CA2293489C - Inhibitors of dna immunostimulatory sequence activity - Google Patents

Inhibitors of dna immunostimulatory sequence activity Download PDF

Info

Publication number
CA2293489C
CA2293489C CA002293489A CA2293489A CA2293489C CA 2293489 C CA2293489 C CA 2293489C CA 002293489 A CA002293489 A CA 002293489A CA 2293489 A CA2293489 A CA 2293489A CA 2293489 C CA2293489 C CA 2293489C
Authority
CA
Canada
Prior art keywords
odn
iss
oligonucleotide
autoantigen
iis
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Expired - Fee Related
Application number
CA002293489A
Other languages
French (fr)
Other versions
CA2293489A1 (en
Inventor
Eyal Raz
Mark Roman
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
University of California
Dynavax Technologies Corp
Original Assignee
University of California
Dynavax Technologies Corp
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Family has litigation
First worldwide family litigation filed litigation Critical https://patents.darts-ip.com/?family=21956478&utm_source=google_patent&utm_medium=platform_link&utm_campaign=public_patent_search&patent=CA2293489(C) "Global patent litigation dataset” by Darts-ip is licensed under a Creative Commons Attribution 4.0 International License.
Application filed by University of California, Dynavax Technologies Corp filed Critical University of California
Publication of CA2293489A1 publication Critical patent/CA2293489A1/en
Application granted granted Critical
Publication of CA2293489C publication Critical patent/CA2293489C/en
Anticipated expiration legal-status Critical
Expired - Fee Related legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C07K14/4703—Inhibitors; Suppressors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/117—Nucleic acids having immunomodulatory properties, e.g. containing CpG-motifs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55561—CpG containing adjuvants; Oligonucleotide containing adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
    • C12N2310/111—Antisense spanning the whole gene, or a large part of it
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/17—Immunomodulatory nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/18—Type of nucleic acid acting by a non-sequence specific mechanism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/315—Phosphorothioates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/33—Chemical structure of the base
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/33—Chemical structure of the base
    • C12N2310/334—Modified C
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/35—Nature of the modification
    • C12N2310/351—Conjugate
    • C12N2310/3513—Protein; Peptide
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

The invention consists of oligonucleotides which inhibit the immunostimulato ry activity of ISS-ODN (immunostimulatory sequence oligodeoxynucleotides) as well as methods for their identification and use. The oligonucleotides of the invention are useful in controlling therapeutically intended ISS-ODN adjuvant activity as well as undesired ISS- ODN activity exerted by recombinant expression vectors, such as those used for gene therapy and gene immunization. The oligonucleotides of the invention also have anti-inflammatory activity useful in reducing inflammation in response to infection of a host with ISS- ODN containing microbes, in controlling autoimmune disease and in boosting host Th2 type immune responses to an antigen. The invention also encompasses pharmaceutically useful conjugates of the oligonucleotides of the invention (including conjugate partners such as antigens and antibodies).

Description

INHIBITORS OF DNA IMMUNOSTIMULATORY SEQUENCE ACTIVITY

BACKGROUND FOR THE INVENTION
1. Field of the Invention The invention relates to immunostimulatory sequences in DNA. The invention further relates to recombinant expression vectors for use in gene therapy.
2. History of the Related Art Recombinant expression vectors are the tools which researchers and clinicians use to achieve the goals of gene therapy and gene immunization. In gene therapy, viral and non-viral vectors are used to deliver an expressible gene into a host to replace a missing or defective gene, or to otherwise supply the host with a therapeutically beneficial polypeptide. In gene immunization, mostly non-viral vectors are used to induce an immune response by the host to an expressed antigen.

One of the obstacles to successful clinical practice of both gene therapy and gene immunization has been the often transient nature of the gene expression achieved in vivo.
Transient gene expression is less problematic in gene immunization, where immune responses sufficient for certain immunization schemes may be stimulated by even short-term exposure to expressed antigen. In addition, several options are available to boost the host immune response to antigen, including use of the vector itself as an adjuvant for the desired immune response by exposing the host to non-coding, immunostimulatory nucleotide sequences (ISS-ODN) present in the vector (Sato, et al., Science, 273:352-354 (1996)).

WO 98r55609 PCT. i:598/11391 However, in a gene therapy protocol, premature loss of gene expression deprives the host of the potential benefits of the therapy (Friedmann, Scientific American, "Making Gene Therapy Work" (June 1997)). Repetitive dosing to extend exposure of the host to a therapeutic polypeptide can require that different vectors be used to deliver each dose so the host immune response to vector antigens is minimized (Tripathy, et al., Nature Med., 2:545-550 (1996)).

One potential source of vector immunogenicity are ISS-ODN in the genome of the microbial species used to construct recombinant expression vectors. To explain, the CpG
motifs which characterize ISS-ODN are present in bacteria and viruses (including retroviruses) at a much greater frequency than is seen in vertebrate genomes.
One consequence of ISS-ODN activity is the ISS-ODN induced production of cvtokines such as interferon-a (INF-a), INF-y and interleukin-12 (IL-12). This ISS-ODN
induced inflammation is believed to be defensive against microbial infection in vertebrates and is also believed to be produced in response to ISS-ODN introduced into a host as oligonucleotides or as part of a recombinant expression vector.

SUMMARY OF THE INVENTION

The invention provides compounds consisting of oligodeoxynucleotides, ribonucleotides or analogs thereof which specifically inhibit the immunostimulatory activity of ISS-ODN.

ISS-ODN induced secretion of INF-a in particular can suppress recombinant gene expression and directly impedes mRNA and protein synthesis in transfected cells. Thus, inhibition of ISS-ODN activity substantially avoids ISS-ODN induced loss of gene expression, thereby prolonging the availability of the expressed polypeptide to a host undergoing gene therapy or gene immunization with an ISS-ODN containing recombi-nant expression vector. Further, the need for repetitive dosing to prolong availability of expressed proteins and for extensive reengineering of recombinant expression vectors to eliminate ISS-ODN sequences is avoided through use of the compounds of the invention.

The compounds of the invention are also useful in modulating the immunostimulatory activity of ISS-ODN administered as adjuvants to boost host immune responses to antigen in, for example, immunotherapy. In this respect, the compounds of the invention permit exquisite control over the effect of ISS-ODN based adjuvants in a host.

Further, the compounds of the invention reduce host inflammation generated in response to an infection by an ISS-ODN containing bacteria or virus. Advantageously, the compounds of the invention can be administered to inhibit ISS-ODN activity exerted by a microbe even if the identity of the particular ISS-ODN present in the microbe is unknown. Thus, the compounds of the invention can be considered to be broad spectrum anti-inflammatory agents.

In one aspect, the ISS-ODN inhibitory compounds of the invention are synthesized oligonucleotides (I-ON) which are comprised of the following general primary structures:

5'-Purine-Purine-[Y]-[Z]-Pyrmidine-Pyrimidine-3' or 5'-Purine-Purine-[Y]-[Z]-Pymlidine-pPyrimidine-3' where Y is any naturally occurring or synthetic nucleotide except cytosine and is preferably guanosine or inosine (for RNA I-ON). In general, Z is any naturally occurring or synthetic nucleotide or repeat of the same nucleotide. Preferably, when Y
is inosine, Z is inosine or one or more guanosine(s). Where Y is guanosine, Z is preferably guanosine or one or more urunethylated cytosine(s). However, when Y is not guanosine or inosine, Z is guanosine or inosine. Most preferably, the 5' purines are the same nucleotide, as are the 3' pyrimidines. For example, where ** is YZ, the 5' purines and 3' pyrunidines may be AA**TT, AG**TT, GA**TT, GG**TT, AA**TC, AG**TC, and so forth. Any sequences present which flank the hexamer core sequence are constructed to substantially match the flanking sequences present in any known ISS-ODN.
Inhibitory I-ON of the invention are prepared in a pharmaceutically acceptable composition for use in a host. I-ON may be mixed into the composition singly, in multiple copies or in a cocktail of different I-ON. Alternatively, the inhibitory I-ON of the invention may be incorporated into a recombinant expression vector. The inhibitory I-ON can also be provided in the form of a kit, comprising inhibitory I-ON and recombinant expression vector constructs which contain, or are susceptible to insertion of, a gene of interest.

A particular advantage of the I-ON of the invention is that they can be used to target ISS-ODN in any ISS-ODN containing recombinant expression vector or microbe, whether or not the nucleotide composition of the vector or microbe is known. Indeed, it is not necessary that the existence, identity or location of ISS-ODN in the vector or microbe be known. If ISS-ODN are not present in the vector or microbe, the I-ON of the invention will simply have no effect. However, if ISS-ODN are present in the vector or microbe, it can be expected that their immunostimulatory activity will be inhibited in a dose-dependent manner by the I-ON even if the specific structure or location of the ISS-ODN in the vector or microbe is not known.

Thus, in another aspect, the invention provides a simple and effective alternative to the arduous task of eliminating ISS-ODN activity from recombinant expression vectors by identifying all ISS-ODN present in the vector and removing them.

Further in this regard, the invention provides methods for screening recombinant expression vectors for the presence of ISS-ODN and for identifying additional inhibitory I-ON. In the former respect, the presence of ISS-ODN in a recombinant expression vector is confirmed by incubating the vector in a population of lymphocytes with an I-ON of known inhibitory activity and comparing the difference, if any, in the level of ISS-induced cytokine production by the lymphocytes before and after I-ON
incubation.

In the latter respect, additional inhibitory I-ON having the characteristics disclosed herein are identified by their ability to inhibit the immunostimulatory activity of a known ISS-containing polynucleotide or recombinant expression vector.

In yet another aspect, the invention further provides a useful anti-inflammatory agent for use in inhibiting the immunostimulatory activity of any ISS-ODN present in an infectious bacterium or virus.

In addition, the invention provides useful means for modulating the immunostimulatory activity of ISS-ODN supplied to a host for immunostimulation (e.g., as an adjuvant).
BRIEF DESCRIPTION OF THE DRAWINGS

FIGURE 1 is a graph which represents in vivo inhibition of ISS-ODN
immunostimulatory activity by inhibitory I-ON of the invention (I-ON DY1019 and DY1041 (having hexamer regions consisting of, respectively, AAGGTT and AAGCTT)).

Lymphocyte proliferation stimulated in a murine model by the ISS-ODN (DY1038, having a hexamer region consisting of AACGTT) was compared in the presence or absence of the I-ON. A decline in measured counts-per-minute (CPM; vertical axis) represents inhibition of ISS-ODN immunostimulatory activity in the Figure.
Dosages for each I-ON tested are shown along the horizontal axis. DY1039 (an ISS-ODN
with the cytosine), DY1040, DY1042 and DY1043 (all with CC dinucleotides in place of the CG dinucleotide of DY1038) served as controls. To confirm the location of potential competition with DY1038, all of the oligonucleotides were identical to DY1038 except for the hexamer regions identified and DY 1043 (an irrelevant sequence control) .
FIGURE 2 is a graph which confirms in vivo dose dependent inhibition of ISS-ODN
immunostimulatory activity by the DY1019 and DY1041 I-ON of the invention.
Lymphocyte proliferation stimulated in a murine model by a different ISS-ODN
than the one tested in the experiment of FIGURE 1(DY1018) was compared in the presence or absence of the I-ON. A decline in measured counts-per-minute (CPM; vertical axis) represents inhibition of ISS-ODN immunostimulatory activity in the Figure.
Dosages for each I-ON tested are shown along the horizontal axis. Inhibitory activity of I-ON
DY1019 and DY-1041 increased with dosage, with the increase in activity of being proportional to the increase in dosage. To confirm the location of potential competition with DY1018, DY1019 and DY1041 are identical to DY1018 except for the hexamer regions identified.

FIGURE 3 is a graph which represents in vivo dose dependent inhibition of ISS-ODN
immunostimulatory activity by several inhibitory I-ON of the invention.
Lymphocyte proliferation stimulated in a murine model by DY1038 was compared in the presence or absence of the I-ON. A decline in measured counts-per-minute (CPM; vertical axis) represents inhibition of ISS-ODN immunostimulatory activity in the Figure.
Dosages for each I-ON tested are shown along the horizontal axis. In descending order, the most inhibitory activity was displayed by I-ON DY1019, DY-1041, DY1048, DY1050 and DY1060 (the latter have hexamer regions consisting of, respectively, AGGGTT, GAGGTC and TTGCAA). DY1039 (an ISS-ODN with the cytosine methylated), DY1040 and DY1043 (the latter with CC dinucleotides in place of the CG
dinucleotide of DY1038) served as controls. To confirm the location of potential competition with DY1038, all of the oligonucleotides were identical to DY1038 except for the hexamer regions identified and DY1043 (an irrelevant sequence control) .

FIGURE 4 is a graph which represents in vivo dose dependent inhibition of ISS-ODN
immunostimulatory activity by inhibitory I-ON of the invention. INF-y production stimulated by DY1018 ISS-ODN in a murine model was compared in the presence or absence of the I-ON. A decline in measured INF-y (vertical axis) represents inhibition of ISS-ODN immunostimulatory activity in the Figure. Dosages for each I-ON
tested are shown along the horizontal axis. Some inhibitory activity was observed for all but one I-ON, with the most activity being displayed by I-ON DY1019 and DY-1041, as well as DY 1042 (having a hexamer region consisting of TTCCTT). The insert separates out the data for inhibition of INF-y production by DY1019. To confirm the location of potential competition with DY1018, all of the oligonucleotides were identical to DY1018 except for the hexamer regions identified and DY 1043 (an irrelevant sequence control) Figure 5 is a graph which represents the adjuvant properties of IIS-ODN, whereby a Th2-type cellular inunune response in antigen (P-galactosidase) inununized mice is induced by coadministration of the antigen and IIS-ODN DY1019 (identified in the Figure as P-gal/M-ODIv'). TH2 responses are represented by IgE levels measured post-boosting. The values obtained are compared to IgE levels measured in mice immunized with antigen and the ISS-ODN composition b-gal/ISS-ODN (5'-AATTCAACGTTCGC-3'), pKISS-3 (a plasmid having three copies of the AACGTT ISS-ODN hexmer in the backbone) and pKISS-0 (a plasmid having no copies of the AACGTT ISS-ODN hexamer in the backbone). as well as mice which received only saline. Potent IgE responses (Th2-type responses) above 1000 CPM were obtained only in the mice which received saline (approximately 1200 CPM at I week post-boosting) and P-gal/M-ODN
(approximately 1750 CPM at I week post-boosting).

------DETAILED DESCRIPTION OF THE INVENTION

A. Activity and Structure of IIS-ON

1. IIS-ONActivity and Screening Assay The IIS-ON of the invention reduce the immunostimulatory effect of ISS-ODN.
Structurally, ISS-ODN are non-coding oligonucleotides 6 mer or greater in length which may include at least one unmethylated CG motif. The relative position of each CG
sequence in ISS-ODN with immunostimulatory activity in certain mammalian species (e.g., rodents) is 5'-CG-3' (i.e., the C is in the 5' position with respect to the G in the 3' position). Many known ISS-ODN flank the CG motif with at least two purine nucleotides (e.g., GA or AA) and at least two pyrimidine nucleotides (e.g., TC
or TT) to enhance the B lymphocyte stimulatory activity of the immunostimulatory polynucleotide see, e.g., Krieg, et al., Nature, 374:546-549, 1995).

Functionally, ISS-ODN enhance the cellular and humoral immune responses in a host, particularly lymphocyte proliferation and the release of cytokines (including IFN) by host monocytes and natural killer (NK) cells. Bacterial DNA contains unmethylated CpG dinucleotides at a frequency of about one per every 16 bases. These dinucleotides are also present in certain viral species, but are notably underrepresented in vertebrate species.

It is believed that the ability of mycobacteria as well as other bacterial and viral species to stimulate lymphocyte proliferation, IL-12-production, tumor necrosis factor (TNF) production, natural killer (NK) cell activity and IFN-y secretion is owed to the presence of ISS-ODN in bacterial and viral DNA see, e.g., Krieg, Trends in Microbiology, 4:73-76 (1996)). In contrast, CpG suppression and methylation in vertebrates may be an evolutionary response to the threat of bacterial and viral infection.
Interestingly, a CpG
containing oligonucleotide comparable to bacterial ISS-ODN has also recently been implicated in the onset and exacerbation of autoimmune disease through an IL-dependent pathway (Segal, et al., J.Immunol., 158:5087 (1997)).

Immuostimulation by synthetic ISS-ODN in vivo occurs by contacting host lymphocytes with, for example, ISS-ODN oligonucleotides, ISS-ODN
oligonucleotide-conjugates and ISS-containing recombinant expression vectors (data regarding the activity of ISS-ODN conjugates and ISS-ODN vectors are set forth in commonly assigned International PCT Publications W098/16247 and WO 97/28259;
- data from which demonstrate ISS-ODN immunostimulatory activity in vivo).
Thus, while native microbial ISS-ODN stimulate the host immune system to respond to infection, synthetic analogs of these ISS-ODN may be useful therapeutically to modulate the host immune response not only to microbial antigens, but also to tumor antigens, allergens and other substances (id.).

Although the invention is not limited by any theory regarding the mechanism of action ofthe IIS-ON, it is believed that they compete with ISS-ODN for binding to the cellular membrane of host lymphocytes. The region of ISS-ODN which confers their immunostimulatory activity is believed to be the 6 mer or greater length of nucleotides which include an unrnethylated dinucleotide (e.g., CpG). Therefore, it is believed that the presence of a region of about 6 mer or greater length having at least one competing dinucleotide (defined as [Y]-[Z] and [Y]-poly[Z] in the formulae set forth below) therein confers ISS-inhibitory activity on the US-ON oÃthe invention.

Thus, the inhibitory compounds of the invention are synthesized oligonucleotides (iIS-ON) which inhibit the immunostimulatory activity of ISS-ODN in vertebrates and vertebrate immune cells.

To identify IIS-ON from a pool of synthesized candidate IIS-ONs, the following steps provide a simple and efficient means of rapidly screening the candidate pool:
a. A population of cultured, antigen stimulated lymphocytes and/or monocytes is contacted with an ISS-ODN to induce lymphocyte proliferation, IFNP, IFN-a, IFN-y, IL-12 and IL-18 cytokine secretion and/or IgG2 antibody production.

b. Any change in the number of lymphocytes, levels of secreted IFNP, IFN-a, IFN-y, IL-12 and IL-18 cytokines, IgGI or IgG2 antibody levels or IgE antibody levels in the cell culture after contact with the ISS-ODN is measured.

c. The cells are contacted with the candidate IIS-ON.

d. Any change in the number of lymphocytes, levels of secreted cytokines, IgG2 antibody levels or IgE antibody levels in the population of cells after contact with the oligonucleotide is measured.

A decline in any of these values (except IgG 1 and IgE antibodies) as compared to the measurements taken in step (2) indicates that the candidate oligonucleotide is an IIS-ON
of the invention; i.e., it inhibits the immunostimulatory activity of ISS-ODN.

Alternatively, a rise in measured levels of IgGI or IgE antibodies is an indirect indicator of a rise in a Th2-type lymphocyte response, indicating that the Th 1 stimulatory activity of ISS-ODN has declined in the presence of the IIS-ODN. Assay techniques suitable for use in performing the steps above are illustrated in the Examples below. In view of the teaching of this disclosure, other assay techniques for measuring changes in ISS-ODN
induced lymphocyte proliferation or cytokine secretion will be apparent to those of ordinary skill in the art.

The screening method can also be used to detect ISS-ODN in a sample of immune cells taken from the host. This aspect of the invention is useful in confirming the presence of ISS-ODN containing antigens (e.g., microbial antigens) and autoantigens in the host. To this end, the steps of the above-described screening method are modified to include the steps of:

a. Obtaining a sample of immune cells from the host, which cells are believed to been exposed to an antigen or autoantigen.

b. Measuring the levels of lymphocyte proliferation in; IFNP, IFN-a, IFN-y, IL-12 and IL-18 cytokine secretion from; IgGl and IgG2 antibody production by; or IgE antibody production by, the sample of host immune cells.

c. Contacting the sample of host immune cells with an IIS-ON.

d. Measuring any change in the number of lymphocytes or levels of secreted IFNP, IFN-a, IFN-y, IL-12 and IL-18 cytokines and/or levels of IgE or IgG1 antibodies in the sample of host immune cells after contact with the IIS-ON, wherein a decline in any of the measured values for lymphocyte proliferation, cytokine secretion or IgG2 antibody production, as well as an increase in IgGI and IgE
antibody production, as compared to the measurements taken in step (b) indicates that an ISS-ODN subject to inhibition by the IIS-ON is present in the sample of host immune cells.

2. Exemplary IIS' ON Structure Particular IIS-ON which inhibit the activity of CpG motif-containing ISS-ODN
include those oligonucleotides which are comprised of the following general primary structure:

5'-Purine-Purine-[Y]-[Z]-Pyrmidine-Pyrimidine-3' or '-Purine-Purine-[Y] -[Z] -Pyrimidine-polyPyrimidine-3' 5 where Y is any naturally occurring or synthetic nucleotide except cytosine and is preferably guanosine, adenosine or inosine (for RNA IIS-ON), most preferably guanosine. In general, Z is any naturally occurring or synthetic nucleotide or repeat of the same nucleotide. Preferably, where Y is inosine, Z is inosine or one or more guanosine(s). Where Y is guanosine, Z is preferably guanosine or one or more unmethylated cytosine(s). Where Y is adenosine, Z is preferably guanosine.
However, when Y is not guanosine, adenosine or inosine, Z is guanosine, adenosine or inosine.
Most preferably, the 5' purines are the same nucleotide, as are the 3' pyrimidines. For example, where ** is YZ, the 5' purines and 3' pyrimidines may be AA**TT, AG**TT, GA**TT, GG**TT, AA**TC, AG**TC, and so forth.

The core hexamer structure of the foregoing IIS-ON may be flanked upstream and/or downstream by any number or composition of nucleotides or nucleosides.
However, IIS-ON will preferably be either 6 mer in length, or between 6 and 45 mer in length, to enhance uptake of the IIS-ON and to minimize non-specific interactions between the IIS-ON and the target recombinant expression vector or host cells. Preferably, any IIS-ON
flanking sequences present are constructed to match the flanking sequences present in any known ISS-ODN (such as the flanking sequence DY1038 (TTGACTGTG* * * * * *AGAGATGA), where * * * * * * is the immunostimulatory hexamer sequence. Those of ordinary skill in the art will be familiar with, or can readily identify, reported nucleotide sequences of known ISS-ODN . For ease of reference in this regard, the following sources are especially helpful:

Yaniamoto, et al., Microbiol.Imniunol., 36:983 (1992) Ballas, et al., J.Immunol., 157:1840 (1996) Klinman, et al., J.Immunol., 158:3635 (1997) Sato, et al., Science, 273:352 (1996) Particular inhibitory US-ON of the invention include those having the following hexamer sequences:

1. IIS-ODN having "GG" dinucleotides: AAGGTT, AGGGTT, GGGGTT, GGGGTC, AAGGTC, AAGGCC, AGGGTT, AGGGTC, GAGGTT, GAGGTC, GAGGCC, GGGGCT and so forth_ 2. IIS-ODN having "GC" dinucleotides: AAGCTT, AGGCTC, AGGCCC, GAGCTT, GAGCTC, GAGCCC, GGGCTT, GGGCTC, GGGCCC, AAGCCC, AAGCCT, AGGCCT, GAGCCT and so forth.

3. Inosine and/or adenosine subsitutions for nucleotides in the foregoing hexamer sequences made according to the formulae set forth above.
IIS-ON hexamers with especially strong expected inhibitory activity are those with GG
and GC competing dinucleotides, particularly AAGGTT (DY1019 in the Figures), AAGCTT (DY1041 in the Figures), AGGGCT, and GAGGTT (including their 3' Pyrimidine-pPyrimidine analogs).

IIS-ON may be single-stranded or double-stranded DNA, single or double-stranded RNA
and/or oligonucleosides. The nucleotide bases of the IIS-ON which flank the competing dinucleotides may be the known naturally occurring bases or synthetic non-natural bases (e.g., TCAG or, in RNA, UACGI). Oligonucleosides may be incorporated into the internal region and/or termini of the IIS-ON using conventional techniques for use as attachment points for other compounds (e.g., peptides). The base(s), sugar moiety, phosphate groups and termini of the IIS-ON may also be modified in any manner known to those of ordinary skill in the art to construct an IIS-ON having properties desired in addition to the inhibitory activity of the IIS-ON. For example, sugar moieties may be attached to nucleotide bases of IIS-ON in any steric configuration. In addition, backbone phosphate group modifications (e.g., methylphosphonate, phosphorothioate, phosphoroamidate and phosphorodithioate internucleotide linkages) can confer anti-microbial activity on the IIS-ON, making them particuarly useful in therapeutic applications.

The techniques for making these phosphate group modifications to oligonucleotides are known in the art and do not require detailed explanation. For review of one such useful technique, the an intermediate phosphate triester for the target oligonucleotide product is prepared and oxidized to the naturally occurring phosphate triester with aqueous iodine or with other agents, such as anhydrous 'amines. The resulting oligonucleotide phosphoramidates can be treated with sulfer to yield phophorothioates. The same general technique (excepting the sulfer treatment step) can be applied to yield methylphosphoamidites from methylphosphonates. For more details conceming phosphate group modification techniques, those of ordinary skill in the art may wish to.
consult U.S. Patent Nos. 4,425,732; 4,458,066; 5,218,103 and 5,453,496, as well as Tetrahedron Lett. at 21:4149 (1995), 7:5575 (1986), 25:1437 (1984) and Journal Am.ChemSoc., 93:6657 (1987), illustrating the standard level of knowledge in the art concerning preparation of these compounds.

A particularly useful phosphate group modification is the conversion to the phosphorothioate or phosphorodithioate forms of the IIS-ON oligonucleotides.
In addition to their potentially anti-microbial properties, phosphorothioates and phosphorodithioates are more resistant to degradation in vivo than their unmodified oligonucleotide counterparts, making the IIS-ON of the invention more available to the host.

IIS-ON can be synthesized using techniques and nucleic acid synthesis equipment which are well-known in the art. For reference in this regard, see, e.g., Ausubel, et al., Current Protocols in Molecular Biology, Chs. 2 and 4 (Wiley Interscience, 1989);
Maniatis, et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Lab., New York, 1982); U.S. Patent No. 4,458,066 and U.S. Patent No. 4,650,675.

Alternatively, IIS-ON can be obtained by mutation of isolated microbial ISS-ODN to substitute a competing dinucleotide for the naturally occurring CpG motif.
Screening procedures which rely on nucleic acid hybridization make it possible to isolate any polynucleotide sequence from any organism, provided the appropriate probe or antibody is available. Oligonucleotide probes, which correspond to a part of the sequence encoding the protein in question, can be synthesized chemically. This requires that short, oligo- peptide stretches of amino acid sequence must be known. The DNA
sequence encoding the protein can also be deduced from the genetic code, however, the degeneracy of the code must be taken into account.

For example, a cDNA library believed to contain an ISS-containing polynucleotide of interest can be screened by injecting various mRNA derived from cDNAs into oocytes, allowing sufficient time for expression of the cDNA gene products to occur, and testing for the presence of the desired cDNA expression product, for example, by using antibody specific for a peptide encoded by the polynucleotide of interest or by using probes for the repeat motifs and a tissue expression pattern characteristic of a peptide encoded by the polynucelotide of interest. Alternatively, a cDNA library can be screened indirectly for expression of peptides of interest having at least one epitope using antibodies specific for ...~_... .

WO 98/55609 PCT/Ã1S98/1; 91 - 1b-the peptides. Such antibodies can be either polyclonally or monoclonally derived and used to detect expression product indicative of the presence of cDNA of interest.

Once the ISS-containing polynucleotide has been obtained, it can be shortened to the desired length by, for example, enzymatic digestion using conventional techniques. The CpG motif in the ISS-ODN oligonucleotide product is then mutated to substitute a competing dinucleotide for the CpG motif. Techniques for making substitution mutations at particular sites in DNA having a known sequence are well known, for example primer mutagenesis through PCR. Because the IIS-ON is non-coding, there is no concern about maintaining an open reading frame in making the substitution mutation.
However, for in vivo use, the polynucleotide starting material, ISS-ODN oligonucleotide intermediate or IIS-ON mutation product should be rendered substantially pure (i.e., as free of naturally occurring contaminants and LPS as is possible using available techniques known to and chosen by one of ordinary skill in the art).

The US-ON of the invention may be used alone or may be incorporated in cis or in trans into a recombinant expression vector (plasmid, cosmid, virus or retrovirus) which may in turn code for any therapeutically beneficial protein deliverable by a recombinant expression vector. For the sake of convenience, the IIS-ON are preferably administered without incorporation into an expression vector. However, if incorporation into an expression vector is desired, such incorporation may be accomplished using conventional techniques which do not require detailed explanation to one of ordinary skill in the art.
For review, however, those of ordinary skill may wish to consult Ausubel, Current Protocols in Molecular Biology, supra.

Briefly, construction of recombinant expression vectors employs standard ligation techniques. For analysis to confirm correct sequences in vectors constructed, the ligation mixtures may be used to transform a host cell and successful transformants selected by antibiotic resistance where appropriate. Vectors from the transformants are prepared, analyzed by restriction and/or sequenced by, for example, the method of Messing, et al., WO 98/55609 PCT/US9s. 1391 (Nucleic Acids Res., 9:309, 198 i), the method of Maxam, et al., (Methods in Enzymolo-gy, 65:499, 1980), or other suitable methods which will be known to those skilled in the art. Size separation of cleaved fragments is performed using conventional gel electrophoresis as described, for example, by Maniatis, et al., (Molecular Cloning, pp.
133-134, 1982).

Host cells may be transformed with the expression vectors of this invention and cultured in conventional nutrient media modified as is appropriate for inducing promoters, selecting transformants or amplifying genes. The culture conditions, such as tempera-ture, pH and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.

If a recombinant expression vector is utilized as a carrier for the IIS-ON of the invention, plasmids and cosmids are particularly preferred for their lack of pathogenicity. However, plasmids and cosmids are subject to degradation in vivo more quickly than viruses and therefore may not deliver an adequate dosage of IIS-ON to substantially inhibit ISS-ODN
immunostimulatory activity exerted by a systemically administered gene therapy vector.
Of the viral vector alternatives, adeno-associated viruses would possess the advantage of low pathogenicity. The relatively low capacity of adeno-associated viruses for insertion of foreign genes would pose no problem in this context due to the relatively small size in which IIS-ON of the invention can be synthesized.

Other viral vectors that can be utilized in the invention include adenovirus, adeno-associated virus, herpes virus, vaccinia or an RNA virus such as a retrovirus.
Retroviral vectors are preferably derivatives of a murine, avian or human HIV retrovirus.
Examples of retroviral vectors in which a single foreign gene can be inserted include, but are not limited to: Moloney murine leukemia virus (MoMuLV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), and Rous Sarcoma Virus (RSV).
A number of additional retroviral vectors cart incorporate multiple genes. All of these vectors can transfer or incorporate a gene for a selectable marker so that transduced cells can be identified and generated.

Since recombinant retroviruses are defective, they require assistance in order to produce infectious vector particles. This assistance can be provided, for example, by using helper cell lines that contain plasmids encoding all of the structural genes of the retrovirus under the control of regulatory sequences within the LTR. These plasmids are missing a nucleotide sequence that enables the packaging mechanism to recognize an RNA
transcript for encapsidation. Helper cell lines that have deletions of the packaging signal include, but are not limited to, T2, PA317 and PA 12, for example. These cell lines produce empty virions, since no genome is packaged. If a retroviral vector is introduced into such helper cells in which the packaging signal is intact, but the structural genes are replaced by other genes of interest, the vector can be packaged and vector virion can be produced.

By inserting one or more sequences of interest into the viral vector, along with another gene which encodes the ligand for a receptor on a specific target cell, for example, the vector can be rendered target specific. Retroviral vectors can be made target specific by inserting, for example, a polynucleotide encoding a sugar, a glycolipid, or a protein.
Preferred targeting is accomplished by using an antibody to target the retroviral vector.
Those of skill in the art will know of, or can readily ascertain without undue experimentation, specific polynucleotide sequences which can be inserted into the retroviral genome to allow target specific delivery of the retroviral vector containing the polynucleotides of interest.

Altematively, a colloidal dispersion system may be used for targeted delivery.
Colloidal dispersion systems include macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. The preferred colloidal system of this invention is a liposome.

WO 98/55609 ' "'T/US98/11391 Liposomes are artificial membrane vesicles which are useful as delivery vehicles in vitro and in vivo. It has been shown that large unilamellar vesicles (LUV), which range in size from 0.2-4.0 um can encapsulate a substantial percentage of an aqueous buffer containing large macromolecules. RNA, DNA and intact virions can be encapsulated within the aqueous interior and be delivered to cells in a biologically active form (Fraley, et al., Trends Biochem. Sci., 6:77, 1981). In addition to mammalian cells, liposomes have been used for delivery of polynucleotides in plant, yeast and bacterial cells. In order for a liposome to be an efficient gene transfer vehicle, the following characteristics should be present: (1) encapsulation of the genes encoding the antisense polynucleotides at high efficiency while not compromising their biological activity; (2) preferential and substantial binding to a target cell in comparison to non-target cells; (3) delivery of the aqueous contents of the vesicle to the target cell cytoplasm at high efficiency; and (4) accurate and effective expression of genetic information (Mannino, et al., Biotechniques, 6:682, 1988).

The composition of the liposome is usually a combination of phospholipids, particularly high-phase-transition-temperature phospholipids, usually in combination with steroids, especially cholesterol. Other phospholipids or other lipids may also be used.
The physical characteristics of liposomes depend on pH, ionic strength, and the presence of divalent cations.

Examples of lipids useful in liposome production include phosphatidyl compounds, such as phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidyletha-nolamine, sphingolipids, cerebrosides, and gangliosides. Particularly useful are diacylphosphatidylglycerols, where the lipid moiety contains from 14-18 carbon atoms, particularly from 16-18 carbon atoms, and is saturated. Illustrative phospholipids include egg phosphatidylcholine, dipalmitoylphosphatidylcholine and distearoylphos-phatidylcholine.

The targeting of liposomes can be classified based on anatomical and mechanistic factors. Anatomical classification is based on the level of selectivity, for example, organ-specific, cell-specific, and organelle-specific. Mechanistic targeting can be distinguished based upon whether it is passive or active. Passive targeting utilizes the natural tendency of liposomes to distribute to cells of the reticulo-endothelial system (RES) in organs which contain sinusoidal capillaries. Active targeting, on the other hand, involves alteration of the liposome by coupling the liposome to a specific ligand such as a monoclonal antibody, sugar, glycolipid, or protein, or by changing the composition or size of the liposome in order to achieve targeting to organs and cell types other than the naturally occurring sites of localization.

The surface of the targeted delivery system may be modified in a variety of ways. In the case of a liposomal targeted delivery system, lipid groups can be incorporated into the lipid bilayer of the liposome in order to maintain the targeting ligand in stable association with the liposomal bilayer. Various well known linking groups can be used for joining the lipid chains to the targeting ligand (see, e.g., Yanagawa, et al., Nuc.Acids Symp.Ser., 19:189 (1988); Grabarek, et al., Ar1: Biochem., 185:131 (1990); Staros, et al., Anal.Biochem., 156:220 (1986) and Bcet al., Proc.Natl.Acad.Sci. USA, 90:5728 (1993), the disclosures of which illustrate the standard level of knowledge in the art concerning conjugation of oligonucleotides to lipids).

Targeted delivery of US-ON can also be achieved by conjugation of the US-ON to a the surface of viral and non-viral recombinant expression vectors, to an antigen or other ligand, to a monoclonal antibody or to any molecule which has the desired binding specificity. A particular IIS-ODN conjugate of interest is one in which an autoantigen or autoantibody is the IIS-ODN conjugate partner. IIS-ODN autoantigen conjugates are useful in boosting host Th2 type immune responses to the autoantigen (suppressing the Thl responses induced by the autoantigen itself; see, e.g., Conboy, et al., J.Ezp.Med., 185:439-451 (1997)), while IIS-ODN autoantibody conjugates are useful in inducing passive immunity in a host suffering from an autoimmune condition. Specific methods for delivery of such conjugates, as well as IIS-ON in general, are described in greater detail infra.

Those of ordinary skill in the art will be familiar with, or can readily determine, sources for autoantigens and autoantibodies useful as IIS-ON conjugates. Examples of such conjugate materials include myelin basic piotein (see, e.g., sequence and sourcing information provided in Segal, et al., J.lmmunol., 158:5087 (1997); Matsuo, et al., Am.J.Pathol., 150:1253 (1997); and Schluesener, FEMS Immunol.Med.Microbiol., 17:179 (1997)); Sjorgen's syndrome autoantigen see, e.g., Hanjei, et al., Science, 276:604 (1997)); hemochromatosis autoantigen (see, e.g., Ruddy, et al., Genome Res., 7:441 (1997)), La/SSB protein (see, e.g., Castro, et al., Cell Calcium, 20:493 (1996));
HsEg5 lupus autoantigen see, e.g., Whitehead, et al., Arthritis Rheuni., 39:1635 (1996));
Ki nuclear lupus autoantigen see, e.g., Paesen and Nuttal, Biochem.Biophys.Acta, 1309:9 (1996)); and antibodies thereto (see, e.g., Menon, et al., J.Autoimmun., 10:43 (1997) and Rahman, et al., Semin.Arthritis Rheum., 26:515 (1996) [human antiphospholipid (anti-DNA) monoclonal antibodies]; and, Kramers, et al., JAutoimmun., 9:723 (1997) [monoclonal anti-nucleosome lupus autoantibodies]). Each of the cited references illustrate the level of knowledge and skill in the art concerning the identity, activity and structure of autoantigens and autoantibodies.

Examples of other useful conjugate partners include any immunogenic antigen (including allergens, live and-attenuated viral particles and tumor antigens), targeting peptides (such as receptor ligands, antibodies and antibody fragments, hormones and enzymes), non-peptidic antigens (coupled via a peptide linkage, such as lipids, polysaccharides, glycoproteins, gangliosides and the like) and cytokines (including interleukins, interferons, erythorpoietin, tumor necrosis factor and colony stimulating factors). Such conjugate partners can be prepared according to conventional techniques (e.g., peptide synthesis) and many are commercially available.

Those of ordinary skill in the art will also be familiar with, or can readily determine, methods useful in preparing oligonucleotide-peptide conjugates. Conjugation can be accomplished at either termini of the IIS-ON or at a suitably modified base in an internal position (e.g., a cytosine or uracil). For reference, methods for conjugating oligonucleotides to proteins and to oligosaccharide moieties of Ig are known (see, e.g., O'Shannessy, et al., J.Applied Biochem., 7:347 (1985), the disclosure of which illustrates the standard level of knowledge in the art concerning oligonucleotide conjugation). Another useful reference is Kessler: "Nonradioactive Labeling Methods for Nucleic Acids", in Kricka (ed.) Nonisotopic DNA Probe Techniques (Acad.
Press, 1992)).

Briefly, examples of known, suitable conjugation methods include: conjugation through 3' attachment via solid support chemistry s e e.g., Haralambidis, et al., Nuc.Acids Res., 18:493 (1990) and Haralambidis, et al., Nuc.Acids Res., 18:501 (1990) [solid support synthesis of peptide partner]; Zuckermann, et al., Nuc.Acids Res., 15:5305 (1987), Corey, et al., Science, 238:1401 (1987) and Nelson, et al., Nuc. Acids Res., 17:1781 (1989) [solid support synthesis of oligonucleotide partner]). Amino-amino group linkages may be performed as described in Benoit, et al., Neuromethods, 6:43 (1987), while thiol-carboxyl group linkages may be performed as described in Sinah, et al., Oligonucleotide Analogues:A Practical Approach (IRL Press, 1991). In these latter methods, the oligonucleotide partner is synthesized on a solid support and a linking group comprising a protected amine, thiol or carboxyl group opposite a phosphoramidite is covalently attached to the 5'-hydroxyl see e.g., U.S. Patent Nos. 4,849,513; 5,015,733;
5,118,800 and 5,118,802).

Linkage of the oligonucleotide partner to a peptide may also be made via incorporation of a linker arm (e.g., amine or carboxyl group) to a modified cytosine or uracil base see e.g., Ruth, 4th Annual Congress for Recombinant DNA Research at 123). Affinity linkages (e.g., biotin-streptavidin) may also be used see e.g., Roget, et al., Nuc.Acids Res., 17:7643 (1989)).

Methods for linking oligonucleotides to lipids are also known and include synthesis of oligo-phospholipid conjugates (see, e.g., Yanagawa, et al., Nuc. Acids Symp.Ser., 19:189 (1988)), synthesis of oligo-fatty acids conjugates (see, e.g., Grabarek, et al., Anal. Biochem., 185:131 (1990)) and oligo-sterol conjugates (see, e.g., Boujrad, et al., Proc.Natl.Acad.Sci USA, 90:5728 (1993)).

Each of the foregoing references _ illustrate the level of knowledge and skill in the art with respect to oligonucleotide conjugation methods.

If to be delivered without use of a vector or other delivery system, IIS-ON
will be prepared in a pharmaceutically acceptable composition. Pharmaceutically acceptable carriers preferred for use with the IIS-ON of the invention may include sterile aqueous of non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/
aqueous solutions, emulsions or suspensions, including saline and buffered media.
TM
Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils. Intravenous vehicles include fluid and TM
nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, antioxidants, chelating agents, and inert gases and the like.
A composi-tion of IIS-ON may also be lyophilized using means well known in the art, for subsequent reconstitution and use according to the invention.

B. Methods for Administering and Using IIS-ON of the Invention The IIS-ON of the invention are useful in inhibiting the immunostimulatory activity of ISS, wherever present. Thus, IIS-ON are useful as, for example, anti-inflammatory agents for reducing host immune responses to ISS-ODN in bacteria and viruses.
IIS-ON

_,_ ._.,_._...~.:,~.._ ...... _ _...........3,.,~..w..~._ . ... . .....
.......,.~..._._,,_..,.~........1...,~..,~w....._.,.-.~.__..,~..

WO 98/55609 PCTlUS98/11391 are also useful as agents for suppressing the immunostimulatory activity of any ISS-ODN, known or unknown, present in recombinant expression vectors, especially those used for gene therapy and immunization. In addition, IIS-ODN are useful in inhibiting host autoimmune responses stimulated by microbial ISS-ODN and in boosting Th2 type responses to antigen.

In this context, "inhibition" refers to a reduction in the host immune response as compared to the level of ISS-ODN stimulated host immune response prior to IIS-ODN
administration. Because ISS-ODN stimulate secretion of certain cytokines (e.g., IL-12, IL-18 and IFNs) and tend to shift the host cellular immune response to the Thl repertoire, measurements of cytokine levels, cytokine-stimulated lymphocyte prolifera-tion, IgG2 antibody levels (the production of which is indicative of a Thl lymphocyte response), IgE levels (the suppression of which is indicative of a Thl lymphocyte response) and IgGl antibody levels (the production of which is indicative of a Th2 lymphocyte response) are all suitable values for use in detecting IIS-ODN
inhibitory activity. Specific examples and details of methods for determining such values are described further infra.

With respect to shifts in the Thl/Th2 repertrtoire and consequent changes in cytokine levels, it is helpful to recall that CD4+ lymphocytes generally fall into one of two distinct subsets; i.e., the Thi and Th2 cells. Thl cells principally secrete IL-2, IFNy and TNFP (the latter two of which mediate macrophage activation and delayed type hypersensitivity) while Th2 cells principally secrete IL-4 (which stimulates production of IgE antibodies), IL-5, IL-6 and IL-10. These CD4+ subsets exert a negative influence on one another; i.e., secretion of Thl lymphokines inhibits secretion of Th2 lymphokines and vice versa. In addition, it is believed that exposure of Th2 cells to cytotoxic T
lymphocytes (CTLs) also suppresses TH2 cell activity.

Factors believed to favor Thl activation resemble those induced by viral infection and include intracellular pathogens, exposure to IFN-(3, IFN-a, IFNy, IL-12 and IL-18, as well as the presence of APCs and exposure to low doses of antigen. Thl type immune responses also predominate in autoimmune disease. Factors believed to favor Th2 activation include exposure to IL-4 and IL- 10, APC activity on the part of B
lymphocytes and high doses of antigen. Active Thl (IFNy) cells enhance cellular immunity and are therefore of particular value in responding to intracellular infections, while active Th2 cells enhance antibody production and are therefore of value in responding to extracellular infections (albeit at the risk of anaphylactic events associated with IL-4 stimulated induction of IgE antibody production). Thus, the ability to shift host immune responses from the Thl to the Th2 repertoire and vice versa has substantial clinical significance for enhancing and controlling host immunity against infection and allergy.
Further, control over Thl/Th2 mediated cytokine release enables one to control host immune responses to self-antigens (having clinical significance for treatment of autoimmune disease) and to recombinant expression vector antigens (having clinical significance for control of gene expression for gene therapy and gene immunization).

For use in modulating the immunogenicity of a recombinant expression vector, the IIS-ON of the invention will be administered according to any means and route by which the target recombinant expression vector is administered to a host, including in vivo and ex vivo routes. Uptake of IIS-ON by host cells occurs at least as robustly as does uptake of therapy and immunization vectors, if not more so due to the small size of IIS-ON as compared to the total dimensions of plasmid, viral and retroviral nucleic acids.

A particular goal of IIS-ON administration in, this context is the inhibition of ISS-ODN
stimulated, Tlil mediated cytokine production. Thus, a measurable reduction of such cytokine levels in a treated host constitutes IIS-ON therapeutic activity in this embodiment of the invention. IIS-ON therapeutic activity is also demonstrated in this context by prolongation of gene expression as compared to expression levels obtained in the absence of IIS-ON. Those of ordinary skill in the gene therapy and immunization arts will be very familiar with, or can readily ascertain, clinically acceptable means and routes for administration of therapy and immunization vectors and, by extension, IIS-ON.

For use as anti-inflammatory agents, IIS-ON and IIS-ON conjugates will be administered according to any means and route by which known anti-inflammatories and antibiotics are administered. A particular goal of IIS-ODN administration in this context is the inhibition of ISS-ODN stimulated, Thl mediated cytokine production. Thus, a measurable reduction of such cytokine levels in a treated host constitutes IIS-ODN
therapeutic activity in this embodiment of the invention. Those of ordinary skill in the art of treating infectious disease will be very familiar with, or can retLdily ascertain, clinically acceptable means and routes for administration of anti-inflammatories and antibiotics and, by extension, US-ON and their conjugates.

For use as autoimmune modulators, IIS-ON and IIS-ON autoantigen or autoantibody conjugates will be administered according to any means and route by which known therapies for autoimmune disease are practiced. A particular goal of IIS-ODN
administration in this context is the inhibition Qf ISS-ODN stimulated, Thl mediated IL-12 production. Thus, a measurable reduction of IL-12 levels in an autoimmune host constitutes IIS-ODN therapeutic activity in this embodiment of the invention.
Those of ordinary skill in the art of treating autoimmune disease will be very familiar with, or can readily ascertain, clinically acceptable means and routes for administration of US-ON
and their conjugates.

For use as modulators of ISS-ODN administered as immunostimulants, the IIS-ON
and IIS-ON conjugates of the invention will be administered according to any means and route by which the target ISS-ODN is administered to a host, including in vivo and ex vivo routes. For example, where ISS-ODN are administered as adjuvants in an immunization protocol see, commonly assigned International PCT Publications W098/16247 and WO 97/28259), it may be desirable to be able to subsequently reduce or eliminate the ISS-ODN immunostimulatory activity to modify the course of therapy.
In this context, therefore, IIS-ON serve as ISS-ODN "off' switches, whereby IIS-ON
and IIS-ON conjugate activity is demonstrated by a measured reduction in ISS-ODN stimulated cytokine production, ISS-ODN stimulated lymphocyte production, or a shift away from the Thl lymphocyte repertoire.

For use as adjuvants for Th2 immune responses to extracellular antigen, the IIS-ON of the invention will be administered according to any means and route by which antigen-based vaccines may be administered to a host. Shifts away from the Thl lymphocyte repertoire are a measure of efficacy for use of IIS-ON and IIS-ON conjugates as Th2 lymphocyte stimulatory adjuvants in the presence of antigen.

A particular advantage of the IIS-ON of the invention is their capacity to exert an ISS-ODN inhibitory activity even at relatively low dosages. Although the dosage used will vary depending on the clinical goals to be achieved, a suitable dosage range is one which provides up to about 1-200 g of IIS-ON/ml of carrier in a single dosage. In view of the teaching provided by this disclosure, those of ordinary skill in the clinical arts will be familiar with, or can readily ascertain, suitable parameters for administration of IIS-ON according to the invention.

In this respect, the inhibitory activity of IIS-ON is essentially dose-dependent.
Therefore, to increase IIS-ON potency by a magnitude of two, each single dose is doubled in concentration. For use in inhibiting ISS-ODN activity (including activity of ISS-ODN in recombinant expression vectors), it is useful to administer the IIS-ON and target ISS-ODN or vector in equivalent dosages, then increase the dosage of IIS-ON as needed to achieve the desired level of inhibition. For use as an anti-inflammatory agent, it is useful to administer the IIS-ON in a low dosage (e.g., about I g/ml to about 50 g/ml), then increase the dosage as needed to achieve the desired therapeutic goal.
Alternatively, a target dosage of IIS-ON can be considered to be about 1-10 M
in a sample of host blood drawn within the first 24-48 hours after administration of IIS-ON.

To maximize the effectiveness of IIS-ON to inhibit ISS-ODN immunostimulatory activity, the IIS-ON are preferably co-administered with the target ISS-ODN or recombinant expression vector. In addition, IIS-ON may be pre-incubated with the target recombinant expression vector prior to administration to the host to reduce the latter's capacity to present ISS-ODN immunostimulatory activity in the host during treatment in a therapy or immunization regime. For use as an anti-inflammatory, the IIS-ON may be co-administered with, or otherwise taken by a host treated with, other anti-inflamma-tory pharmaceuticals.

To these ends, IIS-ON are conveniently supplied in single dose vials and/or in kits together with suitable dosages of ISS-ODN , recombinant expression vectors or anti-inflammatory agents. In kits including recombinant expression vectors, the IIS-ON and vectors can be pre-mixed in single dosage vials. Means for administering each dosage to a host (e.g., syringes, transdermal patches, iontophoresis devices and inhalers), if required, are included in each kit.

Examples illustrating the immunoinhibitory activity of IIS-ON are set forth below. The examples are for purposes of reference only and should not be construed to limit the invention, which is to be defined by the appended claims. All abbreviations and terms used in the examples have their expected and ordinary meaning unless otherwise specified.

EXAMPLE I
ASSAY TO CONFIRM IIS-ON INHIBITORY ACTIVITY
AS MEASURED BY A REDUCTION IN LYMPHOCYTE PROLIFERATION
Splenocytes from immunologically naive female Balb/c mice (6-8 weeks of age) were harvested from each animal. Supematants of the harvested splenocytes were incubated with 1 g/ml of the DY1018 ISS-ODN or 1 g/ml of the DY1038 ISS-ODN in normal saline (all oligonucleotide sequences are set forth in the legend to the FIGURES and in the Description of Drawings). The backbones of both DY1018 and DY1038 were modified as phosphorothioates. In this context, the ISS-ODN served as non-specific adjuvants for in vitro stimulation of the immune system.

Within 4 hours of ISS-ODN contact, the supematants were incubated with various concentrations of IIS-ON or a control. DY1039 (an ISS with the cytosine methylated), DY1040 and DY 1043 (the latter with CC dinucleotides in place of the CG
dinucleotide of DY1018 and DY1038) served as controls. To confirm the location of potential competition with DY1018 and DY1038, all of the oligonucleotides were identical to DY 103 8 (FIGURES 1 and 3) or DY 1018 (FIGURE 2) except for the hexamer regions identified in the FIGURES and DY 1043 (an irrelevant sequence control).

Lymphocyte proliferation pre- and post-IIS-ODN administration was measured (as a function of counts per minute) using conventional assay techniques. Any observable changes in lymphocyte proliferation among the supematants were noted. Values shown in FIGURES 1 through 3 are averages for each group of mice tested.

The results of these assays are shown in FIGURES 1 through 3. With respect to both DY1038 (FIGURES 1 and 3) and DY1018 (FIGURE 2), the strongest inhibition of ISS
immunostimulatory activity by inhibitory IIS-ON of the invention in these experiments was demonstrated by IIS-ON DY1019 (having a hexamer region consisting of AAGGTT). Other strongly inhibitory IIS-ON tested were DY1048 (hexamer re-gion=GAGGTC), DY1050 (hexamer region=AGGGCT), DY1060 (hexamer re-gion=TTGCAA) and DY1041 (hexamer region=AAGCTT) (FIGURE 3). Inhibitory strength was dose-dependent in a generally proportional relationship of dosage to reduction in lymphocyte proliferation measured.

EXAMPLE II
ASSAY TO CONFIRM IIS-ON INHIBITORY ACTIVITY
AS MEASURED BY A REDUCTION IN INF-y SECRETION

Groups of mice were immunized as described in Example I, sacrificied and their splenocytes harvested. Supematants of harvested splenocytes was incubated with I
g/ml of DY1018 ISS-ODN in saline as described in Example I. Within 4 hours, the supematants were incubated with various concentrations of IIS-ON or a control.
DY1039 (an ISS with the cytosine methylated), DY1040 and DY1043 (the latter with CC
dinucleotides in place of the CG dinucleotide of DY1018) served as controls (all oligonucleotide sequences are set forth in the legend to the FIGURES and in the Description of Drawings). To confirm the location of potential competition with DY 1018, all of the oligonucleotides were identical to DY 1018 except for the hexamer regions identified and DY 1043 (an irrelevant sequence control).

IFN-y levels were measured pre- and post- IIS-ODN contact. Any observable changes in IFN-y secretion (pg/mi supematants) among the supematants were noted.
Values shown in FIGURE 4 are averages for each group of mice tested.

The results of these assays are shown in FIGURE 4. Again, the strongest inhibition of ISS immunostimulatory activity by inhibitory IIS-ON of the invention in these experiments was demonstrated by IIS-ON DY 1019 (having a hexamer region consisting of AAGGTT). DY1041 (hexamer region=AAGCTT) was also strongly inhibitory, even at low dosage (1 g/mi saline). At a higher dosage (10 g/ml), INF-y levels began to decline in control mice as well.

EXAMPLE III
IIS-ODN BOOSTING OF Th2 TYPE IMMUNE RESPONSES
TO ANTIGEN

Groups of four Balb/c mice were co-immunized with 10 g (3-galactosidase antigen and 50 g (in 50 l normal saline) of IIS-ODN DY 1019 (identified in the Figure as P-gal/M-ODN), the ISS-ODN composition (3-gal/ISS-ODN (5'-AATTCAACGTTCGC-3'), the (3-TM
gal antigen and pKISS-3 (a plasmid having three copies of the AACGTT ISS-ODN
TM
hexamer in tlie backbone), the (i-gal antigen and pKISS-0 (a control plasmid having no copies of the AACGTT ISS-ODN hexamer in the backbone), or saline alone. Th2 responses in each group of mice were measured by ELISA as a function of IgE
levels obtained post-boosting. As shown in FIGURE 5, potent Th2-type responses (above CPM) were obtained only in the mice which received saline (approximately 1200 CPM
at I week post-boosting) and 0-gal/M-ODN (approximately 1750 CPM at I week post-boosting).

Further, high levels of IgG2a antibodies and low levels of IgG 1 antibodies (Th1 and Th2 type responses, respectively) were induced in response to antigen in the ISS-ODN treated mice, while the opposite responses were obtained in the IIS-ON treated mice, thus showing a shift toward the Th2 repertoire in the latter group.

The invention having been fully described, modifications of the disclosed embodiments may become apparent to those of ordinary skill in the art. All such modifications are considered to be within the scope of the invention, which is defined by the appended claims.

_ ........._.. ._._ .. , ._._.__...,~....~....,.-~-w... ,. __._..-_.
.....,.....,-.~-,~p......_,...........,...._..-._.-.~~.m_.._. ...~...-....._-..,~_.._._....._.. _ SEQUENCE LISTING
(1) GENERAL INFORMATION:

(i) APPLICANT: Raz, Eyal, Roman Mark (ii) TITLE OF INVENTION: Inhibitors of DNA Immunostimulatory Sequence Activity (iii) NUMBER OF SEQUENCES: 11 (iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: MBM & Co.
(B) STREET: P.O. Box 809, Stn. B
(C) CITY: Ottawa (D) Province: ON
(E) COUNTRY: Canada (F) Postal Code: K1P 5P9 (v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: DISKETTE, 3.5 INCH, 1.44 Mb STORAGE
(B) COMPUTER: IBM (emachines) (C) OPERATING SYSTEM: Window XP
(D) SOFTWARE: WORDPERFECT

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER: 2,293,489 (B) FILING DATE: June 5, 1998 (C) CLASSIFICATION: C12N-15/00 (vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: 60/048,794 (B) FILING DATE: 1997-06-06 (A) APPLICATION NUMBER: PCT/US98/11391 (B) FILING DATE: 1998-06-05 (viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: SWAIN, Margaret (B) REGISTRATION NUMBER: 10926 (C) REFERENCE/DOCKET NUMBER: 524-107 (ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: 613-567-0762 (B) TELEFAX: 613-563-7671 - -- -------- ---(2)INFORMATION FOR SEQ ID NO: 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 (B) TYPE: Nucleic Acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (iv) ANTI-SENSE: No (vi) ORIGINAL SOURCE: Artificial sequence (ix) FEATURE:
(D) OTHER INFORMATION: Oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:

(2) INFORMATION FOR SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 (B) TYPE: Nucleic Acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (iv) ANTI-SENSE: No (vi) ORIGINAL SOURCE: Artificial sequence (ix) FEATURE:
(D) OTHER INFORMATION: Oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

(2) INFORMATION FOR SEQ ID NO: 3:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 (B) TYPE: Nucleic Acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (iv) ANTI-SENSE: No (vi) ORIGINAL SOURCE: Artificial sequence (ix) FEATURE:
(D) OTHER INFORMATION: Oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:

(2) INFORMATION FOR SEQ ID NO: 4:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 (B) TYPE: Nucleic Acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (iv) ANTI-SENSE: No (vi) ORIGINAL SOURCE: Artificial sequence (ix) FEATURE:
(D) OTHER INFORMATION: Oligonucleotide (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 4:

(2) INFORMATION FOR SEQ ID NO: 5:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 (B) TYPE: Nucleic Acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (iv) ANTI-SENSE: No (vi) ORIGINAL SOURCE: Artificial sequence (ix) FEATURE:
(D) OTHER INFORMATION: Oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:

(2) INFORMATION FOR SEQ ID NO: 6:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 (B) TYPE: Nucleic Acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (iv) ANTI-SENSE: No (vi) ORIGINAL SOURCE: Artificial sequence (ix) FEATURE:
(D) OTHER INFORMATION: Oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:

(2) INFORMATION FOR SEQ ID NO: 7:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 (B) TYPE: Nucleic Acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (iv) ANTI-SENSE: No (vi) ORIGINAL SOURCE: Artificial sequence (ix) FEATURE:
(D) OTHER INFORMATION: Oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:

(2) INFORMATION FOR SEQ ID NO: 8:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 (B) TYPE: Nucleic Acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (iv) ANTI-SENSE: No (vi) ORIGINAL SOURCE: Artificial sequence (ix) FEATURE:
(D) OTHER INFORMATION: Oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:

(2) INFORMATION FOR SEQ ID NO: 9:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 (B) TYPE: Nucleic Acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (iv) ANTI-SENSE: No (vi) ORIGINAL SOURCE: Artificial sequence (ix) FEATURE:
(D) OTHER INFORMATION: Oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:

(2) INFORMATION FOR SEQ ID NO: 10:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 (B) TYPE: Nucleic Acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (iv) ANTI-SENSE: No (vi) ORIGINAL SOURCE: Artificial sequence (ix) FEATURE:
(D) OTHER INFORMATION: Oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:

(2) INFORMATION FOR SEQ ID NO: 11:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 (B) TYPE: Nucleic Acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (iv) ANTI-SENSE: No (vi) ORIGINAL SOURCE: Artificial sequence (ix) FEATURE:
(D) OTHER INFORMATION: Oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:

Claims (26)

THE EMBODIMENTS OF THE INVENTION FOR WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. A pharmaceutical composition comprising:
a) an immunoinhibitory oligonucleotide which comprises a nucleotide sequence of the formula 5'-Purine-Purine-[Y]-[Z]-Pyrimidine-Pyrimidine-3' or 5' Purine-Purine-[Y]-[Z]-poly(Pyrimidine)-3'; wherein Y is any naturally occurring or synthetic nucleotide other than cytosine and Z is any naturally occurring or synthetic nucleotide and wherein when Y is not guanosine or inosine, Z is guanosine or inosine, wherein the oligonucleotide is present in an amount effective to inhibit immunostimulation by an immunostimulatory sequence oligodeoxynucleotide (ISS-ODN), wherein the ISS-ODN
includes at least one unmethylated 5'-CG-3' motif;
wherein said immunoinhibitory oligonucleotide is other than an oligonucleotide comprising a CpG motif; and b) a pharmaceutically acceptable carrier.
2. The pharmaceutical composition of claim 1 where Y is guanosine or inosine.
3. The pharmaceutical composition of claim 1 or 2 where Y is inosine and Z is inosine or guanosine.
4. The pharmaceutical composition of claim 1 or 2 where Y is guanosine and Z
is guanosine or an unmethylated cytosine.
5. The pharmaceutical composition according to claim 1 wherein said nucleotide sequence is AGGGCT, GAGGTT, AAGCTT, AGGCTC, GAGCTT, GGGCTT, AAGCTC, AGGCTC, GAGCTC, GGGCTC, AAGCCC, AGGCCC, GAGCCC, GGGCCC, AGGCCT, GAGCCT, GGGGCT, AATGTT, GGGGTT, GAGGTC, or AAGCCC.
6. The composition of any one of claims 1 to 5 wherein the oligonucleotide is nucleotides to 45 nucleotides in length.
7. The composition of any one of claims 1 to 6 wherein said oligonucleotide comprises one or more phosphate backbone modifications.
8. The composition of claim 7 wherein the phosphate backbone comprises a phosphorothioate linkage.
9. The pharmaceutical composition of any one of claims 1 to 8 wherein the oligonucleotide is conjugated to a peptide.
10. A kit for use in immunomodulation, comprising a composition according to any one of claims 1 to 9 in a sterile vial.
11. Use of an immunoinhibitory oligonucleotide as defined in any one of claims 1 to 9 in the preparation of a medicament to boost a Th2 type immune response to an antigen in an individual.
12. Use of an effective amount of an immunoinhibitory oligonucleotide as defined in any one of claims 1 to 9 to boost a Th2 type immune response to an antigen in an individual.
13. Use of an immunoinhibitory oligonucleotide as defined in any one of claims 1 to 9 in the preparation of a medicament to reduce Th1-mediated cytokine production in an individual.
14. Use of an effective amount of an immunoinhibitory oligonucleotide as defined in any one of claims 1 to 9 to reduce Th1-mediated cytokine production in an individual.
15. Use according to claim 13 or 14 wherein the Th1-mediated cytokine is IL-12, interferon-.gamma., or tumor necrosis factor-.beta..
16. Use of an immunoinhibitory oligonucleotide as defined in any one of claims 1 to 9 in the preparation of a medicament to reduce a Th1 immune response to an autoantigen.
17. Use of an effective amount of an immunoinhibitory oligonucleotide as defined in any one of claims 1 to 9 to reduce a Th1 immune response to an autoantigen.
18. Use according to claim 16 or 17 wherein the autoantigen is myelin basic protein, Sjogren syndrome autoantigen, hemochromatosis autoantigen, HsEg5 systemic lupus erythematosus (SLE) autoantigen, or Ki nuclear SLE autoantigen.
19. A pharmaceutical composition comprising:
an autoantigen conjugated to an immunoinhibitory nucleic acid oligonucleotide comprising a nucleotide sequence of the formula 5'-Purine-Purine-[Y]-[Z]-Pyrimidine-Pyrimidine-3' or 5'-Purine-Purine-[Y]-[Z]-poly(Pyrimidine)-3', where Y is any naturally occurring or synthetic nucleotide other than cytosine and Z is any naturally occurring or synthetic nucleotide and wherein when Y is not guanosine or inosine, Z is guanosine or inosine; and a pharmaceutically acceptable carrier.
20. The pharmaceutical composition of claim 19 wherein said nucleic acid comprises one or more phosphate backbone modifications.
21. The pharmaceutical composition of claim 20 wherein the phosphate backbone comprises a phosphorothioate linkage.
22. The pharmaceutical composition of claim 19 wherein said nucleotide sequence is AGGGCT, GAGGTT, AAGCTT, AGGCTC, GAGCTT, GGGCTT, AAGCTC, AGGCTC, GAGCTC, GGGCTC, AAGCCC, AGGCCC, GAGCCC, GGGCCC, AGGCCT, GAGCCT, GGGGCT, AATGTT, GGGGTT, GAGGTC, or AAGCCC.
23. The pharmaceutical composition of claim 19 wherein the oligonucleotide is nucleotides to 45 nucleotides in length.
24. Use of the pharmaceutical composition according to claim 19 in the preparation of a medicament to reduce a Th1 immune response to an autoantigen.
25. Use of an effective amount of the pharmaceutical composition according to claim 19 to reduce a Th1 immune response to an autoantigen.
26. Use according to claim 24 or 25 wherein the autoantigen is myelin basic protein, Sjogren syndrome autoantigen, hemochromatosis autoantigen, HsEg5 systemic lupus erythematosus (SLE) autoantigen, or Ki nuclear SLE autoantigen.
CA002293489A 1997-06-06 1998-06-05 Inhibitors of dna immunostimulatory sequence activity Expired - Fee Related CA2293489C (en)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US4879397P 1997-06-06 1997-06-06
US60/048,793 1997-06-06
PCT/US1998/011391 WO1998055609A1 (en) 1997-06-06 1998-06-05 Inhibitors of dna immunostimulatory sequence activity

Publications (2)

Publication Number Publication Date
CA2293489A1 CA2293489A1 (en) 1998-12-10
CA2293489C true CA2293489C (en) 2009-09-29

Family

ID=21956478

Family Applications (2)

Application Number Title Priority Date Filing Date
CA2291483A Expired - Lifetime CA2291483C (en) 1997-06-06 1998-06-05 Immunostimulatory oligonucleotides, compositions thereof and methods of use thereof
CA002293489A Expired - Fee Related CA2293489C (en) 1997-06-06 1998-06-05 Inhibitors of dna immunostimulatory sequence activity

Family Applications Before (1)

Application Number Title Priority Date Filing Date
CA2291483A Expired - Lifetime CA2291483C (en) 1997-06-06 1998-06-05 Immunostimulatory oligonucleotides, compositions thereof and methods of use thereof

Country Status (13)

Country Link
US (4) US6225292B1 (en)
EP (3) EP1003850B1 (en)
JP (2) JP4101888B2 (en)
AT (2) ATE432348T1 (en)
AU (2) AU753172B2 (en)
CA (2) CA2291483C (en)
CY (1) CY1109287T1 (en)
DE (2) DE69819150T3 (en)
DK (1) DK1003850T3 (en)
ES (1) ES2326848T3 (en)
HK (1) HK1024701A1 (en)
PT (1) PT1003850E (en)
WO (2) WO1998055609A1 (en)

Families Citing this family (199)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6727230B1 (en) 1994-03-25 2004-04-27 Coley Pharmaceutical Group, Inc. Immune stimulation by phosphorothioate oligonucleotide analogs
US6239116B1 (en) 1994-07-15 2001-05-29 University Of Iowa Research Foundation Immunostimulatory nucleic acid molecules
US20030026782A1 (en) * 1995-02-07 2003-02-06 Arthur M. Krieg Immunomodulatory oligonucleotides
US7935675B1 (en) * 1994-07-15 2011-05-03 University Of Iowa Research Foundation Immunostimulatory nucleic acid molecules
US6207646B1 (en) 1994-07-15 2001-03-27 University Of Iowa Research Foundation Immunostimulatory nucleic acid molecules
US6429199B1 (en) * 1994-07-15 2002-08-06 University Of Iowa Research Foundation Immunostimulatory nucleic acid molecules for activating dendritic cells
EP0909323B1 (en) 1996-01-04 2007-02-28 Novartis Vaccines and Diagnostics, Inc. Helicobacter pylori bacterioferritin
US6610661B1 (en) 1996-10-11 2003-08-26 The Regents Of The University Of California Immunostimulatory polynucleotide/immunomodulatory molecule conjugates
EP0855184A1 (en) 1997-01-23 1998-07-29 Grayson B. Dr. Lipford Pharmaceutical composition comprising a polynucleotide and an antigen especially for vaccination
AU738513B2 (en) 1997-02-28 2001-09-20 University Of Iowa Research Foundation, The Use of nucleic acids containing unmethylated CpG dinucleotide in the treatment of LPS-associated disorders
US6406705B1 (en) 1997-03-10 2002-06-18 University Of Iowa Research Foundation Use of nucleic acids containing unmethylated CpG dinucleotide as an adjuvant
US6287591B1 (en) 1997-05-14 2001-09-11 Inex Pharmaceuticals Corp. Charged therapeutic agents encapsulated in lipid particles containing four lipid components
US6589940B1 (en) 1997-06-06 2003-07-08 Dynavax Technologies Corporation Immunostimulatory oligonucleotides, compositions thereof and methods of use thereof
ATE432348T1 (en) * 1997-06-06 2009-06-15 Univ California INHIBITORS OF IMMUNO-STIMULATIVE DNA SEQUENCE ACTIVITY
US6221882B1 (en) * 1997-07-03 2001-04-24 University Of Iowa Research Foundation Methods for inhibiting immunostimulatory DNA associated responses
JP4663113B2 (en) * 1997-09-05 2011-03-30 ザ リージェンツ オブ ザ ユニバーシティ オブ カリフォルニア Use of immunostimulatory oligonucleotides to prevent or reduce antigen-stimulated granulocyte-mediated inflammation
DE69932717T2 (en) 1998-05-22 2007-08-09 Ottawa Health Research Institute, Ottawa METHODS AND PRODUCTS FOR INDUCING MUCOSAL IMMUNITY
US6562798B1 (en) 1998-06-05 2003-05-13 Dynavax Technologies Corp. Immunostimulatory oligonucleotides with modified bases and methods of use thereof
US7049302B1 (en) 1998-08-10 2006-05-23 Antigenics Inc. Compositions of CPG and saponin adjuvants and uses thereof
AU6425999A (en) * 1998-10-09 2000-05-01 Dynavax Technologies Corporation Anti hiv compositions comprising immunostimulatory polynucleotides and hiv antigens
PT1154790E (en) * 1999-02-26 2005-03-31 Chiron Srl REFORCO OF THE ACTIVITY BACTERICIDE OF ANTIGEN AGAINST NEISSERIA WITH OLIGONUCLEOTIDOS CONTAINING REASONS CG
WO2000050006A2 (en) 1999-02-26 2000-08-31 Chiron Corporation Microemulsions with adsorbed macromoelecules and microparticles
GB2348132B (en) * 1999-03-02 2004-08-04 Nedaa Abdul-Ghani Nasif Asthma/allergy therapy that targets t-lymphocytes and/or eosinophils
WO2000054803A2 (en) * 1999-03-16 2000-09-21 Panacea Pharmaceuticals, Llc Immunostimulatory nucleic acids and antigens
FR2790955B1 (en) * 1999-03-19 2003-01-17 Assist Publ Hopitaux De Paris USE OF STABILIZED OLIGONUCLEOTIDES AS ANTI-TUMOR ACTIVE INGREDIENT
EP1176966B1 (en) 1999-04-12 2013-04-03 THE GOVERNMENT OF THE UNITED STATES OF AMERICA, as represented by THE SECRETARY, DEPARTMENT OF HEALTH AND HUMAN SERVICES Oligodeoxynucleotide and its use to induce an immune response
US6977245B2 (en) 1999-04-12 2005-12-20 The United States Of America As Represented By The Department Of Health And Human Services Oligodeoxynucleotide and its use to induce an immune response
PL203951B1 (en) 1999-04-19 2009-11-30 Smithkline Beecham Biolog Vaccines
US6558670B1 (en) 1999-04-19 2003-05-06 Smithkline Beechman Biologicals S.A. Vaccine adjuvants
AP1891A (en) * 1999-05-06 2008-09-23 The Immune Response Corp HIV immunogenic compositions and methods.
US6737066B1 (en) 1999-05-06 2004-05-18 The Immune Response Corporation HIV immunogenic compositions and methods
EP1204425B1 (en) 1999-08-19 2009-01-07 Dynavax Technologies Corporation Methods of modulating an immune response using immunostimulatory sequences and compositions for use therein
AU780535B2 (en) * 1999-08-27 2005-03-24 Inex Pharmaceuticals Corp. Compositions for stimulating cytokine secretion and inducing an immune response
US6949520B1 (en) * 1999-09-27 2005-09-27 Coley Pharmaceutical Group, Inc. Methods related to immunostimulatory nucleic acid-induced interferon
ES2541830T3 (en) 1999-10-29 2015-07-27 Novartis Vaccines And Diagnositics S.R.L. Neisseria antigenic peptides
US7223398B1 (en) 1999-11-15 2007-05-29 Dynavax Technologies Corporation Immunomodulatory compositions containing an immunostimulatory sequence linked to antigen and methods of use thereof
EP1322655B1 (en) * 2000-01-14 2007-11-14 The Government of the United States of America, as represented by the Secretary of the Department of Health and Human Services Oligodeoxynucleotide and its use to induce an immune response
AU3108001A (en) * 2000-01-20 2001-12-24 Coley Pharmaceutical Group, Inc. Immunostimulatory nucleic acids for inducing a th2 immune response
US20040131628A1 (en) * 2000-03-08 2004-07-08 Bratzler Robert L. Nucleic acids for the treatment of disorders associated with microorganisms
US7157437B2 (en) * 2000-03-10 2007-01-02 Dynavax Technologies Corporation Methods of ameliorating symptoms of herpes infection using immunomodulatory polynucleotide sequences
US20030129251A1 (en) * 2000-03-10 2003-07-10 Gary Van Nest Biodegradable immunomodulatory formulations and methods for use thereof
US20020028784A1 (en) * 2000-03-10 2002-03-07 Nest Gary Van Methods of preventing and treating viral infections using immunomodulatory polynucleotide sequences
US20020098199A1 (en) * 2000-03-10 2002-07-25 Gary Van Nest Methods of suppressing hepatitis virus infection using immunomodulatory polynucleotide sequences
US20010046967A1 (en) 2000-03-10 2001-11-29 Gary Van Nest Methods of preventing and treating respiratory viral infection using immunomodulatory polynucleotide
US20020107212A1 (en) * 2000-03-10 2002-08-08 Nest Gary Van Methods of reducing papillomavirus infection using immunomodulatory polynucleotide sequences
US7129222B2 (en) 2000-03-10 2006-10-31 Dynavax Technologies Corporation Immunomodulatory formulations and methods for use thereof
ES2206424T3 (en) 2000-06-08 2004-05-16 Intercell Biomedizinische Forschungs- Und Entwicklungs Ag IMMUNO STIMULANT OLIGODESOXINUCLEOTIDOS.
JP2005503320A (en) * 2000-08-25 2005-02-03 イエダ・リサーチ・アンド・デベロツプメント・カンパニー・リミテツド Methods for treating or preventing autoimmune diseases with CpG-containing polynucleotides
CA2425303A1 (en) 2000-10-27 2002-05-02 John Telford Nucleic acids and proteins from streptococcus groups a & b
ES2307568T3 (en) * 2000-12-08 2008-12-01 Coley Pharmaceutical Gmbh CPG TYPE NUCLEIC ACIDS AND SAME USE METHODS.
DE60142410D1 (en) * 2000-12-27 2010-07-29 Dynavax Tech Corp IMMUNOMODULATORY POLYNUCLEOTIDES AND METHOD FOR THE USE THEREOF
CA2448031A1 (en) 2001-05-21 2002-11-28 Intercell Ag Method for stabilising of nucleic acids
EP2305699B1 (en) 2001-06-05 2014-08-13 CureVac GmbH Stabilised mRNA with increased G/C content which is optimised for translation in its coded areas for the vaccination against sleeping sickness, leishmaniosis and toxoplasmosis
GB0115176D0 (en) 2001-06-20 2001-08-15 Chiron Spa Capular polysaccharide solubilisation and combination vaccines
CN100334228C (en) 2001-06-21 2007-08-29 戴纳瓦克斯技术公司 Cimeric immunomodulatory compounds and methods of using the same
GB0118249D0 (en) 2001-07-26 2001-09-19 Chiron Spa Histidine vaccines
US7666674B2 (en) 2001-07-27 2010-02-23 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Use of sterically stabilized cationic liposomes to efficiently deliver CPG oligonucleotides in vivo
GB0121591D0 (en) 2001-09-06 2001-10-24 Chiron Spa Hybrid and tandem expression of neisserial proteins
WO2003012061A2 (en) * 2001-08-01 2003-02-13 Coley Pharmaceutical Gmbh Methods and compositions relating to plasmacytoid dendritic cells
WO2003014316A2 (en) 2001-08-07 2003-02-20 Dynavax Technologies Corporation Immunomodulatory compositions, formulations, and methods for use thereof
US7354909B2 (en) * 2001-08-14 2008-04-08 The United States Of America As Represented By Secretary Of The Department Of Health And Human Services Method for rapid generation of mature dendritic cells
EP2196217A1 (en) 2001-09-14 2010-06-16 Cytos Biotechnology AG Packaging of immunostimulatory substances into virus-like particles: method of preparation and use
AR045702A1 (en) 2001-10-03 2005-11-09 Chiron Corp COMPOSITIONS OF ASSISTANTS.
EP2301552A1 (en) 2001-11-21 2011-03-30 The Board Of Trustees Of The Leland Stanford Junior University Polynucleotide therapy
DE10162480A1 (en) 2001-12-19 2003-08-07 Ingmar Hoerr The application of mRNA for use as a therapeutic agent against tumor diseases
US8466116B2 (en) 2001-12-20 2013-06-18 The Unites States Of America As Represented By The Secretary Of The Department Of Health And Human Services Use of CpG oligodeoxynucleotides to induce epithelial cell growth
AU2002366710A1 (en) * 2001-12-20 2003-07-09 The Government Of The United States Of America As Represented By The Secretary Of The Department Of USE OF CpG OLIGODEOXYNUCLEOTIDES TO INDUCE ANGIOGENESIS
EP2572707A3 (en) 2002-02-20 2013-11-06 Novartis Vaccines and Diagnostics, Inc. Microparticles with adsorbed polypeptide-containing molecules
CA2388049A1 (en) 2002-05-30 2003-11-30 Immunotech S.A. Immunostimulatory oligonucleotides and uses thereof
US20040009949A1 (en) * 2002-06-05 2004-01-15 Coley Pharmaceutical Group, Inc. Method for treating autoimmune or inflammatory diseases with combinations of inhibitory oligonucleotides and small molecule antagonists of immunostimulatory CpG nucleic acids
US20040053880A1 (en) 2002-07-03 2004-03-18 Coley Pharmaceutical Group, Inc. Nucleic acid compositions for stimulating immune responses
DE10229872A1 (en) 2002-07-03 2004-01-29 Curevac Gmbh Immune stimulation through chemically modified RNA
US7807803B2 (en) 2002-07-03 2010-10-05 Coley Pharmaceutical Group, Inc. Nucleic acid compositions for stimulating immune responses
AU2003247880B2 (en) * 2002-07-03 2010-09-02 Coley Pharmaceutical Group, Inc. Nucleic acid compositions for stimulating immune responses
KR20050042134A (en) * 2002-07-15 2005-05-04 길리애드 사이언시즈, 인코포레이티드 Combination therapies with l-fmau for the treatment of hepatitis b virus infection
NZ538628A (en) 2002-08-12 2008-06-30 Dynavax Tech Corp Immunomodulatory compositions, methods of making, and methods of use thereof
GB0220194D0 (en) 2002-08-30 2002-10-09 Chiron Spa Improved vesicles
US8263091B2 (en) * 2002-09-18 2012-09-11 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Method of treating and preventing infections in immunocompromised subjects with immunostimulatory CpG oligonucleotides
MXPA05004588A (en) 2002-10-29 2005-12-14 Coley Pharmaceutical Group Ltd Use of cpg oligonucleotides in the treatment of hepatitis c virus infection.
JP4976653B2 (en) * 2002-11-01 2012-07-18 ザ ガバメント オブ ザ ユナイテッド ステイツ オブ アメリカ アズ リプレゼンティッド バイ ザ セクレタリー オブ ザ デパートメント オブ ヘルス アンド ヒューマン サービシス Method for preventing infections caused by bioterrorism pathogens using immunostimulatory CpG oligonucleotides
EP2279746B1 (en) 2002-11-15 2013-10-02 Novartis Vaccines and Diagnostics S.r.l. Surface proteins in neisseria meningitidis
US7811813B2 (en) * 2002-11-21 2010-10-12 The Board Of Trustees Of The Leland Stanford Junior University Methods and immune modulatory nucleic acid compositions for preventing and treating disease
GB0227346D0 (en) 2002-11-22 2002-12-31 Chiron Spa 741
AU2003300919A1 (en) 2002-12-11 2004-06-30 Coley Pharmaceutical Gmbh 5' cpg nucleic acids and methods of use
US8158768B2 (en) 2002-12-23 2012-04-17 Dynavax Technologies Corporation Immunostimulatory sequence oligonucleotides and methods of using the same
JP2006516099A (en) 2002-12-23 2006-06-22 ダイナバックス テクノロジーズ コーポレイション Branched immunomodulatory compounds and methods of using the compounds
CN100546998C (en) 2002-12-23 2009-10-07 戴纳伐克斯技术股份有限公司 Immunostimulatory sequence oligonucleotides and using method
WO2004060396A2 (en) 2002-12-27 2004-07-22 Chiron Corporation Immunogenic compositions containing phospholpid
ES2391770T3 (en) 2003-01-21 2012-11-29 Novartis Vaccines And Diagnostics, Inc. Use of triptantrin compounds for immune potentiation
ES2385933T3 (en) 2003-02-20 2012-08-03 University Of Connecticut Health Center METHODS FOR THE PRODUCTION OF ALFA ANTIGEN MOLECULES COMPLEXES (2) MACROGLOBULIN.
CA2517839A1 (en) 2003-03-26 2004-10-07 Martin F. Bachmann Melan-a peptide analogue-virus-like-particle conjugates
US7537767B2 (en) 2003-03-26 2009-05-26 Cytis Biotechnology Ag Melan-A- carrier conjugates
ES2423800T3 (en) 2003-03-28 2013-09-24 Novartis Vaccines And Diagnostics, Inc. Use of organic compounds for immunopotentiation
US20040235770A1 (en) * 2003-04-02 2004-11-25 Coley Pharmaceutical Group, Ltd. Immunostimulatory nucleic acid oil-in-water formulations and related methods of use
WO2004100965A1 (en) * 2003-05-15 2004-11-25 Japan Science And Technology Agency Immunostimulant
CA2525966A1 (en) * 2003-05-15 2004-12-23 The Board Of Trustees Of The Leland Stanford Junior University Novel immunomodulating peptide
WO2005020964A1 (en) 2003-06-02 2005-03-10 Chiron Corporation Immunogenic compositions based on microparticles comprising adsorbed toxoid and a polysaccharide-containing antigen
US20060035242A1 (en) 2004-08-13 2006-02-16 Michelitsch Melissa D Prion-specific peptide reagents
US20050239733A1 (en) * 2003-10-31 2005-10-27 Coley Pharmaceutical Gmbh Sequence requirements for inhibitory oligonucleotides
JP4817599B2 (en) * 2003-12-25 2011-11-16 独立行政法人科学技術振興機構 Immune activity enhancer and method for enhancing immune activity using the same
JP3976742B2 (en) 2004-02-27 2007-09-19 江守商事株式会社 Immunostimulatory oligonucleotides that induce interferon alpha
TWI235440B (en) * 2004-03-31 2005-07-01 Advanced Semiconductor Eng Method for making leadless semiconductor package
CA2571710A1 (en) 2004-06-24 2006-11-02 Nicholas Valiante Small molecule immunopotentiators and assays for their detection
EP1765313A2 (en) 2004-06-24 2007-03-28 Novartis Vaccines and Diagnostics, Inc. Compounds for immunopotentiation
WO2006028742A2 (en) 2004-09-01 2006-03-16 Dynavax Technologies Corporation Methods and conpositions for inhibition of innate immune responses and autoimmunity
JP2008000001A (en) * 2004-09-30 2008-01-10 Osaka Univ Immune stimulating oligonucleotide and use in pharmaceutical
US20060135906A1 (en) * 2004-11-16 2006-06-22 Akihiko Matsumura Iontophoretic device and method for administering immune response-enhancing agents and compositions
WO2006066003A2 (en) * 2004-12-17 2006-06-22 Dynavax Technologies Corporation Methods and compositions for induction or promotion of immune tolerance
EP2433647A3 (en) 2005-01-27 2012-06-06 Children's Hospital & Research Center at Oakland GNA1870-based vesicle vaccines for broad spectrum protection against diseases caused by Neisseria meningitidis
CN101160401A (en) * 2005-02-24 2008-04-09 科勒制药集团公司 Immunostimulatory oligonucleotides
NZ561144A (en) * 2005-03-04 2009-09-25 Dynavax Tech Corp Vaccines comprising oligonucleotides having immunostimulatory sequences (ISS) wherein the ISS are conjugated to antigens and stabilized by buffer conditions and further excipients
WO2006110655A2 (en) 2005-04-08 2006-10-19 Chimerix, Inc. Compounds, compositions and methods for the treatment of poxvirus infections
US20070003608A1 (en) * 2005-04-08 2007-01-04 Almond Merrick R Compounds, compositions and methods for the treatment of viral infections and other medical disorders
EP1928539A1 (en) * 2005-09-30 2008-06-11 Tti Ellebeau, Inc. Functionalized microneedles transdermal drug delivery systems, devices, and methods
US20070083186A1 (en) * 2005-09-30 2007-04-12 Darrick Carter Transdermal drug delivery systems, devices, and methods employing novel pharmaceutical vehicles
KR20080066712A (en) * 2005-09-30 2008-07-16 티티아이 엘뷰 가부시키가이샤 Functionalized microneedles transdermal drug delivery systems, devices, and methods
AU2006325225B2 (en) 2005-12-14 2013-07-04 Cytos Biotechnology Ag Immunostimulatory nucleic acid packaged particles for the treatment of hypersensitivity
RU2008135318A (en) * 2006-02-01 2010-03-10 Дзе Джонс Хопкинс Юниверсити (Us) POLYEPEPTIDE-NUCLEIC ACID CONJUGATE FOR IMMUNOPROPHYLAXIS OR IMMUNOTHERAPY OF NEOPLASTIC OR INFECTIOUS DISEASES
EP2405002B1 (en) * 2006-02-15 2014-09-24 AdiuTide Pharmaceuticals GmbH Compositions and methods for oligonucleotide formulations
CA2638760A1 (en) 2006-03-07 2007-09-13 Vaxinnate Corporation Compositions that include hemagglutinin, methods of making and methods of use thereof
CA2653939C (en) 2006-05-31 2013-01-22 Toray Industries, Inc. Immunostimulatory oligonucleotides and use thereof in pharmaceuticals
CA2655108C (en) 2006-06-12 2019-05-07 Cytos Biotechnology Ag Processes for packaging oligonucleotides into virus-like particles of rna bacteriophages
CA2655327A1 (en) * 2006-06-13 2007-12-21 Bayhill Therapeutics, Inc. Methods and immune modulatory nucleic acid compositions for preventing and treating disease
AU2007280690C1 (en) 2006-07-31 2012-08-23 Curevac Gmbh Nucleic acid of formula (I): GIXmGn, or (II): CIXmCn, in particular as an immune-stimulating agent/adjuvant
DE102006035618A1 (en) * 2006-07-31 2008-02-07 Curevac Gmbh New nucleic acid useful as immuno-stimulating adjuvant for manufacture of a composition for treatment of cancer diseases e.g. colon carcinomas and infectious diseases e.g. influenza and malaria
CA2669137A1 (en) 2006-11-09 2008-06-19 Dynavax Technologies Corporation Long term disease modification using immunostimulatory oligonucleotides
AU2008283802A1 (en) * 2007-07-31 2009-02-05 The Board Of Supervisors Of Louisiana State University And Agricultural And Mechanical College Polypeptide-nucleic acid conjugate for immunoprophylaxis or immunotherapy for neoplastic or infectious disorders
CA2693266C (en) * 2007-08-01 2015-06-30 Idera Pharmaceuticals, Inc. Novel synthetic agonists of tlr9
WO2009030254A1 (en) 2007-09-04 2009-03-12 Curevac Gmbh Complexes of rna and cationic peptides for transfection and for immunostimulation
WO2009055076A2 (en) 2007-10-26 2009-04-30 Dynavax Technologies Corporation Methods and compositions for inhibition of immune responses and autoimmunity
DK2222697T3 (en) 2007-11-01 2013-03-11 Perseid Therapeutics Llc Immunosuppressive polypeptides and nucleic acids
JP2011506334A (en) 2007-12-07 2011-03-03 ノバルティス アーゲー Composition for inducing an immune response
CN101977610B (en) * 2008-01-25 2012-05-16 奇默里克斯公司 Methods of treating viral infections
EP2265640B1 (en) 2008-03-10 2015-11-04 Children's Hospital & Research Center at Oakland Chimeric factor h binding proteins (fhbp) containing a heterologous b domain and methods of use
JP2011519828A (en) 2008-04-18 2011-07-14 バクシネート コーポレーション Flagellin deletion mutants and methods of use
AU2009246169B2 (en) 2008-05-15 2015-01-22 Dynavax Technologies Corporation Long term disease modification using immunostimulatory oligonucleotides
GB0809476D0 (en) * 2008-05-23 2008-07-02 Cambridge Entpr Ltd Treatment of neurodegenerative disorders
JP5659332B2 (en) 2008-06-27 2015-01-28 ゾエティス・エルエルシー Novel adjuvant composition
WO2010037408A1 (en) 2008-09-30 2010-04-08 Curevac Gmbh Composition comprising a complexed (m)rna and a naked mrna for providing or enhancing an immunostimulatory response in a mammal and uses thereof
EP2376108B1 (en) 2008-12-09 2017-02-22 Pfizer Vaccines LLC IgE CH3 PEPTIDE VACCINE
CN102307477B (en) 2009-01-05 2015-07-29 埃皮托吉尼西斯股份有限公司 Adjunvant composition and using method
AU2010229835B2 (en) 2009-03-25 2015-01-15 The Board Of Regents Of The University Of Texas System Compositions for stimulation of mammalian innate immune resistance to pathogens
US8614200B2 (en) 2009-07-21 2013-12-24 Chimerix, Inc. Compounds, compositions and methods for treating ocular conditions
PE20120817A1 (en) 2009-07-30 2012-07-07 Pfizer Vaccines Llc ANTIGENIC TAU PEPTIDES AND USES OF THEM
US20110053829A1 (en) 2009-09-03 2011-03-03 Curevac Gmbh Disulfide-linked polyethyleneglycol/peptide conjugates for the transfection of nucleic acids
CA2846746A1 (en) 2009-09-03 2011-03-10 Pfizer Vaccines Llc Pcsk9 vaccine
CN102640001A (en) 2009-11-05 2012-08-15 诺瓦提斯公司 Biomarkers predictive of progression of fibrosis
KR20120120185A (en) 2009-12-22 2012-11-01 셀덱스 쎄라퓨틱스, 인크. Vaccine compositions
PT2534150T (en) 2010-02-12 2017-05-02 Chimerix Inc Methods of treating viral infection
JP2013521002A (en) 2010-03-05 2013-06-10 プレジデント アンド フェロウズ オブ ハーバード カレッジ Induced dendritic cell composition and use thereof
EP2552942B1 (en) 2010-03-30 2017-12-27 Children's Hospital & Research Center at Oakland Factor h binding proteins (fhbp) with altered properties and methods of use thereof
WO2011139709A2 (en) 2010-04-26 2011-11-10 Chimerix, Inc. Methods of treating retroviral infections and related dosage regimes
WO2011154878A1 (en) 2010-06-07 2011-12-15 Pfizer Vaccines Llc Ige ch3 peptide vaccine
EP2576613A1 (en) 2010-06-07 2013-04-10 Pfizer Inc. Her-2 peptides and vaccines
WO2011159958A2 (en) 2010-06-16 2011-12-22 Dynavax Technologies Corporation Methods of treatment using tlr7 and/or tlr9 inhibitors
DK2449113T3 (en) 2010-07-30 2016-01-11 Curevac Ag Complex formation of nucleic acids with the disulfide cross-linked cationic components for transfection and immunostimulation
CA2807552A1 (en) 2010-08-06 2012-02-09 Moderna Therapeutics, Inc. Engineered nucleic acids and methods of use thereof
EP4108671A1 (en) 2010-10-01 2022-12-28 ModernaTX, Inc. Modified nucleosides, nucleotides, and nucleic acids, and uses thereof
US20140004142A1 (en) 2011-03-02 2014-01-02 Pfizer Inc. Pcsk9 vaccine
AU2012236099A1 (en) 2011-03-31 2013-10-03 Moderna Therapeutics, Inc. Delivery and formulation of engineered nucleic acids
US9464124B2 (en) 2011-09-12 2016-10-11 Moderna Therapeutics, Inc. Engineered nucleic acids and methods of use thereof
CN103974724B (en) 2011-10-03 2019-08-30 现代泰克斯公司 Nucleosides, nucleotide and nucleic acid of modification and application thereof
CA2859387A1 (en) 2011-12-16 2013-06-20 Moderna Therapeutics, Inc. Modified nucleoside, nucleotide, and nucleic acid compositions
WO2013113326A1 (en) 2012-01-31 2013-08-08 Curevac Gmbh Pharmaceutical composition comprising a polymeric carrier cargo complex and at least one protein or peptide antigen
US9283287B2 (en) 2012-04-02 2016-03-15 Moderna Therapeutics, Inc. Modified polynucleotides for the production of nuclear proteins
US9878056B2 (en) 2012-04-02 2018-01-30 Modernatx, Inc. Modified polynucleotides for the production of cosmetic proteins and peptides
US9572897B2 (en) 2012-04-02 2017-02-21 Modernatx, Inc. Modified polynucleotides for the production of cytoplasmic and cytoskeletal proteins
AU2013243951A1 (en) 2012-04-02 2014-10-30 Moderna Therapeutics, Inc. Modified polynucleotides for the production of secreted proteins
PE20142406A1 (en) 2012-05-04 2015-01-23 Pfizer ANTIGENS ASSOCIATED WITH PROSTATE AND VACCINE-BASED IMMUNOTHERAPY REGIMES
US9228184B2 (en) 2012-09-29 2016-01-05 Dynavax Technologies Corporation Human toll-like receptor inhibitors and methods of use thereof
US9868955B2 (en) 2012-09-29 2018-01-16 Dynavax Technologies Corporation Human toll-like receptor inhibitors and methods of use thereof
JP6144355B2 (en) 2012-11-26 2017-06-07 モデルナティエックス インコーポレイテッドＭｏｄｅｒｎａＴＸ，Ｉｎｃ． Chemically modified mRNA
EP2948469A4 (en) 2013-01-23 2016-11-02 Univ Leland Stanford Junior Stabilized hepatitis b core polypeptide
ES2708650T3 (en) 2013-03-14 2019-04-10 Andes Biotechnologies Global Inc Antisense oligonucleotides for the treatment of tumor stem cells
CA2902560A1 (en) 2013-03-14 2014-09-25 President And Fellows Of Harvard College Nanoparticle-based compositions
EP2970364B1 (en) 2013-03-14 2019-04-24 Andes Biotechnologies Global, Inc. Methods for detecting and treating multiple myeloma
WO2014151994A1 (en) 2013-03-15 2014-09-25 Kambiz Shekdar Genome editing using effector oligonucleotides for therapeutic treatment
US8980864B2 (en) 2013-03-15 2015-03-17 Moderna Therapeutics, Inc. Compositions and methods of altering cholesterol levels
WO2015132619A1 (en) 2013-05-15 2015-09-11 The Governors Of The University Of Alberta E1e2 hcv vaccines and methods of use
MX369469B (en) 2013-08-21 2019-11-08 Curevac Ag Respiratory syncytial virus (rsv) vaccine.
KR102257743B1 (en) 2013-09-19 2021-05-28 조에티스 서비시즈 엘엘씨 Oil-based adjuvants
WO2015048744A2 (en) 2013-09-30 2015-04-02 Moderna Therapeutics, Inc. Polynucleotides encoding immune modulating polypeptides
EA201690675A1 (en) 2013-10-03 2016-08-31 Модерна Терапьютикс, Инк. POLYNUCLEOTES ENCODING THE RECEPTOR OF LOW DENSITY LIPOPROTEINS
ES2715890T3 (en) 2013-11-01 2019-06-06 Pfizer Expression vectors of prostate associated antigens
ES2806575T3 (en) 2013-11-01 2021-02-18 Curevac Ag Modified RNA with decreased immunostimulatory properties
EP3104877B1 (en) 2014-02-11 2020-01-22 The USA, as represented by The Secretary, Department of Health and Human Services Pcsk9 vaccine and methods of using the same
CA2936286A1 (en) 2014-04-01 2015-10-08 Curevac Ag Polymeric carrier cargo complex for use as an immunostimulating agent or as an adjuvant
US10286065B2 (en) 2014-09-19 2019-05-14 Board Of Regents, The University Of Texas System Compositions and methods for treating viral infections through stimulated innate immunity in combination with antiviral compounds
PL3244920T3 (en) 2015-01-16 2023-09-25 Zoetis Services Llc Foot-and-mouth disease vaccine
KR20180014009A (en) 2015-05-29 2018-02-07 머크 샤프 앤드 돔 코포레이션 A combination of a PD-1 antagonist and a CPG-C type oligonucleotide for treating cancer
BR112018013967A2 (en) 2016-01-19 2019-02-05 Pfizer cancer vaccines
CN114807152A (en) * 2016-06-08 2022-07-29 哈佛学院院长及董事 Engineered viral vectors reduce induction of inflammation and immune responses
AU2017356673B2 (en) 2016-11-09 2023-11-09 Pulmotect, Inc. Methods and compositions for adaptive immune modulation
KR20200085812A (en) * 2017-11-08 2020-07-15 프레지던트 앤드 펠로우즈 오브 하바드 칼리지 Compositions and methods for inhibiting viral vector-induced inflammatory response
US11633471B2 (en) 2018-03-06 2023-04-25 Unm Rainforest Innovations Compositions and methods for reducing serum triglycerides
WO2022147373A1 (en) 2020-12-31 2022-07-07 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Antibody-guided pcsk9-mimicking immunogens lacking 9-residue sequence overlap with human proteins
WO2022196699A1 (en) 2021-03-16 2022-09-22 国立大学法人大阪大学 Follicular helper t (tfh) cells specific to sars-cov-2 virus
JPWO2022196701A1 (en) 2021-03-16 2022-09-22
WO2023081486A1 (en) 2021-11-08 2023-05-11 Nurix Therapeutics, Inc. Toll-like receptor therapy combinations with cbl-b inhibitor compounds

Family Cites Families (46)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US1234718A (en) 1914-03-21 1917-07-31 Int Harvester Canada Buckle.
US2811896A (en) 1953-10-05 1957-11-05 Welsh Mfg Co Ophthalmic mounting
US3725545A (en) 1971-02-03 1973-04-03 R Maes Enhancement of antibody production by nucleic acid-polycation complexes
US3906092A (en) 1971-11-26 1975-09-16 Merck & Co Inc Stimulation of antibody response
US4458066A (en) 1980-02-29 1984-07-03 University Patents, Inc. Process for preparing polynucleotides
US4425732A (en) 1981-06-17 1984-01-17 Kania Tadeusz E Animal trap
US4650675A (en) 1983-08-18 1987-03-17 The Children's Medical Center Corporation Oligonucleotide conjugates
US5118800A (en) 1983-12-20 1992-06-02 California Institute Of Technology Oligonucleotides possessing a primary amino group in the terminal nucleotide
US4849513A (en) 1983-12-20 1989-07-18 California Institute Of Technology Deoxyribonucleoside phosphoramidites in which an aliphatic amino group is attached to the sugar ring and their use for the preparation of oligonucleotides containing aliphatic amino groups
US5015733A (en) 1983-12-20 1991-05-14 California Institute Of Technology Nucleosides possessing blocked aliphatic amino groups
US5118802A (en) 1983-12-20 1992-06-02 California Institute Of Technology DNA-reporter conjugates linked via the 2' or 5'-primary amino group of the 5'-terminal nucleoside
DE3583564D1 (en) * 1984-08-23 1991-08-29 Hans Joachim Wolf DNA SEQUENCES OF THE EBV GENOME, RECOMBINANT DNA MOLECULES, METHOD FOR THE PRODUCTION OF EBV-RELATED ANTIGENS, DIAGNOSTIC COMPOSITIONS AND PHARMACEUTICAL COMPOSITIONS CONTAINING THE SAME ANTIGENS.
US5110906A (en) * 1986-08-21 1992-05-05 The Trustees Of Columbia University In The City Of New York Derivatives of soluble T-4
US5276019A (en) 1987-03-25 1994-01-04 The United States Of America As Represented By The Department Of Health And Human Services Inhibitors for replication of retroviruses and for the expression of oncogene products
US5268365A (en) 1988-03-11 1993-12-07 Rudolph Frederick B Nucleotides, nucleosides, and nucleobases in immune function restoration enhancement or maintenance
US5218103A (en) 1988-05-26 1993-06-08 University Patents, Inc. Nucleoside thiophosphoramidites
US5278302A (en) 1988-05-26 1994-01-11 University Patents, Inc. Polynucleotide phosphorodithioates
US5703055A (en) 1989-03-21 1997-12-30 Wisconsin Alumni Research Foundation Generation of antibodies through lipid mediated DNA delivery
WO1991003251A1 (en) * 1989-09-08 1991-03-21 California Institute Of Biological Research Cr2 ligand compositions and methods for modulating immune cell functions
US5331090A (en) * 1989-09-08 1994-07-19 California Institute Of Biological Research CR2 ligand compositions and methods for modulating immune cell functions
US5514577A (en) * 1990-02-26 1996-05-07 Isis Pharmaceuticals, Inc. Oligonucleotide therapies for modulating the effects of herpes viruses
EP0527818A4 (en) 1990-04-30 1993-09-15 Isis Pharmaceuticals, Inc. Oligonucleotide modulation of arachidonic acid metabolism
EP0468520A3 (en) * 1990-07-27 1992-07-01 Mitsui Toatsu Chemicals, Inc. Immunostimulatory remedies containing palindromic dna sequences
US5286365A (en) 1992-01-15 1994-02-15 Beckman Instruments, Inc. Graphite-based solid state polymeric membrane ion-selective electrodes
EP0656950B1 (en) 1992-08-21 1998-11-04 Biogen, Inc. Tat-derived transport polypeptides
US5523389A (en) * 1992-09-29 1996-06-04 Isis Pharmaceuticals, Inc. Inhibitors of human immunodeficiency virus
US5552390A (en) * 1993-12-09 1996-09-03 The Board Of Regents Of The University Of Nebraska Phosphorothioate inhibitors of metastatic breast cancer
WO1995026204A1 (en) 1994-03-25 1995-10-05 Isis Pharmaceuticals, Inc. Immune stimulation by phosphorothioate oligonucleotide analogs
ES2267100T5 (en) * 1994-07-15 2011-04-08 The University Of Iowa Research Foundation IMMUNOMODULATING OLIGONUCLEOTIDES.
US6207646B1 (en) * 1994-07-15 2001-03-27 University Of Iowa Research Foundation Immunostimulatory nucleic acid molecules
US5665347A (en) * 1995-02-02 1997-09-09 Genetics Institute IL-12 inhibition of B1 cell activity
US5968909A (en) * 1995-08-04 1999-10-19 Hybridon, Inc. Method of modulating gene expression with reduced immunostimulatory response
US5840839A (en) * 1996-02-09 1998-11-24 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Alternative open reading frame DNA of a normal gene and a novel human cancer antigen encoded therein
WO1998010066A1 (en) * 1996-09-04 1998-03-12 Onyx Pharmaceuticals, Inc. Modulators of brca1 activity
US6610661B1 (en) * 1996-10-11 2003-08-26 The Regents Of The University Of California Immunostimulatory polynucleotide/immunomodulatory molecule conjugates
AU738513B2 (en) 1997-02-28 2001-09-20 University Of Iowa Research Foundation, The Use of nucleic acids containing unmethylated CpG dinucleotide in the treatment of LPS-associated disorders
EP1005368B1 (en) 1997-03-10 2009-09-02 Ottawa Hospital Research Institute Use of nucleic acids containing unmethylated CpG dinucleotide in combination with alum as adjuvants
EP0983289A4 (en) * 1997-05-19 2001-04-25 Merck & Co Inc Oligonucleotide adjuvant
US6613881B1 (en) * 1997-05-20 2003-09-02 Corixa Corporation Compounds for immunotherapy and diagnosis of tuberculosis and methods of their use
EP1003531B1 (en) 1997-05-20 2007-08-22 Ottawa Health Research Institute Processes for preparing nucleic acid constructs
ATE432348T1 (en) * 1997-06-06 2009-06-15 Univ California INHIBITORS OF IMMUNO-STIMULATIVE DNA SEQUENCE ACTIVITY
EP1355664A4 (en) 2001-01-04 2005-10-26 Vascular Biogenics Ltd Methods employing and compositions containing plaque associated molecules for prevention and treatment of atherosclerosis
BR0212366A (en) * 2001-09-07 2004-07-27 Univ Boston Method and composition for treating disorders associated with the immune complex
US7811813B2 (en) * 2002-11-21 2010-10-12 The Board Of Trustees Of The Leland Stanford Junior University Methods and immune modulatory nucleic acid compositions for preventing and treating disease
US20050239733A1 (en) * 2003-10-31 2005-10-27 Coley Pharmaceutical Gmbh Sequence requirements for inhibitory oligonucleotides
CA2655327A1 (en) * 2006-06-13 2007-12-21 Bayhill Therapeutics, Inc. Methods and immune modulatory nucleic acid compositions for preventing and treating disease

Also Published As

Publication number Publication date
EP0986572A2 (en) 2000-03-22
EP2085090A2 (en) 2009-08-05
HK1024701A1 (en) 2000-10-20
JP4101888B2 (en) 2008-06-18
AU753172B2 (en) 2002-10-10
EP2085090A3 (en) 2012-05-02
WO1998055609A1 (en) 1998-12-10
US20110182927A1 (en) 2011-07-28
JP2002517156A (en) 2002-06-11
DE69819150T3 (en) 2007-12-20
JP2002505580A (en) 2002-02-19
DK1003850T3 (en) 2009-09-07
US8729039B2 (en) 2014-05-20
CY1109287T1 (en) 2014-07-02
WO1998055495B1 (en) 1999-07-22
DE69840850D1 (en) 2009-07-09
AU7811398A (en) 1998-12-21
JP4280309B2 (en) 2009-06-17
US20110034541A1 (en) 2011-02-10
PT1003850E (en) 2009-08-13
WO1998055495A2 (en) 1998-12-10
DE69819150D1 (en) 2003-11-27
AU7817898A (en) 1998-12-21
ATE432348T1 (en) 2009-06-15
US20020086839A1 (en) 2002-07-04
ATE252596T1 (en) 2003-11-15
EP1003850A1 (en) 2000-05-31
ES2326848T3 (en) 2009-10-20
AU755322B2 (en) 2002-12-12
WO1998055495A3 (en) 1999-05-27
EP1003850A4 (en) 2005-07-06
CA2291483C (en) 2012-09-18
EP1003850B1 (en) 2009-05-27
US6225292B1 (en) 2001-05-01
CA2293489A1 (en) 1998-12-10
EP0986572B1 (en) 2003-10-22
EP0986572B2 (en) 2007-06-13
CA2291483A1 (en) 1998-12-10
DE69819150T2 (en) 2004-07-22

Similar Documents

Publication Publication Date Title
CA2293489C (en) Inhibitors of dna immunostimulatory sequence activity
US7851454B2 (en) Short immunomodulatory oligonucleotides
US10144933B2 (en) Chiral nucleic acid adjuvant having immunity induction activity, and immunity induction activator
JP4874801B2 (en) Stabilized immunomodulatory oligonucleotide
KR100421753B1 (en) Adeno-associated viral(AAV) liposomes and methods related thereto
JP3468773B2 (en) Immunomodulatory oligonucleotide
US20030125279A1 (en) Covalently closed nucleic acid molecules for immunostimulation
LV13641B (en) Immunostimulatory oligonucleotide multimers
JP2007500018A6 (en) Stabilized immunomodulatory oligonucleotide
CA2302805A1 (en) Use of immunostimulatory oligonucleotides for preventing or reducing antigen-stimulated, granulocyte-mediated inflammation
US20070093439A1 (en) Short immunomodulatory oligonucleotides
KR100689429B1 (en) Cancer cell vaccine
WO2007050059A2 (en) Short immunolomodulatory oligonucleotides
AU2007200536A1 (en) Therapeutically useful synthetic oligonucleotides

Legal Events

Date Code Title Description
EEER Examination request
MKLA Lapsed

Effective date: 20160606