CN1852917A - Synergisitic stimulation of the immune system using immunostimulatory oligonucleotides and/or immunomer compounds in conjunction with cytokines and/or chemotherapeutic agents or radiation therapy - Google Patents

Abstract

The invention provides optimized methods and compositions for enhancing the immune response caused by immunostimulatory compounds used for the treatment of disease such as, but not limited to, treatment of cancer, autoimmune disorders, asthma, respiratory allergies, food allergies and infectious diseases in a patient. The optimized methods according to the invention provide synergy between the therapeutic effects of immunostimulatory oligonucleotides and immunomer compounds in accordance with the invention, and the therapeutic effect of cytokine immunotherapy and/or chemotherapeutic agents and/or radiation.

Description

Use immunostimulatory oligonucleotide and/or immune sub-compound in conjunction with cytokine and/or chemotherapeutics or radiotherapy to immune collaborative hormesis

Related application

It is the U.S. Provisional Application No.60/487 on July 15th, 2003 that the application requires the applying date, 529, and the U.S. Provisional Application No.60/503 on September 15th, 2003,242 right of priority, it is incorporated herein by reference in full.

Background of invention

Invention field

The present invention relates to the application of immunity son (immunomer) compound and immunostimulatory oligonucleotide as therapeutical agent.

The correlation technique summary

Recently, some investigators are verified use oligonucleotide is as immunostimulant effectiveness of application in immunotherapy.The discovery that phosphodiester and thiophosphatephosphorothioate oligonucleotide can induction of immunity stimulate has caused the interest that these compounds is developed as treatment tool.These effort have concentrated on the thiophosphatephosphorothioate oligonucleotide that contains natural dinucleotides CpG.Kuramoto etc., Jpn.J.Cancer Res.83:1128-1131 (1992) point out to contain the phosphodiester oligonucleotide of the palindromic sequence that comprises the CpG dinucleotides and can induce the synthetic of α and IFN-and strengthen natural killer cell activity.Krieg etc., Nature 371:546-549 (1995) disclose the thiophosphatephosphorothioate oligonucleotide that contains CpG and have had immunostimulating.Liang etc., J.Clin.Invest.98:1119-1129 (1996) discloses this oligonucleotide and has activated human B cell.The thiophosphatephosphorothioate oligonucleotide that Moldoveanu etc., Vaccine 16:1216-124 (1998) point out to contain CpG has strengthened the immunne response to influenza virus.McCluskie and Davis, the oligonucleotide that J.Immunol.161:4463-4466 (1998) points out to contain CpG has strengthened the immunne response to hepatitis B surface antigen as the albumen adjuvant.

Other modification that contains CpG thiophosphatephosphorothioate oligonucleotide also can influence their abilities as immune response modifier.For example, visible Zhao etc., Biochem.Pharmacol. (1996) 51:173-182; Zhao etc., Biochem Pharmacol. (1996) 52:1537-1544; Zhao etc., Antisense Nucleic Acid Drug Dev. (1997) 7:495-502; Zhao etc., Bioorg.Med.Chem.Lett. (1999) 9:3453-3458; Zhao etc., Bioorg.Med.Chem.Lett. (2000) 10:1051-1054; Yu etc., Bioorg.Med.Chem.Lett. (2000) 10:2585-2588; Yu etc., Bioorg.Med.Chem.Lett. (2001) 11:2263-2267; And Kandimalla etc., Bioorg.Med.Chem. (2001) 9:807-813.U.S. Patent No. 6,426,334 have shown the prospect of these compounds as antitumor and anticancer agent.

Another method of regulating immunne response is to use by the therapeutic of cytokine.Cytokine is the soluble molecule of other iuntercellular reaction of regulation and control of immune system cell generation.Therefore, cytokine is the conditioning agent of body fluid and cellular immunization.It is crucial that the understanding that how the T cell is mediated immunne response is replied for adjusting.Auxiliary (Th) cell of CD4+T is according to Th1 or the differentiation of Th2 approach.The Th1 approach is important and shows as generation for producing cell-mediated immunity, for example, and gamma-interferon and interleukin-2 (IL-2).Th2 replys for producing humoral immunization and is important and shows as generation, for example, and IL-4 and IL-5.Known Th1 replys for opposing and infects, and for example, the immunity system of virus infection and the supervision of the immunity system of body are very crucial to remove tumour cell.

Krieg, A., M. etc. (U.S. Patent No. 6,429,199) and Krieg, A., M. etc. (U.S. Patent No. 6,218,371) are intended to instruct the Combined Preparation of immunostimulating CpG oligonucleotide and cytokine, particularly GM-CSF.Decker etc. (Experimental Hematology 28:558-565 (2000)) have proved that the Combined Preparation of IL-2 and CpG oligonucleotide has increased the generation of middle TNF-α of B-chronic lymphocytic (B-CLL) and IL-6, is not then having in normal B-cell.

The clear and definite result of treatment of immunostimulatory oligonucleotide in disease treatment and the antitumour activity of enhancing immunity pungency oligonucleotide of also needing to be optimized of these reports.

Summary of the invention

The invention provides method, composition and medicine for treatment method for the optimization that strengthens the immunne response that causes by the immunostimulating compound, to be used for the treatment of patient disease, for example, but be not limited to treatment cancer, autoimmune disease, asthma, respiratory system allergy, food anaphylaxis and communicable disease.The method of optimization of the present invention provides between the therapeutic action of the result of treatment of immunostimulatory oligonucleotide of the present invention and cytokine immunotherapy and/or chemotherapeutics and acts synergistically.The modification of optimizing the immunostimulatory oligonucleotide of presenting 5 ' end has obviously strengthened its antitumour activity.This oligonucleotide is expressed as " immunity " in this article, and it can contain one or more immunostimulatory oligonucleotides.

Therefore, aspect first, the invention provides and in the cancer patients, treat method for cancer, comprise that to immunostimulatory oligonucleotide and/or the immune sub-compound of patient's administration wherein immunostimulatory oligonucleotide and/or immune sub-compound and chemotherapeutics produce synergistic therapeutic effect in conjunction with chemotherapeutics.

On the other hand, the invention provides the method for the collaborative patient of stimulation immunne response.This method comprises IL-2 (and/or the reagent of inducing IL-2 to produce in position at least a the present invention's sub-compound of immunity of patient's drug treatment effectively collaborative (synergistic) amount or immunostimulatory oligonucleotide and the effectively collaborative amount of treatment, for example dna vaccination or express the expression vector of IL-2) combination, the collaborative patient's production of cytokines that stimulates of the described combination of administration wherein.The collaborative stimulated cells factor of preferred the present invention is selected from IL-12 and interferon-(IFN-γ), IFN-α, IFN-β or its combination.

Among the present invention, " immunity " means any compound that contains at least two oligonucleotide, described two oligonucleotide are directly at its 3 ' end, or directly by connecting between Nucleotide, or directly connect at functional nucleotide base or glycosyl, or link together by the non-nucleotide linker indirectly, wherein at least one oligonucleotide in the context of the sub-compound of immunity, the immunostimulatory oligonucleotide of come-at-able for having (accessible) 5 ' end.In the context of the present invention, immunostimulatory oligonucleotide is the oligonucleotide that contains at least one immunostimulating CpG dinucleotides, immunostimulating structural domain or other immunostimulating part.Herein, 5 ' end of term " come-at-able 5 ' end " expression oligonucleotide is abundant available, thereby makes identification and sub-compound of binding immunoassay and immunostimulatory oligonucleotide, and the factor of stimulating immune system can be near this 5 ' end.Described immunostimulatory oligonucleotide may comprise secondary structure, as long as this 5 ' terminal maintenance is come-at-able.

In certain embodiments, the immunostimulatory oligonucleotide and/or the immune sub-compound that are used for the method for the invention comprise the immunostimulation dinucleotide, and it is selected from CpG, C ^*PG, CpG ^*, or C ^*PG ^*, wherein C be cytidine or 2 '-Deoxyribose cytidine, C ^*It is 2 '-deoxythymidine, the pectinose cytidine, the pectinose cytidine of 2 '-deoxidation-2 '-replacement, the pectinose cytidine that 2 '-O-replaces, 2 '-deoxidation-5-hydroxyl cytidine, 2 '-'-deoxy-n 4-alkyl-cytidine, 2 '-deoxidation-4-thiourdine, other non-natural pyrimidine nucleoside, or 1-(2 '-deoxidation-β-D-ribofuranosyl)-2-oxo-7-denitrogenation-8-methyl-purine; G be guanosine or 2 '-pancreatic desoxyribonuclease, G ^*It is 2 ' deoxidation-7-denitrogenation guanosine, 2 '-deoxidation-6-sulfo-guanosine, the pectinose guanosine, 2 '-deoxidation-2 ' replaces-the pectinose guanosine, 2 '-O-replaces-the pectinose guanosine, perhaps other non-natural purine nucleoside, and p is to be selected between the Nucleotide of phosphodiester, thiophosphatephosphorothioate or phosphorodithioate to connect.In some preferred embodiment, the immunostimulation dinucleotide is not CpG.

In certain embodiments, immunostimulatory oligonucleotide and/or the immune sub-compound that is used for the method for the invention comprises the immunostimulating structural domain shown in the formula (III):

5’-Nn-N1-Y-Z-N1-Nn-3’ (III)

Wherein:

Y is a cytidine, 2 '-deoxythymidine, 2 '-Deoxyribose cytidine, Arabic cytidine, the pectinose cytidine of 2 '-deoxidation-2 '-replacement, the pectinose cytidine that 2 '-O-replaces, 2 '-deoxidation-5-hydroxyl cytidine, 2 '-'-deoxy-n 4-alkyl-cytidine, 2 '-deoxidation-4-thiourdine, other non-natural pyrimidine nucleoside, perhaps 1-(2 '-deoxidation-β-D-ribofuranosyl)-2-oxo-7-denitrogenation-8-methyl-purine;

Z be guanosine or 2 '-pancreatic desoxyribonuclease, be 2 ' deoxidation-7-denitrogenation guanosine, 2 '-deoxidation-6-sulfo-guanosine, the pectinose guanosine, 2 '-deoxidation-2 ' replaces-the pectinose guanosine, and 2 '-O replaces-the pectinose guanosine, 2 '-Hypoxanthine deoxyriboside, perhaps other non-natural purine nucleoside.

N1, the each appearance, be preferably naturally occurring or synthetic nucleosides or be selected from no base (abasic) nucleosides, the pectinose nucleosides, 2 '-deoxyuridine, α-dezyribonucleoside, the immunostimulating part of β-L-dezyribonucleoside, and be connected the nucleosides that 3 ' end is connected to adjacent nucleosides between nucleosides by phosphodiester or modification, connect between the nucleosides of modifying and be selected from but be not limited to, have linker from 2 dusts to about 200 angstrom lengths, C2-C18 alkyl linker, poly-(ethylene glycol) linker, the amino butyl-1 of 2-, the ammediol linker, the glyceryl linker connects between 2 '-5 ' nucleosides, and thiophosphatephosphorothioate, phosphorodithioate perhaps connects between the methylphosphonate nucleosides;

Nn, the each appearance, be naturally occurring nucleosides or immunostimulating part independently, be preferably selected from the alkali-free yl nucleosides, the pectinose nucleosides, 2 '-deoxyuridine, α-dezyribonucleoside, 2 '-ribonucleoside that O-replaces, and nucleosides is connected at 3 ' end by connecting between the nucleosides of modifying with adjacent nucleosides, connects between the Nucleotide of modification to be selected from amino linker, C2-C18 alkyl linker, poly-(ethylene glycol) linker, the amino butyl-1 of 2-, ammediol linker, glyceryl linker, connect between 2 '-5 ' nucleosides, and connect between the methylphosphonate nucleosides;

Condition is that at least one N1 or Nn are the immunostimulating part;

Wherein n is the numeral of 0-30;

Wherein 3 ' nucleosides randomly directly or by the non-nucleotide linker is connected to another oligonucleotide, and it can have or not have immunostimulating.

In second aspect, the invention provides treatment cancer patients method for cancer, comprise with immunostimulatory oligonucleotide and/or immune sub-binding substances (its as described above comprise immunostimulatory oligonucleotide and/or immune sub-compound) with at position and immunostimulatory oligonucleotide and/or immune sub-compound bonded cancer antigen except that come-at-able 5 ' end, with the chemotherapeutics combination medicine-feeding.

In the third aspect, the invention provides pharmaceutical preparation, comprise immunostimulatory oligonucleotide of the present invention or immunostimulatory oligonucleotide binding substances and/or immune sub-compound or immune sub-binding substances, chemotherapeutics and physiology can be accepted carrier.

In fourth aspect, the invention provides the method for cancer cells that make to ionizing rays sensitization.The method of this aspect of the present invention comprises and is administered to Mammals immunostimulatory oligonucleotide of the present invention or immune sub-compound and with this animal of ionization radiation therapy.

Aspect the 5th, the invention provides the method for the collaborative patient's of stimulation immunne response, comprise the sub-compound of at least a immunity or the immunostimulatory oligonucleotide combination (and the antigen of choosing any one kind of them) of the IL-2 of the effectively collaborative amount of treatment together that are administered to the effectively collaborative amount of patient treatment, the administration of wherein said combination is worked in coordination with stimulates patient's production of cytokines.The collaborative stimulated cells factor of preferred the present invention is selected from IL-12 and interferon-, IFN-α, IFN-β or its combination.In some embodiment of second aspect present invention, antigen randomly links to each other in the position except come-at-able 5 ' end with the sub-compound of immunity.

In a sixth aspect of the present invention, at least a is not that the immunostimulatory oligonucleotide of immune sub-compound and the IL-2 of treatment significant quantity unite use with selectivity and the collaborative patient of stimulation production of cytokines.The collaborative stimulated cells factor of preferred the present invention is selected from IL-12 and IFN-γ, IFN-α, IFN-β or its combination.According to the present invention, preferred is not that the immunostimulatory oligonucleotide of immune sub-compound comprises that those contain the immunostimulatory oligonucleotide of at least one immunostimulating CpG dinucleotides, and wherein C be that cytosine(Cyt) or deoxidation cytosine(Cyt) and/or G are not guanosine or 2-pancreatic desoxyribonuclease.Other preferred of the present invention be not that the immunostimulatory oligonucleotide of immune sub-compound is those of alternate immunostimulating part that contain non-CpG.The example of this alternate immunostimulating part includes but not limited to contain the nucleosides of base that non-natural exists and/or glycosyl and makes the secondary structure of the oligonucleotide that oligonucleotide is stable itself such as hairpin structure.

Aspect the 7th, the invention provides therapeutic composition, it comprises the sub-compound of at least a immunity or the immunostimulatory oligonucleotide of the effectively collaborative amount of treatment, IL-2 (and/or the reagent of inducing IL-2 to produce in position of the effectively collaborative amount of treatment, for example dna vaccination or express the expression vector of IL-2) and optionally antigen, the wherein collaborative patient's production of cytokines that stimulates of the described combination of administration.The collaborative stimulated cells factor of preferred the present invention is selected from IL-12 and IFN-γ, IFN-α, IFN-β or its combination.

The method and composition of all aspects of the invention effectively is used for the treatment of the mankind or veterinary disease, comprises immune system and based on the treatment of immunity.Particularly preferred disease target comprises cancer, transmissible disease, asthma and allergy.

Accompanying drawing mainly illustrates

Fig. 1 is the diagram of the immune sub-compound of representativeness of the present invention.

Fig. 2 has described the sub-compound of several representational immunity of the present invention.

Fig. 3 has described one group of representational small molecules linker of the synthetic the present invention's immunity of suitable linearity.

Fig. 4 has described the one group of representational small molecules linker that is fit to parallel synthetic the present invention's immunity.

Fig. 5 is the synthetic synoptic diagram of the sub-compound of linear synthetic the present invention's immunity.DMTr=4,4 '-dimethoxytrityl; The CE=cyanoethyl.

Fig. 6 is the synthetic synoptic diagram of the sub-compound of parallel synthetic the present invention's immunity.DMTr=4,4 '-dimethoxytrityl; The CE=cyanoethyl.

Fig. 7 A is oligonucleotide (Oligo) 1 and immune sub-2-3 induce IL-12 in BALB/c mouse spleen cell cultures thing a graphical representation.It is stronger IL-12 inductor that immunity 2 of these data presentation contain come-at-able 5 '-end is compared with monomer oligonucleotide 1, and immunity 3 that does not contain come-at-able 5 '-end is compared with oligonucleotide 1 has the immunostimulating ability of that equate or more weak generation.

Fig. 7 B is Oligol and immune sub-2-3 (respectively by the top to the bottom) induce IL-6 in BALB/c mouse spleen cell cultures thing a graphical representation.It is stronger IL-6 inductor that immunity 2 of these data presentation contain come-at-able 5 '-end is compared with monomer oligonucleotide 1, and immunity 3 that does not contain come-at-able 5 '-end is compared with oligonucleotide 1 has the immunostimulating ability of that equate or more weak generation.

Fig. 7 C is oligonucleotide 1 and immune sub-2-3 (respectively by the top to the bottom) induce IL-10 in BALB/c mouse spleen cell cultures thing a graphical representation.

Fig. 8 A is the graphical representation by BALB/c mouse splenocyte propagation in immunity the 5 and 6 inducing cell cultures of non-come-at-able and the come-at-able 5 '-end of having respectively of different concns.

Fig. 8 B is the graphical representation of the BALB/c mouse splenomegaly that causes by oligonucleotide 4 and immune sub-5-6 (it has the immunogenicity chemically modified in CpG 5 '-flanking sequence partly).In addition, the immune sub-compound (6) with come-at-able 5 '-end is compared the ability with stronger increase splenomegaly with the immunity sub 5 that does not have come-at-able 5 '-end with monomer oligonucleotide 4.

Fig. 9 A is oligonucleotide 4 and immunity 7 and 8 graphical representations of inducing IL-12 in BALB/c mouse spleen cell cultures thing by different concns.

Fig. 9 B is oligonucleotide 4 and immunity 7 and 8 graphical representations of inducing IL-6 in BALB/c mouse spleen cell cultures thing by different concns.

Fig. 9 C is oligonucleotide 4 and immunity 7 and 8 graphical representations of inducing IL-10 in BALB/c mouse spleen cell cultures thing by different concns.

Figure 10 A is the graphical representation by immunity 14,15 and 16 inducing cell propagation in BALB/c mouse spleen cell cultures thing.

Figure 10 B is the graphical representation of the IL-12 inducing cell propagation that different concns immunity 14 and 16 causes in the BALB/c mouse spleen cell cultures thing.

Figure 10 C is the graphical representation of the IL-6 inducing cell propagation that different concns immunity 14 and 16 causes in the BALB/c mouse spleen cell cultures thing.

Figure 11 A is the graphical representation by oligonucleotide 4 and 17 and immune sub 19 and 20 inducing cell propagation in BALB/c mouse spleen cell cultures thing.

Figure 11 B is for inducing the graphical representation of the cell proliferation of IL-12 in BALB/c mouse spleen cell cultures thing by different concns oligonucleotide 4 and 17 and immune sub 19 and 20.

Figure 11 C is for inducing the graphical representation of the cell proliferation of IL-6 in BALB/c mouse spleen cell cultures thing by different concns oligonucleotide 4 and 17 and immune sub 19 and 20.

Figure 12 is the BALB/c mouse splenomegaly graphical representation with oligonucleotide 4 and immunity 14,23 and 24.

Figure 13 is presented at the effect of the method for the invention aspect tumor growth in the prostate cancer nude mouse model.

Figure 14 show the method for the invention to institute with rat in the effect aspect the body weight.

Figure 15 A is the graphical representation that proves with the synergy aspect the IL-12 generation behind oligonucleotide 1 and the IL-2 treatments B ALB/c splenocyte.

Figure 15 B is the graphical representation that proves with the synergy aspect the IL-12 generation behind oligonucleotide 2 and the IL-2 treatments B ALB/c splenocyte.

Figure 15 C is the graphical representation that proves with the synergy aspect the IL-12 generation behind oligonucleotide 3 and the IL-2 treatments B ALB/c splenocyte.

Figure 15 D is the graphical representation that proves with the synergy aspect the IL-12 generation behind oligonucleotide 4 and the IL-2 treatments B ALB/c splenocyte.

Figure 16 A is the graphical representation that proves with the effect aspect the IL-6 generation behind oligonucleotide 1 and the IL-2 treatments B ALB/c splenocyte.

Figure 16 B is the graphical representation that proves with the effect aspect the IL-6 generation behind oligonucleotide 2 and the IL-2 treatments B ALB/c splenocyte.

Figure 16 C is the graphical representation that proves with the effect aspect the IL-6 generation behind oligonucleotide 3 and the IL-2 treatments B ALB/c splenocyte.

Figure 16 D is the graphical representation that proves with the effect aspect the IL-6 generation behind oligonucleotide 4 and the IL-2 treatments B ALB/c splenocyte.

Figure 17 is the graphical representation that proves with the synergy aspect the IL-12 generation behind oligonucleotide 5 and the IL-2 treatments B ALB/c splenocyte.

Figure 18 A is the graphical representation that proves with the effect aspect IFN-γ generation behind oligonucleotide 1 and the IL-2 treatments B ALB/c splenocyte.

Figure 18 B is the graphical representation that proves with the effect aspect IFN-γ generation behind oligonucleotide 2 and the IL-2 treatments B ALB/c splenocyte.

Figure 18 C is the graphical representation that proves with the effect aspect IFN-γ generation behind oligonucleotide 3 and the IL-2 treatments B ALB/c splenocyte.

Figure 18 D is the graphical representation that proves with the effect aspect IFN-γ generation behind oligonucleotide 4 and the IL-2 treatments B ALB/c splenocyte.

Figure 19 is the graphical representation that proves with the effect aspect IFN-γ generation behind oligonucleotide 5 and the IL-2 treatments B ALB/c splenocyte.

Detailed description of the preferred embodiments

The present invention relates to strengthen the method and composition of the optimization of the immunne response that causes by the immunostimulating compound that is used for immunotherapy.The method of optimization of the present invention causes the immunostimulating compound, as the synergy between the therapeutic action of the therapeutic action of immunostimulatory oligonucleotide and immune sub-compound and cytokine immunotherapy and/or chemotherapeutics.The patent of the mandate that this paper quotes, patent application, and reference at this by with reference to quoting, its effect is equal to be quoted each all special and independent being indicated as being by reference.When between in that instruct in any reference that this paper quoted and this specification sheets inconsistent situation being arranged, adopt the latter for the purposes of the present invention.

The invention provides enhancing is used for treatment for cancer by the method that is used for the anticancer effect that immunostimulating compound that immunotherapy uses causes.According to method of the present invention, immunostimulatory oligonucleotide and/or immune sub-compound provide synergistic therapeutic action when uniting use with chemotherapeutics.Surprisingly immunostimulatory oligonucleotide and immune sub-compound cause the cell fission of immune system cell, but chemotherapeutics can kill usually and enlivens the splitted cell.

Aspect first, the invention provides and in the cancer patients, treat method for cancer, comprise that associating chemotherapy preparation is to patient's administration immunostimulatory oligonucleotide and/or immune sub-compound, the latter is contained at least two interconnective oligonucleotide, thereby the sub-compound of described immunity has more than come-at-able a 5 ' end, and wherein at least one in the oligonucleotide is immunostimulatory oligonucleotide.Herein, 5 ' end of term " come-at-able 5 ' end " expression oligonucleotide is fully come-at-able, thereby makes that the identification and the factor of sub-compound of binding immunoassay and stimulating immune system can be near this 5 ' ends.Choose wantonly, 5 ' OH can connect phosphoric acid ester, thiophosphatephosphorothioate or phosphorodithioic acid ester moiety, fragrant or fatty linker, and cholesterol, or do not hinder another entity of its come-at-able accessibility.Immunostimulatory oligonucleotide and immune sub-compound induce immune response when being administered to vertebrates.When with the chemotherapeutics combined utilization, obtain synergistic therapeutic action.

The chemotherapeutics that preferably is used for the method for the invention includes but not limited to gemcitabine, methotrexate, vincristine(VCR), adriamycin, cis-platinum, the inferior acylurea of not sacchariferous chloroethyl, 5 FU 5 fluorouracil, ametycin, bleomycin, Zorubicin, Dacarbazine, taxol, fragyline, MeglamineGLA, valrubicin, Carmustine and poliferposan, MMI270, BAY 12-9566, RAS famesyl transferase inhibitor, the famesyl transferase inhibitor, MMP, MTA/LY231514, LY264618/Lometexol, Glamolec, CI-994, TNP-470, Hycamtin/ Hycamtin, PKC412, valspodar/PSC833, Novantrone/Mitroxantrone, Metaret/ suramin, Batimastat, E7070, BCH-4556, CS-682,9-AC, AG3340, AG3433, Incel/VX-710, VX-853, ZD0101, ISI641, ODN 698, TA 2516/Marmistat, BB2516/Marmistat, CDP 845, D2163, PD183805, DX8951f, Lemonal DP 2202, FK 317, molten chain bacterium (Picibanil)/OK-432, AD 32/ valrubicin, strontium chloride (Metastron)/strontium derivative, Temodal/ Temozolomide, Evacet (Evacet)/lipid Zorubicin, Yewtaxan/Placlitaxel, taxol/taxol, xeloda (Xeload)/capecitabine, Furtulon/doxifluridine, the oral taxol of Cyclopax/, oral Taxan, SPU-077/ cis-platinum, HMR1275/Flavopiridol, CP-358 (774)/EGFR, CP-609 (754)/RAS oncogene inhibitor, the oral platinum of BMS-182751/, UFT (Ftorafur/uridylic), LEVAMISOLE HCL/LEVAMISOLE HCL, eniluracil/776C85/5FU toughener, Rinotecan/LEVAMISOLE HCL, the Camptosar/ Rinotecan, Tumodex/Ralitrexed, cladibrine/CldAdo, Paxex/ taxol, Doxil (Doxil)/lipid Zorubicin, Caelyx/ lipid Zorubicin, Fludara/ fludarabine, Pharmarubicin/ epirubicin, DepoCyt, ZD1839, LU 79553/Bis-Naphtalimide, LU103793/ dolastatin, Caetyx/ lipid Zorubicin, (Gemzar)/2, strong pool, 2-difluoro deoxycytidine (Gemcitabine), ZD 0473/Anormed, YM 116, the lodine seed, CDK4 andCDK2 inhibitor, PARP inhibitor, D4809/Dexifosamide, Ifes/ Mesnex (Mesnex)/ifosfamide is defended and is sprouted (Vumon)/teniposide, Paraplatin/carbon platinum, cis-platinum/cis-platinum, etoposide/etoposide, ZD 9331, taxotere/many Xi Taqi, the prodrug of guanine cytosine arabinoside, 10-deacetyltaxol, Nitrosourea is such as the alkylating reagent of melphalan and endoxan, aminoglutethimide, Asparaginase, busulfan, carbon platinum, Chlorombucil, Spongocytidine-hydrochloride, gengshengmeisu, daunorubicin hydrochloride, the estramustine phosphate sodium phosphate, etoposide (VP16-213), Fluracil, Fluracil (5-FU), Drogenil, hydroxyl urea (hydroxyurea), ifosfamide, Intederon Alpha-2a, α-2b, leuprorelin acetate (LHRH-releasing factor analogs), lomustine (CCNU), mustine hydrochlcride (mustargen), the aggressiveness mercaptopurine, mesna, mitotane (o.p '-DDD), mitoxantrone hydrochloride, Sandostatin, Plicamycin, procarbazine hydrochloride, streptozocin, the tamoxifen citrate, Tioguanine, plug is for group, vinealeucoblastine(VLB) sulfuric ester, amsacrine (m-AMSA), azacytidine, Erthropoietin, hexamethyl melamine (HMM), interleukin-22, Methyl GAG (methyl-GAG; Methyl-glyoxal two-methyl-GAG; MGBG), pentostatin (2 ' deoxycoformycin), Semustine (Semustine), teniposide (VM-26) and vindesine sulfuric ester.

According to the method for this aspect of the present invention, immunostimulatory oligonucleotide and/or immune sub-compound can include but not limited to by any suitable way administration, parenterai administration, oral administration, sublingual administration, percutaneous dosing, topical, intranasal administration, aerosol, eye drops, tracheae administration, rectal administration, vagina administration, by particle gun, skin patch or local paste or eye drops or mouth wash shua form.The administration of the therapeutic composition of immunostimulatory oligonucleotide and/or immune sub-compound can be carried out with known method with the dosage and the time cycle of the surrogate markers of effective minimizing symptom or disease.When the whole body administration, therapeutic composition is preferably to reach from about 0.0001 micromole to about 10 micromolar immunostimulatory oligonucleotides and/or the enough dose administration of the blood levels of immune sub-compound.For topical, can be effectively than this much lower concentration, and high a lot of concentration also can be accepted.Preferably, the total dose of immunostimulatory oligonucleotide and/or immune sub-compound from each patient's every day about 0.0001mg to per kilogram of body weight 200mg every day.May need administration simultaneously, perhaps as independent treatment incident one or more therapeutic compositions of the present invention of drug treatment significant quantity in proper order.

Purpose for this aspect of the present invention, term " associating " " in combination with " is meant in the process of the identical patient's of treatment identical disease, comprise with any order administration immunostimulatory oligonucleotide and/or immune sub-compound and/or chemotherapeutics, comprise administration simultaneously, and order is separated several days the timed interval of as many as.Described combination therapy can comprise immunostimulatory oligonucleotide and/or immune sub-compound, and/or chemotherapeutics more than a kind of individually dosed independently.Immunostimulatory oligonucleotide and/or immune sub-compound and/or chemotherapeutics can pass through identical or different administration.

In certain embodiments, the immune sub-compound that is used for the method for the invention contains two or more immunostimulatory oligonucleotides, and (in the context of immunity), it can be identical or different.Preferably, each this immunostimulatory oligonucleotide has at least one come-at-able 5 ' end.

In certain embodiment of the method for the invention, except immunostimulatory oligonucleotide, immune sub-compound also contains at least one oligonucleotide with gene complementation.Herein, term " complementation " is meant the area hybridization of oligonucleotide and gene under physiological condition.In certain embodiments, the expression of oligonucleotide down-regulated gene.Described downward modulation oligonucleotide is preferably selected from antisense oligonucleotide, ribozyme oligonucleotide, little inhibitory RNA and trick (decoy) oligonucleotide.Applied as this paper, term " down-regulated gene " is meant the translation of transcribing of suppressor gene or gene product.Therefore, the immune sub-compound that is used for the method for the invention can be used for the one or more special disease targets of target, and the while is stimulating immune system also.

In certain embodiments, immunostimulatory oligonucleotide in the inventive method and/or immune sub-compound comprise ribozyme or decoy oligonucleotide.Applied as this paper, term " ribozyme " is meant the oligonucleotide with catalytic activity, and preferably, ribozyme is bonded to special target set nucleic acid and cracking target.Herein, term " decoy oligonucleotide " is meant the oligonucleotide that is bonded to transcription factor and inhibition transcriptional activity in the sequence-specific mode.Preferably, ribozyme or decoy oligonucleotide show secondary structure, include but not limited to stem ring or hairpin structure.In certain embodiments, at least a oligonucleotide contains poly (I)-poly (dC).In certain embodiments, at least one serial Nn contains the ribose of 3 to 10 dGs and/or Gs or 2 '-replacement or the string of pectinose Gs.

For the purposes of the present invention, term " oligonucleotide " is meant the polymerized nucleoside that forms from the nucleosides unit of many connections.Described oligonucleotide can comprise among genome or the cDNA obtaining from existing nucleic acid resource, still preferably by synthetic method production.In a preferred embodiment, each nucleosides unit comprises heterocyclic base and furan pentose base (pentofuranosyl), trehalose, pectinose, the pectinose of 2 '-deoxidation-2 '-replacement, pectinose or hexose group that 2 '-O-replaces.The nucleosides residue can be by any the mutual pairing in connecting between many known nucleosides.Connect between described nucleosides and include but not limited to phosphodiester, thiophosphatephosphorothioate, phosphorodithioate, alkyl phosphate, the alkylthio phosphoric acid ester, phosphotriester, phosphoramidate, siloxanes, carbonic ether, carbalkoxy (carboalkoxy), acetyl aminate (acetamidate), carbamate, morpholino, borine (borano), thioether, bridging phosphoramidate, the bridging methene phosphonate ester, the epithio substituted phosphate, and connect between the sulfone nucleosides.Term " oligonucleotide " also comprises having multinuclear glycosides (for example, (R that connects between one or more stereospecificity nucleosides _P)-or (S _P)-thiophosphatephosphorothioate, phosphonate ester or phosphotriester connect).Applied as this paper, term " oligonucleotide " clearly is intended to comprise to have multinuclear glycosides and the dinucleotide that is connected between arbitrary described nucleosides with " dinucleotides ", and no matter whether this connection comprises bound phosphate groups.In certain preferred embodiment, connection can be phosphodiester between these nucleosides, thiophosphatephosphorothioate, and phosphorodithioate, methylphosphonate connects, or its combination.

In certain embodiments, immune sub-compound comprises that each has from about 3 oligonucleotide to about 35 nucleosides residues, preferably from about 4 to about 30 nucleosides residues, more preferably from about 4 to about 20 residues.In certain embodiments, oligonucleotide has from about 5 or 6 to about 18, or from about 5 or 6 to about 14 nucleosides residues.Applied as this paper, term " about " means that definite numerical value is not strict.Therefore, for the purposes of the present invention, the quantity of the nucleosides residue in the oligonucleotide is not strict, has one or two less nucleosides residue or one are considered to above-mentioned each example to the oligonucleotide of the nucleosides residue of several increases equivalent.In certain embodiments, one or more oligonucleotide have 11 nucleosides.

Term " oligonucleotide " also comprises having extra substituting group, and described substituting group includes but not limited to, protein group, lipophilic group, intercalator, diamine (diamine), folic acid, the multinuclear glycosides of cholesterol and diamantane.Term " oligonucleotide " also comprises other the nucleotide base that contains polymkeric substance (nucleobase) arbitrarily, include but not limited to, peptide nucleic acid(PNA) (PNA), peptide nucleic acid(PNA) (PHONA) with bound phosphate groups, the nucleic acid of locking (LNA), morpholino-backbone oligonucleotides, and oligonucleotide with the main chain part that has alkyl linker or amino linker.

The immunostimulatory oligonucleotide and/or the immune sub-compound that are used for the method for the invention can comprise naturally occurring nucleosides, the nucleosides of modification, or its mixing.Applied as this paper, term " nucleosides of modification " is for comprising the heterocyclic base of modification, the glycosyl part of modification, or the nucleosides of its combination.In certain embodiments, the nucleosides of modification is non-natural pyrimidine or purine nucleoside, as described herein.In certain embodiments, the nucleosides of modification be 2 '-ribonucleoside, pectinose nucleosides or 2 '-deoxidation-2 '-fluorine pectinose nucleosides of replacing.

For the purposes of the present invention, the oh group that comprises of term " 2 '-ribonucleoside that replaces " in 2 ' position of pentose part be substituted with produce '-ribonucleoside of the ribonucleoside that O-replaces.Preferably, this replacement replaces wherein said alkyl with the low-grade alkyl group that contains 1-6 saturated or unsaturated carbon atom or with the aromatic yl group with 6-10 carbon atom; or aromatic yl group can not be substituted maybe and can be substituted, and for example, uses halogen; hydroxyl, trifluoromethyl, cyano group; nitro, acyl group, acyloxy; alkoxyl group; carboxyl, carbonyl alkoxyl group (carboalkoxy), perhaps amino group.Described 2 '-example of the ribonucleoside that O-replaces includes, but are not limited to 2 '-O-methylribonucleotide and 2 '-O-methoxy ethyl ribonucleoside.

Term " 2 '-ribonucleoside that replaces " also comprises wherein with containing 1-6 saturated or unsaturated carbon atom, or with amino or halogen group alternative 2 '-ribonucleoside of oh group.The example of the ribonucleoside of described 2 '-replacement includes but not limited to 2 '-amino, 2 '-fluorine, 2 '-allyl group, and 2 '-propargyl ribonucleoside.

Term " oligonucleotide " comprises heterozygosis and or chimeric oligonucleotide." chimeric oligonucleotide " is to have the oligonucleotide that connects between the nucleosides of more than one types.A preferred example of described chimeric oligonucleotide is for containing thiophosphatephosphorothioate, the chimeric oligonucleotide of phosphodiester or phosphorodithioate zone and the nonionic connection that is connected such as alkyl phosphate or alkyl thio-phosphonate (for example as seen, U.S. Patent number .5 such as Pederson, 635,377 and 5,366,878).

" heterozygosis oligonucleotide " is the oligonucleotide with more than one type nucleosides.A preferred example of described hybridization oligonucleotide comprise ribonucleotide or 2 '-the ribonucleoside acid region that replaces, and deoxyribonucleotide zone (for example as seen, Metelev and Agrawal, U.S. Patent No.5,652,355,6,346,614 and 6,143,881).

For the purposes of the present invention, term " immunostimulatory oligonucleotide " is meant oligonucleotide as indicated above, and it ought be administered to vertebrates, such as fish, and induce immune response when bird or Mammals.Herein, term " Mammals " includes but not limited to rat, mouse, cat, dog, horse, ox, milk cow, pig, rabbit, non-human primate, and people.Useful immunostimulatory oligonucleotide can be at Agrawal etc., and WO is open on November 5th, 98/49288,1998; WO 01/12804, and February 22 calendar year 2001 is open; WO 01/55370, and August 2 calendar year 2001 is open; PCT/US01/13682, application on April 30 calendar year 2001; And PCT/US01/30137, find in the description of application on September 26 calendar year 2001.Preferably, immunostimulatory oligonucleotide comprises at least a phosphodiester, thiophosphatephosphorothioate, and methylphosphonate, or connect between the phosphorodithioate nucleosides.

On the other hand, the invention provides the method for the collaborative patient's of stimulation immunne response.This method comprises the sub-compound of immunity at least a of the present invention of the effectively collaborative amount of administration patient treatment or IL-2 (and/or the reagent of inducing IL-2 to produce in position of immunostimulatory oligonucleotide and the effectively collaborative amount of treatment, the for example combination of the expression vector of dna vaccination or expression IL-2), the collaborative patient's production of cytokines that stimulates of the administration of wherein said combination.Preferably, the collaborative stimulated cells factor of the present invention is selected from IL-12 and interferon-(IFN-γ), IFN-α, IFN-β or its combination.

The applied term of this paper " effectively collaborative amount " is expressed as the known concentration of one section administration working lipe sub-compound of immunity or immunostimulatory oligonucleotide and IL-2, its make the effect of stimulation of combination of immune sub-compound or immunostimulatory oligonucleotide and IL-2 be better than its add and, promptly Zu He effect of stimulation is greater than the total effect of stimulation based on the expection of single effect of stimulation sum calculating.

Term " cytokine " herein " mean that immune system cell produces be used for regulating and control and other cell between any one of many shla molecules of reacting.Term " cytokine " comprises that for example, interleukin is (as IL-1, IL-2, IL-3, IL-6, IL-10, IL12 etc.), Interferon, rabbit (as IFN-α., IFN-β., IFN-γ .), chemokine, hemopoieticgrowth factor (as erythropoietin), tumour necrosis factor, G CFS is (as G-CSF, M-CSF, GM-CSF) and transforming growth factor (TGF-α).

Among the present invention, " immunity " means the compound that contains at least two oligonucleotide, described oligonucleotide is directly at its 3 ' end, or directly by connecting between Nucleotide, or directly connect at functionalization nucleic acid base or glycosyl, or link together indirectly, wherein at least one oligonucleotide by the non-nucleotide linker, in the context of the sub-compound of immunity, for having come-at-able 5 ' terminal immunostimulatory oligonucleotide.In the context of the invention, immunostimulatory oligonucleotide is the oligonucleotide that contains at least one immunostimulation " CpG " dinucleotides, immunostimulation structural domain or other immunostimulation part.Herein, 5 ' end of term " come-at-able 5 ' end " expression oligonucleotide is fully available, thereby makes that the identification and the factor of the sub-compound of binding immunoassay and immunostimulatory oligonucleotide and stimulating immune system can be near this 5 ' ends.

In some instances, at least one immunostimulatory oligonucleotide of immune sub-compound contain formula 5 '-immunostimulatory dinucleotide of Pyr-Pur-3 ', wherein Pyr is natural or the synthetic pyrimidine nucleoside, Pur is natural or the synthetic purine nucleoside.Herein, to mean the base component of nucleosides be the nucleosides of pyrimidine bases to term " pyrimidine nucleoside ".Similarly, to mean the base component of nucleosides be the nucleosides of purine radicals to term " purine nucleoside ".For the purposes of the present invention, " synthetic " pyrimidine or purine nucleoside comprise pyrimidine or the purine bases that non-natural produces, the sugar moieties that non-natural exists, or its combination.

In the inventive method in used immunostimulatory oligonucleotide and/or the immune sub-compound preferred pyrimidine nucleoside have 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-withdrawing group and electron-donating group;

A is hydrogen bond receptor or hydrophilic group;

A ' is selected from hydrogen bond receptor, hydrophilic group, hydrophobic group, electron-withdrawing group and electron-donating group;

X is carbon or nitrogen; With

S ' is pentose or hexose ring, or the sugar of non-natural existence.

Preferably, sugar ring is suitable for pyrimidine nucleoside is derived with the linking group that other nucleosides or nucleoside analog are connected with the phosphonate moiety of phosphonate moiety, modification or other.

Preferred hydrogen bond donor includes, but not limited to-NH--NH ₂,-SH and-OH.Preferred hydrogen bond receptor includes, but not limited to C=O, and the theheterocyclic nitrogen atom of C=S and aromatic heterocycle is as the N3 of cytosine(Cyt).

In some instances, the base portion in (I) is the pyrimidine bases that non-natural exists.The example of the pyrimidine bases that preferred non-natural exists includes, but not limited to 5-hydroxyl cytosine(Cyt), 5-hydroxymethyl cytosine, N4-alkyl cytosine(Cyt), preferred N4-ethyl cytosine(Cyt) and 4-sulfo-uridylic.In some instances, (I) middle sugar moieties S ' is the sugar moieties of non-natural existence.For the purposes of the present invention, " naturally occurring sugar moieties " is the natural sugar moieties that exists as a Nucleotide part, for example ribose and 2 '-ribodesose, " non-natural exist sugar moieties " be not for being natural in the existence of a Nucleotide part, but can be used in any sugar in the oligonucleotide main chain, for example hexose.Pectinose and pectinose derivative are the examples of preferred sugar moieties.

In the inventive method in used immunostimulatory oligonucleotide and/or the immune sub-compound preferred purine nucleoside analogs have structure (II):

Wherein:

D is a hydrogen bond donor;

D ' is selected from hydrogen, hydrogen bond donor or hydrophilic group;

A is hydrogen bond receptor or hydrophilic group;

X is carbon or nitrogen;

Each L independently is selected from C, O, N or S; With

S ' is the sugar of pentose or hexose ring or non-natural existence.

Preferred sugar encircles with the phosphonate moiety of phosphonate moiety, modification or other and is suitable for pyrimidine nucleoside is derived with the linking group that other nucleosides or nucleoside analog are connected.

Preferred hydrogen bond donor includes, but not limited to-NH--NH ₂,-SH and-OH.Preferred hydrogen bond receptor includes, but not limited to C=O, C=S ,-NO ₂With the theheterocyclic nitrogen atom of aromatic heterocycle, as the N1 of guanine.

In some instances, (II) middle base portion is the purine bases that non-natural exists.The example of the purine bases that preferred non-natural exists includes, but not limited to 6-thioguanine and 7-deazaguanine.In some instances, (II) the sugar moieties S ' in is the glycosyl of natural generation, described in front structure (I).

In the preferred embodiment, the immunostimulatory dinucleotide in employed immunostimulatory oligonucleotide of the inventive method and/or the immune sub-compound is selected from CpG, C ^*PG, CpG ^*Or C ^*PG ^*, wherein C be cytidine(C or 2 '-deoxycytidine, C ^*It is 2 '-deoxythymidine, the arabinosylcytosine nucleosides, 2 '-deoxythymidine, 2 '-deoxidation-2 '-replacement arabinosylcytosine nucleosides, 2 '-O-replaces the arabinosylcytosine nucleosides, 2 '-deoxidation-5-hydroxyl cytidine(C, 2 '-'-deoxy-n 4-alkyl-cytidine(C, 2 '-deoxidation-4-thiourdine, other non-natural pyrimidine nucleoside, or 1-(2 '-deoxidation-β-D-ribofuranosyl)-2-oxo-7-denitrogenation-8-methyl-purine; G be guanosine or 2 '-pancreatic desoxyribonuclease, G ^*Is 2 ' deoxidation-7-denitrogenation guanosine, 2 '-deoxidation-6-sulfo-guanosine, the pectinose guanosine, 2 '-deoxidation-2 ' replacement-pectinose guanosine, 2 '-O-replacement-pectinose guanosine, 2 '-Hypoxanthine deoxyriboside, or other non-natural purine nucleoside, p connects between nucleosides, is selected from phosphodiester, thiophosphatephosphorothioate and phosphorodithioate.In certain preferred examples, immunostimulatory dinucleotide is not CpG.

Immunostimulatory oligonucleotide can all contain the immunostimulation part in the one or both ends of immunostimulatory dinucleotide.Thereby in some instances, immunostimulatory oligonucleotide contains the immunostimulation structural domain of structure (III):

5’-Nn-N1-Y-Z-N1-Nn-3’ (III)

Wherein:

Y be cytidine, 2 ' deoxythymidine, 2 ' Deoxyribose cytidine, cytosine arabinoside, 2 '-deoxidation-2 '-replacement pectinose cytidine, 2 '-deoxythymidine, 2 '-O-replace pectinose cytidine, 2 '-deoxidation-5-hydroxyl cytidine, 2 '-'-deoxy-n 4-alkyl-cytidine, 2 '-deoxidation-4-thiourdine, other non-natural pyrimidine nucleoside or 1-(2 '-deoxidation-β-D-ribofuranosyl)-2-oxo-7-denitrogenation-8-methyl-purine;

Z be guanosine or 2 '-pancreatic desoxyribonuclease, 2 ' deoxidation-7 denitrogenation guanosine, 2 '-deoxidation-6-sulfo-guanosine, pectinose guanosine, 2 '-deoxidation-2 ' replacement-pectinose guanosine, 2 '-O-replacement-pectinose guanosine, 2 ' Hypoxanthine deoxyriboside or other non-natural purine nucleoside;

N1, when occurring at every turn, be preferably natural existence or synthetic nucleosides or immunostimulation part, it is selected from the alkali-free yl nucleosides, the pectinose nucleosides, 2 '-deoxyuridine, α-dezyribonucleoside, β-L-dezyribonucleoside, nucleosides is connected at 3 ' end by connecting between the nucleosides of phosphodiester or modification with adjacent nucleosides, connect between the nucleosides of modifying and be selected from, but be not limited to, about 2 dusts are to the long linker of about 200 dusts, C2-C18 alkyl linker, the polyoxyethylene glycol linker, the amino butyl-1 of 2-, the ammediol linker, the glyceryl linker, connect and thiophosphatephosphorothioate between 2 '-5 ' nucleosides, connect between phosphorodithioate or methylphosphonate nucleosides;

Nn, when occurring at every turn, be preferably the nucleosides or the immunostimulation part of natural appearance, its be selected from alkali-free yl nucleosides, pectinose nucleosides, 2 '-deoxyuridine, α-dezyribonucleoside, 2 '-O-replaces ribonucleoside, with hold the nucleosides that is connected by being connected between the nucleosides of modifying 3 ' with adjacent nucleosides, connect between the nucleosides of modifying and be preferably selected from amino linker, C2-C18 alkyl linker, polyoxyethylene glycol linker, the amino butyl-1 of 2-, connect between ammediol linker, glyceryl linker, 2 '-5 ' nucleosides with the methylphosphonate nucleosides between be connected;

As long as at least one N1 or Nn are the immunostimulation part;

Wherein each n independently is from 0 to 30 a numeral; With

Wherein, under the situation of the sub-compound of immunity, 3 ' end directly or by the non-nucleotide linker is connected with other oligonucleotide, and described other oligonucleotide can be with or without immunostimulating.

In some preferred examples, YZ is pectinose cytidine or 2 '-deoxidation-2 '-replacement pectinose cytidine and pectinose guanosine or 2 ' deoxidation-2 '-replacement pectinose guanosine.Preferred immunostimulation partly is included in the modification in the phosphate backbone, comprise, but be not limited to, methylphosphonate, methyl thiophosphatephosphorothioate, phosphotriester, phosphorothioate triesters, thiophosphatephosphorothioate, phosphorodithioate, three ester prodrugs, sulfone, sulphonamide, sulfamate, formacetal, N-methyl hydroxylamine, carbonic ether, carboxylamine, morpholino, borine phosphonic acid ester (boranophosphonate), phosphoramidate, particularly first amino-phosphoramidate, N3 phosphoramidate and N5 phosphoramidate, stereospecific connection is (as (R _P)-or (S _P)-thiophosphatephosphorothioate, alkyl phosphonates or phosphotriester connect).

Preferred immunostimulating of the present invention part further comprises and has glycosyl modified nucleosides, includes but not limited to the 2 '-pentose glycosyl that replaces, include but not limited to 2 '-O-methylribose, 2 '-O-methoxy ethyl ribose, 2 '-O-propargyl ribose and 2 '-deoxidation-2 '-fluorine ribose; The pentose glycosyl of 3 '-replacement includes but not limited to 3 '-O-methylribose; 1 ', 2 '-dideoxy ribose; Pectinose; The pectinose that replaces includes but not limited to 1 '-methyl pectinose, 3 '-methylol pectinose, 4 '-methylol-pectinose, and the pectinose glycosyl of 2 '-replacement; The hexose glycosyl includes but not limited to 1, the anhydrous hexitol of 5-; And alpha-anomer.Modifying glycosyl and be 3 '-dezyribonucleoside or 3 '-embodiment of the ribonucleoside that O-replaces in, the immunostimulating part is connected to close nucleosides by ways of connecting between 2 '-5 ' nucleosides.

Preferably be used for the immunostimulatory oligonucleotide of method of the present invention and/or the immunostimulating part of immune sub-compound and further comprise oligonucleotide with other carbohydrate backbone modifications and replacement, comprise peptide nucleic acid(PNA) (PNA), the peptide nucleic acid(PNA) (PHONA) that contains bound phosphate groups, the nucleic acid of locking (LNA), the morpholino backbone oligonucleotides, and the oligonucleotide that contains the main chain linker part that has from about 2 dusts to about 200 angstrom lengths, include but not limited to alkyl linker or amino linker.The alkyl linker can be side chain or unbranched, and is replacement or unsubstituted, and chiral purity or racemic mixture.Most preferred, described alkyl linker has from about 2 to about 18 carbon atoms.Described in some preferred embodiments alkyl linker has from about 3 to about 9 carbon atoms.Some alkyl linkers comprise one or more hydroxyls that are selected from, amino, sulfydryl, thioether, ether, acid amides, sulphamide, ester, the functional group of urea or thioether.Some described functional alkyl linkers are formula-O-(CH ₂-CH ₂-O-) _n(n=1-9) polyoxyethylene glycol connects.Some other functional alkyl linker is peptide or amino acid.

Preferably be used for the immunostimulatory oligonucleotide of method of the present invention and/or the immunostimulating part of immune sub-compound and further comprise the DNA isomer, include but not limited to β-L-dezyribonucleoside and α-dezyribonucleoside.Preferred immunostimulating has partly mixed 3 ' modifier, and further comprises and have link position between non-natural nucleosides, include but not limited to 2 '-5 ', 2 '-2 ' and, 3 '-3 ' and 5 '-5 ' nucleosides that is connected.

The immunostimulating part that preferably is used for the immunostimulatory oligonucleotide of method of the present invention and/or immune sub-compound further comprises the nucleosides of the heterocyclic base with modification, includes but not limited to 5-hydroxyl cytosine(Cyt), 5-hydroxymethyl cytosine, N4-alkyl cytosine(Cyt), preferred N4-ethyl cytosine(Cyt), 4-thiouracil, the 6-Tioguanine, the 7-deazaguanine, inosine, nitro-pyrrole, C5-propine pyrimidine, and diaminopurine, include but not limited to 2,6-diaminopurine.

By the mode of special illustration and the mode that does not limit, for example, in the immunostimulating zone of structure (III), between the methylphosphonate nucleosides of position N1 or Nn, be connected to the immunostimulating part, has linker from about 2 dusts to about 200 angstrom lengths, at the C2-C18 of position X1 alkyl linker is the immunostimulating part, and is the immunostimulating part at β-L-dezyribonucleoside of position X1.As follows representative position in the table 1 of face and immunostimulating the part structure.Known with linker be expressed as immunostimulating at specific position partly be meant the nucleosides residue of this position 3 '-the hydroxyl place pass through shown in linker replace, therefore between nucleosides residue and adjacent 3 ' side nucleosides, produce between the nucleosides of modifying and connect.Similarly, connect between the nucleosides of modification the immunostimulating be expressed as specific position partly be meant the nucleosides residue of this position by shown in mode of connection be connected to the adjacent nucleosides of 3 ' end.

Table 1

The position	Typical case's immunostimulating part
		N1	Naturally occurring nucleosides, the alkali-free yl nucleosides, the pectinose nucleosides, 2 '-deoxyuridine, β-L-dezyribonucleoside C2-C18 alkyl connects, and polyoxyethylene glycol connects, the amino butyl-1 of 2-, ammediol connects (the amino connection), connects between 2 '-5 ' nucleosides, connects between the methylphosphonate nucleosides
Nn	Naturally occurring nucleosides, the alkali-free yl nucleosides, the pectinose nucleosides, 2 '-deoxyuridine, 2 '-ribonucleoside that O-replaces, connect between 2 '-5 ' nucleosides, connect between the methylphosphonate nucleosides, as long as N1 not all is connected for no base with N2

Table 2 is presented at has interior immunostimulating representative position and the structure partly of immunostimulatory oligonucleotide that the upstream strengthens the zone.Applied as this paper, term " interval body 9 " is meant formula-O-(CH ₂CH ₂-O) _n-the polyoxyethylene glycol linker, wherein n is 3.Term " Spacer 18 " is meant formula-O-(CH ₂CH ₂-O) _n-the polyoxyethylene glycol linker, wherein n is 6.Applied as this paper, term " C2-C18 alkyl linker " is meant formula-O-(CH ₂) _qThe linker of-O-, wherein q is from 2 to 18 integer.Correspondingly, term " C3-linker " and " C3-alkyl linker " are meant formula-O-(CH ₂) ₃The linker of-O-.To each interval body 9, interval body 18, and C2-C18 alkyl linker, linker passes through phosphodiester, thiophosphatephosphorothioate, phosphorodithioate or methylphosphonate ways of connecting are connected to adjacent nucleosides.

Table 2

The position	Typical immunostimulating part
		5’N2	Naturally occurring nucleosides, the amino butyl-1 of 2-, ammediol linker
5’N1	Naturally occurring nucleosides, β-L-dezyribonucleoside, C2-C18 alkyl linker, polyoxyethylene glycol, no base linker, the amino butyl-1 of 2-, the ammediol linker
		3’N1	Naturally occurring nucleosides, 1 ', 2 '-dideoxy ribose, 2 '-O-methyl-ribonucleoside, C2-C18 alkyl linker, interval body 9, interval body 18
3’N2	Naturally occurring nucleosides, 1 ', 2 '-dideoxy ribose, 3 '-dezyribonucleoside, β-L-dezyribonucleoside, 2 '-O-propargyl-ribonucleoside, C2-C18 alkyl linker, interval body 9, interval body 18 connects between the methylphosphonate nucleosides
		3’N3	Naturally occurring nucleosides, 1 ', 2 '-dideoxy ribose, C2-C18 alkyl linker, interval body 9, interval body 18, connect between the methylphosphonate nucleosides, connect d (G) n, the poly-dC of poly-I-between 2 '-5 ' nucleosides
3’N2+3’N3	1 ', 2 '-dideoxy ribose, β-L-dezyribonucleoside, C2-C18 alkyl linker, d (G) n, the poly-dC of poly-I-
		3’N3+3’N4	2 '-O-methoxy ethyl-ribonucleoside connects between the methylphosphonate nucleosides, d (G) n, the poly-dC of poly-I-
3’N5+3’N6	1 ', 2 '-dideoxy ribose, C2-C18 alkyl linker, d (G) n, the poly-dC of poly-I-
		5’N1+3’N3	1 ', 2 '-dideoxy ribose, d (G) n, the poly-dC of poly-I-

Table 3 is presented at has interior immunostimulating representative position and the structure partly of immunostimulatory oligonucleotide that the downstream strengthens the zone.

Table 3

The position	Typical case's immunostimulating part
		5’N2	Connect between the methylphosphonate nucleosides
5’N1	Connect between the methylphosphonate nucleosides
		3’N1	1 ', 2 '-dideoxy ribose connects between the methylphosphonate nucleosides, 2 '-O-methyl
3’N2	1 ', 2 '-dideoxy ribose, β-L-dezyribonucleoside, C2-C18 alkyl linker, interval body 9, interval body 18, the amino butyl-1 of 2-, the ammediol linker connects between the methylphosphonate nucleosides, 2 '-O-methyl
		3’N3	3 '-dezyribonucleoside, 3 '-ribonucleoside that O-replaces, 2 '-O-propargyl-ribonucleoside
3’N2+3’N3	1 ', 2 '-dideoxy ribose, β-L-dezyribonucleoside

The immune sub-compound that is used for the inventive method contains at least two directly or the oligonucleotide that connects by the non-nucleotide linker.For the purposes of the present invention, " non-nucleotide linker " is for being connected to the arbitrary portion of oligonucleotide by covalent linkage or non covalent bond mode.The length of preferred described linker is from about 2 dusts to about 200 dusts.Several examples of preferred linker are set forth below.Non-covalent connection includes but not limited to, electrostatic interaction, hydrophobic interaction, π-accumulation, and hydrogen bond.Term " non-nucleotide linker " is not intended to represent to connect between nucleosides, as described above, for example, phosphodiester, phosphorus substituted phosphate, or phosphorodithioate functional groups, its directly connect 3 of two nucleosides '-oh group.For the purposes of the present invention, described direct 3 '-3 ' connection is called as " Nucleotide connection ".

In certain embodiments, the non-nucleotide linker is a metal, includes but not limited to gold grain.In some other embodiment, the non-nucleotide linker is soluble or insoluble biodegradable polymer beads.

In some other embodiment, the non-nucleotide linker is the organic moiety with the functional groups that allows to be attached to oligonucleotide.Described adhere to preferred by firm covalently bound.

In certain embodiments, the non-nucleotide linker is a biomolecules, includes but not limited to polypeptide, antibody, lipid, antigen, allergen, and oligosaccharides.In certain embodiments, the non-nucleotide linker is a small molecules.For the purposes of the present invention, small molecules is for having less than 1 the organic moiety of the molecular weight of 000Da.In certain embodiments, small molecules has the molecular weight less than 750Da.

In certain embodiments, small molecules is fat or aromatic hydrocarbon, and it is optional can contain or in the straight chain that connects oligonucleotide or additional thereon one or more hydroxyls that are selected from, amino, sulfydryl, thioether, ether, acid amides, sulphamide, ester, urea, and the functional groups of thiocarbamide.Small molecules can be cyclic ether, ether, acid amides, sulphamide, ester, urea, and the functional groups of thiocarbamide.Small molecules can be for cyclic or acyclic.The example of small molecules linker includes but not limited to, amino acid, carbohydrate, cyclodextrin, diamantane, cholesterol, haptens and microbiotic.Therefore, in order to describe the purpose of non-nucleotide linker, term " small molecules " does not mean that and comprises nucleosides.

In certain embodiments, the small molecules linker is glycerine or formula HO-(CH ₂) _o-CH (OH)-(CH ₂) _pThe glycerine homologue of-OH, wherein o and p independently are from 1 to about 6, from 1 to about 4, from 1 to about 3 integer.Among other the embodiment, the small molecules linker is 1 at some, the derivative of 3-diamino-2-hydroxy propane.Some described derivatives have HO-(CH ₂) _m-C (O) NH-CH ₂-CH (OH)-CH ₂-NHC (O)-(CH ₂) _mThe chemical formula of-OH, wherein m is from 0 to about 10, from 0 to about 6, from 2 to about 6, or from 2 to about 4 integer.

Some non-nucleotide linkers that are used for the immune sub-compound of the inventive method allow to connect the Nucleotide more than two, as illustrated in Fig. 1.For example, small molecules linker glycerine have three can covalently bound oligonucleotide oh group.Therefore, the sub-compound of some immunity of the present invention contains the oligonucleotide that is connected to the non-nucleotide linker at its 3 ' end more than two.The sub-compound of some described immunity contains at least two immunostimulatory oligonucleotides, and each all has 5 come-at-able ' end.

The immunostimulatory oligonucleotide and/or the immune sub-compound that are used for method of the present invention can be synthetic easily with phosphoramidite method described in automatic synthesizer and Fig. 5 and 6 and that further describe in an embodiment.In certain embodiments, immunostimulatory oligonucleotide and/or immune sub-compound are by the synthetic (see figure 5) of linear synthesis mode.Applied as this paper, term " straight line is synthetic " is meant from an end of the sub-compound of immunity and begins to proceed to linearly the synthetic of the other end.Straight line is synthetic allow or identical or different (length, base composition and/or chemically modified mix aspect) monomeric unit be incorporated into immunostimulatory oligonucleotide and/or immune sub-compound.

The selectable synthesis mode of the sub-compound of immunity is " parallel synthetic ", wherein synthesizes and carries out (see figure 6) from connection portion, center abduction.The linker that is attached to solid support can be used for parallel synthesizing, as U.S. Patent number 5,912, described in 332.Selectable, can use general solid support, such as the phosphoric acid ester of the glass support that is attached to controllable bore diameter.

Parallel synthetic the comparing of the sub-compound of immunity has several advantages linear synthesizing: unitary the mixing of (1) parallel synthetic permission same monomer; (2) with linear synthetic different, two or all monomeric units are synthetic at one time, so the quantity of synthesis step is identical with monomeric unit with synthetic required time; And the reduction in (3) synthesis step has increased sub-degree of purity of production of final immunity and productive rate.

By or the synthetic of linear synthetic or parallel synthesis step last; if mix the nucleosides of modification, being used for the immunostimulatory oligonucleotide of the method for the invention or immune sub-compound can be with spissated ammonia soln or the deprotection of being recommended according to phosphoramidite supplier easily.The oligonucleotide of product immunostimulating and/or immune sub-compound preferably pass through the reversed-phase HPLC purifying, detritylation, and desalination and dialysis come purifying.

Immunostimulatory oligonucleotide is suitable for using as the component of immune sub-compound, perhaps according to a fourth aspect of the present invention, is described in following United States Patent (USP) and pending trial U.S. Patent application, be incorporated herein by reference: U.S. Patent number 6,426,334 and 6,476,000; And Application No. 09/770,602,09/845,623,09/965,116,60/440,587,10/361,111,60/471,247,60/477.Preferred immunostimulatory oligonucleotide of the present invention and immune sub-compound are described in the pending trial Application No. 10/279,684.Table 4 shows the representative immune sub-compound that is used for the method for the invention.Other immune sub-compound is at embodiment and U.S. Patent application No.10/279, describes in 684.

The example of the immune subsequence of table 4.

L=C3-alkyl linker; X=1 ', 2 '-dideoxy ribose; Y= ^5OHDC:R=7-denitrogenation-dG

R=pectinose guanosine; X ₁=glycerine connects;

Another aspect of the present invention provides the immunostimulatory nucleic acid that contains at least two oligonucleotide, and wherein immunostimulatory nucleic acid has secondary structure.In certain embodiments, immunostimulatory nucleic acid have by and complementary sequence between 3 '-end stem ring secondary structure of constituting of hydrogen bond.In certain embodiments, the active nucleic acid that reduces of immunostimulating by and complementary sequence between hydrogen bond constitute 5 '-end stem ring secondary structure.In this respect, immunostimulatory nucleic acid contains suc as formula structure detailed in (I).

Structural domain A-structural domain B-domain C (I)

Structural domain length can be from about 2 to about 12 Nucleotide.Structural domain A can be 5 '-3 ' or 3 '-5 ' or 2 '-5 ' DNA, RNA, and RNA-DNA, DNA-RNA, it has or does not have the palindrome or self complementary structure territory, contains or do not contain at least a CpG of being selected from, C ^*PG, C ^*PG ^*Or CpG ^*Dinucleotides, wherein C is cytidine or 2 '-Deoxyribose cytidine, G is uridine or 2 '-deoxyuridine, C ^*Be 2 '-deoxythymidine, 1-(2 '-deoxidation-β-D-ribose furyl)-2-oxo-7-denitrogenation-8-methyl-purine, 2 '-dideoxy-5-halo cytosine(Cyt), 2 '-deoxidation-5-nitro cytosine(Cyt), the pectinose cytidine, 2 '-deoxidation-2 '-the pectinose cytidine that replaces, 2 '-pectinose cytidine that O-replaces, 2 '-deoxidation-5-hydroxyl cytidine, 2 '-'-deoxy-n 4-alkyl-cytidine, 2 '-deoxidation-4-thiourdine, or other pyrimidine nucleoside analoys, G ^*Be 2 '-deoxidation-7-denitrogenation guanosine, 2 '-deoxidation-6-sulfo-guanosine, the pectinose guanosine, 2 '-deoxidation-2 ' replacement-the pectinose guanosine, 2 '-the O-replacement-the pectinose guanosine, 2 '-Hypoxanthine deoxyriboside, or other purine nucleoside analogs, and p is for being selected from phosphodiester, and thiophosphatephosphorothioate and is connected between the nucleosides of phosphorodithioate.In certain preferred embodiment, the immunostimulation dinucleotide is not CpG.

In certain embodiments, structural domain A will have more than one and be selected from CpG, C ^*PG, C ^*PG ^*Or CpG ^*Dinucleotides.

Structural domain B, " X " is described as following usefulness, is the linker of syndeton territory A and C, and it can be 3 '-' 5 ' connection, 2 '-5 ' connects, and 3 '-3 ' connects bound phosphate groups, nucleosides, or non-nucleosides linker, it can be fatty, fragrance, aromatic base, ring, chirality achiral, peptide, carbohydrate, lipid, lipid acid, single-, three-or six polyoxyethylene glycol, or heterocyclic moiety.

Domain C can be for having or not having 5 '-3 ' or 3 '-5 ', 2 '-5 ' DNA that the palindrome or self complementary sequence are arranged, RNA, and RNA-DNA, DNA-RNA, it can have or not have the CpG of being selected from, C ^*PG, C ^*PG ^*And CpG ^*Dinucleotides, wherein C is cytidine or 2 '-Deoxyribose cytidine, G is guanosine or 2 '-pancreatic desoxyribonuclease, C ^*Be 2 '-deoxythymidine, 1-(2 '-deoxidation-β-D-ribose furyl)-2-oxo-7-denitrogenation-8-methyl-purine, 2 '-dideoxy-5-halogen cytosine(Cyt), 2 '-deoxidation-5-halogen cytosine(Cyt), the pectinose cytidine, 2 '-deoxidation-2 '-the pectinose cytidine that replaces, 2 '-pectinose cytidine that O-replaces, 2 '-deoxidation-5-hydroxyl cytidine, 2 '-'-deoxy-n 4-alkyl-cytidine, 2 '-deoxidation-4-thiourdine, other pyrimidine nucleoside analoys, G ^*Be 2 '-deoxidation-7-denitrogenation guanosine, 2 '-deoxidation-6-thio uridine, the pectinose guanosine, 2 '-deoxidation-2 ' replacement-the pectinose guanosine, 2 '-the O-replacement-the pectinose guanosine, 2 '-Hypoxanthine deoxyriboside, or other purine nucleoside analogs, and p is for being selected from phosphodiester, and thiophosphatephosphorothioate and is connected between the nucleosides of phosphorodithioate.In certain preferred embodiment, the immunostimulation dinucleotide is not CpG.In certain embodiments, structural domain B is preferably the non-nucleotide linker of syndeton territory A and domain C, and it is called as " immunity ".In certain preferred embodiment, domain C does not contain dinucleotides CpG, C ^*PG, C ^*PG ^*Or CpG ^*

By the mode of limiting examples, among certain embodiment in this respect, immunostimulatory nucleic acid will have suc as formula structure detailed in (II).

Those skilled in the art will appreciate that the secondary structure assembly that has intramolecularly stem loop type at the end of molecule.

By the mode of limiting examples, immunostimulatory nucleic acid will have suc as formula structure detailed in (III) among certain embodiment in this respect.

Structure detailed is called as " terminal dimmer (dim aggressiveness) " in the formula (III) at this paper, because the end of two molecules is owing to the complementary intermolecular hydrogen bonding that produces of the sequence at two ends is blocked.In addition, structural domain A and A ' can for or can not be identical, structural domain B and B ' can for or can not be identical and domain C and C ' can for or can be not for identical.

By the mode of limiting examples, among certain embodiment in this respect, immunostimulatory nucleic acid will have the structure of formula (IV).

The end that one of skill in the art will appreciate that described molecule has secondary structure, because its terminal complementary sequence combines with this zone by hydrogen bond.In certain embodiments, thus can be attached to end such as the molecule of part promotes cell to absorb or strengthen the stability of molecule.

The nonrestrictive example of nucleic acid molecule more of the present invention is listed in the table 5.

Table 5

^*: capitalization-PS; Small letter-PO; Runic-2 '-O-methyl-ribonucleotide (in 116 and 117);

G-2 '-deoxidation-7-denitrogenation-G (in 118); G-araG (in 119); X-C3-linker (in 127 and 128).

Selectable, nucleic acid molecule of the present invention can be two immunity that connect by the non-nucleotide linker.The nonrestrictive representational example of these molecules is listed in the table 6.

Table 6

^*Capitalization-PS; Small letter-PO, the X-C3-linker; Y-TEG chain; Z-six ethylene glycol linkers, runic-2 '-O-methylribose nucleic acid (in 134 and 147); G-2 '-deoxidation-7-denitrogenation-G (in 135).

Selectable, further, nonrestrictive representative is listed in the table 7.

Table 7.

Oblique line is represented phosphodiester bond, if there is not other to illustrate that other key is a thiophosphatephosphorothioate

Underscore=2 '-OMe-nucleosides; The X=C3 linker

R=2 '-deoxidation-7-denitrogenation guanosine G ₁=2 '-deoxidation-7-denitrogenation guanoise

Another aspect of the present invention provides immunostimulatory nucleic acid, and wherein the sequence of immunostimulatory oligonucleotide and/or immunity is to small part self complementation.Applied self complementary sequence of this paper is meant base sequence, and it is by suitable arrangement, can form intramolecularly or, more typical intermolecular G-C, A-T, A-U and/or G-U base pairing wave pairing.Self complementary scope is at least 50% in one embodiment.For example at least 50% self complementary 8 aggressiveness aggressiveness can have and can form 4,5,6,7, or 8 G-C, A-T, and A-U and/or G-U wave the sequence of base pairing.Described base pairing can but must not relate to the base of any one end points that is positioned at self complementary immunostimulatory oligonucleotide and/or immunity.When nucleic acid stability is may be to immunostimulatory oligonucleotide and/or immune son very important, has benefit with one or two end points of double-strandednucleic acid or by base pairing or by other suitable method " folder " arbitrarily together.Self complementary degree can depend on the arrangement (alignment) between immunostimulatory oligonucleotide and/or the immune son, and described arrangement can or can not comprise single-or many-nucleosides overhang.In other embodiments, self complementary degree is at least 60%, and at least 70%, at least 80%, at least 90%, perhaps even 100%.

By the mode of limiting examples, immunostimulatory nucleic acid will have as the formula V structure detailed among certain embodiment in this respect.

One of skill in the art will appreciate that the sub-compound of described immunity has secondary structure, because the complementary intermolecular hydrogen bonding that produces of the sequence of structural domain.Structural domain A and A ' can for or can not be identical, structural domain A and C can for or can not be identical, structural domain A and C ' can for or can not be identical, structural domain A ' and C can for or can not be identical, structural domain A ' and C ' can for or can not be identical, structural domain B and B ' can for or can not be identical and domain C and C ' can for or can be not for identical.In addition, additional immune son can produce the chain of the present invention's immunity thus by the intermolecular hydrogen bonding combination, or polymer.N can be any number of the immune sub-compound of continuous self complementation.

Applied as this paper, term " complementation " is meant the ability that has with nucleic acid hybridization.Described hybridization is the result of the hydrogen bonded between the complementary strand normally, is preferably formed watson-Crick or Hoogsteen base pair, although the hydrogen bond of other form and base stacking also can produce hybridization.

Applied as this paper, term " secondary structure " is meant that intermolecular hydrogen bonding connects.Intermolecular hydrogen bonding causes the formation of double chain acid molecule.

Nonrestrictive representational nucleic acid molecule is listed in the table 8.

Table 8

173	5’-TCG1AACG1TTCG1-X-G1CTTG1CAAG1CT-5’
		174	5’-TCG1AACG1TTCG-X-GCTTG1CAAG1CT-5’
175	5’-TCTCACCTTC1-X-TCTTCCACTCT-5’
		176	5’-TCG2AACG2TTCG2-X-G2CTTG2CAAG2CT-5’
177	5’-TCG2AACG2TTCG-X-GCTTG2CAAG2CT-5’
		178	5’-TCG1TCGlAACG1TTCG1AGATGAT-3’
179	5’-TCG2TCG2AACG2TTCG2AGATGAT-3’
		180	5’-TCG3TCG3AACG3TTCG3AGATGAT-3’
181	5’-TC1GTC1GAAC1GTTC1GAGATGAT-3’
		182	5’-TC2GTC2GAAC2GTTC2GAGATGAT-3’
183	5’-TC3GTC3GAAC3GTTC3GAGAfGAT-3’
		184	5’-TCG1AACG1TTC-X-CTTG1CAAG1CT-5’

184	5’-TCG1AACG1TTC-X-CTTG1CAAG1CT-5’
		185	5’-TCG1TTCG1AACG1-X-G1CAAG1CTTG1CT-5’
186	5’-TCCAACCTTCG-X-GCTTCCAACCT-5’
		187	5’-TCG1TTG1CAACG1-X-G1CAACG1TTG1CT-5’
188	5’-TCG2AACG2TTCT-X-TCTTG2CAAG2CT-5’
		189	5’-TCG1AACG2TTCG1-X-G1CTTG2CAAG1CT-5’
190	5’-TCG1AAC1GTTCG1-X-G1CTTGC1AAG1CT-5’

On behalf of thiophosphatephosphorothioate, standard state (normal phase) connect

G ₁=2 '-deoxidation-7-denitrogenation guanosine

G ₂=pectinose guanosine

G ₃=2 '-Hypoxanthine deoxyriboside

C ₁=1-(2 '-deoxidation-β-D-ribose furyl)-2-oxo-7-denitrogenation-8-methyl purine

C ₂=Arabic cytidine

C ₃=2 '-deoxidation-5-hydroxyl cytidine

The X=C3 linker

The sub-compound of particularly preferred immunity that is used for the inventive method has following structure.

Be used for the treatment of relative disease according to the method and composition of all aspects of the present invention, wherein treatment comprises immune system and based on the treatment of immunity.Particularly preferred disease target comprises cancer, communicable disease and transformation reactions.

In some instances, methods of treatment is to be used for treatment for cancer.Cancer or tumour include but not limited to cholangiocarcinoma; The cancer of the brain; Mammary cancer; Cervical cancer; Choriocarcinoma; Colorectal carcinoma; Carcinoma of endometrium; Esophagus cancer; Cancer of the stomach; Last intracutaneous early stage of pyogenic infection of skin; Lymphoma; Liver cancer; Lung cancer (as minicell and non-small cell); Melanoma; Neuroblastoma; Oral carcinoma; Ovarian cancer; Carcinoma of the pancreas; Prostate cancer; The rectum cancer; Sarcoma; Skin carcinoma; Carcinoma of testis; Thyroid carcinoma and kidney, and other cancer and sarcoma.

In some instances, methods of treatment is the treatment that is used to infect.As the example of indefiniteness, be found the virus that infects the mankind and include but not limited to: (for example the human immunodeficiency virus (also is expressed as HTLV-III as HIV-1 to Retroviridae, LAV or HTLV-III/LAV, or HIV-III) and other conivium, as HIV-LP; Picornaviridae (poliovirus for example, hepatitis A virus (HAV); Enterovirus, human coxsackievirus, rhinovirus, ECHO virus); The Calciviridae strain of gastroenteritis (as cause); Alphaherpesvirinae (as equine encephalitis virus, rubella virus); Flaviridae (as dengue fever virus, encephalitis, yellow fever virus); Coronoviridae (as coronavirus); Rhabdoviridae (as stomatitis herpesvirus, rabies virus); Coronaviridae (as coronavirus); Rhabdoviridae (as stomatitis herpesvirus, rabies virus); Filoviridae (as Ebola virus); Paramyxoviridae (as parainfluenza virus, mumps virus, Measles virus, respiratory syncytial virus); Orthomyxoviridae family (as influenza virus); Bungaviridae (as Hantaan virus, bunga virus phleboviruses and Nairo virus); Arena Viraceae (hemorrhagic fever virus); Reoviridae (as reovirus, orbiviurses and rotavirus); Double-core ribonucleic acid virus section; Hepadnaviridae (hepatitis B virus); Parvoviridae (parvovirus group); Papovaviridae (papilloma virus, polyomavirus); Adenoviridae (most of adenovirus); Herpetoviridae (hsv (HSV) 1 and 2, varicella zoster virus (CMV), simplexvirus; Poxviridae (variola virus, vaccinia virus, poxvirus); And Iridoviridae (as afirican swine fever virus); With non-classified virus (as the virulence factor of spongiform encephalopathy, the virulence factor of hepatitis D (thinking the defective comitative aspect of hepatitis B virus), the virulence factor of non-first type, non-hepatitis B (Class1=interior propagation; Type 2=non-intestinal transmitted (being hepatitis C); Norwalk agent and relevant virus and Astrovirus).

In some instances, methods of treatment of the present invention is to be used for hypersensitive treatment." anaphylactogen " be meant can be in come-at-able individuality induced hypersensitivity or or the material (antigen) of asthma reaction.The list of anaphylactogen is huge, can comprise pollen, insect venom, animal scurf, fungal spore and medicine (as penicillin).The example of natural animal and phytosensitinogen includes but not limited to be specific to the albumen of following kind: Canidae (Canis familiaris); Dermatophagoides (as Dermatophagoides farinae); Felis (Felisdomesticus); Ambrosia (Ambrosia artemiisfolia); Lolium (as Lolium perenne or Lolium multiflorum); Japanese cypress (Crypto aggressiveness ia japonica); Alternaria (Alternaria alternata); Alder; Alder (Alnus gultinoasa); Betula (Betulaverrucosa); Oak belongs to (Quercus alba); Olive (Olea europa); Artemisia (Artemisiavulgaris); Plantago (as Plantago lanceolata); Parietaria (as Parietaria officinalis or Parietaria judaica); Blatella (e.g.Blattella germanica); Apis (as Apismultiflorum); Cupressus (as Cupressus sempervirens, Cupressus arizonica and Cupressus macrocarpa); Chinese juniper belongs to (as Juniperus sabinoides, Juniperus virginiana, Juniperus communis and Juniperus ashei); Thuya (e.g.Thuya orientalis); Chamaecyparis (as Chamaecyparis obtusa); Periplaneta (as Periplaneta a aggressiveness icana); Agropyron (as Agropyron repens); Secale (as Secale cereale); Triticum (as Triticumaestivum); Dactylis (as Dactylis glo aggressiveness ata); Festuca (as Festuca elatior); Poa (as Poa pratensis or Poa compressa); Avena (as Avena sativa); Holcus (as Holcus lanatus); Chrysanthemum thatch (as Anthoxanthum odoratum); Arrhenatherum (as Arrhenatherum elatius); Agrostis (as Agrostis alba); Phleum (as Phleumpratense); Phalaris (as Phalaris arundinacea); Paspalum (as Paspalum notatum); Chinese sorghum thing (as Sorghum halepensis); And Bromus (as Bromus inermis).Special anaphylactogen can be buied (as INDOOR Biotechnologies Inc., Charlottesville, VA 22903) by commercial sources.

Aspect second, the invention provides treatment cancer patients method for cancer, comprise binding immunoassay pungency oligonucleotide and/or immune sub-binding substances administration patient chemotherapeutics, described immunostimulatory oligonucleotide and/or immune sub-binding substances contain aforesaid immunostimulatory oligonucleotide and/or immune sub-compound and position and immunostimulatory oligonucleotide and/or immune sub-compound bonded antigen beyond come-at-able 5 ' end.In some instances, the non-nucleotide linker comprises the antigen relevant with cancer, and it combines with oligonucleotide.In other the example, antigen combines with oligonucleotide in 3 ' terminal position in addition at some.In some instances, antigen produces vaccine effect.For the purposes of the present invention, term " is correlated with " and represents that antigen exists when cancer is arranged, but does not exist when no cancer, or exists with the amount that reduces.

Covalently bound at immunostimulatory oligonucleotide and/or immune sub-compound and antigen, perhaps other is operationally related with antigen.Herein, " operationally related " any association that keeps immunostimulatory oligonucleotide and/or immune sub-compound and antigenic activity simultaneously of expression.This operationally related non-limitative example comprises a part that becomes same liposome or other this delivery vector or reagent.In addition, the nucleic acid molecule of coding for antigens can be cloned in the expression vector, and binding immunoassay pungency oligonucleotide and/or immune sub-compound administration.Herein, term " carrier " expression can be transmitted the nucleic acid molecule of other connected nucleic acid.Preferred carrier is can self-replacation and express connected nucleic acid those (as episomes).Can instruct the carrier with its genetic expression that can be operatively connected to be expressed as " expression vector " in this article.Usually, the expression vector that is used for recombinant DNA technology often is the form of " plasmid ", its general representative ring shape double-stranded DNA ring, and it does not combine with karyomit(e) at carrier format.In this manual, " plasmid " and " carrier " can replace use, because plasmid is the most general application form of carrier.Yet, this invention is intended to comprise that other form exercises the form of identical functions, it is known in the prior art so far of this area.

In the covalently bound example of immunostimulatory oligonucleotide and/or immune sub-compound and antigen, this covalently bound come-at-able 5 ' any position holding of preferably removing immunostimulatory oligonucleotide on immunostimulatory oligonucleotide and/or the immune sub-compound.For example, antigen can be attached to connect between nucleic acid and maybe can be attached to the non-nucleotide linker.Optionally, antibody itself can be this non-nucleotide linker.

Aspect the 3rd, the invention provides pharmaceutical formulations, it contains immunostimulatory oligonucleotide of the present invention and/or immunostimulatory oligonucleotide binding substances and/or immune sub-compound or immune sub-binding substances, chemotherapeutics and physiology acceptable carrier.Herein, term " physiology is acceptable " represents that this material does not disturb the effect of immune sub-compound, and and biosystem, as cell, cell culture, tissue or organism compatibility.Preferably, the organism of biosystem for living is as vertebrates.Preferred chemotherapeutics includes, but not limited to the gemcitabine Methotrexate, vincristine(VCR), Zorubicin, cis-platinum, sugar-free chlorethylnitrosourea, 5 FU 5 fluorouracil, ametycin, bleomycin, adriamycin, dacarbazine, taxol, fragyline, urografic acid methylglucamine salt (Meglamine) GLA, valrubicin, carmustine and poliferposan, MMI270, BAY 12-9566, RAS famesyl transferase inhibitor, the famesyl transferase inhibitor, MMP, MTA/LY231514, LY264618/Lometexol, Glamolec, CI-994, TNP-470, with U.S. new/Hycamtin, PKC412, valspodar/PSC833, Novantrone/Mitroxantrone, the Metaret/ Suramine, Batimastat, E7070, BCH-4556, CS-682,9-AC, AG3340, AG3433, Incel/VX-710, VX-853, ZD0101, ISI641, ODN 698, TA2516/Marmistat, BB2516/Marmistat, CDP 845, D2163, PD183805, DX8951f, Lemonal (Lemonal) DP 2202, FK 317, molten chain bacterium/OK-432, AD 32/ valrubicin, strontium chloride/strontium derivative, temodox/Temozolomide, Evacet/Mycocet, Yewtaxan/Placlitaxel, safe element/taxol, xeloda/capecitabine, Furtulon/doxifluridine, the oral taxol of Cyclopax/, oral Taxan, the SPU-077/ cis-platinum, HMR 1275/Flavopiridol, CP-358 (774)/EGFR, CP-609 (754)/RAS oncogene inhibitor, the oral platinum of BMS-182751/, UFT (Tegafur/uridylic), levamisole hydrochloride/Tramisol, eniluracil/776C85/5FU toughener, Rinotecan/Tramisol, Camptosar (Camptosar)/irinotecan, Tumodex/Ralitrexed, cladibrine/CldAdo, the Paxex/ taxol, Doxil/Mycocet, Caelyx/ Mycocet, Fuda China/fludarabine, the Pharmarubicin/ epirubicin, DepoCyt (DepoCyt), ZD1839, LU79553/Bis-Naphtalimide, LU 103793/ dolastatin, the Caetyx/ Mycocet is good for and is selected/gemcitabine ZD 0473/Anormed, YM 116, the lodine seed, CDK4 and CDK2 inhibitor, PARP inhibitor, D4809/Dexifosamide, Ifes/ Mesnex/ifosfamide, teniposide/Vumon, Paraplatin/carboplatin, cis-platinum/cis-platinum, etoposide/etoposide, ZD 9331, taxotere/Docetaxel, Arabinoside guanosine prodrug, 10-deacetyltaxol, nitrosourea, alkylating agent such as melphelan and endoxan, aminoglutethimide, asparaginase, busulfan, carboplatin, Chlorombucil, Spongocytidine-hydrochloride, gengshengmeisu, cerubidine, estramustine phosphate sodium, etoposide (VP16-213), fluorine glycosides, Ro 2-9757 (5-FU), flutamide, hydroxyurea (hydroxycarbamide), ifosfamide, Intederon Alpha-2a, α-2b, the sharp special acetate (LHRH releasing factor analogs) of bright dried meat, lomustine (CCNU), mustine hydrochlcride (mustargen), purinethol, mesna, rice holder load (o.p '-DDD), mitoxantrone hydrochloride, Sostatin, Plicamycin, procarbazine hydrochloride, streptozotocin, the tamoxifen Citrate trianion, Tioguanine, thiophene is for group, vinblastine sulfate, amsacrine (m-AMSA), azacitidine, Erthropoietin, Hexamethylmelamine (HMM), interleukin-22, Methyl GAG (methyl-GAG; Methyl-gloxal bis guanyl hydrazone, (methyl glyoxal bis-guanyl hydrazone) MGBG), pentostatin (2 ' deoxycoformycin), semustine (Semustine), Vumon (VM-26) and vindesine vitriol.

In another example again, preparation comprises Theratope, and it is selected from EFG, the anti-idiotype Theratope, Gp75 antigen, GMK Melacine, MGV Sphingolipids,sialo combined vaccine, Her2/new, Ovarex, M-Vax, O-Vax, L-Vax, the STn-KHL Theratope, BLP25 (MUC-1), liposome Idiotype vaccine, Melacine, the peptide antigen vaccine, toxin/antigen vaccine, MVA-vased vaccine, PACIS, the BCG vaccine, TA-HPV, TA-CIN, DISC-virus and ImmunCyst/TheraCys.

Further, the invention provides treatment cancer patients method for cancer, it comprises monoclonal antibody is combined the administration patient with immunostimulatory oligonucleotide as herein described and/or immune sub-compound.With antibody, especially the passive immunization therapy of monoclonal antibody form is a large amount of cancer therapy drug research and development problem.The antibody molecule of the single molecular components of used herein term " monoclonal antibody " expression.Monoclonal antibody combination shows single binding specificity and the avidity to special antigenic determinant.Therefore, term " human monoclonal antibodies " expression shows the antibody of single binding specificity, and it has the variable and constant zone of derived from human racial immunity sphaeroprotein sequence.The example of cancer therapy drug comprises, but be not limited to, Panorex (Glaxo-Welcome), Rituxan (IDEC/Genentech/Hoffman la Roche), Mylotarg (Wyeth), Campath (Millennium), Zevalin (IDEC and Schering AG), Bexxar (Corixa/GSK), Erbitux (Imclone/BMS), Avastin (Genentech) and Herceptin (Genentech/Hoffman la Roche).As if antibody also can be used to use effective immunotherapy of anti-id AB, and it is simulation (on the immunology meaning) cancer antigen.Monoclonal antibody can produce by recombinant DNA technology field those of ordinary skill known method.

Herein, term " carrier " comprises any vehicle, thinner, filler, salt, damping fluid, stablizer, solubilizing agent, lipid or other material that is used for pharmaceutical formulations well known in the art.Obviously the character of carrier, vehicle or thinner depends on the approach of administration in the special applications.The preparation that the pharmacy that contains these materials can be accepted preparation is recorded in the Sciences as Remington ' s Pharmaceutical, 18thEdition, ed.A.Gennaro, Mack Publishing Co., Easton, PA, 1990.

Toll sample acceptor (TLRs) function is induced congenital and activation adaptive immune response for the transmitter in infecting.TLRs discerns plurality of ligand, is called pathogenic agent associated molecular pattern (PAMPs).Behind the conservative pathogen-associated molecular product of identification, TLRs replys by the defence that its intracellular signal zone, Toll/ interleukin 1 receptor (TIR) zone and downstream adaptin MyD88 activate the host.Dendritic cell and scavenger cell normal response Toll sample acceptor (TLR) part and the cytokine that also produces (interleukin 1 β for example by their; IL-6 and tumour necrosis factor, TNF); Natural killer (NK) cell and T cell are also participated.After the bacterium compound stimulated TLR, the congenital immunity cell discharged a large amount of cytokines.The example of some TLR parts comprises, but be not limited to lipoprotein, peptidoglycan, zymosan (TLR2), double-stranded RNA, polyI:polyC (TLR3), lipopolysaccharides, heat shock protein(HSP), taxol (TLR4), flagellin (TLR5) and imidazoquinolines-R848, resiquimod, Imiquimod, ssRNA (TLR7/8).

Aspect the 4th, the invention provides the method for cancer cells that make to ionizing rays sensitization.The method of this aspect of the present invention comprises administration Mammals immunostimulatory oligonucleotide of the present invention or immune sub-compound and passes through the ionizing radiation treatment animal.In some preferred examples, to use γ-radiation in 1.56Gy/ minute.In some preferred examples, use radiotherapy from about 0.1 to about 10.0Gy, preferably from about 0.25 to about 8.0Gy, more preferably from about 0.5 to about 5.0Gy, or be the radiation of 3.0Gy, perhaps biweekly or Thursday time or at the the 2nd, the 4 and the 9th day three times (three times on Days2,4 and 9).In some instances, be 2 to about 6 hours of γ-radiation precontract with the pre-treatment of immunostimulatory oligonucleotide or immune sub-compound.

Aspect the 5th, the invention provides the method for the collaborative patient of stimulation immunne response, it comprises IL-2 and the antigen administration patient who treatment is effectively worked in coordination with the effectively collaborative amount of immune sub-compound combined treatment of amount, wherein production of cytokines among the collaborative stimulation of the described combination of the administration patient.The preferred stimulated cells factor of the present invention includes but not limited to IL-12, and interferon-is one or more among IFN-α and the IFN-β.

In some instances, treatment method for cancer and antigen-specific in or be associated with a kind of cancer.In some instances, method is used for the treatment of infection, and antigen is and infects relevant antigen.In some instances, method is to be used for the treatment of allergy, and antigen is relevant with allergy.Herein, term " is correlated with " and represents that antigen exists when cancer, allergy or transmissible disease is arranged, but does not exist when no cancer, allergy or transmissible disease, or exists with the amount that reduces.

Herein, term " antigen " expression is by antibody or by T cell antigen receptor specific recognition and bonded material.Antigen comprises peptide, albumen, glycoprotein, polysaccharide, Sphingolipids,sialo and lipid, its part with and the combination.Antigen can be found in nature or is synthetic.Haptens is included in the scope of " antigen ".Haptens is a low-molecular weight compound, and itself does not have immunogenicity, but shows immunogenicity when combining with the immunogenic molecules that contains antigenic determinant.

In some instances, effective antigens is the antigen of the relevant and/or tumour-specific of tumour in the inventive method and the composition.Non-limitative example comprises prostate specific antigen (PSA) and prostate acid phosphatase (PAP), and it, can raise when prostate cancer occurring to be present in the mark in the blood on a small quantity for normal; Cancer antigen 125 (CA-125), its level in ovarian cancer patients raises, and raises sometimes when other cancer occurring; CA 15-3 and CA 27-29, its follow the mammary cancer process and to the treatment reply in be useful; CA 19-9, it generally is used to check the diffusion of carcinoma of the pancreas, and also raises in colon, stomach, cholangiocarcinoma patient; Carcinomebryonic antigen (CEA), it is normally to exist on a small quantity but can raise in the blood samples of patients of multiple cancer; Alpha-fetoprotein, it is the mark of liver cell and sexual cell (nonseminoma) cancer; And galactosyltransferase II, it is the isozyme of galactosyltransferase, its multiple pernicious, mainly be to raise in the gastrointestinal cancer.Known in those skilled in the art, tumour is relevant can buy by commercial sources with tumour specific antigen.The present invention expects that also these can produce as peptide by means commonly known in the art by the antigen and/or the synthetic antigen of recombinant nucleic acid technology generation.

In some example aspect the 5th of the present invention, provide a kind of treatment cancer patients method for cancer, it comprise will the effectively collaborative amount of treatment IL-2 in conjunction with the immune sub-binding substances that contains the sub-compound of aforementioned immunity, and antigen administration patient.In some instances, antigen combines in the position except that come-at-able 5 ' end with immune sub-compound.In some instances, the non-nucleotide linker of immune sub-compound contains the antigen relevant with cancer.In some instances, antigen combines in the position except that its 5 ' end with immune sub-compound.In some instances, antigen produces vaccine effect.For the purposes of the present invention, term " is correlated with " and represents that antigen exists when cancer is arranged, but does not exist when no cancer, or exists with the amount that reduces.

In some embodiment of a fifth aspect of the present invention, immune sub-compound is covalently bound to antigen, and perhaps it can be operatively connected antigen by other mode.Applied as this paper, term " can be operatively connected " and be meant and keep immune sub-compound and antigenic active any connection.This non-limiting instance that effectively connects comprises the part as identical liposome or other this transmission carrier or reagent.Immune therein sub-compound is covalently bound to antigenic embodiment, described covalently bound preferred optional position at the immune sub-compound except the 5 come-at-able ' end of the sub-compound of immunity.For example, antigen can connect to be connected maybe between nucleosides and can be connected to the non-nucleotide linker.Selectable, antigen itself is the non-nucleotide linker.

In a sixth aspect of the present invention, at least a IL-2 that is not used for the combined therapy significant quantity for the immunostimulatory oligonucleotide of immune sub-compound comes selectivity and the collaborative patient of stimulation production of cytokines.The collaborative stimulated cells factor of preferred the present invention is selected from IL-12 and IFN-γ, IFN-α, IFN-β or its combination.According to the present invention, preferably, the immunostimulatory oligonucleotide of immune sub-compound do not contain the oligonucleotide of at least a immunostimulating CpG dinucleotides for comprising those, and wherein C is not that cytosine(Cyt) or deoxidation cytosine(Cyt) and/or G are not guanosine or 2-pancreatic desoxyribonuclease.Other is preferred of the present inventionly not to comprise the immunostimulating oligonucleotide partly of the non-CpG of alternate for the immunostimulatory oligonucleotide of immune sub-compound for those.The example of described alternate immunostimulating part includes but not limited to contain the nucleosides of the secondary structure of oligonucleotide such as the hairpin structure of base that non-natural exists and/or glycosyl and stable oligonucleotide own, described in following United States Patent (USP) and interim U.S. Patent application, be incorporated herein by reference: U.S. Patent number 6,426,334 and 6,476,000; And Application No. 09/770,602,09/845,623,09/965,116,60/440,587,10/361,111,60/471,247,60/477,608.

In certain embodiment of the present invention, each immune sub-compound or immunostimulatory oligonucleotide and IL-2 mix with pharmaceutical acceptable carrier before being administered to the patient.In certain embodiments, immune sub-compound or immunostimulatory oligonucleotide mixed with pharmaceutical acceptable carrier before administration, perhaps as the part combination as medicament composition described in a fourth aspect of the present invention.Applied as this paper, term " carrier " comprises vehicle arbitrarily, thinner, weighting agent, salt, buffer reagent, stablizer, solvating agent, lipid, or other material that is used for pharmaceutical preparation well known in the art.Need know, for special application, carrier, the character of vehicle or thinner will depend on the approach of administration.The preparation that pharmacy can be accepted preparation for example is included in, Remington:The Scienceand Practice of Pharmacy, 20 ^ThEdition, ed.A.L.Gennaro, Lippincott Williams﹠amp; Wilkins Publishing Co., Philadelphia, the material of describing among the PA, 19106 (ISBN:0683306472).

Aspect the 7th, the invention provides the therapeutic composition that contains pharmaceutical acceptable carrier, the immune sub-compound or the immunostimulatory oligonucleotide of the effectively collaborative amount of treatment, the IL-2 of the effectively collaborative amount of treatment and optional antigen, the collaborative patient's production of cytokines that stimulates of the administration of wherein said therapeutic composition.Preferably be selected from IL-12 and interferon-by the collaborative stimulated cells factor, IFN-α, IFN-β or its combination according to of the present invention.

All aspects of the present invention are effectively to the treatment disease, and especially to cancer, and communicable disease and hypersensitive treatment based on immunity are useful." processing " or " treatment " as the applied term disease of this paper comprising: the prevention of disease, the alleviating or eliminate of the S or S of morbidity back disease, and the prevention of the recurrence of disease.

According to method of the present invention, can include but not limited to by any suitable pathways with the immune sub-compound of IL-2 combination or the administration of immunostimulatory oligonucleotide, parenterai administration, oral administration, sublingual administration, percutaneous dosing, topical, intranasal administration, aerosol, eye drops, tracheae administration, rectal administration, vagina administration, by particle gun, skin patch or eye drops or mouth wash shua form.The sub-compound of immunity, immunostimulatory oligonucleotide, the administration of IL-2 or its therapeutic composition can adopt the effectively collaborative amount of treatment to carry out with known method with the treatment effective time period of disease.

Term " combination " is meant in the process of the identical patient's of treatment identical disease, comprises with any order administration sub-compound of immunity and/or immunostimulatory oligonucleotide and/or IL-2, comprises administration simultaneously, and separates several days the timed interval of as many as.Described combined therapy can comprise sub-compound of immunity of administration more than once and/or immunostimulatory oligonucleotide, and/or the independently administration of IL-2.Sub-compound of immunity and IL-2 can pass through identical or different administration.

Those skilled in the art will recognize or immune sub-compound or immunostimulatory oligonucleotide, the described synergy that IL-2 or the two all have will be organized along with the present invention, organ, the patient's of special disease or needs treatment difference may be for different.Further, those skilled in the art will recognize that or immune sub-compound or immunostimulatory oligonucleotide that perhaps the meticulous adjusting and the change of the amount that the effectively collaborative amount of the treatment of IL-2 may be by other component reduce or increase.

When the whole body administration, immune sub-compound is preferably to reach the enough dose administration of the blood levels from about 0.0001 micromole to the sub-compound of about 10 micromolar immunity.For topical, can be effectively than this much lower concentration, and high much can accepting of concentration.Preferably, the total dose of immunostimulatory oligonucleotide and/or immune sub-compound from each patient's every day about 0.0001mg to per kilogram of body weight 200mg every day.May wish administration simultaneously, the sub-compound of every kind of immunity or the IL-2 that perhaps will treat effective collaborative amount are administered to individuality as treating event sequence separately.Preferably, IL-2 is with the about 750 amount administrations to about 75,000 units.

The invention provides and contain cytokine and/or chemotherapeutics, and the test kit of immunostimulatory oligonucleotide and/or immune sub-compound, the latter comprises at least two oligonucleotide that link together, so that immune sub-compound has 5 ' the come-at-able end more than, wherein at least one oligonucleotide is an immunostimulatory oligonucleotide.On the other hand, test kit comprises according to immunostimulatory oligonucleotide of the present invention and/or immunostimulatory oligonucleotide binding substances and/or immune sub-compound or immune sub-binding substances, and cytokine and/or chemotherapeutics and physiology can be accepted carrier.Test kit also comprises a series of operation instructions usually.

Following embodiment is used for further illustrating preferred implementation of the present invention, and is not used in the scope of the present invention that limits.

Embodiment

Embodiment 1: the synthetic oligonucleotide that contains the immunomodulatory part

Or the parallel synthesis program synthetic according to Fig. 5 with 6 linearities that outline, (Expedite 8909 to use automatic dna synthesizer; PerSeptive Biosystems, Framingham, MA) oligonucleotide of synthetic 1 μ mol size.

(Foster City CA) obtains the thymus nucleic acid phosphoramidite from Applied Biosystems.1 ', 2 '-dideoxy ribose phosphoramidite, propyl group-1-phosphoramidite, 2-deoxyuridine phosphoramidite, 1,3-two-[5-(4,4 '-dimethoxytrityl) phenyl amide base]-2-propyl alcohol phosphoramidite (1,3-bis-[5-(4,4 '-dimethoxytrityl) pentylamidyl] α-propanol phosphoramidite) and methyl phosphoramidite available from Glen Research (Sterling, VA).β-L-2 '-deoxyribonucleoside phosphoramidite, α-2 '-deoxyribonucleoside phosphoramidite, list-DMT-glycerine phosphoramidite and two-DMT-glycerine phosphoramidite available from ChemGenes (Ashland, MA).(4-amino butyl)-1, the ammediol phosphoramidite available from Clontech (Palo Alto, CA).Pectinose cytidine phosphoramidite, pectinose guanosine, pectinose thymidine and pectinose uridine available from Reliable Pharmaceutical (St.Louis, MO).By Hybridon, Inc. (Cambridge, MA) synthetic pectinose guanosine phosphoramidite, pectinose thymidine phosphoramidite and pectinose uridine phosphoramidite (Noronha et al. (2000) Biochem., 39:7050-7062).

All nucleoside phosphoramidites are all used ³¹P and ¹H NMR spectral characterization.The Nucleotide of modifying mixes with the normal coupling cycle at special site.After synthetic, oligonucleotide is with spissated ammoniacal liquor deprotection, and by the reverse hplc purifying, then dialyses.The freeze-drying before use of the oligonucleotide of the sodium-salt form of purifying.Detect purity by CGE and MALDi-TOF MS.

Embodiment 2: the analysis of splenocyte propagation

The standard program of describing before using (see, as Zhao etc., Biochem Pharma51:173-182 (1996)) breed at the analyzed in vitro splenocyte.The results are shown among Fig. 8 A.This result shows with not having come-at-able 5 ' terminal immunity 5 or have single come-at-able 5 ' terminal oligonucleotide 4 and compares to have two come-at-able 5 ' terminal immunity sons 6 and produce more splenocyte propagation in higher concentration.Immunity 6 has also caused more splenocyte propagation than LPS positive control.

Embodiment 3: splenomegaly experiment in the body

In order to test the suitability of in vitro results, with selected oligonucleotide administration mouse and measure the index of the degree of splenomegaly as the immunostimulatory activity level to the body inner model.(female, 4-6 week is big, Harlan Sprague Dawley Inc, Baltic, intraperitoneal administration CT) to BALB/c mouse with the single dose of 5mg/kg.The oligonucleotide administration was put to death mouse after 72 hours, and the taking-up spleen is also weighed.The results are shown among Fig. 8 B.This result shows that having two come-at-able 5 ' terminal immunity sons 6 has obviously better immunostimulating effect than oligonucleotide 4 or immunity 5.

Embodiment 4: cytokine analysis

Measure vertebrate cells by sandwich ELISA, the secretion of IL-12 and IL-6 among preferred BALC/c mouse boosting cell or the human PBMC.Required reagent (comprising cytokine antibodies and cytokine standard) is from PharMingen, San Diego, and CA buys.ELISA target (Costar) is incubated overnight under 4 ℃ with the suitable antibodies of 5 μ g/mL in PBSN damping fluid (PBS/0.05% sodiumazide, pH 9.6), then blocks 30 minutes down at 37 ℃ with PBS/1%BSA.Cell culture supernatant and cytokine standard suitable with PBS/10%FBS dilution, it was triplicate to be added to target, 25 ℃ of incubations 2 hours.Target covers with the suitable biotinylated antibody of 1 μ g/mL, and 25 ℃ of incubations 1.5 hours.Target is with PBS-T damping fluid (PBS/0.05%Tween 20) thorough washing then, and add streptavidin bonded peroxidase (Sigma, St.Louis, MO) after further incubation 1.5 hours in 25 ℃.Target develops with Sure Blue (Kirkegaard and Perry) colouring reagents, and reaction stops by adding Stop Solution (Kirkegaard and Perry).Measure change in color with Ceres 900 HDI spectrophotometers (Bio-Tek Instruments).The results are shown among the following table 5A.

By Ficoll-Paque density gradient centrifugation (Histopaque-1077, Sigma, St.Louis, MO) separation of human peripheral blood lymphocytes (PBMCs) from the peripheral blood of healthy premenopausal volunteers.Briefly, heparinized blood places on the Histopaque-1077 (equal-volume) in point end whizzer, with 400xg centrifugal at room temperature 30 minutes.Careful shifting out contains monocytic buffy coat, passes through with centrifugal 10 minutes washed twice of 250xg with isotonic phosphate buffer salt solution (PBS).Then the cell mass that obtains is suspended in again RPMI 1640 substratum that contain L-glutaminate (MediaTech, Inc., Herndon, VA) in, and additional with the FCS and the penicillin-Streptomycin sulphate (100U/ml) of 10% heat inactivation.Exist or do not exist under the situation of oligonucleotide, cell is with 1 * 10 ⁶Cultivate the different time periods in 24 orifice plates in individual cell/ml/ hole.Behind cultivation stage, collect supernatant liquor and freezing being stored in-70 and ℃ analyze various cytokines until being used for sandwich ELISA, comprise IL-6 (BD Pharmingen, San Diego, CA), IL-10 (BD Pharmingen), IL-12 (BioSource International, Camarillo, CA), IFN-α (BioSource International) and-γ (BD Pharmingen) and TNF-α (BDPharmingen).The results are shown among following table 9 and the 9A.

In all examples, the level of IL-12 and IL-6 all is to calculate according to the typical curve of IL-12 that makes up under the same experimental conditions and IL-6 respectively in the cell culture supernatant liquid.The level of IL-10, IFN-γ and TNF-α is to calculate according to the typical curve of IL-10, IFN-γ and TNF-α under the same experimental conditions respectively in the cell culture supernatant liquid.

Immune minor structure of table 9 and the immunostimulatory activity in human PBMC's culture

Oligonucleotide No.	Sequence and modification (5 '-3 ')	Oligonucleotide length/or every chain	IL-12 (pg/mL)	IL-6(pg/mL)
					D1 D2	D1 D2

D1 and D2 are donor 1 and 2.

Table 9A immunity minor structure and the immunostimulatory activity in BALB/c mouse spleen cell cultures thing

Italic represents that phosphodiester connects.

In addition, Fig. 7 A-C result displayed shows, with have one or do not have come-at-able 5 ' terminal oligonucleotide 1 or immunity 3 to compare respectively, have two come-at-able 5 ' terminal immunity sons 2 and improve IL-12 and IL-6, but do not improve IL-10 in lower concentration.

Embodiment 5: the immunostimulatory activity that contains the immune sub-compound of non-natural pyrimidine or non-natural purine nucleoside

As show shown in the 10-12, the immune sub-compound that partly has the different lengths of non-natural pyrimidine nucleoside or non-natural pyrimidine nucleoside at immunostimulatory dinucleotide has still kept immunostimulatory activity.

Immune minor structure of table 10 and immunostimulatory activity

Immune minor structure of table 11 and immunostimulatory activity

Immune minor structure of table 12 and immunostimulatory activity

Embodiment 6: linker influences immunostimulatory activity

Be the influence of the linker length that detect to connect two oligonucleotide, syntheticly contain identical oligonucleotide but the immune sub-compound of different linkers and be used to test immunostimulatory activity.Result in the table 13 shows that linker length has influence to the immunostimulatory activity of the sub-compound of immunity.Obtained best immunostimulating effect when using C3-to C6-alkyl linker or having the no base linker of phosphoric acid salt electric charge of distribution.

Immune minor structure of table 13 and immunostimulatory activity

Embodiment 7: the oligonucleotide main chain is to the influence of immunostimulatory activity

Generally speaking, to compare immunostimulating lower for the immunostimulatory oligonucleotide that contains the natural phosphodiester main chain and the oligonucleotide with thiophosphatephosphorothioate main chain of same length.This lower level immunostimulatory activity can part owing to the quick degraded of phosphodiester oligonucleotide under the experiment condition.The degraded of oligonucleotide mainly by 3 '-exonuclease causes, this enzyme is since 3 ' end oligonucleotide of degrading.Immune sub-compound does not contain free 3 ' end in this example.Therefore, the immune sub-compound with phosphodiester backbone is compared under experiment condition with corresponding monomer oligonucleotide to have the longer transformation period, and therefore demonstrates better immunostimulatory activity.Result in the table 14 has proved this influence, and immunity son 84 and 85 has shown immunostimulatory activity, shows as inducing of cytokine in the BALB/c mouse spleen cell cultures thing.

Immune minor structure of table 14 and immunostimulatory activity

The L=C3-linker

Embodiment 8: immune sub-compound is in conjunction with the vivo antitumor activity of chemotherapeutics

At the 90%Ham ' s that contains 10% foetal calf serum (FBS), in the F12K substratum, under the condition that has 100U/ml penicillin and 100 μ g/ml Streptomycin sulphates, cultivate the PC3 cell to set up human prostata cancer model (PC3).(Frederick Cancer Research andDevelopment Center, Frederick MD) adapt to 6 days to conform earlier to all big male athymic nude mice of 4-6 before research.The PC3 cell that results are cultivated from the monolayer culture thing is used Ham ' s, and F12K substratum (10%FBS) is washed twice, again be suspended in the Ham ' s of no FBS, F 12K substratum: Matrigel basement membrane matrix (BectonDickinson Labware, Bedford, MA) (5: 1; V/V), subcutaneous injection (5 * 10 ⁶Individual cell, cumulative volume 0.2ml) to the left inguinal region territory of every mouse.Animal is monitored by clinic observation, body weight and the tumor growth of routine.Adopt calipers to measure two perpendicular diameter of implant with the monitoring growth of tumor.With formula 1/2a * b ²Calculate the quality (is weight with the gram) of tumour, wherein ' a ' is long diameter (cm), and ' b ' is short diameter (cm).When the average tumor size reaches～80mg, the animal with human tumour heterogeneity graft is divided into treatment group and control group (5 animal/groups) at random.Control group only imposes disinfectant physiological saline (0.9%NaCl).Aseptic be dissolved in immunity son 26 or 194 in the physiological saline by subcutaneous injection with 0.5 or 1.0mg/kg/ days dosed administration, 3 dosage/week.With the dosage of 160mg/kg the 0th day and the 3rd day twice peritoneal injection gemcitabine hydrochloride HCL (Eli Lilly and Company, Indianapolis, IN).Detailed treatment progress chart is as follows:

G1: salt solution

G2: gemcitabine (160mg/kg/ days, IP, the 0th day and the 3rd day)

G3:26 (1.0mg/kg/ days, SC, 3 dosage/week, 6 weeks)

G4:26 (0.5mg/kg/ days, SC, 3 dosage/week, 6 weeks)

G5:194 (1.0mg/kg/ days, SC, 3 dosage/week, 6 weeks)

G6:194 (0.5mg/kg/ days, SC, 3 dosage/week, 6 weeks)

G7:26 (0.5mg/kg/ days, SC, 3 dosage/week, 6 weeks)+gemcitabine (160mg/kg/ days, the 0th day and the 3rd day)

G8:194 (0.5mg/kg/ days, SC, 3 dosage/week, 6 weeks)+gemcitabine (160mg/kg/ days, the 0th day and the 3rd day)

It among table 15 and Figure 13 the measurement of tumor value after the different treatments.Compare with saline control, in the animals of useful immunity son 26 and 194 treatments growth of tumor obviously be suppressed (p＜0.5).The tendency (Figure 13) that dose response relation is arranged in these treatment groups.Not significantly difference (table 15) between 26 and 194.

The tumor quality of table 15 tumor-bearing mice with 26,194, behind gemcitabine or the combined therapy

My god	Salt solution	SD	SE	Gemcitabine	SD	SE	26	SD	SE	26	SD	SE
																	160mg/kg			1mg/kg			0.5mg/kg
0	82.7	16.7	7.5	82.6	15.7	7.0	80.1	10.6	4.7	80.4	10.5	4.7
													3	81.9	13.3	5.9	73.0	3.4	1.5	67.5	8.1	3.6	54.3	8.4	3.7
6	80.5	11.5	5.2	50.4	11.7	5.2	50.4	9.0	4.0	45.3	5.5	2.5
													9	87.7	8.2	3.7	35.7	6.3	2.8	40.9	5.1	2.3	43.9	9.3	4.2
12	97.6	18.6	8.3	36.2	3.3	1.5	41.3	6.2	2.8	46.5	3.8	1.7
													15	112.0	21.5	9.6	31.7	4.1	1.8	42.8	12.8	5.7	50.0	14.1	6.3
18	126.3	17.3	7.7	40.8	8.4	3.7	54.9	7.6	3.4	59.3	6.7	3.0
													21	152.5	25.5	11.4	47.4	9.8	4.4	62.5	10.4	4.6	71.0	16.7	7.5
24	187.0	29.2	13.1	56.5	5.2	2.3	79.5	24.1	10.8	100.1	9.7	4.3
													27	245.2	24.1	10.8	68.0	14.8	6.6	94.1	28.9	12.9	124.5	21.1	9.5
30	343.6	63.9	28.6	89.4	11.1	5.0	119.8	18.7	8.3	162.4	37.5	16.8
													33	438.5	107.1	47.9	106.5	14.1	6.3	176.6	43.8	19.6	213.6	66.7	29.8
36	614.4	185.1	82.8	144.2	48.2	21.6	248.7	47.0	21.0	325.3	106.2	47.5
													39	866.8	237.4	106.2	175.3	61.4	27.5	320.1	64.2	28.7	416.8	154.5	69.1
42	1136.9	205.9	92.1	269.1	78.8	35.2	417.8	78.7	35.2	546.9	139.1	62.2
													45				383.8	146.4	65.5	550.8	134.2	60.0	667.6	284.9	127.4
48				538.6	260.1	116.3	736.0	197.3	88.2	852.8	399.3	178.6

My god	194	SD	SE	194	SD	SE	26+GEM	SD	SE	194+GEM	SD	SE
														1mg/kg			0.5mg/kg			0.5/160mg/kg			0.5/160mg/kg
0	80.4	11.0	4.9	79.9	10.3	4.6	79.4	10.1	4.5	78.7	12.0	5.4
													3	52.3	9.3	4.2	64.7	9.0	4.0	45.1	8.2	3.7	44.6	8.7	3.9

6	38.8	4.6	2.1	46.9	14.7	6.6	31.2	5.9	2.6	34.7	4.4	2.0
													9	34.5	9.5	4.3	43.5	13.6	6.1	22.1	4.8	2.1	23.0	3.2	1.5
12	35.8	9.4	4.2	43.0	15.9	7.1	15.0	3.8	1.7	11.9	2.2	1.0
													15	36.6	8.7	3.9	48.6	15.4	6.9	18.0	3.1	1.4	12.4	3.5	1.6
18	45.1	14.6	6.5	62.0	20.2	9.0	17.9	3.1	1.4	15.5	1.7	0.8
													21	53.5	12.3	5.5	73.6	20.5	9.2	18.3	2.8	1.2	14.8	2.1	1.0
24	72.6	22.7	10.1	93.6	23.0	10.3	23.6	4.5	2.0	23.0	1.5	0.7
													27	86.5	13.7	6.1	119.3	17.3	7.8	27.8	4.1	1.8	25.9	3.7	1.7
30	114.5	22.8	10.2	157.1	49.0	21.9	33.6	5.0	2.2	36.9	6.5	2.9
													33	161.4	44.1	19.7	218.1	81.2	36.3	43.8	10.9	4.9	47.7	16.1	7.2
36	198.3	43.5	19.4	313.2	104.6	46.8	50.3	13.6	6.1	46.4	16.4	7.3
													39	249.8	77.9	34.9	420.2	199.4	89.2	67.3	29.4	13.2	59.4	28.7	12.9
42	366.5	110.5	49.4	527.5	219.0	98.0	77.2	28.0	12.5	82.1	29.1	13.0
													45	490.2	122.2	54.7	620.3	258.1	115.4	104.9	57.9	25.9	110.7	46.3	20.7
48	683.4	144.6	64.7	759.1	223.0	99.7	128.2	77.7	34.7	133.4	62.6	28.0
													51							177.9	109.6	49.0	177.3	68.0	30.4
54							233.1	143.5	64.2	224.0	79.8	35.7
													57							297.7	190.7	85.3	289.7	121.9	54.5

Table 16 and Figure 14 represent to treat the measured body weight value of back different time.Compared with the control, weight increase is obviously not different in separately with 26 or 194.The animal of gemcitabine treatment recovers in a week subsequently at first all weight loss.The side effect that does not change gemcitabine with 26 or 194 combinations shows.In all groups, all there be not the clinical unusual or dead of other.

Table 16 tumor-bearing mice with 26,194 or brine treatment after body weight

My god	Salt solution	SD	SE	Gemcitabine	SD	SE	26	SD	SE	26	SD	SE
																	160mg/kg			1mg/kg			0.5mg/kg
0	24.1	2.5	1.1	23.5	0.9	0.4	23.2	1.4	0.6	23.0	2.4	1.1
													7	25.8	3.0	1.3	20.7	4.4	2.0	25.2	2.4	1.1	24.8	2.8	1.2
14	26.8	3.2	1.4	25.2	4.0	1.8	26.3	2.0	0.9	26.0	2.9	1.3
													21	28.2	3.3	1.5	27.1	3.9	1.7	27.8	2.0	0.9	27.6	2.8	1.2
28	29.4	3.5	1.6	28.1	4.3	1.9	28.6	2.6	1.1	28.0	2.7	1.2
													35	30.6	3.7	1.6	29.4	2.9	1.3	29.5	2.3	1.0	28.6	2.8	1.3
42	31.1	3.7	1.7	30.3	3.0	1.4	30.2	2.3	1.0	29.4	3.9	1.7

My god	194	SD	SE	194	SD	SE	26+GEM	SD	SE	194+GEM	SD	SE
														1mg/kg			0.5mg/kg			0.5/160 mg/kg			0.5/160 mg/kg
0	22.5	1.3	0.6	24.1	1.6	0.7	21.9	1.7	0.7	23.0	0.8	0.4
													7	24.3	0.9	0.4	25.6	2.0	0.9	19.1	2.0	0.9	22.3	3.3	1.5
14	25.1	1.3	0.6	27.0	2.1	0.9	24.6	1.6	0.7	25.9	2.7	1.2
													21	26.1	1.3	0.6	27.8	1.5	0.7	26.8	1.6	0.7	27.1	2.6	1.2
28	27.2	1.5	0.7	28.3	2.2	1.0	27.2	1.6	0.7	27.7	3.2	1.4
													35	28.0	1.4	0.6	29.1	2.3	1.0	27.7	2.1	1.0	28.0	2.4	1.1
42	28.9	1.5	0.7	29.8	2.2	1.0	28.4	2.8	1.2	28.1	3.4	1.5

In a word, 26 and 194 obviously suppress to have growth of tumor in the nude mouse of human prostate cancer PC3 heterograft, do not have significant side effects.When 26 or 194 combination gemcitabines were used jointly, each compound had all obviously increased the result of treatment of gemcitabine, and does not change its side effect performance.In addition, the dose response tendency that has 26 or 194 treatments.

Embodiment 9 immune sub-compounds are in conjunction with the vivo antitumor activity of chemotherapeutics

Substitute the experiment that gemcitabine repeats embodiment 8 with taxotere.Taxotere was the 0th day and administration in the 7th day.165 administrations 5 days weekly.26 and 194 the 0th, 2,4,7, administration in 9 and 11 days.Following table 17 display result.This result shows the synergy between immune sub-compound and the taxotere clearly.

The immune sub-compound of table 17 is in conjunction with the vivo antitumor activity of other chemotherapeutics

My god	Salt solution	SD	SE	Taxotere (16mg/kg)	SD	SE	165(20 mg/kg)	SD	SE	26(1 mg/kg)	SD	SE
													0.00 3.00 6.00 9.00 12.00 15.00	56.93 196.42 708.85 1370.95 2222.96 3303.04	7.92 22.48 32.64 239.99 300.65 672.86	3.54 10.05 14.60 107.33 134.45 300.91	56.64 128.51 320.63 598.89 924.91 1589.08	7.94 20.83 136.80 196.60 297.89 578.38	3.55 9.32 61.18 87.92 133.22 258.66	57.93 95.79 285.71 534.93 994.10 1601.73	5.56 16.04 68.70 225.19 474.89 576.19	2.49 7.18 30.72 100.71 212.38 257.68	56.74 87.12 250.36 450.46 814.21 1465.87	7.79 6.64 52.58 92.25 197.16 348.37	3.48 2.97 23.51 41.26 68.17 155.80

Taxotere+166	SD	SE	Taxotere+26 ( ** mg/kg)	SD	SE	194(1mg/kg)	SD	SE
									55.51 78.47 211.52 302.66 496.20 686.47	9.55 21.79 88.59 178.36 342.69 385.97	4.27 9.74 39.62 79.76 153.25 172.61	56.59 80.14 216.85 307.53 510.18 703.50	8.91 21.59 89.40 184.05 351.16 394.65	3.99 9.65 39.98 82.31 157.04 176.49	55.28 91.01 303.00 512.30 884.12 1479.21	10.89 23.60 61.33 110.16 308.22 416.64	4.87 10.55 27.43 49.26 137.84 186.33

The administration of embodiment 10 immunostimulatory oligonucleotides and IL-2

As above-mentioned from the BALB/c mouse separating Morr. cell, with 5 * 10 ⁶The density of cell/mL places 24 orifice plates.Be dissolved in TE damping fluid (10mM Tris-HCI, pH7.5,1mM EDTA) CpG oligonucleotide with 0.03,0.1,0.3,1.0,3.0, or the ultimate density of 10.0 μ g/mL adds in the mouse boosting cell culture.In order to study the effect of IL-2 in CpG oligonucleotide inductive time-dependent manner cytokine secretion, adding concentration when the experiment beginning is the recombinant human il-2 (Sigma) of 10U/ml.Under the condition that has the experiment oligonucleotide, cell was cultivated 4,8,24 and 48 hours in 37 ℃, collected supernatant liquor and carry out elisa assay.There is not the cell (only adding IL-2) handled in contrast.

Measure mouse IL-12, the secretion of IL-6 and IFN-γ by sandwich ELISA.Required reagent (comprising cytokine antibodies and standard) is buied from PharMingen.Capture antibody suitable in ELISA target (Costar) and PBSN (PBS/0.05% sodiumazide, the pH 9.6) damping fluid is incubated overnight under 4 ℃, then blocks 30 minutes down at 37 ℃ with PBS/1%BSA.Cell culture supernatant and cytokine standard suitable with PBS/10%BSA dilution, be added to target, triplicate, 25 ℃ of incubations 2 hours.The washing target and with suitable biotinylated antibody 25 ℃ of incubations 1.5 hours.Target is with PBS/0.05%Tween 20 washing, adds behind the streptavidin bonded peroxidase (Sigma) further in 25 ℃ incubation 1.5 hours.Target Sure Blue ^TM(Kirkegaard and Perry) colouring reagents develops, and reaction stops by adding Stop Solution (Kirkegaard and Perry).Be determined at the colour-change of 450nm with Ceres 900HDI Spectrophotometer (Bio-Tek Instruments).IL-12 in the cell culture supernatant liquid, the level of IL6 and IFN-γ is respectively according to IL-12 under the same experimental conditions, the typical curve of IL6 and IFN-γ calculates.

Table 18 shows the oligonucleotide that uses in this research.

Table 18

SEQ	The ID sequence	Chemistry
			NO： 86 87 88 89 90	5’-CTATCTGACGTTCTCTGT -3’ (5’-TCTGACRTTCT)2S (5’-TCTGACGTTCT)2S (5’-XXCTGACGTTCTCTGT)2 S (5’-TCTGAYGTTCT)2S	PS-oligonucleotides R=7-denitrogenation-dG, PS-oligonucleotides PS-oligonucleotides PO-oligonucleotides Y=R*, the PS-oligonucleotide

The results are shown among Figure 15-19.The experiment that does not have to show shows that independent SEQ ID NOs 86-90 is inappreciable to the stimulation that IFN-γ produces.This result has proved and has produced IL-6, the synergy in IL-12 and the IFN-γ secretion between SEQ ID NOs 86-90 and the IL-2.

Equivalent

Though aforementioned invention is described in detail for purpose clear and that be convenient to understand, those skilled in the art can understand on the various forms and the variation on the details does not break away from the true scope of the present invention and accessory claim by reading this specification sheets.

Claims (18)

1. the method for treatment mammalian cancer comprises to the Mammals that suffers from tumour giving immunostimulatory oligonucleotide or immune sub-compound, and treats animal with ionizing radiation.

2. the described method of claim 1, wherein γ-radiation was used with 1.56Gy/ minute.

3. the described method of claim 1 is wherein with twice weekly of the radiation quantity of 3Gy, carry out radiotherapy on every Thursdays time or at the 2nd, 4 and 9 day three times.

4. the described method of claim 1, wherein Mammals before γ-radiation from about 2 to about 6 hours with immunostimulatory oligonucleotide or immune sub-compound pre-treatment.

5. stimulate the method for patient's immunne response, comprise to the patient and treat the sub-compound of at least a immunity of effectively collaborative amount or the IL-2 of immunostimulatory oligonucleotide and the effectively collaborative amount of treatment.

6. stimulate the method for patient's immunne response, comprise to the patient and treat the sub-compound of at least a immunity or the immunostimulating Nucleotide of effectively collaborative amount and the combination for the treatment of the IL-2 of effectively collaborative amount, wherein give the generation of the collaborative stimulating cytokine of described combination.

7. stimulate the method for patient's immunne response, comprise the combination for the treatment of the sub-compound of at least a immunity or the immunostimulatory oligonucleotide and the effectively collaborative IL-2 that measures of treatment of effectively collaborative amount to the patient, give wherein that described combination is collaborative to stimulate one or more to be selected from IL-12 and IFN-γ, IFN-α, the production of cytokines of IFN-β or its combination.

8. the described method of claim 7, wherein immune sub-compound has following structure:

9. claim 5,6 or 7 method, it further comprises to the patient and gives antigen.

10. the method for claim 9, wherein said antigen is the antigen relevant with cancer, communicable disease or allergy.

11. composition, it comprises the sub-compound of at least a immunity of the effectively collaborative amount of treatment or the IL-2 of immunostimulatory oligonucleotide and the effectively collaborative amount of treatment, give wherein that described composition is collaborative to stimulate one or more to be selected from IL-12 and IFN-γ, IFN-α, the production of cytokines of IFN-β or its combination.

12. treatment patient method for cancer, comprise to the patient and treat the sub-compound of at least a immunity of effectively collaborative amount or the IL-2 of immunostimulatory oligonucleotide and the effectively collaborative amount of treatment, give wherein that described composition is collaborative to stimulate one or more to be selected from IL-12 and IFN-γ, IFN-α, the production of cytokines of IFN-β or its combination.

13. the described method of claim 12 further comprises giving the antigen relevant with cancer.

14. the hypersensitive method of treatment patient, comprise the sub-compound of at least a immunity or immunostimulatory oligonucleotide and the effectively collaborative IL-2 that measures of treatment that give the effectively collaborative amount of patient treatment, give wherein that described composition is collaborative to stimulate one or more to be selected from IL-12 and IFN-γ, IFN-α, the production of cytokines of IFN-β or its combination.

15. the described method of claim 14 further comprises giving the antigen relevant with described allergy.

16. the method for treatment patient communicable disease, comprise to the patient and treat the sub-compound of at least a immunity of effectively collaborative amount or the IL-2 of immunostimulatory oligonucleotide and the effectively collaborative amount of treatment, give wherein that described composition is collaborative to stimulate one or more to be selected from IL-12 and IFN-γ, IFN-α, the production of cytokines of IFN-β or its combination.

17. the described method of claim 16 further comprises giving the antigen relevant with described communicable disease.

18. stimulate the method for patient's immunne response, comprise the immunostimulatory oligonucleotide that gives the effectively collaborative amount of patient treatment, it comprises at least a immunostimulating CpG dinucleotides, wherein C is not that cytosine(Cyt) or deoxidation cytosine(Cyt) and/or G are not guanosine or 2-pancreatic desoxyribonuclease, and the IL-2 of the effectively collaborative amount of treatment.

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