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Definitions

• the present invention relates generally to oligonucleotides and more specifically to oligonucleotides which have a sequence including at least one unmethylated CpG dinucleotide which are immunostimulatory.
• poly (I,C) which is a potent inducer of LFN production as well as a macrophage activator and inducer of NK activity (Talmadge, J.E., J.
• bZip basic region/leucine zipper
• All cells appear to express one or more CREB/ATF proteins, but the members expressed and the regulation of mRNA splicing appear to be tissue-specific. Differential splicing of activation domains can determine whether a particular CREB/ATF protein will be a transcriptional inhibitor or activator. Many CREB/ATF proteins activate viral transcription, but some splicing variants which lack the activation domain are inhibitory.
• the invention provides an isolated immunostimulatory nucleic acid sequence contains a CpG motif represented by the formula: wherein at least one nucleotide separates consecutive CpGs; X ⁇ is selected from the group consisting of GpT, GpG, GpA, ApT and ApA; X 3 X 4 is selected from the group consisting of TpT or CpT; N is any nucleotide and N, + N 2 is from about 0-26 bases with the proviso that that N, and N 2 do not contain a CCGG quadmer or more than one CCG or CGG trimer; and the nucleic acid sequence is from about 8-30 bases in length.
• a CpG motif represented by the formula: wherein at least one nucleotide separates consecutive CpGs; X ⁇ is selected from the group consisting of GpT, GpG, GpA, ApT and ApA; X 3 X 4 is selected from the group consisting of TpT or CpT; N is any nucleo
• Figure 4A is a graph plotting dose-dependent inhibition of CpG-induced IgM production by anti-IL-6.
• Splenic B-cells from DBA/2 mice were stimulated with CpG ODN 5 TCCAAGACGTTCCTGATGCT 3' (SEQ ID No: 9) in the presence of the indicated concentrations of neutralizing anti-IL-6 (u) or isotype control Ab (1) and IgM levels in culture supernatants determined by ELISA.
• the anti-IL-6 Ab had no effect on IgM secretion (n).
• Figure 9 is a graph plotting lung lavage cell count over time. The graph shows that when the mice are initially injected with Schistosoma mansoni eggs “egg", which induces a Th2 immune response, and subsequently inhale Schistosoma mansoni egg antigen "SEA" (open circle), many inflammatory cells are present in the lungs. However, when the mice are initially given CpG oligo (SEQ ID NO. 10) along with egg, the inflammatory cells in the lung are not increased by subsequent inhalation of SEA (open triangles).
• Figure 10 is a graph plotting lung lavage eosinophil count over time.
• Figure 13 is a graph plotting interleukin 4 (IL-4) production (pg/ml) in mice over time in response to injection of egg, then SEA (open diamond); egg and SEQ ID No. 10, then SEA (open circle); or saline, then saline (open square).
• the graph shows that the resultant inflammatory response correlates with the levels of the Th2 cytokine IL-4 in the lung.
• Figure 14 is a bar graph plotting interleukin 12 (IL-12) production (pg/ml) in mice over time in response to injection of saline; egg, then SEA; or SEQ ID No. 10 and egg, then SEA.
• the graph shows that administration of an oligonucleotide containing an unmethylated CpG motif can actually redirect the cytokine response of the lung to production of IL-12, indicating a Thl type of immune response.
• Figure 15 is a bar graph plotting interferon gamma (IFN- ⁇ ) production (pg/ml) in mice over time in response to injection of saline; egg, then saline; or SEQ ID No. 10 and egg, then SEA. The graph shows that administration of an oligonucleotide containing an unmethylated CpG motif can also redirect the cytokine response of the lung to production of IFN-g, indicating a Thl type of immune response.
• IFN- ⁇ interferon gamma
• an “immune system deficiency” shall mean a disease or disorder in which the subject's immune system is not functioning in normal capacity or in which it would be useful to boost a subject's immune response for example to eliminate a tumor or cancer (e.g. tumors of the brain, lung (e.g. small cell and non-small cell), ovary, breast, prostate, colon, as well as other carcinomas and sarcomas) or an infection in a subject.
• a tumor or cancer e.g. tumors of the brain, lung (e.g. small cell and non-small cell), ovary, breast, prostate, colon, as well as other carcinomas and sarcomas
• an infection in a subject e.g. tumors of the brain, lung (e.g. small cell and non-small cell), ovary, breast, prostate, colon, as well as other carcinomas and sarcomas) or an infection in a subject.
• infectious bacteria examples include: Helicobacter pyloris, Borelia burgdorferi, Legionella pneumophilia, Mycobaderia sps (e.g. M. tuberculosis, M. avium, M. intracellulare, M. kansaii, M.
• infectious fungi examples include: Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis,Chlamydia trachomatis, Candida albicans.
• Other infectious organisms i.e., protists
• Plasmodium falciparum and Toxoplasma gondii examples include: Plasmodium falciparum and Toxoplasma gondii.
• an "immunostimulatory nucleic acid molecule” refers to a nucleic acid molecule, which contains an unmethylated cytosine, guanine dinucleotide sequence (i.e.
• the nucleic acid sequences of the invention are also useful for stimulating natural killer cell (NK) lytic acitivity in a subject such as a human.
• NK natural killer cell
• Specific, but non-limiting examples of such sequences include:TCGTCGTTGTCGTTGTCGTT (SEQ ID NO: 57), TCGTCGTTTTGTCGTTTTGTCGTT (SEQ ID NO:58 ), TCGTCGTTGTCGTTGTCGTT (SEQ ID NO:59), GCGTGCGTTGTCGTTGTCGTT (SEQ ID NO: ), TGTCGTTTGTCGTTTGTCGTT (SEQ ID NO: ),
• nucleic acid sequences of the invention are also useful for stimulating B cell proliferation in a subject such as a human.
• Specific, but non-limiting examples of such sequences include: TCCTGTCGTTCCTTGTCGTT(SEQ ID NO:62),TCCTGTCGTTTTTTGTCGTT (SEQ ID NO:63),
• TCGTCGCTGTCTGCCCTTCTT (SEQ LD NO:64),TCGTCGCTGTTGTCGTTTCTT (SEQ ID NO:65),TCGTCGTTTTGTCGTTTTGTCGTT (SEQ ID NO:66),TCGTCGTTGTCGTTTTGTCGTT (SEQ ID NO:67 ) and TGTCGTTGTCGTTGTCGTT (SEQ ID NO:68 ).
• nucleic acid sequences of the invention are useful as an adjuvant for use during antibody production in a mammal.
• specific, but non-limiting examples of such sequences include: TCCATGACGTTCCTGACGTT (SEQ LD NO.10), GTCG(T/C)T and TGTCG(T/C)T.
• the claimed nucleic acid sequences can be administered to treat or prevent the symptoms of an asthmatic disorder by redirecting a subject's immune response from Th2 to Thl.
• An exemplary sequence includes TCCATGACGTTCCTGACGTT (SEQ LD NO.10).
• the immunostimulatory CpG DNA be capable of effectively inducing cytokine secretion by monocytic cells and/or Natural Killer (NK) cell lytic activity.
• nucleic acid molecules can be obtained from existing nucleic acid sources (e.g. genomic or cDNA), but are preferably synthetic (e.g. produced by oligonucleotide synthesis).
• “Palindromic sequence” shall mean an inverted repeat (i.e., a sequence such as ABCDEE'D'C'B'A' in which A and A' are bases capable of forming the usual Watson-Crick base pairs. In vivo, such sequences may form double stranded structures.
• phosphorothioate-modified nucleic acid molecules can increase the extent of immune stimulation of the nucleic acid molecule, which contains an unmethylated CpG dinucleotide as shown herein.
• ODN 1 and 2 were synthesized. These ODNs, including the two original “controls” (ODN 1 and 2) and two originally synthesized as “antisense” (ODN 3D and 3M; Krieg, A.M. J. Immunol. 143-.244S (1989)), were then examined for in vitro effects on spleen cells (representative sequences are listed in Table 1).
• ODN that contained CpG dinucleotides induced B cell activation and IgM secretion; the magnitude of this stimulation typically could be increased by adding more CpG dinucleotides (Table 1 ; compare ODN 2 to 2a or 3D to 3Da and 3Db). Stimulation did not appear to result from an antisense mechanism or impurity. ODN caused no detectable proliferation of ⁇ or other T cell populations.
• the optimal stimulatory motif was determined to consist of a CpG flanked by two 5' purines (preferably a GpA dinucleotide) and two 3' pyrimidines (preferably a TpT or TpC dinucleotide). Mutations of ODN to bring the CpG motif closer to this ideal improved stimulation (e.g. Table 1, compare ODN 2 to 2e; 3M to 3Md) while mutations that disturbed the motif reduced stimulation (e.g. Table 1, compare ODN 3D to 3Df; 4 to 4b, 4c and 4d).
• octamer ODN containing a 6 base palindrome with a TpC dinucleotide at the 5' end were also active (e.g. Table 1, ODN 4b,4c).
• Other dinucleotides at the 5' end gave reduced stimulation (e.g., ODN 4f; all sixteen possible dinucleotides were tested).
• the presence of a 3' dinucleotide was insufficient to compensate for the lack of a 5' dinucleotide (e.g., Table 1, ODN 4g).
• Disruption of the palindrome eliminated stimulation in octamer ODN (e.g., Table 1, ODN 4h), but palindromes were not required in longer ODN.
• L-LME L-leucyl-L-leucine methyl ester
• Results are representative using PBMC from different donors.
• CAT assays were performed after stimulation with various concentrations of CpG or non- CpG ODN. As shown in Figure 5, CpG ODN induced increased CAT activity in dose- dependent manner while non-CpG ODN failed to induce CAT activity. This confirms that CpG induces the transcriptional activity of the IL-6 promoter.
• the lymphocyte stimulating effects of these ODN were tested at three concentrations (3.3, 10, and 30 ⁇ M) by measuring the total levels of RNA synthesis (by ⁇ uridine incorporation) or DNA synthesis (by ⁇ thymidine incorporation) in treated spleen cell cultures (Example 10).
• O-ODN (0/0 phosphorothioate modifications) bearing a CpG motif caused no spleen cell stimulation unless added to the cultures at concentrations of at least 10 ⁇ M (Example 10).
• this sequence was modified with two S linkages at the 5' end and at least three S linkages at the 3' end, significant stimulation was seen at a dose of 3.3 ⁇ M.
• the level of stimulation showed a progressive increase as the number of 3' modified bases was increased, until this reached or exceeded six, at which point the stimulation index began to decline.
• the optimal number of 3' S linkages for spleen cell stimulation was five. Of all three concentrations tested in these experiments, the S-ODN was less stimulatory than the optimal chimeric compounds.
• Target Effector Target ODN 50:1 100:1 50:1 100:1
• Immune activation by CpG motifs may depend on bases flanking the CpG, and the number and spacing of the CpGs present within an ODN. Although a single CpG in an ideal base context can be a very strong and useful immune activator, superior effects can be seen with ODN containing several CpGs with the appropriate spacing and flanking bases.
• the optimal CpG motif is TGACGTT. The following studies were conducted to identify optimal ODN sequences for stimulation of human cells by examining the effects of changing the number, spacing, and flanking bases of CpG dinucleotides.
• ODNs with internal CpG motifs are generally less potent stimulators than those in which a GTCGCT motif immediately follows the 5' TC (e.g., ODN 1967 and 1968).
• ODN 1968 which has a second GTCGTT motif in its 3' half, was consistently more stimulatory than ODN 1967, which lacks this second motif.
• ODN 1967 was slightly more potent than ODN 1968 in experiments 1 and 3, but not in experiment 2.
• ODN 2005 which has a third GTCGTT motif, induced slightly higher NK activity on average than 1968.
• CpG DNA can directly activate highly purified B cells and monocytic cells. There are many similarities in the mechanism through which CpG DNA activates these cell types. For example, both require NFkB activation as explained further below.
• oligo 1668 with the best mouse B cell motif, is a strong inducer of both B cell and natural killer (NK) cell activation, while oligo 1758 is a weak B cell activator, but still induces excellent NK responses (Table 14).
• CpG dinucleotides are underlined; oligonucleotides were synthesized with phosphorothioate modified backbones to improve their nuclease resistance. 'Measured by H thymidine incorporation after 48 hr culture with ohgodeoxynucleotides at a 200 nM concentration as described in Example 1. : Measured in lytic units.
• Vertebrate DNA is highly methylated and CpG dinucleotides are underrepresented.
• the stimulatory CpG motif is common in microbial genomic DNA, but quite rare in vertebrate DNA.
• bacterial DNA has been reported to induce B cell proliferation and immunoglobulin (lg) production, while mammalian DNA does not (Messina, J.P. et al, J. Immunol. 147:1159 (1991)).
• Example 3 in which methylation of bacterial DNA with CpG methylase was found to abolish mitogenicity, demonstrates that the difference in CpG status is the cause of B cell stimulation by bacterial DNA. This data supports the following conclusion: that unmethylated CpG dinucleotides present within bacterial DNA are responsible for the stimulatory effects of bacterial DNA.
• lymphocyte activation by the CpG motif represents an immune defense mechanism that can thereby distinguish bacterial from host DNA.
• Host DNA which would commonly be present in many anatomic regions and areas of inflammation due to apoptosis (cell death), would generally induce little or no lymphocyte activation due to CpG suppression and methylation.
• the presence of bacterial DNA containing unmethylated CpG motifs can cause lymphocyte activation precisely in infected anatomic regions, where it is beneficial. This novel activation pathway provides a rapid alternative to T cell dependent antigen specific B cell activation.
• B cells bearing antigen receptor specific for bacterial antigens would receive one activation signal through cell membrane lg and a second signal from bacterial DNA, and would therefore tend to be preferentially activated.
• the interrelationship of this pathway with other pathways of B cell activation provide a physiologic mechanism employing a polyclonal antigen to induce antigen-specific responses.
• B cell activation would not be totally nonspecific.
• B cells bearing antigen receptors specific for bacterial products could receive one activation signal through cell membrane lg, and a second from bacterial DNA, thereby more vigorously triggering antigen specific immune responses.
• the response to bacterial DNA could have undesirable consequences in some settings.
• autoimmune responses to self antigens would also tend to be preferentially triggered by bacterial infections, since autoantigens could also provide a second activation signal to autoreactive B cells triggered by bacterial DNA. Indeed the induction of autoimmunity by bacterial infections is a common clinical observance.
• the autoimmune disease systemic lupus erythematosus which is: i) characterized by the production of anti-DNA antibodies; ii) induced by drugs which inhibit DNA methyltransferase ( Cornacchia, E.J. et al, J. Clin. Invest. 92:38 (1993)); and iii) associated with reduced DNA methylation ( Richardson, B., L. et al, Arth. Rheum 35:641 (1992)), is likely triggered at least in part by activation of DNA-specific B cells through stimulatory signals provided by CpG motifs, as well as by binding of bacterial DNA to antigen receptors.
• sepsis which is characterized by high morbidity and mortality due to massive and nonspecific activation of the immune system may be initiated by bacterial DNA and other products released from dying bacteria that reach concentrations sufficient to directly activate many lymphocytes. Further evidence of the role of CpG DNA in the sepsis syndrome is described in Cowdery, J., et. al., (1996) The Journal of Immunology 156:4570-4575.
• CpG-ODN did not induce any detectable Ca 2+ flux, changes in protein tyrosine phosphorylation, or IP 3 generation.
• Flow cytometry with FITC-conjugated ODN with or without a CpG motif was performed as described in Zhao, Q et al,( Antisense Research and Development 3:53-66 (1993)), and showed equivalent membrane binding, cellular uptake, efflux, and intracellular localization. This suggests that there may not be cell membrane proteins specific for CpG ODN.
• the dye only sample in Panel A of the figure shows the background level of cells positive for the dye at 28.6%.
• this level of reactive oxygen species was greatly increased to 80% in the cells treated for 20 minutes with PMA and ionomycin, a positive control (Panel B).
• the cells treated with the CpG oligo also showed an increase in the level of reactive oxygen species such that more than 50% of the cells became positive (Panel D).
• cells treated with an oligonucleotide with the identical sequence except that the CpG was switched did not show this significant increase in the level of reactive oxygen species (Panel E).
• Chloroquine, monensin, and bafilomycin also appear to block the activation of NFkB by CpG DNA as well as the subsequent proliferation and induction of cytokine secretion.
• a class of medications effective in the treatment of lupus is antimalarial drugs, such as chloroquine. While the therapeutic mechanism of these drugs has been unclear, they are known to inhibit endosomal acidification.
• Leukocyte activation by CpG DNA is not mediated through binding to a cell surface receptor, but requires cell uptake, which occurs via adso ⁇ tive endocytosis into an acidified chloroquine-sensitive intracellular compartment. This suggested the hypothesis that leukocyte activation by CpG DNA may occur in association with acidified endosomes, and might even be pH dependent. To test this hypothesis specific inhibitors of DNA acidicification were applied to determine whether B cells or monocytes could respond to CpG DNA if endosomal acidification was prevented.
• ROS reactive oxygen species
• TNF- ⁇ TNF- ⁇
• IL-12 24 hr
• ELISA for IL-12 or TNF- ⁇ (pg/ml) was performed on the supernatants essentially as descnbed (A. K. Kneg, A.-K. Yi, S.
• CpG motifs may contribute to the pathogenesis of the autoimmune disease systemic lupus erythematosus, which is associated with elevated levels of circulating hypomethylated CpG DNA.
• Chloroqume and related antimalarial compounds are effective therapeutic agents for the treatment of systemic lupus erythematosus and some other autoimmune diseases, although their mechanism of action has been obscure.
• NFKB is known to be an important regulator of gene expression, it's role in the transcriptional response to CpG DNA was uncertain. To determine whether this NFKB activation was required for the CpG mediated induction of gene expression cells were activated with CpG DNA in the presence or absence of pyrrolidine dithiocarbamate (PDTC), an inhibitor of I ⁇ B phosphorylation. These inhibitors of NFKB activation completely blocked the CpG- induced expression of protooncogene and cytokine mRNA and protein, demonstrating the essential role of NFKB as a mediator of these events. None of the inhibitors reduced cell viability under the experimental conditions used in these studies.
• PDTC pyrrolidine dithiocarbamate
• CT calf thymus
• EC E. coli
• mEC methylated E. coli
• a doublestranded ODN containing a consensus NFKB site was 5' radiolabeled and used as a probe for EMS A essentially as described (J. D. Dignam, R. M. Lebovitz and R. G. Roeder, Nucleic Acids Res. 11, 1475 (1983); M. Briskin, M. Damore, R. Law, G. Lee, P. W. Kincade, C. H. Sibley, M. Kuehl and R. Wall, Mol. Cell. Biol. 10, 422 (1990)).
• the position of the p50/p65 heterodimer was determined by supershifting with specific Ab to p65 and p50 (Santa Cruz Biotechnology, Santa Cruz, CA).
• Chloroquine inhibition of CpG-induced but not LPS-induced NFKB activation was established using J774 cells. The cells were precultured for 2 hr in the presence or absence of chloroquine (20 ⁇ g/ml) and then stimulated as above for 1 hr with either EC DNA, CpG ODN, non-CpG ODN or LPS (1 ⁇ g/ml). Similar chloroquine sensitive CpG-induced activation of NFKB was seen in a B cell line, WEHI-231 and primary spleen cells. These experiments were performed three times over a range of chloroquine concentrations from 2.5 to 20 ⁇ g/ml with similar results.
• J774 cells (2xl0 6 cells/ml) were cultured for 2 hr in the presence or absence of chloroquine (2.5 ⁇ g/ml [ ⁇ 5 ⁇ M]) or N-tosyl-L- phenylalanine chlorometryl ketone (TPCK; 50 ⁇ M), a serine/threonine protease inhibitor that prevents I ⁇ B proteolysis and thus blocks NFKB activation. Cells were then stimulated with the addition of E.
• chloroquine 2.5 ⁇ g/ml [ ⁇ 5 ⁇ M]
• TPCK N-tosyl-L- phenylalanine chlorometryl ketone
• RNA was prepared using RNAzol following the manufacturer' s protocol.
• Multi-probe RNase protection assay was performed as described (A.-K. Yi, P. Hornbeck, D. E. Lafrenz and A. M. Krieg, J. Immunol, 157, 4918-4925 (1996).
• Co ⁇ parable a ⁇ ounts of RNA were loaded into each lane by using riboso ⁇ al ⁇ RNA as a loading control (L32). These experi ⁇ ents were perfor ⁇ ed three ti ⁇ es with si ⁇ ilar results.
• the pH dependent step may be the transport or processing of the CpG DNA, the ROS generation, or some other event.
• ROS are widely thought to be second messengers in signaling pathways in diverse cell types, but have not previously been shown to mediate a stimulatory signal in B cells.
• eiectrophoretic mobility shift assays were used with 5' radioactively labeled oligonucleotides with or without CpG motifs. A band was found that appears to represent a protein binding specifically to single stranded oligonucleotides that have CpG motifs, but not to oligonucleotides that lack CpG motifs or to oligonucleotides in which the CpG motif has been methylated.
• NFkB or related protein is a component of a protein or protein complex that binds the stimulatory CpG oligonucleotides.
• the CpG nucleic acids may bind to one of the TRAF proteins that bind to the cytoplasmic region of CD40 and mediate NFkB activation when CD40 is cross-linked.
• TRAF proteins include TRAF-2 and TRAF-5.
• nucleic acids can be synthesized de novo using any of a number of procedures well known in the art. For example, the b-cyanoethyl phosphoramidite method (S.L. Beaucage and M.H. Caruthers, (1981) Tet. Let. 22:1859); nucleoside H-phosphonate method (Garegg et al, (1986) Tet. Let. 27: 4051-4054; Froehler et al, (1986) Nucl Acid. Res. 14: 5399-5407; Garegg et al, (1986) Tet. Let.
• oligonucleotides can be prepared from existing nucleic acid sequences (e.g. genomic or cDNA) using known techniques, such as those employing restriction enzymes, exonucleases or endonucleases.
• 4,469,863; and alkylphosphotriesters in which the charged oxygen moiety is alkylated as described in U.S. Patent No. 5,023,243 and European Patent No. 092,574 can be prepared by automated solid phase synthesis using commercially available reagents. Methods for making other DNA backbone modifications and substitutions have been described (Uhlmann, E. and Peyman, A. (1990) Chem. Rev. 90:544; Goodchild, J. (1990) Bioconjugate Chem. 1:165). 2 * -O-methyl nucleic acids with CpG motifs also cause immune activation, as do ethoxy-modified CpG nucleic acids. In fact, no backbone modifications have been found that completely abolish the CpG effect, although it is greatly reduced by replacing the C with a 5-methyl C.
• nucleic acid molecules containing at least one unmethylated CpG dinucleotide can be administered to a subject in vivo to treat an "immune system deficiency".
• nucleic acid molecules containing at least one unmethylated CpG dinucleotide can be contacted with lymphocytes (e.g. B cells, monocytic cells or NK cells) obtained from a subject having an immune system deficiency ex vivo and activated lymphocytes can then be re-implanted in the subject.
• lymphocytes e.g. B cells, monocytic cells or NK cells
• IL-6 As reported herein, in response to unmethylated CpG containing nucleic acid molecules, an increased number of spleen cells secrete IL-6, IL-12, IFN- ⁇ , IFN- ⁇ , IFN- ⁇ , IL-1, IL-3, IL-10, TNF- ⁇ , TNF- ⁇ , GM-CSF, RANTES, and probably others.
• the increased IL-6 expression was found to occur in B cells, CD4 + T cells and monocytic cells.
• Immunostimulatory nucleic acid molecules can also be administered to a subject in conjunction with a vaccine to boost a subject's immune system and thereby effect a better response from the vaccine.
• the immunostimulatory nucleic acid molecule is administered slightly before or at the same time as the vaccine.
• a conventional adjuvant may optionally be administered in conjunction with the vaccine, which is minimally comprised of an antigen, as the conventional adjuvant may further improve the vaccination by enhancing antigen abso ⁇ tion.
• the vaccine is a DNA vaccine
• the antigen encoded by the vaccine determines the specificity of the immune response.
• the backbone of the plasmid contains CpG motifs, it functions as an adjuvant for the vaccine.
• CpG DNA acts as an effective "danger signal” and causes the immune system to respond vigorously to new antigens in the area. This mode of action presumably results primarily from the stimulatory local effects of CpG DNA on dendritic cells and other "professional" antigen presenting cells, as well as from the co-stimulatory effects on B cells.
• an immunostimulatory oligonucleotide can be administered prior to, along with or after administration of a chemotherapy or immunotherapy to increase the responsiveness of the malignant cells to subsequent chemotherapy or immunotherapy or to speed the recovery of the bone marrow through induction of restorative cytokines such as GM-CSF.
• CpG nucleic acids also increase natural killer cell lytic activity and antibody dependent cellular cytotoxicity (ADCC). Induction of NK activity and ADCC may likewise be beneficial in cancer immunotherapy, alone or in conjunction with other treatments.
• Another use of the described immunostimulatory nucleic acid molecules is in desensitization therapy for allergies, which are generally caused by IgE antibody generation against harmless allergens.
• the cytokines that are induced by unmethylated CpG nucleic acids are predominantly of a class called "Thl" which is most marked by a cellular immune response and is associated with IL-12 and IFN- ⁇ .
• Th2 immune response The other major type of immune response is termed a Th2 immune response, which is associated with more of an antibody immune response and with the production of IL-4, IL-5 and IL-10.
• Th2 immune response which is associated with more of an antibody immune response and with the production of IL-4, IL-5 and IL-10.
• allergic diseases are mediated by Th2 type immune responses and autoimmune diseases by Thl immune response.
• an effective dose of an immunostimulatory nucleic acid (or a vector containing a nucleic acid) alone or in conjunction with an allergen can be administered to a subject to treat or prevent an allergy.
• Nucleic acids containing unmethylated CpG motifs may also have significant therapeutic utility in the treatment of asthma.
• Th2 cytokines especially IL-4 and IL-5 are elevated in the airways of asthmatic subjects. These cytokines promote important aspects of the asthmatic inflammatory response, including IgE isotype switching, eosinophil chemotaxis and activation and mast cell growth.
• Thl cytokines especially IFN- ⁇ and IL-12, can suppress the formation of Th2 clones and production of Th2 cytokines.
• oligonucleotides containing an unmethylated CpG motif i.e., TCCATGACQTTCCTGACGTT; SEQ ID NO. 10
• TCCATGAGCTTCCTGAGTCT a control oligonucleotide
• a nucleic acid alone or as a nucleic acid delivery complex can be administered in conjunction with a pharmaceutically acceptable carrier.
• pharmaceutically acceptable carrier is intended to include substances that can be coadministered with a nucleic acid or a nucleic acid delivery complex and allows the nucleic acid to perform its indicated function. Examples of such carriers include solutions, solvents, dispersion media, delay agents, emulsions and the like. The use of such media for pharmaceutically active substances are well known in the art. Any other conventional carrier suitable for use with the nucleic acids falls within the scope of the instant invention.
• B cells were purified from spleens obtained from 6-12 wk old specific pathogen free DBA 2 or BXSB mice (bred in the University of Iowa animal care facility; no substantial strain differences were noted) that were depleted of T cells with anti-Thy-1.2 and complement and centrifugation over lymphocyte M (Cedarlane Laboratories, Hornby, Ontario, Canada) ("B cells"). B cells contained fewer than 1% CD4 + or CD8 + cells.
• 8xl0 4 B cells were dispensed in triplicate into 96 well microtiter plates in 100 ⁇ l RPMI containing 10% FBS (heat inactivated to 65°C for 30 min.), 50 ⁇ M 2-mercaptoethanol, 100 U/ml penicillin, 100 ug/ml streptomycin, and 2 mM L-glutamate.
• 20 ⁇ M ODN were added at the start of culture for 20 h at 37°C, cells pulsed with 1 ⁇ Ci of 3 H uridine, and harvested and counted 4 hr later. Ig secreting B cells were enumerated using the ELISA spot assay after culture of whole spleen cells with ODN at 20 ⁇ M for 48 hr.
• B cells were incubated for 6 hrs on anti-Ig coated microtiter plates. The lg they produced (>99%
• IgM was detected using phosphatase-labeled anti-Ig (Southern Biotechnology Associated,
• DBA/2 B cells were cultured with either no additive, 50 ⁇ g/ml LPS or the ODN 1 ; la; 4; or 4a at 20 uM. Cells were cultured and harvested at 4, 8, 24 and 48 hours. BXSB cells were cultured as in Example 1 with 5, 10, 20, 40 or 80 ⁇ M of ODN 1 ; la; 4; or 4a or LPS. In this experiment, wells with no ODN had 3833 cpm. Each experiment was performed at least three times with similar results. Standard deviations of the triplicate wells were ⁇ 5%.
• Percent specific lysis was determined by calculating the ratio of the 5! Cr released in the presence of effector cells minus the 51 Cr released when the target cells are cultured alone, over the total counts released after cell lysis in 2% acetic acid minus the 51 Cr cpm released when the cells are cultured alone.
• mice were weighed and injected IP with 0.25 ml of sterile PBS or the indicated phophorothioate ODN dissolved in PBS. Twenty four hours later, spleen cells were harvested, washed, and stained for flow cytometry using phycoerythrin conjugated 6B2 to gate on B cells in conjunction with biotin conjugated anti Ly-6A/E or anti-Ia d (Pharmingen, San Diego, CA) or anti-Bla-1 (Hardy, R.R. et al, J. Exp. Med. 159:1169 (1984). Two mice were studied for each condition and analyzed individually.
• B cells were cultured with phosphorothioate ODN with the sequence of control ODN la or the CpG ODN Id and 3Db and then either pulsed after 20 hr with 3 H uridine or after 44 hr with 3 H thymidine before harvesting and determining cpm.
• WEHI-231 cells (5 x 10 4 /well) were cultured for 1 hr. at 37 C in the presence or absence of LPS or the control ODN la or the CpG ODN Id and 3Db before addition of anti-IgM (l ⁇ /ml). Cells were cultured for a further 20 hr. before a 4 hr. pulse with 2 ⁇ Ci/well 3 H thymidine. In this experiment, cells with no ODN or anti-IgM gave 90.4 x 10 3 cpm of 3 H thymidine inco ⁇ oration by addition of anti-IgM.
• the phosphodiester ODN shown in Table 1 gave similar protection, though with some nonspecific suppression due to ODN degradation. Each experiment was repeated at least 3 times with similar results.
• Example 8 In vivo Induction of Murine IL-6
• mice were killed by cervical dislocation. Single cell suspensions were prepared aseptically from the spleens from mice. T cell depleted mouse splenocytes were prepared by using anti -Thy- 1.2 and complement and centrifugation over lymphocyte M (Cedarlane Laboratories, Hornby, Ontario, Canada) as described (Krieg, A. M. et al, (1989) A role for endogenous retroviral sequences in the regulation of lymphocyte activation. J. Immunol 143:2448).
• E. coli and calf thymus DNA were digested with DNase I (2U/ ⁇ g of DNA) at 37°C for 2 hr in IX SSC with 5mM MgC12.
• DNase I 2U/ ⁇ g of DNA
• IX SSC IX SSC with 5mM MgC12.
• E. coli DNA was treated with CpG methylase (M. Sssl; 2U/ ⁇ g of DNA) in NEBuffer 2 supplemented with 160 ⁇ M S-adenosyl methionine and incubated overnight at 37°C.
• Methylated DNA was purified as above. Efficiency of methylation was confirmed by Hpa II digestion followed by analysis by gel electrophoresis.
• LPS level in ODN was less than 12.5 ng mg and E. coli and calf thymus DNA contained less than 2.5 ng of LPS/mg of DNA by Limulus assay.
• cells were treated with CpG O-ODN along with various concentrations (1-10 ⁇ g/ml) of neutralizing rat IgGl antibody against murine EL-6 (hybridoma MP5-20F3) or control rat IgGl mAb to E. coli 6-galactosidase (hybridoma GL113; ATCC, Rockville, MD) (20) for 5 days.
• culture supernatant fractions were analyzed by ELISA as below.
• mice were injected intravenously (iv) with PBS, calf thymus DNA (200 ⁇ g/100 ⁇ l PBS/mouse), E. coli DNA (200 ⁇ g/100 ⁇ l PBS/mouse), or CpG or non-CpG S-ODN (200 ⁇ g/100 ⁇ l PBS/mouse).
• Mice two/group were bled by retroorbital puncture and sacrificed by cervical dislocation at various time points. Liver, spleen, thymus, and bone marrow were removed and RNA was prepared from those organs using RNAzol B (Tel-Test, Friendswood, TX) according to the manufacturers protocol.
• RNAzol B Tel-Test, Friendswood, TX
• the plates were washed and 100 ⁇ l/well of biotinylated rat anti-mouse IL-6 monoclonal antibodies (MP5-32C11, Pharmingen, San Diego, CA) (l ⁇ g/ml in 10% FCS) or biotinylated anti-mouse lg (Sigma, St. Louis, MO) were added and incubated for 45 min. at room temperature following washes with TPBS. Horseradish peroxidase (HRP) conjugated avidin (Bio-rad Laboratories, Hercules, CA) at 1 :4000 dilution in 10% FCS (100 ⁇ l/well) was added and incubated at room temperature for 30 min.
• HRP horseradish peroxidase
• RNA synthesis and IL-6 PCR was done essentially as described by Montgomery and Dallman (Montgomery, R.A. and M.S. Dallman (1991), Analysis of cytokine gene expression during fetal thymic ontogeny using the polymerase chain reaction (J. Immunol.) 147:554).
• cDNA synthesis and IL-6 PCR was done essentially as described by Montgomery and Dallman (Montgomery, R.A. and M.S. Dallman (1991), Analysis of cytokine gene expression during fetal thymic ontogeny using the polymerase chain reaction (J.
• the gel was dried and prehybridized at 47°C for 2 hr. hybridization buffer (5X SSPE, 0.1 % SDS) containing 10 ⁇ g/ml denatured salmon sperm DNA. The gel was hybridized with 2xl0 6 cpm/ml g 3 - ⁇ P]ATP end-labeled internal oligonucleotide probe for IL- ⁇ (5'CATTTCCACGATTTCCCA3') SEQ ID. No. 56) overnight at 47°C, washed 4 times (2X SSC, 0.2% SDS) at room temperature and autoradiographed. The results are shown in Figure 3.
• DBA/2 mice spleen B cells (5x10 4 cells/100 were treated with media, CpG or non-CpG S-ODN (0.5 ⁇ M) or O-ODN (20 ⁇ M) for 24 hr at
• WEHI-231 cells (10 7 cells) were e with 20 ⁇ g of control or human IL-6 promoter-CAT construct (kindly provided by S. Manolagas,
• Chloramphenicol acetyltransferase (CAT) activity was measured by a solution assay (Seed, B. and J.Y. Sheen (1988) A single phase-extraction assay for chloramphenicol acetyl transferase activity. Gene 76:271) 16 hr. after transfection. The results are presented in Figure 5.
• ODN were synthesized on an Applied Biosystems Inc. (Foster City, CA) model 380A, 380B, or 394 DNA synthesizer using standard procedures (Beacage and Caruthers (1981) Deoxynucleoside phosphoramidites-- A new class of key intermediates for deoxypolynucleotide synthesis. Tetrahedron Letters 22, 1859-1862.).
• Phosphodiester ODN were synthesized using standard beta-cyanoethyl phosphoramidite chemistry. Phosphorothioate linkages were introduced by oxidizing the phosphite linkage with elemental sulfur instead of the standard iodine oxidation.
• the four common nucleoside phosphoramidites were purchased from Applied Biosystems. All phosphodiester and thioate containing ODN were deprotected by treatment with concentrated ammonia at 55°C for 12 hours. The ODN were purified by gel exclusion chromatography and lyophilized to dryness prior to use. Phosphorodithioate linkages were introduced by using deoxynucleoside S-(b-benzoylmercaptoethyl) pyrrolidino thiophosphoramidites (Wiesler, W.T. et al, (1993) In Methods in Molecular Biology: Protocols for Oligonucleotides and Analogs- Synthesis and Properties, Agrawal, S. (ed.), Humana Press, 191 -206.). Dithioate containing ODN were deprotected by treatment with concentrated ammonia at 55°C for 12 hours followed by reverse phase HPLC purification.
• the methylphosphinodiester is treated with the sulfurizing reagent (5% elemental sulfur, 100 millimolar N,N-diamethylaminopyridine in carbon disulfide/pyridine/triethylamine), four consecutive times for 450 seconds each to produce methylphosphonothioates.
• the methylphosphinodiester is treated with standard oxidizing reagent (0.1 M iodine in tetrahydrofuran/2,6-lutidine/water).
• silica gel bound oligomer was treated with distilled pyridine/concentrated ammonia, 1:1, (v/v) for four days at 4 degrees centigrade.
• the supernatant was dried in vacuo, dissolved in water and chromatographed on a G50/50 Sephadex column.
• O-ODN refers to ODN which are phosphodiester; S-ODN are completely phosphorothioate modified; S-O-ODN are chimeric ODN in which the central linkages are phosphodiester, but the two 5' and five 3' linkages are phosphorothioate modified; S 2 -O-ODN are chimeric ODN in which the central linkages are phosphodiester, but the two 5' and five 3' linkages are phosphorodithioate modified; and MP-O-ODN are chimeric ODN in which the central linkages are phosphodiester, but the two 5' and five 3' linkages are methylphosphonate modified.
• the ODN sequences studied include:
• mice DBA/2, or BXSB mice obtained from The Jackson Laboratory (Bar Harbor, ME), and maintained under specific pathogen-free conditions were used as a source of lymphocytes at 5- 10 wk of age with essentially identical results.
• mouse spleen cells (5xl0 4 cells/100 ⁇ l/well) were cultured at 37°C in a 5% CO 2 humidified incubator in RPMI-1640 supplemented with 10% (v/v) heat inactivated fetal calf serum (heated to 65°C for experiments with O-ODN, or 56°C for experiments using only modified ODN), 1.5 ⁇ M L-glutamine, 50 ⁇ M 2-mercaptoethanol, 100 U/ml penicillin and 100 ⁇ g/ml streptomycin for 24 hr or 48 hr as indicated.
• Phosphodiester ODN were purchased from Operon Technologies (Alameda, CA).
• Virus-free, 4-6 week old, DBA/2, C57BL/6 (B6) and congenitally athymic BALB/C mice were obtained on contract through the Veterans Affairs from the National Cancer Institute (Bethesda, MD). C57BL/6 SCID mice were bred in the SPF barrier facility at the University of Iowa Animal Care Unit.
• PBMC peripheral mononuclear blood leukocytes
• 1 LU was defined as the number of cells needed to effect 30% specific lysis.
• neutralizing antibodies against IFN- ⁇ Lee Biomolecular, San Diego, CA
• EL-12 C15.1, C15.6, C17.8, and C17.15; provided by Dr. Giorgio Trinchieri, The Wistar Institute, Philadelphia, PA
• isotype controls were added at the initiation of cultures to a concentration of 10 ⁇ g/ml.
• 10 ⁇ g of each of the 4 MAB (or isotype controls) were added simultaneously.
• Recombinant human IL-2 was used at a concentration of 100 U/ml.
• Schistosoma mansoni eggs contain an antigen (Schistosoma mansoni egg antigen (SEA)) that induces a Th2 immune response (e.g. production of IgE antibody).
• SEA Schostosoma mansoni egg antigen
• IgE antibody production is known to be an important cause of asthma.
• mice were then treated with oligonucleotides (30 ⁇ g in 200 ⁇ l saline by i.p.injection), which either contained an unmethylated CpG motif (i.e., TCCATGACGTTCCTGACGTT; SEQ ID NO.10) or did not (i.e., control, TCCATGAGCTTCCTGAGTCT; SEQ ID NO.11).
• Soluble SEA (lO ⁇ g in 25 ⁇ l of saline) was administered by intranasal instillation on days 14 and 21. Saline was used as a control.
• mice were sacrificed at various times after airway challenge.
• Whole lung lavage was performed to harvest airway and alveolar inflammatory cells.
• Cytokine levels were measured from lavage fluid by ELISA.
• RNA was isolated from whole lung for Northern analysis and RT-PCR studies using CsCl gradients. Lungs were inflated and perfused with 4% paraformaldehyde for histologic examination.
• Figure 9 shows that when the mice are initially injected with the eggs i.p., and then inhale the egg antigen (open circle), many inflammatory cells are present in the lungs. However, when the mice are initially given a nucleic acid containing an unmethylated CpG motif along with the eggs, the inflammatory cells in the lung are not increased by subsequent inhalation of the egg antigen (open triangles).
• Figure 10 shows that the same results are obtained when only eosinophils present in the lung lavage are measured.
• Eosinophils are the type of inflammatory cell most closely associated with asthma.
• Figure 11 shows that when the mice are treated with a control oligo at the time of the initial exposure to the egg, there is little effect on the subsequent influx of eosinophils into the lungs after inhalation of SEA.
• mice inhale the eggs on days 14 or 21 they develop an acute inflammatory response in the lungs.
• giving a CpG oligo along with the eggs at the time of initial antigen exposure on days 0 and 7 almost completely abolishes the increase in eosinophils when the mice inhale the egg antigen on day 14.
• Figure 12 shows that very low doses of oligonucleotide ( ⁇ lO ⁇ g) can give this protection.
• Figure 13 shows that the resultant inflammatory response correlates with the levels of the Th2 cytokine IL-4 in the lung.
• Figure 14 shows that administration of an oligonucleotide containing an unmethylated CpG motif can actually redirect the cytokine response of the lung to production of 11-12, indicating a Thl type of immune response.

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