A novel antagonist of Toll-like receptors 7, 8 and 9 suppresses lupus disease-associated parameters in NZBW/F1 mice.

Systemic Lupus Erythematosus is an autoimmune disease characterized by production of autoantibodies against nucleic acid-associated antigens. Endogenous DNA and RNA associated with these antigens stimulate inflammatory responses through Toll-like receptors (TLRs) and exacerbate lupus disease pathology. We have evaluated an antagonist of TLR7, 8 and 9 as a therapeutic agent in lupus-prone NZBW/F1 mice. NZBW/F1 mice treated with the antagonist had lower serum levels of autoantibodies targeting DNA, RNP, Smith antigen, SSA and SSB than did untreated mice. Reduction in blood urea nitrogen and proteinuria and improvements in kidney histopathology were observed in antagonist-treated mice. The antagonist treatment also reduced serum IL-12 and IL-1ß and increased IL-10 levels. Levels of mRNA for IL-6, iNOS and IL-1ß were lower in the kidneys and spleen of antagonist-treated mice than in those of untreated mice. Levels of mRNA for IP-10, TNFRSF9 and FASL were lower and IL-4 mRNA were higher in spleens of antagonist-treated mice than in spleens of untreated mice. mRNA for the inflammasome component NLRP3 was lower and mRNA for the antioxidant enzymes, catalase and glutathione peroxidase 1 was higher in the kidneys of antagonist-treated mice than in those of untreated mice. These results show that the antagonist of TLR7, 8 and 9 effectively inhibits inflammatory pathways involved in the development of lupus in NZBW/F1 mice and constitutes a potential therapeutic approach for the treatment of lupus and other autoimmune diseases.

A Toll-like receptor 7, 8, and 9 antagonist inhibits Th1 and Th17 responses and inflammasome activation in a model of IL-23-induced psoriasis.

Psoriasis is a chronic inflammatory skin disease that involves the induction of T-helper 1 (Th1) and T-helper 17 (Th17) cell responses and the aberrant expression of proinflammatory cytokines, including IL-1ß. Copious evidence suggests that abnormal activation of Toll-like receptors (TLRs) contributes to the initiation and maintenance of psoriasis. We have evaluated an antagonist of TLR7, 8, and 9 as a therapeutic agent in an IL-23-induced psoriasis model in mice. Psoriasis-like skin lesions were induced in C57BL/6 mice by intradermal injection of IL-23 in the ear or dorsum. IL-23-induced increase in ear thickness was inhibited in a dose-dependent manner by treatment with antagonist. Histological examination of ear and dorsal skin tissues demonstrated a reduction in epidermal hyperplasia in mice treated with the antagonist. Treatment with antagonist also reduced the induction of Th1 and Th17 cytokines in skin and/or serum, as well as dermal expression of inflammasome components, NLRP3 and AIM2, and antimicrobial peptides. These results indicate that targeting TLR7, 8, and 9 may provide a way to neutralize multiple inflammatory pathways that are involved in the development of psoriasis. The antagonist has the potential for the treatment of psoriasis and other autoimmune diseases.

Design of synthetic oligoribonucleotide-based agonists of Toll-like receptor 3 and their immune response profiles in vitro and in vivo.

Double-stranded RNA of viral origin and enzymatically synthesized poly I:C act as agonists of TLR3 and induce immune responses. We have designed and synthesized double-stranded synthetic oligoribonucleotides (dsORNs) which act as agonists of TLR3. Each strand of dsORN contains two distinct segments, namely an alignment segment composed of a heteronucleotide sequence and an oligo inosine (I) or an oligo cytidine (C) segment. We report here the results of studies of dsORNs containing varying lengths and compositions of alignment and oligo I/oligo C segments. dsORNs of 50-mer length with a 15-mer alignment segment and a 35-mer oligo I/oligo C segment form stable duplexes under physiological conditions and induce TLR3-mediated immune responses. dsORNs activated the IRF3 signaling pathway in J774 cells, induced production of cytokines, including IFN-ß, IFN-α, IP-10, IL-12 and IL-6, in murine and human cell-based assays and also induced multiple cytokines following systemic administration in mice and non-human primates.

Novel oligonucleotides containing two 3'-ends complementary to target mRNA show optimal gene-silencing activity.

Oligonucleotides are being employed for gene-silencing activity by a variety of mechanisms, including antisense, ribozyme, and siRNA. In the present studies, we designed novel oligonucleotides complementary to targeted mRNAs and studied the effect of 3'-end exposure and oligonucleotide length on gene-silencing activity. We synthesized both oligoribonucleotides (RNAs) and oligodeoxynucleotides (DNAs) with phosphorothioate backbones, consisting of two identical segments complementary to the targeted mRNA attached through their 5'-ends, thereby containing two accessible 3'-ends; these compounds are referred to as gene-silencing oligonucleotides (GSOs). RNA and/or DNA GSOs targeted to MyD88, VEGF, and TLR9 mRNAs had more potent gene-silencing activity than did antisense phosphorothioate oligonucleotides (PS-oligos) in cell-based assays and in vivo. Of the different lengths of GSOs evaluated, 19-mer long RNA and DNA GSOs had the best gene-silencing activity both in vitro and in vivo. These results suggest that GSOs are novel agents for gene silencing that can be delivered systemically with broader applicability.

Antitumor activity and immune response induction of a dual agonist of Toll-like receptors 7 and 8.

Viral and synthetic single-stranded RNAs are the ligands for Toll-like receptors 7 and 8 (TLR7 and TLR8). We have reported a novel class of synthetic oligoribonucleotides, referred to as stabilized immune-modulatory RNA compounds, which act as agonists of TLR7, TLR8, or both TLR7 and TLR8 depending on the sequence composition and the presence of specific chemical modifications. In the present study, we evaluated the antitumor activity of a dual TLR7/8 agonist in tumor-bearing mice with peritoneal disseminated CT26.CL25 colon and 3LL-C75 lung carcinomas. Peritoneal administration of dual TLR7/8 agonist in mice bearing CT26.CL25 colon carcinomas had potent dose-dependent antitumor activity, which was associated with a marked decrease in CD4(+)CD25(+)Foxp3(+) T regulatory cells and a significant increase in tumor antigen-specific IFN-gamma-secreting effector cell responses in splenocytes and local tumor-infiltrating cells. In 3LL-C75 lung carcinoma, dual TLR7/8 agonist induced strong immune responses and antitumor effects in C57BL/6 and TLR9(-/-) mice, but not in TLR7(-/-) and MyD88(-/-) mice, indicating that the agonist induces immune responses via TLR7 and through the MyD88-dependent signaling pathway. TLR8 is not functional in mice. Additionally, s.c. administration of TLR7/8 agonist effectively prevented lung metastasis of tumors in the CT26.CL25 pulmonary metastasis model. These studies show that the dual TLR7/8 agonist induced Th1-type immune responses and potent antitumor activity in mice via TLR7 and through the MyD88-dependent pathway.

Synthesis and immunological activities of novel agonists of toll-like receptor 9.

Novel agonists of TLR9 with two 5'-ends and synthetic immune stimulatory motifs, referred to as immune modulatory oligonucleotides (IMOs) are potent agonists of TLR9. In the present study, we have designed and synthesized 15 novel IMOs by incorporating specific chemical modifications and studied their immune response profiles both in vitro and in vivo. Analysis of the immunostimulatory profiles of these IMOs in human and NHP cell-based assays suggest that changes in the number of synthetic immunostimulatory motifs gave only a subtle change in immune stimulation of pDCs as indicated by IFN-alpha production and pDC maturation while the addition of self-complementary sequences produced more dramatic changes in both pDC and B cell stimulation. All IMOs induced cytokine production in vivo immediately after administration in mice. Representative compounds were also compared for the ability to stimulate cytokine production in vivo (IFN-alpha and IP-10) in rhesus macaques after intra-muscular administration.

On-chip magnetic separation of superparamagnetic beads for integrated molecular analysis.

We have demonstrated a postprocessed complementary metal oxide semiconductor (CMOS) integrated circuit (IC) capable of on-chip magnetic separation, i.e., removing via magnetic forces the nonspecifically bound magnetic beads from the detection area on the surface of the chip. Initially, 4.5 mum wide superparamagnetic beads sedimenting out of solution due to gravity were attracted to the detection area by a magnetic concentration force generated by flowing current through a conductor embedded in the IC. After sedimentation, the magnetic beads that did not bind strongly to the functionalized surface of the IC through a specific biochemical complex were removed by a magnetic separation force generated by flowing current through another conductor placed laterally to the detection area. As the spherical bead pivoted on the surface of the chip, the lateral magnetic force was further amplified by mechanical leveraging, and 50 mA of current flowing through the separation conductor placed 18 mum away from the bead resulted in 7.5 pN of tensile force on the biomolecular tether immobilizing the bead. This force proved high enough to break nonspecific interactions while leaving specific antibody-antigen bonds intact. A sandwich capture immunoassay on purified human immunoglobulin G showed strong correlation with a control enzyme linked immunosorbent assay and a detection limit of 10 ngml or 70 pM. The beads bound to the detection area after on-chip magnetic separation were detected optically. To implement a fully integrated molecular diagnostics platform, the on-chip magnetic separation functionality presented in this work can be readily combine with state-of-the art CMOS-based magnetic bead detection technology.

Oligodeoxyribonucleotide-based antagonists for Toll-like receptors 7 and 9.

Oligodeoxyribonucleotides containing unmethylated CpG motifs act as TLR9 agonists. In this study, we evaluated oligonucleotides containing an unmethylated CpG motif in which two nucleotides adjacent to the CpG dinucleotide were substituted with 2'-O-methylribonucleotides, resulting in TLR7 and TLR9 antagonists. In mouse and human cell cultures, antagonists did not stimulate immune activation but inhibited TLR7 and TLR9 agonist-induced activity. In mice, antagonists inhibited immune responses induced by TLR9 agonists for up to several days, and the inhibition was dose-dependent. Antagonists also inhibited immune responses induced by an RNA-based TLR7/8 agonist but not TLRs 2, 3, 4, or 5 agonists in mice. Additionally, antagonist inhibited TLR9 agonist-induced IL-6 in lupus-prone MRL/lpr mouse spleen cell cultures. These results indicate that antagonists described herein can suppress immune responses induced by TLR7 and TLR9 agonists. Antagonists may be suitable candidates for treating inflammatory and autoimmune diseases where inappropriate or uncontrolled TLR activation has been implicated.

Agonists of Toll-like receptor 9 containing synthetic dinucleotide motifs.

Oligodeoxynucleotides (ODNs) containing unmethylated CpG motifs activate Toll-like receptor 9 (TLR9). Our previous studies have shown that ODNs containing two 5'-ends are more immunostimulatory than those with one 5'-end. In the present study, to understand the role of functional groups in TLR9 recognition and subsequent immune response, we substituted C or G of a CpG dinucleotide with 5-OH-dC, 5-propyne-dC, furano-dT, 1-(2'-deoxy-beta- d-ribofuranosyl)-2-oxo-7-deaza-8-methyl-purine, dF, 4-thio-dU, N(3)-Me-dC, N (4)-Et-dC, Psi-iso-dC, and arabinoC or 7-deaza-dG, 7-deaza-8-aza-dG, 9-deaza-dG, N(1)-Me-dG, N(2)-Me-dG, 6-Thio-dG, dI, 8-OMe-dG, 8-O-allyl-dG, and arabinoG in ODN containing two 5'-ends. Agonists of TLR9 containing cytosine or guanine modification showed activity in HEK293 cells expressing TLR9, mouse spleen, and human cell-based assays and in vivo in mice. The results presented here provide insight into which specific chemical modifications at C or G of the CpG motif are recognized by TLR9 and the ability to modulate immune responses substituting natural C or G in immune modulatory oligonucleotides.

Stabilized immune modulatory RNA compounds as agonists of Toll-like receptors 7 and 8.

Viral and synthetic single-stranded RNAs are the ligands for Toll-like receptor (TLR)7 and TLR8. However, single-stranded RNA is rapidly degraded by ubiquitous RNases, and the studies reported to date have used RNA with lipid carriers. To overcome nuclease susceptibility of RNA, we have synthesized several RNAs incorporating a range of chemical modifications. The present study describes one pool of RNA compounds, referred to as stabilized immune modulatory RNA (SIMRA) compounds, in which two RNA segments are attached through their 3' ends. SIMRA compounds showed greater stability in human serum compared with linear RNA and activated human TLR8, but not TLR7, in HEK293 cells without using lipid carriers. Interestingly, another set of SIMRA compounds containing 7-deazaguanosine substituted for natural guanosine activated human TLR7 and TLR8. Additionally, TLR7- and TLR8-activating compounds, but not the compounds that activated only TLR8, stimulated mouse immune cells in vitro and in vivo and produced dose-dependent T helper 1-type cytokines. Both types of compounds activated human peripheral blood mononuclear cells, but only TLR7- and TLR8-activating compounds activated plasmacytoid dendritic cells and produced high levels of IFN-alpha. In monkeys, s.c. administration of both types of SIMRA compounds induced transient changes in peripheral blood monocytes and neutrophils, and activated T lymphocytes, monocytes, and NK cells. Both types of compounds induced IFN-gamma-inducible protein 10, but only the 7-deazaguanosine-containing compound that activated both TLR7 and TLR8 induced IFN-alpha in monkeys. This is a comprehensive study of RNA-based compounds containing structures and synthetic stimulatory motifs in mouse, monkey, and human systems without using lipid carriers.

Immunomodulatory oligonucleotides containing a cytosine-phosphate-2'-deoxy-7-deazaguanosine motif as potent toll-like receptor 9 agonists.

Bacterial DNA and synthetic oligomers containing CpG dinucleotides activate the immune system through Toll-like receptor (TLR) 9. Here, we compare the immunostimulatory activity of three immunomers with different nucleotide sequences containing a synthetic cytosine-phosphate-2'-deoxy-7-deazaguanosine dinucleotide (CpR), called immunomodulatory oligonucleotides (IMOs), in mouse, human, and monkey systems. IMOs induced IL-12 and IFN-gamma secretion more than a control non-CpG IMO in mice. All three IMOs activated HEK293 cells expressing TLR9 but not TLR3, -7, or -8. IMOs induced human B-cell proliferation and enhanced expression of CD86 and CD69 surface markers on B cells. The three IMOs induced CD86 expression on human plasmacytoid dendritic cells, but only IMOs that contained a 5'-terminal TCR nucleotide sequence induced IFN-alpha secretion. A sequence that forms a duplex structure also was required for IFN-alpha induction in human peripheral blood mononuclear cell cultures. IMOs induced chemokine and cytokine gene expression in human peripheral blood mononuclear cells. In monkeys, all three IMOs induced transient changes in peripheral blood leukocytes and lymphocytes and activated B and T lymphocytes. All three IMOs induced IFN-alpha in vivo in monkeys; the IMO sequence that forms a stable secondary structure induced the highest levels of IFN-alpha. These studies are, to our knowledge, the first comprehensive studies to compare the activity of IMOs containing synthetic stimulatory CpR dinucleotides in mouse, monkey, and human systems. These results suggest that IMOs induce strong and rapid immunostimulation and that the CpR dinucleotide is recognized by TLR9, leading to immune-cell activation and cytokine secretion in vitro and in vivo.

Oral administration of second-generation immunomodulatory oligonucleotides induces mucosal Th1 immune responses and adjuvant activity.

CpG DNA induces potent Th1 immune responses through Toll-like receptor 9. In the present study, we used oligonucleotides consisting of a novel 3'-3'-linked structure and synthetic stimulatory motifs, referred as second-generation immunomodulatory oligonucleotides (IMOs). The stimulatory motifs included: CpR, YpG, or R'pG (R = 2'-deoxy-7-deazaguanosine, Y = 2'-deoxy-5-hydroxy-cytidine, and R' = 1-[2'-deoxy-beta-d-ribofuranosyl]-2-oxo-7-deaza-8-methyl-purine). We evaluated the stability of orally administered IMOs in the gastrointestinal (GI) environment and their ability to induce mucosal immune responses in mice, and compared these characteristics with those of a conventional CpG DNA. The IMOs were significantly more stable than CpG DNA following oral administration, and IMOs induced stronger local and systemic immune responses as determined by MIP-1beta, MCP-1, IP-10, and IL-12 production. Mice orally immunized with ovalbumin (OVA) and IMO had higher levels of IgG2a antibodies in serum and IgA antibodies in intestinal mucosa than did mice immunized with OVA and CpG DNA. These studies demonstrate that IMOs are more stable than CpG DNA in the GI tract and can induce more potent mucosal Th1 adjuvant responses. IMOs may prove to be effective oral adjuvants, able to promote strong systemic and mucosal immune responses to oral vaccines and antigens for therapeutic and prophylactic applications.

Modulation of ovalbumin-induced Th2 responses by second-generation immunomodulatory oligonucleotides in mice.

Oligodeoxynucleotides containing unmethylated CpG dinucleotides (CpG DNAs) prevent development of T-helper type 2 (Th2) immune responses and reverse established allergic responses in mouse models. We recently reported that second-generation immunomodulatory oligonucleotides (IMOs) containing novel structures (immunomers) and a synthetic immunostimulatory CpR (R=2'-deoxy-7-deazguanosine) motif induce the production of distinct cytokine secretion profiles in vitro and in vivo. In the present study, we evaluated IMOs containing CpG and CpR motifs to modulate allergen-induced Th2 immune responses in prevention and treatment models. Mice sensitized and challenged with ovalbumin (OVA) were treated with a CpG DNA or an IMO by administration either at the time of OVA sensitization (co-administration; prevention) or after establishment of an allergic response (treatment). Spleens, blood, and lungs were collected and analyzed for immune responses. Spleen-cell cultures harvested from OVA-sensitized mice showed a significant decrease in Th2 cytokine levels with a concomitant increase in Th1 cytokine levels only when CpG DNA or IMOs were co-administered with OVA. The co-administration of CpG DNA or IMOs during OVA sensitization significantly reduced serum OVA-specific and total IgE levels in mice. The mice who received CpG DNA or IMOs co-administered with OVA showed a small reduction in serum OVA-specific and total IgG1 levels and a significant increase in serum OVA-specific and total IgG2a levels. Similar results were found in mice with established allergic responses who received IMO treatment. IMO treatment also resulted in strong inhibition of inflammatory cell infiltration and goblet cell hyperplasia in the lungs compared with untreated mice lungs. These data demonstrate that IMOs prevent antigen-induced Th2 immune responses when co-administered to mice during OVA sensitization and that IMOs reverse established allergic responses induced by OVA.

A dinucleotide motif in oligonucleotides shows potent immunomodulatory activity and overrides species-specific recognition observed with CpG motif.

Bacterial and synthetic DNAs containing CpG dinucleotides in specific sequence contexts activate the vertebrate immune system through Toll-like receptor 9 (TLR9). In the present study, we used a synthetic nucleoside with a bicyclic heterobase [1-(2'-deoxy-beta-d-ribofuranosyl)-2-oxo-7-deaza-8-methyl-purine; R] to replace the C in CpG, resulting in an RpG dinucleotide. The RpG dinucleotide was incorporated in mouse- and human-specific motifs in oligodeoxynucleotides (oligos) and 3'-3-linked oligos, referred to as immunomers. Oligos containing the RpG motif induced cytokine secretion in mouse spleen-cell cultures. Immunomers containing RpG dinucleotides showed activity in transfected-HEK293 cells stably expressing mouse TLR9, suggesting direct involvement of TLR9 in the recognition of RpG motif. In J774 macrophages, RpG motifs activated NF-kappa B and mitogen-activated protein kinase pathways. Immunomers containing the RpG dinucleotide induced high levels of IL-12 and IFN-gamma, but lower IL-6 in time- and concentration-dependent fashion in mouse spleen-cell cultures costimulated with IL-2. Importantly, immunomers containing GTRGTT and GARGTT motifs were recognized to a similar extent by both mouse and human immune systems. Additionally, both mouse- and human-specific RpG immunomers potently stimulated proliferation of peripheral blood mononuclear cells obtained from diverse vertebrate species, including monkey, pig, horse, sheep, goat, rat, and chicken. An immunomer containing GTRGTT motif prevented conalbumin-induced and ragweed allergen-induced allergic inflammation in mice. We show that a synthetic bicyclic nucleotide is recognized in the C position of a CpG dinucleotide by immune cells from diverse vertebrate species without bias for flanking sequences, suggesting a divergent nucleotide motif recognition pattern of TLR9.

Divergent synthetic nucleotide motif recognition pattern: design and development of potent immunomodulatory oligodeoxyribonucleotide agents with distinct cytokine induction profiles.

Unmethylated CpG dinucleotides present within certain specific sequence contexts in bacterial and synthetic DNA stimulate innate immune responses and induce cytokine secretion. Recently, we showed that CpG DNAs containing two 5'-ends, immunomers, are more potent in both regards. In this study, we show that an immunomer containing a synthetic CpR motif (R = 2'-deoxy-7-deazaguanosine) is a potent immunostimulatory agent. However, the profile of cytokine induction is different from that with immunomers containing a natural CpG motif. In general, a CpR immunomer induced higher interleukin (IL)-12 and lower IL-6 secretion. Compared with conventional CpG DNAs, both types of immunomers showed a rapid and enhanced activation of the transcription factor NF-kappaB in J774 cells. NF-kappaB activation by CpG DNA corresponded to degradation of IkappaBalpha in J774 cells. All three immunostimulatory oligonucleotides activated the p38 mitogen-activated protein kinase pathway as expected. Immunomers containing CpG and CpR motifs showed potent reversal of the antigen-induced Th2 immune response towards a Th1 type in antigen-sensitized mouse spleen cell cultures. Immunomers containing a CpR motif showed significant antitumor activity in nude mice bearing MCF-7 human breast cancer and U87MG glioblastoma xenografts. These studies suggest the ability for a divergent synthetic nucleotide motif recognition pattern of the receptor involved in the immunostimulatory pathway and the possibility of using synthetic nucleotides to elicit different cytokine response patterns.

CpG penta- and hexadeoxyribonucleotides as potent immunomodulatory agents.

We demonstrate a new design for immunomodulatory CpG DNA containing two sequences each with as few as five or six-nucleotides joined together via 3(')-3(') linkers. These do not require the -PuPu(Py)CGPyPy- hexameric motif generally found essential for CpG DNA immune stimulation. These novel, short-immunomers show potent immunostimulatory activity manifested by IL-12 and IL-6 secretion in murine spleen cell and PBMC cultures and splenomegaly in vivo. Short-immunomers show strong activation of NF-kappaB and stress-activated signaling pathways and induce cytokines in J774 cell cultures. The same sequences also induce cytokines in healthy human PBMC cultures whereas conventional CpG DNA requires different optimal sequences for murine and human immune cells. Additionally, short-immunomers inhibit IL-5 secretion and induce IFN-gamma secretion in conalbumin-sensitized mouse spleen cell cultures, suggesting reversal of established Th2 responses to Th1 type responses. Short-immunomer also inhibits growth of MCF-7 human tumor xenograft in nude mice. This is the first report of activity with such short DNA sequences and also of sequences lacking hexameric motifs proposed in earlier studies.

'Immunomers'--novel 3'-3'-linked CpG oligodeoxyribonucleotides as potent immunomodulatory agents.

Oligodeoxyribonucleotides containing CpG dinucleotides (CpG DNAs) are currently being evaluated as novel immunomodulators in clinical trials. Recently, we showed that an accessible 5' end is required for immunostimulatory activity and blocking the 5' end of CpG DNA by conjugation of certain ligands abrogates immunostimulatory activity. Based on these results, we designed and synthesized 3'-3'-linked CpG DNAs that contained two or more identical CpG DNA segments, referred to here as 'immunomers'. The use of solid support bearing diDMT-glyceryl-linker permitted convenient synthesis of immunomers with both segments synthesized simultaneously, giving better yields and purity. The in vitro and in vivo studies suggest that as a result of accessibility to two 5' ends for recognition, immunomers show an enhanced immunostimulatory activity compared with linear CpG DNAs. We also studied the suitability of a number of different linkers for attaching the two segments of immunomers. A C3-linker was found to be optimal for joining the two segments of immunomers. Incorporation of multiple linkers between the two segments of immunomers resulted in different cytokine profiles depending on the nature and number of linkers incorporated. Additionally, the length of immunomer also plays a significant role in inducing immune responses. An immunomer containing 11 nt in each segment showed the highest activity and an 11mer linear CpG DNA failed to stimulate an immune response. These results suggest that immunomers have several advantages over conventional linear CpG DNAs for immunomodulatory activity studies.

Design, synthesis, and immunostimulatory properties of CpG DNAs containing alkyl-linker substitutions: role of nucleosides in the flanking sequences.

Bacterial and synthetic DNA containing unmethylated CpG dinucleotides activate the innate immune system and promote Th1-like immune responses. Recently, a receptor, TLR9, has been shown to recognize CpG DNA and activate immune cascade. But there have been no reports on the molecular mechanisms of recognition between CpG DNA and the receptor(s). Our earlier studies described a number of the chemical and structural characteristics of CpG dinucleotide and the sequences flanking the CpG dinucleotide that are critical for immunostimulatory activity. In the present study, we examined the effect of the presence and absence of a nucleoside in the flanking sequences by replacing one or two natural deoxyribonucleosides at various positions with one or more alkyl- (C2-C12), branched alkyl- (glyceryl or aminobutyryl-propanediol), or ethyleneglycol- (tri or hexa) linkers. The results suggest that a linker substitution at the first two nucleoside positions adjacent to the CpG dinucleotide on the 5'- or the 3'-side neutralizes the immunostimulatory activity, as determined by in vitro mouse spleen cell proliferation, cytokine secretion, and in vivo mouse spleen enlargement. The same substitutions placed about three to six nucleotides away from the CpG dinucleotide either did not affect or potentiated immunostimulatory activity compared with parent CpG-DNA without modifications. Substitution of deoxyribonucleosides with a C3 or C4 alkyl-linker was found to be optimal for potentiating immunostimulatory activity.

Conjugation of ligands at the 5'-end of CpG DNA affects immunostimulatory activity.

Bacterial DNA and synthetic oligonucleotides containing unmethylated CpG dinucleotides (CpG DNA) activate the vertebrate immune system and promote Th1-like immune responses. Several CpG DNAs are currently being tested in clinical trials as either alone or in combination with vaccines, antibodies, and allergens separately or as conjugates for a number of disease indications including cancers, allergies, and asthma. In this paper, we show that conjugation of an oligonucleotide and a CpG DNA through their 5'-ends (5'-5'-linked DNA) significantly reduces the immunostimulatory activity of the CpG DNA. In addition, we found that the reduction in immunostimulatory activity of 5'-5'-linked CpG DNA depends on the size of the oligonucleotide conjugated to CpG DNA. Conjugation of a smaller group or molecule, such as a phosphorothioate group, at the 5'-end of CpG DNA has an insignificant effect on immunostimulatory activity. However, conjugation of a mononucleotide, tetra- or longer oligonucleotide or a fluorescein molecule to the 5'-end of a CpG DNA (5'-5'-linked DNA) significantly suppresses the immunostimulatory activity of CpG DNA. Surprisingly, conjugation of an oligonucleotide or a ligand through the 3'-end of CpG DNA (3'-3'-linked DNA) has an insignificant effect on immunostimulatory activity. Studies of cellular uptake and activation of transcription factor NF-kappaB in J774 cells using fluorescein-conjugated CpG DNAs suggest that the differences in the immune stimulation of 5'- and 3'-end-conjugated CpG DNAs is not as a result of differences in their cellular uptake properties. These results suggest that for optimal immunostimulatory activity, ligands should not be attached at the 5'-end of the CpG DNA.