US 20100166780A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2010/0166780 A1 Debelak et al. (43) Pub. Date: Jul. 1, 2010

(54) CPG OLGONUCLEOTDE ANALOGS Related U.S. Application Data CONTAINING HYDROPHOBC TANALOGS WITHENHANCED IMMUNOSTIMULATORY (60) Provisional application No. 60/847,811, filed on Sep. ACTIVITY 27, 2006. (75) Inventors: Harald Debelak, Hilden (DE); Publication Classification Eugen Uhlmann, Glashuetten (51) Int. Cl. (DE); Marion Jurk, Dormagen A6139/00 (2006.01) (DE) C7H 9/06 (2006.01) Correspondence Address: C7H 9/16 (2006.01) PHARMACA & UPJOHN C07H 19/052 (2006.01) 7000 Portage Road, KZO-300-104 C7H 9/056 (2006.01) KALAMAZOO, MI 49001 (US) A63L/702 (2006.01) A6IP3L/2 (2006.01) (73) Assignee: Pfizer Inc (52) U.S. Cl...... 424/184.1:536/28.4:536/27.21; (21) Appl. No.: 12/442,295 536/28.53: 536/28.8:536/28.7; 514/44 R (22) PCT Fled: Sep. 27, 2007 (57) ABSTRACT (86) PCT NO.: PCT/B2007/004389 The invention relates to oligonucleotides including at least one lipophilic Substituted analog and a pyrimi S371 (c)(1), dine- dinucleotide. The invention also relates to phar (2), (4) Date: Dec. 7, 2009 maceutical compositions and methods of use thereof.

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CPG OLGONUCLEOTIDE ANALOGS induces IFN-O.; this class has been termed the C-class. The CONTAINING HYDROPHOBC TANALOGS C-class CpG nucleic acids, as first characterized, typically are WITHENHANCED IMMUNOSTIMULATORY fully stabilized, include a B class-type sequence and a GC ACTIVITY rich palindrome or near-palindrome. This class has been described in co-pending U.S. provisional patent application FIELD OF THE INVENTION 60/313,273, filed Aug. 17, 2001 and U.S. Ser. No. 10/224,523 filed on Aug. 19, 2002 and related PCT Patent Application 0001. The present invention relates generally to the field of PCT/US02/26468 published under International Publication immunology. More specifically the invention relates to thera Number WO O3/O15711. peutic oligonucleotides with enhanced immunostimulatory capacity. SUMMARY OF THE INVENTION BACKGROUND OF THE INVENTION 0005. The invention relates to an oligonucleotide which comprises one or more modifications that elicits enhanced 0002 Bacterial DNA has immune stimulatory effects to immunostimulatory capacity. In particular, the invention is activate B cells and natural killer cells, but vertebrate DNA based on the finding that specific Sub-classes of oligonucle does not (Tokunaga, T., et al., 1988. Jpn. J. Cancer Res. otides having at least one lipophilic Substituted nucleotide 79:682-686: Tokunaga, T., et al., 1984, JNCI 72:955-962: analog are highly effective in mediating immune response. Messina, J. P., et al., 1991, J. Immunol. 147: 1759-1764; and These oligonucleotides are useful therapeutically and pro reviewed in Krieg, 1998. In: Applied Oligonucleotide Tech phylactically for inducing an immune response and for treat nology, C. A. Stein and A. M. Krieg, (Eds.), John Wiley and ing diseases and disorders such as cancer and viral infections. Sons, Inc., New York, N.Y., pp. 431-448). It is now under 0006. In one aspect, the invention is a composition com stood that these immune stimulatory effects of bacterial DNA prising the sequence: RYZR, wherein RandR representa are a result of the presence of unmethylated CpG dinucle lipophilic Substituted nucleotide analog (L), a nucleotide, and otides in particular base contexts (CpG motifs), which are a linkage, wherein at least one of R and R is a lipophilic common in bacterial DNA, but methylated and underrepre Substituted nucleotide analog (L), wherein Y is a sented in vertebrate DNA (Kriegetal, 1995 Nature 374:546 nucleotide and wherein Z is a purine, a pyrimidine, or an 549; Krieg, 1999 Biochim. Biophys. Acta 93321:1-10). The abasic residue. immune stimulatory effects of bacterial DNA can be mim icked with synthetic oligodeoxynucleotides (ODN) contain 0007. In some embodiments, L comprises a 5- or 6-mem ing these CpG motifs. Such CpG ODN have highly stimula bered ring analog. tory effects on human and murine leukocytes, inducing B cell 0008. In other embodiments of the aspect of the invention, proliferation; cytokine and immunoglobulin secretion; natu L is a group of formula I. ral killer (NK) cell lytic activity and IFN-y secretion; and activation of dendritic cells (DCs) and other antigen present ing cells to express costimulatory molecules and secrete Formula I cytokines, especially the Th1-like cytokines that are impor tant in promoting the development of Th1-like T cell airls1. responses. These immune stimulatory effects of native phos BSSn1 - -D phodiester backbone CpG ODN are highly CpG specific in that the effects are dramatically reduced if the CpG motif is methylated, changed to a GpC, or otherwise eliminated or altered (Krieg et al., 1995 Nature 374:546-549; Hartmann et having the following elements: A, B, X, D, E, and F are C al, 1999 Proc. Natl. Acad. Sci. USA96:9305-10). (carbon) or N (nitrogen) optionally bearing hydrogen or a 0003. In early studies, it was thought that the immune substituent; n is 0 or 1; the dotted lines indicate optional stimulatory CpG motif followed the formula purine-purine double bonds; wherein at least one substituent is not chosen CpG-pyrimidine-pyrimidine (Krieg et al., 1995 Nature 374: from the group consisting of oxo, thio, hydroxy, mercapto, 546-549; Pisetsky, 1996.J. Immunol. 156:421-423; Hackeret imino, amino, methyl and hydrogen, and that the total of A, B, al., 1998 EMBO.J. 17:6230-6240; Lipford et al., 1998 Trends X, D, E and Fatoms is not more than 3 nitrogens (N). In some in Microbiol. 6:496-500). However, it is now clear that mouse cases, n is 1, and in other cases n is 0. In some embodiments, lymphocytes respond quite well to phosphodiester CpG all atoms A, B, X, D, E, F are carbon (C). In some embodi motifs that do not follow this “formula” (Yi et al., 1998 J. ments, one, two or three of the atoms A, B, X, D, E, F are Immunol. 160:5898-5906) and the same is true of human B nitrogen (N). According to some embodiments, at least one of cells and dendritic cells (Hartmann et al., 1999 Proc. Natl. the atoms A, B, X, D, E, F is substituted by one of the Acad. Sci. USA 96:9305-10; Liang, 1996 J. Clin. Invest. following: F, Cl, Br, I, alkyl, alkenyl, alkinyl, halogenated 98:1119-1129). alkyl, halogenated alkenyl, cycloalkyl, O-alkyl, O-alkenyl, 0004 Several different classes of CpG nucleic acids has —NH-alkyl, - N(alkyl). —S-alkyl, -SO-alkyl, -SO recently been described. One class is potent for activating B alkyl, nitro, cyano, carboxylester, phenyl, thiophenyl, benzyl, cells but is relatively weak in inducing IFN-O. and NK cell oXo, thio, hydroxy, mercapto, and imino, wherein at least one activation; this class has been termed the B class. The B class Substituent is not oXo, thio, hydroxy, mercapto, imino, amino CpG nucleic acids typically are fully stabilized and include an or methyl. According to yet other embodiments, one of the unmethylated CpG dinucleotide within certain preferred base two atoms A or E is substituted by one of the following: F, Cl, contexts. See, e.g., U.S. Pat. Nos. 6,194,388; 6.207,646: Br, I, C-C-alkyl, alkenyl, alkinyl, halogenated alkyl, halo 6.214,806; 6,218,371; 6,239,116; and 6,339,068. Another genated alkenyl, cycloalkyl, O-alkyl, O-alkenyl, - NH-alkyl, class of CpG nucleic acids activates B cells and NK cells and —N(alkyl). —S-alkyl, -SO-alkyl, -SO-alkyl, nitro, US 2010/01 66780 A1 Jul. 1, 2010

cyano, carboxylester, phenyl, thiophenyl, benzyl, or methyl, acid, Saturated or unsaturated fatty acid, folate, a hexadecyl provided that if methyl then A, B, X, D, E, and F are all C. glycerol or dihexadecyl-glycerol group, an octadecyl-glyc 0009. In some embodiments formula I comprises a substi erol or dioctadecyl-glycerol group, a vitamin E group. In tuted pyrimidine, , toluene, imidazole or pyrazole or other embodiments, the oligonucleotide of the invention fur triazole. According to other embodiments, formula I is ther comprises a non-nucleotidic brancher moiety or a selected from the following: 5-chloro-uracil, 5-bromo-uracil, nucleotidic brancher moiety. In some embodiments, the oli 5-iodo-uracil, 5-ethyl-uracil, 5-propyl-uracil, 5-propinyl gonucleotide includes a brancher moiety, wherein the oligo uracil, (E)-5-(2-bromovinyl)-uracil, and 2,4-difluoro-tolu has at least two 5'-ends. ene. According to one embodiment of the invention, formula 0017. According to the invention, some embodiments I is fused with a 3- to 6-membered aromatic or aliphatic ring include at least two nucleotides of the oligonucleotide have a system. According to other embodiments, formula I is linked stabilized linkage, including: phosphorothioate, phospho to a 5- to 6-membered Sugar moiety, including a pentose or rodithioate, methylphosphonate, methylphosphonothioate hexose. In some cases, the pentose is a furanose and hexose is boranophosphonate, phosphoramidate, or a dephospho link a pyranose, which can optionally be substituted by F. amino, age, eitheras enantiomeric mixture or as enantiomeric pure S alkoxy, alkoxy-ethoxy, amonipropyl, alkenyl, alkinyl, or a or R-configuration. O2.C4-alkylene bridge In other cases, the furanose is (0018 Yet in some embodiments, the YZ of RYZR has a or deoxyribose. phosphodiester linkage or a phosphorothioate linkage. In 0010. According to some embodiments of the invention, some cases, the RY and or the ZR of RYZR has a phos RandR are both L. In some embodiments, R is Land R is phorothioate linkage. In some embodiments, all other nucle a nucleotide. Alternatively, in some cases R is a L and R is otides have a phosphorothioate linkage. a linkage. Such that the oligonucleotide comprises a structure 0019. According to some embodiments of the invention, 5' RCG 3'. Other embodiments include oligonucleotide the oligonucleotide is free of a microcarrier, including a lipid wherein R is L and R is a linkage, and wherein a R is 5' to carrier. RYZ, such that the oligonucleotide comprises a structure 5' RRYZ3'. In some embodiments, R is Land R is a linkage, 0020. According to the invention, the oligonucleotides and wherein a second R is 5' to RYZ spaced by one nucle may be an A class oligonucleotide, a B class oligonucleotide, otide N. Such that the oligonucleotide comprises a structure a C class oligonucleotide, a P class oligonucleotide or a T 5'RNRYZ 3'. In some cases, the oligonucleotide may class oligonucleotide. For the B class oligonucleotide of the include two 5' RNRYZ 3' motifs. invention, some embodiments include the sequence 5' TCN 0011. According to some embodiments. The oligonucle TXXCGXX3', wherein X is G or A: X is T.G, or A: X otide includes Y that is one of the following : is T or Cand X is T or C; and N is any nucleotide, and N and , 5-methyl-cytosine, 5-hydroxy-cytosine, 5-hy N are sequences of about 0-25 N’s each. droxymethyl-cytosine, 5-halogeno-cytosine,2-thio-cytosine, 0021. According to some embodiments of the invention, 4-thio-cytosine, N3-methyl-cytosine, N4-alkyl-cytosine or a the oligonucleotide comprises at least one 3'-3' linkage and or 6-substituted cytosine. at least one 5'-5' linkage. 0012. According to some embodiments, the oligonucle 0022. In another aspect the invention is a composition of otide includes Z that is a purine nucleotide including: gua the oligonucleotides described herein in combination with an nine, 7-deaza-, , 7-deaza-hypoxan antigen or other therapeutic compound, such as an anti-mi thine, 2-amino-purine, 4-thio-purine, 2,6-diamino-purine, crobial agent. The anti-microbial agent may be, for instance, 8-oxo-7,8-dihydroguanine, 7-thia-8-oxo-7,8-dihydrogua an anti-viral agent, an anti-parasitic agent, an anti-bacterial nine, 7-allyl-8-oxo-7,8-dihydroguanine, 7-deaza-8-aza-gua agent or an anti-fungal agent. nine, 8-aza-guanine, N1-methyl-guanine or purine. In other 0023. A composition of a sustained release device includ embodiments, Z is a pyrimidine nucleotide, including T. ing the oligonucleotides described herein is provided accord 0013. According to some embodiments of the invention, ing to another aspect of the invention. R is L and R is a nucleotide. 0024. The composition may optionally include a pharma 0014. According to some embodiments, the oligonucle ceutical carrier and/or be formulated in a delivery device. In otide is between 3-100 nucleotides in length; for example, the some embodiments the delivery device is selected from the oligonucleotide is 3-6 nucleotides in length, 3-100 nucle group consisting of cationic lipids, cell permeating proteins, otides in length, or 7-100 nucleotides in length. In some and Sustained release devices. In one embodiment the Sus circumstances, the oligonucleotide is T-rich, Such that at least tained release device is a biodegradable polymer or a micro 80% of the nucleotides are T. particle. 0015 The invention includes embodiments comprising at 0025. According to another aspect of the invention a least one palindromic sequence. For example, in Some cases, method of stimulating an immune response is provided. The the oligonucleotide includes two palindromic sequences. method involves administering an oligonucleotide to a Sub 0016. According to the invention, some embodiments ject in an amount effective to induce an immune response in include one to four unmethylated CG dinucleotides. In some the subject. Preferably the oligonucleotide is administered embodiments, the oligonucleotide may include at least one orally, locally, in a Sustained release device, mucosally, sys (G)m sequence, wherein m is 4 to 10. In some cases, at least temically, parenterally, or intramuscularly. When the oligo one but up to all CG dinucleotides are unmethylated. Accord nucleotide is administered to the mucosal Surface it may be ing to some embodiments, the oligonucleotide may addition delivered in an amount effective for inducing a mucosal ally comprise a non-nucleotidic modification. The non immune response or a systemic immune response. In pre nucleotidic modifications include but are not limited to: ferred embodiments the mucosal surface is selected from the Co-Cas-polyethyleneglycol, Cs-Co-alkane-diol, Ca-Cis group consisting of an oral, nasal, rectal, vaginal, and ocular alkylamino linker, C-Cls-alkylthiol linker, cholesterol, bile Surface. US 2010/01 66780 A1 Jul. 1, 2010

0026. In some embodiments the method includes expos The method involves administering to a Subject having or at ing the Subject to an antigen wherein the immune response is risk of having a viral or retroviral infection, an effective an antigen-specific immune response. In some embodiments amount for treating the viral or retroviral infection of any of the antigen is selected from the group consisting of a tumor the compositions of the invention. In some embodiments the antigen, a viral antigen, a bacterial antigen, a parasitic antigen virus is caused by a hepatitis virus e.g., hepatitis B, hepatitis and a peptide antigen. C, HIV, herpes virus, or papillomavirus. 0027. The oligonucleotides are useful for treating cancer 0033. A method for treating a bacterial infection is pro in a subject having cancer or in a Subject at risk of developing vided according to another aspect of the invention. The a cancer (e.g., reducing a risk of developing cancer). The method involves administering to a Subject having or at risk of cancer may be selected from the group consisting of biliary having a bacterial infection, an effective amount for treating tract cancer, breast cancer, cervical cancer, choriocarcinoma, the bacterial infection of any of the compositions of the inven colon cancer, endometrial cancer, gastric cancer, intraepithe tion. In one embodiment the bacterial infection is due to an lial neoplasms, lymphomas, liver cancer, lung cancer (e.g. intracellular bacteria. Small cell and non-Small cell), melanoma, neuroblastomas, 0034. In another aspect the invention is a method for treat oral cancer, ovarian cancer, pancreatic cancer, prostate can ing a parasite infection by administering to a subject having or cer, rectal cancer, sarcomas, thyroid cancer, and renal cancer, at risk of having a parasite infection, an effective amount for as well as other carcinomas and sarcomas. In some important treating the parasite infection of any of the compositions of embodiments, the cancer is selected from the group consist the invention. In one embodiment the parasite infection is due ing of bone cancer, brain and CNS cancer, connective tissue to an intracellular parasite. In another embodiment the para cancer, esophageal cancer, eye cancer, Hodgkin’s lymphoma, site infection is due to a non-helminthic parasite. larynx cancer, oral cavity cancer, skin cancer, and testicular 0035. In some embodiments the subject is a human and in CaCC. other embodiments the subject is a non-human vertebrate 0028. The oligonucleotides may also be used for increas selected from the group consisting of a dog, cat, horse, cow, ing the responsiveness of a cancer cell to a cancer therapy pig, turkey, goat, fish, monkey, chicken, rat, mouse, and (e.g., an anti-cancer therapy), optionally when the CpG sheep. immunostimulatory oligonucleotide is administered in con 0036. In another aspect, the invention relates to a method junction with an anti-cancer therapy. The anti-cancer therapy for treating autoimmune disease by administering to a subject may be a chemotherapy, a vaccine (e.g., an in vitro primed having orat risk of having an autoimmune disease an effective dendritic cell vaccine or a cancer antigen vaccine) or an amount for treating or preventing the autoimmune disease of antibody based therapy. This latter therapy may also involve any of the compositions of the invention. administering an antibody specific for a cell Surface antigen 0037. The invention in some aspects is a method for treat of for example, a cancer cell, wherein the immune response ing airway remodeling, asthma orallergy comprising: admin results in antibody dependent cellular cytotoxicity (ADCC). istering to a Subject any of the compositions of the invention, In one embodiment, the antibody may be selected from the in an effective amount to treat airway remodeling asthma or group consisting of Ributaxin, Herceptin, Quadramet, Pan allergy in the Subject. In one embodiment the Subject has orex, IDEC-Y2B8, BEC2, C225. Oncolym, SMART M195, asthma, chronic obstructive pulmonary disease, or is a ATRAGEN, Ovarex, Bexxar, LDP-03, ior té, MDX-210, smoker. In other embodiments the subject is free of symptoms MDX-11, MDX-22, OV103, 3622W94, anti-VEGF, Zena of asthma. pax, MDX-220, MDX-447, MELIMMUNE-2, MELIM 0038. Use of an oligonucleotide of the invention for stimu MUNE-1, CEACIDE, Pretarget, NovoMAb-G2, TNT, lating an immune response is also provided as an aspect of the Gliomab-H, GNI-250, EMD-72000, LymphoCide, CMA invention. 676, Monopharm-C, 4B5, ioregfr3, ior c5. BABS, anti-FLK 0039. A method for manufacturing a medicament of an 2, MDX-260, ANA Ab, SMART 1D10 Ab, SMARTABL 364 oligonucleotide of the invention for stimulating an immune Ab and ImmuRAIT-CEA. response is also provided. 0029. Thus, according to some aspects of the invention, a 0040. Each of the limitations of the invention can encom Subject having cancer or at risk of having a cancer is admin pass various embodiments of the invention. It is, therefore, istered an oligonucleotide and an anti-cancertherapy. In some anticipated that each of the limitations of the invention embodiments, the anti-cancer therapy is selected from the involving any one element or combinations of elements can group consisting of a chemotherapeutic agent, an immuno be included in each aspect of the invention. This invention is therapeutic agent and a cancer vaccine. not limited in its application to the details of construction and 0030 The invention in other aspects relates to methods for the arrangement of components set forth in the following preventing disease in a Subject. The method involves admin description or illustrated in the drawings. The invention is istering to the Subject an oligonucleotide on a regular basis to capable of other embodiments and of being practiced or of promote immune system responsiveness to prevent disease in being carried out in various ways. Also, the phraseology and the Subject. Examples of diseases or conditions sought to be terminology used herein is for the purpose of description and prevented using the prophylactic methods of the invention should not be regarded as limiting. The use of “including.” include microbial infections (e.g., sexually transmitted dis “comprising.” or “having.” “containing”, “involving, and eases) and anaphylactic shock from food allergies. variations thereof herein, is meant to encompass the items 0031. In other aspects, the invention is a method for induc listed thereafter and equivalents thereofas well as additional ing an innate immune response by administering to the Sub items. ject an oligonucleotide in an amount effective for activating an innate immune response. BRIEF DESCRIPTION OF THE DRAWINGS 0032. According to another aspect of the invention a 0041 FIG. 1 is two drawings illustrating the structure of method for treating a viral or retroviral infection is provided. the modified bases of the invention. FIG. 1a shows a section US 2010/01 66780 A1 Jul. 1, 2010

of a CpG hexamer motif (GTCGTT). FIG. 1b shows the 0048 FIG. 8 is two graphs demonstrating the results of a incorporated hydrophobic shape analogs of 2'-deoxythymi luciferase assay and a PBMC assay with modified A class dine: 2,4-Difluorotoluene (FF), 5-bromouridine (BU) and ODN. The activity of JU-modified SEQ ID NO:35-37 was 5-iodouridine (JU). compared to that of the unmodified parent sequence (SEQID NO:43) and to unmodified B-class ODNSEQID NO:1. FIG. 0042 FIG. 2 is a graph showing results of a luciferase 8a shows TLR9 activity and FIG. 8b shows IFN-alpha pro assay with B-class oligonucleotides (ODN) modified with duction. Shown is the mean+/-SEM of three donors. The shape analog 2,4-difluorotoluene (FF). The activity x-axes are log ODN concentration (FIG. 8a) or ODN concen of FF-modified ODN (SEQID NO:3-9) was compared to that tration (FIG. 8b) in LM and the y-axes are the relative stimu of the unmodified B-class parent sequence (SEQ ID NO: 1), lation index (FIG. 8a) or IFN-alpha concentration in pg/ml fully PSparent sequence (SEQID NO:2), and a third unmodi (FIG. 8b). fied B-class ODN (SEQID NO:37). htLR9-LUC-293 cells 0049 FIG. 9 is a graph demonstrating the results of a were stimulated with indicated amounts of ODN and NF-KB luciferase assay with modified C class ODN. The activity of stimulation was determined by measuring luciferase activity JU-modified C-class ODNSEQID NO:27-28 and 44-45 was 16h later. The x-axis is log ODN concentrationinuM and the compared to that of the unmodified parent sequence SEQID y-axis is the relative stimulation index. NO:45 and to an unmodified B-class ODN (SEQID NO:37). 0043 FIG. 3 is a graph demonstrating the results of a The x-axis is ODN concentration in LM and the y-axis is the luciferase assay with modified B-class ODN. (T) relative stimulation index. was substituted with 5-bromo-2'-deoxyuridine (BU) (SEQID 0050 FIG. 10 is a graph demonstrating the results of a NO:10-12) and 5-iodo-2'-deoxyuridine (JU) (SEQ ID luciferase assay with modified P class ODN. The activity of NO:13-15). Their activity was compared to that of the JU-modified SEQID NO:31-33 was compared to that of the unmodified B-class parent sequence (SEQID NO:1), fully PS unmodified parent sequence (SEQID NO:52). The x-axis is parent sequence (SEQ ID NO:2), and a third unmodified log ODN concentration in LM and the y-axis is the relative B-class ODN (SEQID NO:37). htLR9-LUC-293 cells were stimulation index. stimulated with indicated amounts of ODN and NF-KB 0051 FIG. 11 is a graph demonstrating the results of a stimulation was determined by measuring Luciferase activity luciferase assay with modified T class ODN. The activity of 16h later. The x-axis is log ODN concentrationinuM and the JU-modified SEQ ID NO:47-50 and U-modified SEQ ID y-axis is the relative stimulation index. NO:51 was compared to that of unmodified parent sequence 0044 FIG. 4 is a graph demonstrating the results of a SEQID NO:25. The x-axis is log ODN concentration in uM luciferase assay with modified B-class ODN. 2'-deoxythymi and the y-axis is the relative stimulation index. dine (T) was substituted with 2'-deoxyuridine (U) (SEQ ID 0.052 FIG. 12 is a graph demonstrating the results of a NO:16-18). The activity of the U-modified ODN was com luciferase assay with short ODN. The activity of JU-modified pared to that of the unmodified B-class parent sequence (SEQ short ODN SEQID NO:39-40 was compared to that of the ID NO:1), fully PS parent sequence (SEQ ID NO:2), and a unmodified parent sequence SEQ ID NO:38 and to the third unmodified B-class ODN (SEQ ID NO:37). htLR9 B-class ODN SEQ ID NO:37. ODN were formulated with LUC-293 cells were stimulated with indicated amounts of and without DOTAP. The x-axis is log ODN concentration in ODN and NF-kB stimulation was determined by measuring uM and the y-axis is the relative stimulation index. Luciferase activity 16h later. The x-axis is log ODN concen 0053 FIG. 13 is four graphs showing the results of an tration in LM and the y-axis is the relative stimulation index. ELISA assay measuring cytokine concentration in splenocyte 0045 FIG. 5 is two graphs demonstrating the results of a culture supernatants where BALB/c mouse splenocytes were luciferase assay and a PBMC assay with modified B class cultured with different ODNs. Culture supernatants were har ODN. The relative activity of an ODN with 5-Ethyl-2'-deox vested at 6 hr (for TNF-alpha) or 24 hr (for IL-6, IL-10 and yuridine (EU) (SEQ ID NO:42), 2'-deoxyuridine (U) (SEQ IL-12). The activities of a JU-modified B-class ODN (SEQID ID NO:16), 5-iodo-2'-deoxyuridine (JU) (SEQ ID NO:13), NO:13), an unmodified B-class ODN (SEQID NO:37), and a 5-bromo-2'-deoxyuridine (BU) (SEQ ID NO:10), and non-CpG negative control ODN (SEQID NO:26) were com 5-Chloro-2'-deoxyuridine (CU) (SEQ ID NO:41) was com pared. FIGS. 13a-d show TNF-alpha, IL-6, IL-10, and IL-12 pared to that of the parent sequence (SEQID NO:1). FIG.5a concentration, respectively. The x-axes are ODN concentra shows TLR9 activity and FIG. 5b shows IFN-alpha produc tion in ug/ml and the y-axes are cytokine concentration in tion. Shown is the mean+/-SEM of three donors. The X-axes pg/ml. are ODN concentration in uM and the y-axes are the relative 0054 FIG. 14 is a graph showing the results of FACS stimulation index (FIG. 5a) or IFN-alpha concentration in analysis of B cell proliferation. CFSE stained BALB/c mouse pg/ml (FIG.5b). splenocytes (4x10/well) were incubated with 0.001, 0.01, 0046 FIG. 6 is a graph demonstrating the results of a 0.1.0.3, 1.3 or 10 g/ml of ODN. At 72 hours post incubation, luciferase assay with EU-modified ODN. The activity of EU cells were stained for CD19 and B-cell proliferation was modified ODNSEQ ID NO:29, 30, and 42 was compared to determined by FACS followed by analysis by ModFit Soft that of the parent sequence (SEQ ID NO: 1) and another ware. The activities of a JU-modified B-class ODN (SEQ ID unmodified B-class ODN (SEQ ID NO:37). The x-axis is NO:13), an unmodified B-class ODN (SEQID NO:37), and a ODN concentration in LM and the y-axis is the relative stimu non-CpG negative control ODN (SEQID NO:26) were com lation index. pared. The x-axis is ODN concentration in lug/ml and the 0047 FIG. 7 is a graph demonstrating the results of a y-axis is relative B cell proliferation. luciferase assay with modified B class ODN. The activity of 0055 FIG. 15 is two graphs showing in vivo cytokine JU-modified SEQ ID NO:19-24 was compared to that of production as measured by ELISA. BALB/c mice (5 per parent sequence SEQID NO:37. The x-axis is ODN concen group) were injected SC with 10, 50 or 100 ug of ODN. tration in LM and the y-axis is the relative stimulation index. Control group received 100 ul of PBS alone. Animals were US 2010/01 66780 A1 Jul. 1, 2010 bled by cardiac puncture at 1 hour (for TNF-alpha) or 3 hour log in addition to a lipophilic Substituted nucleotide analog (for IP-10) post injection and plasma assayed for TNF-alpha (SEQ ID NO:138, 7-deaza-dG: SEQ ID NO:139, : and IP-10 by ELISA. The activities of a JU-modified B-class SEQ ID NO:140, 5-methyl-dC). The activity of these ODN ODN (SEQ ID NO:13) and an unmodified B-class ODN was compared to that of the parent sequence (SEQID NO:1) (SEQIDNO:37) were compared. FIG. 15a shows TNF-alpha and the same sequence with a lipophilic Substituted nucle concentration and FIG. 15b shows IP-10 concentration. The otide analog only (SEQ ID NO:13). htLR9-LUC-293 cells X-axes are ODN dose in ug and the y-axes are cytokine con were incubated with indicated amounts of nucleic acids and centration in pg/ml. NF-kB activation was determined 16 h later by measuring 0056 FIG. 16 is a graph showingTLR9-mediated NF-kB luciferase activity. The x-axis is log of ODN concentration in activation by a B-class ODN with a universal base (6-ni uM and the y-axis is IFN-C concentration in pg/ml. trobenzimidazol) (SEQID NO:178) in place of thymidine in 0063 FIG. 23 is a graph showing hTLR9-mediated NF the parent sequence (SEQID NO:1). htLR9-LUC-293 cells KB activation by T-class ODN with a lipophilic substituted were incubated with indicated amounts of nucleic acids and nucleotide analog (SEQ ID NO:132-134). The activity of NF-kB activation was determined 16 h later by measuring these was compared to that of an immunostimulatory C-class luciferase activity. The x-axis is log of ODN concentration in ODN (SEQ ID NO:198). htLR9-LUC-293 cells were incu uM and the y-axis is IFN-C concentration in pg/ml. bated with indicated amounts of nucleic acids and NF-KB 0057 FIG. 17 is a graph showingTLR9-mediated NF-kB activation was determined 16 h later by measuring luciferase activation by B-class ODN with 5-(2-bromovinyl)- activity. The x-axis is log of ODN concentration in LM and (SEQ ID NO:153 and 154) in place of thymine in the parent the y-axis is IFN-C concentration in pg/ml. sequence (SEQID NO:1). htLR9-LUC-293 cells were incu 0064 FIG. 24 is two graphs showing hTLR9-mediated bated with indicated amounts of nucleic acids and NF-KB NF-kB activation by P-class ODN with a lipophilic substi activation was determined 16 h later by measuring luciferase tuted nucleotide analog (SEQID NO:58-63). FIG. 24a shows activity. The x-axis is log of ODN concentration in uM and the activity of SEQ ID NO:58-61 compared to that of a the y-axis is IFN-C concentration in pg/ml. B-class positive control (SEQID NO:55) and an unmodified 0058 FIG. 18 is a graph showingTLR9-mediated NF-kB P-class ODN (SEQID NO:56). FIG.24b shows the activity of activation by B-class ODN with a sugar modification (2'-O- SEQID NO:62-63 compared to that of the same positive and methylguanosine) in addition to a lipophilic Substituted negative controls. htLR9-LUC-293 cells were incubated nucleotide analog (SEQ ID NO:111-113). The activity of with indicated amounts of nucleic acids and NF-kBactivation these ODN was compared to that of the parent sequence (SEQ was determined 16 h later by measuring luciferase activity. ID NO: 1) and the same sequence with a lipophilic substituted The x-axis is log of ODN concentration in uM and the y-axis nucleotide analog only (SEQ ID NO:13). htLR9-LUC-293 is the relative stimulation index. cells were incubated with indicated amounts of nucleic acids 0065 FIG. 25 is a graph showing hTLR9-mediated NF and NF-kB activation was determined 16h later by measuring KB activation by P-class ODN with a lipophilic substituted luciferase activity. The x-axis is log of ODN concentration in nucleotide analog (SEQ ID NO:64, 66-67). The activity of uM and the y-axis is IFN-C concentration in pg/ml. these is compared to that of a B-class positive control (SEQ 0059 FIG. 19 is a graph showingTLR9-mediated NF-kB ID NO:55), a C-class ODN (SEQID NO:68) and an unmodi activation by branched B-class ODN with multiple 5' acces fied P-class ODN (SEQ ID NO:57). htLR9-LUC-293 cells sible ends. The activity of the branched ODN (SEQ ID were incubated with indicated amounts of nucleic acids and NO:96, 97, 101, and 102) was compared to that of SEQ ID NF-kB activation was determined 16 h later by measuring NO:1.hTLR9-LUC-293 cells were incubated with indicated luciferase activity. The x-axis is log of ODN concentration in amounts of nucleic acids and NF-kB activation was deter uM and the y-axis is the relative stimulation index. mined 16 h later by measuring luciferase activity. The x-axis 0.066 FIG. 26 is two graphs showing induction of IFN-O. is log of ODN concentration in LM and the y-axis is IFN-C. by P-class ODN with a lipophilic substituted nucleotide ana concentration in pg/ml. log (SEQID NO:58-63). FIG. 26a shows the activity of SEQ 0060 FIG. 20 is a graph showingTLR9-mediated NF-kB ID NO:58-61 compared to that of a B-class positive control activation by a short unmodified B-class ODN (SEQ ID (SEQ ID NO:55) and an unmodified P-class ODN (SEQ ID NO:38) and an ODN of the same sequence with a lipophilic NO:56). FIG. 26b shows the activity of SEQ ID NO:62-63 substituted nucleotide analog and a lipophilic 3' tag (SEQID compared to that of the same positive and negative controls. NO:126). Both were formulated with and without DOTAP. Human PBMC were incubated with the indicated ODN for 48 hTLR9-LUC-293 cells were incubated with indicated hours. IFN-C. was then determined in the cell culture super amounts of nucleic acids and NF-kB activation was deter natants by ELISA. The x-axes are ODN concentration in uM mined 16 h later by measuring luciferase activity. The x-axis and the y-axes are IFN-C concentration in pg/ml. is log of ODN concentration in LM and the y-axis is IFN-C. 0067 FIG. 27 is a graph showing induction of IFN-O by concentration in pg/ml. P-class ODN with a lipophilic substituted nucleotide analog 0061 FIG. 21 is a graph showing TLR9-mediated NF-KB (SEQID NO:64, 66-67). The activity of these is compared to activation by two B-class ODN with 5-proynyl-dU (SEQID that of a B-class positive control (SEQID NO:55), a C-class NO: 116 and 117) in place of thymine of the parent sequence ODN (SEQ ID NO:68) and an unmodified P-class ODN (SEQID NO:1). htLR9-LUC-293 cells were incubated with (SEQ ID NO:57). Human PBMC were incubated with the indicated amounts of nucleic acids and NF-KB activation was indicated ODN for 48 hours. IFN-C was then determined in determined 16 h later by measuring luciferase activity. The the cell culture supernatants by ELISA. The x-axes are ODN x-axis is log of ODN concentration in LM and the y-axis is concentration in LM and the y-axes are IFN-C concentration IFN-C concentration in pg/ml. in pg/ml. 0062 FIG. 22 is a graph showing hTLR9-mediated NF 0068 FIG. 28 is two graphs showing IL-6 induction by KB activation by B-class ODN with a second nucleotide ana P-class ODN with a lipophilic substituted nucleotide analog US 2010/01 66780 A1 Jul. 1, 2010

(SEQ ID NO:58, 60-62, FIG. 28a) (SEQID NO:64 and 67, latory oligonucleotides tested. These modified oligonucle FIG. 28b). The activity was compared to that of an unmodi otides with enhanced stimulatory capacity have been termed fied B-class ODN (SEQID NO:55), and unmodified C-class E class oligonucleotides. ODN (SEQ ID NO:54), a negative control ODN (SEQ ID 0073. The E class modified oligonucleotides of the instant NO:53), and an unmodified P-class ODN (SEQID NO:56). invention have in some instances enhanced capacity for PBMC from three donors were incubated with the ODN for inducing an immune response. An induction of an immune 24 hours and the Supernatants were analyzed by lumineX. response refers to any increase in number or activity of an Shown is the mean+/-SEM. The X-axes are ODN concentra immune cell, oran increase in expression or absolute levels of tion in LM and the y-axes are IL-6 concentration in pg/ml. an immune factor, Such as a cytokine. Immune cells include, 0069 FIG. 29 is two graphs showing B-cell proliferation but are not limited to, NK cells, CD4+ T lymphocytes, CD8+ after treatment with P-class class ODN with a lipophilic sub T lymphocytes, B cells, dendritic cells, macrophage and other stituted nucleotide analog (SEQID NO:58, 60-62, FIG. 29a) antigen-presenting cells. Cytokines include, but are not lim (SEQ ID NO:64 and 67, FIG. 29b). The activity was com ited to, interleukins, TNF-C. IFN-O.B and Y, Flt-ligand, and pared to that of an unmodified B-class ODN (SEQ ID co-stimulatory molecules. NO:55), an unmodified C-class ODN (SEQ ID NO:54), a 0074. It is known that oligonucleotides containing unm negative control ODN (SEQ ID NO:53), an unmodified ethylated CpG motifs are able to stimulate immune responses P-class ODN (SEQ ID NO:56), LPS, R-848, SEB, and a through the Toll-like receptor 9 (TLR9) pathway. The induc polyIJ: CODN. CFSE-labeled PBMC from three donors tion of many cytokines correlates with TLR9 activation. Thus were incubated with the ODN for 5 days and then stained with induction increases as TLR9 stimulation increases. However a CD19 antibody. The percentage of B cells with reduced there is generally an inverse correlation between TLR9 and CFSE staining was determined. The x-axes are ODN concen IFN-C. induction for CpG ODN. It was discovered that some tration in uM and the y-axes are 96 of B cells with reduced of the modifications of the invention can produce a modified staining after division. signaling pattern Such that a more direct correlation, rather 0070 FIG. 30 is a graph showing induction of murine than an inverse correlation between TLR9 activation and IFN-O by P-class ODN with a lipophilic substituted nucle IFN-Cl is observed. otide analog (SEQID NO:58, 60-62, 64, and 67). The activity 0075. The inventors set out to investigate the impact of the of these is compared to that of a B-class positive control (SEQ lipophilic residues in region Surrounding the CpG motif. As ID NO:55) and a negative control (SEQID NO:26). BALB/c described in the examples below several different types of mice (5 per group) were injected SC with differing doses of lipophilic substituted nucleotide analogs, such as 2,4-difluo ODN. Animals were bled at 3 hr post injection and plasma rotoluene, 5-bromouracil and 5-iodouracil were incorporated tested for IFN-alpha by ELISA. The X axis is ODN dose in lug into a CpG oligonucleotide on either the 5' or 3' side of the and the y-axis is IFN-C concentration in pg/ml. CpG motif. Unexpectedly, incorporation of these lipophilic (0071 FIG.31 is two graphs showing the effect of ODN on Substituted nucleotide analogs led to an unusually strong tumor volume in the mouse SA1N tumor model. Female A/J increase in hTLR9 activity as well as IFN-C. induction in mice (10 per group) were injected SC with 5x10 Sal/N tumor human PBMC's. Substitution with a non-lipophilic nucle cells on day 0. Mice were treated with 35ug (FIG.31a) or 100 otide Such as a uracil residue (which is structurally similar to ug (FIG. 31b) P-class ODN with a lipophilic substituted a thymine but lacking a methyl group) produced a strong nucleotide analog (SEQID NO:60, 64, and 67), an unmodi decrease in hTLR9 stimulation. In the oligonucleotides fied C-class ODN, an unmodified B-class ODN (SEQ ID tested, the increase in TLR9 stimulation appeared to be better NO:55), or PBS alone given SC once weekly starting on day if the lipophilic substituted nucleotide analog is positioned 5' 8 post tumor induction. Animals were monitored for survival to the CpG motif than when it was positioned 3' to the motif. and tumor volume. Tumor size (the length and the width) was Double substitution (i.e. a 5' and 3' lipophilic substituted measured using a digital Vernier caliper. Tumor Volume was nucleotide analog Substitution) resulted in most potent stimu calculated by using the formula: Tumor volume=(0.4) (ab2), lation of those tested. In contrast, Substitution of guanine or where a=large diameter and b-smaller diameter. The x-axes cytosine by 2,4-difluorotoluene at the CpG motifled in both show days post tumor induction and the y-axes show tumor cases to a strong decrease of the TLR9 stimulation index. volume in mm. 0076. The lipophilic substituted nucleotide analogs modi fication resulted in a strong enhancement of IFN-C. induction. DETAILED DESCRIPTION Especially, for the 5-bromouracil and 5-iodouracil modified ODN, there appeared to be a good correlation between TLR9 0072 The invention is based in part on CpG oligonucle stimulation and IFN-O. nduction. As mentioned above, this otides that show enhanced immunostimulatory capacity. CpG observation was unexpected, since (i) the parent molecule oligonucleotides are known to stimulate the immune system, 21317 is virtually inactive in inducing IFN-O. and (ii) there is for example through interaction with toll-like receptor 9 usually an inverse correlation between TLR9 and IFN-C. (TLR9). Stimulation of TLR9 has many effects including induction for CpG ODN which do not contain these modifi stimulation of a Th1 biased immune response, NK cell acti cations. vation and B cell activation. The invention is related in some 0077. In some aspects of the invention the oligonucleotide aspects to the identification of immunostimulatory oligo has the sequence RYZR. The oligonucleotide may be nucleotides with altered structure that affects their interaction include one or more Such motifs. RandR are independently with TLR9. It was discovered by the inventors that oligo any one of lipophilic Substituted nucleotide analog (L), a nucleotides with lipophilic Substituted nucleotide analogs nucleotide, or a linkage. It is preferred, however, that at least outside the CpG motif have enhanced ability to stimulate one of R and R is a lipophilic Substituted nucleotide analog interferon-C. (IFN-C) production and induce TLR9 activation. (L). In some instances R and R2 are both L. As shown in the This effect has been observed in all classes of immunostimu examples section below oligonucleotides having an L both 5' US 2010/01 66780 A1 Jul. 1, 2010

and 3' to the CpG motif were particularly stimulatory. How lthiol linker, cholesterol, bile acid, saturated or unsaturated ever sometimes only one R is an L. For instance R may be L fatty acid, folate, hexadecyl-glycerol, dihexadecyl-glycerol and R is a nucleotide or vice versa. Alternatively R may be group, an octadecyl-glycerol or dioctadecyl-glycerol group a L and R may be a linkage, such that the oligonucleotide or a vitamin E group. comprises a structure 5' RCG 3'. I0085. The lipophilic substituted nucleotide analogs can be 0078. In some instances the oligonucleotide has the incorporated into any immunostimulatory oligonucleotide. In sequence RNYZNR whereinN, and N are nucleotides of Some embodiments of the invention the immunostimulatory 0-3 nucleotides in length. Other possible variations include oligonucleotides include immunostimulatory motifs which structures such as 5' RNRYZ N, 3',5' RRYZ 3 and RZN.R. are “CpG dinucleotides'. A CpG dinucleotide can be methy 0079 Y is a pyrimidine nucleotide. Z is a purine, a pyri lated or unmethylated. An immunostimulatory nucleic acid midine, or an abasic residue. In some embodiments Z is containing at least one unmethylated CpG dinucleotide is a preferably a purine. nucleic acid molecule which contains an unmethylated cytosine-guanine dinucleotide sequence (i.e., an unmethy 0080 L is a lipophilic substituted nucleotide analog which lated 5' followed by 3" and linked by a may be, for instance, a 5- or 6-membered ring nucleobase phosphate bond) and which activates the immune system; analog. An example of a 5- or 6-membered ring nucleobase Such an immunostimulatory nucleic acid is a CpG nucleic analog is shown in the following group of formula I. acid. CpG nucleic acids have been described in a number of issued patents, published patent applications, and other pub Formula I lications, including U.S. Pat. Nos. 6,194,388; 6.207,646: 6.214,806; 6,218,371; 6,239,116; and 6,339,068. An immu nostimulatory nucleic acid containing at least one methylated 1Nth CpG dinucleotide is a nucleic acid which contains a methy B. su - D lated cytosine-guanine dinucleotide sequence (i.e., a methy n1 lated 5' cytidine followed by a 3" guanosine and linked by a phosphate bond) and which activates the immune system. In other embodiments the immunostimulatory oligonucleotides 0081 A, B, X, D, E, and Fare independently any one of C are free of CpG dinucleotides. These oligonucleotides which (carbon) or N (nitrogen) optionally bearing hydrogen or a are free of CpG dinucleotides are referred to as non-CpG substituent such as for instance, but not limited to, F, Cl, Br, I, oligonucleotides, and they have non-CpG immunostimula alkyl, alkenyl, alkinyl, halogenated alkyl, halogenated alk tory motifs. Preferably these are T-rich ODN, such as ODN enyl, cycloalkyl, O-alkyl, O-alkenyl, —NH-alkyl, N(alkyl) having at least 80% T. 2. – S-alkyl, -SO-alkyl, -SO-alkyl, nitro, cyano, car I0086. The E class ODNs of the invention may include boxylester, phenyl, thiophenyl, benzyl, Oxo, thio, hydroxy, motifs and properties of other CpG ODN classes such as A mercapto, and imino. In some instances, at least one substitu class, B call, C class, T class and P class as long as they ent is not oXo, thio, hydroxy, mercapto, imino, amino or include lipophilic substituted nucleotide analogs 5' and/or 3' methyl. n is 0 or 1. The dotted lines indicate optional double of a YGZ motif. bonds. However, at least one substituent is not chosen from I0087. A class' CpG immunostimulatory nucleic acids the group consisting of oxo, thio, hydroxy, mercapto, imino, have been described in U.S. Non-Provisional patent applica amino, methyl and hydrogen. Additionally the total of A, B, tion Ser. No. 09/672,126 and published PCT application X, D, E and Fatoms is not more than 3 nitrogens (N). In some PCT/US00/26527 (WO 01/22990), both filed on Sep. 27, embodiments all atoms A, B, X, D, E, F are carbon (C). 2000. These nucleic acids are characterized by the ability to Alternatively, at least one, two, or three of the atoms A, B, X, induce high levels of interferon-alpha while having minimal D, E, F is nitrogen (N). effects on B cell activation. The A class CpG immunostimu 0082. The compound of formula may be, for example, any latory nucleic acid do not necessarily contain a hexamer of the following lipophilic Substituted nucleotide analogs: a palindrome GACGTC, AGCGCT, or AACGTT described by substituted pyrimidine, a substituted uracil, a substituted tolu Yamamoto and colleagues. Yamamoto S et al. J. Immunol ene, a Substituted imidazole or pyrazole, a Substituted triaz 148:4072-6 (1992). ole, 5-chloro-uracil, 5-bromo-uracil, 5-iodo-uracil, 5-ethyl I0088 Exemplary sequences of A class immunostimula uracil, 5-propyl-uracil, 5-propinyl-uracil, (E)-5-(2- tory nucleic acids are described in U.S. Non-Provisional bromovinyl)-uracil, or 2,4-difluoro-toluene. patent application Ser. No. 09/672,126 and published PCT 0083. The lipophilic substituted nucleotide analog may be application PCT/US00/26527 (WO 01/22990), both filed on separate or it may be fused with another compound. For Sep. 27, 2000. instance is may be fused to a 3-to-6-membered aromatic or I0089 “B class' ODN are potent at activating B cells but aliphatic ring system. It may also be linked to a 5- to 6-mem are relatively weak in inducing IFN-O. and NK cell activation. bered Sugar moiety Such as for instance a pentose or hexose. The B class CpG nucleic acids typically are fully stabilized An example of a pentose is a furanose Such as a ribose or and include an unmethylated CpG dinucleotide within certain deoxyribose and an example of a hexose is a pyranose. The preferred base contexts. See, e.g., U.S. Pat. Nos. 6,194,388; pentose or hexose can optionally be substituted by F. amino, 6,207,646; 6,214,806; 6,218,371; 6,239,116; and 6,339,068. alkoxy, alkoxy-ethoxy, amonipropyl, alkenyl, alkinyl, or a Another class is potent for inducing IFN-C. and NK cell O2.C4-alkylene bridge. activation but is relatively weak at stimulating B cells; this 0084. The oligonucleotide may also include a non-nucleo class has been termed the “A class'. The A class CpG nucleic tidic modification Such as a C-Cas-polyethyleneglycol, acids typically have stabilized poly-G sequences at 5' and 3' Cs-Co-alkane-diol, C-Cls-alkylamino linker, C-Cls-alky ends and a palindromic phosphodiester CpG dinucleotide US 2010/01 66780 A1 Jul. 1, 2010 containing sequence of at least 6 nucleotides. See, for lipid with a special DNA condensing enhancer) and SUPER example, published patent application PCT/US00/26527 FECTTM (a novel acting dendrimeric technology). Liposomes 0090. Yet another class of CpG nucleic acids activates B are commercially available from Gibco BRL, for example, as cells and NK cells and induces IFN-O.; this class has been LIPOFECTINTM and LIPOFECTACETM, which are formed termed the C-class. The “C class' immunostimulatory of cationic lipids such as N-1-(2, 3 dioleyloxy)-propyl-N, nucleic acids contain at least two distinct motifs have unique N,N-trimethylammonium chloride (DOTMA) and dimethyl and desirable stimulatory effects on cells of the immune dioctadecylammonium bromide (DDAB). Methods for mak system. Some of these ODN have both a traditional “stimu ing liposomes are well known in the art and have been latory’ CpG sequence and a “GC-rich' or “B-cell neutraliz described in many publications. Liposomes also have been ing' motif. These combination motif nucleic acids have reviewed by Gregoriadis G. (1985) Trends Biotechnol 3:235 immune stimulating effects that fall somewhere between 241. those effects associated with traditional “class B’ CpG ODN, 0094. In other embodiments the immunostimulatory ODN which are strong inducers of B cell activation and dendritic are not formulated in cationic liposomes. Due to the lipophilic cell (DC) activation, and those effects associated with a more nature of the modified analogs within the ODN even short recently described class of immune stimulatory nucleic acids ODN such as 3 nucleotides in length may not require formu ("class A' CpG ODN) which are strong inducers of IFN-C. lation to efficiently function in vivo. and natural killer (NK) cell activation but relatively poor 0095. In one embodiment the immunostimulatory ODN of inducers of B-cell and DC activation. Krieg A Metal. (1995) the invention are in the form of covalently closed, dumbbell Nature 374:546-9: Ballas Z Ket al. (1996).J Immunol 157: shaped molecules with both primary and secondary structure. 1840-5: Yamamoto Set al. (1992) J Immunol 148:4072-6. In one embodiment Such cyclic oligoribonucleotides include While preferred class BCpGODN often have phosphorothio two single-stranded loops connected by an intervening ate backbones and preferred class A CpG ODN have mixed or double-stranded segment. In one embodiment at least one chimeric backbones, the C class of combination motif single-stranded loop includes an immunostimulatory DNA immune stimulatory nucleic acids may have either stabilized, motif of the invention. Other covalently closed, dumbbell e.g., phosphorothioate, chimeric, or phosphodiester back shaped molecules of the invention include chimeric DNA: bones, and in some preferred embodiments, they have semi RNA molecules in which, for example, the double-stranded soft backbones. This class has been described in U.S. patent segment is at least partially DNA (e.g., either homodimeric application U.S. Ser. No. 10/224,523 filed on Aug. 19, 2002, dsDNA or heterodimeric DNA:RNA) and at least one single the entire contents of which is incorporated herein by refer stranded loop includes an immunostimulatory DNA motif of CCC. the invention. Alternatively, the double stranded segment of 0091. The “P class' immunostimulatory oligonucleotides the chimeric molecule is DNA. have several domains, including a 5'TLRactivation domain, 2 0096. In certain embodiments the immunostimulatory duplex forming regions and an optional spacer and 3' tail. This ODN is isolated. An isolated molecule is a molecule that is class of oligonucleotides has the ability in some instances to substantially pure and is free of other substances with which induce much higher levels of IFN-C secretion than the it is ordinarily found in nature or in in Vivo systems to an C-Class. The P-Class oligonucleotides have the ability to extent practical and appropriate for its intended use. In par spontaneously self-assemble into concatamers either in vitro ticular, the immunostimulatory ODN are sufficiently pure and and/or in vivo. Without being bound by any particular theory are sufficiently free from other biological constituents of cells for the method of action of these molecules, one potential So as to be useful in, for example, producing pharmaceutical hypothesis is that this property endows the P-Class oligo preparations. Because an isolated immunostimulatory ODN nucleotides with the ability to more highly crosslink TLR9 of the invention may be admixed with a pharmaceutically inside certain immune cells, inducing a distinct pattern of acceptable carrier in a pharmaceutical preparation, the immu immune activation compared to the previously described nostimulatory ODN may comprise only a small percentage classes of CpG oligonucleotides. Cross-linking of TLR9 by weight of the preparation. The immunostimulatory ODN receptors may induce activation of stronger IFN-C. Secretion is nonetheless Substantially pure in that it has been Substan through the type IIFNR feedback loop in plasmacytoid den tially separated from the substances with which it may be dritic cells. P class oligonucleotides are described at least in associated in living systems. U.S. application Ser. No. 1 1/706,561. 0097. The immunostimulatory nucleic acid molecules 0092. The “T class’ oligonucleotides induce secretion of may have a chimeric backbone. For purposes of the instant lower levels of IFN-alpha when not modified as in the ODNs invention, a chimeric backbone refers to a partially stabilized of the invention and IFN-related cytokines and chemokines backbone, wherein at least one internucleotide linkage is than B class or C class oligonucleotides, while retaining the phosphodiester or phosphodiester-like, and wherein at least ability to induce levels of IL-10 similar to B class oligonucle one other internucleotide linkage is a stabilized internucle otides. T class oligonucleotides are described at least in U.S. otide linkage, wherein the at least one phosphodiester or patent application Ser. No. 1 1/099,683, the entire contents of phosphodiester-like linkage and the at least one stabilized which are hereby incorporated by reference. linkage are different. Since boranophosphonate linkages have 0093. In one embodiment the immunostimulatory ODN of been reported to be stabilized relative to phosphodiester link the invention is advantageously combined with a cationic ages, for purposes of the chimeric nature of the backbone, lipid. In one embodiment the cationic lipid is DOTAP (N-1- boranophosphonate linkages can be classified either as phos (2,3-dioleoyloxy)propy-1-N.N.N-trimethylammonium phodiester-like or as stabilized, depending on the context. For methyl-sulfate). Other agents with similar properties includ example, a chimeric backbone according to the instant inven ing trafficking to the endosomal compartment can be used in tion could in one embodiment include at least one phosphodi place of or in addition to DOTAP. Other lipid formulations ester (phosphodiester or phosphodiester-like) linkage and at include, for example, as EFFECTENETM (a non-liposomal least one boranophosphonate (Stabilized) linkage. In another US 2010/01 66780 A1 Jul. 1, 2010

embodiment a chimeric backbone according to the instant 0101. In some embodiments the oligonucleotides may be invention could include boranophosphonate (phosphodiester Soft or semi-soft oligonucleotides. A Soft oligonucleotide is or phosphodiester-like) and phosphorothioate (stabilized) an immunostimulatory oligonucleotide having a partially sta linkages. A 'stabilized internucleotide linkage' shall mean an bilized backbone, in which phosphodiester or phosphodi internucleotide linkage that is relatively resistant to in vivo ester-like internucleotide linkages occur only within and degradation (e.g., via an exo- or endo-nuclease), compared to immediately adjacent to at least one internal pyrimidine a phosphodiester internucleotide linkage. Preferred stabilized purine dinucleotide (YZ). PreferablyYZ is YG, a pyrimidine internucleotide linkages include, without limitation, phos guanosine (YG) dinucleotide. The at least one internal YZ phorothioate, phosphorodithioate, methylphosphonate, and dinucleotide itself has a phosphodiester or phosphodiester methylphosphorothioate. Other stabilized internucleotide like internucleotide linkage. A phosphodiester or phosphodi linkages include, without limitation: peptide, alkyl, dephos ester-like internucleotide linkage occurring immediately pho, and others as described above. adjacent to the at least one internal YZ dinucleotide can be 5'. 0098 Modified backbones such as phosphorothioates 3', or both 5' and 3' to the at least one internal YZ dinucleotide. may be synthesized using automated techniques employing 0102. In particular, phosphodiester orphosphodiester-like either phosphoramidate or H-phosphonate chemistries. Aryl internucleotide linkages involve “internal dinucleotides'. An and alkyl-phosphonates can be made, e.g., as described in internal dinucleotide in general shall mean any pair of adja U.S. Pat. No. 4,469,863; and alkylphosphotriesters (in which cent nucleotides connected by an internucleotide linkage, in the charged oxygen moiety is alkylated as described in U.S. which neither nucleotide in the pair of nucleotides is a termi Pat. No. 5,023.243 and European Patent No. 092.574) can be nal nucleotide, i.e., neither nucleotide in the pair of nucle prepared by automated Solid phase synthesis using commer otides is a nucleotide defining the 5' or 3' end of the oligo cially available reagents. Methods for making other DNA nucleotide. Thus a linear oligonucleotide that is n nucleotides backbone modifications and substitutions have been long has a total of n-1 dinucleotides and only n-3 internal described. Uhlmann E et al. (1990) Chem Rev. 90:544: Good dinucleotides. Each internucleotide linkage in an internal child J (1990) Bioconjugate Chem 1:165. Methods for pre dinucleotide is an internal internucleotide linkage. Thus a paring chimeric oligonucleotides are also known. For linear oligonucleotide that is n nucleotides long has a total of instance patents issued to Uhlmann et al have described Such n-1 internucleotide linkages and only n-3 internal inter techniques. nucleotide linkages. The strategically placed phosphodiester or phosphodiester-like internucleotide linkages, therefore, 0099 Mixed backbone modified ODN may be synthe refer to phosphodiester or phosphodiester-like internucle sized using a commercially available DNA synthesizer and otide linkages positioned between any pair of nucleotides in standard phosphoramidite chemistry. (F. E. Eckstein, “Oligo the nucleic acid sequence. In some embodiments the phos nucleotides and Analogs—A Practical Approach IRL Press, phodiester or phosphodiester-like internucleotide linkages Oxford, UK, 1991, and M. D. Matteucci and M. H. Caruthers, Tetrahedron Lett. 21,719 (1980)). After coupling, PS linkages are not positioned between either pair of nucleotides closest are introduced by Sulfurization using the Beaucage reagent to the 5' or 3' end. (R. P. Iyer, W. Egan, J. B. Regan and S. L. Beaucage, J. Am. 0103 Preferably a phosphodiester or phosphodiester-like Chem. Soc. 112, 1253 (1990)) (0.075 M in acetonitrile) or internucleotide linkage occurring immediately adjacent to the phenyl acetyl disulfide (PADS) followed by capping with at least one internal YZ dinucleotide is itself an internal inter acetic anhydride, 2.6-lutidine in tetrahydrofurane (1:1:8; V:v: nucleotide linkage. Thus for a sequence NYZ N, wherein V) and N-methylimidazole (1.6% in tetrahydrofurane). This N and N are each, independent of the other, any single capping step is performed after the Sulfurization reaction to nucleotide, the YZ dinucleotide has a phosphodiester orphos minimize formation of undesired phosphodiester (PO) link phodiester-like internucleotide linkage, and in addition (a)N ages at positions where a phosphorothioate linkage should be and Y are linked by a phosphodiester or phosphodiester-like located. In the case of the introduction of a phosphodiester internucleotide linkage when N is an internal nucleotide, (b) linkage, e.g. at a CpG dinucleotide, the intermediate phos Z and N are linked by a phosphodiester or phosphodiester phorous-III is oxidized by treatment with a solution of iodine like internucleotide linkage when N is an internal nucleotide, in water/pyridine. After cleavage from the Solid Support and or (c) N and Y are linked by a phosphodiester orphosphodi final deprotection by treatment with concentrated ammonia ester-like internucleotide linkage when N is an internal (15 hrs at 50° C.), the ODN are analyzed by HPLC on a nucleotide and Z and N are linked by a phosphodiester or Gen-Pak Fax column (Millipore-Waters) using a NaCl-gra phosphodiester-like internucleotide linkage when N is an dient (e.g. buffer A: 10 mM NaH2PO in acetonitrile/wa internal nucleotide. ter=1:4/v:v pH 6.8; buffer B: 10 mM NaHPO, 1.5 M NaCl 0104. In the oligonucleotide of the invention at least one in acetonitrile/water-1:4/v:v; 5 to 60% B in 30 minutes at 1 YZ of RYZR may have a phosphodiester linkage. Alterna ml/min) or by capillary gel electrophoresis. The ODN can be tively the YZ of RYZR may have a phosphorothioate link purified by HPLC or by FPLC on a Source High Performance age. In some embodiments the RY and or ZR of RYZR column (Amersham Pharmacia). HPLC-homogeneous frac have a phosphorothioate linkage. tions are combined and desalted via a C18 column or by 0105 Soft oligonucleotides according to the instant inven ultrafiltration. The ODN was analyzed by MALDI-TOF mass tion are believed to be relatively susceptible to nuclease spectrometry to confirm the calculated mass. cleavage compared to completely stabilized oligonucle 0100. The nucleic acids of the invention can also include otides. Without meaning to be bound to a particular theory or other modifications. These include nonionic DNA analogs, mechanism, it is believed that soft oligonucleotides of the Such as alkyl- and aryl-phosphates (in which the charged invention are cleavable to fragments with reduced or no phosphonate oxygen is replaced by an alkyl or aryl group), immunostimulatory activity relative to full-length soft oligo phosphodiester and alkylphosphotriesters, in which the nucleotides. Incorporation of at least one nuclease-sensitive charged oxygen moiety is alkylated. Nucleic acids which internucleotide linkage, particularly near the middle of the contain diol. Such as tetraethyleneglycol or hexaethylenegly oligonucleotide, is believed to provide an “off switch' which col, at either or both termini have also been shown to be alters the pharmacokinetics of the oligonucleotide So as to Substantially resistant to nuclease degradation. reduce the duration of maximal immunostimulatory activity US 2010/01 66780 A1 Jul. 1, 2010 of the oligonucleotide. This can be of particular value in particularly preferred when it is important to reduce the tissue tissues and in clinical applications in which it is desirable to half-life of the oligonucleotide. avoid injury related to chronic local inflammation or immu 0110. A phosphodiester-like internucleotide linkage is a nostimulation, e.g., the kidney. phosphorus-containing bridging group that is chemically 0106 A semi-soft oligonucleotide is an immunostimula and/or diastereomerically similar to phosphodiester. Mea tory oligonucleotide having a partially stabilized backbone, sures of similarity to phosphodiester include susceptibility to in which phosphodiester or phosphodiester-like internucle nuclease digestion and ability to activate RNAse H. Thus for otide linkages occur only within at least one internal pyrimi example phosphodiester, but not phosphorothioate, oligo dine-purine (YZ) dinucleotide. Semi-soft oligonucleotides nucleotides are Susceptible to nuclease digestion, while both generally possess increased immunostimulatory potency phosphodiester and phosphorothioate oligonucleotides acti relative to corresponding fully stabilized immunostimulatory vate RNAse H. In a preferred embodiment the phosphodi oligonucleotides. Due to the greater potency of semi-soft ester-like internucleotide linkage is boranophosphate (or oligonucleotides, semi-soft oligonucleotides may be used, in equivalently, boranophosphonate) linkage. U.S. Pat. No. Some instances, at lower effective concentations and have 5,177, 198: U.S. Pat. No. 5,859,231; U.S. Pat. No. 6,160,109: lower effective doses than conventional fully stabilized U.S. Pat. No. 6,207,819; Sergueev et al., (1998).JAm Chem immunostimulatory oligonucleotides in order to achieve a Soc 120:9417-27. In another preferred embodiment the phos desired biological effect. phodiester-like internucleotide linkage is diasteromerically 0107. It is believed that the foregoing properties of semi pure Rp phosphorothioate. It is believed that diasteromeri Soft oligonucleotides generally increase with increasing cally pure Rp phosphorothioate is more Susceptible to “dose” of phosphodiester or phosphodiester-like internucle nuclease digestion and is better at activating RNAse H than otide linkages involving internal YZ dinucleotides. Thus it is mixed or diastereomerically pure Sp phosphorothioate. Ste believed, for example, that generally for a given oligonucle reoisomers of CpG oligonucleotides are the subject of co otide sequence with five internal YZ dinucleotides, an oligo pending U.S. patent application Ser. No. 09/361,575 filed Jul. nucleotide with five internal phosphodiester or phosphodi 27, 1999, and published PCT application PCT/US99/17100 ester-like YZ internucleotide linkages is more (WO 00/06588). It is to be noted that for purposes of the immunostimulatory than an oligonucleotide with four inter instant invention, the term “phosphodiester-like internucle nal phosphodiester or phosphodiester-like YG internucle otide linkage' specifically excludes phosphorodithioate and otide linkages, which in turnis more immunostimulatory than methylphosphonate internucleotide linkages. an oligonucleotide with three internal phosphodiester or 0111. As described above the soft and semi-soft oligo phosphodiester-like YZ internucleotide linkages, which in nucleotides of the invention may have phosphodiester like turnis more immunostimulatory than an oligonucleotide with linkages between C and G. One example of a phosphodiester two internal phosphodiester orphosphodiester-like YZ inter like linkage is a phosphorothioate linkage in an Rp confor nucleotide linkages, which in turn is more immunostimula mation. Oligonucleotide p-chirality can have apparently tory than an oligonucleotide with one internal phosphodiester opposite effects on the immune activity of a CpG oligonucle or phosphodiester-like YZ internucleotide linkage. Impor otide, depending upon the time point at which activity is tantly, inclusion of even one internal phosphodiester orphos measured. At an early time point of 40 minutes, the R, but not phodiester-like YZ internucleotide linkage is believed to be the S. stereoisomer of phosphorothioate CpG oligonucle advantageous over no internal phosphodiester or phosphodi otide induces INK phosphorylation in mouse spleen cells. In ester-like YZ internucleotide linkage. In addition to the num contrast, when assayed at a late time point of 44 hr, the S, but ber of phosphodiester orphosphodiester-like internucleotide not the R stereoisomer is active in stimulating spleen cell linkages, the position along the length of the nucleic acid can proliferation. This difference in the kinetics and bioactivity of also affect potency. the R, and Sestereoisomers does not result from any differ 0108. The soft and semi-soft oligonucleotides will gener ence in cell uptake, but rather most likely is due to two ally include, in addition to the phosphodiester or phosphodi opposing biologic roles of the p-chirality. First, the enhanced ester-like internucleotide linkages at preferred internal posi activity of the Rp stereoisomer compared to the Sp for stimu tions, 5' and 3' ends that are resistant to degradation. Such lating immune cells at early time points indicates that the Rp degradation-resistant ends can involve any Suitable modifica may be more effective at interacting with the CpG receptor, tion that results in an increased resistance against exonuclease TLR9, or inducing the downstream signaling pathways. On digestion over corresponding unmodified ends. For instance, the other hand, the faster degradation of the Rp PS-oligo the 5' and 3' ends can be stabilized by the inclusion there of at nucleotides compared to the Sp results in a much shorter least one phosphate modification of the backbone. In a pre duration of signaling, so that the Sp PS-oligonucleotides ferred embodiment, the at least one phosphate modification of appear to be more biologically active when tested at later time the backbone at each end is independently a phosphorothio points. ate, phosphorodithioate, methylphosphonate, or methylphos 0112 A surprisingly strong effect is achieved by the phorothioate internucleotide linkage. In another embodi p-chirality at the CpG dinucleotide itself. In comparison to a ment, the degradation-resistant end includes one or more Stereo-random CpG oligonucleotide the congener in which nucleotide units connected by peptide or amide linkages at the single CpG dinucleotide was linked in Rp was slightly the 3' end. more active, while the congener containing an Splinkage was 0109) A phosphodiester internucleotide linkage is the type nearly inactive for inducing spleen cell proliferation. of linkage characteristic of nucleic acids found in nature. The 0113. The terms “nucleic acid' and "oligonucleotide' also phosphodiester internucleotide linkage includes a phospho encompass nucleic acids or oligonucleotides with Substitu rus atom flanked by two bridging oxygen atoms and bound tions or modifications, such as in the bases and/or Sugars. For also by two additional oxygen atoms, one charged and the example, they include nucleic acids having backbone Sugars other uncharged. Phosphodiester internucleotide linkage is that are covalently attached to low molecular weight organic US 2010/01 66780 A1 Jul. 1, 2010 groups other thana hydroxyl group at the 2' position and other (C-C)—O-alkylester, (C-C)alkylphosphonate and/or than a phosphate group or hydroxy group at the 5' position. (C-C)arylphosphonate bridges, (C7-C)-O-hydroxym Thus modified nucleic acids may include a 2'-O-alkylated ethyl-aryl (e.g., disclosed in WO95/01363), wherein (C- ribose group. In addition, modified nucleic acids may include C.)aryl, (C-C)aryl and (C-C)aryl are optionally sub Sugars such as arabinose or 2'-fluoroarabinose instead of stituted by halogen, alkyl, alkoxy, nitro, cyano, and where R' ribose. Thus the nucleic acids may be heterogeneous in back and Rare, independently of each other, hydrogen, (C-Cs)- bone composition thereby containing any possible combina alkyl, (C-C)-aryl, (C-C)-aryl-(C-Cs)-alkyl, preferably tion of polymer units linked together Such as peptide-nucleic hydrogen, (C-C)-alkyl, preferably (C-C)-alkyl and/or acids (which have an amino acid backbone with nucleic acid methoxyethyl, or R' and R form, together with the nitrogen bases). atom carrying them, a 5-6-membered heterocyclic ring which 0114. Nucleic acids also include substituted and can additionally contain a further heteroatom from the group pyrimidines such as C-5 propyne pyrimidine and 7-deaza-7- O, S and N. substituted purine modified bases. Wagner RW et al. (1996) 0.124. The replacement of a phosphodiester bridge located Nat Biotechnol 14:840-4. Purines and pyrimidines include at the 3' and/or the 5' end of a nucleotide by a dephospho but are not limited to , cytosine, guanine, thymine, bridge (dephospho bridges are described, for example, in 5-methylcytosine, 5-hydroxycytosine, 5-fluorocytosine, Uhlmann E and Peyman A in “Methods in Molecular Biol 2-aminopurine, 2-amino-6-chloropurine, 2,6-diaminopurine, ogy”, Vol. 20, “Protocols for Oligonucleotides and Analogs”. hypoxanthine, and other naturally and non-naturally occur S. Agrawal, Ed., Humana Press, Totowa 1993, Chapter 16, pp. ring , Substituted and unsubstituted aromatic 355 ff), wherein a dephospho bridge is for example selected moieties. Other such modifications are well knownto those of from the dephospho bridges formacetal, 3'-thioformacetal, skill in the art. methylhydroxylamine, oxime, methylenedimethyl-hydrazo, 0115 The immunostimulatory oligonucleotides of the dimethylenesulfone and/or silyl groups. instant invention can encompass various chemical modifica 0.125. A sugar phosphate unit (i.e., a B-D-ribose and phos tions and Substitutions, in comparison to natural RNA and phodiester internucleotide bridge together forming a Sugar DNA, involving a phosphodiester internucleotide bridge, a phosphate unit) from the Sugar phosphate backbone (i.e., a B-D-ribose unit and/or a natural nucleotide base (adenine, Sugar phosphate backbone is composed of Sugar phosphate guanine, cytosine, thymine, uracil). Examples of chemical units) can be replaced by another unit, wherein the other unit modifications are known to the skilled person and are is for example suitable to build up a “morpholino-derivative' described, for example, in Uhlmann E et al. (1990) Chem Rev oligomer (as described, for example, in Stirchak EP et al. 90:543; “Protocols for Oligonucleotides and Analogs' Syn (1989) Nucleic Acids Res 17:6129-41), that is, e.g., the thesis and Properties & Synthesis and Analytical Techniques, replacement by a morpholino-derivative unit; or to build up a S. Agrawal, Ed, Humana Press, Totowa, USA 1993; Crooke S polyamide nucleic acid (“PNA'; as described for example, in Tetal. (1996) Annu Rev Pharmacol Toxicol 36:107-129; and Nielsen PE et al. (1994) Bioconjug Chem 5:3-7), that is, e.g., Hunziker J et al. (1995) Mod Synth Methods 7:331-417. An the replacement by a PNA backbone unit, e.g., by 2-amino oligonucleotide according to the invention may have one or ethylglycine. more modifications, wherein each modification is located at a 0.126 A B-ribose unit or a B-D-2'-deoxyribose unit can be particular phosphodiester internucleotide bridge and/or at a replaced by a modified Sugar unit, wherein the modified Sugar particular B-D-ribose unit and/or at a particular natural nucle unit is for example selected from B-D-ribose, C-D-2'-deox otide base position in comparison to an oligonucleotide of the yribose, L-2'-deoxyribose, 2'-F-2'-deoxyribose, 2'-F-arabi same sequence which is composed of natural DNA or RNA. nose. 2'-O (C-C)alkyl-ribose, preferably 2'-O-(C-C) 0116 For example, the invention relates to an oligonucle alkyl-ribose is 2'-O-methylribose, 2'-O (C-C)alkenyl otide which may comprise one or more modifications and ribose, 2O (C-C)alkyl-O (C-C)alkyl-ribose, wherein each modification is independently selected from: 2'-NH-2'-deoxyribose, B-D-xylo-furanose, C-arabinofura 0117 a) the replacement of a phosphodiester internucle nose, 2,4-dideoxy-B-D-erythro-hexo-pyranose, and carbocy otide bridge located at the 3' and/or the 5' end of a nucle clic (described, for example, in Froehler J (1992) Am Chem otide by a modified internucleotide bridge, SoC 114:8320) and/or open-chain Sugar analogs (described, 0118 b) the replacement of phosphodiester bridge located for example, in Vandendriessche et al. (1993) Tetrahedron at the 3' and/or the 5' end of a nucleotide by a dephospho 49:7223) and/or bicyclosugar analogs (described, for bridge, example, in Tarkov Metal. (1993) Helv Chim Acta 76:481). 0119 c) the replacement of a sugar phosphate unit from I0127. In some preferred embodiments the sugar is 2'-O- the Sugar phosphate backbone by another unit, methylribose, particularly for one or both nucleotides linked 0120 d) the replacement of a B-D-ribose unit by a modi by a phosphodiester or phosphodiester-like internucleotide fied Sugar unit, and linkage. 0121 e) the replacement of a natural nucleotide base by a I0128 Nucleic acids also include substituted purines and modified nucleotide base. pyrimidines such as C-5 propyne pyrimidine and 7-deaza-7- 0122) More detailed examples for the chemical modifica substituted purine modified bases. Wagner RW et al. (1996) tion of an oligonucleotide are as follows. Nat Biotechnol 14:840-4. Purines and pyrimidines include 0123. A phosphodiester internucleotide bridge located at but are not limited to adenine, cytosine, guanine, and thym the 3' and/or the 5' end of a nucleotide can be replaced by a ine, and other naturally and non-naturally occurring nucleo modified internucleotide bridge, wherein the modified inter bases, Substituted and unsubstituted aromatic moieties. nucleotide bridge is for example selected from phospho I0129. A modified base is any base which is chemically rothioate, phosphorodithioate, NR'R''-phosphoramidate, distinct from the naturally occurring bases typically found in boranophosphate, C-hydroxybenzyl phosphonate, phos DNA and RNA such as T, C, G, A, and U, but which share phate-(C-C)—O-alkyl ester, phosphate-(C-C)aryl basic chemical structures with these naturally occurring US 2010/01 66780 A1 Jul. 1, 2010

bases. The modified nucleotide base may be, for example, K-base), an aromatic ring system (e.g. benzimidazole or selected from hypoxanthine, uracil, dihydrouracil, pseudou dichloro-benzimidazole, 1-methyl-1H-1,2,4-triazole-3-car racil, 2-thiouracil, 4-thiouracil, 5-aminouracil, 5-(C-C)- boxylic acid amide) or a hydrogen atom (dSpacer). alkyluracil, 5-(C-C)-alkenyluracil, 5-(C-C)-alkynylu 0.132. The oligonucleotides may have one or more acces racil, 5-(hydroxymethyl)uracil, 5-chlorouracil, sible 5' ends. It is possible to create modified oligonucleotides 5-fluorouracil, 5-bromouracil, 5-hydroxycytosine, 5-(C- having two such 5' ends. This may be achieved, for instance C)-alkylcytosine, 5-(C-C)-alkenylcytosine, 5-(C-C)- by attaching two oligonucleotides through a 3'-3' linkage to alkynylcytosine, 5-chlorocytosine, 5-fluorocytosine, 5-bro generate an oligonucleotide having one or two accessible 5' mocytosine, N-dimethylguanine, 2,4-diamino-purine, ends. The 3'3'-linkage may be a phosphodiester, phospho 8-azapurine, a substituted 7-deazapurine, preferably 7-deaza 7-substituted and/or 7-deaza-8-substituted purine, 5-hy rothioate or any other modified internucleotide bridge. Meth droxymethylcytosine, N4-alkylcytosine, e.g., N4-ethylcy ods for accomplishing Such linkages are known in the art. For tosine, 5-hydroxydeoxycytidine, instance, such linkages have been described in Seliger, H., et 5-hydroxymethyldeoxycytidine, N4-alkyldeoxycytidine, al., Oligonucleotide analogs with terminal 3'-3'- and 5'-5'- e.g., N4-ethyldeoxycytidine, 6-thiodeoxyguanosine, and internucleotidic linkages as antisense inhibitors of viral gene of nitropyrrole, C5-propynylpyrimi expression, Nucleotides & Nucleotides (1991), 10(1-3), 469 dine, and diaminopurine e.g., 2,6-diaminopurine, inosine, 77 and Jiang, et al., Pseudo-cyclic oligonucleotides: in vitro 5-methylcytosine, 2-aminopurine, 2-amino-6-chloropurine, and in vivo properties, Bioorganic & Medicinal Chemistry hypoxanthine or other modifications of a natural nucleotide (1999), 7(12), 2727-2735. bases. This list is meant to be exemplary and is not to be 0.133 Additionally, 3'3'-linked nucleic acids where the interpreted to be limiting. linkage between the 3'-terminal nucleotides is not a phos 0130. In particular formulas described herein a set of phodiester, phosphorothioate or other modified bridge, can be modified bases is defined. For instance the letter Y is used to prepared using an additional spacer, such as tri- or tetra refer to pyrimidine and in some embodiments a nucleotide ethylenglycol phosphate moiety (Durand, M. et al. Triple containing a cytosine or a modified cytosine. A modified helix formation by an oligonucleotide containing one (dA)12 cytosine as used herein is a naturally occurring or non-natu and two (dT)12 sequences bridged by two hexaethylene gly rally occurring pyrimidine base analog of cytosine which can colchains, (1992), 31(38),9197-204, U.S. Pat. replace this base without impairing the immunostimulatory No. 5,658,738, and U.S. Pat. No. 5,668,265). Alternatively, activity of the oligonucleotide. Modified include the non-nucleotidic linker may be derived from ethanediol, but are not limited to 5-substituted cytosines (e.g. 5-methyl propanediol, or from an abasic deoxyribose (dSpacer) unit cytosine, 5-fluoro-cytosine, 5-chloro-cytosine, 5-bromo-cy (Fontanel, Marie Laurence et al., Sterical recognition by T4 tosine, 5-iodo-cytosine, 5-hydroxy-cytosine, 5-hydroxym polynucleotide kinase of non-nucleosidic moieties 5'-at ethyl-cytosine, 5-difluoromethyl-cytosine, and unsubstituted tached to oligonucleotides; Nucleic Acids Research (1994), or Substituted 5-alkynyl-cytosine), 6-substituted cytosines, 22(11), 2022-7) using standard phosphoramidite chemistry. N4-substituted cytosines (e.g. N4-ethyl-cytosine), 5-aza-cy The non-nucleotidic linkers can be incorporated once or mul tosine, 2-mercapto-cytosine, isocytosine, pseudo-isocy tiple times, or combined with each other allowing for any tosine, cytosine analogs with condensed ring systems (e.g. desirable distance between the 3'-ends of the two ODNs to be N,N'-propylene cytosine or phenoxazine), and uracil and its linked. derivatives (e.g. 5-fluoro-uracil, 5-bromo-uracil, 5-bromovi I0134. The oligonucleotides are partially resistant to deg nyl-uracil, 4-thio-uracil, 5-hydroxy-uracil, 5-propynyl radation (e.g., are stabilized). A “stabilized oligonucleotide uracil). Some of the preferred cytosines include 5-methyl molecule' shall mean an oligonucleotide that is relatively cytosine, 5-fluoro-cytosine, 5-hydroxy-cytosine, resistant to in vivo degradation (e.g. via an exo- or endo 5-hydroxymethyl-cytosine, and N4-ethyl-cytosine. In nuclease). Nucleic acid stabilization can be accomplished via another embodiment of the invention, the cytosine base is backbone modifications. Oligonucleotides having phospho substituted by a universal base (e.g. 3-nitropyrrole, P-base), rothioate linkages provide maximal activity and protect the an aromatic ring system (e.g. fluorobenzene or difluoroben oligonucleotide from degradation by intracellular exo- and Zene) or a hydrogen atom (dSpacer). endo-nucleases. Other modified oligonucleotides include 0131 The letter Z is used to refer to a purine, pyrimidine, phosphodiester modified nucleic acids, combinations of or abasic and in some embodiments a guanine or a modified phosphodiester and phosphorothioate nucleic acid, meth guanine base. A modified guanine as used herein is a naturally ylphosphonate, methylphosphorothioate, phosphorodithio occurring or non-naturally occurring purine base analog of ate, p-ethoxy, and combinations thereof. guanine which can replace this base without impairing the 0.135 Modified backbones such as phosphorothioates immunostimulatory activity of the oligonucleotide. Modified may be synthesized using automated techniques employing include but are not limited to 7-deazaguanine, either phosphoramidate or H-phosphonate chemistries. Aryl 7-deaza-7-substituted guanine (such as 7-deaza-7-(C2-C6) and alkyl-phosphonates can be made, e.g., as described in alkynylguanine), 7-deaza-8-substituted guanine, hypoxan U.S. Pat. No. 4,469,863; and alkylphosphotriesters (in which thine, N2-substituted guanines (e.g. N2-methyl-guanine), the charged oxygen moiety is alkylated as described in U.S. 5-amino-3-methyl-3H,6H-thiazolo 4.5-dpyrimidine-2,7- Pat. No. 5,023,243 and European Patent No. 092.574) can be dione, 2,6-diaminopurine, 2-aminopurine, purine, indole, prepared by automated Solid phase synthesis using commer adenine, Substituted (e.g. N6-methyl-adenine, cially available reagents. Methods for making other DNA 8-oxo-adenine) 8-Substituted guanine (e.g. 8-hydroxygua backbone modifications and substitutions have been nine and 8-bromoguanine), and 6-thioguanine. In another described (e.g., Uhlmann, E. and Peyman, A., Chem. Rev. embodiment of the invention, the guanine base is Substituted 90:544, 1990; Goodchild, J., Bioconjugate Chem. 1:165, by a universal base (e.g. 4-methyl-indole, 5-nitro-indole, and 1990). US 2010/01 66780 A1 Jul. 1, 2010

0136. Other stabilized oligonucleotides include: nonionic DNA analogs, such as alkyl- and aryl-phosphates (in which the charged phosphonate oxygen is replaced by an alkyl or Formula IV aryl group), phosphodiester and alkylphosphotriesters, in which the charged oxygen moiety is alkylated. Nucleic acids which contain diol. Such as tetraethyleneglycol or hexaethyl eneglycol, at either or both termini have also been shown to be Substantially resistant to nuclease degradation. 0.137 In some embodiments the oligonucleotide com prises one or more palindromic sequences. As used herein, “palindrome' and, equivalently, “palindromic sequence' shall refer to an inverted repeat, i.e., a sequence Such as ABCDEED'C'B'A' in which A and A, B and B', etc., are bases capable of forming the usual Watson-Crick base pairs. X Nu X Nul In some cases the palindrome is GC-rich. A GC-rich palin drome is a palindrome having a base composition of at least 0140. The modified oligoribonucleotide analog may con two-thirds G’s and Cs. In some embodiments the GC-rich tain a doubler or trebler unit (Glen Research, Sterling, Va.), in domain is preferably 3' to the “B cell stimulatory domain”. In particular those modified oligodeoxyribonucleotide analogs the case of a 10-base long GC-rich palindrome, the palin with a 3'-3' linkage. A doubler unit in one embodiment can be drome thus contains at least 8 G's and Cs. In the case of a based on 1,3-bis-(5-(4,4'-dimethoxytrityloxy)pentylamido 12-base long GC-rich palindrome, the palindrome also con propyl-2-(2-cyanoethyl)-(N,N-diisopropyl)-phosphora tains at least 8 G's and Cs. In the case of a 14-mer GC-rich midite. A trebler unit in one embodiment can be based on palindrome, at least ten bases of the palindrome are G’s and incorporation of Tris-2.2.2-3-(4,4'-dimethoxytrityloxy)pro C’s. In some embodiments the GC-rich palindrome is made pyloxymethylethyl-(2-cyanoethyl)-(N,N-diisopropyl)- up exclusively of G's and Cs. In some embodiments the phosphoramidite. Branching of the modified oligoribonucle oligonucleotide contains more than one palindromic otide analogs by multiple doubler, trebler, or other multiplier Sequence. units leads to dendrimers which are a further embodiment of this invention. Branched modified oligoribonucleotide ana 0138 DNA is a polymer of deoxyribonucleotides joined logs may lead to crosslinking of receptors particularly for through 3'-5' phosphodiester linkages. Units of the polymer combinations of immunostimulatory RNA and DNA such as of the invention can also be joined through 3'-5' phosphodi TLR3, TLR7, TLR8, and TLR9 with distinct immune effects ester linkages. However, the invention also encompasses compared to non-branched forms of the analogs. In addition, polymers having unusual internucleotide linkages, including the synthesis of branched or otherwise multimeric analogs specifically 5'-5', 3'-3',2'-2', 2'-3', and 2'-5' internucleotide may stabilize DNA against degradation and may enable weak linkages. In one embodiment such unusual linkages are or partially effective DNA sequences to exert a therapeuti excluded from the immunostimulatory DNA motif, even cally useful level of immune activity. The modified oligode though one or more of Such linkages may occur elsewhere oxyribonucleotide analogs may also contain linker units within the polymer. For polymers having free ends, inclusion resulting from peptide modifying reagents or oligonucleotide of one 3'-3' internucleotide linkage can result in a polymer modifying reagents (Glen Research). Furthermore, the modi having two free 5' ends. Conversely, for polymers having free fied oligodeoxyribonucleotide analogs may contain one or ends, inclusion of one 5'-5' internucleotide linkage can result more natural or unnatural amino acid residues which are in a polymer having two free 3' ends. connected to the polymer by peptide (amide) linkages. 0.139. An immunostimulatory composition of this inven 0.141. The 3'-5", 5'-5',3'-3', 2'-2',2'-3', and 2'-5' internucle tion can contain two or more immunostimulatory DNA otide linkages can be director indirect. Direct linkages in this motifs which can be linked through a branching unit. The context refers to a phosphate or modified phosphate linkage internucleotide linkages can be 3'-5, 5'-5, 3'-3, 2-2,2'-3 or as disclosed herein, without an intervening linker moiety. An 2'-5' linkages. Thereby, the nomenclature 2'-5" is chosen intervening linker moiety is an organic moiety distinct from a according to the carbon atom of deoxyribose. However, if phosphate or modified phosphate linkage as disclosed herein, unnatural Sugar moieties are employed, such as ring-ex which can include, for example, polyethylene glycol, trieth panded Sugar analogs (e.g., hexanose, cylohexene or pyra ylene glycol, hexaethylene glycol, dSpacer (i.e., an abasic nose) or bi- or tricyclic Sugar analogs, then this nomenclature deoxynucleotide), doubler unit, or trebler unit. changes according to the nomenclature of the monomer. The 0142. The linkages are preferably composed of C, H, N, O, unusual internucleotide linkage can be a phosphodiester link S. B. P. and Halogen, containing 3 to 300 atoms. An example age, but it can alternatively be modified as phosphorothioate with 3 atoms is an acetal linkage (ODN1-3'-O CH-O-3'- or any other modified linkage as described herein. Formula ODN2) connecting e.g. the 3'-hydroxy group of one nucle IV shows a general structure for branched DNA oligomers otide to the 3'-hydroxy group of a second oligonucleotide. An and modified oligoribonucleotide analogs of the invention via example with about 300 atoms is PEG-40 (tetraconta poly a nucleotidic branching unit. Thereby Nu, Nu, and Nu can ethyleneglycol). Preferred linkages are phosphodiester, phos be linked through 3'-5',5'-5',3'-3', 2'-2',2'-3', or 2'-5'-linkages. phorothioate, methylphosphonate, phosphoramidate, bora Branching of DNA oligomers can also involve the use of nophosphonate, amide, ether, thioether, acetal, thioacetal, non-nucleotidic linkers and abasic spacers. In one embodi urea, thiourea, sulfonamide, Schiff Base and disulfide link ment, Nu, Nu, and Nu represent identical or different ages. It is also possible to use the Solulink BioConjugation immunostimulatory DNA motifs. System i.e., (www.trilinkbiotech.com). US 2010/01 66780 A1 Jul. 1, 2010

0143. If the oligonucleotide is composed of two or more asthma but that don’t have the active disease during the CpG sequence parts, these parts can be identical or different. Thus, immunostimulatory oligonucleotide treatment as well as Sub in an oligonucleotide with a 3'3'-linkage, the sequences can jects that are considered to be at risk of developing these be identical 5'-ODN1-3'3'-ODN1-5" or different 5'-ODN1 diseases because of genetic or environmental factors. 3'3'-ODN2-5". Furthermore, the chemical modification of the 0147 A subject at risk of developing a cancer is one who various oligonucleotide parts as well as the linker connecting has a high probability of developing cancer. These Subjects them may be different. Since the uptake of short oligonucle include, for instance, Subjects having a genetic abnormality, otides appears to be less efficient than that of long oligonucle the presence of which has been demonstrated to have a cor otides, linking of two or more short sequences results in relative relation to a higher likelihood of developing a cancer improved immune stimulation. The length of the short oligo and Subjects exposed to cancer causing agents such as nucleotides is preferably 2-20 nucleotides, more preferably tobacco, asbestos, or other chemical toxins, or a subject who 3-16 nucleotides, but most preferably 5-10 nucleotides. Pre has previously been treated for cancer and is in apparent ferred are linked oligonucleotides which have two or more remission. When a Subject at risk of developing a cancer is unlinked 5'-ends. treated with an antigen specific for the type of cancerto which 0144. The oligonucleotide partial sequences may also be the Subject is at risk of developing and a CpG immunostimu linked by non-nucleotidic linkers. A “non-nucleotidic linker' latory oligonucleotide, the subject may be able to kill the as used herein refers to any linker element that is not a nucle cancer cells as they develop. If a tumor begins to form in the otide or polymer thereof (i.e., a polynucleotide), wherein a Subject, the Subject will develop a specific immune response nucleotide includes a purine or pyrimidine nucleobase and a against the tumor antigen. Sugar phosphate, in particular abasic linkers (dSpacers), tri 0.148. In addition to the use of the CpG immunostimula etyhlene glycol units or hexaethylene glycol units. Further tory oligonucleotides for prophylactic treatment, the inven preferred linkers are alkylamino linkers, such as C3, C6, C12 tion also encompasses the use of the CpG immunostimulatory aminolinkers, and also alkylthiol linkers, such as C3 or C6 oligonucleotides for the treatment of a Subject having an thiol linkers. The oligonucleotides can also be linked by aro infection, an allergy, asthma, or a cancer. matic residues which may be further substituted by alkyl or 0149. A subject having an infection is a subject that has Substituted alkyl groups. been exposed to an infectious pathogen and has acute or 0145 For facilitating uptake into cells, the immunostimu chronic detectable levels of the pathogen in the body. The latory oligonucleotides are in Some embodiments in the range CpG immunostimulatory oligonucleotides can be used with of 3 to 100 bases in length. In some embodiments the oligo or Without an antigen to mountanantigen specific Systemic or nucleotides are 7-100 bases in length. Typically, nucleic acids mucosal immune response that is capable of reducing the of any size greater than 6 nucleotides (even many kb long) are level of or eradicating the infectious pathogen. An infectious capable of inducing an immune response according to the disease, as used herein, is a disease arising from the presence invention if sufficient immunostimulatory motifs are present. of a foreign microorganism in the body. It is particularly However, the improved immunostimulatory capacity of the important to develop effective vaccine strategies and treat modified oligonucleotides of the invention provides for ments to protect the body's mucosal Surfaces, which are the immunostimulatory molecules of much shorter length. In primary site of pathogenic entry. Some embodiments the immunostimulatory oligonucleotides 0150. A subject having an allergy is a subject that has or is are 3-6 bases in length. at risk of developing an allergic reaction in response to an 0146 The CpG immunostimulatory oligonucleotides are allergen. An allergy refers to acquired hypersensitivity to a useful in some aspects of the invention as a vaccine for the Substance (allergen). Allergic conditions include but are not treatment of a subject at risk of developing allergy or asthma, limited to eczema, allergic rhinitis or coryza, hay fever, con an infection with an infectious organism or a cancer in which junctivitis, bronchial asthma, urticaria (hives) and food aller a specific cancer antigen has been identified. The CpG immu gies, and other atopic conditions. nostimulatory oligonucleotides can also be given without the 0151. Allergies are generally caused by IgE antibodygen antigen orallergen for protection against infection, allergy or eration against harmless allergens. The cytokines that are cancer, and in this case repeated doses may allow longer term induced by Systemic or mucosal administration of CpG protection. A subject at risk as used herein is a Subject who has immunostimulatory oligonucleotides are predominantly of a any risk of exposure to an infection causing pathogen or a class called Th1 (examples are IL-12, IP-10. IFN-O. and IFN cancer or an allergen or a risk of developing cancer. For Y) and these induce both humoral and cellular immune instance, a subject at risk may be a subject who is planning to responses. The other major type of immune response, which travel to an area where a particular type of infectious agent is is associated with the production of IL-4 and IL-5 cytokines, found or it may be a subject who through lifestyle or medical is termed a Th2 immune response. In general, it appears that procedures is exposed to bodily fluids which may contain allergic diseases are mediated by Th2 type immune infectious organisms or directly to the organism or even any responses. Based on the ability of the CpG immunostimula Subject living in an area where an infectious organism or an tory oligonucleotides to shift the immune response in a Sub allergen has been identified. Subjects at risk of developing ject from a predominant Th2 (which is associated with pro infection also include general populations to which a medical duction of IgE antibodies and allergy) to a balanced Th2/Th1 agency recommends vaccination with a particular infectious response (which is protective against allergic reactions), an organism antigen. If the antigen is an allergen and the Subject effective dose for inducing an immune response of a CpG develops allergic responses to that particular antigen and the immunostimulatory oligonucleotide can be administered to a Subject may be exposed to the antigen, i.e., during pollen Subject to treat or prevent asthma and allergy. season, then that Subject is at risk of exposure to the antigen. 0152 Thus, the CpG immunostimulatory oligonucle A Subject at risk of developing allergy or asthma includes otides have significant therapeutic utility in the treatment of those Subjects that have been identified as having an allergy or allergic and non-allergic conditions such as asthma. Th2 US 2010/01 66780 A1 Jul. 1, 2010 cytokines, especially IL-4 and IL-5 are elevated in the air 0157. The methods in which a subject is passively exposed ways of asthmatic Subjects. These cytokines promote impor to an antigen can be particularly dependent on timing of tant aspects of the asthmatic inflammatory response, includ administration of the CpG immunostimulatory oligonucle ing IgE isotope Switching, eosinophil chemotaxis and otide. For instance, in a subject at risk of developing a cancer activation and mast cell growth. Th1 cytokines, especially or an infectious disease or an allergic or asthmatic response, IFN-Y and IL-12, can suppress the formation of Th2 clones the subject may be administered the CpG immunostimulatory and production of Th2 cytokines. Asthma refers to a disorder oligonucleotide on a regular basis when that risk is greatest, of the respiratory system characterized by inflammation, nar i.e., during allergy season or after exposure to a cancer caus rowing of the airways and increased reactivity of the airways ing agent. Additionally the CpG immunostimulatory oligo nucleotide may be administered to travelers before they travel to inhaled agents. Asthma is frequently, although not exclu to foreign lands where they are at risk of exposure to infec sively associated with atopic or allergic symptoms. tious agents. Likewise the CpG immunostimulatory oligo 0153. A subject having a cancer is a subject that has detect nucleotide may be administered to soldiers or civilians at risk able cancerous cells. The cancer may be a malignant or non of exposure to biowarfare to induce a systemic or mucosal malignant cancer. Cancers or tumors include but are not lim immune response to the antigen when and if the Subject is ited to biliary tract cancer, brain cancer, breast cancer, exposed to it. cervical cancer, choriocarcinoma; colon cancer, endometrial 0158 An antigen as used herein is a molecule capable of cancer, esophageal cancer, gastric cancer, intraepithelial neo provoking an immune response. Antigens include but are not plasms; lymphomas; liver cancer; lung cancer (e.g. Small cell limited to cells, cell extracts, proteins, polypeptides, peptides, and non-Small cell); melanoma; neuroblastomas; oral cancer, polysaccharides, polysaccharide conjugates, peptide and ovarian cancer, pancreas cancer, prostate cancer, rectal can non-peptide mimics of polysaccharides and other molecules, cer; sarcomas; skin cancer, testicular cancer; thyroid cancer, Small molecules, lipids, glycolipids, carbohydrates, viruses and renal cancer, as well as other carcinomas and sarcomas. In and viral extracts and multicellular organisms such as para one embodiment the cancer is hairy cell leukemia, chronic sites and allergens. The term antigen broadly includes any myelogenous leukemia, cutaneous T-cell leukemia, multiple type of molecule which is recognized by a host immune myeloma, follicular lymphoma, malignant melanoma, squa system as being foreign. Antigens include but are not limited mous cell carcinoma, renal cell carcinoma, prostate carci to cancer antigens, microbial antigens, and allergens. noma, bladder cell carcinoma, or colon carcinoma. 0159. A cancer antigenas used herein is a compound, such 0154) A subject shall mean a human or vertebrate animal as a peptide or protein, associated with a tumor or cancer cell including but not limited to a dog, cat, horse, cow, pig, sheep, Surface and which is capable of provoking an immune goat, turkey, chicken, primate, e.g., monkey, and fish (aquac response when expressed on the Surface of an antigen pre ulture species), e.g. salmon. Thus, the invention can also be senting cell in the context of an MHC molecule. Cancer used to treat cancer and tumors, infections, and allergy/ antigens can be prepared from cancer cells either by preparing asthma in non human Subjects. Cancer is one of the leading crude extracts of cancer cells, for example, as described in causes of death in companion animals (i.e., cats and dogs). Cohen, et al., 1994, Cancer Research, 54:1055, by partially 0155 As used herein, the term treat, treated, or treating purifying the antigens, by recombinant technology, or by de when used with respect to an disorder Such as an infectious novo synthesis of known antigens. Cancer antigens include disease, cancer, allergy, or asthma refers to a prophylactic but are not limited to antigens that are recombinantly treatment which increases the resistance of a subject to devel expressed, an immunogenic portion of, or a whole tumor or opment of the disease (e.g., to infection with a pathogen) or, cancer. Such antigens can be isolated or prepared recombi in other words, decreases the likelihood that the subject will nantly or by any other means known in the art. develop the disease (e.g., become infected with the pathogen) 0160 A microbial antigenas used herein is an antigen of a as well as a treatment after the subject has developed the microorganism and includes but is not limited to virus, bac disease in order to fight the disease (e.g., reduce or eliminate teria, parasites, and fungi. Such antigens include the intact the infection) or prevent the disease from becoming worse. microorganism as well as natural isolates and fragments or 0156. In the instances when the CpG oligonucleotide is derivatives thereof and also synthetic compounds which are administered with an antigen, the Subject may be exposed to identical to or similar to natural microorganism antigens and the antigen. As used herein, the term exposed to refers to induce an immune response specific for that microorganism. either the active step of contacting the Subject with an antigen A compound is similar to a natural microorganism antigen if or the passive exposure of the Subject to the antigen in vivo. it induces an immune response (humoral and/or cellular) to a Methods for the active exposure of a Subject to an antigen are natural microorganism antigen. Such antigens are used rou well-known in the art. In general, an antigen is administered tinely in the art and are well known to those of ordinary skill directly to the Subject by any means such as intravenous, in the art. intramuscular, oral, transdermal, mucosal, intranasal, 0.161 Viruses are small infectious agents which generally intratracheal, or Subcutaneous administration. The antigen contain a nucleic acid core and a protein coat, but are not can be administered systemically or locally. Methods for independently living organisms. Viruses can also take the administering the antigen and the CpG immunostimulatory form of infectious nucleic acids lacking a protein. A virus oligonucleotide are described in more detail below. A subject cannot survive in the absence of a living cell within which it is passively exposed to an antigen if an antigen becomes can replicate. Viruses enter specific living cells either by available for exposure to the immune cells in the body. A endocytosis or direct injection of DNA (phage) and multiply, Subject may be passively exposed to an antigen, for instance, causing disease. The multiplied virus can then be released and by entry of a foreign pathogen into the body or by the devel infect additional cells. Some viruses are DNA-containing opment of a tumor cell expressing a foreign antigen on its viruses and others are RNA-containing viruses. DNA viruses Surface. include Pox, Herpes, Adeno, Papova, Parvo, and Hepadna. US 2010/01 66780 A1 Jul. 1, 2010

RNA viruses include Picorna, Calici, Astro, Toga, Flavi, are carriers of the hepatitis B virus with which it co-associ Corona, Paramyxo, Orthomyxo, Bunya, Arena, Rhabdo, Filo, ates. Hepatitis E is similar to viral hepatitis A and is generally Borna, Reo, and Retro. In some aspects, the invention also associated with poor sanitation. intends to treat diseases in which prions are implicated in 0.165 Both gram negative and gram positive bacteria serve disease progression Such as for example bovine Spongiform as antigens invertebrate animals. Such gram positive bacteria encephalopathy (i.e., mad cow disease, BSE) or scrapie infec include, but are not limited to, Pasteurella species, Staphylo tion in animals, or Creutzfeldt-Jakob disease in humans. cocci species, and Streptococcus species. Gram negative bac 0162 Viruses include, but are not limited to, enteroviruses teria include, but are not limited to, Escherichia coli, (including, but not limited to, viruses that the family picor Pseudomonas species, and Salmonella species. Specific naviridae, such as polio virus, Coxsackie virus, echo Virus), examples of infectious bacteria include but are not limited to, rotaviruses, adenovirus, and hepatitis virus, such as hepatitis Helicobacter pyloris, Borelia burgdorferi, Legionella pneu A, B, CD and E. Specific examples of viruses that have been mophilia, Mycobacteria sps (e.g. M. tuberculosis, M. avium, found in humans include but are not limited to: Retroviridae M intracellulare, M kansai, MgOrdonae), Staphylococcus (e.g., human immunodeficiency viruses, such as HIV-1 (also aureus, Neisseria gonorrhoeae, Neisseria meningitidis, List referred to as HTLV-III, LAV or HTLV-III/LAV, or HIV-III; eria monocytogenes, Streptococcus pyogenes (Group A and other isolates, such as HIV-LP: Picornaviridae (e.g., polio Streptococcus), Streptococcus agalactiae (Group B Strepto viruses, hepatitis. A virus; enteroviruses, human Coxsackie coccus), Streptococcus (viridans group), Streptococcus viruses, rhinoviruses, echoviruses); Calciviridae (e.g., strains faecalis, Streptococcus bovis, Streptococcus (anaerobic sps.), that cause gastroenteritis); Togaviridae (e.g., equine encepha Streptococcus pneumoniae, pathogenic Campylobacter sp., litis viruses, rubella viruses); Flaviviridae (e.g., dengue Enterococcus sp., Haemophilus influenzae, Bacillus antracis, viruses, encephalitis viruses, yellow fever viruses); Coro corynebacterium diphtheriae, corynebacterium sp., Ery naviridae (e.g., coronaviruses); Rhabdoviridae (e.g., vesicu sipelothrix rhusiopathiae, Clostridium perfingers, lar stomatitis viruses, rabies viruses); Filoviridae (e.g., ebola Clostridium tetani, Enterobacter aerogenes, Klebsiella pneu viruses); Paramyxoviridae (e.g., parainfluenza viruses, moniae, Pasturella multocida, Bacteroides sp., Fusobacte mumps virus, measles virus, respiratory syncytial virus); rium nucleatum, Streptobacillus moniliformis, Treponema Orthomyxoviridae (e.g., influenza viruses); Bunyaviridae pallidium, Treponema pertenue, Leptospira, Rickettsia, and (e.g., Hantaan viruses, bunya viruses, phleboviruses and Actinomyces israeli. Nairo viruses); Arenaviridae (hemorrhagic fever viruses): 0166 Examples of fungi include Cryptococcus neofor Reoviridae (e.g., reoviruses, orbiviurses and rotaviruses); mans, Histoplasma capsulatum, Coccidioides immitis, Blas Birnaviridae: Hepadnaviridae (Hepatitis B virus); Parvoviri tomyces dermatitidis, Chlamydia trachomatis, Candida albi dae (parvoviruses); Papovaviridae (papillomaviruses, CCS. polyoma viruses); Adenoviridae (most adenoviruses); Herp 0167. Other infectious organisms (i.e., protists) include esviridae (herpes simplex virus (HSV) 1 and 2, varicella Plasmodium spp. Such as Plasmodium falciparum, Plasmo Zoster virus, cytomegalovirus (CMV)); Poxyiridae (variola dium malariae, Plasmodium ovale, and Plasmodium vivax viruses, vaccinia viruses, pox viruses); Iridoviridae (e.g., and Toxoplasma gondii. Blood-borne and/or tissues parasites African swine fever virus); and other viruses acute laryn include Plasmodium spp., Babesia microti, Babesia diver gotracheobronchitis virus, Alphavirus, Kaposi's sarcoma-as gens, Leishmania tropica, Leishmania spp., Leishmania bra sociated herpesvirus, Newcastle disease virus, Nipah virus, Ziliensis, Leishmania donovani, Trypanosoma gambiense and Norwalk virus, Papillomavirus, parainfluenza virus, avian Trypanosoma rhodesiense (African sleeping sickness), Try influenza, SARS virus, West Nile virus. panosoma Cruzi (Chagas disease), and Toxoplasma gondii. 0163 The methods of the invention are particularly useful, 0168 Other medically relevant microorganisms have been in some embodiments, for the treatment of Human immuno described extensively in the literature, e.g., see C. G. A Tho deficiency virus (HIV) and hepatitis virus. HIV, a species of mas, Medical Microbiology, Bailliere Tindall, Great Britain retrovirus also known as human T-cell lymphotropic virus III 1983, the entire contents of which is hereby incorporated by (HTLV III), is responsible for causing the deterioration reference. resulting in the disorder known as AIDS. HIV infects and 0169. An allergen refers to a substance (antigen) that can destroys T-cells, upsetting the overall balance of the immune induce an allergic or asthmatic response in a Susceptible system, resulting in a loss in the patients ability to combat Subject. The list of allergens is enormous and can include other infections and predisposing the patient to opportunistic pollens, insect venoms, animal dander dust, fungal spores and infections which frequently prove fatal. drugs (e.g. penicillin). Examples of natural, animal and plant 0164 Viral hepatitis is an inflammation of the liver which allergens include but are not limited to proteins specific to the may produce Swelling, tenderness, and sometimes permanent following genuses: Canine (Canis familiaris); Dermatopha damage to the liver. If the inflammation of the liver continues goides (e.g. Dermatophagoides farinae); Felis (Felis domes at least six months or longer, it is referred to as chronic ticus); Ambrosia (Ambrosia artemisfolia, Lolium (e.g. hepatitis. There are at least five different viruses known to Lolium perenne or Lolium multiflorum); Cryptomeria (Cryp cause viral hepatitis, include hepatitis A, B, C D and E. tomeria japonica); Alternaria (Alternaria alternata); Alder, Hepatitis A is generally communicated through food or drink Alnus (Alnus guiltinoasa); Betula (Betula verrucosa); Ouer ing water contaminated with human feces. Hepatitis B gen cus (Ouercus alba); Olea (Olea europa); Artemisia (Artemi erally is spread through bodily fluids such as blood. For sia vulgaris); Plantago (e.g. Plantago lanceolata); Parietaria instance, it may be spread from mother to child at birth, (e.g. Parietaria officinalis or Parietaria judaica); Blattella through sexual contact, contaminated blood transfusions and (e.g. Blattella germanica): Apis (e.g. Apis multiflorum); needles. Hepatitis C is quite common and like Hepatitis B is Cupressus (e.g. Cupressus sempervirens, Cupressus ari often spread through blood transfusions and contaminated zonica and Cupressus macrocarpa); Juniperus (e.g. Junipe needles. Hepatitis D is found most often in IV drug users who rus Sabinoides, Juniperus virginiana, Juniperus communis US 2010/01 66780 A1 Jul. 1, 2010 and Juniperus ashei). Thuya (e.g. Thuya Orientalis); idazole, niclosamide, nifurtimox, oxaminiquine, paromomy Chamaecyparis (e.g. Chamaecyparis obtusa); Periplaneta cin, pentamidine isethionate, piperazine, praziquantel, pri (e.g. Periplaneta americana); Agropyron (e.g. Agropyron maquine phosphate, proguanil, pyrantel pamoate, repens); Secale (e.g. Secale cereale); Triticum (e.g. Triticum pyrimethanmine-Sulfonamides, pyrimethanmine-Sulfadox aestivum); Dactylis (e.g. Dactylis glomerata); Festuca (e.g. ine, quinacrine HCl, quinine Sulfate, quinidine gluconate, Festuca elation); Poa (e.g. Poa pratensis or Poa compressa); spiramycin, Stibogluconate sodium (Sodium antimony glu Avena (e.g. Avena sativa); Holcus (e.g. Holcus lanatus); conate), Suramin, tetracycline, doxycycline, thiabendazole, Anthoxanthum (e.g. Anthoxanthum Odoratum); Arrhen timidazole, trimethroprim-Sulfamethoxazole, and tryparsa atherum (e.g. Arrhenatherum elatius); Agrostis (e.g. Agrostis mide some of which are used alone or in combination with alba); Phleum (e.g. Phleum pratense); Phalaris (e.g. others. Phalaris arundinacea); Paspalum (e.g. Paspalum notatum); 0173 Antibacterial agents kill or inhibit the growth or Sorghum (e.g. Sorghum halepensis); and Bromus (e.g. Bro function of bacteria. A large class of antibacterial agents is mus inermis). antibiotics. Antibiotics, which are effective for killing or 0170 The term substantially purified as used herein refers inhibiting a wide range of bacteria, are referred to as broad to a polypeptide which is substantially free of other proteins, spectrum antibiotics. Other types of antibiotics are predomi lipids, carbohydrates or other materials with which it is natu nantly effective against the bacteria of the class gram-positive rally associated. One skilled in the art can purify viral or or gram-negative. These types of antibiotics are referred to as bacterial polypeptides using standard techniques for protein narrow spectrum antibiotics. Other antibiotics which are purification. The substantially pure polypeptide will often effective against a single organism or disease and not against yield a single major band on a non-reducing polyacrylamide other types of bacteria, are referred to as limited spectrum gel. In the case of partially glycosylated polypeptides or those antibiotics. Antibacterial agents are sometimes classified that have several start codons, there may be several bands on based on their primary mode of action. In general, antibacte a non-reducing polyacrylamide gel, but these will form a rial agents are cell wall synthesis inhibitors, cell membrane distinctive pattern for that polypeptide. The purity of the viral inhibitors, protein synthesis inhibitors, nucleic acid synthesis or bacterial polypeptide can also be determined by amino or functional inhibitors, and competitive inhibitors. terminal amino acid sequence analysis. Other types of anti 0.174 Antiviral agents are compounds which prevent gens not encoded by a nucleic acid vector Such as polysac infection of cells by viruses or replication of the virus within charides, Small molecule, mimics etc are included within the the cell. There are many fewer antiviral drugs than antibacte invention. rial drugs because the process of viral replication is so closely 0171 The oligonucleotides of the invention may be related to DNA replication within the host cell, that non administered to a Subject with an anti-microbial agent. An specific antiviral agents would often be toxic to the host. anti-microbial agent, as used herein, refers to a naturally There are several stages within the process of viral infection occurring or synthetic compound which is capable of killing which can be blocked or inhibited by antiviral agents. These or inhibiting infectious microorganisms. The type of anti stages include, attachment of the virus to the host cell (immu microbial agent useful according to the invention will depend noglobulin or binding peptides), uncoating of the virus (e.g. upon the type of microorganism with which the Subject is amantadine), synthesis or translation of viral mRNA (e.g. infected or at risk of becoming infected. Anti-microbial interferon), replication of viral RNA or DNA (e.g. nucleotide agents include but are not limited to anti-bacterial agents, analogs), maturation of new virus proteins (e.g. protease anti-viral agents, anti-fungal agents and anti-parasitic agents. inhibitors), and budding and release of the virus. Phrases such as “anti-infective agent”, “anti-bacterial agent'. 0.175 Nucleotide analogs are synthetic compounds which 'anti-viral agent”, “anti-fungal agent”, “anti-parasitic agent' are similar to nucleotides, but which have an incomplete or and “parasiticide” have well-established meanings to those of abnormal deoxyribose or ribose group. Once the nucleotide ordinary skill in the art and are defined in standard medical analogs are in the cell, they are phosphorylated, producing the texts. Briefly, anti-bacterial agents kill or inhibit bacteria, and triphosphate formed which competes with normal nucle include antibiotics as well as other synthetic or natural com otides for incorporation into the viral DNA or RNA. Once the pounds having similar functions. Antibiotics are low molecu triphosphate form of the nucleotide analog is incorporated lar weight molecules which are produced as secondary into the growing nucleic acid chain, it causes irreversible metabolites by cells, such as microorganisms. In general, association with the viral polymerase and thus chain termi antibiotics interfere with one or more bacterial functions or nation. Nucleotide analogs include, but are not limited to, structures which are specific for the microorganism and acyclovir (used for the treatment of herpes simplex virus and which are not present in host cells. Anti-viral agents can be Varicella-Zoster virus), gancyclovir (useful for the treatment isolated from natural Sources or synthesized and are useful for of cytomegalovirus), idoxuridine, ribavirin (useful for the killing or inhibiting viruses. Anti-fungal agents are used to treatment of respiratory syncitial virus), dideoxyinosine, treat Superficial fungal infections as well as opportunistic and dideoxycytidine, Zidovudine (azidothymidine), imiquimod, primary systemic fungal infections. Anti-parasite agents kill and resimiquimod. or inhibit parasites. 0176 The interferons are cytokines which are secreted by 0172 Examples of anti-parasitic agents, also referred to as virus-infected cells as well as immune cells. The interferons parasiticides useful for human administration include but are function by binding to specific receptors on cells adjacent to not limited to albendazole, amphotericin B, benznidazole, the infected cells, causing the change in the cell which pro bithionol, chloroquine HCl, chloroquine phosphate, clinda tects it from infection by the virus. C. and B-interferon also mycin, dehydroemetine, diethylcarbamazine, diloxanide induce the expression of Class I and Class II MHC molecules furoate, eflornithine, furazolidaone, glucocorticoids, halo on the Surface of infected cells, resulting in increased antigen fantrine, iodoquinol, ivermectin, mebendazole, mefloquine, presentation for host immune cell recognition. C. and B-inter meglumine antimoniate, melarsoprol, metrifonate, metron ferons are available as recombinant forms and have been used US 2010/01 66780 A1 Jul. 1, 2010

for the treatment of chronic hepatitis Band C infection. At the covered that both systemic and mucosal immunity are dosages which are effective for anti-viral therapy, interferons induced by mucosal delivery of CpG nucleic acids. Thus, the have severe side effects such as fever, malaise and weight oligonucleotides may be administered in combination with loss. other mucosal adjuvants. 0177 Anti-viral agents useful in the invention include but 0182 Immune responses can also be induced or aug are not limited to immunoglobulins, amantadine, interferons, mented by the co-administration or co-linear expression of nucleotide analogs, and protease inhibitors. Specific cytokines (Bueler & Mulligan, 1996; Chow et al., 1997: Gei examples of anti-virals include but are not limited to Aceman ssler et al., 1997: Iwasaki et al., 1997: Kim et al., 1997) or B-7 nan: Acyclovir; Acyclovir Sodium: Adefovir; Alovudine: co-stimulatory molecules (Iwasaki et al., 1997: Tsuji et al., Alvircept Sudotox: Amantadine Hydrochloride; Aranotin: 1997) with the CpG immunostimulatory oligonucleotides. Arildone: Atevirdine Mesylate; Avridine: Cidofovir; Cipam The term cytokine is used as a generic name for a diverse fylline; Cytarabine Hydrochloride; Delavirdine Mesylate: group of soluble proteins and peptides which act as humoral Desciclovir, Didanosine; Disoxaril: Edoxudine; Enviradene; regulators at nano- to picomolar concentrations and which, Enviroxime; Famciclovir, Famotine Hydrochloride; Fiacit either under normal or pathological conditions, modulate the abine; Fialuridine; Fosarilate; Foscarnet Sodium; Fosfonet functional activities of individual cells and tissues. These Sodium; Ganciclovir, Ganciclovir Sodium; Idoxuridine; proteins also mediate interactions between cells directly and Kethoxal; Lamivudine; Lobucavir; Memotine Hydrochlo regulate processes taking place in the extracellular environ ride; Methisazone; Nevirapine; Penciclovir; Pirodavir; Rib ment. Examples of cytokines include, but are not limited to avirin; Rimantadine Hydrochloride: Saquinavir Mesylate: IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12, IL-15, IL-18, Somantadine Hydrochloride: Sorivudine: Statolon; Stavu granulocyte-macrophage colony stimulating factor (GM dine; Tilorone Hydrochloride; Trifluridine; Valacyclovir CSF), granulocyte colony stimulating factor (G-CSF), inter Hydrochloride: : Vidarabine Phosphate: Vidara feron-Y (Y-IFN), IFN-C. tumor necrosis factor (TNF), TGF-B, bine Sodium Phosphate; Viroxime; Zalcitabine; Zidovudine: FLT-3 ligand, and CD40 ligand. and Zinviroxime. 0183 The oligonucleotides are also useful for redirecting 0.178 Anti-fungal agents are useful for the treatment and an immune response from a Th2 immune response to a Th1 prevention of infective fungi. Anti-fungal agents are some immune response. This results in the production of a rela times classified by their mechanism of action. Some anti tively balanced Th1/Th2 environment. Redirection of an fungal agents function as cell wall inhibitors by inhibiting immune response from a Th2 to a Th1 immune response can glucose synthase. These include, but are not limited to, basi be assessed by measuring the levels of cytokines produced in ungin/ECB. Other anti-fungal agents function by destabiliz response to the nucleic acid (e.g., by inducing monocytic cells ing membrane integrity. These include, but are not limited to, and other cells to produce Th1 cytokines, including IL-12, immidazoles, such as clotrimazole, Sertaconzole, flucona IFN-Y and GM-CSF). The redirection or rebalance of the Zole, itraconazole, ketoconazole, miconazole, and Voricona immune response from a Th2 to a Th1 response is particularly cole, as well as FK 463, amphotericin B, BAY 38-9502, MK useful for the treatment or prevention of asthma. For instance, 991, pradimicin, UK 292, butenafine, and terbinafine. Other an effective amount for treating asthma can be that amount; anti-fungal agents function by breaking down chitin (e.g. useful for redirecting a Th2 type of immune response that is chitinase) or immunosuppression (501 cream). associated with asthma to a Th1 type of response or a bal 0179 CpG immunostimulatory oligonucleotides can be anced Th1/Th2 environment. Th2 cytokines, especially IL-4 combined with other therapeutic agents such as adjuvants to and IL-5 are elevated in the airways of asthmatic subjects. The enhance immune responses. The CpG immunostimulatory CpG immunostimulatory oligonucleotides of the invention oligonucleotide and other therapeutic agent may be adminis cause an increase in Th1 cytokines which helps to rebalance tered simultaneously or sequentially. When the other thera the immune system, preventing or reducing the adverse peutic agents are administered simultaneously they can be effects associated with a predominately Th2 immune administered in the same or separate formulations, but are response. administered at the same time. The other therapeutic agents 0.184 The oligonucleotides of the invention may also be are administered sequentially with one another and with CpG useful for treating airway remodeling. Airway remodeling immunostimulatory oligonucleotide, when the administra results from smooth muscle cell proliferation and/or submu tion of the other therapeutic agents and the CpG immuno cosal thickening in the airways, and ultimately causes nar stimulatory oligonucleotide is temporally separated. The rowing of the airways leading to restricted airflow. The oli separation in time between the administration of these com gonucleotides of the invention may prevent further pounds may be a matter of minutes or it may be longer. Other remodeling and possibly even reduce tissue buildup resulting therapeutic agents include but are not limited to adjuvants, from the remodeling process. cytokines, antibodies, antigens, etc. 0185. The oligonucleotides are also useful for improving 0180. The compositions of the invention may also be Survival, differentiation, activation and maturation of den administered with non-nucleic acid adjuvants. A non-nucleic dritic cells. The CpG immunostimulatory oligonucleotides acid adjuvant is any molecule or compound except for the have the unique capability to promote cell survival, differen CpG immunostimulatory oligonucleotides described herein tiation, activation and maturation of dendritic cells. which can stimulate the humoral and/or cellular immune 0186 CpG immunostimulatory oligonucleotides also response. Non-nucleic acid adjuvants include, for instance, increase natural killer cell lytic activity and antibody depen adjuvants that create a depo effect, immune stimulating adju dent cellular cytotoxicity (ADCC). ADCC can be performed vants, and adjuvants that create a depo effect and stimulate the using a CpG immunostimulatory oligonucleotide in combi immune system. nation with an antibody specific for a cellular target. Such as 0181. The CpG immunostimulatory oligonucleotides are a cancer cell. When the CpG immunostimulatory oligonucle also useful as mucosal adjuvants. It has previously been dis otide is administered to a Subject in conjunction with the US 2010/01 66780 A1 Jul. 1, 2010 antibody the subject's immune system is induced to kill the fosamide, Ifes/Mesnex/Ifosamide, Vumon/Teniposide, Para tumor cell. The antibodies useful in the ADCC procedure platin/Carboplatin, Plantinol/cisplatin, Vepeside/Etoposide, include antibodies which interact with a cell in the body. ZD 9331, Taxotere/Docetaxel, prodrug of guanine arabino Many such antibodies specific for cellular targets have been side, Taxane Analog, nitrosoureas, alkylating agents such as described in the art and many are commercially available. melphelan and cyclophosphamide, Aminoglutethimide, 0187. The CpG immunostimulatory oligonucleotides may Asparaginase, BuSulfan, Carboplatin, Chlorombucil, Cytara also be administered in conjunction with an anti-cancer bine HCl, Dactinomycin, Daunorubicin HCl, Estramustine therapy. Anti-cancer therapies include cancer medicaments, phosphate sodium, Etoposide (VP16-213), Floxuridine, radiation and Surgical procedures. As used herein, a "cancer Fluorouracil (5-FU), Flutamide, Hydroxyurea (hydroxycar medicament” refers to a agent which is administered to a bamide), Ifosfamide, Interferon Alfa-2a, Alfa-2b, Leuprolide Subject for the purpose of treating a cancer. As used herein, acetate (LHRH-releasing factor analog), Lomustine “treating cancer includes preventing the development of a (CCNU), Mechlorethamine HCl (nitrogen mustard), Mer cancer, reducing the symptoms of cancer, and/or inhibiting captopurine, Mesna, Mitotane (op'-DDD), Mitoxantrone the growth of an established cancer. In other aspects, the HCl, Octreotide, Plicamycin, Procarbazine HCl, Streptozo cancer medicament is administered to a Subject at risk of cin, Tamoxifen citrate. Thioguanine. Thiotepa, Vinblastine developing a cancer for the purpose of reducing the risk of sulfate, Amsacrine (m-AMSA), AZacitidine, Erthropoietin, developing the cancer. Various types of medicaments for the Hexamethylmelamine (HMM), Interleukin 2, MitoguaZone treatment of cancer are described herein. For the purpose of (methyl-GAG; methyl glyoxal bis-guanylhydraZone; this specification, cancer medicaments are classified as che MGBG), Pentostatin (2' deoxycoformycin), Semustine (me motherapeutic agents, immunotherapeutic agents, cancer thyl-CCNU), Teniposide (VM-26) and Vindesine sulfate, but vaccines, hormone therapy, and biological response modifi it is not so limited. CS. 0190. The immunotherapeutic agent may be selected from 0188 Additionally, the methods of the invention are the group consisting of Ributaxin, Herceptin, Quadramet, intended to embrace the use of more than one cancer medi Panorex, IDEC-Y2B8, BEC2, C225, Oncolym, SMART cament along with the CpG immunostimulatory oligonucle M195, ATRAGEN, Ovarex, Bexxar, LDP-03, ior té, MDX otides. As an example, where appropriate, the CpG immuno 210, MDX-11, MDX-22, OV103, 3622W94, anti-VEGF, stimulatory oligonucleotides may be administered with both Zenapax, MDX-220, MDX-447, MELIMMUNE-2, a chemotherapeutic agent and an immunotherapeutic agent. MELIMMUNE-1, CEACIDE, Pretarget, NovoMAb-G2, Alternatively, the cancer medicament may embrace an immu TNT, Gliomab-H, GNI-250, EMD-72000, LymphoCide, notherapeutic agent and a cancer vaccine, or a chemothera CMA 676, Monopharm-C, 4B5, ior egfr3, ior c5, BABS, peutic agent and a cancer vaccine, or a chemotherapeutic anti-FLK-2, MDX-260, ANA Ab, SMART 1D10 Ab, agent, an immunotherapeutic agent and a cancer vaccine all SMART ABL 364 Ab and ImmuRAITCEA, but it is not so administered to one subject for the purpose of treating a limited. Subject having a cancer or at risk of developing a cancer. 0191 The cancer vaccine may be selected from the group 0189 The chemotherapeutic agent may be selected from consisting of EGF, Anti-idiotypic cancer vaccines, Gp75 anti the group consisting of methotrexate, Vincristine, adriamy gen, GMK melanoma vaccine, MGV ganglioside conjugate cin, cisplatin, non-Sugar containing chloroethylnitrosoureas, vaccine, Her2/neu, Ovarex, M-Vax, O-Vax, L-Vax, STn-KHL 5-fluorouracil, mitomycin C, bleomycin, doxorubicin, dacar theratope, BLP25 (MUC-1), liposomal idiotypic vaccine, bazine, taxol, fragyline, Meglamine GLA, valrubicin, car Melacine, peptide antigen vaccines, toxinfantigen vaccines, mustaine and poliferposan, MMI270, BAY 12-9566, RAS MVA-based vaccine, PACIS, BCG vacine, TA-HPV, farnesyl transferase inhibitor, farnesyl transferase inhibitor, TA-CIN, DISC-virus and ImmuCyst/TheraCys, but it is not MMP, MTA/LY231514, LY264618/Lometexol, Glamolec, so limited. CI-994, TNP-470, Hycamtin/Topotecan, PKC412, Valspo 0.192 The use of CpG immunostimulatory oligonucle dar/PSC833, Novantrone/Mitroxantrone, Metaret/Suramin, otides in conjunction with immunotherapeutic agents such as Batimastat, E7070, BCH-4556, CS-682, 9-AC, AG3340, monoclonal antibodies is able to increase long-term Survival AG3433, Incel/VX-710, VX-853, ZD0101, ISI641, ODN through a number of mechanisms including significant 698, TA 2516/Marmistat, BB2516/Marmistat, CDP 845, enhancement of ADCC (as discussed above), activation of D2163, PD183805, DX8951f. Lemonal DP 2202, FK 317, natural killer (NK) cells and an increase in IFNC. levels. The Picibanil/OK-432, AD 32/Valrubicin, Metastron/strontium nucleic acids when used in combination with monoclonal derivative, Temodal/Temozolomide, Evacet/liposomal doxo antibodies serve to reduce the dose of the antibody required to rubicin, Yew taxan/Paclitaxel, Taxol/Paclitaxel, Xeload/ achieve a biological result. Capecitabine. Furtulon/Doxifluridine, Cyclopax/oral pacli 0193 As used herein, the terms "cancer antigen’ and taxel, Oral Taxoid, SPU-077/Cisplatin, HMR 1275/ "tumor antigen” are used interchangeably to refer to antigens Flavopiridol, CP-358 (774)/EGFR, CP-609 (754)/RAS which are differentially expressed by cancer cells and can oncogene inhibitor, BMS-182751/oral platinum, UFT thereby be exploited in order to target cancer cells. Cancer (Tegafur/Uracil), Ergamisol/Levamisole, Eniluracil/776C85/ antigens are antigens which can potentially stimulate appar 5FU enhancer, Campto/Levamisole, Camptosar/Irinotecan, ently tumor-specific immune responses. Some of these anti TumodeX/Ralitrexed, Leustatin/Cladribine, Paxex/Pacli gens are encoded, although not necessarily expressed, by taxel, Doxil/liposomal doxorubicin, Caelyx/liposomal doxo normal cells. These antigens can be characterized as those rubicin, Fludara/Fludarabine, Pharmarubicin/Epirubicin, which are normally silent (i.e., not expressed) in normal cells, DepoCyt, ZD1839, LU 79553/Bis-Naphtalimide, LU those that are expressed only at certain stages of differentia 103793/Dolastain, Caetyx/liposomal doxorubicin, Gemzar/ tion and those that are temporally expressed such as embry Gemcitabine, ZD 0473/Anormed, YM 116, iodine seeds, onic and fetal antigens. Other cancer antigens are encoded by CDK4 and CDK2 inhibitors, PARP inhibitors, D4809/Dexi mutant cellular genes, such as oncogenes (e.g., activated ras US 2010/01 66780 A1 Jul. 1, 2010 20 oncogene), Suppressor genes (e.g., mutant p53), fusion pro spectrum resistance to infectious challenge using the CpG teins resulting from internal deletions or chromosomal trans immunostimulatory oligonucleotides. The term antigen non locations. Still other cancer antigens can be encoded by viral specific innate immune activation as used herein refers to the genes such as those carried on RNA and DNA tumor viruses. activation of immune cells other than B cells and for instance 0194 The CpG immunostimulatory oligonucleotides are can include the activation of NK cells, T cells or other also useful for treating and preventing autoimmune disease. immune cells that can respond in an antigen independent Autoimmune disease is a class of diseases in which an Sub fashion or some combination of these cells. Abroad spectrum ject's own antibodies react with host tissue or in which resistance to infectious challenge is induced because the immune effector T cells are autoreactive to endogenous self immune cells are in active form and are primed to respond to peptides and cause destruction of tissue. Thus an immune any invading compound or microorganism. The cells do not response is mounted against a Subject's own antigens, have to be specifically primed against a particular antigen. referred to as self antigens. Autoimmune diseases include but This is particularly useful in biowarfare, and the other cir are not limited to rheumatoid arthritis, Crohn's disease, mul cumstances described above such as travelers. tiple Sclerosis, systemic lupus erythematosus (SLE), autoim 0198 The CpG immunostimulatory oligonucleotides may mune encephalomyelitis, myasthenia gravis (MG), Hashimo be directly administered to the subject or may be adminis to's thyroiditis, Goodpasture's syndrome, pemphigus (e.g., tered in conjunction with a nucleic acid delivery complex. A pemphigus Vulgaris), Grave's disease, autoimmune nucleic acid delivery complex shall mean a nucleic acid mol hemolytic anemia, autoimmune thrombocytopenic purpura, ecule associated with (e.g. ionically or covalently bound to; or Scleroderma with anti-collagen antibodies, mixed connective encapsulated within) a targeting means (e.g. a molecule that tissue disease, polymyositis, pernicious anemia, idiopathic results in higher affinity binding to target cell. Examples of Addison's disease, autoimmune-associated infertility, glom nucleic acid delivery complexes include nucleic acids asso erulonephritis (e.g., crescentic glomerulonephritis, prolifera ciated with a sterol (e.g. cholesterol), a lipid (e.g. a cationic tive glomerulonephritis), bullous pemphigoid, Sjögren's Syn lipid, Virosome or liposome), or a target cell specific binding drome, insulin resistance, and autoimmune diabetes mellitus. agent (e.g. a ligand recognized by target cell specific recep 0.195 A “self-antigen” as used herein refers to an antigen tor). Preferred complexes may be sufficiently stable in vivo to of a normal host tissue. Normal host tissue does not include prevent significant uncoupling prior to internalization by the cancer cells. Thus an immune response mounted against a target cell. However, the complex can be cleavable under self-antigen, in the context of an autoimmune disease, is an appropriate conditions within the cell so that the oligonucle undesirable immune response and contributes to destruction otide is released in a functional form. and damage of normal tissue, whereas an immune response (0199 Delivery vehicles or delivery devices for delivering mounted against a cancer antigen is a desirable immune antigen and oligonucleotides to Surfaces have been described. response and contributes to the destruction of the tumor or The CpG immunostimulatory oligonucleotide and/or the cancer. Thus, in Some aspects of the invention aimed at treat antigen and/or other therapeutics may be administered alone ing autoimmune disorders it is not recommended that the (e.g., in Saline or buffer) or using any delivery vehicles known CpG immunostimulatory nucleic acids be administered with in the art. For instance the following delivery vehicles have self antigens, particularly those that are the targets of the been described: Cochleates; Emulsomes, ISCOMs: Lipo autoimmune disorder. Somes; Live bacterial vectors (e.g., Salmonella, Escherichia 0196. In other instances, the CpG immunostimulatory coli, Bacillus calmatte-guerin, Shigella, Lactobacillus); Live nucleic acids may be delivered with low doses of self-anti viral vectors (e.g., Vaccinia, adenovirus, Herpes Simplex); gens. A number of animal studies have demonstrated that Microspheres; Nucleic acid vaccines; Polymers; Polymer mucosal administration of low doses of antigen can result in rings; Proteosomes; Sodium Fluoride; Transgenic plants; a state of immune hyporesponsiveness or “tolerance.” The Virosomes: Virus-like particles. Other delivery vehicles are active mechanism appears to be a cytokine-mediated immune known in the art and some additional examples are provided deviation away from a Th1 towards a predominantly Th2 and below in the discussion of vectors. Th3 (i.e., TGF-B dominated) response. The active suppres 0200. The term effective amount of a CpG immunostimu sion with low dose antigen delivery can also Suppress an latory oligonucleotide refers to the amount necessary or Suf unrelated immune response (bystander Suppression) which is ficient to realize a desired biologic effect. For example, an of considerable interest in the therapy of autoimmune dis effective amount of a CpG immunostimulatory oligonucle eases, for example, rheumatoid arthritis and SLE. Bystander otide administered with an antigen for inducing mucosal Suppression involves the secretion of Th1-counter-regulatory, immunity is that amount necessary to cause the development Suppressor cytokines in the local environment where proin of IgA in response to an antigen upon exposure to the antigen, flammatory and Th1 cytokines are released in either an anti whereas that amount required for inducing systemic immu gen-specific or antigen-nonspecific manner. "Tolerance' as nity is that amount necessary to cause the development of IgG used herein is used to refer to this phenomenon. Indeed, oral in response to an antigen upon exposure to the antigen. Com tolerance has been effective in the treatment of a number of bined with the teachings provided herein, by choosing among autoimmune diseases in animals including: experimental the various active compounds and weighing factors such as autoimmune encephalomyelitis (EAE), experimental potency, relative bioavailability, patient body weight, severity autoimmune myasthenia gravis, collagen-induced arthritis of adverse side-effects and preferred mode of administration, (CIA), and insulin-dependent diabetes mellitus. In these an effective prophylactic or therapeutic treatment regimen models, the prevention and Suppression of autoimmune dis can be planned which does not cause Substantial toxicity and ease is associated with a shift in antigen-specific humoral and yet is entirely effective to treat the particular subject. The cellular responses from a Th1 to Th2/Th3 response. effective amount for any particular application can vary 0197) The invention also includes a method for inducing depending on Such factors as the disease or condition being antigen non-specific innate immune activation and broad treated, the particular CpG immunostimulatory oligonucle US 2010/01 66780 A1 Jul. 1, 2010 otide being administered the size of the subject, or the severity pharmaceutical composition of the present invention may be of the disease or condition. One of ordinary skill in the art can accomplished by any means known to the skilled artisan. empirically determine the effective amount of a particular Preferred routes of administration include but are not limited CpG immunostimulatory oligonucleotide and/orantigen and/ to oral, parenteral, intramuscular, intranasal, Sublingual, or other therapeutic agent without necessitating undue intratracheal, inhalation, ocular, vaginal, and rectal. experimentation. 0205 For oral administration, the compounds (i.e., CpG 0201 Subject doses of the compounds described herein immunostimulatory oligonucleotides, antigens and other for mucosal or local delivery typically range from about 0.1 therapeutic agents) can be formulated readily by combining ug to 10 mg per administration, which depending on the the active compound(s) with pharmaceutically acceptable application could be given daily, weekly, or monthly and any carriers well known in the art. Such carriers enable the com other amount of time therebetween. More typically mucosal pounds of the invention to be formulated as tablets, pills, or local doses range from about 10 ug to 5 mg per adminis dragees, capsules, liquids, gels, syrups, slurries, Suspensions tration, and most typically from about 100 ug to 1 mg, with and the like, for oral ingestion by a subject to be treated. 2-4 administrations being spaced days or weeks apart. More Pharmaceutical preparations for oral use can be obtained as typically, immune stimulant doses range from 1 Jug to 10 mg Solid excipient, optionally grinding a resulting mixture, and per administration, and most typically 10 ug to 1 mg, with processing the mixture of granules, after adding Suitableaux daily or weekly administrations. Subject doses of the com iliaries, if desired, to obtain tablets or dragee cores. Suitable pounds described herein for parenteral delivery for the pur excipients are, in particular, fillers such as Sugars, including pose of inducing an antigen-specific immune response, lactose, Sucrose, mannitol, or Sorbitol; cellulose preparations wherein the compounds are delivered with an antigen but not Such as, for example, maize starch, wheat starch, rice starch, another therapeutic agent are typically 5 to 10,000 times potato starch, gelatin, gum tragacanth, methyl cellulose, higher than the effective mucosal dose for vaccine adjuvant or hydroxypropylmethyl-cellulose, sodium carboxymethylcel immune stimulant applications, and more typically 10 to lulose, and/or polyvinylpyrrolidone (PVP). If desired, disin 1,000 times higher, and most typically 20 to 100 times higher. tegrating agents may be added, such as the cross-linked poly Doses of the compounds described herein for parenteral vinyl pyrrolidone, agar, oralginic acid or a salt thereofsuch as delivery for the purpose of inducing an innate immune Sodium alginate. Optionally the oral formulations may also be response or for increasing ADCC or for inducing an antigen formulated in saline or buffers, i.e. EDTA for neutralizing specific immune response when the CpG immunostimulatory internal acid conditions or may be administered without any oligonucleotides are administered in combination with other carriers. therapeutic agents or in specialized delivery vehicles typi 0206. Also specifically contemplated are oral dosage cally range from about 0.1 ug to 10 mg per administration, forms of the above component or components. The compo which depending on the application could be given daily, nent or components may be chemically modified so that oral weekly, or monthly and any other amount of time therebe delivery of the derivative is efficacious. Generally, the chemi tween. More typically parenteral doses for these purposes cal modification contemplated is the attachment of at least range from about 10 ug to 5 mg per administration, and most one moiety to the component molecule itself, where said typically from about 100 ug to 1 mg, with 2-4 administrations moiety permits (a) inhibition of proteolysis; and (b) uptake being spaced days or weeks apart. In some embodiments, into the blood stream from the stomach or intestine. Also however, parenteral doses for these purposes may be used in desired is the increase in overall stability of the component or a range of 5 to 10,000 times higher than the typical doses components and increase in circulation time in the body. described above. Examples of Such moieties include: polyethylene glycol, 0202 For any compound described herein the therapeuti copolymers of ethylene glycol and propylene glycol, car cally effective amount can be initially determined from ani boxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl mal models. A therapeutically effective dose can also be pyrrolidone and polyproline. Abuchowski and Davis, 1981, determined from human data for CpG oligonucleotides which “Soluble Polymer-Enzyme Adducts In: Enzymes as Drugs, have been tested in humans (human clinical trials have been Hocenberg and Roberts, eds., Wiley-Interscience, New York, initiated) and for compounds which are known to exhibit N.Y., pp. 367-383: Newmark, et al., 1982, J. Appl. Biochem. similar pharmacological activities, such as other adjuvants, 4:185-189. Other polymers that could be used are poly-1,3- e.g., LT and other antigens for vaccination purposes. Higher dioxolane and poly-1,3,6-tioxocane. Preferred for pharma doses may be required for parenteral administration. The ceutical usage, as indicated above, are polyethylene glycol applied dose can be adjusted based on the relative bioavail moieties. ability and potency of the administered compound. Adjusting 0207 For the component (or derivative) the location of the dose to achieve maximal efficacy based on the methods release may be the stomach, the Small intestine (the duode described above and other methods as are well-known in the num, the jejunum, or the ileum), or the large intestine. One art is well within the capabilities of the ordinarily skilled skilled in the art has available formulations which will not artisan. dissolve in the stomach, yet will release the material in the 0203 The formulations of the invention are administered duodenum or elsewhere in the intestine. Preferably, the in pharmaceutically acceptable solutions, which may rou release will avoid the deleterious effects of the stomach envi tinely contain pharmaceutically acceptable concentrations of ronment, either by protection of the oligonucleotide (or salt, buffering agents, preservatives, compatible carriers, derivative) or by release of the biologically active material adjuvants, and optionally other therapeutic ingredients. beyond the stomach environment, such as in the intestine. 0204 For use in therapy, an effective amount of the CpG 0208. To ensure full gastric resistance a coating imperme immunostimulatory oligonucleotide can be administered to a able to at least pH 5.0 is essential. Examples of the more subject by any mode that delivers the oligonucleotide to the common inertingredients that are used as enteric coatings are desired Surface, e.g., mucosal, systemic. Administering the cellulose acetate trimellitate (CAT), hydroxypropylmethyl US 2010/01 66780 A1 Jul. 1, 2010 22 cellulosephthalate (HPMCP), HPMCP50, HPMCP 55, poly 0216 Glidants that might improve the flow properties of vinyl acetate phthalate (PVAP), Eudragit L30D, Aquateric, the drug during formulation and to aid rearrangement during cellulose acetate phthalate (CAP), Eudragit L., Eudragit S, compression might be added. The glidants may include and Shellac. These coatings may be used as mixed films. starch, talc, pyrogenic silica and hydrated silicoaluminate. 0209. A coating or mixture of coatings can also be used on 0217. To aid dissolution of the therapeutic into the aque tablets, which are not intended for protection against the ous environment a surfactant might be added as a wetting stomach. This can include Sugar coatings, or coatings which agent. Surfactants may include anionic detergents such as make the tablet easier to Swallow. Capsules may consist of a Sodium lauryl Sulfate, dioctyl sodium SulfoSuccinate and dio hard shell (such as gelatin) for delivery of dry therapeutic i.e. ctyl Sodium sulfonate. Cationic detergents might be used and powder; for liquid forms, a softgelatin shell may be used. The could include benzalkonium chloride or benzethomium chlo shell material of cachets could be thick starch or other edible ride. The list of potential non-ionic detergents that could be paper. For pills, lozenges, molded tablets or tablet triturates, included in the formulation as Surfactants are lauromacrogol moist massing techniques can be used. 400, polyoxyl 40 stearate, polyoxyethylene hydrogenated 0210. The therapeutic can be included in the formulation castor oil 10, 50 and 60, glycerol monostearate, polysorbate as fine multi-particulates in the form of granules or pellets of 40, 60, 65 and 80, sucrose fatty acid ester, methyl cellulose particle size about 1 mm. The formulation of the material for and carboxymethyl cellulose. These surfactants could be capsule administration could also be as a powder, lightly present in the formulation of the oligonucleotide orderivative compressed plugs or even as tablets. The therapeutic could be either alone or as a mixture in different ratios. prepared by compression. 0218. Pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin, as well as 0211 Colorants and flavoring agents may all be included. soft, sealed capsules made of gelatin and a plasticizer, Such as For example, the oligonucleotide (or derivative) may be for glycerol or Sorbitol. The push-fit capsules can contain the mulated (such as by liposome or microsphere encapsulation) active ingredients in admixture with filler Such as lactose, and then further contained within an edible product, such as a binders such as starches, and/or lubricants such as talc or refrigerated beverage containing colorants and flavoring magnesium Stearate and, optionally, stabilizers. In soft cap agents. Sules, the active compounds may be dissolved or Suspended in 0212. One may dilute or increase the volume of the thera Suitable liquids, such as fatty oils, liquid paraffin, or liquid peutic with an inert material. These diluents could include polyethylene glycols. In addition, stabilizers may be added. carbohydrates, especially mannitol, a-lactose, anhydrous lac Microspheres formulated for oral administration may also be tose, cellulose, Sucrose, modified dextrans and starch. Certain used. Such microspheres have been well defined in the art. All inorganic salts may be also be used as fillers including cal formulations for oral administration should be in dosages cium triphosphate, magnesium carbonate and Sodium chlo Suitable for Such administration. ride. Some commercially available diluents are Fast-Flo, 0219 For buccal administration, the compositions may Emdex, STA-RX 1500, Emcompress and Avicell. take the form of tablets or lozenges formulated in conven 0213 Disintegrants may be included in the formulation of tional manner. the therapeutic into a solid dosage form. Materials used as 0220. For administration by inhalation, the compounds for disintegrates include but are not limited to starch, including use according to the present invention may be conveniently the commercial disintegrant based on starch, Explotab. delivered in the form of an aerosol spray presentation from Sodium starch glycolate, Amberlite, sodium carboxymethyl pressurized packs or a nebulizer, with the use of a suitable cellulose, ultramylopectin, Sodium alginate, gelatin, orange propellant, e.g., dichlorodifluoromethane, trichlorofluo peel, acid carboxymethyl cellulose, natural sponge and ben romethane, dichlorotetrafluoroethane, carbon dioxide or tonite may all be used. Another form of the disintegrants are other suitable gas. In the case of a pressurized aerosol the the insoluble cationic exchange resins. Powdered gums may dosage unit may be determined by providing a valve to deliver be used as disintegrants and as binders and these can include a metered amount. Capsules and cartridges of e.g. gelatin for powdered gums such as agar, Karaya or tragacanth. Alginic use in an inhaler or insufflator may be formulated containing acid and its Sodium salt are also useful as disintegrants. a powder mix of the compound and a suitable powder base 0214 Binders may be used to hold the therapeutic agent Such as lactose or starch. together to form a hard tablet and include materials from 0221) Also contemplated herein is pulmonary delivery of natural products such as acacia, tragacanth, starch and gela the oligonucleotides (or derivatives thereof). The oligonucle tin. Others include methyl cellulose (MC), ethyl cellulose otide (or derivative) is delivered to the lungs of a mammal (EC) and carboxymethyl cellulose (CMC). Polyvinyl pyrroli while inhaling and traverses across the lung epithelial lining done (PVP) and hydroxypropylmethyl cellulose (HPMC) to the blood stream. Other reports of inhaled molecules could both be used in alcoholic solutions to granulate the include Adjei et al., 1990, Pharmaceutical Research, 7:565 therapeutic. 569; Adjei et al., 1990, International Journal of Pharmaceu 0215. An anti-frictional agent may be included in the for tics, 63:135-144 (leuprolide acetate); Braquet et al., 1989, mulation of the therapeutic to prevent sticking during the Journal of Cardiovascular Pharmacology, 13 (suppl. 5): 143 formulation process. Lubricants may be used as a layer 146 (endothelin-1); Hubbard et al., 1989, Annals of Internal between the therapeutic and the die wall, and these can Medicine, Vol. III, pp. 206-212 (a1-antitrypsin); Smith et al., include but are not limited to; Stearic acid including its mag 1989, J. Clin. Invest. 84:1145-1146 (a-1-proteinase); Oswein nesium and calcium salts, polytetrafluoroethylene (PTFE), et al., 1990, Aerosolization of Proteins’. Proceedings of liquid paraffin, vegetable oils and waxes. Soluble lubricants Symposium on Respiratory Drug Delivery II, Keystone, may also be used such as sodium lauryl Sulfate, magnesium Colo., March, (recombinant human growth hormone); Debs lauryl Sulfate, polyethylene glycol of various molecular et al., 1988, J. Immunol. 140:3482-3488 (interferon-g and weights, Carbowax 4000 and 6000. tumor necrosis factor alpha) and Platz et al., U.S. Pat. No. US 2010/01 66780 A1 Jul. 1, 2010

5.284,656 (granulocyte colony stimulating factor). A method sity for deposition of the product in the lung. Formulations for and composition for pulmonary delivery of drugs for sys nasal delivery include those with dextran or cyclodextran. temic effect is described in U.S. Pat. No. 5,451,569, issued 0229. For nasal administration, a useful device is a small, Sep. 19, 1995 to Wong et al. hard bottle to which a metered dose sprayer is attached. In one 0222 Contemplated for use in the practice of this inven embodiment, the metered dose is delivered by drawing the tion are a wide range of mechanical devices designed for pharmaceutical composition of the present invention Solution pulmonary delivery of therapeutic products, including but not into a chamber of defined volume, which chamber has an limited to nebulizers, metered dose inhalers, and powder aperture dimensioned to aerosolize and aerosol formulation inhalers, all of which are familiar to those skilled in the art. by forming a spray when a liquid in the chamber is com 0223 Some specific examples of commercially available pressed. The chamber is compressed to administer the phar devices suitable for the practice of this invention are the maceutical composition of the present invention. In a specific Ultravent nebulizer, manufactured by Mallinckrodt, Inc., St. embodiment, the chamber is a piston arrangement. Such Louis, Mo.; the Acorn II nebulizer, manufactured by Mar devices are commercially available. quest Medical Products, Englewood, Colo.; the Ventolin 0230. Alternatively, a plastic squeeze bottle with an aper metered dose inhaler, manufactured by Glaxo Inc., Research ture or opening dimensioned to aerosolize an aerosol formu Triangle Park, N.C.; and the Spinhaler powder inhaler, manu lation by forming a spray when Squeezed is used. The opening factured by Fisons Corp., Bedford, Mass. is usually found in the top of the bottle, and the top is gener 0224 All such devices require the use of formulations ally tapered to partially fit in the nasal passages for efficient suitable for the dispensing of oligonucleotide (or derivative). administration of the aerosol formulation. Preferably, the Typically, each formulation is specific to the type of device nasal inhaler will provide a metered amount of the aerosol employed and may involve the use of an appropriate propel formulation, for administration of a measured dose of the lant material, in addition to the usual diluents, adjuvants drug. and/or carriers useful in therapy. Also, the use of liposomes, 0231. The compounds, when it is desirable to deliver them microcapsules or microspheres, inclusion complexes, or systemically, may be formulated for parenteral administra other types of carriers is contemplated. Chemically modified tion by injection, e.g., by bolus injection or continuous infu oligonucleotide may also be prepared in different formula Sion. Formulations for injection may be presented in unit tions depending on the type of chemical modification or the dosage form, e.g., in ampoules or in multi-dose containers, type of device employed. with an added preservative. The compositions may take Such 0225. Formulations suitable for use with a nebulizer, forms as suspensions, solutions or emulsions in oily or aque either jet or ultrasonic, will typically comprise oligonucle ous vehicles, and may contain formulatory agents such as otide (or derivative) dissolved in water at a concentration of Suspending, stabilizing and/or dispersing agents. about 0.1 to 25 mg of biologically active oligonucleotide per 0232 Pharmaceutical formulations for parenteral admin mL of solution. The formulation may also include a buffer istration include aqueous solutions of the active compounds and a simple Sugar (e.g., for oligonucleotide stabilization and in water-soluble form. Additionally, suspensions of the active regulation of osmotic pressure). The nebulizer formulation compounds may be prepared as appropriate oily injection may also contain a surfactant, to reduce or prevent Surface suspensions. Suitable lipophilic solvents or vehicles include induced aggregation of the oligonucleotide caused by atomi fatty oils such as sesame oil, or synthetic fatty acid esters, Zation of the Solution in forming the aerosol. Such as ethyl oleate or triglycerides, or liposomes. Aqueous 0226 Formulations for use with a metered-dose inhaler injection Suspensions may contain Substances which increase device will generally comprise a finely divided powder con the Viscosity of the Suspension, Such as Sodium carboxym taining the oligonucleotide (or derivative) Suspended in a ethyl cellulose, sorbitol, or dextran. Optionally, the suspen propellant with the aid of a surfactant. The propellant may be sion may also contain Suitable stabilizers or agents which any conventional material employed for this purpose, such as increase the solubility of the compounds to allow for the a chlorofluorocarbon, a hydrochlorofluorocarbon, a hydrof preparation of highly concentrated Solutions. luorocarbon, or a hydrocarbon, including trichlorofluo 0233. Alternatively, the active compounds may be in pow romethane, dichlorodifluoromethane, dichlorotetrafluoroet der form for constitution with a suitable vehicle, e.g., sterile hanol, and 1,1,1,2-tetrafluoroethane, or combinations pyrogen-free water, before use. thereof. Suitable surfactants include sorbitan trioleate and 0234. The compounds may also be formulated in rectal or Soya lecithin. Oleic acid may also be useful as a surfactant. vaginal compositions such as Suppositories or retention 0227. Formulations for dispensing from a powder inhaler enemas, e.g., containing conventional Suppository bases Such device will comprise a finely divided dry powder containing as cocoa butter or other glycerides. oligonucleotide (or derivative) and may also include a bulk 0235. In addition to the formulations described previously, ing agent, such as lactose, Sorbitol. Sucrose, or mannitol in the compounds may also be formulated as a depot prepara amounts which facilitate dispersal of the powder from the tion. Such long acting formulations may be formulated with device, e.g., 50 to 90% by weight of the formulation. The Suitable polymeric or hydrophobic materials (for example as oligonucleotide (or derivative) should most advantageously an emulsion in an acceptable oil) or ion exchange resins, or as be prepared in particulate form with an average particle size sparingly soluble derivatives, for example, as a sparingly of less than 10 mm (or microns), most preferably 0.5 to 5mm, soluble salt. for most effective delivery to the distal lung. 0236. The pharmaceutical compositions also may com 0228 Nasal delivery of a pharmaceutical composition of prise Suitable Solid or gel phase carriers or excipients. the present invention is also contemplated. Nasal delivery Examples of Such carriers or excipients include but are not allows the passage of a pharmaceutical composition of the limited to calcium carbonate, calcium phosphate, various present invention to the blood stream directly after adminis Sugars, starches, cellulose derivatives, gelatin, and polymers tering the therapeutic product to the nose, without the neces Such as polyethylene glycols. US 2010/01 66780 A1 Jul. 1, 2010 24

0237 Suitable liquid or solid pharmaceutical preparation EXAMPLES forms are, for example, aqueous or saline Solutions for inha lation, microencapsulated, encochleated, coated onto micro Materials and Methods scopic gold particles, contained in liposomes, nebulized, Oligodeoxynucleotides (ODN) and Reagents aerosols, pellets for implantation into the skin, or dried onto a 0242 All ODN were synthesized following standard sharp object to be scratched into the skin. The pharmaceutical phosphoramidite chemistry protocols and controlled for compositions also include granules, powders, tablets, coated identity and purity by Coley Pharmaceutical GmbH and had tablets, (micro)capsules, suppositories, syrups, emulsions, undetectable endotoxin levels (<0.1 EU/ml) measured by the Suspensions, creams, drops or preparations with protracted Limulus assay (BioWhittaker, Verviers, Belgium). ODN were release of active compounds, in whose preparation excipients suspended in sterile, endotoxin-free Tris-EDTA (Sigma, and additives and/or auxiliaries such as disintegrants, binders, Deisenhofen, Germany), and stored and handled under asep coating agents, Swelling agents, lubricants, flavorings, Sweet tic conditions to prevent both microbial and endotoxin con eners or solubilizers are customarily used as described above. tamination. All dilutions were carried out using endotoxin The pharmaceutical compositions are Suitable for use in a free Tris-EDTA. variety of drug delivery systems. For a brief review of meth ods for drug delivery, see Langer, Science 249:1527-1533, TLR Assays 1990, which is incorporated herein by reference. 0243 HEK293 cells were transfected by electroporation 0238. The CpG immunostimulatory oligonucleotides and with vectors expressing the respective human TLR and a optionally other therapeutics and/or antigens may be admin 6xNF-kB-luciferase reporter plasmid. Stable transfectants istered per se (neat) or in the form of a pharmaceutically (3x10" cells/well) were incubated indicated amounts of ODN acceptable salt. When used in medicine the salts should be for 16 hat 37°C. in a humidified incubator. Each data point pharmaceutically acceptable, but non-pharmaceutically was done in triplicate. Cells were lysed and assayed for acceptable salts may conveniently be used to prepare phar luciferase gene activity (using the BriteLite kit from Perkin maceutically acceptable salts thereof. Such salts include, but Elmer, Zaventem, Belgium). Stimulation indices were calcu are not limited to, those prepared from the following acids: lated in reference to reporter gene activity of medium without hydrochloric, hydrobromic, Sulphuric, nitric, phosphoric, addition of ODN. maleic, acetic, salicylic, p-toluene Sulphonic, tartaric, citric, methane sulphonic, formic, malonic, succinic, naphthalene Cell Purification 2-sulphonic, and benzene Sulphonic. Also, Such salts can be 0244 Peripheral blood buffy coat preparations from prepared as alkaline metal or alkaline earth salts, such as healthy human donors were obtained from the Blood Bank of Sodium, potassium or calcium salts of the carboxylic acid the University of Düsseldorf (Germany) and PBMC were group. purified by centrifugation over Ficoll-Hypaque (Sigma). Cells were cultured in a humidified incubator at 37° C. in 0239 Suitable buffering agents include: acetic acid and a RPMI 1640 medium supplemented with 5% (v/v) heat inac salt (1-2% w/v); citric acid and a salt (1-3% w/v); boric acid tivated human AB serum (BioWhittaker) or 10% (v/v) heat and a salt (0.5-2.5% w/v); and phosphoric acid and a salt inactivated FCS, 2 mM L-glutamine, 100U/ml penicillin and (0.8-2% w/v). Suitable preservatives include benzalkonium 100 g/ml streptomycin (all from Sigma). chloride (0.003-0.03% w/v); chlorobutanol (0.3-0.9% w/v); parabens (0.01-0.25% w/v) and thimerosal (0.004-0.02% Cytokine Detection and Flow Cytometric Analysis w/v). 0240. The pharmaceutical compositions of the invention 0245 PBMC were resuspended at a concentration of contain an effective amount of a CpG immunostimulatory 5x10° cells/ml and added to 96 well round-bottomed plates oligonucleotide and optionally antigens and/or other thera (2500/well). PBMC were incubated with ODN and culture peutic agents optionally included in a pharmaceutically-ac supernatants (SN) were collected after the indicated time ceptable carrier. The term pharmaceutically-acceptable car points. If not used immediately, SN were stored at -20° C. rier means one or more compatible Solid or liquid filler, until required. diluents or encapsulating Substances which are suitable for 0246 Amounts of cytokines in the SN were assessed using administration to a human or other vertebrate animal. The an in-house ELISA for IFN-Cl developed using commercially term carrier denotes an organic or inorganic ingredient, natu available antibody (PBL, New Brunswick, N.J., USA) or on ral or synthetic, with which the active ingredient is combined the Luminex multiplex system (LumineX Corporation, 12212 to facilitate the application. The components of the pharma Technology Boulevard, Austin, Tex. 78727-6115). ceutical compositions also are capable of being comingled with the compounds of the present invention, and with each Animals other, in a manner Such that there is no interaction which 0247. Female BALB/c mice (6-8 weeks of age) were pur would substantially impair the desired pharmaceutical effi chased from Charles River Canada (Quebec, Canada) and ciency. housed in micro-isolators in the Animal Care Facility at Coley 0241 The present invention is further illustrated by the Pharmaceutical Group Canada. All studies were conducted in following Examples, which in no way should be construed as accordance with the Animal Care Committee of Coley further limiting. The entire contents of all of the references Canada under the guidance of the Canadian Council on Ani (including literature references, issued patents, published mal Care. All animals were naive to CpG ODNs. patent applications, and co-pending patent applications) cited 0248 SA1N tumor model: Female A/J mice (10 per throughout this application are hereby expressly incorporated group) were injected SC with 5x10 Sal/N tumor cells on day by reference. 0. Mice were treated with 100 ug ODN or PBS alone given SC US 2010/01 66780 A1 Jul. 1, 2010

once weekly starting on day 8 post tumor induction. Animals ine base shape analogs, such as 2,4-difluorotoluene (FF) were monitored for survival and tumor volume. Tumor size (SEQ ID NO:3-9), 5-bromo-2'-deoxyuridine (BU) and (the length and the width) was measured using a digital ver 5-iodo-2'-deoxyuridine (JU), were incorporated outside of nier caliper. Tumor Volume was calculated by using the for the CpG motif (see Table 1 and FIGS. 2-3). Surprisingly, mula: Tumor volume=(0.4) (ab2), where a=large diameter incorporation of all tested hydrophobic thymine analogs led and b=Smaller diameter. to an unusually strong increase in hTLR9 activity, while substitution by uracil residues (thymine with lacking methyl In Vitro Assays group, FIG. 4) led to a strong decrease inhTLR9 stimulation. 0249 Naive BALB/c mouse splenocytes (from pools of The increase in TLR9 stimulation was pronounced when the 3-5 animals) were used for in vitro assays. Animals were modification was 5' to the CpG motif. Double substitution anaesthetized with isoflurane and euthanized by cervical dis with 5-iodouracil (JU) 5' and 3' of the CpG motif resulted in location. Spleens were removed under aseptic conditions and most potent stimulation of those tested. In contrast, Substitu placed in PBS+0.2% bovine serumalbumin (Sigma Chemical tion of guanine and cytosine by 2,4-difluorotoluene at the Company). Spleens were then homogenized and splenocytes CpG motifled in both cases to a strong decrease of the TLR9 were re-suspended in RPMI 1640 (Life Technologies, Grand stimulation index. Island, N.Y.) tissue culture medium supplemented with 2% 0253 Incorporation of hydrophobic T analogs also normal mouse serum (Cedarlane Laboratories, Ontario, resulted in a strong enhancement of IFN-alpha induction in Canada), penicillin-Streptomycin solution (final concentra human PBMCs. Unexpectedly, modification of an ODN tion of 1000 U/ml and 1 mg/ml respectively; Sigma Chemical (SEQ ID NO: 1) that is virtually inactive in inducing IFN Company), and 5x10-5 M b-mercaptoethanol (Sigma alpha with 5-bromouridine and 5-iodouridine in particular Chemical Company). resulted in increased TLR9 stimulation and IFN-alpha induc tion. There is usually an inverse correlation between TLR9 B Cell Proliferation Assays and IFN-alpha induction for CpG ODN which do not contain these modifications. 0250 Caboxy-florescein diacetate, succimidyl ester (CFSE) (Invitrogen, Eugene, Oreg., USA) stained BALB/c mouse splenocytes (4x10/well) were incubated with differ TABLE 1. ent concentrations of ODNina humidified 5% CO incubator Examples of modified oligonucleotides with at 37° C. for 5 days. Cells were then stained with PE conju hydrophobic thymine base shape analogs near gated anti-CD19 antibody (BD Pharmingen, San Diego, the CpG motif Calif., USA) for CD19 and B-cell proliferation was deter Seq mined by FACS followed by analysis by ModFit Software ID Description/class V3.0 (Verity Software House Inc., Topsham, Me., USA). Noi. Oligonucleotide sequence derived from Example 1 1 Tik G*T* C-G-k T. T. T. Tk Tik TKT + 1xPO of SEQ ID NO: 2

Investigation of Structure Activity Relationship at the CpG Motif 0251. It is known that oligonucleotides containing unm 5' FF derivative of SEQ ethylated CpG motifs are able to stimulate immune responses through the Toll-like receptor 9 (TLR9) pathway. In order to identify oligonucleotides with the greatestability to stimulate 3' FF derivative of SEQ the TLR9 pathway, comprehensive structure activity relation ID NO : 1 ship (SAR) study at the CpG motif was performed. The kTikTikTikTik 3" and is F derivative results showed that Substitution of guanine by hypoxanthine of SEQ ID NO: 1 and 6-thioguanine leads to a similar activity in hTLR9 assay, while purine, 2-aminopurine, 2,6-diaminopurine, 8-oxo-7,8- dihydroguanine and 7-deazaguanine Substitution resulted in a 40-80% reduction in hTLR9 stimulation. Further, modifica tion at C5 and N4 resulted in no stimulation of the hTLR9 pathway. These observations resulted in a SAR model in which guanine is recognized via the Hoogsteen site while cytosine binds at the CH-Edge to the TLR9 receptor (see FIG.1a). Thus, no modification at the Hoogsteen recognition 3' FF derivative of SEQ site of guanine as well as the CH-edge of the cytosine was ID NO : 1 possible without significant loss in hTLR9 activity. None of 5'BU derivative of SEQ the investigated base modifications at the dinucleotide motif was more active than the unmodified CpG motif. 11 T kG kT k 3'BU derivative of SEQ r ID NO : 1 Example 2 12 TikG kBU k C-G-kBU kTikTikTikTikTik 3" and s "BU derivative The Effect of Hydrophobic Thymine Base Shape of SEQ ID NO: 1 Analogs Near the CpG Motif 5"JU derivative of SEQ 0252) To investigate the impact of the dT residues in ID NO : 1 neighborhood to the CpG motif, several hydrophobic thym US 2010/01 66780 A1 Jul. 1, 2010 26

TABLE 1 - continued TABLE 2 Examples of modified oligonucleotides with Examples of modified oligonucleotides with hydrophobic thymine base shape analogs near lipophilic base analog substitutions the CpG motif Seq ID Description/class D Description/class Noi. Oligonucleotide sequence derived from Noi. Oligonucleotide sequence derived from 1 T kG kT k C-G-kT kT kT kT kT kT kT k Unmodified

41 TK Gk CUkC-G*T*T*T* Tk Tk Tk CI derivative of SEQ ID NO : 1 is TikG kJU k C-G-kU kTikTikTikTikTik 3" and is "JU derivative T*T*T*T*T*T*T* T+ T of SEQ ID NO: 1 10 TKGKBUkC-Gk TKT kT k Tk Tk Tk 5 'BU derivative of SEQ ID NO : 1

13 TK Gk JUK C-G-k TKT kT k Tk Tk Tk 5 'JU derivative of SEQ ID NO : 1

16 T k Gk UK C-G-k Tk Tk Tk Tk TKT kT K U derivative of SEQ ID NO: 1 8 TikG kU k C-G-kU kTikTikTikTikTikTik 3" and is 'U derivative T*T*T*T*T*T* T+ T of SEQ ID NO: 1 41 TK Gk CUkC-G*T*T*T* Tk Tk Tk CU derivative of SEQ ID NO : 1 phosphorothioate internucleotide linkage -phosphodiester internucleotide linkage 42 T-kick EUkC-G*T*T*T* Tk Tk Tk EU derivative of SEQ ID NO : 1 Example 3 29 TikG kTik C-G-kEU kTikTikTikTikTik 3" EU derivative of SEQ ID NO: 1 Activation of TLR9 with Lipophilic Base Shape 3 O TikG kEU k C-G-kEU kTikTikTikTikTik is '3' EU derivative of Substitutions SEQ ID NO: 1 phosphorothioate internucleotide linkage 0254. Since different types of lipophilic substitution of the -phosphodiester internucleotide linkage base 5' to the CpG motif caused significant increases in Stimu lation of hTLR9, other base analogs, such as 5-chloro-uracil, 5-trifluoromethyl-uracil, phenyl, aryl and substituted aryl Example 4 residues were investigated for their ability to stimulate hTLR9 (Table 3). To investigate activation of human TLR9 by Lipophilic Substitution on Oligonucleotides of A, B, B-class oligonucleotides modified with various lipophilic C, P, and T Classes base analogs, B-class ODNSEQID NO:1 was modified with 5-Chloro-2'-deoxyuridine (CU), 5-Bromo-2'-deoxyuridine 0256 To investigate the effects of lipophilic base analog (BU), 5-Iodo-2'-deoxyuridine (JU) and 5-Ethyl-2'-deoxyuri substitution on the different classes of ODN, modifications dine (EU). htLR9-NFkB-293 cells were incubated with the were made on A class, B class, C class, P class, and T class indicated ODN (FIG. 5a) for 16 hours. Cells were then lysed oligonucleotides. Some examples of these oligonucleotides and luciferase activity was determined. CU-modified (SEQ are given in Table 3. ID NO:41), BU-modified (SEQ ID NO:10) JU-modified (SEQ ID NO:13) and EU-modified (SEQ ID NO:42) oligo TABLE 3 nucleotides all showed greater stimulation of TLR9 activity JU-modified oligonucleotides of A, B, C, P, and over control (SEQ ID NO:1). SEQ ID NO:16 with uridine T class modification showed dramatically decreased activity. In a second experiment IFN-alpha production was measured Seq (FIG.5b). Human PBMC were incubated with the modified ID Oligo ODN as indicated for 24h, after which the supernatants were No. Modified Oligonucleotide Class tested by ELISA. JU-modified, BU-modified, and EU-modi fied ODN resulted in the greatest increase in IFN-alpha over control. These data demonstrate that 5'-substitution of dU on a B-class ODN increases TLR9 activity and IFN-alpha pro duction. 0255. To further investigate the effect of EU modification on TLR9 activation, the experiment was repeated with modi fied oligonucleotides having EU modifications 5' of the CpG (SEQID NO:42), 3' of the CpG (SEQID NO:29), and 5' and 3' of the CpG (SEQ ID NO:30). SEQ ID NOs 42 and 30 showed a significant increase in TLR9 activation over unmodified SEQID NO:1 and unmodified B class ODNSEQ ID NO:37 (FIG. 6). US 2010/01 66780 A1 Jul. 1, 2010 27

TABLE 3 - continued TABLE 3 - continued

JU-modified oligonucleotides of A, B, C, P, and JU-modified oligonucleotides of A, B, C, P, and T class T class Seq Seq ID Oligo ID Oligo No. Modified Oligonucleotide Class No. Modified Oligonucleotide Class

21 B

22 phosphorothioate internucleotide linkage -phosphodiester internucleotide linkage

23 T: C kg kJUk C. kog kJUk T+ T 0257 To investigate activation of human TLR9 by modi T. T. Tk T fied B class oligonucleotides, 5-iodo-2'-deoxyuridine-modi 24 fied B-class derivatives of SEQID NO:37 were evaluated in a luciferase assay for their ability to activate TLR9 (see mate rials and methods). All modified B-class oligonucleotides 25 showed a significant increase in TLR9 activation over unmodified SEQID NO:37 (FIG. 7). 26 Non CpG ODN 0258 To investigate activation of human TLR9 by modi fied A-class oligonucleotides, 5-iodo-2'-deoxyuridine-modi fied A-class derivatives of SEQ ID NO:43 were tested for 27 their ability to activate TLR9 in a luciferase assay (FIG. 8a) and a PBMC assay (FIG. 8b) as in FIG. 5. The increase in 28 TLR9 stimulation was pronounced when the modification was 5' to the CpG motif, although double substitution with 31 5-iodouracil (JU) 5' and 3' of the CpG motif resulted in most potent stimulation. 0259. To investigate the activation of human TLR9 by 32 modified C class oligonucleotides, 5-iodo-2'-deoxyuridine modified C class derivatives of SEQ ID NO:46, SEQ ID 33 NO:44 and 45, were tested for their ability to activate TLR9. A class sequences SEQID NO:43 (unmodified) and SEQID NO:35 and 36 were tested simultaneously. As shown in FIG. 34 9, modified ODNSEQ ID NO:35, 36, 44, and 45 all showed 35 Tk C-G-A-C- G- JU- C - G - T- G- GGGG increased stimulation of TLR9 above unmodified A and C class in a luciferase assay. To investigate the activation of 36 Tk C-G-A-C- G- JU- C - G - JU- G - GGGG human TLR9 by modified P class oligonucleotides, 5-iodo 37 2'-deoxyuridine-modified P class derivatives of SEQ ID NO:46 were tested for their ability to activate TLR9 in a luciferase assay. As shown in FIG. 10, modified ODNSEQID 43 NO: 31-33 showed an increased stimulation of TLR9 over 44 unmodified ODN. 0260 To investigate the activation of human TLR9 by modified T class oligonucleotides, 5-iodo-2'-deoxyuridine 45 modified T class derivatives of unmodified T class ODNSEQ ID NO:52 were tested for their ability to activate TLR9. As 46 shown in FIG. 11, modified ODNSEQID NOs 47-50 showed an increased stimulation of TLR9 over unmodified T class ODN in a luciferase assay. The uridine derivative SEQ ID 47 NO:51 showed reduced Stimulation of TLR9. 0261. As the above examples demonstrate, substitution of 48 lipophilic T-analogs 5' to the CpG motif results in a strong increase in TLR9 activation in all classes tested, and resulted in an increased ability to induce IFN-alpha production. 49 Example 5 SO Stimulation of TLR9 by Short Modified Oligonucle otides 51 0262. As the modified CpG ODN of 20 nucleotides in length showed an unusual affinity for TLR9 activation, very short CpG ODN were investigated for their ability to activate US 2010/01 66780 A1 Jul. 1, 2010 28

TLR9. Very short oligonucleotides would be a great advan BALB/c mice with modified SEQ ID NO:13 resulted in tage overlonger oligonucleotides for use intreatment because higher TNF-alpha and IP-10 production than the non-modi of the increased ease in uptake by cells, as well as the potential fied SEQ ID NO:37, demonstrating that the lipophilic base a simpler formulation, without the use of DOTAP. Three short shape substituted ODN of the invention result in greater CpG ODN (shortmers) were investigated (Table 3): a 6-mer immune stimulation in vivo than unmodified immune stimu CpG motif hexamer (SEQID NO:38), a 5'JU modification of latory ODN. the hexamer (SEQ ID NO:39), and a 5'3"JU modification of the hexamer (SEQ ID NO:40) (Table 4). The activity of the TABLE 5 shortimers was compared to the unmodified B class oligo nucleotide SEQID NO:37 in a luciferase assay. As shown in Oligonucleotides tested in vivo FIG. 12, most particularly with SEQ ID NO:40, the use of Seq modified shortimers shows great potential as an improved ID immunotherapy medicament. No. Sequence Modification 13 T kG kJU k C-G-kT kT kT kT kT kT kT kT kT kT k 5 'JU derivative TABL E 4. T*T*T* T+ T of SEQ ID NO: 1

Modified short oligonucleotides 37 T kC kG kT k C-k G'k TikT kT kT kT kC kG kG kT k Unmodified B C kg+ T k T+ T k T class Seq ID No. Shortmer sequence Modification

38 G'kT k C-G-kT kT Unmodified phosphorothioate internucleotide linkage -phosphodiester internucleotide linkage 39 G'kJUk C-G-kT kT s' JU 4 O Gik U:k C- Giku UtkT 5 and 3 JU Example 7 37 T kC kG kT k C+G'k TikT kT kT kT kC kG kG kT kC kG k Unmodified T. T. Tk T B class Oligonucleotides with Additional Modifications

phosphorothioate internucleotide linkage 0266 ODN with lipophilic base analogs were tested for -phosphodiester internucleotide linkage their ability to induce TLR9-mediated NF-kB activity in a luciferase assay (see materials and methods). FIGS. 16-23 show the activity of ODN with additional modifications (see Example 6 table 6). Activation of TLR9 Pathway In Vivo by Modified 0267 In order to test the activity of other base analogs, the Oligonucleotides activity of 6-nitro-benzimidazol (6NB)-modified ODNSEQ ID NO:178 and unmodified parent sequence SEQ ID NO:1 0263. In order to determine the efficacy of the modified was compared. As shown in FIG. 12, SEQ ID NO: 178 was ODN of the invention in vivo, ODN with lipophilic Tanalogs able to activate TLR9-mediated NF-kB to a degree compa were tested in isolated mouse splenocytes. BALB/c mouse rable with the unmodified parent sequence. Next the activity splenocytes were isolated and incubated with modified B of 5-(2-bromovinyl)-uridine modified ODN (SEQ ID class (SEQID NO:13), unmodified B class (SEQID NO:37), NO:153-154) was compared to that of unmodified parent and a non-CpG ODN (SEQ ID NO:26) (Table 5). Culture sequence SEQID NO:1. As shown in FIG. 17, both modified supernatants were collected at 6 hour (TNF-alpha) or 24 ODN were more active in the assay than the parent sequence. hours (IL-6, IL-10, IL-12) and cytokine concentration was Next the activity of two B-class ODN with 5-proynyl-dU measured by ELISA. As shown in FIG. 13, incubation with (SEQID NO:116 and 117) in place of thymidine of the parent modified SEQID NO:13 resulted in dramatically increased sequence (SEQID NO:1). As shown in FIG. 21, both modi levels of all cytokines tested. fied ODN had activity comparable to that of the parent 0264 ODN were then tested their ability to induce B cell sequence. The activity of SEQ ID NO: 116, in which the proliferation in splenocytes. CFSE-stained BALB/c mouse modification is 5' to the CG dinucleotide, was slightly splenocytes (4x10/well) were incubated with 0.001, 0.01, improved over the parent sequence. 0.1, 0.3, 1, 3 or 10 ug/ml of the indicated ODN (FIG. 14). At 0268. In order to test the effect of a second type of modi 72 hours post-incubation, cells were stained for cell surface fication on JU-modified ODN, 2O-methylguanosines were marker CD19 and B-cell proliferation was determined by incorporated into JO-modified ODN. The activity of 2'-O- FACS followed by analysis by ModFit Software. As shown in methylguanosine/JU ODN SEQ ID NO:111-113 was com FIG. 14, incubation with modified SEQID NO:13 resulted in pared to that of parent SEQID NO:1 and JU only modified a marked increase in B-cell proliferation. The increase was SEQ ID NO:13. As shown in FIG. 18, all JU-modified ODN most pronounced even at lower ODN concentration. were more active than the parent ODN. ODN with the 2'O- 0265. To measure the effect of modified ODN in vivo, methylguanosine modification 3' of the CG dinucleotide BALB/c mice (5 per group) were injected subcutaneously (SEQ ID NO:112-113) were slightly more active than the (SC) with 10, 50 or 100 ug of SEQ ID NO:13 or 100 g of ODN with the 2'O-methylguanosine modification 5' of the SEQID NO:37 in a total volume of 100 ul SC. Control group CG dinucleotide (SEQ ID NO:111) or the ODN modified received 100 ul of PBS alone. Animals were bled by cardiac with JU alone (SEQ ID NO:13). puncture at 1 hour post injection (TNF-alpha) or 3 hours post 0269. Next the activity of the JU-modified branched ODN injection (IP-10). Plasma samples were assayed ELISA for (SEQ ID NO:96, 97, 101, and 102) was compared to that of TNF-alpha (FIG. 15a) and IP-10 (FIG. 15b). Injection of SEQID NO:1. As shown in FIG. 19, the branched ODN with US 2010/01 66780 A1 Jul. 1, 2010 29 two accessible 5' ends were all as active or more active than the unmodified SEQID NO:1 in the assay. SEQID NO:101 TABLE 6-continued and 102, with the triethylenglycol phosphate spacer, were more active than SEQ ID NO:96 AND 97 with the 3'-O- Lipophilic substituted oligonucleotides with Methyl-G spacer. additional modifications Seq (0270. Next the activity of a short unmodified B-class ODN D (SEQ ID NO:38) and an ODN of the same sequence with a No. Sequence Type and modification lipophilic Substituted nucleotide analog and a lipophilic 3' tag 26 GUk C- GUkT-ex B-class derivative of (SEQID NO:126) was compared. Both were formulated with 38 with JU and hexa and without DOTAP As shown in FIG. 20, the addition of the decylglyceryl 3" tag JU-modification and the lipophilic tag greatly enhanced the activity of the ODN, as did the addition of DOTAP. (0271 Next the activity of B-class ODN with a second nucleotide analogin addition to a lipophilic Substituted nucle otide analog (SEQIDNO: 138,7-deaza-dG: SEQIDNO:139, inosine; SEQID NO:140, 5-methyl-dC) was compared to that of the parent sequence (SEQIDNO: 1) and the same sequence with a lipophilic substituted nucleotide analog only (SEQID NO:13). As shown in FIG. 22, all modified ODN were more 38 TikG kJU k C-E kTikTikTikTikTikTik B-class with 7-deaza active in the assay than the parent ODN dG (E) (0272. Next the activity of T-class ODN with a lipophilic 39 T-ko-kJU k C-I k Tx Tyk Tx T- T - T - B class with inosine substituted nucleotide analog (SEQ ID NO:132-134) was r ( ) compared to that of a C-class ODN (SEQID NO:198) known to be immunostimulatory. As shown in FIG. 23, all modified ODN showed much greater activity in the assay than the 53 Tk G'kBVU k C- Gk Tk Tk Tk Tk Tk Tk B-class with 5-(2- unmidified C-class ODN. bromo- vinyl)- uridine (BWU) TABLE 6 54 Tk Gik Tk C- GkBWUk Tk Tk Tk Tk Tk B-class with 5-(2- Lipophilic substituted oligonucleotides with bromo- vinyl)- additional modifications uridine (BWU) Seq 78 TikGik 6NB:k C-G-kTikTikTikTikTikTik B-class with ID 6-nitro -benzimidazol No. Sequence Type and modification (6N) 1 TikG kTik C-G-kTikTikTikTikTikTikTik unmodified B-class 98 C kg kG'k C-k G'k C-k C-kT k C-kg C-class T*T*T*T*T*T* T+ T phosphorothioate internucleotide linkage 13 TikG kJU k C-G-kTikTikTikTikTikTik B-class : "JU deriva -phosphodiester internucleotide linkage

38 G'kT k C-G-kT kT Unmodified B-class Example 8 96 (TikG#JU k C-G-k Tik Tsk Lk) 3'3" -branched 2 doub-3mG Activity of Modified P-Class Oligonucleotides 97 (JU k C# G# Tik Tk C# G# Lk) 3'3" -branched 0273 P-class ODN with lipophilic base analogs were 2 doub-3mG tested for the ability to activate the NF-kB pathway through O1 (T + G*JU k C-G-k Tik Tsk Lk) 3'3" -branched TLR9 as measured by luciferase assay. The activity of P-class 2 doub-teg ODN with a lipophilic substituted nucleotide analog (SEQID NO:58-61) was compared to that of a B-class positive control O2 (JU k C# G# Tik Tk C# G# Lk) 3'3" -branched 2 doub-teg (SEQ ID NO:55) and an unmodified P-class ODN (SEQ ID NO:56). As shown in FIG. 24, all modified P-class ODN showed increased TLR9 stimulation compared to the con trols. FIG. 24a shows JU-modified P-class ODN and 24b shows EU-modified P-class ODN. (0274) Next the activity of modified P-class ODN (SEQID NO:64 (EU-modified), 66-67 (JU-modified) was compared to that of a B-class positive control (SEQ ID NO:55), a C-class ODN (SEQ ID NO:68) and an unmodified P-class ODN (SEQ ID NO:57). As shown in FIG. 25, all modified ODN showed a higher degree of TLR9 stimulation than the unmodified P class ODN. SEQ ID NO:66, with the phos phodiester bond in the CG dinucleotide, showed reduced activity compared to the fully phosphorothioate SEQ ID NO:67. US 2010/01 66780 A1 Jul. 1, 2010 30

0275 Next the modified P-class ODN were tested for their NO:54), a negative control ODN (SEQ ID NO:53), an ability to induce expression of IFN-alpha. The activity of unmodified P-class ODN (SEQ ID NO:56), LPS, R-848, P-class ODN with a lipophilic substituted nucleotide analog SEB, and a polyII:IC ODN. CFSE-labeled PBMC from (SEQID NO:58-61) was compared to that of a B-class posi three donors were incubated with the ODN for 5 days and tive control (SEQIDNO:55) and an unmodified P-class ODN then stained with a CD19 antibody. The percentage of B cells (SEQID NO:56) as measured by an ELISA assay. As shown with reduced CFSE staining was determined. Treatment with in FIG. 26, all modified P-class ODN showed an increase in the B-class ODN resulted in the highest percentage of B cells IFN-alpha induction. FIG. 26a shows JU-modified P-class after division. Treatment with the JU-modified ODN resulted ODN and 26b shows EU-modified P-class ODN. in a higher percentage of B cells than the EU-modified ODN. 0279. In order to determine the effect of the modified (0276 Next the modified P-class ODN (EU-modified), P-class ODN in vivo, BALB/c mice (5 per group) were 66-67 (JU-modified) was compared to that of a B-class posi injected SC with differing doses of ODN. Animals were bled tive control (SEQ ID NO:55), a C-class ODN (SEQ ID at 3 hr post injection and plasma tested for IFN-alpha by NO:68) and an unmodified P-class ODN (SEQID NO:57) for ELISA. The activity of modified P-class ODN (SEQ ID the ability to induce IFN-alpha as measured by an ELISA NO:58, 60-62, 64, and 67) was compared to that of a B-class assay. As shown in FIG. 27, the modified P-class ODN negative control (SEQ ID NO:55) and a negative control showed enhanced ability to induce IFN-alpha. As in FIG. 24, (SEQ ID NO:26). As shown in FIG. 30, treatment with the SEQID NO:66 showed reduced activity compared to SEQID JU-modified ODN SEQ NO:58, 60, and 61 resulted in NO:67. slightly higher IFN-alpha induction than the EU-modified (0277 Next the modified P-class ODN were tested for the ODNSEQID NO:64. The B-class ODNSEQID NO:55 did ability to induce IL-6 in human PBMC. PBMC from three not induce much murine IFN-alpha, as expected. donors were incubated with ODN at concentrations as indi 0280 Next the modified P-class ODN were evaluated for cated for 24 h, followed by luminex 25-plex analysis of the their ability to reduce tumor volume mouse SA1N tumor supernatants for IL-6. The activity of modified P-class ODN model. Female A/J mice (10 pergroup) were injected SC with (SEQ ID NO:58, 60-62, FIG. 28a) (SEQID NO:64 and 67, 5x10 Sal/N tumor cells on day 0. Mice were treated with 35 FIG. 28b) was compared to that of an unmodified B-class ug (FIG. 31a) or 100 ug (FIG. 31b) P-class ODN with a ODN (SEQID NO:55), and unmodified C-class ODN (SEQ lipophilic substituted nucleotide analog (SEQID NO:60, 64. ID NO:54), a negative control ODN (SEQID NO:53), and an and 67), an unmodified C-class ODN, an unmodified B-class unmodified P-class ODN (SEQID NO:56). The JU-modified ODN (SEQ ID NO:55), or PBS alone. ODN were given SC ODN (SEQ ID NO:58, 60-61 and 67) showed a slightly once weekly starting on day 8 post tumor induction. Animals higher activation of IL-6 than did the EU-modified ODN were monitored for survival and tumor volume. As shown in (SEQ ID NO:62 and 64). All modified ODN showed FIG. 31a, at the lower dosage treatment with the modified increased activity compared to unmodified ODN. P-class ODN showed the greatest reduction in tumor volume, (0278 Next the activity of modified P-class class ODN suggesting that these ODN would be effective in treating (SEQ ID NO:58, 60-62, FIG. 29a) (SEQID NO:64 and 67, cancer. At the higher dosage in 31b, all modified P-class ODN FIG. 29b) was compared to that of an unmodified B-class and the C-class ODN were effective in reducing tumor vol ODN (SEQID NO:55), an unmodified C-class ODN (SEQID le.

TABLE 7

Modified P-class oligonucleotides

Seq Type and ID No. Sequence modification

neg control US 2010/01 66780 A1 Jul. 1, 2010 31

TABLE 7-continued Modified P-class oligonucleotides US 2010/01 66780 A1 Jul. 1, 2010 32

TABLE 8- continued Seq ID No

92

93

94

95

96

97

98

99

OO

O2

21

22

23

24

25 GUk C- GkTikT-ex

26 GUk C- GUkT-ex

91 27 GEUk C- GEUkT-ex US 2010/01 66780 A1 Jul. 1, 2010

TABLE 8- continued Seq ID No Oligonucleotide sequence

55

56

58

59

6 O

61

62

63

64

65

66

67

68

69

70

71.

72

73

74

76

77

78

79 US 2010/01 66780 A1 Jul. 1, 2010 34

TABLE 8- continued TABLE 8- continued

Seq ID Seq ID No H. Oligonucleotide sequence No H. Oligonucleotide sequence

phosphorothioate internucleotide linkage -phosphodiester internucleotide linkage teg Spacer 9 (triethylenglycol phosphate) hex hexadecylglyceryl 3mG 3'-O-Methyl-rC iT inverse nucleotide (3 and 5 switched) 2doub Doubler2 (Chemgenes FF 2, 4-difluorotoluene BU 5-bromo-2'-deoxyuridine JU 5-iodo-2'-deoxyuridine U Uridine CU 5-chloro-2'-deoxyuridine EU 5-fluoro-dU EU 5-ethyl-2'-deoxyuridine SNB 3-nitro-benzimidazol PU 5-proynyl-dU I inosine Z 5-methyl-dC E 7-deaza-dC FT aaa-trifluoro-dT BVU 5- (d-bromo-vinyl)-uridine NI nitroindol NP nitropyrrol F 5-fluoro-dU L. Spacer 18 (hexaethylenglycol phosphate) EQUIVALENTS 0282. The foregoing written specification is considered to be sufficient to enable one skilled in the art to practice the invention. The present invention is not to be limited in scope by examples provided, since the examples are intended as a single illustration of one aspect of the invention and other functionally equivalent embodiments are within the scope of the invention. Various modifications of the invention in addi tion to those shown and described herein will become appar ent to those skilled in the art from the foregoing description and fall within the scope of the appended claims. The advan tages and objects of the invention are not necessarily encom passed by each embodiment of the invention.

tgtcgtttitt tttittttittt

SEO ID NO 2 LENGTH: 2O TYPE: DNA ORGANISM: Artificial Sequence FEATURE; US 2010/01 66780 A1 Jul. 1, 2010 35

- Continued OTHER INFORMATION: Synthetic Oligonucleotide

SEQUENCE: 2 tgtcgtttitt tttitttittitt

SEQ ID NO 3 LENGTH: 2O TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Synthetic Oligonucleotide FEATURE: NAMEAKEY: misc feature LOCATION: (3) ... (3) OTHER INFORMATION: wherein n is 2, 4-difluorotoluene SEQUENCE: 3 tgnogtttitt tttitttittitt

SEQ ID NO 4 LENGTH: 2O TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Synthetic Oligonucleotide FEATURE: NAMEAKEY: misc feature LOCATION: (6) . . (6) OTHER INFORMATION: wherein n is 2, 4-difluorotoluene SEQUENCE: 4 tgtcgnttitt tttitttittitt

SEO ID NO 5 LENGTH: 2O TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Synthetic Oligonucleotide FEATURE: NAMEAKEY: misc feature LOCATION: (3) ... (3) OTHER INFORMATION: wherein n is 2, 4-difluorotoluene FEATURE: NAMEAKEY: misc feature LOCATION: (6) . . (6) OTHER INFORMATION: wherein n is 2, 4-difluorotoluene

SEQUENCE: 5 tgnognttitt tttitttittitt

SEQ ID NO 6 LENGTH: 2O TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Synthetic Oligonucleotide FEATURE: NAMEAKEY: misc feature LOCATION: (4) ... (4) OTHER INFORMATION: wherein n is 2, 4-difluorotoluene

SEQUENCE: 6 tgtngtttitt tttitttittitt

SEO ID NO 7 US 2010/01 66780 A1 Jul. 1, 2010 36

- Continued

&211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (5) . . (5) <223> OTHER INFORMATION: wherein n is 2, 4-difluorotoluene <4 OO > SEQUENCE: 7 tgtcntttitt tttitttittitt

<210s, SEQ ID NO 8 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (2) ... (2) <223> OTHER INFORMATION: wherein n is 2, 4-difluorotoluene <4 OOs, SEQUENCE: 8 tncgttittitt tttitttittitt

<210s, SEQ ID NO 9 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (7) . . (7) <223> OTHER INFORMATION: wherein n is 2, 4-difluorotoluene

<4 OOs, SEQUENCE: 9 tgtcgtnttt tttitttittitt

<210s, SEQ ID NO 10 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (3) ... (3) <223> OTHER INFORMATION: wherein n is 5-bromo-2 prime-deoxyuridine

<4 OOs, SEQUENCE: 10 tgnogtttitt tttitttittitt

<210s, SEQ ID NO 11 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (6) . . (6) <223> OTHER INFORMATION: wherein n is 5-bromo-2prime-deoxyuridine

<4 OOs, SEQUENCE: 11 US 2010/01 66780 A1 Jul. 1, 2010 37

- Continued tgtcgnttitt tttitttittitt 2O

<210s, SEQ ID NO 12 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (3) ... (3) <223> OTHER INFORMATION: wherein n is 5-bromo-2 prime-deoxyuridine 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (6) . . (6) <223> OTHER INFORMATION: wherein n is 5-bromo-2 prime-deoxyuridine <4 OOs, SEQUENCE: 12 tgnognttitt tttitttittitt 2O

<210s, SEQ ID NO 13 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (3) ... (3) <223> OTHER INFORMATION: wherein n is 5-iodo-2 prime-deoxyuridine <4 OOs, SEQUENCE: 13 tgnogtttitt tttitttittitt 2O

<210s, SEQ ID NO 14 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (6) . . (6) <223> OTHER INFORMATION: wherein n is 5-iodo-2 prime-deoxyuridine

<4 OOs, SEQUENCE: 14 tgtcgnttitt tttitttittitt 2O

<210s, SEQ ID NO 15 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (3) ... (3) <223> OTHER INFORMATION: wherein n is 5-iodo-2 prime-deoxyuridine 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (6) . . (6) <223> OTHER INFORMATION: wherein n is 5-iodo-2 prime-deoxyuridine

<4 OOs, SEQUENCE: 15 tgnognttitt tttitttittitt 2O

<210s, SEQ ID NO 16 US 2010/01 66780 A1 Jul. 1, 2010 38

- Continued

LENGTH: 2O TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Synthetic Oligonucleotide FEATURE: NAMEAKEY: misc feature LOCATION: (3) ... (3) OTHER INFORMATION: wherein n is uridine

SEQUENCE: 16 tgnogtttitt tttitttittitt

SEO ID NO 17 LENGTH: 2O TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Synthetic Oligonucleotide FEATURE: NAMEAKEY: misc feature LOCATION: (6) . . (6) OTHER INFORMATION: wherein n is uridine

SEQUENCE: 17 tgtcgnttitt tttitttittitt

SEQ ID NO 18 LENGTH: 2O TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Synthetic Oligonucleotide FEATURE: NAMEAKEY: misc feature LOCATION: (3) ... (3) OTHER INFORMATION: wherein n is uridine FEATURE: NAMEAKEY: misc feature LOCATION: (6) . . (6) OTHER INFORMATION: wherein n is uridine

SEQUENCE: 18 tgnognttitt tttitttittitt

SEQ ID NO 19 LENGTH: 21 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Synthetic Oligonucleotide FEATURE: NAMEAKEY: misc feature LOCATION: (1) ... (1) OTHER INFORMATION: wherein n is 5-iodo-2 prime-deoxyuridine

SEQUENCE: 19 incgt.cgttitt toggtcgttt t 21

SEQ ID NO 2 O LENGTH: 21 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Synthetic Oligonucleotide FEATURE: NAMEAKEY: misc feature LOCATION: (4) ... (4) US 2010/01 66780 A1 Jul. 1, 2010 39

- Continued <223> OTHER INFORMATION: wherein n is 5-iodo-2 prime-deoxyuridine <4 OOs, SEQUENCE: 2O tcqncgttitt toggtcgttt t 21

<210s, SEQ ID NO 21 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (15) . . (15) <223> OTHER INFORMATION: wherein n is 5-iodo-2 prime-deoxyuridine <4 OOs, SEQUENCE: 21 tcqtcqttitt toggnogttt t 21

<210s, SEQ ID NO 22 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: wherein n is 5-iodo-2 prime-deoxyuridine 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (4) ... (4) <223> OTHER INFORMATION: wherein n is 5-iodo-2 prime-deoxyuridine

<4 OOs, SEQUENCE: 22 incgncgttitt toggtcgttt t 21

<210s, SEQ ID NO 23 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (4) ... (4) <223> OTHER INFORMATION: wherein n is 5-iodo-2 prime-deoxyuridine 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (7) . . (7) <223> OTHER INFORMATION: wherein n is 5-iodo-2 prime-deoxyuridine

<4 OOs, SEQUENCE: 23 tcqncgnttt toggtcgttt t 21

<210s, SEQ ID NO 24 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (15) . . (15) <223> OTHER INFORMATION: wherein n is 5-iodo-2 prime-deoxyuridine 22 Os. FEATURE: <221 > NAMEAKEY: misc feature LOCATION: (18) ... (18) US 2010/01 66780 A1 Jul. 1, 2010 40

- Continued <223> OTHER INFORMATION: wherein n is 5-iodo-2 prime-deoxyuridine <4 OOs, SEQUENCE: 24 tcqtcqttitt toggnogntt t 21

<210s, SEQ ID NO 25 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide

<4 OOs, SEQUENCE: 25 tottttttgt cqttttittitt tt 22

<210s, SEQ ID NO 26 &211s LENGTH: 24 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide

<4 OOs, SEQUENCE: 26 tgctgcttitt gtgcttttgt gctt 24

<210s, SEQ ID NO 27 &211s LENGTH: 24 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: wherein n is 5-iodo-2 prime-deoxyuridine

<4 OOs, SEQUENCE: 27 incgt.cgttitt acggcgc.cgit gcc.g 24

<210s, SEQ ID NO 28 &211s LENGTH: 24 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (4) ... (4) <223> OTHER INFORMATION: wherein n is 5-iodo-2 prime-deoxyuridine

<4 OOs, SEQUENCE: 28 tcgn.cgttitt acggcgc.cgit gcc.g 24

<210s, SEQ ID NO 29 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (6) . . (6) <223> OTHER INFORMATION: wherein n is 5-ethyl-2 prime-deoxyuridine

<4 OOs, SEQUENCE: 29 US 2010/01 66780 A1 Jul. 1, 2010 41

- Continued tgtcgnttitt tttitttittitt 2O

<210s, SEQ ID NO 3 O &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (3) ... (3) <223> OTHER INFORMATION: wherein n is 5-ethyl-2 prime-deoxyuridine 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (6) . . (6) <223> OTHER INFORMATION: wherein n is 5-ethyl-2 prime-deoxyuridine

<4 OOs, SEQUENCE: 30 tgnognttitt tttitttittitt 2O

<210s, SEQ ID NO 31 &211s LENGTH: 23 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: wherein n is 5-iodo-2 prime-deoxyuridine <4 OOs, SEQUENCE: 31 incgt.cgacga t cqgcgc.gcg cc.g 23

<210s, SEQ ID NO 32 &211s LENGTH: 23 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (4) ... (4) <223> OTHER INFORMATION: wherein n is 5-iodo-2 prime-deoxyuridine

<4 OOs, SEQUENCE: 32

<210s, SEQ ID NO 33 &211s LENGTH: 23 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: wherein n is 5-iodo-2 prime-deoxyuridine 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (3) ... (3) <223> OTHER INFORMATION: wherein n is 5-iodo-2 prime-deoxyuridine 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (4) ... (4) <223> OTHER INFORMATION: n is a, c, g, or t <4 OOs, SEQUENCE: 33 US 2010/01 66780 A1 Jul. 1, 2010 42

- Continued incgn.cgacga t cqgcgc.gcg cc.g 23

<210s, SEQ ID NO 34 &211s LENGTH: 15 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: wherein n is 5-iodo-2 prime-deoxyuridine <4 OOs, SEQUENCE: 34 incgacgt.cgt gigggg 15

<210s, SEQ ID NO 35 &211s LENGTH: 15 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (7) . . (7) <223> OTHER INFORMATION: wherein n is 5-iodo-2 prime-deoxyuridine <4 OOs, SEQUENCE: 35 togacgncgt ggggg 15

<210s, SEQ ID NO 36 &211s LENGTH: 15 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (7) . . (7) <223> OTHER INFORMATION: wherein n is 5-iodo-2 prime-deoxyuridine 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (10) ... (10) <223> OTHER INFORMATION: wherein n is 5-iodo-2 prime-deoxyuridine

<4 OOs, SEQUENCE: 36 tCgacgn.cgn gigggg 15

<210s, SEQ ID NO 37 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide

<4 OO > SEQUENCE: 37 tcqtcqttitt toggtcgttt t 21

<210s, SEQ ID NO 38 &211s LENGTH: 6 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide

<4 OOs, SEQUENCE: 38 US 2010/01 66780 A1 Jul. 1, 2010 43

- Continued gtcgtt 6

<210s, SEQ ID NO 39 &211s LENGTH: 6 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (2) ... (2) <223> OTHER INFORMATION: wherein n is 5-iodo-2 prime-deoxyuridine <4 OOs, SEQUENCE: 39 gncgtt 6

<210s, SEQ ID NO 4 O &211s LENGTH: 6 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (2) ... (2) <223> OTHER INFORMATION: wherein n is 5-iodo-2 prime-deoxyuridine 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (5) . . (5) <223> OTHER INFORMATION: wherein n is 5-iodo-2 prime-deoxyuridine <4 OOs, SEQUENCE: 4 O gncgnt 6

<210s, SEQ ID NO 41 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (3) ... (3) <223> OTHER INFORMATION: wherein n is 5-chloro-2 prime-deoxyuridine

<4 OOs, SEQUENCE: 41 tgnogtttitt tttitttittitt 2O

<210s, SEQ ID NO 42 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (3) ... (3) <223> OTHER INFORMATION: wherein n is 5-ethyl-2 prime-deoxyuridine

<4 OOs, SEQUENCE: 42 tgnogtttitt tttitttittitt 2O

<210s, SEQ ID NO 43 &211s LENGTH: 15 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: US 2010/01 66780 A1 Jul. 1, 2010 44

- Continued <223> OTHER INFORMATION: Synthetic Oligonucleotide

<4 OOs, SEQUENCE: 43 tCgacgt.cgt gigggg 15

<210s, SEQ ID NO 44 &211s LENGTH: 24 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: wherein n is 5-iodo-2 prime-deoxyuridine 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (3) ... (3) <223> OTHER INFORMATION: wherein n is 5-iodo-2 prime-deoxyuridine 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (4) ... (4) <223> OTHER INFORMATION: n is a, c, g, or t <4 OOs, SEQUENCE: 44 incgn.cgttitt acggcgc.cgit gcc.g 24

<210s, SEQ ID NO 45 &211s LENGTH: 24 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (4) ... (4) <223> OTHER INFORMATION: wherein n is 5-iodo-2 prime-deoxyuridine 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (7) . . (7) <223> OTHER INFORMATION: wherein n is 5-iodo-2 prime-deoxyuridine

<4 OOs, SEQUENCE: 45 tcgn.cgnttt acggcgc.cgit gcc.g 24

<210s, SEQ ID NO 46 &211s LENGTH: 24 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide

<4 OOs, SEQUENCE: 46 tcgt.cgttitt acggcgc.cgit gcc.g 24

<210s, SEQ ID NO 47 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (10) ... (10) <223> OTHER INFORMATION: wherein n is 5-iodo-2 prime-deoxyuridine

<4 OOs, SEQUENCE: 47 US 2010/01 66780 A1 Jul. 1, 2010 45

- Continued tottttittgn cgttttittitt tt 22

<210s, SEQ ID NO 48 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (10) ... (10) <223> OTHER INFORMATION: wherein n is 5-iodo-2 prime-deoxyuridine 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (13) . . (13) <223> OTHER INFORMATION: wherein n is 5-iodo-2 prime-deoxyuridine <4 OOs, SEQUENCE: 48 tottttittgn cgntttittitt tt 22

<210s, SEQ ID NO 49 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: wherein n is 5-iodo-2 prime-deoxyuridine <4 OOs, SEQUENCE: 49 ncttittttgt cqttttittitt tt 22

<210s, SEQ ID NO 50 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: wherein n is 5-iodo-2 prime-deoxyuridine

<4 OOs, SEQUENCE: 50 ncttittttgt cqttttittitt tt 22

<210s, SEQ ID NO 51 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (10) ... (10) <223> OTHER INFORMATION: wherein n is uridine

<4 OOs, SEQUENCE: 51 tottttittgn cgttttittitt tt 22

<210s, SEQ ID NO 52 &211s LENGTH: 23 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: US 2010/01 66780 A1 Jul. 1, 2010 46

- Continued <223> OTHER INFORMATION: Synthetic Oligonucleotide

<4 OOs, SEQUENCE: 52

23

<210s, SEQ ID NO 53 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide

<4 OOs, SEQUENCE: 53 tccagg actt citcticaggitt

<210s, SEQ ID NO 54 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide

<4 OOs, SEQUENCE: 54

22

<210s, SEQ ID NO 55 &211s LENGTH: 24 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide

<4 OO > SEQUENCE: 55 tcqtcqttitt gtcgttttgt cqtt 24

<210s, SEQ ID NO 56 &211s LENGTH: 23 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide

<4 OOs, SEQUENCE: 56

23

<210s, SEQ ID NO 57 &211s LENGTH: 23 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide

<4 OO > SEQUENCE: 57

23

<210s, SEQ ID NO 58 &211s LENGTH: 23 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1) . . (1) US 2010/01 66780 A1 Jul. 1, 2010 47

- Continued <223> OTHER INFORMATION: wherein n is 5-iodo-2 prime-deoxyuridine <4 OOs, SEQUENCE: 58 incgacgt.cga t cqgcgc.gcg cc.g 23

<210s, SEQ ID NO 59 &211s LENGTH: 23 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: wherein n is 5-iodo-2 prime-deoxyuridine 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1) ... (23) <223> OTHER INFORMATION: wherein all the internucleotide linkages are phosphorothioate linkages

<4 OO > SEQUENCE: 59 incgacgt.cga t cqgcgc.gcg cc.g 23

<210s, SEQ ID NO 60 &211s LENGTH: 24 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: wherein n is 5-iodo-2 prime-deoxyuridine

<4 OOs, SEQUENCE: 60 incgacgt.cga t cqgcgc.gcg ccgt. 24

<210s, SEQ ID NO 61 &211s LENGTH: 24 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: wherein n is 5-iodo-2 prime-deoxyuridine 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1) ... (24) <223> OTHER INFORMATION: wherein all the internucleotide linkages are phosphorothioate linkages

<4 OOs, SEQUENCE: 61 incgacgt.cga t cqgcgc.gcg ccgt. 24

<210s, SEQ ID NO 62 &211s LENGTH: 23 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: wherein n is 5-ethyl-2 prime-deoxyuridine US 2010/01 66780 A1 Jul. 1, 2010 48

- Continued <4 OOs, SEQUENCE: 62 incgacgt.cga t cqgcgc.gcg cc.g 23

<210s, SEQ ID NO 63 &211s LENGTH: 23 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: wherein n is 5-ethyl-2 prime-deoxyuridine

<4 OOs, SEQUENCE: 63 incgacgt.cga t cqgcgc.gcg cc.g 23

<210s, SEQ ID NO 64 &211s LENGTH: 23 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: wherein n is 5-ethyl-2 prime-deoxyuridine < 4 OO > SEQUENCE: 64 incgacgt.cga t cqgcgc.gcg cc.g 23

<210s, SEQ ID NO 65 &211s LENGTH: 23 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1) ... (23) <223> OTHER INFORMATION: wherein all the internucleotide linkages are phosphorothioate linkages 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: wherein n is 5-ethyl-2 prime-deoxyuridine

<4 OOs, SEQUENCE: 65 incgacgt.cga t cqgcgc.gcg cc.g 23

<210s, SEQ ID NO 66 &211s LENGTH: 24 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: wherein n is 5-ethyl-2 prime-deoxyuridine

<4 OOs, SEQUENCE: 66 incgt.cgacga t cqgcggc.cg ccgt. 24

<210s, SEQ ID NO 67 LENGTH: 24 US 2010/01 66780 A1 Jul. 1, 2010 49

- Continued

&212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: 223 OTHER INFORMATION: Synthetic Oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature &222s. LOCATION: (1) . . <223> OTHER INFORMATION: wherein n is 5-iodo-2 prime-deoxyuridine

<4 OOs, SEQUENCE: 67 incgt.cgacga t cqgcggc.cg ccgt. 24

<210s, SEQ ID NO 68 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: 223 OTHER INFORMATION: Synthetic Oligonucleotide

<4 OOs, SEQUENCE: 68

22

<210s, SEQ ID NO 69 &211s LENGTH: 23 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: 223 OTHER INFORMATION: Synthetic Oligonucleotide

<4 OOs, SEQUENCE: 69 tcgt.cgacgt toggcgc.gcg cc.g 23

<210s, SEQ ID NO 70 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: 223 OTHER INFORMATION: Synthetic Oligonucleotide

<4 OOs, SEQUENCE: 70 tcggacgttc ggcgc.gc.gcc g 21

<210s, SEQ ID NO 71 &211s LENGTH: 19 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: 223 OTHER INFORMATION: Synthetic Oligonucleotide

<4 OOs, SEQUENCE: 71.

19

<210s, SEQ ID NO 72 &211s LENGTH: &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: 223 OTHER INFORMATION: Synthetic Oligonucleotide

<4 OOs, SEQUENCE: 72

<210s, SEQ ID NO 73 &211s LENGTH: US 2010/01 66780 A1 Jul. 1, 2010 50

- Continued

&212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide

<4 OO > SEQUENCE: 73

<210s, SEQ ID NO 74 &211s LENGTH: 18 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide

<4 OOs, SEQUENCE: 74

18

<210s, SEQ ID NO 75 &211s LENGTH: 18 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide

<4 OO > SEQUENCE: 75

18

<210s, SEQ ID NO 76 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide

<4 OO > SEQUENCE: 76

22

<210s, SEQ ID NO 77 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (3) ... (3) <223> OTHER INFORMATION: wherein n is 5-fluoro- du

<4 OO > SEQUENCE: 77 tgnogtttitt tttitttittitt

<210s, SEQ ID NO 78 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (6) . . (6) <223> OTHER INFORMATION: wherein n is 5-fluoro- du

<4 OO > SEQUENCE: 78 tgtcgnttitt tttitttittitt US 2010/01 66780 A1 Jul. 1, 2010 51

- Continued

SEO ID NO 79 LENGTH: 2O TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Synthetic Oligonucleotide FEATURE: NAMEAKEY: misc feature LOCATION: (3) ... (3) OTHER INFORMATION: wherein n is 5-fluoro- du FEATURE: NAMEAKEY: misc feature LOCATION: (6) . . (6) OTHER INFORMATION: wherein n is 5-fluoro- du

SEQUENCE: 79 tgnognttitt tttitttittitt

SEQ ID NO 8O LENGTH: 2O TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Synthetic Oligonucleotide FEATURE: NAMEAKEY: misc feature LOCATION: (3) ... (3) OTHER INFORMATION: wherein n is uridine FEATURE: NAMEAKEY: misc feature LOCATION: (6) . . (6) OTHER INFORMATION: wherein n is uridine

SEQUENCE: 8O tgnognttitt tttitttittitt

SEQ ID NO 81 LENGTH: 2O TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Synthetic Oligonucleotide FEATURE: NAMEAKEY: misc feature LOCATION: (5) . . (5) OTHER INFORMATION: wherein n is 6-nitro-benzimidazol

SEQUENCE: 81 tgtcntttitt tttitttittitt

SEQ ID NO 82 LENGTH: 2O TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Synthetic Oligonucleotide FEATURE: NAMEAKEY: misc feature LOCATION: (4) ... (4) OTHER INFORMATION: wherein n is 6-nitro-benzimidazol FEATURE: NAMEAKEY: misc feature LOCATION: (4) . . (5) OTHER INFORMATION: wherein the linkages is a phosphdiester linkage

SEQUENCE: 82 tgtngtttitt tttitttittitt US 2010/01 66780 A1 Jul. 1, 2010 52

- Continued

<210s, SEQ ID NO 83 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (4) ... (4) <223> OTHER INFORMATION: wherein n is 6-nitro-benzimidazol 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (4) . . (6) <223> OTHER INFORMATION: wherein the linkages are phosphodiester linkages

<4 OOs, SEQUENCE: 83 tgtngtttitt tttitttittitt 2O

<210s, SEQ ID NO 84 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: wherein n is 5-iodo-2 prime-deoxyuridine <4 OOs, SEQUENCE: 84 ngtogtttitt tttitttittitt 2O

<210s, SEQ ID NO 85 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: wherein n is 5-iodo-2 prime-deoxyuridine 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (3) ... (3) <223> OTHER INFORMATION: wherein n is 5-iodo-2 prime-deoxyuridine

<4 OOs, SEQUENCE: 85 ngncgtttitt tttitttittitt 2O

<210s, SEQ ID NO 86 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (7) . . (7) <223> OTHER INFORMATION: wherein n is 5-iodo-2 prime-deoxyuridine

<4 OOs, SEQUENCE: 86 tgtcgtnttt tttitttittitt 2O

<210s, SEQ ID NO 87 US 2010/01 66780 A1 Jul. 1, 2010 53

- Continued

LENGTH: 2O TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Synthetic Oligonucleotide FEATURE: NAMEAKEY: misc feature LOCATION: (3) ... (3) OTHER INFORMATION: wherein n is a a? a -trifluoro - d.T SEQUENCE: 87 tgnogtttitt tttitttittitt

SEO ID NO 88 LENGTH: 2O TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Synthetic Oligonucleotide FEATURE: NAMEAKEY: misc feature LOCATION: (6) . . (6) OTHER INFORMATION: wherein n is a a? a -trifluoro - d.T SEQUENCE: 88 tgtcgnttitt tttitttittitt

SEO ID NO 89 LENGTH: 2O TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Synthetic Oligonucleotide FEATURE: NAMEAKEY: misc feature LOCATION: (3) ... (3) OTHER INFORMATION: wherein n is a a? a -trifluoro - d.T FEATURE: NAMEAKEY: misc feature LOCATION: (6) . . (6) OTHER INFORMATION: wherein n is a a? a -trifluoro - d.T

SEQUENCE: 89 tgnognttitt tttitttittitt

SEO ID NO 9 O LENGTH: 2O TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Synthetic Oligonucleotide FEATURE: NAMEAKEY: misc feature LOCATION: (3) ... (3) OTHER INFORMATION: wherein n is 5-chloro-2 prime-deoxyuridine

SEQUENCE: 9 O tgnogtttitt tttitttittitt

SEQ ID NO 91 LENGTH: 2O TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Synthetic Oligonucleotide FEATURE: NAMEAKEY: misc feature LOCATION: (6) . . (6) US 2010/01 66780 A1 Jul. 1, 2010 54

- Continued <223> OTHER INFORMATION: wherein n is 5-chloro-2 prime-deoxyuridine <4 OOs, SEQUENCE: 91 tgtcgnttitt tttitttittitt 2O

<210s, SEQ ID NO 92 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (3) ... (3) <223> OTHER INFORMATION: wherein n is 5-chloro-2 prime-deoxyuridine 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (6) . . (6) <223> OTHER INFORMATION: wherein n is 5-chloro-2 prime-deoxyuridine <4 OOs, SEQUENCE: 92 tgnognttitt tttitttittitt 2O

<210s, SEQ ID NO 93 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (2) ... (2) <223> OTHER INFORMATION: wherein n is 5-iodo-2 prime-deoxyuridine

<4 OOs, SEQUENCE: 93 tncgttittitt tttitttittitt 2O

<210s, SEQ ID NO 94 &211s LENGTH: 9 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (3) ... (3) <223> OTHER INFORMATION: wherein n is 5-iodo-2 prime-deoxyuridine

<4 OOs, SEQUENCE: 94 tgnogttitt 9

<210s, SEQ ID NO 95 &211s LENGTH: 15 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (3) ... (3) <223> OTHER INFORMATION: wherein n is 5-iodo-2 prime-deoxyuridine

<4 OO > SEQUENCE: 95 tgnogttttgtcgtt 15

<210s, SEQ ID NO 96 US 2010/01 66780 A1 Jul. 1, 2010 55

- Continued

&211s LENGTH: 10 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (3) ... (3) <223> OTHER INFORMATION: wherein n is 5-iodo-2 prime-deoxyuridine 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (8) ... (8) <223> OTHER INFORMATION: wherein n is hexaethylenglycol phosphate 22 Os. FEATURE <221 > NAMEAKEY: misc feature <222s. LOCATION: (9) ... (9) <223> OTHER INFORMATION: wherein n is Doubler2 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (10) ... (10) <223> OTHER INFORMATION: wherein n is 3 prime-O-Methyl-ro <4 OOs, SEQUENCE: 96 tgnogttnnn 10

<210s, SEQ ID NO 97 &211s LENGTH: 10 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: n is a, c, g, or t 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (3) ... (3) <223> OTHER INFORMATION: wherein n is 5-iodo-2 prime-deoxyuridine 22 Os. FEATURE <221 > NAMEAKEY: misc feature <222s. LOCATION: (8) ... (8) <223> OTHER INFORMATION: wherein n is hexaethylenglycol phosphate 22 Os. FEATURE <221 > NAMEAKEY: misc feature <222s. LOCATION: (9) ... (9) <223> OTHER INFORMATION: wherein n is Doubler2 22 Os. FEATURE <221 > NAMEAKEY: misc feature <222s. LOCATION: (10) ... (10) <223> OTHER INFORMATION: wherein n is 3 prime-O-Methyl-ro

<4 OO > SEQUENCE: 97 incgttcginnin 10

<210s, SEQ ID NO 98 &211s LENGTH: 18 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (3) ... (3) <223> OTHER INFORMATION: wherein n is 5-iodo-2 prime-deoxyuridine

<4 OOs, SEQUENCE: 98 ttncgt.cgtt togtogtt 18

<210s, SEQ ID NO 99 US 2010/01 66780 A1 Jul. 1, 2010 56

- Continued

&211s LENGTH: 15 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: wherein n is 5-bromo-2 prime-deoxyuridine <4 OOs, SEQUENCE: 99 incgacgt.cgt gigggg 15

<210s, SEQ ID NO 100 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (3) ... (3) <223> OTHER INFORMATION: wherein n is 5-iodo-2 prime-deoxyuridine <4 OOs, SEQUENCE: 1.OO tgngctttitt tttitttittitt 2O

<210s, SEQ ID NO 101 &211s LENGTH: 10 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (3) ... (3) <223> OTHER INFORMATION: wherein n is 5-iodo-2 prime-deoxyuridine 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (8) ... (8) <223> OTHER INFORMATION: wherein n is hexaethylenglycol phosphate 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (9) ... (9) <223> OTHER INFORMATION: wherein n is Doubler2 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (10) ... (10) <223> OTHER INFORMATION: wherein n is triethylenglycol phosphate

<4 OOs, SEQUENCE: 101 tgnogttnnn 10

<210s, SEQ ID NO 102 &211s LENGTH: 10 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: n is a, c, g, or t 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (3) ... (3) <223> OTHER INFORMATION: wherein n is 5-iodo-2 prime-deoxyuridine 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (8) ... (8) US 2010/01 66780 A1 Jul. 1, 2010 57

- Continued <223> OTHER INFORMATION: wherein n is hexaethylenglycol phosphate 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (9) ... (9) <223> OTHER INFORMATION: wherein n is Doubler2 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (10) ... (10) <223> OTHER INFORMATION: wherein n is triethylenglycol phosphate

<4 OOs, SEQUENCE: 102 incgttcginnin 10

<210s, SEQ ID NO 103 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: wherein n is 5-iodo-2 prime-deoxyuridine <4 OOs, SEQUENCE: 103

<210s, SEQ ID NO 104 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (4) ... (4) <223> OTHER INFORMATION: wherein n is 5-iodo-2 prime-deoxyuridine

<4 OOs, SEQUENCE: 104

<210s, SEQ ID NO 105 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (10) ... (10) <223> OTHER INFORMATION: wherein n is 5-iodo-2 prime-deoxyuridine

<4 OOs, SEQUENCE: 105 tcgt.cgtttin cggcgc.gc.gc cg 22

<210s, SEQ ID NO 106 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: wherein n is 5-iodo-2 prime-deoxyuridine 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (15) . . (15) US 2010/01 66780 A1 Jul. 1, 2010 58

- Continued <223> OTHER INFORMATION: wherein n is 5-iodo-2 prime-deoxyuridine <4 OOs, SEQUENCE: 106 incgt.cgttitt toggnogttt t 21

<210s, SEQ ID NO 107 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (4) ... (4) <223> OTHER INFORMATION: wherein n is 5-iodo-2 prime-deoxyuridine 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (15) . . (15) <223> OTHER INFORMATION: wherein n is 5-iodo-2 prime-deoxyuridine <4 OOs, SEQUENCE: 107 tcqncgttitt toggnogttt t 21

<210s, SEQ ID NO 108 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (3) ... (3) <223> OTHER INFORMATION: wherein n is 5-iodo-2 prime-deoxyuridine 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (16) ... (16) <223> OTHER INFORMATION: wherein n is 5-iodo-2 prime-deoxyuridine

<4 OOs, SEQUENCE: 108 tgnogtttitt ttittgnogtt 2O

<210s, SEQ ID NO 109 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (3) ... (3) <223> OTHER INFORMATION: wherein n is 5-iodo-2 prime-deoxyuridine

<4 OOs, SEQUENCE: 109 tgnogtttitt tttitttittitt 2O

<210s, SEQ ID NO 110 &211s LENGTH: 15 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: wherein n is 5-iodo-2 prime-deoxyuridine 22 Os. FEATURE: <221 > NAMEAKEY: misc feature LOCATION: (13) . . (13) US 2010/01 66780 A1 Jul. 1, 2010 59

- Continued

<223> OTHER INFORMATION: wherein n is 7- deaza-dC

<4 OOs, SEQUENCE: 110 incgacgt.cgt ggingg 15

<210s, SEQ ID NO 111 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (2) ... (2) <223> OTHER INFORMATION: wherein n is 3 prime-O-Methyl-ro 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (3) ... (3) <223> OTHER INFORMATION: wherein n is 5-iodo-2 prime-deoxyuridine <4 OOs, SEQUENCE: 111 tnincgtttitt tttitttittitt 2O

<210s, SEQ ID NO 112 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (3) ... (3) <223> OTHER INFORMATION: wherein n is 5-iodo-2 prime-deoxyuridine 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (5) . . (5) <223> OTHER INFORMATION: wherein n is 3 prime-O-Methyl-ro

<4 OOs, SEQUENCE: 112 tgnontttitt tttitttittitt 2O

<210s, SEQ ID NO 113 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (2) ... (2) <223> OTHER INFORMATION: wherein n is 3 prime-O-Methyl-ro 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (3) ... (3) <223> OTHER INFORMATION: wherein n is 5-iodo-2 prime-deoxyuridine 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (5) . . (5) <223> OTHER INFORMATION: wherein n is 3 prime-O-Methyl-ro

<4 OOs, SEQUENCE: 113 tnchittittt ttt tttitt tt 2O

<210s, SEQ ID NO 114 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: US 2010/01 66780 A1 Jul. 1, 2010 60

- Continued <223> OTHER INFORMATION: Synthetic Oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: wherein n is 5-iodo-2 prime-deoxyuridine 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (4) ... (4) <223> OTHER INFORMATION: wherein n is 5-iodo-2 prime-deoxyuridine <4 OOs, SEQUENCE: 114 incgncgttitt toggtcgttt t 21

<210s, SEQ ID NO 115 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: wherein n is 5-iodo-2 prime-deoxyuridine 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1) ... (21) <223> OTHER INFORMATION: wherein all internucleotide linkages are phosphorothioate internucleotide linkages 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (4) ... (4) <223> OTHER INFORMATION: wherein n is 5-iodo-2 prime-deoxyuridine

<4 OOs, SEQUENCE: 115 incgncgttitt toggtcgttt t 21

<210s, SEQ ID NO 116 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (3) ... (3) <223> OTHER INFORMATION: wherein n is 5-proynyl-dU

<4 OOs, SEQUENCE: 116 tgnogtttitt tttitttittitt 2O

<210s, SEQ ID NO 117 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (6) . . (6) <223> OTHER INFORMATION: wherein n is 5-proynyl-dU <4 OOs, SEQUENCE: 117 tgtcgnttitt tttitttittitt 2O

<210s, SEQ ID NO 118 &211s LENGTH: 15 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence US 2010/01 66780 A1 Jul. 1, 2010 61

- Continued

22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: wherein n is 5-bromo-2 prime-deoxyuridine <4 OOs, SEQUENCE: 118 incgacgt.cgt gigggg 15

<210s, SEQ ID NO 119 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (3) ... (3) <223> OTHER INFORMATION: wherein n is 5-iodo-2 prime-deoxyuridine

<4 OOs, SEQUENCE: 119 tgnogtttitc ggcgc.gc.gcc g 21

<210s, SEQ ID NO 120 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (2) ... (2) <223> OTHER INFORMATION: wherein n is 5-iodo-2 prime-deoxyuridine

<4 OOs, SEQUENCE: 120

21

<210s, SEQ ID NO 121 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (2) ... (2) <223> OTHER INFORMATION: wherein n is 5-ethyl-2 prime-deoxyuridine

<4 OOs, SEQUENCE: 121 tncgttittitt tttitttittitt

<210s, SEQ ID NO 122 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (3) ... (3) <223> OTHER INFORMATION: wherein n is 5-ethyl-2 prime-deoxyuridine

<4 OOs, SEQUENCE: 122 tgngctttitt tttitttittitt US 2010/01 66780 A1 Jul. 1, 2010 62

- Continued <210s, SEQ ID NO 123 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: wherein n is 5-iodo-2 prime-deoxyuridine

<4 OOs, SEQUENCE: 123 incgt.cgttitt toggtcgttt t 21

<210s, SEQ ID NO 124 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: wherein n is 5-ethyl-2 prime-deoxyuridine

<4 OOs, SEQUENCE: 124 incgt.cgttitt toggtcgttt t 21

<210 SEQ ID NO 125 &211s LENGTH: 7 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (2) ... (2) <223> OTHER INFORMATION: wherein n is 5-iodo-2 prime-deoxyuridine 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (7) . . (7) <223> OTHER INFORMATION: wherein n is hexadecylglyceryl

<4 OOs, SEQUENCE: 125 gncgttn 7

<210s, SEQ ID NO 126 &211s LENGTH: 7 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (2) ... (2) <223> OTHER INFORMATION: wherein n is 5-iodo-2 prime-deoxyuridine 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (5) . . (5) <223> OTHER INFORMATION: wherein n is 5-iodo-2 prime-deoxyuridine 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (7) . . (7) <223> OTHER INFORMATION: wherein n is hexadecylglyceryl <4 OOs, SEQUENCE: 126 gncgntn 7 US 2010/01 66780 A1 Jul. 1, 2010 63

- Continued <210s, SEQ ID NO 127 &211s LENGTH: 7 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (2) ... (2) <223> OTHER INFORMATION: wherein n is 5-ethyl-2 prime-deoxyuridine 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (5) . . (5) <223> OTHER INFORMATION: wherein n is 5-ethyl-2 prime-deoxyuridine 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (7) . . (7) <223> OTHER INFORMATION: wherein n is hexadecylglyceryl

<4 OOs, SEQUENCE: 127 gncgntn 7

<210s, SEQ ID NO 128 &211s LENGTH: 24 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: wherein n is 5-ethyl-2 prime-deoxyuridine

<4 OOs, SEQUENCE: 128 incgt.cgttitt acggcgc.cgit gcc.g 24

<210s, SEQ ID NO 129 &211s LENGTH: 24 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (4) ... (4) <223> OTHER INFORMATION: wherein n is 5-ethyl-2 prime-deoxyuridine

<4 OOs, SEQUENCE: 129 tcgn.cgttitt acggcgc.cgit gcc.g 24

<210s, SEQ ID NO 130 &211s LENGTH: 23 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: wherein n is 5-ethyl-2 prime-deoxyuridine

<4 OOs, SEQUENCE: 130 incgt.cgacga t cqgcgc.gcg cc.g 23

<210s, SEQ ID NO 131 &211s LENGTH: 17 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence US 2010/01 66780 A1 Jul. 1, 2010 64

- Continued

22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: wherein n is 5-iodo-2 prime-deoxyuridine <4 OOs, SEQUENCE: 131 nott tttittt tttittitt 17

<210s, SEQ ID NO 132 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: wherein n is 5-iodo-2 prime-deoxyuridine

<4 OOs, SEQUENCE: 132 incttittittitt cgtttitttitt tt 22

<210s, SEQ ID NO 133 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (10) ... (10) <223> OTHER INFORMATION: wherein n is 5-iodo-2 prime-deoxyuridine

<4 OOs, SEQUENCE: 133 tottttitttn cgtttitttitt tt 22

<210s, SEQ ID NO 134 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: wherein n is 5-iodo-2 prime-deoxyuridine 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (10) ... (10) <223> OTHER INFORMATION: wherein n is 5-iodo-2 prime-deoxyuridine

<4 OOs, SEQUENCE: 134 ncttttitttn cgttttittitt tt 22

<210s, SEQ ID NO 135 &211s LENGTH: 24 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: wherein n is 5-iodo-2 prime-deoxyuridine <4 OOs, SEQUENCE: 135 US 2010/01 66780 A1 Jul. 1, 2010 65

- Continued incgt.cgtttc gtcgttttgt cqtt 24

<210s, SEQ ID NO 136 &211s LENGTH: 24 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (2O) . . (2O) <223> OTHER INFORMATION: wherein n is 5-iodo-2 prime-deoxyuridine

<4 OOs, SEQUENCE: 136 tcqtcqtttic gtcgttittgn cgtt 24

<210s, SEQ ID NO 137 &211s LENGTH: 24 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: wherein n is 5-iodo-2 prime-deoxyuridine 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (2O) . . (2O) <223> OTHER INFORMATION: wherein n is 5-iodo-2 prime-deoxyuridine

<4 OOs, SEQUENCE: 137 incgt.cgtttc gtcgttittgn cgtt 24

<210s, SEQ ID NO 138 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (3) ... (3) <223> OTHER INFORMATION: wherein n is 5-iodo-2 prime-deoxyuridine 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (5) . . (5) <223> OTHER INFORMATION: wherein n is 7- deaza-dC

<4 OOs, SEQUENCE: 138 tgnontttitt tttitttittitt 2O

<210s, SEQ ID NO 139 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (3) ... (3) <223> OTHER INFORMATION: wherein n is 5-iodo-2 prime-deoxyuridine 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (5) . . (5) <223> OTHER INFORMATION: wherein n is inosine

<4 OOs, SEQUENCE: 139 US 2010/01 66780 A1 Jul. 1, 2010 66

- Continued tgnontttitt tttitttittitt 2O

<210s, SEQ ID NO 140 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (3) ... (3) <223> OTHER INFORMATION: wherein n is 5-iodo-2 prime-deoxyuridine 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (4) ... (4) <223> OTHER INFORMATION: wherein n is 5-methyl-dC <4 OOs, SEQUENCE: 140 tgningtttitt tttitttittitt 2O

<210s, SEQ ID NO 141 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (8) ... (8) <223> OTHER INFORMATION: wherein n is 5-iodo-2 prime-deoxyuridine

<4 OOs, SEQUENCE: 141 tgtcgttntt tttitttittitt 2O

<210s, SEQ ID NO 142 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (9) ... (9) <223> OTHER INFORMATION: wherein n is 5-iodo-2 prime-deoxyuridine

<4 OOs, SEQUENCE: 142 tgtcgtttnt tttitttittitt 2O

<210s, SEQ ID NO 143 &211s LENGTH: 23 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: wherein n is 5-iodo-2 prime-deoxyuridine <4 OOs, SEQUENCE: 143 incgt.cgttitt C9gcgc.gc.gc ct 23

<210s, SEQ ID NO 144 &211s LENGTH: 23 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence US 2010/01 66780 A1 Jul. 1, 2010 67

- Continued

22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: wherein n is 5-ethyl-2 prime-deoxyuridine

<4 OOs, SEQUENCE: 144 incgt.cgttitt C9gcgc.gc.gc ct 23

<210s, SEQ ID NO 145 &211s LENGTH: 23 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (4) ... (4) <223> OTHER INFORMATION: wherein n is 5-ethyl-2 prime-deoxyuridine

<4 OOs, SEQUENCE: 145

<210s, SEQ ID NO 146 &211s LENGTH: 23 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (10) ... (10) <223> OTHER INFORMATION: wherein n is 5-iodo-2 prime-deoxyuridine

<4 OOs, SEQUENCE: 146 tcgt.cgtttin cggcgc.gc.gc cgt. 23

<210s, SEQ ID NO 147 &211s LENGTH: 23 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (10) ... (10) <223> OTHER INFORMATION: wherein n is 5-ethyl-2 prime-deoxyuridine

<4 OOs, SEQUENCE: 147 tcgt.cgtttin cggcgc.gc.gc cgt. 23

<210s, SEQ ID NO 148 &211s LENGTH: 23 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: wherein n is 5-ethyl-2 prime-deoxyuridine 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (10) ... (10) <223> OTHER INFORMATION: wherein n is 5-ethyl-2 prime-deoxyuridine

<4 OOs, SEQUENCE: 148