EP1108017A2 - Oligonucleotides a motif g et leurs utilisations - Google Patents

Oligonucleotides a motif g et leurs utilisations

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Publication number
EP1108017A2
EP1108017A2 EP99946118A EP99946118A EP1108017A2 EP 1108017 A2 EP1108017 A2 EP 1108017A2 EP 99946118 A EP99946118 A EP 99946118A EP 99946118 A EP99946118 A EP 99946118A EP 1108017 A2 EP1108017 A2 EP 1108017A2
Authority
EP
European Patent Office
Prior art keywords
oligonucleotide
composition
cells
present
odn
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.)
Withdrawn
Application number
EP99946118A
Other languages
German (de)
English (en)
Inventor
Hermann Wagner
Grayson B. Lipford
Klaus Heeg
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.)
Coley Pharmaceutical GmbH
Original Assignee
Coley Pharmaceutical GmbH
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
Application filed by Coley Pharmaceutical GmbH filed Critical Coley Pharmaceutical GmbH
Priority to EP99946118A priority Critical patent/EP1108017A2/fr
Publication of EP1108017A2 publication Critical patent/EP1108017A2/fr
Withdrawn legal-status Critical Current

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Classifications

    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • 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
    • 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
    • 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
    • C12N2310/18—Type of nucleic acid acting by a non-sequence specific mechanism
    • 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

Definitions

  • the present invention relates to a composition
  • a composition comprising an oligonucleotide comprising (a) the sequence N ⁇ - N 2 - G - N 3 - G, wherein Ni represents any nucleotide if N 2 and N 3 are G; N 2 represents any nucleotide if N T and N 3 are G; and N 3 represents any nucleotide if IS and N 2 are G, or (b) the sequence of (a), wherein at least one nucleotide is replaced by a corresponding nucleotide analog or derivative.
  • the present invention also relates to an oligonucleotide having (a) a sequence selected from the group consisting of the sequences of SEQ ID NOs: 1 to 19, or (b) a sequence of (a), wherein at least one nucleotide is replaced by a corresponding nucleotide analog or derivative.
  • a vector comprising an oligonucleotide of the present invention, a host cell comprising the vector of the present invention, a method for the production of the oligonucleotide of the present invention as well as a kit comprising the composition, the oligonucleotide, the vector, and/or the host cell of the present invention are described.
  • the present invention further relates to the use of the composition, and/or the oligonucleotide of the present invention for the production of a pharmaceutical composition for preventing or treating septic shock, inflammation, autoimmune diseases, T H 1 -mediated diseases, bacterial infections, parasitic infections, viral infections, spontaneous abortions, and/or tumors.
  • compositions, and/or the oligonucleotide of the present invention to inhibit activation of antigen-presenting cells, to inhibit the uptake of DNA by a cell, to stimulate natural killer cells, to co- stimulate cytotoxic T-lymphocytes or natural killer cells, to enhance the production of antibodies directed against an antigen, to enhance the uptake of an agent by a cell, and/or to induce proliferation of bone marrow cells in vitro or in vivo.
  • Pathogen challenge by supplying danger stimuli, mobilizes the immune system to respond. Most typically these responses result in the clearance of the challenging pathogen but can also result in overshoot or misdirected responses leading to conditions such as allergy or autoimmune diseases.
  • the immune system can be enhanced, e.g., by vaccination or cytokine therapies, to bolster favorable host responses, or can be suppressed, e.g., by drug intervention or cytokine therapies, to limit unfavorable responses.
  • the functioning of the immune system is based on two distinct recognition systems: innate (myeloid cells, NK cells etc.) or nonclonal host defense and adaptive (T and B cells) or clonal host defense.
  • the innate immune systems receptors recognize conserved molecular structures shared by a large group of pathogens, termed pattern recognition receptors (PRRs) (reviewed in Fearon DT, Locksley RM, Science 1996; 272: 50; Medzhitov R, Janeway CA, Jr., Curr Opin Immunol 1997; 9: 4).
  • PRRs pattern recognition receptors
  • the main difference between PRRs and clonally-distributed antigen receptors of the adaptive system is that their specificities are germline encoded. Thus a parameter imposed on PRRs is recognition of non-self structural patterns.
  • T and B cell receptors through their specificity and diversity, represent the crowning achievement of the vertebrate adaptive immune system they do not distinguish self from non-self.
  • the innate system controls the initiation of the adaptive immune response by regulating the expression of costimulatory activity on antigen presenting cells (APCs), and instructs the adaptive immune system to develop a particular effector response (humoral, cytotoxic, Th1 versus Th2) by releasing effector cytokines.
  • APCs antigen presenting cells
  • the adaptive immune system of vertebrates consists of several interacting components. They can be divided into humoral and cellular branches. Humoral immunity involves antibodies, proteins which are produced and secreted by B cells into the body fluids and which directly recognize an antigen. The cellular system, in contrast, relies on T cells which recognize cells presenting foreign antigens and then responding by proliferation, cytotoxicity or secretion of cytokines. This basic functional division reflects two different strategies of immune defense. Humoral immunity is mainly directed at antigens which are whole proteins, the cellular system responds to antigens which are actively processed and presented by either APC or pathogen infected cells (see, e.g., Paul WE, Cell 1989; 57: 521).
  • Antibody molecules the effectors of humoral immunity, are secreted by B cells in response to antigen receptor stimulation, co-receptor stimulation and cytokines.
  • Antibodies can bind to and inactivate antigen directly (neutralizing antibodies) or activate other cells of the immune system to destroy the antigen depending on isotype; IgM, lgG1 , etc.
  • Isotype class switching in B cells is controlled by the cytokine milieu. Abnormalities in antigen response, co-receptor engagement or cytokine milieu can lead to suboptimal immune responses, tolerance or autoimmunity.
  • the cytokine milieu can push the isotype repertoire to reflect either Th1 (lgG2) or Th2 (lgG1 and IgE) responses which can have beneficial or detrimental effects dependent on the target antigen and challenge source.
  • T cells Cellular immune recognition is mediated by a special class of lymphoid cells, T cells. These cells do not recognize whole antigens but instead they respond to degraded peptide fragments thereof which appear on the surface of the target cell bound to proteins called major histocompatibility complex (MHC) molecules. Essentially all nucleated cells have MHC class I molecules whereas MHC II are restricted to immune cells with special presenting qualities.
  • MHC major histocompatibility complex
  • Proteins produced within the cell are continually degraded to peptides as part of normal cellular metabolism. These fragments are bound to the MHC I molecules and are transported to the cell surface.
  • the cellular immune system is constantly monitoring the spectra of proteins produced by all cells in the body and is poised to eliminate any cells producing foreign antigens or abnormal self-antigens. This is a function of cytolytic T cells.
  • APCs take up antigens from the environment, process them to peptides, and present them in the context of MHC II to helper T cells (Th cells) which respond by producing cytokines. These responses can be divided into Th1 or Th2, as defined by the cytokine pattern. Th1 or Th2 responses are generated dependent on the pathogen challenge and/or the contextual engagement through which the response is generated. Again, Th1 or Th2 biased responses can have either beneficial or detrimental outcomes dependent on the target antigen and the intensity of response.
  • APCs such as dendritic cells and macrophages
  • APCs represent a decisive interface between the innate and adaptive immune system (Banchereau J, Steinman RM, Nature 1998; 392: 245).
  • Signals induced by pattern recognition can be grouped into three categories: first, inflammatory responses including IL-1 , TNF, IL-6, type I IFN and chemokines; second, costimulators of T cell activation including B7.1 (CD80), B7.2 (CD86) and CD40; and third, effector cytokines including IL-10, IL-12, TNF- ⁇ and IFN- ⁇ .
  • APCs need first to be activated thus acquiring the ability to instigate productive T and B cell activation through the above mentioned mediators.
  • TNF Tumor necrosis factor
  • TNF TNF-associated fibrosis .
  • infectious e.g., sepsis syndrome, bacterial meningitis, cerebral malaria, and AIDS
  • autoimmune e.g., rheumatoid arthritis, Crohn's disease, sarcoidosis, multiple sclerosis, Kawasaki syndrome, graft-versus host disease and transplant rejection
  • organ failure e.g., adult respiratory distress syndrome
  • Bacterial DNA can also sensitize mice for the action of LPS and more importantly, bacterial DNA or oligonucleotides (in the following abbreviated by the letters "ODN”) comprising (a) CpG-dinucleotide(s) synergise with LPS in inducing macrophage cytokines in vitro and toxic amounts of TNF- ⁇ in vivo (Sparigan T, Montgomeryhke T, Lipford G, et al., Eur J Immunol 1997; 27: 1671 ; Cowdery JS, Chace JH, Yi AK, Krieg AM, J Immunol 1996; 156: 4570).
  • ODN bacterial DNA or oligonucleotides
  • Th1 response patterns have been demonstrated in a variety of diseases: infectious (e.g., streptococcal induced arthritis, and Lyme arthritis), and autoimmune (e.g., rheumatoid arthritis, chronic inflammatory bowel disease, psoriasis vulgaris, experimental allergic encephalomyelitis (EAE), insulin-dependent diabetes mellitus (IDDM), spontaneous abortion, graft-versus host disease and transplant rejection).
  • Th2 responses are beneficial in various helminth infection and bacterial responses. Thus, in the context of vaccination the ability to direct responses toward Th2 for certain diseases would be beneficial.
  • Vaccination is the process of preparing an animal to respond to an antigen. Vaccination is more complex than immune recognition and involves not only B cells and cytotoxic T cells but other types of lymphoid cells as well. During vaccination, cells, which recognize the antigen (B cells or cytotoxic T cells), are clonally expanded. In addition, the population of ancillary cells (helper T cells which provide co-receptor and cytokine stimulation) specific for the antigen also increase. Vaccination also involves specialized antigen presenting cells, which can process the antigen and display it in a form, which can stimulate one of the two pathways (macrophages and dendritic cells).
  • a foreign antigen is introduced into an animal where it activates specific B cells by binding to surface immunoglobulins. It is also taken up by antigen processing cells, wherein it is degraded, and appears in fragments on the surface of these cells bound to Class II MHC molecules. Peptides bound to class II molecules are capable of stimulating the helper class of T cells. Both helper T cells and activated B cells are required to produce active humoral immunization. Cellular immunity is stimulated by a similar mechanism but entry into the MHC I presentation pathway of antigen presenting cells is typically by intracellular pathogen replication and not achieved by injection of protein antigen only.
  • Standard vaccination schemes nearly always produce a humoral immune response.
  • the humoral system protects a vaccinated individual from subsequent challenge from a pathogen and can prevent the spread of an intracellular infection if the pathogen goes through an extracellular phase during its life cycle; however, it can do relatively little to eliminate intracellular pathogens.
  • Cytotoxic immunity complements the humoraf system by eliminating the infected cells and cancer cells. Thus effective vaccination should activate both types of immunity.
  • cytotoxic T cell response is necessary to remove intracellular pathogens such as viruses as well as malignant cells.
  • pathogens such as viruses as well as malignant cells.
  • a major problem exists in the prior art with respect to the presention of an exogenously administered antigen in adequate concentrations in conjunction with Class I molecules to assure an adequate response.
  • mmune adjuvant refers to compounds which when administered to an individual or tested in vitro, increase the immune response to an antigen.
  • immune adjuvants are known in the art. These include classical adjuvants such as complete Freund's adjuvant or more recently developed compounds like liposomes. Many of these adjuvants, however, have adverse side effects such as local inflammation after administration, or their production is time consuming and/or requires complicated standardized production protocols, in particular when vaccination of humans is envisaged.
  • the present invention relates to a composition
  • a composition comprising an oligonucleotide comprising
  • Ni represents any nucleotide if N 2 and N 3 are G
  • N 2 represents any nucleotide if Ni and N 3 are G
  • N 3 represents any nucleotide if Ni and N 2 are G
  • composition denotes a solution, e.g., a buffer, comprising at least one of the oligonucleotides of the present invention.
  • composition denotes a formulation comprising at least one of the oligonucleotides of the present invention and at least one further compound. Said formulation may be of liquid, solid or vaporous nature.
  • nucleotide analog or derivative denotes any nucleoside phosphate the nucleoside of which deviates in its chemical structure from the nucleosides guanosine, adenosine, thymidine, uridine or cytidine.
  • modified nucleosides are well known to the person skilled in the art and comprise, e.g., 5,6-dihydrouridine, ribothymidine, inosine or 1-methylguanosine (see, e.g. Lewin B, Genes, 1983, John Wiley & Sons, Inc., NY).
  • one or more nucleotides of the oligonucleotides of the present invention or the oligonucleotides to be employed in accordance with the composition of the present invention may be replaced by said nucleotide analogs and/or derivatives as long as the modified oligonucleotides remain functionally equivalent to their unmodified counterparts, i.e. as long as they maintain essentially the same portfolio of biological activities that are described in accordance with this invention.
  • "Essentially the same portfolio of biological activities” means that the modified oligonucleotides described above display at least one of the biological activities described in accordance with this invention.
  • the different biological activities of the modified oligonucleotides may be more or less pronounced compared to the corresponding activities of their unmodified counterparts.
  • a potential deleterious effect of foreign DNA and CpG motif(s) containing oligonucleotides derived thereof is the activation of myeloid lineage immune cells and/or lymphoid lineage cells to produce toxic cytokines.
  • the strong Th1 inducing potential of foreign DNA can hyperactivate the immune system leading to potential autoreactivity and tissue damage.
  • the oligonucleotides of the present invention and/or the oligonucleotides to be employed in accordance with the composition of the present invention have the potential to efficiently block the activating effects on antigen-presenting cells of foreign DNA or CpG motif(s) containing oligonucleotides derived therefrom.
  • the experiments performed in accordance with the present invention suggest that foreign DNA uptake is receptor mediated and that blockade of this cell surface DNA receptor with the oligonucleotides of the present invention or the oligonucleotides to be employed in accordance with the composition of the present invention blocks the CpG driven cell activation.
  • T cell costimulation by the oligonucleotides of the present invention and/or the oligonucleotides to be employed in accordance with the composition of the present invention may be advantageously used to promote T cell activation in the apparent absence of APCs, thus circumventing the need of APC mediated cross priming.
  • T cell costimulation mediated by the oligonucleotides of the present invention or the oligonucleotides to be employed in accordance with the composition of the present invention also allows induction of CTLs in vivo.
  • the oligonucleotides of the present invention and/or the oligonucleotides to be employed in accordance with the composition of the present invention may be used to beneficially influence the overall cytokine production of the cells of the immune system in that the production of (co-)stimulatory cytokines is enhanced whereas the production of inflammatory cytokines like, e.g., TNF- ⁇ is suppressed.
  • a further advantage of the oligonucleotides of the present invention and/or the oligonucleotides to be employed in accordance with the composition of the present invention is that, especially if chemically synthesized, they represent compounds which can be easily produced at low costs. Moreover, due to the chemical production, the apparent absence of, e.g., contaminating pathogens, allergens or otherwise harmful agents makes the oligonucleotides of the present invention and/or the oligonucleotides to be employed in accordance with the composition of the present invention superior compounds when used as pharmaceutical compositions, for the preparation of a pharmaceutical composition or in methods for the treatment of diseases.
  • Ni represents G > A > T/U > C if N 2 and N 3 are G
  • N 2 represents G > A > T/U > C if Ni and N 3 are G
  • N 3 represents G > A > T/U > C if Ni and N 2 are G
  • N-i, N 2 , and N 3 represent a nucleotide analog or derivative of the nucleotides of (a).
  • G > A refers to the fact that Gs in the mentioned position usually show an improved biological effect over As. Accordingly, Gs are more preferred in said position than As. This rule mutatis mutandis applies to the above- referenced further relationships between nucleotides.
  • oligonucleotides are most effective if the positions N ⁇ N 2 , and/or N 3 are occupied by Gs.
  • Adenosine phosphates are almost equally preferred. Less preferred are T/Us, and least preferred are Cs.
  • composition of the present invention said oligonucleotide consists of between 10 and 50 nucleotides.
  • said oligonucleotide consists of between 13 and 30 nucleotides. In a most preferred embodiment of the composition of the present invention said oligonucleotide consists of between 17 and 21 nucleotides.
  • the G- rich pentamer motif as defined above represents the 3'-terminus of said oligonucleotide.
  • the present invention also relates to an oligonucleotide consisting of
  • the nucleotides of said oligonucleotide are linked via phosphodiester-, phosphorothioate-, methylphosphonate- or peptide bonds.
  • the oligonucleotides of the present invention and/or the oligonucleotides to be employed in accordance with the composition of the present invention may be peptide nucleic acids (PNAs).
  • PNAs peptide nucleic acids
  • nucleotides of the oligonucleotides of the present invention and/or the oligonucleotides to be employed in accordance with the composition of the present invention via bonds other than phosphodiester bonds may increase their resistance against nucleases.
  • the nucleotides in the preparation of a pharmaceutical composition or in a method for the treatment of a disease, may be advantageously linked via nuclease-resistant bonds which increase the half-life of the corresponding oligonucleotide in, e.g., the organism. This may, in turn, decrease the frequency of administration of the pharmaceutical composition and, thus, may, e.g., contribute to a patient's comfort.
  • oligonucleotide is DNA or RNA.
  • the oligonucleotides of the present invention and/or the oligonucleotides to be employed in accordance with the composition of the present invention may be DNA, cDNA, RNA or (semi)synthetically produced DNA or RNA or a recombinantly produced chimeric nucleic acid molecule.
  • composition or the oligonucleotide of the present invention said oligonucleotide is single-stranded.
  • composition of the present invention is a pharmaceutical composition optionally comprising a pharmaceutically acceptable carrier and/or diluent.
  • Suitable pharmaceutical carriers include phosphate buffered saline solutions, water, emulsions, such as oil/water emulsions, various types of wetting agents, sterile solutions etc.
  • Compositions comprising such carriers can be formulated by well known conventional methods. These pharmaceutical compositions can be administered to the subject at a suitable dose. Administration of the suitable compositions may be effected by different ways, e.g., by intravenous, intraperitoneal, subcutaneous, intramuscular, topical, intradermal, intranasal or intrabronchial administration. The dosage regimen will be determined by the attending physician and clinical factors.
  • dosages for any one patient depends upon many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently.
  • a typical dose can be, for example, in the range of 0.001 to 1000 ⁇ g (or of nucleic acid for expression or for inhibition of expression in this range); however, doses below or above this exemplary range are envisioned, especially considering the aforementioned factors.
  • the regimen as a regular administration of the pharmaceutical composition should be in the range of 1 ⁇ g to 10 mg units per day. If the regimen is a continuous infusion, it should also be in the range of 1 ⁇ g to 10 mg units per kilogram of body weight per minute, respectively.
  • compositions of the invention may be administered locally or systemically.
  • DNA may also be administered directly to the target site, e.g., by biolistic delivery to an internal or external target site or by catheter to a site in an artery.
  • Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions.
  • 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.
  • Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils.
  • Intravenous vehicles include fluid and 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, anti-oxidants, chelating agents, and inert gases and the like.
  • the pharmaceutical composition of the invention may comprise further agents depending on the intended use of the pharmaceutical composition.
  • composition of the present invention is a vaccine.
  • the oligonucleotides of the present invention and/or the oligonucleotides to be employed in accordance with the composition of the present invention may advantageously be used in the development of vaccines against tumor-specific antigens (e.g., on breast or colon cancer cells), and against weakly immunogenic viral proteins (e.g., HIV, Herpes, non-A, non-B hepatitis, CMV and EBV).
  • tumor-specific antigens e.g., on breast or colon cancer cells
  • weakly immunogenic viral proteins e.g., HIV, Herpes, non-A, non-B hepatitis, CMV and EBV.
  • oligonucleotides may advantageously be used to provide such a response against both chronic and latent viral infections and against malignant cells.
  • the oligonucleotides of the present invention and/or the oligonucleotides to be employed in accordance with the composition of the present invention may be used to efficiently enhance the immune response of an individual against an antigen which is weakly immunogenic when administered alone or which is toxic at the concentration which evokes an immune response.
  • the present invention relates to a vector comprising an oligonucleotide of the present invention.
  • the vector of the present invention may be, e.g., a plasmid, cosmid, virus, bacteriophage or another vector used conventionally in genetic engineering, and may comprise further genes such as marker genes which allow for the selection of said vector in a suitable host cell and under suitable conditions.
  • the present invention relates to a host cell comprising the vector of the present invention.
  • the host cell can be any prokaryotic or eukaryotic cell, such as a bacterial, preferably Escherichia coli, insect, fungal, preferably Saccharomyces cerevisiae, plant, animal or human cell.
  • a prokaryotic or eukaryotic cell such as a bacterial, preferably Escherichia coli, insect, fungal, preferably Saccharomyces cerevisiae, plant, animal or human cell.
  • the present invention also relates to a method for the production of the oligonucleotide of the present invention, said method comprising the steps of culturing the host cell of the present invention under conditions that cause production of the oligonucleotide, and recovering said oligonucleotide from the culture.
  • the oligonucleotide of the present invention or the oligonucleotide to be employed in accordance with the composition of the present invention may be chemically synthesized according to methods well known in the art (Finnan et al. Nucleic Acids Symp Ser 7, 1980, 133-145), or may be a (semi)synthetic nucleic acid.
  • (semi)synthetic denotes oligonucleotides which, after their chemical synthesis, have been, e.g., enzymatically modified. Such modifications are well known in the art and comprise, e.g., the introduction of methyl residues into the oligonucleotide.
  • (semi)synthetic also denotes oligonucleotides which comprise a chemically and a biologically synthesized part.
  • the present invention further relates to an oligonucleotide obtainable by the method of the present invention.
  • the present invention relates to a kit comprising the composition, the oligonucleotide, the vector, and/or the host cell of the present invention.
  • kits of the invention may be packaged in containers such as vials, optionally in buffers and/or solutions. If appropriate, one or more of said components may be packaged in one and the same container.
  • the present invention relates to the use of the composition, and/or the oligonucleotide of the present invention, and/or the oligonucleotide to be employed in accordance with the composition of the present invention for the production of a pharmaceutical composition for preventing or treating septic shock, inflammation, autoimmune diseases, TH1 -mediated diseases, bacterial infections, parasitic infections, viral infections, spontaneous abortions, and/or tumors.
  • the present invention relates to a method for preventing or treating septic shock, inflammation, autoimmune diseases, T 1 -mediated diseases, bacterial infections, parasitic infections, viral infections, spontaneous abortions, and/or tumors comprising administering to a subject the composition, and/or the oligonucleotide of the present invention, and/or the oligonucleotide to be employed in accordance with the composition of the present invention.
  • said septic shock is induced by DNA, preferably of non-vertebrate origin
  • said autoimmune diseases are rheumatoid arthritis, Crohn's disease, sarcoidosis, multiple sclerosis, Kawasaki syndrome, graft-versus-host disease, and/or transplant rejection
  • said T H 1 -mediated diseases are streptococcal induced arthritis, Lyme arthritis, chronic inflammatory bowel disease, psoriasis vulgaris, experimental allergic encephalomyelitis (EAE), and/or insulin-dependent diabetes mellitus (IDDM)
  • said parasitic infections are Leishmaniasis or Toxoplasmosis
  • said viral infections are Cytomegalovirus- and/or HIV-infection.
  • Septic shock is inducible not only by DNA but also by, e.g., LPS and superantigens.
  • LPS low-density polypeptide
  • septic shock induced by LPS or superantigens can not be blocked by the oligonucleotides of the present invention.
  • the oligonucleotides of the present invention may be used to suppress the synergistic action of DNA and a second inducer of septic shock.
  • the time point of administration is not crucial for the oligonucleotides of the present invention to effectively block septic shock.
  • the oligonucleotides of the present invention are superior to known blocker of septic shock like, e.g., anti-TNF antibodies where the time point of administration is not only of extraordinary importance but may decide whether the treatment results in a positive or detrimental outcome.
  • the present invention relates to the use of the composition, and/or the oligonucleotide of the present invention, and/or the oligonucleotide to be employed in accordance with the composition of the present invention to inhibit activation of antigen-presenting cells.
  • said antigen- presenting cells are macrophages, dentritic cells and/or B-lymphocytes. Macrophages, dendritic cells and B cells (antigen presenting cells) utilizing PRRs recognize non-self DNA through CpG motifs and initiate inflammatory responses.
  • IFN- ⁇ / ⁇ produced by immunostimulatory ODN stimulated spleen cells possibly came from an adherent cell population (Yamamoto S, Kuramoto E, Shimada S, Tokunaga T., Jpn J Cancer Res 1988; 79: 866).
  • Mac-1 CD11 b/CD18
  • PMNs human polymorphonuclear leukocytes
  • ROS reactive oxygen species
  • the present invention relates to the use of the composition, and/or the oligonucleotide of the present invention, and/or the oligonucleotide to be employed in accordance with the composition of the present invention to inhibit the uptake of DNA by a cell.
  • the present invention relates to the use of the composition, and/or the oligonucleotide of the present invention, and/or the oligonucleotide to be employed in accordance with the composition of the present invention to co-stimulate cytotoxic T-lymphocytes or natural killer cells.
  • Cytotoxic cells e.g. cytolytic T cells or NK cells are responsible for tumor cell and pathogen infected cell removal. The stimulation of either of these immune cells enhances tumor clearance and pathogen clearance.
  • the present invention relates to the use of the composition, and/or the oligonucleotide of the present invention, and/or the oligonucleotide to be employed in accordance with the composition of the present invention to stimulate natural killer cells.
  • the present invention also relates to the use of the composition, and/or the oligonucleotide of the present invention, and/or the oligonucleotide to be employed in accordance with the composition of the present invention to enhance the production of antibodies directed against an antigen.
  • NK cells monitor for the reduction of antigen presenting MHC molecules on the surface of a cell. Often tumors or virally infected cells have reduced MHC molecules on their surface. Thus, NK cells are capable of sensing and killing tumor cells and some virally infected cells. The stimulation of NK cells enhances their surveillance and killing potential and therefore their ability to detect and eliminate tumor cells or virally infected cells.
  • the present invention relates to the use of the composition, and/or the oligonucleotide of the present invention, and/or the oligonucleotide to be employed in accordance with the composition of the present invention to enhance the uptake of an agent by a cell.
  • the oligonucleotides of the present invention may be readily internalized by cells.
  • the oligonucleotide of the present invention or the oligonucleotide to be employed in accordance with the composition of the present invention may be utilized as carriers which make possible or facilitate the internalization of an agent by a cell.
  • Said agent may be, e.g., another nucleic acid molecule, a protein, a peptide, a drug, a hormone, a toxin, etc.
  • said agent may be covalently linked to the oligonucleotide of the present invention.
  • the carrier and the agent may also be linked, e.g., via a protein-protein or protein-nucleic acid interaction.
  • the agent is another nucleic acid molecule, of course, the oligonucleotide of the present invention may also be introduced into said nucleic acid molecule by well known methods. Such methods as well as procedures which may be used to produce, e.g., nucleic acid molecules and/or proteins capable of interacting with each other are described, e.g., in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989), and Ausubel et al., Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, New York, N.Y. (1989).
  • said agent is a nucleic acid or a (poly)peptide.
  • nucleic acid is a gene therapy vector.
  • Gene therapy which is based on introducing therapeutic genes into cells by ex-vivo or in-vivo techniques is one of the most important applications of gene transfer.
  • Suitable vectors and methods for in-vitro or in-vivo gene therapy are described in the literature and are known to the person skilled in the art; see, e.g., Giordano, Nature Medicine 2 (1996), 534-539; Schaper, Circ. Res. 79 (1996), 911-919; Anderson, Science 256 (1992), 808-813; Isner, Lancet 348 (1996), 370-374; Muhlhauser, Circ. Res.
  • the present invention relates to a method for inducing proliferation of bone marrow cells in vitro or in vivo comprising culturing bone marrow cells in the presence of the composition, and/or the oligonucleotide of the present invention, and/or the oligonucleotide to be employed in accordance with the composition of the present invention.
  • the present invention relates to the use of the composition, and/or the oligonucleotide of the present invention, and/or the oligonucleotide to be employed in accordance with the composition of the present invention to induce proliferation of bone marrow cells in vitro or in vivo.
  • said bone marrow cells are macrophage precursor cells.
  • progenitors for all lineages of the cells of the immune system are generated. Descending from pluripotent stem cells progenitors proliferate and differentiate under the influence of hematopoietic growth factors and cytokines. Agents that interfere with the cell cycle or change levels of hematopoietic cytokines have therefore profound effects on the homeostasis of cells generated in the bone marrow. For example, treatment of malignant neoplasias with cytostatic drugs is often limited by severe leukopenia. On the other hand, administration of colony stimulating factors like G-CSF leads to enhanced proliferation of granulocyte precursors and their mobilization into peripheral blood. Situations where cytokines are produced systemically also influence the production of blood cells in the bone marrow and the relative lineage distribution.
  • the oligonucleotide of the present invention and/or the oligonucleotide to be employed in accordance with the composition of the present invention can be advantageously used to induce proliferation of bone marrow cells.
  • the oligonucleotide of the present invention and/or the oligonucleotide to be employed in accordance with the composition of the present invention can be advantageously used to induce proliferation of bone marrow cells.
  • up to the making of the present invention it was not possible to cultivate macrophage precursor cells in vitro. This is mainly due to the fact that all agents that were available so far and suitable for the propagation of macrophage precursor cells in vitro either themselves also induce differentiation of said precursor cells or induce the production of differentiation stimulating cytokines, thereby obviating further proliferation of said precursor cells.
  • the oligonucleotides of the present invention induce neither differentiation of said precursor cells nor the synthesis of the above mentioned cytokines.
  • the present invention enables for the first time the person skilled in the art to propagate macrophage precursor cells in vitro.
  • the oligonucleotides of the present invention do not display the above mentioned and other adverse side effects of cytokines used up to now in such methods and, thus, lead to a substantial and unexpected improvement of the same.
  • oligonucleotides of the invention in combination with cytokines and/or growth factors.
  • the present invention relates to the use of the composition, and/or the oligonucleotide of the present invention, and/or the oligonucleotide to be employed in accordance with the composition of the present invention to induce apoptosis in tumor cells.
  • the oligonucleotides of the present invention fail to induce TNF and inhibit TNF production induced by immunostimulatory DNA.
  • oligonucleotides of the present invention inhibit TNF secretion induced by immunostimulatory DNA.
  • the oligonucleotides of the present invention block entry of bacterial DNA into macrophages and thus block CpG induced signaling.
  • the oligonucleotides of the present invention prevent lethal shock induced by immunostimulatory DNA. Sequence specificity and dose dependency.
  • Figure 6 The oligonucleotides of the present invention do not interfere with Superantigen- or LPS-induced lethal cytokine syndrome.
  • oligonucleotides of the present invention act as adjuvants for generation of antigen-specific cytotoxic T cells in vivo.
  • the oligonucleotides of the present invention induce NK activity in vivo.
  • BMC stimulated with an oligonucleotide of the present invention are responsive to M-
  • G-motif ODN denotes the oligonucleotides of the present invention and/or the oligonucleotides to be employed in accordance with the composition of the present invention.
  • Example 1 G-motif ODN fail to induce TNF and inhibit TNF production induced by immunostimulatory DNA.
  • the macrophage cell line J774 was seeded at 10 5 /culture and stimulated with the reagents indicated in Figure 1A. After 12 h the cell culture supernatant was removed and tested for TNF content by ELISA.
  • the results in Figure 1 A demonstrate that LPS and the immunostimulatory CpG-motif ODN (1668) induced TNF secretion.
  • G-motif ODN PZ1 , PZ2 and PZ3 failed to induced TNF.
  • the control ODN PZ4, PZ5 or poly-G were also ineffective.
  • the macrophage cell line J774 was seeded at 10 5 /culture and stimulated with the reagents indicated in Figure 1 B. To cultures that were stimulated with ODN 1668 at 1 ⁇ M, different amounts of PZ-ODN were added. After 12 h the cell culture supernatant was removed and tested for TNF content by ELISA. The results shown in Figure 1 B demonstrate that the ODN PZ1 , PZ2 and PZ3 inhibited the TNF inducing capacity of ODN 1668 even at very low concentrations. ODN PZ4 and PZ5 and poly-G were only effective at high concentrations.
  • Example 2 G-motif ODN inhibit TNF secretion induced by immunostimulatory DNA.
  • the macrophage cell line J774 was seeded at 10 5 /culture and stimulated with the reagents indicated in Figure 2. After 6 h the cell culture supernatant was removed and tested for TNF content by ELISA.
  • the dose response experiment shown in Figure 2A demonstrates that ODN PZ1 , PZ2 and PZ3 inhibit TNF secretion induced by the immunostimulatory ODN 1668 even at very low concentrations.
  • FIG. 2B shows the ODN PZ1 and PZ2 that were added to cultures stimulated with ODN 1668 at the time points and concentrations indicated.
  • the results reveal that addition of G-motif ODN even after 120 min reduced TNF secretion, i.e. inhibition with PZ ODN can occur post CpG-motif stimulation
  • Figure 2C shows the results of an experiment performed to investigate the time kinetics of G-motif ODN induced inhibition of TNF secretion.
  • the macrophage cell line J774 was seeded at 10 5 /culture and stimulated with the reagents indicated.
  • the G-motif ODN PZ1 , PZ2 and PZ3 were added as inhibitors at the indicated time.
  • Example 3 G-motif ODN block entry of bacterial DNA into macrophages and thus block CpG induced signaling.
  • Example 4 Inhibition of macrophage signal transduction by blockade of CpG ODN.
  • the macrophage cell line, RAW-264 was stably transfected with vectors which utilized a NFkB response element (panel A), the TNF-a promoter (panel B) or the IL- 12 p40 promoter (see panel C of Figure 4) driving the expression of a luciferase gene.
  • the cells were stimulated for 3 h with CpG ODN (closed bars) or LPS (hatched bars) to induce luciferase activity measured as arbitrary light units in the presence or absence of a G-motif ODN.
  • N represents connectednot tested" because RAW 264 cells do not induce IL-12 p40 promoter activity in response to LPS.
  • results are given as percent control which represents the mean arbitrary light units in the presence of inhibitor divided by the uninhibited reading. The values represent mean and standard deviations of 3 determinations. These results demonstrate that blockade with G-motif ODN inhibits macrophage responses to foreign DNA at the promoter level. This includes reduction of NFkB activity, a critical transcription factor which promotes the induction of many inflammatory responses. TNF promoter activity is also reduced, in compliance with the cytokine release data ( Figure 1 and 2). In addition the promoter activity for IL-12 is reduced, a critical cytokine needed for Th1 induction.
  • Example 5 G-motif ODN prevent lethal shock induced by immunostimulatory DNA. Sequence specificity and dose dependency.
  • mice were injected with 5 nmol of ODN 1668 plus D-galactosamine (D-GalN) i.p. (closed circles in Figure 5A). The number of surviving mice is shown.
  • One group of mice received in addition the G-motif ODN PZ2 (closed diamonds in Figure 5A) or a non G-motif ODN control (PZ5) (closed squares in Figure 5A) at 5 nmol.
  • Example 6 G-motif ODN do not interfere with Superantigen- or LPS-induced lethal cytokine syndrome.
  • mice were injected i.p. with 10 ⁇ g SEB and 20 mg D-galactosamine (D-GalN) into BALB/c mice. The number of surviving mice is shown in Figure 6A (open circles). It was then tested whether the G-motif ODN PZ2 would be able to interfere with the superantigen-induced lethal shock. To this PZ2 was injected with SEB at 10 nmol / mouse i.p.(closed circles in Figure 6A).
  • LPS (Endot xin)-mediated shock was induced by injecting 10 ⁇ g LPS and 20 mg D- GalN into BALB/c mice (closed circles in Figure 6B).
  • mice C57BI/6 mice were injected with liposome entrapped Ovalbumin (Ova) into the footpad.
  • Ova liposome entrapped Ovalbumin
  • One group of mice received 10 nmol of ODN PZ2 in addition (see Figure 7B).
  • the draining lymph nodes were removed and the cells prepared. They were then cultured at 3 x 10 6 in 2 ml tissue culture plates with 10U/ml recombinant IL-2. After four days the cells were harvested and tested in a ⁇ Cr- release assay for cytolytic activity against syngeneic target cells pulsed with either the immunodominant Ova-peptide or a third party peptide (VSV).
  • VSV third party peptide
  • Example 8 G-motif ODN induce NK activity in vivo.
  • C57BI/6 mice were injected with 10 nmol of ODN PZ2 into the footpad. After four days the draining lymph nodes were removed and the cells prepared. They were then cultured at 3 x 10 6 in 2 ml tissue culture plates with 10U/ml recombinant IL-2. After four days the cells were harvested and tested in a 51 Cr-release assay for cytolytic activity against NK-sensitive YAC-1 target cells. Prior to the cytotoxicity assay from the effector population CD4 + cells (closed triangle up in Figure 8), CD8 + cells (closed triangle down in Figure 8) or both T cell populations (closed diamond in Figure 8) were removed by magnetic cell sorting.
  • the NK effector cell is CD4 and CD8 negative and resembles a classical NK cell population.
  • Example 9 Sequence dependency of T cell costimulation induced by G-motif ODN.
  • T lymphocytes from lymph node cells from C57 BI/6 mice were negatively selected. To accomplish this cells were first passed over a Sephadex G10 column to remove macrophages and activated lymphocytes. Then the cells were incubated with magnetic beads coupled with anti-mouse IgG and the lgG + B cell population was removed by magnetic cell sorting. The purity of the T cell population exceeded > 97% as determined by cytometrical analyses.
  • T cells were stimulated in cell culture medium containing 10% FCS, antibiotics and indomethacin. Cultures were set up in u-shaped microtiter plates that had been precoated with anti-CD3 monoclonal hamster antibodies. To this plates were incubated with 10 ⁇ g rabbit anti hamster polyclonal antibodies in PBS for 16 h. Thereafter the plates were washed with PBS and the plastic surface was blocked by addition of 2% BSA for 4 h. After additional washing 1 ⁇ g/ml anti-CD3 monoclonal antibody in PBS was added. After incubation for 16h plates were again washed and then used for the cultures. 15,000 selected T cells were added to each culture.
  • Example 10 Single stranded G-motif ODN but not double-stranded or complementary ODN costimulated T cells.
  • T lymphocytes from lymph node cells from C57 BI/6 mice were negatively selected. To this cells were first passed over a Sephadex G10 column to remove macrophages and activated lymphocytes. Then the cells were incubated with magnetic beads coupled with anti-mouse IgG and the lgG + B cell population was removed by magnetic cell sorting. The purity of the T cell population exceeded > 97% as determined by cytometrical analyses.
  • T cells were stimulated in cell culture medium containing 10% FCS, antibiotics and indomethacin. Cultures were set up in u-shaped microtiter plates that had been precoated with anti-CD3 monoclonal hamster antibodies. To this plates were incubated with 10 ⁇ g rabbit anti hamster polyclonal antibodies in PBS for 16 h. Thereafter the plates were washed with PBS and the plastic surface was blocked by addition of 2% BSA for 4 h. After additional washing 1 ⁇ g/ml anti-CD3 monoclonal antibody in PBS was added. After incubation for 16h plates were again washed and then used for the cultures. 15,000 selected T cells were added to each culture.
  • Controls included supplementation with IL-2 (10U/ml), or LPS (10 ⁇ g/ml). Oligonucleotides were added at 5 ⁇ M, 1 ⁇ M or 0.2 ⁇ M. 4 replicate cultures each were set up. After 4 days the cultures were pulsed with 3 H-thymidine and the proliferative response was recorded.
  • the experiment shown in Figure 10A demonstrates that the G-motif ODN PZ1 , PZ2 and PZ3 but not the controls PZ4 or PZ5 costimulate T cells.
  • the DNA- complementary derivatives of PZ1 K, PZ2K and PZ3K are negative.
  • ODN 1628 (a CpG-ODN) was used as a positive control.
  • poly G or poly A ODN do not stimulate T cells.
  • the experiment shown in Figure 10B details that the G-motif ODN are only active in single stranded form (PZ1 , PZ2 and PZ3).
  • the double-stranded form (PZ1 , PZ2 and PZ3 plus PZ1 K, PZ2K and PZ3K, respectively) fail to costimulated T cells.
  • poly G does not stimulate T cells.
  • Example 11 Proliferation of mouse splenocvtes and primary BMC in response to a G-motif ODN.
  • Example 12 Restimulation of BMC cultured for six days with a G-motif ODN.
  • Example 13 G-motif stimulated BMC are responsive to M-CSF and GM-CSF. but not to G-CSF.
  • Example 14 Sequence specificity of the G-motif binding protein.
  • Extracts of J774 cells were incubated with single-stranded, radiolabeled GR1 for 30 min at room temperature. Different G-motif and non-G-motif containing competitors were added in a 150-fold molar excess. Treatment with Proteinase K or RNAseA was performed before the binding reaction was initiated. Free probe was separated from complexes formed by running a 5 % non-denaturing PAGE.
  • RNAse A failed to destroy the target, thus the target is not RNA as would be the case for antisense
  • Example 15 UV-crosslink analysis of binding of single-stranded G-motif ODN to protein extracts prepared from J774 cells. After incubation in hypotonic buffer, J774 cells were lysed by douncing, followed by pelleting of the nuclei. The supernatant was transferred to a new tube and cleared of insoluble material by a 30 min centrifugation at 35,000 x g. 5 ⁇ g of the extract was incubated for at room temperature for 30 min with 3 ng of 33 P-labeled G-motif ODN. To reduce non-specific binding 1 ⁇ g of polydldC was added to the binding reaction.
  • Figure 15 shows that two protein bands become detectable by this method, at approximately 58kD and 25kD. These binding protein are apparently non-nuclear in origin and are specific for the G-motif similar to the protein target detected and discussed in Example 14, supra.
  • Bold lettering represents changed nucleotides from the initial PZ1 ODN.
  • the sequence PZ3 was iteratively modified in the core G-motif. These sequences were tested through a blocking titration range similar to fig. 1 B and fig.2A. A determination of the 50% inhibitory concentration (IC50) is given for each ODN.
  • Table 2A Data arrange to easily show the iterative process. Last column of values from preliminary experiment.
  • Table 2B Data arrange to easily show the affinity rank order of the oligonucleotides of the present invention. Range of affinity differences from highest too lowest greater than 62 fold. These data combined with the uptake data (Fig. 3) demonstrate that a sequence selective receptor on the surface of cells is responsible for DNA uptake. Because the receptor is sequence selective high affinity interacting oligonucleotides can be designed which interfere with the potential inflammatory effects of CpG-motif containing DNA.

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Abstract

L'invention porte sur une composition composée d'un oligonucléotide comprenant (a) la séquence N1-N2-G-N3-G, dans laquelle N1 représente tout nucléotide si N2 et N3 sont G; N2 représente tout nucléotide si N1 et N3 sont G; et N3 représente tout nucléotide si N1 et N2 sont G, ou (b) la séquence de (a), dans laquelle au moins un nucléotide est remplacé par un nucléotide correspondant analogue ou dérivé. L'invention porte en outre sur l'utilisation de la composition, et/ou du susdit pour la production d'une composition pharmaceutique destinée à prévenir ou traiter le choc septique, les inflammations, les maladies auto-immunes, les maladies induites par le TH1-, les infections bactériennes, les infections parasitaires, les infections virales, les avortements spontanés, et/ou les tumeurs.
EP99946118A 1998-09-03 1999-09-03 Oligonucleotides a motif g et leurs utilisations Withdrawn EP1108017A2 (fr)

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Families Citing this family (39)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6207646B1 (en) 1994-07-15 2001-03-27 University Of Iowa Research Foundation Immunostimulatory nucleic acid molecules
US20030026782A1 (en) 1995-02-07 2003-02-06 Arthur M. Krieg Immunomodulatory oligonucleotides
US6239116B1 (en) 1994-07-15 2001-05-29 University Of Iowa Research Foundation Immunostimulatory nucleic acid molecules
US7935675B1 (en) 1994-07-15 2011-05-03 University Of Iowa Research Foundation Immunostimulatory nucleic acid molecules
EP0855184A1 (fr) 1997-01-23 1998-07-29 Grayson B. Dr. Lipford Composition pharmaceutique comprenant un polynucléotide et un antigène notamment pour la vaccination
US6406705B1 (en) 1997-03-10 2002-06-18 University Of Iowa Research Foundation Use of nucleic acids containing unmethylated CpG dinucleotide as an adjuvant
DK1733735T3 (en) 1998-05-22 2017-06-19 Ottawa Hospital Res Inst Methods and products for the induction of mucosal immunity
ES2572834T3 (es) 1999-02-17 2016-06-02 Csl Limited Complejos inmunogénicos y métodos relacionados con los mismos
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
US6949520B1 (en) 1999-09-27 2005-09-27 Coley Pharmaceutical Group, Inc. Methods related to immunostimulatory nucleic acid-induced interferon
EP1322655B1 (fr) 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 Oligonucleotide synthetique et son utilisation pour induire une reaction immunitaire
US7585847B2 (en) 2000-02-03 2009-09-08 Coley Pharmaceutical Group, Inc. Immunostimulatory nucleic acids for the treatment of asthma and allergy
WO2001097843A2 (fr) 2000-06-22 2001-12-27 University Of Iowa Research Foundation Procedes d'amelioration de la lyse cellulaire provoquee par des anticorps et procedes de traitement du cancer
JP2005500806A (ja) 2000-09-15 2005-01-13 コーリー ファーマシューティカル ゲーエムベーハー ＣｐＧに基づく免疫アゴニスト／免疫アンタゴニストの高スループットスクリーニングのためのプロセス
WO2003020884A2 (fr) 2001-08-14 2003-03-13 The Government Of The United States Of America As Represented By The Secretary Of Health And Human Services Procede de generation rapide de cellules dentritique matures
ES2314099T3 (es) 2001-08-17 2009-03-16 Coley Pharmaceutical Gmbh Oligonucleotidos inmunoestimulantes con motivos combinados con actividad mejorada.
EP1450856B1 (fr) 2001-09-14 2009-11-11 Cytos Biotechnology AG Emballage de cpg dans des particules de type virus : procede de preparation et utilisation correspondante
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
CA2480775C (fr) 2002-04-04 2016-05-31 Coley Pharmaceutical Gmbh Oligoribonucleotides immunostimulants contenant de la guanine et de l'uracile
AU2003219383B2 (en) 2002-04-22 2010-08-26 Bioniche Life Sciences Inc. Oligonucleotide compositions and their use for the modulation of immune responses
CA2388049A1 (fr) 2002-05-30 2003-11-30 Immunotech S.A. Oligonucleotides immunostimulateurs et utilisations connexes
US20040053880A1 (en) 2002-07-03 2004-03-18 Coley Pharmaceutical Group, Inc. Nucleic acid compositions for stimulating immune responses
US7807803B2 (en) 2002-07-03 2010-10-05 Coley Pharmaceutical Group, Inc. Nucleic acid compositions for stimulating immune responses
US7569553B2 (en) 2002-07-03 2009-08-04 Coley Pharmaceutical Group, Inc. Nucleic acid compositions for stimulating immune responses
US7576066B2 (en) 2002-07-03 2009-08-18 Coley Pharmaceutical Group, Inc. Nucleic acid compositions for stimulating immune responses
US7605138B2 (en) 2002-07-03 2009-10-20 Coley Pharmaceutical Group, Inc. Nucleic acid compositions for stimulating immune responses
AR040996A1 (es) 2002-08-19 2005-04-27 Coley Pharm Group Inc Acidos nucleicos inmunoestimuladores
CA2503693A1 (fr) 2002-10-29 2004-05-13 Coley Pharmaceutical Group, Ltd. Techniques et produits relatifs au traitement et a la prevention de l'infection par le virus de l'hepatite c
JP2006512927A (ja) 2002-12-11 2006-04-20 コーリー ファーマシューティカル グループ，インコーポレイテッド ５’ｃｐｇ核酸およびその使用方法
RU2351362C2 (ru) 2003-03-26 2009-04-10 Цитос Байотекнолоджи Аг КОНЪЮГАТЫ ПЕПТИДА Melan-A, АНАЛОГА ВИРУСОПОДОБНОЙ ЧАСТИЦЫ
US7537767B2 (en) 2003-03-26 2009-05-26 Cytis Biotechnology Ag Melan-A- carrier conjugates
CN101454451A (zh) 2003-10-30 2009-06-10 科勒制药有限公司 具有增强免疫刺激能力的c类寡核苷酸类似物
CA2572427A1 (fr) 2004-07-18 2006-01-18 Heather L. Davis Complexe immunostimulant et formulations d'oligonucleotides permettant d'induire des reactions a interferon-gamma
MY159370A (en) 2004-10-20 2016-12-30 Coley Pharm Group Inc Semi-soft-class immunostimulatory oligonucleotides
ES2536103T3 (es) 2005-11-25 2015-05-20 Zoetis Belgium S.A. Oligorribonucleótidos inmunoestimuladores
WO2007068747A1 (fr) 2005-12-14 2007-06-21 Cytos Biotechnology Ag Particules emballees avec des acides nucleiques immunostimulateurs pour le traitement de l’hypersensibilite
JP5437797B2 (ja) 2006-06-12 2014-03-12 サイトス バイオテクノロジー アーゲー Ｒｎａバクテリオファージのウイルス様粒子にオリゴヌクレオチドをパッケージ化するための方法
CN101517082B (zh) 2006-09-27 2014-01-22 科勒制药有限责任公司 具有增强的免疫刺激活性的含疏水性T类似物的CpG寡聚核苷酸类似物
US7879812B2 (en) 2007-08-06 2011-02-01 University Of Iowa Research Foundation Immunomodulatory oligonucleotides and methods of use therefor

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
PL321711A1 (en) * 1995-02-09 1997-12-22 Icn Pharmaceuticals Methods of and compositions for controlling the cd28 expression
SK84599A3 (en) * 1996-12-27 2000-07-11 Icn Pharmaceuticals G-rich oligo aptamers and methods of modulating an immune response
EP0855184A1 (fr) * 1997-01-23 1998-07-29 Grayson B. Dr. Lipford Composition pharmaceutique comprenant un polynucléotide et un antigène notamment pour la vaccination

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO0014217A2 *

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