTCT-5’ |
| 21 | 5’-TCTGACGTTCT-X-TCTTGCAGTCT-5’ |
| 22 | 5’-TCTGAC 2GTTCT-X-TCTTGC 2AGTCT-5’ |
| 23 | 5’-TCTGAC 3GTTCT-X-TCTTGC 3AGTCT-5’ |
| 24 | 5’-TCTGAGC 2TTCT-X-TCTTC 2GAGTCT-5 ' (contrast) |
| 25 | 5’-TCTGAGC 3TTCT-X-TCTTC 3GAGTCT-5 ' (contrast) |
| 26 | 5’-TCTGTCGTTCT-X-TCTTGCTGTCT-5’ |
| 27 | 5’-TCTGTC 3GTTCT-X-TCTTGC 3TGTCT-5’ |
| 28 | 5’-TCTGTC 2GTTCT-X-TCTTGC 2TGTCT-5’ |
| 29 | 5 '-ACACACCAACT-X-TCAACCACACA-5 ' (contrast) |
| 30 | 5’-TC 3G 2AAC 3G 3TTC 3G 3-X-G 2C 3TTG 3C 3AAG 2C 3T-5’ |
| 31 | 5’-TC 4G 2AAC 4G 3TTC 4G 2-X-G 2C 4TTG 3C 4AAG 2C 4T-5’ |
| 32 | 5’-TC 3G 2AAC 3G 2TTCG 2-Y-TCTTG 3C 3TGTCT-5’ |
| 33 | 5’-TC 4G 2AAC 4G 2TTC 4G 2-Y-TCTTG 3C 4TGTCT-5’ |
C
1=N
3-methyl-dC; C
2=dF; C
3=Ψ-different-dC; C
4=1-(2 '-deoxidation-β-D-ribofuranosyl)-2-oxo-7-denitrogenation-8-methyl purine; G
1=N
1-methyl-dG; G
2=7-denitrogenation-dG; G
3=arabinose guanosine; X=glycerol joint; The Y=C3 joint
The specific embodiment of this aspect of the present invention provides and comprises the immune regulative oligonucleotide conjugate of immunostimulatory oligonucleotide and chemical compound as mentioned above, and described chemical compound and this immunostimulatory oligonucleotide are in the position coupling that is different from accessible 5 ' end.In some embodiments, chemical compound and the coupling of non-nucleotide joint.In other the embodiment, chemical compound and oligonucleotide are in the position coupling that is different from its 5 ' end at some.Can comprise with the link coupled suitable combination thing of immune regulative oligonucleotide of the present invention, but be not limited to, the Polyethylene Glycol of cholesterol, different length, peptide, antibody, albumen, vaccine, lipoid, antigen and immunostimulation micromolecule arbitrarily, for example, but be not limited to imiquimod (imiquimod), R848, loxoribine (loxoribine), Ai Shatuolibin (isatorbin) and chemotherapeutics.
Antigen includes, but not limited to the antigen relevant with pathogen, the antigen relevant with cancer, the antigen relevant with autoimmune disorder and and other diseases, such as but not limited to the relevant antigen of veterinary or pediatric disease.In some embodiments, antigen produces vaccine effect.For the present invention, term " with ... relevant " represent when pathogen, cancer, autoimmune disorder, food anaphylaxis, respiratory allergies, asthma or other disease exist, antigen also exists, but when pathogen, cancer, autoimmune disorder, food anaphylaxis, respiratory allergies or disease did not exist, antigen did not exist or decrement exists.
Immunostimulatory oligonucleotide and antigen covalency connect, and perhaps it otherwise operationally combines (operatively associated) with antigen.Term used herein " with ... operationally in conjunction with " be meant and keep immunostimulatory oligonucleotide and the two active any combination (association) of antigen.The limiting examples of like this " can operate in conjunction with " comprise constitute same liposome or other this type of deliver carrier or reagent part (being part of the same liposome or other such delivery vehicle orreagent).Covalently bound in antigenic embodiment at immunostimulatory oligonucleotide, this class covalency connects the optional position that is preferably placed on the immunostimulatory oligonucleotide, except the accessible 5 ' end of immunostimulatory oligonucleotide.For example, antigen can attach to connect between nucleoside and maybe can be connected to the non-nucleotide joint.In addition, antigen itself can be the non-nucleotide joint.
Aspect second, the invention provides the pharmaceutical preparation that comprises immune regulative oligonucleotide of the present invention or immune regulative oligonucleotide conjugate and physiologically acceptable carrier.Term used herein " physiologically acceptable " is meant that material does not disturb the effectiveness of immune regulative oligonucleotide, and with biosystem for example cell, cell culture, tissue or biocompatible.Preferably, biosystem is the biology of living, for example vertebrates.
Term used herein " carrier " comprises any excipient, diluent, filler, salt, buffer agent, stabilizing agent, solubilizing agent, lipoid or other material that is used for pharmaceutical preparation well known in the art.The character that should be appreciated that carrier, excipient or diluent will depend on the route of administration at concrete application.The preparation that comprises the pharmaceutically acceptable preparation of these materials is described in, for example, and Remington ' sPharmaceutical Sciences, 18th Edition, ed.A.Gennaro, Mack Publishing Co., Easton, PA, 1990.
Aspect the 3rd, the invention provides the method that in vertebrates, produces immunne response, this method comprises uses immune regulative oligonucleotide of the present invention or immune regulative oligonucleotide conjugate to vertebrates.In some embodiments, vertebrates is a mammal.For the present invention, term " mammal " is intended to comprise the people clearly.In specific embodiment, the immunostimulating vertebrates of needs is used described immune regulative oligonucleotide or immune regulative oligonucleotide conjugate.
In the method for this respect of the present invention, using of immune regulative oligonucleotide or immune regulative oligonucleotide conjugate can be by any suitable way, include but not limited in non-digestive tract, oral, Sublingual, percutaneous, part, mucosa, suction, intranasal, aerosol, ophthalmic, the trachea, internal rectum, vagina, by particle gun, transdermal patches or adopt eye drop or collutory form.Can use known program, carry out using of immune regulative oligonucleotide treatment compositions with the symptom of effective minimizing disease or the dosage and the time period of surrogate markers thing.When the whole body administration, the therapeutic combination of preferably using sufficient dosage is so that the blood levels of immune regulative oligonucleotide reaches from about 0.0001 micromoles per liter (micromolar) to about 10 micromoles per liter.For local application, also may be effectively than this much lower concentration, and much higher concentration also may tolerate.Preferably, the accumulated dose of immune regulative oligonucleotide is from each patient of about 0.001mg every day to the every kg body weight of about 200mg every day.May need simultaneously or continuously to one or more therapeutic combinations of the present invention of individual administering therapeutic effective dose as the single therapy paragraph.
In specific embodiment, co-administered specificity or the size of replying of immune regulative oligonucleotide of the present invention or immune regulative oligonucleotide conjugate and vaccine, antibody, cytotoxic agent, allergen, antibiotic, antisense oligonucleotide, peptide, albumen, gene therapy vector, dna vaccination and/or adjuvant with enhance immunity.In these embodiments, immune regulative oligonucleotide of the present invention can be used as adjuvant and plays a role by different way and/or produce direct immunostimulation.
Immune regulative oligonucleotide or immune regulative oligonucleotide conjugate and/or vaccine can randomly connect immunogenic protein, for example keyhole limpet hemocyanin (KLH), b subunit of cholera toxin or other immunogenic carrier albumen arbitrarily.Can use any kind in the multiple adjuvant, include but not limited to, Freund's complete adjuvant, KLH, single phosphinylidyne lipoid A (MPL), Alumen and saponin comprise QS-21, imiquimod, R848 or their combination.
For the present invention, term " with ... the associating " meaning is in the process of same disease of treatment same patient, comprise with random order and use immune regulative oligonucleotide and/or vaccine and/or adjuvant, comprise and use simultaneously and go up the order of separating (as many as was separated by several days) with the time and use.This therapeutic alliance can also comprise uses the immune regulative oligonucleotide, and/or uses vaccine independently, and/or uses adjuvant independently more than once.Immune regulative oligonucleotide and/or vaccine and/or adjuvant can be used by identical or different approach.
The method of this respect of the present invention can be used for immune scale-model investigation.This method also can be used for the prevention or the treatment of people or Animal diseases.For example, this method can be used for department of pediatrics usefulness and live vaccine is used.
The 4th aspect the invention provides the method that therapeutic treatment suffers from the patient of disease or disease, and this method comprises uses immune regulative oligonucleotide of the present invention or immune regulative oligonucleotide conjugate to the patient.In different embodiments, disease to be treated or disease are the diseases that cancer, autoimmune disorder, airway inflammation, inflammatory disease, allergy, asthma or pathogen cause.Pathogen comprises antibacterial, parasite, fungus, virus, viroid and Protein virus.Description according to third aspect of the present invention is used.
For the present invention, term " allergy " includes, but not limited to food allergy (food anaphylaxis) and respiratory system allergy.Term " airway inflammation " includes, but not limited to asthma.Term used herein " autoimmune disorder " is meant that " self " albumen suffers the disease of immune system attack.This term comprises autoimmune asthma.
Aspect the 5th, the invention provides the method that is used for prevent disease or disease, these methods comprise uses immune regulative oligonucleotide of the present invention or immune regulative oligonucleotide conjugate to the patient.In numerous embodiments, the disease that prevent or disease are the diseases that cancer, autoimmune disorder, airway inflammation, inflammatory disease, allergy, asthma or pathogen cause.Pathogen comprises antibacterial, parasite, fungus, virus, viroid and Protein virus.Description according to third aspect of the present invention is used.
In any means of the present invention aspect this, any other reagent of immunostimulation that immune regulative oligonucleotide or immune regulative oligonucleotide conjugate can not reduced the immune regulative oligonucleotide with being useful on treatment disease or disease is co-administered.In any means of the present invention, treatment disease or disease useful reagent are comprised, but be not limited to, vaccine, antigen, antibody, cytotoxic agent, allergen, antibiotic, antisense oligonucleotide, peptide, albumen, gene therapy vector, dna vaccination and/or the adjuvant of specificity that enhance immunity is replied or size, or costimulatory molecules for example cytokine, chemotactic factor, protein ligands, trans-activating factor, peptide and comprise the peptide of modified amino acid.For example, in treatment for cancer, expection can be co-administered with immune regulative oligonucleotide or immune regulative oligonucleotide conjugate and chemotherapy compound or monoclonal antibody.Perhaps, described reagent can comprise the dna vector of coding for antigens or allergen.In these embodiments, immune regulative oligonucleotide of the present invention can be used as adjuvant and plays a role by different way and/or produce direct immunoregulation effect.
The chemotherapeutics that uses in the method for the present invention includes, but are not limited to gemcitabine (Gemcitabine), methotrexate, vincristin, amycin, cisplatin, the chloroethylnitrosoureas that contains non-sugar, 5-fluorouracil, ametycin, bleomycin, doxorubicin (doxorubicin), dacarbazine (dacarbazine), paclitaxel, Fu Lajilin (fragyline), meglumine GLA (Meglamine GLA), valrubicin (valrubicin), carmustine (carmustaine) and polifeprosan (poliferposan), MMI270, BAY12-9566, the RAS farnesyl transferase inhibitor, farnesyl transferase inhibitor, MMP, MTA/LY231514, LY264618/ lometrexol (Lometexol), glatiramer comes (Glamolec), CI-994, TNP-470, with new (the Hycamtin)/hycamtin (Topotecan) of U.S., PKC412, valspodar (Valspodar)/PSC833, mitoxantrone (Novantrone/Mitroxantrone), suramin six sodium salts (Metaret)/suramin (Suramin), batimastat (Batimastat), E7070, BCH-4556, CS-682,9-AC, AG3340, AG3433, Incel/VX-710, VX-853, ZD0101, ISI641, ODN 698, TA 2516/ Ma Misita (Marmistat), BB2516/ Ma Misita (Marmistat), CDP 845, D2163, PD183805, DX8951f, Lemonal DP2202 (Lemonal DP 2202), FK317, Picibanil (Picibanil)/OK-432, AD 32/ valrubicin, strontium chloride (Metastron)/strontium derivant, Tai Dao (Temodal)/temozolomide (Temozolomide), Evacet (Evacet)/Mycocet, excellent his (Yewtaxan)/taxol (Placlitaxel) that looses, paclitaxel/taxol (Paclitaxel), xeloda (Xeload)/capecitabine (Capecitabine), fortulon (Furtulon)/doxifluridine (Doxifluridine), (Cyclopax)/oral taxol is sent in the Crow, west, oral taxane (Oral Taxoid), the SPU-077/ cisplatin, HMR 1275/ husband's degree of evening up (Flavopiridol), CP-358 (774)/EGFR, CP-609 (754)/RAS oncogene inhibitor, the oral platinum of BMS-182751/, UFT (ftorafur (Tegafur)/uracil), levamisole (Ergamisol)/levamisole (Levamisole), eniluracil (Eniluracil)/776C85/5FU reinforcing agent, Irinotecan (Campto)/levamisole, Camptosar (Camptosar)/Irinotecan (Irinotecan), the special plug of Tu Mode (Tumodex)/Rayleigh (Ralitrexed), cladibrine (Leustatin)/cladribine (Cladribine), the Paxex/ taxol, Doxil (Doxil)/Mycocet, pattern Lay (Caelyx)/Mycocet, fluorine reaches China (Fludara)/fludarabine (Fludarabine), epirubicin (Pharmarubicin)/epirubicin (Epirubicin), DepoCyt (DepoCyt), ZD1839,79553/ pair-naphthalimide of LU (Bis-Naphtalimide), LU 103793/ aplysiatoxin (Dolastain), pattern is for (Caetyx)/Mycocet, hydrochloride for injection gemcitabine (Gemzar)/gemcitabine (Gemcitabine), ZD0473/Anormed, YM 116, etodolac inoculation (lodine seeds), CDK4 and CDK2 inhibitor, the PARP inhibitor, D4809/Dexifosamide, Ifes/ Mesnex (Mesnex)/ifosfamide (Ifosamide), brave and fierce (Vumon)/Buddhist nun moors glycosides (Teniposide), carboplatin (Paraplatin)/carboplatin (Carboplatin), cisplatin (Plantinol)/cisplatin, etoposide (Vepeside)/etoposide (Etoposide), ZD9331, taxotere (Taxotere)/Docetaxel (Docetaxel), guanine arabinose glycosides prodrug, 10-deacetyltaxol, nitroso ureas, alkylating agent is melphalan (melphelan) and cyclophosphamide for example, aminoglutethimide (Aminoglutethimide), asparaginase, busulfan (Busulfan), carboplatin, chlorambucil (Chlorombucil), cytarabine hydrochloride (Cytarabine HCl), dactinomycin (Dactinomycin), daunorubicin hydrochloride (Daunorubicin HCl), estramustine phosphate (Estramustinephosphate sodium), etoposide (VP16-213), floxuridine (Floxuridine), fluorouracil (5-FU), flutamide (Flutamide), hydroxyurea (Hydroxyurea) (hydroxycarbamide (hydroxycarbamide)), ifosfamide (Ifosfamide), Intederon Alpha-2a, α-2b, leuprorelin acetate (Leuprolide acetate) (LHRH-releasing factor analogs), lomustine (Lomustine) (CCNU), hydrochloric acid chlormethine (Mechlorethamine HCl) (chlormethine), mercaptopurine, mesna (Mesna), mitotane (Mitotane) (mitotane (o.p '-DDD)), mitoxantrone hydrochloride (Mitoxantrone HCl), octreotide (Octreotide), plicamycin (Plicamycin), procarbazine hydrochloride (Procarbazine HCl), streptozocin (Streptozocin), Tamoxifen Citrate (Tamoxifencitrate), thioguanine, plug is for sending (Thiotepa), vinblastine (Vinblastine sulfate), amsacrine (Amsacrine) (amsacrine (m-AMSA)), azacitidine (Azacitidine), erythropoietin (Erthropoietin), altretamine (Hexamethylmelamine) (HMM), interleukin II, mitoguazone (Mitoguazone) (methyl-GAG; Methylglyoxal bis (methyl glyoxalbis-guanylhydrazone); MGBG), pentostatin (Pentostatin) (2 '-deoxycoformycin (2 ' deoxycoformycin)), semustine (Semustine) (Semustine), teniposide (Teniposide) (VM-26), vindesine sulfate (Vindesine sulfate), tyrosine kinase inhibitor, for example EGFR and VEGF inhibitor, include but not limited to, Lapatinib (Lapatinib) (the dual tyrosine kinase inhibitor of EGFR and ErbB-2 (Her2/neu) (GSK)), gefitinib (Gefitinib) (ZD1839/Iressa (AstraZeneca)), Ai Luo is for Buddhist nun (Erlotinib) (Ta Saiwa (Tarceva)-EGFR/HER1 inhibitor (Genentech)), Thalidomide (Thalidomide) ((Thalidomide)-anti-angiogenic drugs), imatinib (Imatinib) (Glivec) and Wa Talani (Vatalanib) (VEGFR tyrosine kinase inhibitor), Sorafenib (Sorafenib) (Raf inhibitors of kinases (Bayer)), VX-680 (Aurora inhibitors of kinases), Suo Tan (Sutent) (receptor tyrosine kinase (RTKs) inhibitor (Pfizer)), bortezomib (Bortezomib) ((Velcade) proteasome inhibitor), temozolomide (Temozolomide) (Tai Dao (Temodal) alkylating agent) and interferon-ALPHA (Recombinant Interferon (Intron A), Recomvinated Interferon (Roferon A)).
Antibody, the particularly passive immunotherapy of monoclonal antibody form are the theme of a large amount of research and development as anticarcinogen.Term used herein " monoclonal antibody " is meant single molecular antibody molecule.Monoclonal antibody is formed and is presented single binding specificity of defined epitope and affinity.Therefore, term " human monoclonal antibodies " refers to present the antibody of single binding specificity, and it has variable region and constant region that the ethnic group of being derived from is an immunoglobulin sequences.The example of anticarcinogen comprises, but be not limited to, 17-1A MAB (Panorex) (Glaxo-Welicome), B cell monoclonal antibody (Rituxan) (IDEC/Genentech/Hoffman laRoche), Mai Luota (Mylotarg) (Wyeth), bank Paasche (Campath) (Millennium), Ze Waling (Zevalin) (IDEC and Schering AG), hectogram sand (Bexxar) (Corixa/GSK), Erbitux (Erbitux) (Imclone/BMS), Avastin (Avastin) (Genentech), Trastuzumab (Herceptin) (Genentech/Hoffman la Roche), Cetuximab (Cetuximab) (Imclone) and handkerchief Buddhist nun monoclonal antibody (Panitumumab) (Abgenix/Amgen).Antibody also can be applicable in the active immunity treatment of utilizing anti-idiotype antibody, apparent imitation (on the immunology meaning) the cancer antigen of going up.Monoclonal antibody can prepare by the method known to the skilled in recombinant DNA technology field.
The following examples are intended to further specify specific implementations of the present invention, rather than intention limits scope of the present invention.
Embodiment
Embodiment 1: oligonucleotide synthetic that contains the immunostimulation module
Utilize automatization's dna synthesizer (OligoPilot II, AKTA, (Amersham) and/or Expedite8909 (Applied Biosystem)), according to Fig. 3 with 4 listed linearities are synthetic or parallel synthesis programs, with the scale synthetic oligonucleotide of 1 μ mol to 0.1mM.
5 '-DMT dA, dG, dC and T phosphoramidite available from Proligo (Boulder, CO).5 '-DMT 7-denitrogenation-dG and araG phosphoramidite available from Chemgenes (Wilmington, MA).DiDMT-glycerol joint solid support is available from Chemgenes.1-(2 '-deoxidation-β-D-ribofuranosyl)-2-oxo-7-denitrogenation-8-methyl-purine phosphoramidite (1-(2 '-deoxy-β-D-ribofuranosyl)-2-oxo-7-deaza-8-methyl-purine amidite) available from Glen Research (Sterling, VA), 2 '-O-methylribonucleotide phosphoramidite (2 '-O-methylribonuncleoside amidites) available from Promega (Obispo, CA).All oligonucleotide all are that phosphorothioate backbone is modified.
All nucleoside phosphoramidites are all passed through
31P and
1The H nuclear magnetic resoance spectrum characterizes.The conventional coupling that utilizes supplier to recommend circulates in the nucleoside that specific site mixes modification.After synthetic, utilize dense ammonium hydroxide that oligonucleotide is carried out deprotection, by the reversed-phase HPLC purification, detritylation is succeeded by dialysis then.The oligonucleotide of purification is before use with the sodium-salt form lyophilizing.By CGE and MALDI-TOF MS check purity.Level of endotoxin is measured by lal test and is lower than 1.0EU/mg.
Embodiment 2: the mouse boosting cell culture
Age in 4-8 week, C57BL/6 and BALB/c mouse be available from Taconic Farms, Germantown, and NY, and raise according to the animal rule of operation (Idera ' s IACUC-approved animalprotocol) of the IACUC permission of Idera.All zooscopies of this paper report all follow the IACUC principle of Idera and the rule of operation of permission is carried out.Preparation is cultivated in the RPMI complete medium from the splenocyte of 4-8 BALB/c in age in week or C57BL/6 mice.With mouse boosting cell with 5X10
6Cell/ml is seeded in the 24 hole culture dishs.In cell culture, add be dissolved in the TE buffer (10mM Tris-HCL, pH 7.5, IMOs 1mMEDTA) to final concentration be 0.03,0.1,0.3,1.0,3.0 or 10 μ g/ml.Then cell was hatched 24 hours at 37 ℃, collect supernatant and be used for elisa assay.
Level by IL-12 and IL-6 in the sandwich ELISA analysis supernatant.The results are shown in Fig. 5 A-5D.Required reagent comprises cytokine antibodies and standard substance, all available from BD Pharmingen.Succ-PEG-DSPE-peroxidase and substrate are from KPL.
Embodiment 3: the human PBMC separates
(Histopaque-1077, Sigma) (CBR Laboratories, Boston separate PERIPHERAL BLOOD MONONUCLEAR CELL (PBMCs) in MA) from healthy volunteer's blood of fresh collection to utilize the Ficoll density.
Embodiment 4: cytokine ELISA
With the human PBMC with 5X10
6Cell/ml is seeded in the 48 hole flat boards.In cell culture, add and be dissolved in DPBS (pH 7.4; Mediatech) IMOs to final concentration be 10 μ g/ml.Then cell was hatched 24 hours at 37 ℃, collect supernatant and be used for elisa assay.Experiment is carried out the repetition in the same form 3 holes.Level by IL-6 and IL-10 in the sandwich ELISA analysis supernatant.The results are shown in Fig. 6 A and 6B.Required reagent comprises cytokine antibodies and standard substance, all available from PharMingen.
Embodiment 5:HEK293 cell culture:
(Invivogen, San Diego CA) cultivate at 5%CO with the HEK293/mTLR9 cell
2In the 48 hole flat boards in the incubator, the DMEM of wherein every hole 250 μ l is added with 10% heat-inactivated FBS.
Embodiment 6: reporter gene transforms
When 80% converged, (Invitrogen was under condition CA), with Seap report plasmid (pNifty2-Seap) (the San Diego CA) instantaneous conversion of culture with 400ng/ml 4 μ l/ml lipofection reagent (Lipofectamine) in culture medium.In the culture medium that does not contain serum, dilute plasmid DNA and lipofection reagent respectively, and incubated at room 5 minutes.After hatching, the DNA of mixed diluting and lipofection reagent, again with mixture incubated at room 20 minutes.The 25 μ l DNA/ lipofection reagent mixtures that will contain 100ng plasmid DNA and 1 μ l lipofection reagent are added into each hole of cell culture flat board, continue to cultivate 4 hours.
Embodiment 7: the immune regulative oligonucleotide is handled
After the transfection, culture medium is replaced with fresh culture, individually add zest oligomer (stimulating oligo) immune regulative oligonucleotide, continue to cultivate 18 hours to all cultures.
Embodiment 8:SEAP analyzes
When the processing of oligomer immune regulative oligonucleotide finished, the culture supernatant of getting 30 μ l from each processing was used for the SEAP analysis.Rules (Invivogen) according to manufacturer are carried out described analysis.With microplate reader at the 405nm detection signal.The results are shown in Fig. 7, illustrated that the using of immune regulative oligonucleotide of containing new base produced unique TLR9 activation spectrum.
Embodiment 9: the assessment of mice serum cytokine levels
The female C57BL/6 mice in age in 5-6 week is available from Taconic Farms, Germantown, and NY raises according to the animal rule of operation of the IACUC permission of Idera Pharmaceutical.Give the independent immune regulative oligonucleotide of mice (n=2-3) subcutaneous injection with 25 or 100 μ g dosage or 1mg/kg (single dose).Collect serum by blood sampling behind the socket of the eye after use at the immune regulative oligonucleotide 4 hours, analyze IL-12 with sandwich ELISA.The results are shown in Fig. 8, illustrated that using the immune regulative oligonucleotide that contains new base in the body can produce unique IL-12 spectrum.All reagent comprise cytokine antibodies and standard substance, all available from PharMingen. (San Diego, CA).
Embodiment 10: the mouse boosting cell culture:
Preparation is from the splenocyte of C57BL/6 mice, cultivation is in the RPMI complete medium, (HyClone, Logan UT) form described culture medium by the RPMI 1640 that contains 10% hyclone (FCS), 100U/ml penicillin, 100 μ g/ml streptomycins and 2mM L-glutaminate.With mouse boosting cell with 5X10
6Cell/ml is seeded in the 24 hole flat boards.Adding independent immune regulative oligonucleotide to the final concentration that is dissolved in TE buffer [10mMTris-HCl (pH 7.5) and 1mM EDTA] in cell culture is 3 or 10 μ g/ml.Then cell was hatched 24 hours at 37 ℃, collect supernatant and be used for the cytokine analysis that undertaken by elisa (ELISAs).
By IL-12 and the IL-6 level in the sandwich ELISA measurement supernatant.Required reagent comprises cytokine antibodies and standard substance, all available from BD Pharmingen (San Diego, CA).Succ-PEG-DSPE-peroxidase and tmb substrate respectively from Sigma (St.Louis, MO) and KPL (Gaithersburg, MD).
Embodiment 11: the human B cell proliferation test:
From the about 1x10 of human PBMC s purification
5The B cell stimulated 64 hours with the immune regulative oligonucleotide of variable concentrations, use then 0.75 μ Ci [
3H]-thymidine impulse stimulation (pulse), results after 8 hours.Utilize scintillation counter to measure to mix [
3H]-thymidine, data are expressed as per minute reading (c.p.m.).
Embodiment 12: people's cell multiplex factor ELISA:
With human PBMC s with 5x10
6The concentration of cell/ml is seeded in the 96 hole flat boards.Adding immune regulative oligonucleotide to the final concentration that is dissolved in the phosphate buffered saline (PBS) to cell culture is 10 μ g/ml.Then cell was hatched 24 hours at 37 ℃.Re-use the listed cytokine in the multiple ELISA systematic analysis of the Luminex-supernatant.People's multiple reagent box is available from invitrogen.
Embodiment 13: mice spleen enlargement test
(4-6 week 19-21gm) is divided into three one group with female BALB/c mouse.The immune regulative oligonucleotide DNA is dissolved in aseptic PBS, and subcutaneous (SC) is administered to mice with the dosage of 5mg/kg.After 72 hours, put to death mice, the results spleen is also weighed.The results are shown in Fig. 9, illustrated that using the immune regulative oligonucleotide that contains new base in the body can produce unique immunne response spectrum.
The mouse boosting cell culture test of embodiment 14:OVA sensitization
The BALB/c female mice in age in 4-6 week available from Taconic (Germantown, NY).Gave injection and the blended 20 μ g chicken egg white (OVA that are dissolved among the 100 μ LPBS of 100 μ L ImjectAlum adjuvants (Pierce) in the mouse peritoneum at the 0th day, the 7th day; And carry out intranasal the 14th day and the 21st day with the 10 μ g OVA that are dissolved in 40 μ l PBS and attack (intranasally challenge) Sigma).Attack 72 hours afterwards the last time by sucking CO2 execution mice.
Spleen is downcut, prepare single cell suspension as mentioned above.Immune regulative oligonucleotide with variable concentrations was handled splenocyte 2 hours, succeeded by the processing of 100 μ g/mL OVA.
After 72 hours, collect supernatant, measure IL-5, IL-13, IL-12 and IFN-alpha levels as mentioned above by ELISA.The results are shown in Figure 10 A-10D, even illustrated in the presence of immune system activator (for example ovalbumin), the using of immune regulative oligonucleotide of containing new base also can produce unique cytokine/chemotactic factor spectrum, and its amount and base composition according to the oligonucleotide of using changes.
Embodiment 15: with the vivo antitumor activity of the immune regulative oligonucleotide of chemotherapeutics associating
Can in the presence of 100U/ml penicillin and 100 μ g/ml streptomycins,, cultivate the PC3 cell in the F12K culture medium, to set up human prostata cancer model (PC3) at the 90%Ham ' s that contains 10% hyclone (FBS).Before research, (Frederick Cancer Researchand Development Center, Frederick MD) adapt to 6 days to the environment adjustment can to make male athymic nude mice in 4-6 age in week.Can collect the PC3 cell of cultivating from the monolayer culture thing, use Ham ' s, F12K culture medium (10%FBS) washed twice, be resuspended in the Ham ' s that does not contain FBS, F12K culture medium: matrigel (Matrigel) basement membrane matrix (BectonDickinson Labware, Bedford, MA) (5:1; Volume/volume) in, subcutaneous injection (5X10
6Cell, cumulative volume 0.2ml) to the inguinal region of every mice.Can monitor routine clinical observation, body weight and the tumor growth of animal.Can come the monitoring tumor growth by two perpendicular diameter measuring implant with caliper.Can pass through formula 1/2aXb
2Calculate tumor quality (in the weight of gram), wherein ' a ' is long diameter (cm), and ' b ' is short diameter (cm).When the average tumor size reaches～80mg, the animal of carrier's cancer xenograft can be divided into randomly processed group and matched group (5 animal/groups).Matched group can only be accepted physiological saline solution (0.9%NaCl).Immune regulative oligonucleotide of the present invention sterilely can be dissolved in the normal saline, by the dosage of subcutaneous injection 0.5 or 1.0mg/kg/ days, agent is on every Wendesdays used.Can be by providing chemotherapeutics twice at the 0th day and the 3rd day peritoneal injection 160mg/kg.
Embodiment 16: oligonucleotide synthetic that contains the immune regulative module
Utilize β-cyanoethyl phosphoramidite chemical method, on PerSeptive Biosystem 8909 ExpediteDNA synthesizers, with the synthetic 2 '-deoxidation-pyridine [2 that contains of 2-μ mol scale, 3-d] pyrimidine-2,7 (8H)-diketone (2 '-deoxy-pyrido[2,3-d] pyrimidine-2,7 (8H)-dione) (dF) or 2 '-the false different cytidine of deoxidation (2 '-deoxypseudoisocytidine) (the immune regulative oligonucleotide of Ψ-different-dC) modify.Attach to CPG solid phase support body Di-DMT protection the glyceryl joint available from ChemGenes Corporation (Wilmington, MA).3 of dA, dG, dC and T '-phosphoramidite is available from Applied Biosystems, and the dmf-dG phosphoramidite available from Glen Research (Sterling, VA).The phosphoramidite of dF and Ψ-iso-dC is available from Berry ﹠amp; Associates (Dexter, MI).Use Beaucage reagent as oxidant to obtain the D2EHDTPA backbone modification.The synthetic schemes that uses supplier to recommend carries out dF and Ψ-different-dC phosphoramidite mixes and deprotection.After synthetic; with immune regulative oligonucleotide deprotection; by " band trityl (trityl on) " RP-HPLC purification; detritylation; again to having the flushing usefulness sterilized water (Braun of American Pharmacopeia (UnitedStates Pharmacopea) quality; Irvine CA) dialyses.With the lyophilization of immune regulative oligonucleotide, be dissolved in once more in the distilled water, measure concentration by the UV absorptance of measuring the 260nm place.The purity of all synthetic chemical compounds is measured by degeneration PAGE, and by the auxiliary mastrix-assisted laser desorption ionization time of flight of the substrate that is used for molecular mass (MALDI-TOF) mass spectral characteristi sequence integrity.Synthesized whole immune regulative oligonucleotide (table 4A), and under identical condition, carried out purification so that contaminated with endotoxins minimizes.
Embodiment 17: the immune regulative oligonucleotide activation TLR9 that contains dF or Ψ-different-dC in the CpG motif
With concentration is the immune regulative oligonucleotide of 10 μ g/ml and the HEK293 cell that control compound activates the expression mouse TLR 9.Research contains the ability of dF or the immune regulative oligonucleotide activation TLR9 that Ψ-different-dC modifies in the HEK293 of stably express mouse TLR 9 cell.Utilize people's secreted embryo alkali phosphatase (SEAP) gene as NF-κ B receptor.The result is expressed as with respect to PBS impinging upon the multiple (Figure 11) that increases on the NF-kB activation.Shown in the increase of NF-kB activity, the immune regulative oligonucleotide 27 and 28 (SEQ ID NO 27 and 28) (table 4A) that contains dF or Ψ-different-dC has activated TLR9.These presentation of results dF or Ψ-different-dC be modified at the C-position be toleration and have a function, further proof is used the immune regulative oligonucleotide that contains new base and can be produced unique TLR9 activation spectrum (Figure 11).
Embodiment 18: contain the cytokine secretion in the immune regulative oligonucleotide inducing mouse spleen cell cultures thing of dF or Ψ-different-dC in the CpG motif
Induce the cytokine secretion of C57BL/6 mouse boosting cell culture by IMO.The C57BL/6 mouse boosting cell in independent culture medium (M) or exist in the culture medium of variable concentrations immune regulative oligonucleotide and cultivated 24 hours, is measured the level of excretory IL-12 (Figure 12 A) and IL-6 (Figure 12 B) in the culture supernatant by ELISA.Data presented is when immune regulative oligonucleotide concentration is 3 and 10 μ g/ml (Figure 12 A and 12B).Compare (SEQ ID NO 29) with contrast immune regulative oligonucleotide 29, contain the secretion (Figure 12 A and 12B) that the immune regulative oligonucleotide 27 (SEQ ID NO 27) of dF or Ψ-different-dC and 28 (SEQ ID NO28) have induced IL-12 and IL-6 in the C57BL/6 mouse boosting cell culture.These presentation of results immunocytes are to containing the immune regulative oligonucleotide tolerance that dF or Ψ-different-dC modifies, and contain the immune regulative oligonucleotide that dF or Ψ-different-dC modifies immunocyte is had activity, further specify and use IL-6 and the IL-12 spectrum that the immune regulative oligonucleotide that contains new base can produce uniqueness, its amount and base composition according to the oligonucleotide of using changes.
Embodiment 19: the intravital splenomegaly of immune regulative oligonucleotide inducing mouse and the cytokine that contain dF or Ψ-different-dC in the CpG motif
Accepted the splenomegaly (Figure 13 A) in the C57BL/6 mice of immune regulative oligonucleotide, control compound or PBS of 5mg/kg dosage of subcutaneous administration.After using 72 hours, the immune regulative oligonucleotide measures the variation of spleen weight.After subcutaneous administration 1mg/kg dosage, the immune regulative oligonucleotide has been induced IL-12 (13.B) secretion in the C57BL/6 mice.After using, the immune regulative oligonucleotide collected blood in 4 hours, and by the IL-12 level in the ELISA mensuration serum.After using the CpG oligomer, the increase of mice spleen weight is measuring of immunoregulatory activity.Compare with the mice of accepting contrast immune regulative oligonucleotide 4 or 5, contain the mice of dF or Ψ-different-dC and the increase (Figure 13 A) that human specific immune regulative oligonucleotide all demonstrates spleen.Compare with the mice of having injected human specific immune regulative oligonucleotide 27 (SEQ ID NO 27) or 28 (SEQ ID NO 28), accepted to contain the mice specific immunity adjustment oligonucleotide 22 (SEQ ID NO 22) of dF or Ψ-different-dC or the mice of 23 (SEQIDNO23) and on spleen weight, produced bigger increase.These results also illustrate with having injected and contain immune regulative oligonucleotide 23 (SEQ ID NO 23) that Ψ-different-dC modifies or the mice of 27 (SEQ ID NO27) is compared, and accepted to contain the immune regulative oligonucleotide 22 (SEQ ID NO 22) that dF modifies or the mice of 28 (SEQ IDNO 28) and produced bigger increase respectively on spleen weight.After the immune regulative oligonucleotide is used 4 hours, induce the further check of spectrum to disclose to the cells in vivo factor and contain dF or Ψ-mice of different-dC modification and the rising (Figure 13 B) that human specific immune regulative oligonucleotide has all been induced IL-12 in the mice body.Seen in the splenomegaly test, (SEQ ID NO 23) compares with immune regulative oligonucleotide 23, and mice specific immunity adjustment oligonucleotide 22 (SEQ ID NO 22) has been induced the IL-12 of higher level.Two kinds of modifications of these presentation of results (dF or Ψ-different-dC) all tolerate, and all can activate TLR9, different but the level of immunne response has, use the immune regulative oligonucleotide that contains new base in the body and produced unique immunne response spectrum.
Embodiment 20: the inductive human B cell propagation of immune regulative oligonucleotide
Immune regulative oligonucleotide with variable concentrations stimulates from obtaining from the isolating human B cell of healthy people volunteer's PBMC, measures the 3H-thymidine by scinticounting and takes in (Figure 14).Figure 14 proof is used the immune regulative oligonucleotide that contains new base and has been produced unique cell proliferation spectrum, and its amount and base composition according to the oligonucleotide of using changes.
Embodiment 21: the cytokine/chemotactic factor of immune regulative oligonucleotide is induced
In human PBMC's cell culture, measure immune regulative oligonucleotide 26 (SEQ ID NO 26), 27 (SEQ ID NO 27), 28 (SEQ ID NO 28) or contrast immune regulative oligonucleotide 29 (SEQID NO 29) induce (table 5) to IL-2R, IL-6, IL-8, TNF-α, MIP-1 α, MIP-β and MCP-1.
Table 5
| SEQ?IDNO. | IL-2R(pg/ml) | IL-6(pg/ml) | TNF-a(pg/ml) | MIP-1a(pg/ml) | MIP-b(pg/ml) | MCP-1(pg/ml) | IL-8(pg/ml) |
| Culture medium | 96.05 | 13.66 | 14.48 | 34.18 | 191.42 | 18.51 | 125.28 |
| 26 | 178.21 | 523.42 | 165.00 | 115.41 | 1339.33 | 2036.87 | 1632.89 |
| 27 | 173.69 | 461.86 | 114.53 | 115.01 | 1225.94 | 406.18 | 3324.42 |
| 28 | 197.71 | 403.29 | 119.42 | 108.31 | 1121.74 | 443.07 | 3547.89 |
| 29 | 96.62 | 97.01 | 61.27 | 67.62 | 525.34 | 68.67 | 2769.96 |
Embodiment 22: the immune regulative oligonucleotide activation human PBMC and the B cell that contain dF or Ψ-different-dC in the CpG motif
Further check contains the immune regulative oligonucleotide activation human PBMC of dF or Ψ-different-dC and the ability that the inducing cell factor produces.In these trials, use immune regulative oligonucleotide 27 (SEQ ID NO 27) and 28 (SEQ ID NO 28) (Fig. 4 A) that contain the human specific motif.Compare (SEQ ID NO 29) with contrast 29, immune regulative oligonucleotide 27 (SEQ ID NO 27) and 28 (SEQ ID NO28) have all induced IL-2R, IL-6, IL-8, TNF-α, MIP-1 α, MIP-β and MCP-1 (table 5), two kinds of modifications of this explanation all tolerate, and all can activate people TLR9.29 (SEQ ID NO 29) compare with contrast immune regulative oligonucleotide, and the two has induced dose dependent B cell proliferation (Figure 14) immune regulative oligonucleotide 27 (SEQ ID NO 27) and 28 (SEQ ID NO 28).
Equivalent
Although for the purpose that is aware and understand, foregoing invention has been carried out detailed description to a certain degree, but should understand the various variations that do not break away from the present invention and appended claims essential scope making aspect form and the details by reading this paper those skilled in the art.
Claims (47)
1. immune regulative oligonucleotide, it comprises the immunomodulating dinucleotide that at least one general formula is CG, and wherein C is cytosine, 2 '-deoxidation cytosine, N
3-methyl-dC, dF or Ψ-different-dC, and G is guanosine, 2 '-deoxyguanosine, 2 '-deoxidation-7-denitrogenation guanosine, arabinose guanosine or N
1-methyl-dG;
Condition is that G is N when C is cytosine or 2 '-deoxidation cytosine
1-methyl-dG;
Further condition is that C is N when G is guanosine or 2 '-deoxyguanosine
3-methyl-dC, dF or Ψ-different-dC.
2. according to the immune regulative oligonucleotide of claim 1, it has following institute array structure:
5’-CTATCTGAC
1GTTCTCTGT-3’，
5’-CTATCTGACG
1TTCTCTGT-3’，
5’-CTATCTGTC
1GTTCTCTGT-3’，
5’-CTATCTGTCG
1TTCTCTGT-3’，
5’-TCTGAC
1GTTCT-X-TCTTGC
1AGTCT-5’，
5’-TCTGACG
1TTCT-X-TCTTG
1CAGTCT-5’，
5’-TCTGTC
1GTTCT-X-TCTTGC
1TGTCT-5’，
5’-TCTGTCG
1TTCT-X-TCTTG
1CTGTCT-5’；
5’-TCTGAC
2GTTCT-X-TCTTGC
2AGTCT-5’，
5’-TCTGAC
3GTTCT-X-TCTTGC
3AGTCT-5’，
5’-TCTGTC
3GTTCT-X-TCTTGC
3TGTCT-5’，
5 '-TC
3G
2AAC
3G
3TTC
3G
3-X-G
2C
3TTG
3C
3AAG
2C
3T-5 ' or
5’-TCTGTC
2GTTCT-X-TCTTGC
2TGTCT-5’；
C wherein
1=N
3-methyl-dC; C
2=dF; C
3=Ψ-different-dC, G
1=N
1-methyl-dG; And X=glycerol joint.
3. pharmaceutical preparation, it comprises the oligonucleotide and the physiologically acceptable carrier of claim 1.
4. method that is used for producing vertebrates immunne response, described method comprises the immune regulative oligonucleotide of vertebrates being used claim 1.
5. according to the method for claim 4, wherein route of administration be selected from non-digestive tract, oral, Sublingual, percutaneous, part, mucosa, suction, intranasal, aerosol, ophthalmic, the trachea, internal rectum, vagina, particle gun, transdermal patches, eye drop and collutory.
6. according to the method for claim 4, wherein said immune regulative oligonucleotide is selected from following listed:
5’-CTATCTGAC
1GTTCTCTGT-3’，
5’-CTATCTGACG
1TTCTCTGT-3’，
5’-CTATCTGTC
1GTTCTCTGT-3’，
5’-CTATCTGTCG
1TTCTCTGT-3’，
5’-TCTGAC
1GTTCT-X-TCTTGC
1AGTCT-5’，
5’-TCTGACG
1TTCT-X-TCTTG
1CAGTCT-5’，
5’-TCTGTC
1GTTCT-X-TCTTGC
1TGTCT-5’，
5’-TCTGTCG
1TTCT-X-TCTTG
1CTGTCT-5’；
5’-TCTGAC
2GTTCT-X-TCTTGC
2AGTCT-5’，
5’-TCTGAC
3GTTCT-X-TCTTGC
3AGTCT-5’，
5’-TCTGTC
3GTTCT-X-TCTTGC
3TGTCT-5’，
5 '-TC
3G
2AAC
3G
3TTC
3G
3-X-G
2C
3TTG
3C
3AAG
2C
3T-5 ' or
5’-TCTGTC
2GTTCT-X-TCTTGC
2TGTCT-5’；
C wherein
1=N
3-methyl-dC; C
2=dF; C
3=Ψ-different-dC, G
1=N
1-methyl-dG; And X=glycerol joint.
One kind be used for the treatment of suffer from cancer, the vertebrate method of disease that autoimmune disorder, airway inflammation, inflammatory disease, skin disorder, allergy, asthma or pathogen cause, this method comprises uses immunostimulatory oligonucleotide according to claim 1 to the patient.
8. according to the method for claim 7, wherein route of administration be selected from non-digestive tract, oral, Sublingual, percutaneous, part, intranasal, aerosol, ophthalmic, the trachea, internal rectum, vagina, particle gun, transdermal patches, eye drop and collutory.
9. according to the method for claim 7, wherein said immune regulative oligonucleotide is selected from following listed:
5’-CTATCTGAC
1GTTCTCTGT-3’，
5’-CTATCTGACG
1TTCTCTGT-3’，
5’-CTATCTGTC
1GTTCTCTGT-3’，
5’-CTATCTGTCG
1TTCTCTGT-3’，
5’-TCTGAC
1GTTCT-X-TCTTGC
1AGTCT-5’，
5’-TCTGACG
1TTCT-X-TCTTG
1CAGTCT-5’，
5’-TCTGTC
1GTTCT-X-TCTTGC
1TGTCT-5’，
5’-TCTGTCG
1TTCT-X-TCTTG
1CTGTCT-5’；
5’-TCTGAC
2GTTCT-X-TCTTGC
2AGTCT-5’，
5’-TCTGAC
3GTTCT-X-TCTTGC
3AGTCT-5’，
5’-TCTGTC
3GTTCT-X-TCTTGC
3TGTCT-5’，
5 '-TC
3G
2AAC
3G
3TTC
3G
3-X-G
2C
3TTG
3C
3AAG
2C
3T-5 ' or
5’-TCTGTC
2GTTCT-X-TCTTGC
2TGTCT-5’；
C wherein
1=N
3-methyl-dC; C
2=dF; C
3=Ψ-different-dC, G
1=N
1-methyl-dG; And X=glycerol joint.
10. method that is used for the disease that causes in vertebrates prophylaxis of cancer, autoimmune disorder, airway inflammation, inflammatory disease, skin disorder, allergy, asthma or pathogen, this method comprise uses immunostimulatory oligonucleotide according to claim 1 to vertebrates.
11. according to the method for claim 10, wherein route of administration be selected from non-digestive tract, oral, Sublingual, percutaneous, part, mucosa, suction, intranasal, aerosol, ophthalmic, the trachea, internal rectum, vagina, particle gun, transdermal patches, eye drop and collutory.
12. according to the method for claim 10, wherein said immune regulative oligonucleotide is selected from following listed:
5’-CTATCTGAC
1GTTCTCTGT-3’，
5’-CTATCTGACG
1TTCTCTGT-3’，
5’-CTATCTGTC
1GTTCTCTGT-3’，
5’-CTATCTGTCG
1TTCTCTGT-3’，
5’-TCTGAC
1GTTCT-X-TCTTGC
1AGTCT-5’，
5’-TCTGACG
1TTCT-X-TCTTG
1CAGTCT-5’，
5’-TCTGTC
1GTTCT-X-TCTTGC
1TGTCT-5’，
5’-TCTGTCG
1TTCT-X-TCTTG
1CTGTCT-5’；
5’-TCTGAC
2GTTCT-X-TCTTGC
2AGTCT-5’，
5’-TCTGAC
3GTTCT-X-TCTTGC
3AGTCT-5’，
5’-TCTGTC
3GTTCT-X-TCTTGC
3TGTCT-5’，
5 '-TC
3G
2AAC
3G
3TTC
3G
3-X-G
2C
3TTG
3C
3AAG
2C
3T-5 ' or
5’-TCTGTC
2GTTCT-X-TCTTGC
2TGTCT-5’；
C wherein
1=N
3-methyl-dC; C
2=dF; C
3=Ψ-different-dC, G
1=N
1-methyl-dG; And X=glycerol joint.
13., also comprise antibody, antisense oligonucleotide, albumen, antigen, allergen, chemotherapeutics or adjuvant according to the oligonucleotide of claim 1.
14., also comprise antibody, antisense oligonucleotide, albumen, antigen, allergen, chemotherapeutics or adjuvant according to the pharmaceutical composition of claim 3.
15., also comprise administration of antibodies, antisense oligonucleotide, albumen, antigen, allergen, chemotherapeutics or adjuvant according to the method for claim 4.
16., also comprise administration of antibodies, antisense oligonucleotide, albumen, antigen, allergen, chemotherapeutics or adjuvant according to the method for claim 7.
17., also comprise administration of antibodies, antisense oligonucleotide, albumen, antigen, allergen, chemotherapeutics or adjuvant according to the method for claim 10.
18. an immune regulative oligonucleotide chemical compound, it comprises general formula and is 5 '-pyrimidine-purine-3 ' the immunostimulation dinucleotide, wherein pyrimidine is N
3-methyl-dC, purine are purine nucleosides natural or that modify.
19. an immune regulative oligonucleotide chemical compound, it comprises general formula and is 5 '-pyrimidine-purine-3 ' the immunostimulation dinucleotide, wherein pyrimidine is a pyrimidine nucleoside natural or that modify, purine is N
1-methyl-dG.
20. a pharmaceutical preparation, it comprises according to the oligonucleotide of claim 18 and physiologically acceptable carrier.
21. a pharmaceutical preparation, it comprises according to the oligonucleotide of claim 19 and physiologically acceptable carrier.
22. a method that is used for producing vertebrates immunne response, described method comprise vertebrates is used immunostimulatory oligonucleotide according to claim 18.
23. a method that is used for producing vertebrates immunne response, described method comprise vertebrates is used immunostimulatory oligonucleotide according to claim 19.
24. one kind be used for the treatment of suffer from cancer, the vertebrate method of disease that autoimmune disorder, airway inflammation, inflammatory disease, skin disorder, allergy, asthma or pathogen cause, this method comprises uses immunostimulatory oligonucleotide according to claim 18 to the patient.
25. one kind be used for the treatment of suffer from cancer, the vertebrate method of disease that autoimmune disorder, airway inflammation, inflammatory disease, skin disorder, allergy, asthma or pathogen cause, this method comprises uses immunostimulatory oligonucleotide according to claim 19 to the patient.
26. a method that is used for the disease that causes in vertebrates prophylaxis of cancer, autoimmune disorder, airway inflammation, inflammatory disease, skin disorder, allergy, asthma or pathogen, this method comprise vertebrates is used immunostimulatory oligonucleotide according to claim 18.
27. a method that is used for the disease that causes in vertebrates prophylaxis of cancer, autoimmune disorder, airway inflammation, inflammatory disease, skin disorder, allergy, asthma or pathogen, this method comprise vertebrates is used immunostimulatory oligonucleotide according to claim 19.
28., also comprise antibody, antisense oligonucleotide, albumen, antigen, allergen, chemotherapeutics or adjuvant according to the oligonucleotide of claim 18.
29., also comprise antibody, antisense oligonucleotide, albumen, antigen, allergen, chemotherapeutics or adjuvant according to the pharmaceutical composition of claim 20.
30., also comprise administration of antibodies, antisense oligonucleotide, albumen, antigen, allergen, chemotherapeutics or adjuvant according to the method for claim 22.
31., also comprise administration of antibodies, antisense oligonucleotide, albumen, antigen, allergen, chemotherapeutics or adjuvant according to the method for claim 24.
32., also comprise administration of antibodies, antisense oligonucleotide, albumen, antigen, allergen, chemotherapeutics or adjuvant according to the method for claim 26.
33., also comprise antibody, antisense oligonucleotide, albumen, antigen, allergen, chemotherapeutics or adjuvant according to the oligonucleotide of claim 19.
34., also comprise antibody, antisense oligonucleotide, albumen, antigen, allergen, chemotherapeutics or adjuvant according to the pharmaceutical composition of claim 21.
35., also comprise administration of antibodies, antisense oligonucleotide, albumen, antigen, allergen, chemotherapeutics or adjuvant according to the method for claim 23.
36., also comprise administration of antibodies, antisense oligonucleotide, albumen, antigen, allergen, chemotherapeutics or adjuvant according to the method for claim 25.
37., also comprise administration of antibodies, antisense oligonucleotide, albumen, antigen, allergen, chemotherapeutics or adjuvant according to the method for claim 27.
38. an immunostimulatory oligonucleotide chemical compound, it comprises general formula and is 5 '-pyrimidine-purine-3 ' the immunostimulation dinucleotide, wherein pyrimidine is N
3-methyl-dC, purine are N
1-methyl-dG.
39. a pharmaceutical preparation, it comprises according to the oligonucleotide of claim 38 and physiologically acceptable carrier.
40. a method that is used for producing vertebrates immunne response, described method comprise vertebrates is used immunostimulatory oligonucleotide according to claim 38.
41. one kind be used for the treatment of suffer from cancer, the vertebrate method of disease that autoimmune disorder, airway inflammation, inflammatory disease, skin disorder, allergy, asthma or pathogen cause, this method comprises uses immunostimulatory oligonucleotide according to claim 38 to the patient.
42. a method that is used for the disease that causes in vertebrates prophylaxis of cancer, autoimmune disorder, airway inflammation, inflammatory disease, skin disorder, allergy, asthma or pathogen, this method comprise vertebrates is used immunostimulatory oligonucleotide according to claim 38.
43., also comprise antibody, antisense oligonucleotide, albumen, antigen, allergen, chemotherapeutics or adjuvant according to the oligonucleotide of claim 38.
44., also comprise antibody, antisense oligonucleotide, albumen, antigen, allergen, chemotherapeutics or adjuvant according to the pharmaceutical composition of claim 39.
45., also comprise administration of antibodies, antisense oligonucleotide, albumen, antigen, allergen, chemotherapeutics or adjuvant according to the method for claim 40.
46., also comprise administration of antibodies, antisense oligonucleotide, albumen, antigen, allergen, chemotherapeutics or adjuvant according to the method for claim 41.
47., also comprise administration of antibodies, antisense oligonucleotide, albumen, antigen, allergen, chemotherapeutics or adjuvant according to the method for claim 42.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US75233505P | 2005-12-20 | 2005-12-20 | |
| US60/752,335 | 2005-12-20 | ||
| US60/821,458 | 2006-08-04 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN101378776A true CN101378776A (en) | 2009-03-04 |
Family
ID=40421926
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNA2006800530586A Pending CN101378776A (en) | 2005-12-20 | 2006-12-19 | Novel synthetic agonists of TOll-like receptors containing CG dinucleotide modifications |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101378776A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105051194A (en) * | 2013-01-14 | 2015-11-11 | 萨勒普塔医疗公司 | Inhibitory oligonucleotides and their use in therapy |
-
2006
- 2006-12-19 CN CNA2006800530586A patent/CN101378776A/en active Pending
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105051194A (en) * | 2013-01-14 | 2015-11-11 | 萨勒普塔医疗公司 | Inhibitory oligonucleotides and their use in therapy |
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