JP2005510462A - Production of immunomodulating oligonucleotides and methods of use thereof - Google Patents

Abstract

  The present invention provides new compositions and methods therefor for immunomodulating an individual. Immunomodulation is achieved by administering an immunomodulatory polynucleotide / microcarrier (IMP / MC) complex. The IMP / MC complex may be covalent or non-covalent and features a 7-mer oligonucleotide comprising at least one 5'-CG-3 'sequence attached to a microcarrier or nanocarrier.

Description

  The present invention relates to immunomodulatory compositions comprising oligonucleotides and methods of use thereof. In particular, the present invention relates to an immunomodulatory composition comprising an oligonucleotide that binds to a microparticle having an oligonucleotide length of 7 nucleotides. It further relates to administering an oligonucleotide / microcarrier complex to be modulated with at least one immune response.

  The type of immune response that occurs upon infection or challenge can generally be distinguished by the subset of T helper (The) cells that are normally involved in the response. The Th1 subset is involved in classical cell-mediated functions such as delayed-type hypersensitivity and activation of cytotoxic T cells (CTLs), while the Th2 subset functions more effectively as a helper for B cell activation doing. In general, the type of immune response to an antigen is affected by cytokines produced by cells that respond to the antigen. Differences in cytokines released in Th1 and Th2 cells are thought to reflect differences in biological function in these two subsets. See, for example, Romagnani (2000) Ann. Allergy Asthma Immunol. 85: 9-18.

  The Th1 subset is particularly suitable for viral infections, intracellular damage, and response to tumor cells because it releases IL-2 and INF-γ and activates CTLs. The Th2 subset is particularly suitable for responding to live free bacteria and intestinal microorganisms and can also mediate allergic reactions because IL-4 and IL-5 produce IgE and eosinophil activity This is because it is known to induce morphism. In general, Th1 and Th2 cells release distinctly different forms of cytokines, and one such responding type can moderate the other type of response activity. If the Th1 / Th2 balance shifts, for example, an allergic response or, as an option, an increased CTL response.

  For many infectious diseases such as tuberculosis and malaria, Th2-type responses have little protective value against infection. Presented vaccines using small peptides derived from target antigens, and other newly used antigenic agents that avoid the use of potentially infectious intact virus particles, the immune response required to enhance therapeutic efficacy Does not always provoke. The lack of a therapeutically effective human immunodeficiency virus (HIV) vaccine is a disappointing example of failure. Protein-dependent vaccines typically elicit a Th2-type immune response and are characterized by high titers of neutralizing antibodies, except that there is no significant cell-mediated immunity.

  In addition, some types of antibody responses have certain inappropriate manifestations, especially in allergies, that IgE antibody responses can cause anaphylactic shock. More generally, allergic responses include Th2-type immune responses. Allergic responses, including allergic asthma, are characterized by an early response, occur in seconds to minutes after allergen exposure, and in a late response characterized by cellular degranulation, the response occurs 2 to 24 hours later, And the eosinophil invades the site exposed to the allergen. Especially in the early stage allergic response, allergens cross-links with IgE antibodies in basophils and mast cells, which in turn trigger degranulation, and then from mast cells and basophils Releases histamine and other inclusions of inflammation. In particular, during the late response period, eosinophils invade allergen exposed sites (sites where tissue is damaged and dysfunctional).

  Antigen immunotherapy for allergic diseases involves injecting small amounts of antigen subcutaneously but gradually increasing the amount. Such immunotherapy has the risk of inducing IgE-mediated anaphylaxis and does not effectively address cytokine-mediated events of late allergic responses. Therefore, this method is only limited but successful.

Administration of an immune stimulatory sequence, commonly known as “ISS”, introduces an immune response with a Th1-type bias manifested in the release of cytokines related to Th1 To do. Administration of an immunostimulatory polynucleotide with an antigen results in a Th1-type immune response to the administered antigen. Roman et al. (1997) Nature Med. 3: 849-854. For example, mice are injected subcutaneously with E. coli β-galactosidase (β-Gal) in saline or adjuvant alum, in response to production of specific IgG1 and IgE antibodies, and IL-4 and IL-5 CD4 ⁺ cells that release A but not IFN-γ show that in T cells, the Th2 subset is dominant. However, mice respond by injecting subcutaneously (or using a chin skin scratch applicator) with plasmid DNA (in saline) that encodes β-Gal and contains ISS to produce IgG2a antibodies. CD4 ⁺ cells that release IFN-γ but not IL-4 and IL-5 show that the Th1 subset is dominant in T cells. Furthermore, specific IgE production was reduced by 66-75% by mice injected with plasmid DNA.

See Raz et al. (1996) Proc. Natl. Acad. Sci. USA 93: 5141-5145. In general, the response to bare DNA immunization is characterized by the production of IL-2, TNFα and INF-γ by antigen-stimulated CD4 ⁺ T cells, which manifests a Th1-type response. This is particularly important for the treatment of allergies and asthma, indicated by reducing the production of IgE. It has been demonstrated by bacterial antigens, viral antigens, and allergens that immunostimulatory polynucleotides can stimulate Th1-type immune responses (see, eg, WO 98/55495).

It was previously shown that ISS-containing oligonucleotides bound to microparticles (SEPHAROSE ™ beads) have immunostimulatory activity in vitro.
See Liang et al. (1996), J. Clin. Invest. 98: 1119-1129. However, recent results have shown that oligonucleotides containing ISS bound to gold, latex, and magnetic particles are not active in stimulating 7TD1 cell proliferation but proliferate in response to oligonucleotides containing ISS ( Manzel et al. (1999), Antisense Nucl. Acid Drug Dev. 9: 459-464).

  Other documented ISSs are described by krieg et al. (1989) J. Immunol. 143: 2448-2451; Tokunaga et al. (1992) Microbiol. Immunol. 36: 55-66; Kataoka et al. (1992) Jptz. J. Cancer Res. 83: 244-247; Yamamoto et al. (1992) J. Immunol. 148: 4072-4076; Mojcik et al. (1993) Clin. Immuno. And Imnzutlopathol. 67: 130-136; Branda et al. (1993) Biochem. Pharmacol. 45 : 2037-2043; Pisetsky et al. (1994) Life Sci. 54 (2): 101-107; Yamamoto et al. (1994a) Antisense Research and Development. 4: 119-122; Yamamoto et al. (1994b) Jptz. J. Cancer Res. 85: 775-779; Raz et al. (1994) Proc. Natl. Acad. Sci. USA 91: 9519-9523; Kimura et al. (1994) J. Biochem (Tokyo) 116: 991-994; Krieg et al. (1995) Nature 374 : 546-549; Pisetsky et al. (1995) Ann.NYAcad. Sci. 772: 152-163; Pisetsky (I996a) J. Immunol. 156: 421-423; Pisetsky (1996b) Immunity 5: 303-310; Zhao et al. (1996) Biochem. Pharmacol. 51: 173-182; Yi et al. (1996) J. Immunol. 156: 558-564; Krieg (1996) Trends Microbiol. 4 (2): 73-76; Krieg et al. (1996) Antisense Nucleic Acid Drug Dev. 6: 133-139; Klinman et al. (1996) Proc. Natl. Acad. Sci. USA. 93: 2879-2883: Raz et al. (1996); Sato et al. (1996) Science 273: 352-354; Stacey et al. (1 996) J. Immunol. 157: 2116-2122; Ballas et al. (1996) J. Immunol. 157: 1840-1845; Branda et al. (1996) J. Lab. Clin. Med. 128: 329-338; Sonehara et al. (1996). ) J. Interferon and Cytokine Res. 16: 799-803; Klinman et al. (1997) J. Immunol. 158: 3635-3639; Sparwasser et al. (1997) Eur. J. Immunol. 27: 1671-1679; Roman et al. (1997) Carson et al. (1997) J Exp. Med. 186: 1621-1622; Chace et al. (1997) Clin. Immunol. And Immunopathol. 84: 185; Chu et al. (1997) J. Exp. Med. 186: 1623-1631 Lipford et al. (1997a) Eur. J. Immunol. 27: 2340-2344; Lipford et al. (1997b) Eur. J. Immunol. 27: 3420-3426; Weiner et al. (1997) Proc. Natl. Acad. Sci. USA 94 : 10833-10837; Macfarlane et al. (1997) Immunology 91: 586-593; Schwartz et al. (1997) J. Clin. Invest. 100: 68-73; Stein et al. (1997) Antisense Technology, Ch. 11pp. 241-264, C. Lichtenstein and W. Nellen, Eds., IRL Press; Wooldridge et al. (1997) BLood 89: 2994-2998; Leclerc et al. (1997) Cell.Immunol. 179: 97-106; Kline et al. (1997) J. Invest. Med. 45 (3): 282A; Yi et al. (1998a) J. Immunol. 160: 1240-1245; Yi et al. (1998b) J. Immunol. 160: 4755-4761; Yi et al. (1998c) J. Immunol. 160: 5898-5906; Yi et al. (1998d) J. Immunol. 161: 4493-4497; Krieg (1998) Applied Antisetzse Oligonucleotide Ch. 24, pp. 431-448, CAStein and AM Krieg, Eds., Wiley-Liss, Inc .; krieg et al. (1998a) Trends Microbiol. 6: 23-27; Krieg et al. (1998b) ) J. Immunol. 161: 2428-2434; Krieg et al. (1998c) Proc. Natl. Acad. Sci. USA 95: 12631-12636; Spiegelberg et al. (1998) Allergy 53 (45S): 93-97; Horner et al. 1998) Cell Immunol. 190: 77-82; Jakob et al. (1998) J. Immunol. 161: 3042-3049; Redford et al. (1998) J. Immunol. 161: 3930-3935; Weeratna et al. (1998) Antisense & Nucleic Acid Drug Development 8: 351-356; McCluskie et al. (1998) J. Immunol. 161 (9): 4463-4466; Gramzinski et al. (1998) Mol. Med. 4: 109-118; Liu et al. (1998) Blood 92: 3730 Moldoveanu et al. (1998) Vaccine 16: 1216-1224; Brazolot Milan et al. (1998) Proc. Natl. Acad. Sci. USA 95: 15553-15558; Briode et al. (1998) J. Immunol. 161: 7054-7062. Briode et al. (1999) Int. Arch. Allergy Immunol. 118: 453-456; Kovarik et al. (1999) J. Immunol. 162: 1611-1617; Spiegelberg et al. (1999) Pediatr. Pulmonol. Suppl. 18: 118-121 Martin-Orozco et al (1999) Int.Immunol. 11: 1111-1118; EP 468,520; WO 96/02555; WO 97/28259; WO 98/16247; WO 98/18810; WO 98/3791 9; WO 98/40100; WO 98/52581; WO 98/55495; WO 98/55609 and WO 99/11275.

  See also EIkins et al. (1999) J. Immunol. 162: 2291-2298, WO 98/52962, WO 99/33488, WO 99/33868, WO 99/51259 and WO 99/62923. See also Zimmermann et al. (1998) J. Immunol. 160: 3627-3630; Krieg (1999) Trends Microbiol. 7: 64-65; U.S. Patent Nos. 5,663,153, 5,723,335, 5,849,719 and 6,174,872. See also WO 99/56755, WO 00/06588; WO 00/16804; WO 00/21556; WO 00/67023 and WO 01/12223. Verthelyi et al. (2001) J. Immunol. 166: 2372-2377; WO 00/54803; WO 00/61161; WO 00/54803; WO 01/15726; WO 01/22972; WO 01/22990; WO 01/35991 WO 01/51500; WO 01/54720; US Patent Nos. 6,194,388,6,207,646,6,214,806,6,239,116.

  Furthermore, Godard et al. (1995) Eur. J. Biochem. 232: 404-410 discloses cholesterol-modified antisense oligonucleotides conjugated to poly (isohexyl cyanoacrylate).

  All patents, patent applications, and published references cited herein are hereby incorporated by reference in their entirety.

  The present invention relates to new compositions and methods for modulating immune responses in individuals, particularly in human individuals.

  In one aspect, the present invention relates to a composition comprising an immune modulator / microcarrier (IMP / MC) complex. The IMP / MC complex can be either biologically denatured or not, and a 7-mer oligonucleotide having 5'-CG-3 'bound to a filterable and insoluble microcarrier (MC). Including. The oligonucleotide can be covalently or non-covalently bound to the complexed microcarrier, and the oligonucleotide can be easily modified to form a complex. The microcarriers used in the IMP / MC complex can also be liquid phase microcarriers (e.g. polymers or oils, preferably biologically denatured polymers or oils), but are typically solid phase microcarriers. is there. Microcarriers are usually less than about 150, 120 or 100 μm, more typically less than about 50-60 μm in size, and are possible in sizes from about 10 nm to about 10 μm or 25 nm to 5 μm. In certain instances, the compositions of the present invention comprise an IMP / MC complex and a pharmaceutically acceptable excipient. In certain instances, the compositions of the present invention comprise an antigen and a free IMP / MC complex, ie, the IMP / MC complex did not bind to the antigen (either directly or indirectly). In certain instances, the IMP / MC complex further comprises an antigen.

  The 7-mer oligonucleotide of the IMP / MC complex in certain examples comprises a 5′-TCG-3 ′ sequence and / or a 5′-UCG-3 ′ sequence. The oligonucleotide of the IMP / MC complex in a particular example comprises a 5′-CG-3 ′ sequence, further comprising a 5′-TCG-3 ′ sequence and / or a 5′-UCG-3 ′ sequence.

The oligonucleotides of the IMP / MC complex in specific examples are represented by the following formulas: 5'-TCGX ₁ X ₂ X ₃ X ₄ -3 ', 5'-X ₁ TCGX ₂ X ₃ X ₄ -3' and 5 ' _{1 from} -X ₁ X ₂ TCGX ₃ X ₄ -3 ', where X _1, X ₂ , X ₃ and X ₄ consist of the corresponding sequence being nucleotides.

  In another aspect, the invention provides an immune response within the individual comprising administering to the individual a composition comprising an IMP / MC complex in an amount sufficient to modulate the immune response within the individual. It is related with the method of adjusting. A diseased individual whose immunomodulation by the method of the invention is associated with a TH2-type immune response (e.g., allergy or allergy-derived asthma), therapeutic vaccine (e.g., allergy epitope, mycobacterial epitope, or tumor-associated epitope), Alternatively, it can be performed on individuals, including individuals receiving vaccines such as prophylactic vaccines, individuals with cancer, individuals with infectious diseases, and individuals exposed to infectious substances at risk.

  In a further aspect, the present invention relates to a method for increasing interferon-gamma (IFN-γ) in an individual comprising administering to the individual an effective amount of a composition comprising an IMP / MC complex. Administration of the IMP / MC complex according to the present invention increases IFN-γ in the individual. Appropriate objects for this method include idiopathic pulmonary fibrosis (IPF), scleroderma, skin-induced fibrosis, liver fibers including schistosomiasis-induced liver fibers, kidney fibers, and IFN-γ. These individuals include other symptoms that are improved by administration.

  In another aspect, the present invention relates to a method of ameliorating one or more symptoms of an infectious disease comprising administering to an individual having an infectious disease an effective amount of a composition comprising an IMP / MC complex. . Administration of the IMP / MC complex according to the invention improves one or more symptoms of infectious disease. An infectious disease that can be treated according to the present invention is an infectious disease that can be treated according to the present invention, such as a mycobacterial disease, malaria, leishmaniasis, toxoplasm, schistosomiasis, or liver fluke Including viral diseases, and may or may not include viral diseases.

  Furthermore, the present invention relates to a kit for performing the method of the present invention. The kit of the invention comprises an IMP / MC complex (or a substance for producing an IMP / MC complex as described herein) and optionally, for example, an individual associated with a Th2-type immune response An infectious disease that suffers from a disease (for example, allergy or allergy-induced asthma) and a cancer if you are receiving a vaccine such as a therapeutic vaccine (for example, an allergy epitope, mycobacterial epitope, or tumor-associated epitope) or a prophylactic vaccine Including directing the use of IMP / MC complexes in an individual's immunomodulation if they are exposed to or at risk of exposure to an infectious agent.

Embodiments of the present invention

We have discovered new compositions and methods for modulating immune responses in individuals, including humans, and in specific humans. The composition of the invention comprises a heptamer oligonucleotide comprising a 5′-C, G-3 ′ sequence complexed with a microcarrier (MC). Oligonucleotides of nucleotide length 7 were conjugated with human immune cells and micro-carriers (approximately 1 to 4.5 μm in diameter, less than 2.0 μm or about 1.5 μm) that effectively regulate human immune cells. Preferably, the oligonucleotide of the IMP / MC complex consists of a sequence of 1 of the following formula, ie the sequence is 5'-TCGX ₁ X ₂ X ₃ X ₄ -3 ', 5'-XGX ₁ TCGX ₂ X ₃ X ₄ -3 ′ and 5′-X ₁ X ₂ TCGX ₃ X ₄ -3 ′, where X ₁ , X ₂ , X ₃ and X ₄ are nucleotides. Our findings are of particular interest as human cells allow significant resistance to immune modulation by oligonucleotides than cells from commonly used laboratory animals such as mice, as is well known in the art It is.

  The IMP / MC complex was found to be significantly effective in immunomodulation at lower doses than only free oligonucleotides. IMP / MC complexes in human cells were usually significantly more active than free oligonucleotides in inducing IFN-γ and IFN-α.

  The IMP / MC complex may include or exclude the antigen. In some examples, the invention provides a composition comprising an antigen-free IMP / MC complex, ie, an IMP / MC complex that is not bound to an antigen (directly or indirectly). In another example, the present invention provides a composition comprising an IMP / MC complex mixed with one or more antigens. In another example, the present invention provides a composition comprising an IMP / MC complex conjugated to an antigen.

  We have further found that covalently linked IMP / MC complexes containing nanocarrier particles are highly active immunopreps. It has been shown before technical teaching that immunomodulatory oligonucleotides tightly bound to microparticles and nanoparticles are ineffective (especially Manzel et al.). In light of these technical judgments, we believe that this would be a surprise and unexpected result for those skilled in the art.

  A microcarrier (eg, a carrier having a size of less than 10 μm), wherein the immunomodulatory polynucleotide / microcarrier (IMP / MC) complex of the present invention may be covalent or non-covalent and is insoluble in water and / or filterable. including.

  Microcarriers can be liquid phase microcarriers (e.g. polymers or oils, preferably suspended oils in water containing biologically denatured polymers or oils), but usually solid phases (e.g. high Molecular lactic acid beads). Oligonucleotides can be modified to bind to MC or to increase binding (eg, covalent cross-linking or addition of a hydrophobic moiety such as cholesterol to incorporate a free sulfhydryl group for hydrophobic binding).

  The present invention provides a new composition comprising an oligonucleotide covalently attached to a microcarrier to produce a covalent IMP / MC complex. Binding between the oligonucleotide and MC can be induced (e.g., via a disulfide bond between the sulfhydryl groups of the oligonucleotide and MC), or a component thereof can bind the oligonucleotide to the MC. When separated, they can be joined by a bridging component of one or more atoms.

  Further provided is a composition comprising an oligonucleotide non-covalently bound to a microcarrier to provide a non-covalent IMP / MC complex. As will be appreciated by those skilled in the art, the properties of natural oligonucleotides can be electrostatically bound to a microcarrier such as a cationic poly (lactic acid, glycolic acid) copolymer to bind to the microcarrier. Can be used, but the non-covalent IMP / MC complex has been modified so that it can normally bind to microcarriers (eg, it can add a cholesterol moiety to an oligonucleotide and bind hydrophobically to oil or lipid-based microcarriers) Includes oligonucleotides.

  The present invention further provides a method for modulating an immune response in an individual by administering the IMP / MC complex to the individual. Further provided is a kit for performing the method of the present invention. The kit includes any IMP / MC complex, and / or composition for the IMP / MC complex when properly packaged, and further instructions for administering the IMP / MC complex to immunomodulate the subject. Also included.

General Techniques The practice of the present invention employs molecular biology conventional techniques (including recombinant techniques), bacterial biology, cell biology, biochemistry and immunology that are within the technical scope unless otherwise specified. It will be. These techniques are described in Molecular Cloning: A Laboratory Manual, second edition (Sambrook et al. 1989); Oligonucleotide Synthesis (MJGait, ed., 1984); Animal Cell Culture (RIFreshney, ed., 1987); Handbook of Experimental Immunology (DM Weir & CC Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (JMMiller & MP Cabs, eds., 1987); Current Protocols in Molecular Biology (FMAusubel et.al., eds., 1987); PCR: The Polymerise Chain Reaction, ( Mullis et al., Eds., 1994); Current Protocols in Immunology (JE Coligan et al., Eds., 1991); The Immunoassay Handbook (D. Wild, ed., Stockton Press NY, 1994); Bioconjugate Techniques (Greg T. Hermanson, ed., Academic Press, 1996); and Methods of Immunological Analysis (R. Masseyeff, WHAlbert, and NAStaines, eds., Weinheim: VCH Verlags gesellschaft mbH, 1993) .

Definitions As used herein, the terms `` one (a) '', `` one '' (an), `` the () '' include multiple citations unless otherwise indicated. Yes. For example, an “an” ISS includes one or more ISSs. As used interchangeably herein, the terms `` polynucleotide '' and `` oligonucleotide '' refer to single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), single-stranded RNA (ssRNA), Includes double stranded RNA (dsRNA), modified oligonucleotides and oligonucleosides or combinations thereof. Oligonucleotides can be linear or circular in structure, or oligonucleotides can include both linear or circular fragments. Oligonucleotides can be used for oligonucleotides in other linkages, such as phosphorothioate esters, but are usually polymers of nucleosides linked through phosphate ester linkages. A nucleoside consists of a purine (adenine, guanine or inosine, or derivative thereof) or pyrimidine (thymine, cytosine or olacil, or derivative thereof) base linked to a sugar. The four nucleoside units (or base pairs) in DNA are called deoxyadenosine, deoxyguanosine, deoxythymidine, and deoxycytidine. The nucleotide is a phosphate ester of a nucleoside.

  As used herein, the term “7-mer” oligonucleotide refers to an oligonucleotide that is 7 nucleotides in length, or base pairs (ie, the number of base pairs). In this disclosure, a single letter is used to indicate the base pair of a nucleotide sequence, where A is adenine, G is guanine, C is cytosine, T is thymine, U is uracil, I is inosine, R is purine, and Y Is a pyrimidine.

  As used herein, the term “ISS” refers to a polynucleotide sequence, alone and / or complexed with MC, which is measurable, measured in vitro, in vivo and / or ex vivo. It produces an immune response. Examples of measurable immune responses include the production of antigen-specific antibodies, cytokine release, activation or expansion of lymphocyte populations such as NK cells, CD4 + T lymphocytes, CD8 + T lymphocytes, B lymphocytes Including but not limited to. Preferably, this ISS sequence suitably activates a Th1-type response. The polynucleotide used in the present invention contains at least one ISS. “ISS” as used herein is also an abbreviated term for ISS-containing polynucleotides.

  The term “immunomodulatory polynucleotide” or “IMP” as used herein refers to a polynucleotide comprising at least one ISS. In a particular example, IMP is ISS.

  The term “microcarrier” refers to a particle composition that is insoluble in water and has a size of less than about 150, 120 or 100 μm, more typically less than about 50 to 60 μm, preferably less than about 10, 5, 2.5, 2 or 1.5 μm. It points to a thing. Microcarriers include “nanocarriers”, which are microcarriers having a size less than about 1 μm, preferably less than about 500 nm. Solid phase microcarriers can be particles formed from biologically compatible naturally formed polymers, synthetic polymers or synthetic copolymers, which are agarose or cross-linked agarose, and are well known in the art Microcarriers formed from other biologically denatureable materials may be included or excluded. The microcarrier used in the present invention may be biologically denatureable or nondenaturable. Biologically denatured solid phase microcarriers can be denatured (e.g., poly (lactic acid), poly (glycolic acid) and copolymers thereof) or eroded (e.g., poly (3,9) under physiological conditions in mammals. -Diethylidine-2,4,8,10-tetraoxasuro [5,5] undecane (DETOSU) and other ortho-esters) or sebacic acid poly (anhydrides) and other polymers) Can be generated from

  Bio-unmodified microcarriers are non-erodible and / or non-erosive under animal physical conditions such as polystyrene, polypropylene, silica, ceramic, polyacrylamide, gold, latex, hydroxyapatite, dextran, and ferromacnetec and paramagnetic materials. Alternatively, it can be formed from a material that is non-denaturing. In addition, the microcarriers are antigen-free liposomes, iscoms (an immunostimulatory complex that is an adjuvant-activated saponin in the cholesterol and phospholipid stabilization complex) or oil in water or water and oil. It is also possible in the liquid phase (based on oil or lipid) such as droplets or micelles found in the emulsion. Biologically denatured liquid phase microcarriers typically incorporate biodegradable oils, many of which are known in the art, including squalene and vegetable oils. Microcarriers are typically spherical in shape, but microcarriers that deviate from the spherical shape can also be accepted (eg, oval, rod shape, etc.). Due to its insoluble nature (relative to water), microcarriers can be filtered out of water and water (water soluble) solutions.

  The “size” of the microcarrier is usually the “design size” or the intended size of the particle as described by the manufacturer. The size can be a directly measured dimension, such as an average or maximum diameter, or can be determined by an indirect assay such as a filtration screening assay. Direct measurement of the size of the microcarriers is typically made by a microscope, usually an optical microscope, or a scanning electron microscope (SEM) or on the basis of a micrometer compared to particles of known size. Since the smallest variation in size occurs during the manufacturing process, if the measurement shows that the microcarrier is about ± 5 to 10% of the specified measurement, the microcarrier is considered to be the specified size. Also determined by dynamic light scattering or obscuration techniques. As another option, the microcarrier size can be determined by a filtration screening assay. If microcarriers pass at least 97% of the particles pass through a "screen-type" filter (i.e. retained particles as opposed to a "depth filter" where the retained particles remain in the filter) Suppose that the particles are smaller than the prescribed size), a filter on the surface of a filter, such as a polycarbonate or polyethersulfone filter. The microcarrier is larger than the defined size when at least about 97% of the microcarrier particles are retained in a defined size screen filter. Thus, at least about 97% of the microcarriers of a size of about 10 nm to about 10 μm pass through the 10 μm pore screen filter but are retained by the 10 nm screen filter.

  From the above, the criteria for the size or size range of the microcarriers implicitly includes approximate variations and approximate sizes and / or size ranges. This is the terminology when size and / or size range criteria, and size and / or size range criteria in the absence of a “about” criterion mean that there is no size and / or size range. Reflected by using “about”.

  A microcarrier is considered “biologically denatureable” if it can be denatured or eroded under normal mammalian physiological conditions. Generally, microcarriers can be biologically denatured after being incubated with normal human serum at 37 ° C. for 72 hours and then denatured (ie, at least 5% capacity and / or average polymer length is lost) I believe that. Thus, and conversely, microcarriers are considered “biologically non-denaturable” if they are not denatured or eroded under normal mammalian physiological conditions. In general, if the microcarrier is not denatured after incubation for 72 hours at 37 ° C. in normal human serum (ie less than at least 5% loss of mass and / or average polymer length) I think that there is no possibility of denaturation.

  The term “immunomodulatory polynucleotide / microcarrier complex” or “IMP / MC complex” refers to a complex of a 7-mer oligonucleotide comprising a 5′-CG-3 ′ sequence and a microcarrier of the invention. Yes. The components of the complex may be bound covalently or non-covalently. Non-covalent binding can be mediated by some non-covalent bonding force, including by hydrophobic interaction, ionic (electrostatic) bonding, hydrogen bonding and / or van der Waals attraction. In the case of a hydrophobic bond, the bond is usually via a hydrophobic component (eg, cholesterol) covalently attached to the oligonucleotide.

  As used herein, “immunomodulation” refers to oligonucleotides and / or complexes thereof. Thus, when presented as oligonucleotides only, IPM / MC can exhibit immunomodulatory activity even if the oligonucleotide of IPM / MC has a sequence that does not exhibit comparable immunomodulatory activity. In some instances, the IMP / MC oligonucleotide, when presented alone, has no “isolated immunomodulatory activity” or “poor, isolated immunomodulatory activity” (Ie when compared to IMP / MC). The “isolated immunomodulatory activity” of an oligonucleotide is determined using the same nucleic acid backbone (eg, phosphorothioate) using standard assays that exhibit at least one aspect of the immune response, as described herein. , Phosphodiester, chimeric) by measuring the immunomodulatory activity of the isolated oligonucleotide.

  As used herein, the term “immunomodulation” or “modulate immune response” includes immune stimulatory and immunosuppressive effects. Even though quantitative changes occur in conjunction with immune regulation, immune regulation is primarily a qualitative change in the overall immune response. The immune-regulated immune response according to the present invention is shifted to a “Th1-type” immune response as opposed to a “Th2-type” immune response. A Th1-type response is typically considered a cellular immune system (eg, cytotoxic T cell) response, while a Th2-type response is usually “humoral” or “antibody-based”. A Th1-type immune response is characterized by a “delayed hypersensitivity” response to normal antigens, and IL-6 can be similarly associated with a Th2-type response, but not only IFN-α and IL-6 but also IFN- By increasing the level of Th1-related cytokines such as γ, IL-2, IL-12, and TNF-β, it can be detected at the biochemical level. Th1-type immune responses are usually associated with the production of cytotoxic T cells (CTLs) and low or transient production of antibodies. A Th2-type immune response is usually associated with high levels of antibody production, including IgE production, non-producing or minimal CTL production, and expression of Th2-related cytokines such as IL-4. Thus, immunomodulation according to the present invention can be recognized, for example, by an increase in IFN-γ and / or a decrease in IgE production in individuals treated by the method of the present invention compared to the untreated.

  The term “conjugate” refers to a complex to which an oligonucleotide comprising a 5′-CG-3 ′ sequence and an antigen are linked. Such chain bonds include covalent and / or non-covalent bonds.

  The term “antigen” means a substance that is recognized and bound, in particular, by an antibody or T cell antigen receptor. Antigens can include peptides, proteins, glycoproteins, polypolysaccharides, hydrocarbon complexes, sugars, gangliosides, lipids and phospholipids, portions thereof and combinations thereof. Antigens may be found in nature or may be synthetic. Suitable antigens for administration at the ISS include any molecule capable of eliciting a specific response to B cell or T cell antigens. Preferably, the antigen elicits an antibody response specific for the antigen. Haptens are included within the scope of “antigen”. When a hapten binds to an immunogenic molecule containing an antigenic determinant, it is a low molecular weight compound that does not become an immunogen by itself but is given as an immunogen. There is a need to haptenize small molecules to be given as antigens. Preferably, the antigens of the present invention include peptides, lipids (eg, sterols, fatty acids, and phospholipids), polypolysaccharides such as those used in Hemophilus influenza vaccines, gangliosides, and glycoproteins.

  “Adjuvant” refers to a substance that, when added to an immunogenic agent, such as an antigen, non-specifically enhances or enhances the efficacy of the immune response to the recipient host upon exposure to the mixture. .

  The term “peptide” is a composition that is of sufficient length to undergo a biological response, eg, to produce antibodies or activate cytokines, whether the peptide is a hapten or not. Typically in a peptide there are at least 6 amino acid residues in length. Furthermore, the term “peptide” includes modified amino acids (whether natural or non-naturally occurring), including such modifications as phosphorylation, glycosylation, pegylation, lipidation. , And methylation.

  An “antigenic peptide” is a purified natural peptide, synthetic peptide, recombinant peptide, crude peptide extract, or partly purified or unpurified active state (partially attenuated or inactive virus, cell, or Microorganisms, or fragments of such peptides). Thus, “antigen peptide” or “antigen polypeptide” means all or part of a polypeptide that exhibits one or more antigenic properties. Thus, for example, an “Amb 1 antigen polypeptide” or “Amb 1 polypeptide antigen” is an entire sequence, a partial sequence and / or a modified sequence that exhibits antigenic properties (ie, specifically binds to an antibody or a T cell receptor). It is the amino acid sequence from Ama 1.

  An “inducible molecule” or “induction vehicle” is a chemical moiety that easily, enables and / or enhances the induction of IMP / MC complexes at specific sites and / or at specific times. The induction carrier may or may not additionally stimulate the immune response.

  An “allergic response to an antigen” is usually characterized by the production of eosinophils and / or antigen-specific IgE and the effects obtained therefrom. As is well known in the art, IgE binds to mast cell and basophil IgE receptors. The latter exposure to antigen recognized by IgE includes, but is not limited to, the antigen cross-linking to IgE of mast cells and basophils, causing degranulation of these cells and releasing histamine. The terms “allergic response to antigen”, “allergy” and allergic conditions are understood and intended to be equally suitable for the application of some methods of the invention. Further, it is understood and intended that the methods of the present invention include sources that are equally suitable for preventing allergic responses and treating preexisting allergic symptoms.

  As used herein, the term “allergen” refers to a molecule of an antigen or antigen portion, usually a protein, that elicits an allergic response upon exposure to an object. Typically, the object is allergic to the allergen, as evidenced by, for example, wheal and redness tests or some method known in the art. A molecule is said to be an allergen by only a small subset of objects exhibiting an allergic (eg, IgE) immune response upon exposure to the molecule. Many isolated allergens are known in the art. These include, but are not limited to, those provided in the tables herein.

  The term “desensitization” refers to the process of administration in which the subject increases the dose of allergen that has proven sensitizing. Examples of allergen doses used for desensitization are known in the art, see for example Fornadley (1998) Otolaryngol. Clin. North Am. 31: 111-127.

  “Antigen-specific immunotherapy” refers to any form of immunotherapy that involves an antigen and forms an antigen-specific modulation of the immune response. In the context of allergies, allergen-specific immunotherapy includes but is not limited to desensitization therapy.

  A “solid” is a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, humans, primates, farm animals, sport animals, rodents and pets. Further, vertebrates include, but are not limited to, birds (ie, bird individuals) and reptiles (reptile individuals).

  An “effective amount” or “sufficient amount” of a substance is an amount sufficient to obtain a desired result, including clinical results, and such “effective amount” depends on what it is applied to. In the context of administering a composition that modulates an immune response against an antigen, an effective amount of the IMP / MC complex is an amount sufficient to effect such modulation compared to the immune response obtained when the antigen alone is administered. It is. An effective amount can be administered in one or more administrations.

  The term “co-administration” as used herein refers to the administration of at least two different substances at once in a sufficiently closed form to modulate the immune response. Preferably, it refers to the simultaneous administration of at least two different substances.

  “Synchronization” of an immune response, such as a Th1 response, refers to an increase in response that enhances the initiation and / or enhancement of the response.

An “IgE-related disease” is a physiological symptom in which the level of IgE is partially elevated, characterized in that it may or may not be continuous. IgE-related diseases include, but are not limited to, allergies and allergic reactions, allergy-related diseases (as described below), asthma, rhinitis, conjunctivitis, urticaria, seizures, stabbing allergies, and drug allergies, and infection with parasites. Not. The term further relates to the development of these diseases. Such effects include, but are not limited to, hypotension and stroke. Anaphylaxis is an example of an allergy-related disease where histamine released in the circulatory system causes vasodilation and increases the permeability of the capillaries as a result of significant loss of plasma from the circulatory system. Anaphylaxis can occur as a systemic system due to related effects that occur throughout the body, and can occur locally due to reactions that are limited to specific target tissues or organs.
As used herein, the term “viral disease” refers to a disease that has a virus as an etiology. Examples of viral diseases include hepatitis B, hepatitis C, influenza, acquired immune deficiency syndrome (AIDS), and herpes zoster.

  As used herein and well understood in the art, “treatment” is a method for obtaining beneficial or desired results, including clinical results. For the purposes of the present invention, beneficial or desired clinical results include alleviation or amelioration of one or more symptoms, alleviation of the extent of the disease, stabilization of the disease state (ie, no worsening), prevention of symptom expansion, disease progression Including, but not limited to, delayed or indolent, improved or reduced disease state, and reduced (even partly or totally), even if detectable or not possible. “Treatment” may also mean prolonging survival as compared to expected survival if not receiving treatment.

  “Palliating” a disease or disorder reduces the clinical onset of an expanded and / or undesirable disease or disease state and / or in the course of progression compared to not treating the disease. Means slow and long. Reducing in allergic symptoms, as is particularly well understood by those skilled in the art, may occur by modulation of the immune response to the allergen. Further, relief of symptoms is not necessarily required to occur by administering a single dose, but is often caused by administering a series of doses. Thus, an amount sufficient to alleviate the response or disease can be administered in one or more administrations.

  The “antibody titer” or “antibody amount” “induced” by the IMP / MC complex refers to the amount of a given antibody measured at the time after administration of the IMP / MC complex.

  A “Th1-related antibody” is an antibody whose production and increase in antibodies are associated with a Th1 immune response. For example, IgG2a is a Th1-related antibody in mice. For purposes of the present invention, measurement of Th1-related antibodies allows measurement of one or more such antibodies. For example, in humans, measuring Th1-related antibodies can involve measuring IgG1 and / or IgG3.

  A “Th2-related antibody” is an antibody in which the production and increase of antibodies is associated with a Th2 immune response. For example, IgG1 is a Th2-related antibody in mice. For the purposes of the present invention, measurement of Th2-related antibodies allows measurement of one or more such antibodies. For example, in humans, measuring Th2-related antibodies can involve measuring IgG2 and / or IgG4.

  When “inhibiting” or “inhibiting” a function or activity, such as cytokine production, antibody production, or histamine release, is not in a condition or parameter of interest or choice compared to another condition If the same conditions are compared separately, it is to reduce function or activation. Antigens that inhibit histamine release or IMP / MC complexes administered with that antigen, for example, reduced histamine release compared to histamine release induced by antigen alone.

  As used herein, the term “comprising” and its corresponding series are used in the inclusive sense, ie, are equivalent to the term “including” and its corresponding series.

Compositions of the Invention The present invention provides new compositions for immunomodulating an individual. This new composition is an immunomodulatory polynucleotide / microcarrier (IMP / MC) complex complexed with a microcarrier, which contains a 7-mer oligonucleotide containing a 5′-CG-3 ′ sequence, Either degradable or non-biodegradable may be used. The IMP / MC complex may be a covalent complex, in which the oligonucleotide part of the complex is covalently linked either directly or via a linker (indirectly), or these May be a non-covalent complex.

Oligonucleotides of the IMP / MC complex Oligonucleotides according to the invention may be 7 bases or base pairs long and may contain at least one 5'-cytosine, guanine-3 'sequence and include modifications Contains two or more 5'-CG-3 'sequences. In addition, the oligonucleotide can further comprise 5′-T, C, G-3 ′ sequences (ie, one or more). Thus, the oligonucleotide can comprise 5′-CG-3 ′ and / or 5′-TCG-3 ′. When oligonucleotides are conjugated to microcarriers, they affect measurable immune responses as measured in vitro, in vivo and / or ex vivo. If not conjugated, in some instances the oligonucleotide is inactive or less active in some instances, as measured in vitro, in vivo and / or ex vivo.

  The 5′-cytosine and guanine-3 ′ sequences are usually unmethylated, particularly at the C-5 site. Since the term “IMP / MC” is transmitted and defined, the IMP / MC complex affects the immune response, and in certain instances, the cytosine methylation of such oligonucleotides, eg, N-4 It is possible at the position.

  In some examples, the oligonucleotide comprises the sequence 5′-C, G, pyrimidine, pyrimidine, C, G-3 (such as 5′-CGTTCG-3 ′). In some examples, the oligonucleotide comprises a sequence of 5'-purine, purine, C, G, pyrimidine, pyrimidine-3 '(such as 5'-AACGTT-3'). The oligonucleotide in some examples can comprise the sequence 5'-purine, T, C, G, pyrimidine, pyrimidine-3 '.

In some examples, the oligonucleotide is one of the following sequences:
: GACGCT; GACGTC; GACGTT; GACGCC; GACGCU; GACGUC; GACGUU; GACGUT; GACGTU; AGCGTT;
AGCGCT; AGCGTC; AGCGCC; AGCGUU; AGCGCU; AGCGUC; AGCGUT; AGCGTU; AACGTC; AACGCC; AACGTT;
AACGCT; AACGUC; AACGUU; AACGCU; AACGUT; AACGTU; GGCGTT; GGCGCT; GGCGTC; GGCGCC; GGCGUU;
Includes GGCGCU; GGCGUC; GGCGUT; GGCGTU.

In some examples, the oligonucleotide is one of the following sequences:
GABGCT; GABGTC; GABGTT; GABGCC; GABGCU; GABGUC; GABGUU; GABGUT; GABGTU; AGBGTT; AGBGCT;
AGBGTC; AGBGCC; AGBGUU; AGBGCU: AGBGUC; AGBGUT; AGBGTU; AABGTC; AABGCC; AABGTT; AABGCT;
AAGUC; AABGUU; AABGCU; AABGUT; AABGTU; GGBGTT; GGBGCT: GGBGTC; GGBGCC; GGBGUU; GGBGCU;
It contains the sequence GGBGUC; GGBGUT; GGBGTU, where B is 5-bromocytosine.

In some examples, the oligonucleotide consists of the sequence 5'-TCGX ₁ X ₂ X ₃ X ₄ -3 'or 5'-UCGX ₁ X ₂ X ₃ X ₄ -3', where X ₁ , X ₂ , X ₃ and X ₄ are nucleotides. In some examples, the oligonucleotide consists of any of the following sequences: 5′-TCGTTTT-3 ′: 5′-TCGAAAA-3 ′; 5′-TCGCCCC-3 ′; 5′-TCGGGGG-3 ';5'-TCGUUUU-3';5'-TCGIIII-3';5'UCGTTTT-3';5'-UCGAAAA-3';5'-UCGCCCC-3';5'-UCGGGGG-3'; 5 It consists of '-UCGUUUU-3';5'-UCGIIII-3'. In some examples, the oligonucleotide consists of the sequence 5′-X ₁ TCGX ₂ X ₃ X ₄ -3 ′ or 5′-X ₁ UCGX ₂ X ₃ X ₄ -3 ′, where X ₁ , X ₂ , X ₃ and X ₄ are nucleotides. The oligonucleotide in some examples consists of any of the following sequences: 5'-TTCGTTT-3 ';5'-ATCGATT-3';5'-TUCGTTT-3';5'-AUCGATT-3' . In some examples, the oligonucleotide consists of the sequence 5′-X ₁ X ₂ TCGX ₃ X ₄ -3 ′ or 5′-X ₁ X ₂ UCGX ₃ X ₄ -3 ′, where X ₁ , X ₂ , X ₃ and X ₄ are nucleotides. In some examples, the oligonucleotide consists of any of the following sequences: 5'-TTTCGTT-3 ';5'-AATCGAT-3';5'-TTUCGTT-3';5'-AAUCGAT-3' Consists of.

In some examples, the oligonucleotide consists of the sequence 5′-TCGTCGX ₁ -3 ′, wherein X ₁ is a nucleotide. In some examples, the oligonucleotide consists of any of the following sequences: 5'-TCGTCGA-3;5'-TCGTCGC-3';5'-TCGTCGG-3';5'-TCGTCGT-3 It consists of ';5'-TCGTCGU-3';5'-TCGTCGI-3'. In some examples, the oligonucleotide consists of 5′-TCGUCGX ₁ -3 ′, 5′-UCGTCGX ₁ -3 ′, or 5′-UCGUCGX ₁ -3 ′, where X ₁ is a nucleotide. In some examples, the oligonucleotide consists of any of the following sequences:
That is: 5'-TCGUCGA-3 ';5'-TCGUCGC-3';5'-TCGUCGG-3';5'-TCGUCGT-3';5'-TCGUCGU-3';
5'-TCGUCGI-3 ';5'-UCGTCGA-3';5'-UCGTCGC-3';5'-UCGTCGG-3';5'-UCGTCGT-3';
5'-UCGTCGU-3 ';5'-UCGTCGI-3';5'-UCGUCGA-3';5'-UCGUCGC-3';5'-UCGUCGG-3';
5'-UCGUCGT-3 ';5'-UCGUCGU-3';5'-UCGUCGI-3'.

In some examples, the oligonucleotide consists of the sequence 5'-TmCGX ₁ X ₂ X ₃ X ₄ -3 'or 5'-UMCGX ₁ X ₂ X ₃ X ₄ -3', where X ₁ , X ₂ , X ₃ , X ₄ are nucleotides, and mC is cytosine modified as described herein. In some examples, the oligonucleotide consists of the sequence 5′-X ₁ TmCGX ₂ X ₃ X ₄ -3 ′ or 5′-X ₁ UmCGX ₂ X ₃ X ₄ -3 ′, where X ₁ , X ₂ , X ₃ and X ₄ are nucleotides. In some examples, the oligonucleotide consists of the sequence 5′-X ₁ X ₂ TmCGX ₃ X ₄ -3 ′ or 5′-X ₁ X ₂ UmCGX ₃ X ₄ -3 ′, where X ₁ , X ₂ , X ₃ and X ₄ are nucleotides.

In some examples, the oligonucleotide consists of the sequence 5'-TmCGTCGX ₁ -3 ', 5'-TmCGUCGX ₁ -3' or 5 'UmCGTCGX ₁ -3' or 5 'UmCGUCGX ₁ -3', where X ₁ is a nucleotide. In some examples, the oligonucleotide consists of the sequence 5'-TCGTmCGX ₁ -3 ', 5'-UmCGTmCGX ₁ -3', 5'-TmCGUmGX ₁ -3 'or 5'UmCGUmCGX ₁ -3' X ₁ is a nucleotide. Modified cytosine (mC) as described herein includes the addition of an electron leaving component to C-5 and / or C-6 of cytosine, which includes C-5 halogenated cytosines such as 5-bromocytosine. Including but not limited to.

Thus, in some examples, the oligonucleotide consists of any of the following sequences: 5'-TBGTTTT-3 ';5'-TBGAAAA-3';5'-TBGCCCC-3';5'- TBGGGGG-3 ';
5'-TBGUUUU-3 ';5'-TBGIIII-3';5'-TBGTCGA-3';5'-TBGTCGC-3';5'-TBGTCGG-3';
5'-TBGTCGT-3 ';5'-TBGTCGU-3';5'-TBGTCGI-3';5'-TCGTBGA-3';5'-TCGTBGC-3';
5'-TCGTBGG-3 ';5'-TCGTBGT-3';5'-TCGTBGU-3';5'-TCGTBGI-3';5'-TBGTBGA-3';
5'-TB GTB GC-3 ';5'-TBGTBGG-3';5'-TBGTBGT-3',5'-TBGTBGU-3';5'-TBGTBGI-3'; where B is 5 -Bromocytosine.

  The heptamer oligonucleotide may contain a modification. Modifications of heptamer oligonucleotides include, but are not limited to, well-known techniques such as 3′OH or 5′OH group modification, nucleotide base modification, sugar component modification, and phosphate group modification. These various modifications are described below.

  Cytosine present in the oligonucleotide is not methylated, however, in certain instances, the oligonucleotide can include one or more methylated cytosines. In such an example, it is preferred that the 5′-CG-3 ′ cytosine of the oligonucleotide is not methylated at the C-5 position. However, methylation at the N-4 position is defined as an oligonucleotide with methylated cytosine.

  The heptamer oligonucleotide may be single-stranded or double-stranded and single-stranded or double-stranded RNA, or other modified polynucleotide. The heptamer oligonucleotide may or may not contain one or more circular structure regions, may be present in the motif, or may extend beyond the motif. A heptamer oligonucleotide may include a base formed or modified by nature, a non-naturally formed base, and may include a modified sugar, phosphate and / or terminus. For example, modifications with phosphoric acid include, but are not limited to, methyl phosphorylated, phosphorothioate, phosphoramidite (cross-linked or non-cross-linked), phosphorotriester, and phosphorodithioate, and in any combination Can be used. Other binding components with non-phosphoric acid can also be used.

  Preferably, the oligonucleotide of the present invention comprises a phosphorodiester and / or a phosphorothioate backbone. Sugar modifying components known in the art, such as 2'-alkoxy-RNA analogs, 2'-amino-RNA analogs and 2'-alkoxy- or amino-RNA / DNA chimeras and other components described herein, Furthermore, it can be created and combined by some phosphoric acid modification. Examples of base modifications include adding an electron leaving component at the C-5 and / or C-6 sites in ISS cytosine (eg, 5-bromocytosine, 5-chlorocytosine, 5-fluorocytosine, 5-iodocytosine) Including, but not limited to. See, for example, International Patent Application No. WO 99/62923.

  Heptameric oligonucleotides can be synthesized using techniques well known in the art, including but not limited to enzymatic methods, chemical methods and denaturation of large oligonucleotide sequences and nucleic acid synthesizers. See Ausubel et al. (1987); and Sambrook et al. (1989). Oligonucleotide denaturation can be accomplished through exposure of the oligonucleotide to a nuclease, as set forth in US Pat. No. 4,650,675.

  Circular oligonucleotides can be synthesized via isolation, recombinant methods, or chemically synthesized. Chemical synthesis of small circular oligonucleotides can be performed using any method described in the literature. See, for example, Gao et al. (1995) Nucleic Acids Res. 23: 2025-2029; and Wang et al. (1994) Nucleic Acids Res. 22: 2326-2333.

  Techniques for making oligonucleotides and modified oligonucleotides are known in the art. Naturally formed DNA or RNA containing a phosphodiester bond linearly binds the appropriate nucleoside phosphoramidite to the 5 'hydroxyl group that grows the oligonucleotide bound to the solid support at the 3' end And then the phosphoric acid triester can be synthesized by oxidation of the intermediate of the phosphoric acid triester.

  Once the desired oligonucleotide sequence is synthesized, the oligonucleotide is removed from the carrier, the phosphate triester group is deprotected to a phosphodiester, and the nucleoside base is dissolved in water-soluble ammonia or other Deprotected with base. See, for example, Beaucage (1993) “Oligodeoxyribonucleoide Synthesis” in Protocols for Oligonucletides and Analog, Synthesis and Properties (Agrawal et al.) Humana Press, Totowa, NJ; Wanner et al. (1984) DNA 3: 401 and US Pat. No. 4,458,066.

  In addition, the oligonucleotide can include a phosphate-modified linkage. Furthermore, the synthesis of polynucleotides containing modified phosphate bonds or non-phosphate bonds is known. For a review, see Matteucci (1997) “Oligonucleotide Analogs: an Overview” in Oligonucleotides as Therapeutic Agents, (D.J. Chadwick and G. Cardew, ed.) John Wiley and Sons, New York, NY. Phosphate derivatives (or modified phosphate groups) capable of binding to sugar components and sugar-like components in the oligonucleotides of the present invention are monophosphates, diphosphates, triphosphates, alkyl phosphonates, phosphorothioates , Phosphorodithioate and the like. In addition, the preparation of the above phosphate analogs and their incorporation into nucleotides, modified nucleotides and oligonucleotides are well known per se and need not be described in detail herein. See Peyrottes et al. (1996) Nucleic Acids Res. 24: 1841-1848; Chaturvedi et al. (1996) Nucleic Acids Res. 24: 2318-2323; and Schultz et al. (1996) Nucleic Acids Res. 24: 2966-2973. . For example, the synthesis of phosphorothioate oligonucleotides involves the oxidation step, the sulfurization step (Zon (1993) `` Oligonucleoside Phosphorothioates '' in Protocols for Oligonucleotides and Analogs, Synthesis and Properties (Agrawal et al.) Humana Press, pp.165-190. ) Is similar to the description above for naturally occurring oligonucleotides except that Furthermore, phosphorotriesters (Miller et al. (1971) JACS 93: 6657-6665), non-crosslinked phosphoramidites (lager et al. (1988) Biochem. 27: 7247-7246), N3'to P5 'phosphoramidiates (Nelson et al. (1997) The synthesis of other phosphate analogues such as JOC 62: 7278-7287) and phosphorodithioates (US Pat. No. 5,453,496) has been described. Still other non-phosphate dependent modified oligonucleotides can be used (Stirchak et al. (1989) Nucleic Acids Res. 17: 6129-6141). Oligonucleotides with phosphorothioate backbones are seen to be more immunogenic than those with phosphodiester backbones and to be significantly resistant to denaturation after being injected into the host. See Braun et al. (1988) J. Immunol. 141: 2084-2089; and Latimer et al. (1995) Mol. Immunol. 32: 1057-1064.

  Oligonucleotides used in the present invention can comprise ribonucleotides (including ribose alone or as a major sugar component), deoxyribonucleotides (including deoxyribose as a major sugar component), or known in the art As such, modified sugars or sugar analogs can be incorporated into oligonucleotides. Thus, in the addition of a saccharide component to ribose and deoxyribose, the saccharide component can be a pentose, deoxypentose, hexose, deoxyhexose, glucose, arabinose, xylose, lyxose and sugar “similar” cyclopentyl group. The sugar may be in the form of pyranosyl or furanosyl. The saccharide component in the oligonucleotide is preferably ribose, deoxyribose, arabinose or 2'-O-alkylribose furanoside, and the saccharide is linked to a heterocyclic base in either the α or β anomeric structure, respectively. Can do. Sugar modifications include, but are not limited to, 2'-alkoxy-RNA analogs, 2'-amino-RNA analogs and 2'-alkoxy- or amino-RNA / DNA chimeras. The preparation of each “nucleoside” in which these saccharides or sugar analogs and such saccharides or analogs thereof are linked to a heterocyclic base (nucleobase) is well known per se, and examples of such preparations include There is no need to describe it here, except to the extent that it can. Furthermore, sugar modifications can create or link any phosphate modification in the preparation of the oligonucleotide.

  Heterocyclic bases or nucleobases that are incorporated into oligonucleotides are the main purine and pyrimidine bases that form naturally (i.e., uracil, thymine, cytosine, inosine, adenine as described above), and the natural production of said main bases And by synthetic modification.

  A number of “synthetic” non-natural nucleosides, including various heterocyclic bases, and various sugar components (and sugar analogs) are available in the art and other criteria of the invention are satisfied. Those skilled in the art will appreciate that an oligonucleotide can contain one or several heterocyclic bases other than the five major base components of a naturally-occurring nucleic acid. Preferably, however, the heterocyclic base in the oligonucleotide is uracil-5-yl, cytosine-5-yl, adenine-7-yl, adenine-8-yl, guanine-7-yl, guanine-8-yl, 4 -Aminopyrrolo [2.3-d] pyrimidin-5-yl, including but not limited to 2-amino-4-oxopyrrolo [2.3-d] pyrimidine-3 groups, where purines are linked to ISS sugars via the 9 position The component is attached to the pyrimidine through position 1, to the pyrrolopyrimidine through position 7, and to the pyrazolopyrimidine through position 1.

  The oligonucleotide can comprise at least one modified base as described, for example, in commonly acquired international application WO 99/62923. As used herein, the term “modified base” is synonymous with “base analog”, eg, “modified cytosine” is synonymous with “cytosine analog”. Similarly, “modified” nucleosides or nucleotides are defined herein to be synonymous with “analogs” of nucleosides or nucleotides. Examples of base modifications include, but are not limited to, adding an electron leaving component to C-5 and / or C-6 of ISS cytosine. Preferably, the electron leaving component is halogen. The modified human synthates include azacytosine, 5-bromocytosine, 5-chlorocytosine, chlorinated cytosine, cyclocytosine, cytosine arabinoside, 5-fluorocytosine, fluoropyrimidine, 5,6-dihydrocytosine, 5-iodocytosine, Hydroxyurea, 5-nitrocytosine, 5-hydroxycytosine, and any other pyrimidine analogs or modified pyrimidines can be included, but are not limited thereto.

  Preferably modified uracil is preferably modified with halogen at C-5 and / or C-6, and bromouracil such as 5-bromouracil, chlorouracil such as 5-chlorouracil, fluorouracil such as 5-fluorouracil , But not limited to, iodouracil such as 5-iodouracil and hydroxyuracil. See also Kandimalla et al. 2001, Bioorg. Med. Chem. 9: 807-813. See, for example, International Patent Application No. WO 99/62923. Other examples of base modifications include adding one or more thiol groups to bases including but not limited to 6-thio-guanine, 4-thio-thymine, and 4-thio-uracil. . In addition, some nucleotides contain modified bases such as 7-diazaguanosine instead of any guanosine residue, or any cytosine residue containing 5′-CG-3 ′ cytosine Alternatively, modified cytosine selected from N-4 ethylcytosine or N-4 methylcytosine can be included.

  The preparation of base-modified nucleosides and the synthesis of modified oligonucleotides using the modified nucleosides as precursors are described, for example, in US Pat. Nos. 4,910,300, 4,948,582, and 5,093,231. These base-modified nucleosides were designed so that they could be incorporated by chemical synthesis at either the terminal or internal position of the oligonucleotide. Such base-modified nucleosides present at either the terminal position or the internal position of the oligonucleotide can be used as binding sites for peptides or other antigens. In addition, nucleosides that are modified to these sugar components are described in (eg, including but not limited to, US Pat. Nos. 4,849,513,5,015,733,5,118,800, 5,118,802) and can be used as well.

  ISS is technically described and easily used with standard assays that demonstrate various aspects of the immune response such as cytokine release, antibody production, NK cell activation, and T cell proliferation Can be identified. WO 97/28259; WO 98/16247; WO 99/11275; Kreg et al. (1995) Nature 374: 546-549; Yamamoto et al. (1992a); Ballas et al. (1996); KIinman et al. (I997); Sato et al. (1996) Pisetsky (1996a); Shimada et al. (1986) Jpn. J. Cancer Res. 77: 808-816; Cowdery et al. (1996) J. Immunol. 156: 4570-4575; Roman et al. (1997) and Lirford et al. (I997a) See These methods can be similarly applied to assess the immunomodulatory activity of IMP / MC complexes.

  One property of an oligonucleotide is “isolated immunomodulatory activity” in relation to the nucleotide sequence of the oligonucleotide. Surprisingly, the IPM / MC complex exhibits immunomodulatory activity, even when the oligonucleotide has a sequence that does not exhibit comparable immunomodulatory activity when presented as an oligonucleotide alone, The present invention has been discovered as described above.

  In some examples, the oligonucleotide of the IMP / MC complex does not have “isolated immunomodulatory activity” or “significantly less isolated immunomodulatory activity” (ie, IMP / MC) as described below. As compared to MC complex).

  The “isolated immunomodulatory activity” of an oligonucleotide is determined by measuring the immunomodulatory activity (eg, phosphorothioate, phosphorodiester, chimeric) of an isolated oligonucleotide having the same nucleic acid backbone. It is determined. For example, test oligonucleotides having the same sequence (eg, 5'-TCGTCGA-3 ') and the same backbone structure (eg, phosphorothioate) to determine the independent immunomodulatory activity of the oligonucleotide in the IMP / MC complex Are synthesized using routine methods and their immunomodulatory activity is measured (if possible). Immunomodulatory activity can be determined using standard assays that demonstrate various aspects of the immune response, such as those described herein. For example, the human PBMC assay described herein is used. Assays are typically performed on multiple donors to account for donor changes. The amount of IFN-γ released by the oligonucleotides by the PBMCs contacted with the oligonucleotide is greater than in the absence of the test compound or (in some instances) the presence of an inactive control compound (e.g., 5'-TGACTGTGAACCTTAGAGATGA An oligonucleotide does not have “isolated immunomodulatory activity” if it is not significantly larger (less than about 2-fold in size) in a large number of donors than if it was −3 ′ (SEQ ID NO: 1).

In order to compare the immunomodulatory activity of the IMP / MC complex and the isolated oligonucleotide, the immunomodulatory activity is preferably measured, but it is not necessary to use a human PBMC assay. Usually, the activities of two compounds are compared by evaluating them in parallel under the same conditions (eg using the same cells), usually at a concentration of about 20 μ / ml. Generally the concentration is determined by measuring the absorbance at 260 nm and using a conversion of 0.5 OD ₂₆₀ / ml = 20 μg / ml. This normalizes the amount of total nucleic acid in the test sample. As an option, concentration or weight can be measured by other methods known in the art.

  If the test oligonucleotide is less active than the IMP / MC complex being compared, the oligonucleotide of the IMP / MC complex is characterized as having “poor immunomodulatory activity”. Preferably, the isolated immunomodulatory activity of the test oligonucleotide is no more than about 50%, more preferably no more than about 20% of the activity of the IMP / MC complex, most preferably the IMP / MC complex. About 10% or less, or in some instances, less.

Useful microcarriers in microcarrier present invention, the size is smaller than 150,120, or 100 [mu] m, preferably insoluble in is less than about 10 [mu] m, and pure water sizes. Despite the acceptability of microcarriers that are non-biologically modified, the microcarriers used in the present invention are preferably those that are biologically denatured. Further contemplated are liquid phase microcarriers, such as emulsions with biodegradable polymers or oils in water containing oils, but the microcarriers are generally solid phases such as “beads” or other particles. A variety of bio- and non-bio-denaturing materials that are acceptable for use as microcarriers are known in the art.

  The compositions in the present invention, or microcarriers for use in the methods of the present invention, are typically screen filters having a size of less than about 10 μm (average diameter is less than about 10 μm, or at least about 97% of the particles are 10 μm). ), And nanocarriers (ie, carriers smaller than 1 μm in size). Preferably, the microcarrier is selected from a size within the upper limit of about 9,7,5,2 or 1 μm or 900,800,700,600,500,400,300,250,200 or 100 nm, and about 4,2 or 1 μm or about 800,600,500,400,300,250,200,150,100,50,25, or 10 nm. It is independently selected at the lower limit, where the significantly lower limit is less than the upper limit. In some examples, the microcarrier has a size of about 1.0-1.5 μm, about 1.0-2.0 μm, or about 0.9-1.6 μm. In certain preferred examples, the microcarrier is about 10 nm to about 5 μm or about 25 nm to about 4.5 μm, about 1 μm, about 1.2 μm, about 1.4 μm, about 1.5 μm, about 1.6 μm, about 1.8 μm, about 2.0 μm, about It has a size of 2.5 μm, or about 4.5 μm. When the microcarrier is a nanocarrier, preferred examples include about 25 to about 300 nm, about 50 to about 200 nm, about 50 nm, or about 200 nm.

  Solid-phase bio-denatured microcarriers are poly (lactic acid), poly (glycolic acid), and copolymers thereof (including block copolymers), and block copolymers of poly (lactic acid) and poly (ethylene glycol); Polyorthoesters such as polymers based on 3,9-diethylidene-2,4,8,10-tetraoxapyro [5.5] undecane (DETOSU); poly (anhydride) polymers with relatively hydrophilic monomers such as sebacic acid High polymerization anhydrides; high polymerization anhydrides such as high polymerization anhydride polymers based on sebacic acid-derived monomers incorporating amino acids such as glycine or alanine (ie, bonded to sebacic acid by an imide bond through the amino-terminal nitrogen) Highly polymerized anhydride esters; especially poly (phosphazenes) (Schacht) containing anhydride-sensitive ester groups that can catalyze the modification of the polymer backbone through the formation of carboxylic acid groups. . (1996) Biotechnol Bioeng.1946: 102); and poly (may include a polyamide such as lactic -co- lysine) prepared from, but not limited to biologically modified polymers. In addition, various suitable non-biodegradable materials for producing microcarriers are well known, including but not limited to polystyrene, polypropylene, polyethylene, latex, gold, and ferromanganese or paramagnetic materials. In particular examples, gold, latex and / or magnetic beads are excluded. In a particular example, the microcarrier can be made from a first material (eg, a magnetic material) that is included in a second material (eg, polystyrene).

  Microspheres consisting of a solid phase are prepared using techniques well known in the art. For example, they can be prepared by emulsification, solvent extraction / evaporation techniques. Generally in this technique, biodegradable polymers such as polyanhydrates, poly (alkyl-α-cyanoacrylates) and poly (α-hydroxy esters) such as poly (glycolic acid), poly (D, L-lactic acid-co-glycolic acid), poly (caprolactone), and the like are dissolved in a suitable organic solvent such as methylene chloride so as to constitute the dispersed phase (DP) of the emulsion. By rapidly homogenizing the dispersed phase (DP) into an excess amount of aqueous continuous phase (CP) containing dissolved surfactants such as polyvinyl alcohol (PVA) or polyvinylpyrrolidene (PVP). Emulsified. This is to ensure that the surfactant in the CP will form discrete and appropriately sized emulsified droplets. The organic solvent is then extracted in CP and then evaporated to dryness by raising the temperature of the system. The solid microparticles are then separated by concentration or filtration and dried by lyophilization or application of vacuum, for example, prior to storage at 4 ° C.

  Physicochemical characteristics such as the average size, size distribution and surface electrification of the dried microspheres can be determined. Size characteristics were determined, for example, by dynamic light scattering techniques, and surface charge was determined by measuring the zeta (ζ) potential.

  Liquid phase microcarriers include oil-based particles that incorporate liposomes, micelles, oil droplets and other lipids or biomodified polymers or oils. In particular examples, the biomodified polymer is a surfactant. In another example, the liquid phase microcarrier can be biologically modified by including a biodegradable oil such as squalene or vegetable oil. One preferred liquid phase microcarrier is oil droplets in an emulsion with oil in water. Preferably, the emulsified liquid in water used as a microcarrier contains a biodegradable substituent such as squalene.

Antigen It is possible to prepare an IMP / MC complex containing the antigen or to prepare an IMP / MC complex without the antigen (ie IMP / MC is not bound to the antigen). Either antigen can be used to prepare an IMP / MC complex containing the antigen.

  In some examples, the antigen is an allergen. Examples of recombinant allergens are provided in Table 1. The preparation of many allergens is well known in the art, and ragweed pollen antigen E (Amb aI) (Rafnar et al. (1991) J. Biol. Chem. 266: 1229-1236), major dust mite allergen Der pI and Der PII (Chua et al. (1988) J. Exp. Med. 167: 175-182; Chua et al. (1990) Int. Arch. Allergy Appl. Immunol. 91: 124-129), birch pollen Bet v1 (Breiteneder et al. (1989) EMBO J.8: 1935-1938), house cat allergen Fel d I (Rogers et al. (1993) Mol. Immunol. 30: 559-568), and three pollen protein antigens (Elsayed et al. (1991) Scand.J.Clin.Lab.Invest.Suppl.204: 17-31), but not limited to. As specified, the three types of allergens are well known, including allergens from oak trees, juniper and Japanese cedar. Preparation of protein antigens from grass pollen for administration in Vivo has been described. See Malley (1989) J. Reprod. Immunol. 16: 173-186. Recombinant allergens As Table 1 shows, in some instances the allergen is a food allergen such as a peanut allergen such as Ara h I, and in some examples the allergen is a rye allergen such as Lol p 1 is a grass allergen. Recombinant allergens Table 1 lists the available allergens.

  In some examples, the antigen is derived from protozoa, bacteria, fungi (including single and multiple cells) infectious agents, and viral infectious agents. Examples of suitable viral antigens are described herein and known in the art. Bacteria include Hemophilus influenza, Mycobacterium tuberculosis and Bordetella pertussis. Protozoan infectious agents include malaria parasites, Rishmania species, trypanosoma species, and schistosomes. Fungi include Candida albicans.

  In some examples, the antigen is a viral antigen. Viral polypeptide antigens include HIV proteins such as HIV gag protein (including but not limited to membrane anchoring (MA) protein, core capsid (CA) protein, and nucleocapsid (NC) protein), HIV polymerase, influenza virus matrix (M) protein, and influenza virus nucleocapsid (NP) protein, hepatitis B surface antigen (HBsAg), hepatitis B core protein (HBcAg), hepatitis e protein (HBeAg), hepatitis B DNA polymerase Including, but not limited to, hepatitis C antigen.

  The literature describing influenza vaccination includes Scherle and Gerhard (1988) Proc. Natl. Acad. Sci. USA 85: 4446-4450; Scherle and Gerhard (1986) J. Exp. Med. 164: 1114-1128; Granoff (1993) Vaccine 11: S46-51; Kodihalli et al. (1997) J. Virol. 71: 3391-3396; Ahmeida et al. (1993) Vaccine 11: 1302-1309; Chen et al. (1999) Vaccine 17: 653-659; Govorkova and Smirnov (1997) Acta Virol. (1997) 41: 251-257; Koide et al. (1995) Vaccine 13: 3-5; Mbawuike et al. (1994) Vaccine 12: 1340-1348; Tamura et al. (1994) Vaccine 12: 310-316; Tamura et al. (1992) Eur. J. Immunol. 22: 477-481; Hirabayashi et al. (1990) Vaccine 8: 595-599. Other examples of antigen polypeptides are antigens specific to groups, or subgroups, known as many infectious agents, adenoviruses, herpes simplex viruses, papilloma viruses, respiratory syncytial viruses and Including but not limited to poxviruses.

  Many antigenic peptides and proteins are known in the art and can be utilized, others can be identified using conventional techniques. For immunization against tumor formation or treatment of existing tumors, immunomodulating peptides are tumor cells (live or irradiated), extracts of tumor cells, protein sub-proteins of tumor antigens such as Her-2 / neu. Unit, Mart1, carcinoembryonic antigen (CEA), ganglioside, human milk fat vesicle (HMFG), mucin (mucin (MUC1)), MAGE antigen, BAGE antigen, GAGE antigen, gp100, prostate specific antigen (PSA), and Tyrosinase can be included. Vaccines for contraception based on immunity can be generated by including sperm proteins administered with ISS. See Lea et al. (1996) Biochim. Biophys. Acta 1307: 263.

  Attenuated and inactivated viruses are suitable for use herein as antigens. The preparation of these viruses is well known in the art and many are commercially available (see, eg, Physicians' Desk Reference (1998) 52nd edition, Medical Economics Company, Inc.). For example, polioviruses are IPOLTM (Pasteur Merieux Connaught) and ORIMUNETM (Lederle Laboratories), hepatitis A virus as VAQTATM (Merck), measles virus as ATTENUVAXTM (Merck), mumps virus as MUMPSVAX ™ (Merck) and rubella virus as MERUVAX ™ II (Merck). In addition, attenuated and inactivated viruses such asHIV-1, HN-2, attenuated and inactivated such as herpes simplex virus, hepatitis B virus, donkey virus, human and non-human papilloma virus, and late brain virus The virus produced can provide a peptide antigen.

  In some examples, the antigen includes vaccines, adenoviruses, and viral vectors such as canary pox. Antigens can be isolated from these resources using purification techniques known in the art, and more conveniently can be produced using recombinant methods.

  Antigenic peptides can include purified natural peptides, synthetic peptides, recombinant proteins, crude protein extracts, attenuated or inactive viruses, cells, microorganisms, or fragments of such peptides. The immunomodulating peptide may be natural, or may be chemically or enzymatically synthesized. Any method of chemical synthesis known in the art is suitable. Peptide synthesis in the liquid phase can be used to construct peptides of the appropriate size, or the solid phase can be used to chemically construct the peptides. See Atherton et al. (1981) Hoppe Seylers Z Physiol. Chem. 362: 833-839.

  In addition, proteolytic enzymes can be utilized to bind amino acids and generate peptides. See Kullmann (1987) Enzymatic Peptide Synthesis, CRC Press, Inc. As an option, peptides can be obtained by using the biochemical machinery of the cell or by being isolated from biological resources. Recombinant DNA technology can be used for the production of peptides. See Hames et al. (1987) Transcription and Translation: A Practical Approach, IRL Press. In addition, the peptides can be isolated using standard techniques such as affinity chromatography.

  Preferably, the antigens are peptides, lipids (sterols excluding cholesterol, fatty acids, and phospholipids), polysaccharides such as those used in H. influenza vaccines, gangliosides and glycoproteins.

  These can be obtained through several methods known in the art, including isolation and synthesis using chemical and enzymatic methods. In certain instances, the antigenic portion of the molecule can be obtained from the market, such as for many sterols, fatty acids, and phospholipids. Such antigens include, but are not limited to, antigens derived from HIV envelope glycoproteins, including, but not limited to, gp160, gp120 and gp41. Many sequences of HIV genes and antigens are known. For example, the Los Alamos National Laboratory HIV Sequence Database collects annotates HIV nucleotide and amino acid sequences (curates and annotates). The database can be accessed via the Internet at http://hiv-web.Ianl.gov/, and for annual publications see Human Retroviruses and AIDS Compendium (eg 1998).

  Antigens derived from infectious agents can be produced using methods known in the art, for example natural viruses or bacteria, cells infected with infectious agents, purified polypeptides, recombinantly produced polypeptides And as a synthetic peptide.

  Generation of IMP / MC complexes can be prepared with other immunotherapeutic agents including but not limited to cytokines, adjuvants, and antibodies, such as anti-tumor antibodies and derivatives thereof. The production of these IMP / MC complexes can be prepared with or without antigen.

IMP / MC composite
The IMP / MC complex has a 5′-C, G-3 ′ sequence that is bound to or inserted into the surface of the microcarrier (ie, the 7-mer oligonucleotide is not encapsulated in the MC). Including a 7-mer oligonucleotide, and preferably including a plurality of molecules of oligonucleotide bound to each microcarrier. In certain instances, a mixture of different oligonucleotides can be complexed with a microcarrier, so that the microcarrier is bound to two or more oligonucleotide species. The bond between the 7-mer oligonucleotide and the MC may be covalent or non-covalent. As can be appreciated by those skilled in the art, the 7-mer oligonucleotide can be modified or derivatized, and the composition of the microcarrier can be selected as desired in the desired linkage to form the IMP / MC complex. The mold can be selected and / or modified to apply.

  The present invention provides methods for making IMP / MC complexes and products from such methods. An IMP / MC complex is created by combining oligonucleotide and MC to form a complex. The particular method for conjugating the oligonucleotide and MC to form a complex will of course depend on the type and properties of the MC and the binding form of the oligonucleotide and MC. When the MC is a solid phase MC, the IMP / MC complex is preferably contacted with the oligonucleotide and the MC under conditions that promote complex formation (depending on the type of binding used in the complex). It is preferable to create. When the MC is in the liquid phase, the oligonucleotide can bind to the preformed MC under conditions that promote complex formation, or can bind to the components of the MC prior to forming the MC. In the situation where the oligonucleotide is bound to a component of the liquid phase MC, the process of making the MC can incorporate the oligonucleotide, thus resulting in the simultaneous creation or incorporation of the IMP / MC complex. If not, the process includes an additional step under conditions that promote complex formation.

  It is preferred that the IMP / MC complex according to the present invention is insoluble in pure water and the IMP / MC complex composition is free of acetonitrile, dichloroethane, toluene, and methylene chloride (dichloromethane).

  Covalently bound IMP / MC complexes can be bound using any of the covalent crosslinking techniques known in the art. Typically, an oligonucleotide moiety is modified to incorporate an additional component (e.g., a free amine, carboxyl or sulfhydryl group) or to provide a site where the oligonucleotide moiety can be attached by a microcarrier (e.g. Phosphorothioate) would be modified either by incorporating nucleotide bases. The conjugation between the oligonucleotide and the MC portion of the complex can be made at the 3 ′ or 5 ′ end of the oligonucleotide, or can be made at an appropriately modified base at an internal site of the oligonucleotide. Functional groups that are normally present on microcarriers are also available, but in addition, the microcarrier is incorporated with components that can undergo general modifications to form covalent bonds. Under conditions that allow IMP / MC to form a covalent bond (eg, by using an activated microcarrier that includes an activated moiety that forms a covalent bond with an oligonucleotide in the presence of a teaching agent) Formed by incubating oligonucleotides with microcarriers.

A variety of cross-linking techniques are known in the art and include cross-linking reactions with amino, carboxyl and sulfhydryl groups. As will be apparent to those skilled in the art, the choice of cross-linking agent and cross-linking method will depend on the oligonucleotide and the structure of the microcarrier and the desired final IMP / MC complex. The cross-linking agent may be either a homo-binary function or a hetero-binary function. When a homobifunctional cross-linker is used, the cross-linker is an oligonucleotide and MC (
For example, the aldehyde crosslinker can be coupled so that the oligonucleotide and MC both bind covalently to the oligonucleotide and MC, both of which contain one or more free amines. Heterogeneous crosslinkers include oligonucleotides and MC (eg, maleimide-N-hydroxysuccinimide ester can be used to covalently link free sulfhydryl on the oligonucleotide and free amine on the MC) It is preferred to utilize the different components above and minimize the binding between the microcarriers. In most cases, it is preferred to crosslink via a first cross-linking component on the microcarrier and a second cross-linking component on the oligonucleotide, wherein the second cross-linking component is not present on the microcarrier. One preferred method for generating the IMP / MC complex is to “activate” the microcarrier by incubating with a heterobifunctional cross-linker, and under conditions appropriate for the reaction, the oligonucleotide and This is a method for forming an IMP / MC complex by incubating activated MC. The cross-linking agent can incorporate a “spacer” arm between the reaction components, or can directly bond the two reaction components in the cross-linking agent.

  In preferred examples, the oligonucleotide moiety includes at least one free sulfhydryl (eg, a base or linker modified to a 5′-thiol) to crosslink the microcarrier, but the microcarrier includes a free amino group. . Reaction of heterobifunctional crosslinkers with these two groups such as succinimidyl 4- (N-maleimidomethyl) cyclohexane-1-maloxylate (eg crosslinkers containing maleimide groups and NHS-esters) activates MC Thus, the oligonucleotide is covalently cross-linked to form an IMP / MC complex.

  When the binding pair binds the oligonucleotide and MC, the non-covalent IMP / MC complex is an ionic bond (electrostatic), the non-covalent IMP / MC complex is a hydrophobic interaction, as is normal. , Hydrogen bonds, one-denwald attractive forces, etc., can be combined by any non-covalent bond or interaction or a combination of two or more different interactions.

  Preferred non-covalent IMP / MC complexes are used from the mating pair via hydrophobic or electrostatic (ionic) interactions or combinations thereof (e.g., base pairing of oligonucleotides and polynucleotides bound to MC) ) Is typically combined. Due to the hydrophilic nature of the backbone of the polynucleotide, the IMP / MC complex due to the hydrophobic interaction to form a complex requires modification of the oligonucleotide portion of the complex to incorporate a highly hydrophobic component. Preferably, the hydrophobic component is a biologically compatible non-immunogen and forms naturally in an individual intended for the composition (eg found in mammals, particularly humans). Examples of preferred hydrophobic components include lipids, steroids such as cholesterol, and terpenses. The method of attaching the hydrophobic component to the oligonucleotide will of course depend on the structure of the oligonucleotide and the identification of the hydrophobic component. The hydrophobic component can be added to any conventional site of the oligonucleotide, preferably either 5 'or 3', i.e. when adding a cholesterol component to the oligonucleotide, the cholesterol component is (See Godard et al. (1995) Eur. J. Biochem. 232: 404-410) and is preferably added to the 5 ′ end of the oligonucleotide. Microcarriers for use in IMP / MC composites, preferably linked by hydrophobic bonds, can similarly be used with hydrophobic materials modified to incorporate hydrophobic components, such as oil droplets or hydrophobic polymers Made from a hydrophobic material. If the microcarrier is a liposome or other liquid phase containing lumens, the IMP / MC complex may be conjugated with the oligonucleotide after MC preparation to avoid encapsulation of the oligonucleotide during the MC preparation process. It is formed by mixing with MC.

  The non-covalent IMP / MC complex was bound by an electrical bond that typically indicates a strong negative charge on the polynucleotide backbone. Thus, microcarriers for use in non-covalently bound IMP / MC complexes are usually positively charged (cationic) at physiological pH (eg, about 6.8 to 7.4). Microcarriers can inherently hold a positive charge, but microcarriers made from compounds that normally do not have a positive charge can be derivatized to be positively charged (e.g., cations), and so on It can be modified without it. For example, the polymer used to make the microcarrier can be derivatized to add a positively charged group such as a primary amine. As an option, when the microcarrier being fabricated is formed, a positively charged compound can be incorporated (e.g., a positively charged surfactant can be obtained, e.g., as described in Example 2). It can also be used during the production of poly (lactic acid) / poly (glycolic acid) copolymer to impart a positive charge on the microcarrier particles). Therefore, the microcarrier can contain a positively charged component.

  As described herein, to prepare positively charged microcarriers, positively charged lipids or polymers such as 1,2-dioleyl-1,2,3-trimethylammoniopropane (DOTAP), cetyltrimethyl Ammonium bromide (CTAB) or polylysine is added to either DP or CP for solubility in these phases.

  As described herein, IMP / MC complexes can be performed by adsorption onto cationic microspheres and particles, preferably in aqueous mixtures, upon oligonucleotide incubation. The chin cubation is performed under any desired condition including normal temperature (room temperature) (for example, about 20 ° C.) or under freezing (serving 4 ° C.). Because cationic microspheres and oligonucleotides associate relatively quickly, incubation is possible at any conventional time period, including 5,10,15 minutes or more, including one day or longer incubation. is there. For example, oligonucleotides containing 5′-CG-3 ′ can be adsorbed onto cationic microspheres by incubating the oligonucleotides and particles with aqueous overnight at 4 ° C. However, since cationic microspheres and oligonucleotides associate simultaneously, IMP / MC complexes can be formed by simple co-administration of oligonucleotides and MC. Microspheres are characterized for size before and after oligonucleotide binding, and surface charge.

The selected batch can then be evaluated for activity against appropriate controls as described herein, eg, in established human peripheral blood mononuclear cells (PBMC) and mouse splenocyte assays. The product can be evaluated in an appropriate animal mode. In another example, a binding pair can be used to bind the oligonucleotide and MC to the IMP / MC complex. The binding pair can be either a receptor and ligand, an antibody and an antigen (or epitope), or other binding pair that binds with high affinity (eg, a K _{d of} less than about 10 ⁻⁸ ). One preferred type of binding pair is biotin and step abdin, or biotin and avidin, which form a very strong complex. When using binding base pairs to mediate the binding of the IMP / MC complex, the oligonucleotide is typically derived by covalently binding together one member of the binding pair, and the MC is , Guided by another member of the binding pair. IMP / MC composites are formed by mixing two derivatized compounds.

  Many examples of IMP / MC complexes do not contain antigens, and in particular examples, exclude antigens associated with the disease or disorder that is the purpose of IMP / MC complex therapy. In a further example, the oligonucleotide is also bound to one or more molecules. For example, as described in WO 98/16247, the antigen can be conjugated to the oligonucleotide portion of the IMP / MC complex in a variety of ways, including interacting covalently and / or non-covalently. it can. As an option, the antigen may be bound to a microcarrier (either directly or indirectly). Conjugating an antigen to a microcarrier can be done by any of a number of methods known in the art, including direct covalent linkage, covalent linkage via a bridging moiety (including spacer arms) Non-covalent bonds through specific binding pairs (eg, buotin and avidin), and non-covalent bonds through electrostatic or hydrophobic bonds.

Appropriately modified bases between the antigen and the oligonucleotide in the IMP / MC complex containing the antigen bound to the oligonucleotide, either 3 'or 5' of the oligonucleotide or at an internal position in the oligonucleotide Can be done. If the antigen is a peptide and contains an appropriate reactive group (eg, N-hydroxysuccinimide ester), it can react directly with the N ⁴ amino group of a cytosine residue. Depending on the number and position of the oligonucleotides, specific binding at one or more residues can be achieved.

  As an option, a modified nucleoside or nucleotide known in the art can be incorporated at the terminal or internal position of the oligonucleotide. These can contain blocked functional groups and react with various functional groups that can be present or bound on the associated antigen once the block is released.

  If the antigen is a peptide, this binding moiety can be attached to the 3 ′ end of the oligonucleotide via solid support chemistry. For example, an oligonucleotide moiety can be added to a polypeptide moiety that has been pre-synthesized on a carrier. See Haralambidis et al. (1990a) Nucleic Acids Res. 18: 498-499: and Haralambidis et al. (1990b) Nucleic Acids Res. 18: 501-505. As an option, the oligonucleotide can be synthesized to be attached to the solid support via a cleavable linker extending from the 3 ′ end. Chemical cleavage of the oligonucleotide from the support leaves a terminal thiol group at the 3 ′ end of the oligonucleotide (Zuckermann et al. (1987) Nucleic Acids Res. 15: 5305-5321; and Corey et al. (1987) Science. 238: 1401-1403) or the terminal amino group is left at the 3 ′ end of the oligonucleotide (see Nelson et al. (1989) Nucler. Acids Res. 17: 1781-1794). Coupling with amino-modified oligonucleotides to the amino group of the peptide can be achieved as described in Benoit et al. (1987) Neuromethods 6: 43-72. Binding of the thiol-modified oligonucleotide to the carboxyl group of the peptide can be performed as described in Sinha et al. (1991), pp. 185-210, Oligonucleotide Analogues: A Practical Approach, IRL Press. it can. Further described is the attachment of a maleimide-transferring oligonucleotide attached to the thiol side chain of the cytosine residue of the peptide. See Tung et al. (1991) Bioconjug. Chem. 2: 464-465.

The peptide portion of the conjugate can be attached to the 5 ′ end of the oligonucleotide via an amine, thiol, or carbonyl group that has been incorporated into the oligonucleotide during synthesis. Preferably, the oligonucleotide is immobilized on a solid support and has a protected amine, thiol or carbonyl group at one end and a phosphoramidite at the other end covalently linked to the 5'-hydroxyl group. Combined with Agrawal et al. (1986) Nucleic Acids Res. 14: 6227-6245; Connolly (1985) Nucleic Acids Res. 13: 4485-4502; Kremsky et al. (1987) Nucleic Acids Res. 15: 2891-2909; ConnoIly (1987) Nucleic Acids Res. 15: 3131-3139; Bischoff et al. (1987) Anal. Biochem. 164: 336-344; Blanks et al. (1988) Nucleic Acids Res. 16: 10283-10299; and US Patent Nos. 4,849,513,5,015,733,5,118,800, and 5,118,802 See After deprotection, amine, thiol, or carbonyl functional groups can be used to covalently attach the oligonucleotide to the peptide. See Benoit et al. (1987); and Sinha et al. (1991).
Furthermore, oligonucleotide-antigen bonds can be formed through non-covalent interactions such as ionic bonds, hydrophobic interactions, hydrogen bonds, and / or One Den Waals attractions.

  Non-covalently bound binding agents can include non-covalent interactions such as biotin-streptavidin complexes. A biotinyl group can be attached, for example, to a modified base of an oligonucleotide. See Roget et al. (1989) Nucleic Acids Res. 17: 7643-7651. By incorporating streptavidin into the peptide moiety, a non-covalent complex of streptavidin-conjugated peptide and biotinylated oligonucleotide can be formed.

In addition, non-covalent associations include charged residues that can interact through ionic interactions involving oligonucleotides and residues within the antigen, such as charged amino acids, or by both oligonucleotides and antigens. It can be formed by using a binding moiety. For example, non-covalent bonds can occur between positively charged amino acid residues of normally negatively charged oligonucleotides and peptides such as polylysine, polyarginine, and polyhistidine residues.
Non-covalent bonds between oligonucleotides and antigens can occur due to the DNA-binding motifs of molecules that interact with DNA as their natural ligand. For example, such DNA binding motifs can be found in transcription factors and anti-DNA antibodies.

  The oligonucleotide is attached to the lipid using standard methods. These methods include oligonucleotide and phospholipid linkage (see Yanagawa et al. (1988) Nucleic Acides Symp. Ser. 19: 189-192), oligonucleotide and fatty acid linkage (Grabareck et al. (1990) Anal. Biochem. 185 : 131-135; and Staros et al. (1986) Anal. Biochem. 156: 220-222) and conjugation of oligonucleotides and sterols (Boujrad et al. (1993) Proc. Natl. Acad. Sci. Reference)), but is not limited thereto.

  Oligonucleotide linkages to oligopolysaccharides are formed by standard well-known methods. These methods include, but are not limited to, the synthesis of conjugates of oligonucleotides and oligopolysaccharides, where the oligopolysaccharide is a component of an immunoglobulin. See O'Shannessy et al. (1985) J. Applied Biochem. 7: 347-355.

  Binding of a cyclic oligonucleotide to a peptide or antigen can be formed in several ways. If circular oligonucleotides are synthesized using recombinant or chemical methods, modified nucleosides are suitable. See Ruth (1991), pp. 255-282, in Oligonucleotides and Analogues: A Practical Approach IRL Press. Standard binding techniques can then be used to attach the cyclic oligonucleotide to the antigen or other peptide. See Goodchild (1990) Bioconjug. Chem. 1: 165. Here, when a circular oligonucleotide is isolated or synthesized using recombinant or chemical methods, its linkage is incorporated into an antigen or other peptide, a chemically active or photoactivated reactive group (e.g. Carbene radical).

  Additional methods for conjugating peptides and other molecules to oligonucleotides are described in US Patent No. 5,391,723; Kessier (1992) `` Nonradioactive labeling methods for nucleic acids '' in Kricka (ed.) Nonisotopic DNA Probe Techniques, Academic Press: and Geoghegan et al. (1992) Bioconjug. Chem. 3: 138-146.

Methods of the Invention The invention comprises administering to an individual an IMP / MC complex (typically a complex and a pharmaceutically acceptable excipient) such that the desired modulation of the immune response is made. Methods are provided for modulating an immune response in an individual, preferably a mammal, more preferably a human. Immunomodulation includes stimulating a Th1-type immune response and / or inhibiting or reducing a Th2-type immune response. As described herein, modulation of the immune response may be humoral and / or cellular and is measured using technically standard techniques and as described herein.

  In some examples, immunomodulation includes stimulating (ie, one or more) Th1-related cytokines such as IFN-γ, IL-12 and / or IFN-α. Technically standard methods are used to measure these parameters and are also described herein. As described herein, administration of IMP / MC further includes one or more additional immunotherapeutic agents (i.e., via the immune system) including but not limited to cytokines, adjuvants, and antibodies. Administration) and / or derived from the immune system). Examples of therapeutic antibodies include those used in cancer relations (eg anti-tumor antibodies). Administration of such additional immunotherapeutic agents applies to all methods described herein.

  In certain instances, an individual suffers from a disease associated with a Th2-type immune response, such as allergy or allergy-induced asthma. Administration of the IMP / MC complex results in immune regulation, which may increase the level of one or more Th1-type response-related cytokines, resulting in a decrease in Th2-type response characteristics associated with the individual's response to the allergen.

  Immunomodulating an individual associated with a Th2-type response-related disease reduces or improves symptoms in one or more diseases. If the disease is allergy or allergy-induced asthma, one or more of the following can be ameliorated: rhinitis, allergic conjunctivitis, decreased circulating level of IgE, decreased circulating level of histamine, and Included is a need for “rescue” inhalation therapy (eg, a dose metered inhaler or nebulizer).

  In a further example, the individual's challenge for the immunomodulatory treatment of the present invention is the individual receiving the vaccine. The vaccine may be a prophylactic vaccine or a therapeutic vaccine. Prophylactic vaccines contain one or more epitopes associated with diseases that can be dangerous for an individual (eg, M. tuberculosis as a vaccine to prevent tuberculosis). Therapeutic vaccines include M. tuberculosis or M. bovis surface antigens in tuberculosis patients, antigens that make the individual allergic in allergic subjects (allergy desensitization therapy), tumor cells from individuals with cancer (US Patent No. 5,484,596), or one or more epitopes associated with a particular disease affecting an individual, such as tumor associated cells in cancer patients. The IMP / MC complex may be given in combination with the vaccine (eg, the same injection or simultaneous but separate injection), or the IMP / MC complex may be administered separately (eg, vaccine At least 12 hours before and after administration). In certain instances, the antigen of the vaccine is part of the IMP / MC complex, either covalently or non-covalently bound to the IMP / MC complex. Therapy that administers the IMP / MC complex to individuals receiving the vaccine causes an immune response to the vaccine that shifts towards a Th1-type response compared to individuals receiving the vaccine without the IMP / MC complex. A shift towards a Th1-type response is an increase in cytokines associated with INF-γ and other Th1-type responses, an increase in INF-α, production of CTLs specific for vaccine antigens, and specificity for vaccine antigens Delayed hypersensitivity to antigens in vaccines, such as reduced or reduced levels of IgE, decreased Th2-related antibodies specific to vaccine antigens, and / or increased Th1-related antibodies specific to vaccine antigens Can be recognized by the disease (DTH) response. In the case of therapeutic vaccines, further administration of the IMP / MC complex and vaccine will improve the symptoms of the disease for which the vaccine is intended for treatment. As will be apparent to those skilled in the art, the exact symptoms and methods for these improvements will depend on the disease sought to be treated. For example, a therapeutic vaccine is for tuberculosis, and treatment of the IMP / MC complex with the vaccine alleviates cough, peritoneal or chest wall pain, fever and / or other symptoms known in the art. If the vaccine is an allergen used for allergic desensitization treatment, such treatment may result in one or more allergic symptoms (e.g. rhinitis, allergic conjunctivitis, IgE at the circulating level, and / or histamine at the circulating level). Will decrease).

  Other examples of the present invention relate to immunomodulatory treatment of individuals having a disease or disorder, such as pre-existing cancer or infectious disease. Cancer is an attractive target for immunomodulation because most cancers express tumor-related and / or tumor-specific antigens that are not found in other cells in the body. Stimulating a Th1-type response to tumor cells results in direct and / or indirect killing of tumor cells by the immune system, leading to a reduction in cancer cells and symptoms. Administration of an IMP / MC complex to an individual with cancer will stimulate a Th1-type immune response against tumor cells. Such by either the direct action of cells of the cellular immune system (e.g. CTLs) or the composition of the humoral immune system or by a bystander that affects cells adjacent to cells targeted by the immune system. The immune response can kill tumor cells. Further in the cancer context, administration of the IMP / MC complex includes administration of one or more additional therapeutic agents such as anti-tumor antibodies, chemotherapeutic forms, and / or radiation therapy. Anti-tumor antibody fragments and / or derivatives thereof, and monoclonal anti-tumor antibodies, fragments and / or derivatives thereof, including but not limited to cancer treatments (e.g., RituxanTM (rituximab); Herrceptin ( (Trastuzumab) is known in the art as administering such antibody reagents, administration of one or more additional therapeutic therapeutic agents may be performed before, after, and after administration of the IMP / MC complex. And / or can occur at the same time.

  Furthermore, the immunomodulatory treatment according to the invention is beneficial for individuals suffering from infectious diseases, in particular infectious diseases that resist humoral immune responses (eg diseases caused by mycobacteria and intracellular pathogens). Immunomodulatory therapy can be used for the treatment of infectious diseases caused by cellular pathogens (bacteria or protozoa) or non-cellular pathogens (eg viruses). Treatment with an IMP / MC complex may include tuberculosis (eg, infection with M. tuberculosis and / or M. bovis), lei disease (infection with R. bacterium), M. marinum or ursalan (M. .ulcerans) can be administered to individuals suffering from a mycobacterial disease. In addition, IMP / MC complex therapies include viral infections, including infection with influenza virus, respiratory syncytial virus (RSV), liver virus B, liver virus C, herpes virus, especially herpes simplex virus, and papilloma virus. Useful for treatment.

  Malaria etc. Diseases caused by intracellular parasitic bacteria such as infantum, L. chagasi, and / or L. aethiopica), and toxoplasmosis (ie infection with Toxoplasmosis gondii) can further benefit from IMP / MC complex treatment . Further treatment of IMP / MC includes schistosomiasis (ie, infection by blood fiukes of Schistosoma species such as S. haematobium, S. mansoni, S. japonicum, and S. mekogi), and hepatosomiasis (ie, Clonorchis Treatment of parasitic bacteria such as infection by sinensis is beneficial. Administration of an IMP / MC complex to an individual suffering from an infectious disease will recover one or more symptoms of the infectious disease. As shown herein, in some cases the infectious disease is not a viral disease.

  The present invention further provides a method of increasing at least one Th1-related cytokine in an individual, including IL-2, IL-12, TFN-β, and IFN-γ. In certain instances, the present invention provides a method for increasing IFN-γ in an individual, particularly in an individual in need of increased IFN-γ levels, by administering to the individual an effective amount of an IMP / MC complex To do. Individuals with a disease in which the individual responds to administration of IFN-γ with an increase in IFN-γ. Such diseases include many inflammatory diseases, including but not limited to neoplastic colitis. In addition, these diseases include idiopathic pulmonary fibrosis (IPF), scleroderma, fibers induced by skin radiation, liver fibers including liver fibers induced by schistosomiasis, kidney fibers, and IFN-γ. It includes many fibrotic diseases, including but not limited to other symptoms that can be ameliorated by administration. Administration of the IMP / MC complex according to the present invention increases the level of IFN-γ and improves one or more symptoms, stabilizes one or more symptoms, prevents the progression of disease in response to IFN-γ (Additional damage or reduction or elimination of symptoms). The methods of the invention can be performed in combination with other therapies that constitute criteria for caring for diseases such as administration of anti-inflammatory agents such as systemic corticosteroid therapy (eg, cortisone) in IPF.

  In a specific example, the present invention relates to administering an effective amount of an IMP / MC complex to an individual so that the level of IFN-α is increased, particularly in an individual in need of increased levels of IFN-α. Provide a method to increase -α. An individual in need of an increase in IFN-α is an individual with a disease that responds to administration of IFN-α, including recombinant IFN-α, including but not limited to viral infection and cancer. Thus, administration of an IMP / MC complex according to the present invention results in an increase in IFN-α levels and the improvement of one or more symptoms, stabilization of one or more symptoms, or a disease responsive to IFN-α. Stops progression (eg, reduces or eliminates additional lesions or symptoms). The methods of the invention can be performed in combination with other therapies that constitute standard care for the disease, such as administering an antiviral agent for viral infection.

  Further provided is to reduce the level of IgE, especially its serum level, to an individual with an IgE-related disease by administering an effective amount of IMP / MC complex to the individual so that the level of IgE is reduced It is a method to make it. A decrease in IgE will improve the symptoms of IgE-related diseases. Such symptoms include allergic symptoms such as rhinitis and conjunctivitis in reducing allergen sensitivity, reducing allergic symptoms in individuals with allergies, or reducing the severity of allergic responses.

  As will be apparent to those skilled in the art, the methods of the invention can be performed in combination with other therapies for the specific indication that the IMP / MC complex is administered. For example, therapy with an IMP / MC complex is associated with anti-malarial drugs such as chloroquinine for malaria patients, and with rheumania drugs such as pentamidine and / or allopurinol for patients with rheumania. It can be administered in patients in conjunction with anti-mycobacterial drugs such as isoniaside, rifampin and / or ethambutol or in conjunction with allergen desensitization therapy for atopic (allergy) patients.

Administration and evaluation as an immune response
The IMP / MC complex can be administered in combination with other pharmaceutical agents and / or immunogens and / or immunomodulatory agents and can be combined with its physiologically acceptable carrier.

  Thus, the IMP / MC complex can be administered in conjunction with other immunotherapeutic agents including but not limited to cytokines, adjuvants, and antibodies.

  As long as the IMP / MC is active, the IMP / MC complex can contain any combination of oligonucleotide and MC as described above. In general, in some instances, the IMP / MC complex has a negative control activity of at least about 2, preferably at least about 3, more preferably, in at least one assay of activity. Considered active if it has at least about 5, more preferably at least about 10 (ie, affects a measurable immune response as measured in vitro, in vivo and / or ex vivo) . Methods for assessing a measurable immune response are known in the art and include the human PBMC assay disclosed herein.

Any of the above may be used as the oligonucleotide of the IMP / MC complex. Preferably, the administered 7-mer oligonucleotide comprises the sequence 5′-T, C, G-3 ′. Another preferred example uses a nucleotide comprising the sequence 5′-CG-3 ′ and further comprising the sequence 5′-TCG-3 ′. Another preferred example is 5'-TCGX ₁ X ₂ X ₃ X ₄ -3 ', 5'-X ₁ TCGX ₂ X ₃ X ₄ -3', 5'-X ₁ X ₂ TCGX ₃ X ₄ -3 ' An oligonucleotide is used that consists of a sequence selected from the group consisting of X ₁ , X ₂ , X ₃ , X ₄ being nucleotides.

  Regardless of the immunogenic composition, the immunologically effective amount and method of administering a particular IMP / MC complex molding can be treated by one of ordinary skill in the art and other factors will be apparent. It changes based on the individual. Possible factors depend on the antigenicity, the IMP / MC complex is administered with an adjuvant or transduction molecule, or is covalently linked, the route of administration, and the number of immunization doses administered. , Covalently coupled with it. These factors are well known in the art and are within the technically sufficient range to make such a determination without undue experimentation. An appropriate dosage range is one that provides the desired modulation of the immune response to the antigen. In general, the dosage is determined by the amount of oligonucleotide administered to the patient other than the total amount of IMP / MC complex. For a given derivatized oligonucleotide amount, the beneficial dose range of the IMP / MC complex is from any of the following: 0.1 to 100 μg / kg, 0.1 to 50 μg / kg, 0.1 to 25 μg / kg, 0.1 10μg / kg, 1 to 500μg / kg, 100 to 400μg / kg, 200 to 300μg / kg, 1 to 100μg / kg, 100 to 200μg / kg, 300 to 400μg / kg, 400 to 500μg / kg . As an option, the dose can be any of the following: 0.1 μg, 0.25 μg, 0.5 μg, 1.0 μg, 2.0 μg, 5.0 μg, 10 μg, 25 μg, 50 μg, 75 μg, 100 μg.

  Thus, dose ranges are possible in the following ranges at the lower limit: 0.1 μg, 0.25 μg, 0.5 μg, 1.0 μg, and upper limits at the following: 25 μg, 50 μg, 100 μg. The absolute amount given to each patient depends on pharmacological properties such as bioavailability, tolerance, and route of administration.

  The effective amount and method of administration of a particular IMP / MC complex former will vary based on the individual patient and the stage of disease and other factors apparent to those skilled in the art. Administration routes useful for specific indications will be apparent to those skilled in the art. Routes of administration include, but are not limited to, topical, cutaneous, transdermal, transmucosal, epithelial, parenteral, gastrointestinal, nasopharyngeal and lung, including transbronchial and transgingival pus. A suitable dosage range is one that provides sufficient oligonucleotide composition to achieve a tissue concentration of about 1-10 μM as measured at the level of blood. The absolute amount given to each patient depends on pharmacological properties such as bioavailability, tolerance, and route of administration.

  As described herein, APC and high concentrations of APC tissue are preferred targets for the IMP / MC complex. Thus, when APC is present at relatively high concentrations, it is preferred to administer the IMP / MC complex to mammalian skin and / or mucosa.

  The present invention provides products of IMP / MC complexes suitable for topical application including, but not limited to, physiologically acceptable implants, ointments, creams, rinses and gels. Topical administration can be performed, for example, by placing the introducer system dispersed or wound in a bandage, or by administering the introducer system directly to an incised or open wound, or percutaneously under the relevant site. Depending on the dosing device. Creams, rinses, gels or ointments in which the IMP / MC complex is dispersed are suitable for use as topical ointments or drugs for filling wounds.

  The preferred route of dermal administration is at least an invasive route. Preferred among these means are transdermal transport, epithelial administration, and subcutaneous injection. Of these means, epithelial administration is preferred for significantly higher concentrations of APC predicted to be in subcutaneous tissue.

  Transdermal administration can be performed by applying a cream, rinse, gel, etc. so that the IMP / MC complex can enter the bloodstream through the skin. Compositions suitable for transdermal administration can be applied directly to the skin or incorporated into protective carriers such as subcutaneous administration devices (so-called “patch”), pharmacologically acceptable suspensions, oils, creams , And ointments. Examples of suitable creams, ointments and the like can be found, for example, in the Physician's Desk Reference.

  Ion osmosis is a suitable method for transdermal delivery. Transfer by ion osmosis can be performed using commercially available distribution agents in which the transfer material is continuously produced through unbroken skin for a period of days greater than a few days. Using this method, the transport of the pharmacological composition can be controlled at relatively high concentrations, exudative agents can be combined, and adsorption enhancers can be used simultaneously.

  A typical distribution for use in the present method is LECTRO PATCH ™ as a trademarked product of the General Medical Company of Los Angeles, CA. The product can be applied to provide doses at different concentrations so that the pH of the reservoir electrode is neutral, electrically held, with continuous and / or periodic doses. it can. The preparation of the wrapping and its use should be carried out according to the manufacturer's printed instructions accompanying the LECTRO PATCH product, ie those instructions incorporated herein by reference. Other storable distribution systems are more suitable.

  For transdermal transport, low frequency ultrasound guidance is also a suitable method. See Mitragotri et al. (1995) Science 269: 850-853. Application of a low frequency ultrasound frequency (about 1 MHz) can normally control the transport of therapeutic compositions, including high molecular weight compositions.

  Epithelial administration specifically includes mechanically or chemically stimulating the outermost layer of the epithelium to sufficiently elicit an immune response to the stimulant. In particular, the stimulation should sufficiently attract APC to the stimulation site.

  A typical mechanistic stimulation means is the use of multiple thin, short chins that can be used to stimulate the skin, and attracts APC to the stimulation site and is transported from the end of the chin / imp / Construct MC complex. For example, the MONO-VACC Old Tuberculin Test, created by Pasteur Merieux of Lyon, France, includes a suitable device for introducing a composition containing an IMP / MC complex.

  The device (distributed to the United States by Connaught Laboratories, Ine. Of Swiftwater, PA) consists of a plastic container with a syringe plunger at one end and a chin disc at the other end. Chin discs support multiple thin-diameter chins by scratching the outermost layer of epithelial cells. Each tin in the MONO-VACC kit is coated with old tuberculin, ie, in the present invention, each needle is coated with the pharmacological composition of the product of the IMP / MC complex. In using the device, it is preferable to follow the written instructions of the manufacturer included in the device product. Further, a similar device that can be used in this example is a device that is newly used to perform an allergy test.

  Other suitable methods for administration of the IMP / MC complex from the epithelium elicit a sufficient immune response by using chemical methods that stimulate the outermost layer of the epithelium and thus attract APC to that region . The sample is a keratinocyte agent such as salicylic acid sold in a commercially available topical hair removal cream sold by Noxema Corporation under the trademark NAIR. This method can also be used to administer epithelial to mucosa. In addition, chemical stimuli can be applied in conjunction with mechanical stimuli (eg, as triggers when MONO-VACC type chin is further coated with chemical stimulants). The IMP / MC complex can be suspended in a carrier that also contains a chemical stimulant, or can be co-administered therewith.

  The parenteral route of administration includes electrical (microelectrophoresis) or direct injection methods such as direct injection into the central venous system, intravenous, intramuscular, intraperitoneal, intradermal or subcutaneous injection. Including but not limited to. IMP / MC products suitable for parenteral administration are usually formulated in USP water or water for infusion, and pH buffers, salt bulking agents, preservatives and other pharmacologically acceptable An excipient may further be included. IMP / MC complexes for parenteral injection can be formulated in pharmacologically acceptable sterile, isosmotic solutions such as saline and phosphate buffered saline for injection. .

  The gastrointestinal route of administration includes but is not limited to ingestion and rectum. The invention includes IMP / MC complex formation suitable for gastrointestinal administration, including but not limited to pharmacologically acceptable powders, pills or liquids for ingestion, and suppositories for rectal administration. As will be apparent to those skilled in the art, the pill or suppository further comprises a pharmaceutically acceptable solid, such as starch, to provide a bulk for the composition.

  It includes administration from the nasopharynx, which is performed by inhalation, and to the lungs, and includes inductive routes such as intranasal, transbronchial, and transpulmonary routes. IMP / MC complex products suitable for administration by inhalation including, but not limited to, suspension liquids to form aerosols and powder forms for dry powder inhalation induction systems including. Suitable devices for administering IMP / MC composite moldings by inhalation include, but are not limited to, atomizers, evaporators, nebulizers, and dry powder inhalation induction devices.

  Selection of induction pathways can be used to modulate the immune response elicited. For example, when the influenza virus vector was administered intramuscularly or via the epithelial (gene gun) route, the IgG titer and CTL activity were identical, however, when ingested mainly into muscle, the IgG2a However, most of the epithelial pathway produced IgG1. See Pertmer et al. (1996) J. Virol. 70: 6119-6125. Thus, one skilled in the art can take the slightly different advantages of immunogenicity elicited by different routes of administering the immunomodulatory oligonucleotides of the invention.

  The above compositions and methods of administration are within the stated meaning of the method of administering the IMP / MC complex product of the present invention, but are not so limited. Methods for manufacturing various compositions and devices are within the ability of those skilled in the art and are not described in detail herein.

  Analysis of immune response (both qualitative analysis and quantitative analysis) on the IMP / MC composite molded body is possible by any method known in the art, and the production of antigen-specific antibodies is measured (specific antibodies Activation of specific populations in lymphocytes such as CD4 + T cells or NK cells, IFN-γ, IFN-α, IL-2, IL-4, IL-5, IL-10 Or including but not limited to cytokine production such as IL-12 and / or histamine release. Methods for measuring specific antibody responses include enzyme-linked immunosolvent assays (ELISA) and are known in the art. Measurement of the number of lymphocytes of a specific type such as CD4 + T cells can be performed, for example, with a fluorescence activated cell sorter (FACS). Cytotoxicity tests can be performed as described by Raz et al. (1994) Proc. Natl. Acad. Sci. USA 91: 9519-9523. Cytokine concentrations can be measured, for example, by ELISA. These and other assays for assessing immune responses against immunogens are well known in the art. See, eg, Selected Methods in Cellular Immunology (1980) Mishell and Shiigi, eds., W.H. Freeman and Co.

  Preferably, the Th1-type response is stimulated, i.e. evoked and / or enhanced. In the context of the present invention, stimulating a Th1-type immune response in vitro or ex vivo by measuring cytokines produced from cells treated with ISS compared to those treated without ISS, Can be determined. Methods for determining cellular cytokine production include those described herein and well known in the art. Depending on the type of cytokine produced in response to ISS treatment, the Th1 or Th2 type that is biased by the immune response by the cell is demonstrated. As used herein, the term “Th1-type bias” cytokine product refers to the production of cytokines associated with a Th1-type immune response in the presence of a stimulant compared to the production of cytokines in the absence of stimulation. Mention an increase in measurable product. Examples of such Th1-type bias cytokines include but are not limited to IL-2, IL-12 and IFN-γ. In contrast, “Th2-type biased cytokine” refers to those associated with Th2-type immunity, including but not limited to IL-4, IL-5, and IL-13. Cells useful for the determination of ISS activity include major cells isolated from the host and / or cell line, preferably APCs and lymphocytes, very preferably macrophages and T cells, in cells of the immune system. Further stimulating a Th1-type immune response can be measured in a host treated with the IMP / MC complex product and can be determined by any method known in the art, ie (1 ) Decreased levels of IL-4, or IL-5 measured before and after challenge; or compared to controls treated without ISS, sensitized with antigen or sensitized and challenged, Detecting low levels (or even no) of IL-4 or IL-5 in a host treated with IMP / MC complex; (2) IL-12, IL-18 and / or before and after antigen challenge Or increased levels of IFN (α, β or γ); or treated with IMP / MC complex compared to controls sensitized, sensitized and challenged with ISS and treated without ISS Detect high levels of IL-12 IL-18 and / or IFN (α, β, or γ) in a given host; (3) no ISS "Th1-type bias" antibody production in hosts treated with IMP / MC complex compared to treated controls; and / or (4) levels of antigen-specific IgE as measured before and after antigen administration Or low levels (or even if not) in hosts treated with the IMP / MC complex as compared to controls sensitized with antigen, sensitized and challenged, and treated without ISS. ) Detecting antigen-specific IgE, but is not limited thereto. These various determinations can be performed by APCs and / or lymphocytes, preferably macrophages and / or cells, in vitro or ex vivo using any of the methods described herein or known in the art. This can be done by means of the cytokines produced. Some of these determinations can be made by measuring the species or subspecies of an antigen-specific antibody using any of the methods described herein or known in the art.

  Species and / or subspecies of antigen-specific antibodies generated in response to IMP / MC complex treatment manifest an immune response that is biased by Th1 or Th2 types by the cell. As used herein, the production of the term “Th1-type bias” antibody refers to a measurable increased production of antibodies associated with a Th1-type immune response (ie, a Th1-related antibody). One or more Th1-related antibodies can be measured. Examples of such Th1-type bias antibodies include human IgG1 and / or IgG3 (Widhe et al. (1998) Scand. J. Immunol 47: 575-581 and de Martino et al. (1999) Anr. Allergy Asthma Immunol. 83: 160-164). Reference), and murine IgG2a, but is not limited thereto. In contrast, “Th2-type biased antibody” refers to antibodies associated with a Th2-type immune response, and human IgG2, IgG4 and / or IgE (eg Widhe et al. (1998) and de Martino et al. (1999) and murine (murine). ) IgG1 and / or IgE.

  Induction of cytokines by Th1-type bias resulting from products administered with the IMP / MC complex will enhance the cellular immune response produced by NK cells, cytokine killer cells, Th1 helper cells, and memory cells . In particular, these responses are beneficial for use in vaccines that are protective or therapeutic against viruses, fungi, protozoa, bacteria, allergic diseases and asthma, and tumors.

  In some examples, the Th2 response is suppressed. Inhibition of the Th2 response can be determined, for example, by decreasing levels of Th2-related cytokines such as IL-4 and il-5, and decreasing IgE, decreasing histamine release in response to allergens.

  The present invention provides kits for use in the methods of the present invention. In certain instances, the kits of the invention comprise one or more containers comprising an IMP / MC complex, and optionally intended treatment (e.g., immunomodulation, ameliorate one or more symptoms of an infectious disease, IFN -Increasing γ levels, increasing IFN-α levels, or ameliorating IgE-related diseases) Includes some instructions, usually written instructions, on the use of IMP / MC complexes.

  In a further example, the kit of the invention comprises a container of substances for producing an IMP / MC complex, instructions for producing the IMP / MC complex, and IMP / MC for intended treatment as desired. Includes instructions on the use of the complex. For example, if the IMP / MC composite is a dry product (eg lyophilized or dry powder), a vial with an elastic stopper is usually used, so that the IMP / MC composite is passed through the elastic stopper. It is easily resuspended by injection. Inelastic ampules, removable sealed containers (eg, sealed glass) or elastic stoppers are most conveniently used for the liquid form of the IMP / MC composite. Also conceivable is the overall challenge for use in combination with specific devices such as inhalers, nasal administers (eg nebulizers), or invasive devices such as minipumps.

  The kit containing the substance for producing IMP / MC is supplied in the specific example, where the substance for producing MC is supplied instead of the previously produced MC, but the oligonucleotide and NC are separated. In a container. It is preferred that the oligonucleotide and NC are supplied in a form capable of forming an IMP / MC complex by mixing the supplied oligonucleotide and MC. This structure is preferred when the IMP / MC complex is bound by non-covalent bonds. In addition, if either this oligonucleotide or MC is cross-linked via a heterobifunctional crosslinker, i.e., an "activated" shape (e.g., heterobifunctional bifunctional crosslinks so that oligonucleotides and reactive components can be utilized. More preferably, it is supplied in combination with the agent.

  The IMP / MC complex kit including the solid phase MC preferably includes one or more containers including a substance for generating the solid phase MC. For example, an IMP / MC kit for cationic MC can include one or more containers containing a polymer and a positively charged surfactant. The contents of the container can be mixed in an emulsified form, for example to produce cationic MC, and then mixed with the oligonucleotide. Such materials include poly (lactic acid) / poly (glycolic acid) copolymers, cationic lipid containers such as DOTAP, plus one or more containers in an aqueous solution phase (e.g., a pharmacologically acceptable aqueous buffer). included.

  It is preferable that the kit for the IMP / MC complex including the liquid phase MC includes one or a plurality of containers including a substance for generating the liquid phase MC. For example, an IMP / MC kit for emulsified MC from oil-in-water can include one or more containers containing an oil phase and an aqueous phase. The contents of the container can be emulsified to produce MC and then mixed with the oligonucleotide, and modified oligonucleotides are preferred for incorporation of hydrophobic components. These substances can be oil and water to make an oil-in-water emulsion, or one or more containers (e.g., pharmacologically acceptable) of a lipophilic liposomal component (e.g., phospholipids, cholesterol and surfactant) plus an aqueous phase. Aqueous buffer).

  Instructions regarding the use of the IMP / MC complex for intended treatment generally include information regarding dosage, dosage regimen, and route of administration for the intended treatment. Oligonucleotide containers can be in unit doses, bulk packages (eg, multiple dose packages) or subunit doses. The instructions provided in the kits of the invention are typically written instructions on a label or package insert (e.g., a paper sheet included in the kit), but are mechanically readable instructions (e.g., magnetic Or instructions guided by optical storage discs).

  The following examples provide, but are not limited to, drawings of the present invention.

  Example

Oligonucleotide Synthesis A 7-mer oligonucleotide with a phosphorothioate-binding agent and the sequence 5'-TCGTCGA-3 '(SEQ ID NO: 2) is placed on a Perseptive Biosystems Expedite 8909 automated DNA synthesizer. And synthesized. The manufacturer's method of 15 μmol phosphorothioate DNA was used with the following changes: 1.6 ml of 3% dichloroacetic acid in dichloromethane over 2.5 minutes was used for the detritylation step; 9: 1 acetonitrile : 0.02M 3-amino-1,2,4-dithiazole-5-thione (ADTT) in pyridine over 1.1 minutes and then 1.0 ml of the derivative was used in the sulfurization step over 1.0 minute. Nucleoside phosphoramidite monomer was dissolved in 0.1M concentration in acetonitrile anhydrous. The instrument added nucleotide monomers in the desired order by program execution and the synthesis occurred in the 3 'to 5' direction. The synthesis cycle consists of a tritylation step, a coupling step (phosphoramidite monomer + 1H-tetrazole), a capping step, a sulfurization step, and a final capping step.

  Upon completion of the assembly, the “trityl-one” oligonucleotide was cleaved from the control pore glass and the base was deprotected with concentrated ammonia at 58 ° C. for 16 hours.

  Seven repeat (7-mer) oligonucleotides were purified by RP-HPLC on a Polymer Labs PLRP-S column using 0.1 M triethylammonium acetate with increasing components of acetonitrile. The purified oligonucleotide was concentrated and dried, the 4,4′-dimethoxytrityl group was taken out with 80% aqueous acetic acid, and the compound was further diluted with 3 volumes of isopropanol with 0.6 M aqueous sodium acetate / pH 5 It was precipitated twice from 0.0. The 7-mer oligonucleotide is dissolved in Milli Q water and the product is determined from the absorbance at 260 nm. Finally, the oligonucleotide is lyophilized to a powder.

  Seven-times (7-mer) oligonucleotides were characterized by capillary gel electrophoresis, electrospray mass spectrometry, and RP-HPLC to confirm composition and purity. Further endotoxin content assays (LAL assay, Bio Whittaker) were performed to show that endotoxin levels were <5 EU / mg oligonucleotide.

  Other 7-repeat (7-mer) phosphorothioate oligonucleotides were synthesized using the same method, 5′-ACGTTCG-3 ′ (SEQ ID NO: 3), 5′-ACGTTCG-3 '(SEQ ID NO: 3), 5'-ACGTTCG-3' (SEQ ID NO: 3), 5'-ATCTCGA-3 '(SEQ ID NO: 4), 5'-AGATGAT-3' (SEQ ID NO: 5), 5'-TCGTTTT-3 '(SEQ ID NO: 6), 5'-TCGAAAA-3' (SEQ ID NO: 7), 5'-TCGCCCC-3 '(SEQ ID NO: 8), and 5'-TCGGGGG-3' (SEQ ID NO: 9) is included.

  A seven-repeat (7-mer) phosphorothioate oligonucleotide of sequence 5′-TCGTCGA-3 ′ (SEQ ID NO: 2) can be conjugated to a second on the 5 ′ end to facilitate binding to a microcarrier. Synthesized with sulfide linker. A C62 disulfide linker was dissolved at one end with a 4,4'-O-dimethoxytrityl group, and a cyanoethyl phosphoramidite (Glen Research) was dissolved at the other end in acetonitrile at a concentration of 0.1M. A linker was attached to the 5 'end of the oligonucleotide, and the resulting oligonucleotide was purified and isolated as described above.

  The disulfide group of the oligonucleotide was reduced to a thiol before binding to the microcarrier. The disulfide-oligonucleotide was dissolved at a concentration of 25 mg / ml in 100 mM sodium phosphate / 150 mm sodium chloride / 1 mM ethylenediaminetetraacetic acid (EDTA) / pH 7.5 buffer. Then 5 equivalents of tris (carboxyethyl) phosphine (TCEP; Pierce) hydrochloride was added and the solution was heated at 40 ° C. for 2 hours. Thiol-containing oligonucleotides are purified from small molecule by-products by size exclusion chromatography and either used immediately or stored at −80 ° C. until use.

Preparation of microspheres denatured in organisms Cationic poly (lactic acid, glycolic acid) (PLGA) microspheres were prepared as follows. That is, 0.875 g of a poly (D, L-lactide-co-glycoside) 50:50 polymer having an intrinsic viscosity of 0.41 dl / g (0.1%, chloroform, 25 ° C.) is added to 0.375 g of 7.875 g of methylene chloride. Dissolved with DOTAP at a concentration of 10% w / w. The clean organic phase is then emulsified in 500 ml of PVA aqueous solution (0.35% w / v) by homogenizing at 4000 rpm for 30 minutes at room temperature using a laboratory mixer (Silverson L4R, Silverson Instruments). It was. The system temperature was then raised to 40 ° C. by circulating hot water through the jacket of the mixing vessel. At the same time, the stirring speed was reduced to 1500 rpm and these conditions were held for 2 hours for extraction and the methylene chloride was evaporated. The microsphere suspension is cooled to room temperature with the aid of cooling water circulation.

  Microparticles were separated by centrifugation at 8000 rpm for 10 minutes at room temperature and resuspended by gentle bath sonication in deionized water. Centrifugal washing was repeated two more times to remove excess PVA from the particle surface. The final pellet of particles by centrifugation was suspended in approximately 10 ml of water and freeze-dried overnight. The dried cationic microsphere powder is characterized by an average size (weighed value, μ) = 1.4 and zeta potential (mV) = 32.4 in size and surface charge.

  Unmodified poly (lactic acid, glycolic acid) biodegradable microspheres were synthesized as described above, except that 0.3 g DOTAP was omitted, rinsed and dried. The dried microsphere powder is characterized by an average size (weighed value, μ) = 1.1 and zeta potential (mV) = − 18.1 in size and surface charge.

Preparation of IMP / MC with biologically denatureable microspheres 200 mg of cationic PLGA prepared as described in Example 2 was dispersed in 1.875 ml of 0.1% w / v Tween solution by bath ultrasound. 0.75 ml of an aqueous oligonucleotide solution is added to the microsphere suspension, giving an approximate and theoretical drug amount loading 2% w / w (oligonucleotide to the microsphere). After mixing briefly, the oligonucleotide-microsphere suspension was incubated overnight at 4 ° C. Microspheres are separated by centrifugation in an Eppendorf centrifuge at 14,000 rpm for 30 minutes at room temperature. Supernatant and microspheres are evaluated by standard laboratory analytical techniques for determining free oligonucleotides and bound oligonucleotides, respectively, for oligonucleotide-related efficiency or loading. Oligonucleotide adsorption to the microspheres was confirmed by centrifuging the oligonucleotide and microsphere suspension and then determining the loss of oligonucleotide from the water-soluble supernatant using an absorbance of 260 nm. After the oligonucleotide binding, the preparation was further divided into size and surface charge, average size (weighed number, μ) = 1.6, zeta potential (mV) = 33.3, oligonucleotide binding (drug loading) = 88 (1.78% w / w) is a feature.

Immunomodulation by IPM / MC in human cells
Heptamer oligonucleotides were tested for PLGA microspheres alone and the immunomodulatory activity of the complex in the human PBMC assay. Peripheral blood was collected from healthy volunteers by venipuncture using heparinized injections. The blood was placed in a FICOLL ™ (Amersham Pharmacia Biotech) cushion and centrifuged. PBMCs placed at the FICOLL ™ interface were collected and then washed twice with chilled phosphate buffered saline (PBS). Resuspend the cells and heat-inactivate human AB serum with 50% plus 50 units / mL penicillin, 50 μg / mL streptomycin, 300 μg / mL glutamine, 1 mM sodium biruvate, and 1 × MEM non-essential Cultured in amino acids (NEAA) at 2 × 10 ⁶ cells / mL in RPMI in 24 or 48 well plates.

Cationic and unmodified PLGA microspheres were prepared as described in Example 2. Oligonucleotides were tested as a single agent or in combination with PLGA microspheres (unmodified or cation). The oligonucleotides tested were 5′-TCGTCGA-3 ′ (SEQ ID NO: 2), 5′-ACGTTCG-3 ′ (SEQ ID NO: 3), 5′-ATCTCGA-3 ′ (SEQ ID NO: 4), and 5′-AGATGAT-3 (SEQ ID NO: 5) (control). All oligonucleotides contained contained 100% phosphorothioate binder and were tested at 20 μg / ml. PLGA was added at a concentration of 250 μg / ml. When the oligonucleotide was tested with PLGA, the oligonucleotide and PLGA were added simultaneously to the incubator. SAC (PANSORBIN ™ CalBiochem, diluted 1/5000) and IMP, 5′-TGACTGTGAACGTTCGAGATGA-3 ′ (SEQ ID NO: 10) are used as positive control and control oligonucleotide SEQ ID NO: 1 and Only cells were used as negative controls. In addition, unmodified microcarriers (umm MC) and cationic microcarriers (cat MC) were tested alone. SAC contains Staph Aureus (Cowan I) cell material. Samples were evaluated in duplicate with two healthy donors. IFN-γ and IFN-α were evaluated using the CYTOSCREEN ^™ ELISA kit from BioSource International, Inc, according to the manufacturer's instructions.

In the human PMBC assay, IFN-γ background levels vary significantly from donor to donor. However, IFN-α levels demonstrate a general stable form of activation and routinely demonstrate low background levels under unstimulated conditions.
SEQ ID NO: 2 shows the results of the assay. Results are presented in picograms (pg / ml) per milliliter of interferon-gamma (IFN-γ) or interferon-alpha (IFN-α). Because of the variability between assays with different human donors, the results are presented as an assay and means using different donor cells (each value parenterally).

  As shown below in SEQ ID NO: 2, when used alone, PLGA microspheres (cationic or unmodified) and heptamers had little, if any, activity. However, the 7-mer oligonucleotide SEQ ID NO: 2 is the activity inducing IFN-α and IFN-γ in combination with cationic PLGA microspheres. Cationic PLGA adsorbs oligonucleotides by electrostatic binding and produces oligonucleotide / microcarrier complexes, but does not produce unmodified PLGA. Each value in the table represents the average of two readings enclosed in parentheses.

  The lower side of SEQ ID NO: 3 is a 7-mer oligonucleotide, SEQ ID NO: 2 is the activity in two or more healthy human donors when using cationic microspheres but not when used alone. Met. In this case, the oligonucleotides and cationic microspheres were premixed for 15 minutes at room temperature before they were added to the incubator. Each value in the table represents the average of two readings enclosed in parentheses.

Immunomodulation by IMP / MC complex Heptamer oligonucleotides were tested for immunomodulatory activity in complex with cationic PLGA microspheres, alone and under two different conditions in the human PBMC assay. The human PBMC assay was performed as described in Example 4.

  Oligonucleotides were tested as single agents in combination with other oligonucleotides or in combination with cationic PLGA microspheres. The concentrations of oligonucleotide and microsphere are 20 μg / ml and 250 μg / ml, respectively. When the oligonucleotides were tested in cationic PLGA microspheres, the oligonucleotides and microspheres were added simultaneously to the incubator (at the same time, see “Simultaneous addition” in SEQ ID NOS: 5 and 6). When testing multiple oligonucleotides in combination, equal amounts of each oligonucleotide were premixed and a concentration of 20 μg / ml was used for all. In addition, the oligonucleotide is complexed with cationic PLGA microspheres under the conditions described in Example 3 to obtain 2% w / w addition of oligonucleotide / microspheres (16 hours incubation). In this case, the complex was tested at 2 and 10 μ / ml based on the weight of the oligonucleotide. SAC (PANSORBIN ™ CalBiochem, diluted 1/5000) and IMPs, SEQ ID NO: 100 and 5′-TCGTCGAACGTTCGAGATG-3 ′ (SEQ ID NO: 11) as positive control and control oligonucleotide, SEQ ID NO: Used as 1 and a single cell was used as a negative control. SAC contains Staph Aureus (Cowan I) cell material. Samples were evaluated in duplicate with 2 healthy donors. The data presented below SEQ ID NOs: 4, 5, and 6 show that when used alone, the 7-mer oligonucleotide and the cationic PLGA microspheres have little, if any, activity. do not do. Furthermore, SEQ ID NOs: 4, 5 and 6 also had little activity, if any, of a mixture of 7-mer oligonucleotides. However, consistent with previous experiments, when the 7-mer oligonucleotide, SEQ ID NO: 2, is used under any of the conditions with cationic PLGA microspheres, IFN-α and IFN-β It is active in the induction of Each value in the table represents the average of two readings in parentheses.

Immunomodulation by IMP / MC complex
Heptamer oligonucleotides were tested for immunomodulatory activity of the complex alone and by cationic PLGA microspheres in a human PBMS assay. The oligonucleotide was premixed with cationic PLGA microspheres for 15 minutes at room temperature and the concentrations were 20 μg / ml and 250 μg / ml, respectively. A human PBMC assay is performed as described in Example 4, cationic PLGA microspheres are prepared as described in Example 2, SAC (PANSORBIN ™ CalBiochem, diluted 1/5000) and IMP SEQ ID NO: 10 was used as a positive control, and control oligonucleotide SEQ ID NO: 1 and cells alone were used as negative controls. SAC contains Staph. Aureus (Cowan I) cell material. Samples were evaluated in duplicate with two healthy donors.

  The 7-mer phosphorothioate tested was 5′-TCGTCGA-3 ′ (SEQ ID NO: 2), 5′-TCGTTTT-3 ′ (SEQ ID NO: 6), 5′-TCGAAAA-3 ′ (SEQ ID NO: 6). 7), 5′-TCGCCCC-3 ′ (SEQ ID NO: 8), 5′-TCGGGGG-3 ′ (SEQ ID NO: 9), 5′-AGATGAT-3 ′ (SEQ ID NO: 5) (control) It was.

  As shown in SEQ ID NO: 7, a 7-mer oligonucleotide containing a single 5'TCG is active in inducing IFN-γ and IFN-α in a human PBMC assay when generated in cationic PLGA microspheres It is. Oligonucleotides with four or more guanosines in a row are known for forming a tetraplex structure. SEQ ID NO: 9 contains 5 guanosines in a row and appears to be collected by capillary electrophoresis.

Effect of IMP / MC formation in cell proliferation assay. Human PBMC were isolated from two normal heparanized blood. Some PBMCs were preliminarily retained while the rest were incubated with CD + MACs beads from Miltenyi. These cells are then magnetized and separated into positively selected CD19 + B cells. These populations were determined to be> 98% CD19 + via FACS analysis. The next B cells were cultured at 1x10 ^5/200 [mu] l / well in round-bottom 96-well plate. It was further cultured PBMCs, but 2x10 ^5/200 [mu] l / well. Cells were stimulated in triplicate at 2 μg / ml, either alone or with 100 μg / ml cationic PLGA microspheres (premixed at room temperature for 15 minutes). The culture period was 48 hours at 37 ° C. At the end of the incubation period, ³ H thymidine, 1 μCi / well was added to the plate and incubated for an additional 8 hours. The plates were then collected using standard liquid scintillation techniques, collected within a count, and data was collected as a number per minute.

  As shown in SEQ ID NO: 8 on the lower side, the heptamer, SEQ ID NO: 2, when used alone, induces less proliferation of human B cells and PBMC than the control sequence, SEQ ID NO: 10.

  However, if heptamer SEQ ID NO: 2 is generated by cationic PLGA microspheres, a similar growth amount can be generated as IMP, SEQ ID NO: 1. The table values for experiments 1 and 2 represent the cpm average of 3 wells.

Generation of non-covalent bonds, liquid phase IMP / MC complexes IMP / MC complexes containing modified oligonucleotides and liquid phase MC were generated and tested for complex formation.

  The oligonucleotide is modified by the addition of a cholesterol molecule at the 5 'end of the oligonucleotide using phosphoramidite chemistry. Emulsified oil in water, pH 6.5, using microfluidizer, 4.5% (w / v) squalene, 0.5% (w / v) sorbitan trioleate, 0.5% (w / v) TweenTM It is made by homogenizing a mixture of 80 and 10 mM sodium citrate. This emulsion was inspected and the determined average diameter of the oil droplets was determined.

  The emulsion is mixed with an oligonucleotide modified with cholesterol, or an unmodified version of the same oligonucleotide, and the mixture is then centrifuged to separate the oil and water phases. RP-HPLC is run on samples from each phase to determine the nucleotide content.

Immunomodulation with IMP / MC mixture with non-denaturable microcarriers Mixture of oligonucleotide containing sequence 5'-CG-3 'and control oligonucleotide (without sequence 5'-CG-3') Mixed with product-derived polycarbonate microparticles or nanoparticles (Polysciences, Inc.) and assessed for immunomodulatory activity in mouse spleen cells. The tested particle sizes include those of 50 nm, 200 nm, 1 μm and 4.5 μm.

  A spleen fragment of BALB / c mice was digested with collagenase / despase (0.1 U / mL / 0.8 U / mL) dissolved in phosphate buffered saline (PBS) for 45 minutes at 37 ° C, then The digested pieces were mechanically dispersed by strongly passing through a metal screen. The dispersed spleen cells were pelleted by centrifugation and further fresh medium (RPMI 1640 with 10% fetal calf serum, plus 50 units / mL penicillin, 50 μg / mL streptomycin, 2 mM glutamine, and 0.05 mM Resuspended in β-mercaptoethanol).

4 × 10 ⁵ mouse spleen cells were distributed into wells of a 96-well plate and incubated at 37 ° C. for 1 hour. The cells were incubated with DNA dosages of 5 μg / ml, 1 μg / ml, and 0.1 μg / ml. Test samples at 2x concentration or 100 μL of control were added and the cells were further incubated for 24 hours. Medium was taken from each well and tested for cytokine concentration by ELISA.

  IFN-γ was evaluated using a sandwich-format ELISA. Medium from mouse spleen cell assay is incubated in microtiter plates coated with anti-IFN-γ monoclonal antibody (Nunc). Bound IFN-γ was detected using a biotinylated anti-IFN-γ antibody and a step avidin-radish-peroxidase-conjugated secondary antibody, and the complex was detected in the presence of peroxidase. Detected using the substrate 3,3 ′, 5 <5′-tetramethylbenzidine (TMB) and quantified by measuring absorbance at 450 nm using an Emax precision microplate reader (Molecular Devices). IL-6 and IL-12 were evaluated in the same manner using anti-IL-6 antibody and anti-IL-12 antibody, respectively.

Immunomodulation of mouse cells by IMP / MC complex Oligonucleotides covalently bound to biologically non-denaturing polystyrene beads (designed at 200 nm in size) were tested for immunomodulatory activity in mouse spleen cells.

  Amine-derived polystyrene beads were obtained from Molecular Probe, Inc., and Polysciences, Inc. Three types of beads are used: amine-derived beads (Polysciences, Inc., Catalog No. 15699), excitation / maximum emission 580 and 605 nm ("red beads" Molecular Probes, Inc., Catalog No. F8763). The amine-derived beads bound to the fluorophore, and the amine-derived beads bound to the fluorophore of excitation / maximum emission 505 and 515 nm (“yellow beads” Molecular Probes, Inc., Catalog No. • Activated with SMCC (sulfosuccinimidyl-4- (N-maleimidomethyl) cyclopentane-1-carboxylate, Pierce Chemical Co.). The beads are then bound to thiol-modified oligonucleotides or treated to quench free maleimide groups for use as MC-only controls. The IMP / MC complex was isolated by centrifugation and washed 3 times with phosphate buffered saline to remove excess oligonucleotide.

  The immunomodulatory effect of the IMP / MC complex was tested using the spleen of the mouse of Example 9 above. The cells were incubated with DNA dosages of 20 μg / ml, 5 μg / ml and 1 μ / ml. Immunomodulatory activity is assessed by detection of IL-12, IL-6 and / or IFN-γ in response to the IMP / MC complex.

Immunomodulation of human cells with bionon-denaturing microcarriers and IMP / MC complexes Oligonucleotides covalently bound to bio-non-denaturing polystyrene beads (200 nm design size) are tested for immunomodulatory activity in human PBMC assays . The human PBMC assay is performed as described in Example 4. The cells were incubated with DNA dosages of 20 μg / ml, 10 μg / ml and 2 μ / ml. Immunomodulatory activity is assessed by IFN-γ and / or IFN-α detection in response to the IMP / MC complex.

  While the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity and understanding, it will be apparent to those skilled in the art that certain changes and modifications may be made. Accordingly, the description and examples should not be construed as limiting the scope of the invention, which is set forth in accordance with the appended claims.

Claims (127)

[An oligonucleotide bound to a microcarrier (MC), where the oligonucleotide sequence consists of 5'-TCGX ₁ X ₂ X ₃ X ₄ -3 ', where X ₁ , X ₂ , X ₃ , X ₄ are Nucleotides],
An immunomodulatory polynucleotide / microcarrier (IMP / MC) complex comprising   The oligonucleotide according to claim 1, wherein the oligonucleotide consists of a sequence selected from the group consisting of 5'-TCGAAAA-3 ', 5'-TCGCCCC-3', 5'-TCGGGGG-3 ', 5'-TCGTTTT-3'. IMP / MC complex. The IMP / MC complex of claim 1, wherein the oligonucleotide consists of the sequence 5'-TCGTCGX ₁ -3 ', wherein X ₁ is a nucleotide.   4. The IMP / MC complex according to claim 3, wherein the oligonucleotide consists of the sequence 5'-TCGTCGA-3 '. [An oligonucleotide bound to a microcarrier (MC), where the oligonucleotide sequence consists of 5'-X ₁ TCGX ₂ X ₃ X ₄ -3 ', where X ₁ , X ₂ , X ₃ , X ₄ are Nucleotides],
An immunomodulatory polynucleotide / microcarrier (IMP / MC) complex comprising [An oligonucleotide bound to a microcarrier (MC), where the oligonucleotide sequence consists of 5'-X ₁ X ₂ TCGX ₃ X ₄ -3 ', where X ₁ , X ₂ , X ₃ , X ₄ are Nucleotides],
An immunomodulatory polynucleotide / microcarrier (IMP / MC) complex comprising   The IMP / MC complex according to any one of claims 1 to 6, wherein the oligonucleotide is covalently bound to the microcarrier.   The IMP / MC complex according to any one of claims 1 to 6, wherein the oligonucleotide is non-covalently bound to the microcarrier.   The IMP / MC composite according to any one of claims 1 to 6, wherein the microcarrier is a liquid phase microcarrier.   The IMP / MC complex according to any one of claims 1 to 6, wherein the microcarrier is a solid phase microcarrier.   The IMP / MC composite according to any one of claims 1 to 6, wherein the microcarrier has a size smaller than 10 µm.   12. The IMP / MC composite according to claim 11, wherein the microcarrier has a size of 25 nm to 5 μm.   13. The IMP / MC composite according to claim 12, wherein the microcarrier has a size of 1.0 μm to 2.0 μm.   14. The IMP / MC composite according to claim 13, wherein the microcarrier has a size of 1.4 μm.   7. The IMP / MC composite according to claim 1, wherein the microcarrier is a cation.   The IMP / MC complex according to any one of claims 1 to 6, wherein the complex is free of an antigen.   The IMP / MC complex according to any one of claims 1 to 6, wherein the complex further contains an antigen.   18. The IMP / MC complex according to claim 17, wherein the antigen is an allergen.   The IMP / MC complex according to any one of claims 1 to 6, wherein the oligonucleotide comprises a phosphate backbone modification.   20. The IMP / MC complex according to claim 19, wherein the modification of the phosphate skeleton is phosphorothioate.   21. The IMP / MC complex according to any one of claims 1 to 20, wherein the microcarrier is biodegradable.   21. The IMP / MC complex according to any one of claims 1 to 20, wherein the microcarrier is non-biologically denatureable. [The individual is administered an organism comprising an immunomodulatory polynucleotide / microcarrier (IMP / MC) complex in an amount sufficient to modulate the immune response to the individual, and the complex is transferred to the microcarrier (MC). Including the oligonucleotide to be bound, where the oligonucleotide consists of the sequence 5′-TCGX ₁ X ₂ X ₃ X ₄ -3 ′, where X ₁ , X ₂ , X ₃ , X ₄ are nucleotides]
A method of modulating an immune response in an individual comprising:   The oligonucleotide according to claim 23, wherein the oligonucleotide consists of a sequence selected from the group consisting of 5'-TCGAAAA-3 ', 5'-TCGCCCC-3', 5'-TCGGGGG-3 ', 5'-TCGTTTT-3'. Method. Oligonucleotide consists sequence 5'-TCGTCGX ₁ -3 ', where X ₁ is The method of claim 23 wherein the nucleotide.   26. The method of claim 25, wherein the oligonucleotide consists of the sequence 5'-TCGTCGA-3 '. [The individual is administered an organism comprising an immunomodulatory polynucleotide / microcarrier (IMP / MC) complex in an amount sufficient to modulate the immune response to the individual, and the complex is transferred to the microcarrier (MC). Including the oligonucleotide to be bound, where the oligonucleotide consists of the sequence 5′-X ₁ TCGX ₂ X ₃ X ₄ -3 ′, where X ₁ , X ₂ , X ₃ , X ₄ are nucleotides],
A method of modulating an immune response in an individual comprising: [The individual is administered an organism comprising an immunomodulatory polynucleotide / microcarrier (IMP / MC) complex in an amount sufficient to modulate the immune response to the individual, and the complex is transferred to the microcarrier (MC). Including the oligonucleotide to be bound, where the oligonucleotide consists of the sequence 5′-X ₁ X ₂ TCGX ₃ X ₄ -3 ′, where X ₁ , X ₂ , X ₃ , X ₄ are nucleotides]
A method of modulating an immune response in an individual comprising:   The method according to any one of claims 23 to 28, wherein the microcarrier is a solid phase microcarrier.   The method according to any one of claims 23 to 28, wherein the microcarrier is a liquid phase microcarrier.   29. The method according to any one of claims 23 to 28, wherein the oligonucleotide is covalently bound to the microcarrier.   29. The method according to any one of claims 23 to 28, wherein the oligonucleotide is non-covalently bound to the microcarrier.   29. A method according to any one of claims 23 to 28, wherein the complex is free of antigen.   The method according to any one of claims 23 to 28, wherein the complex further comprises an antigen.   36. The method of claim 35, wherein the antigen is an allergen.   29. The method according to any one of claims 23 to 28, wherein the oligonucleotide comprises a phosphate backbone modification.   37. The method according to claim 36, wherein the modification of the phosphate skeleton is phosphorothioate.   29. The method according to any one of claims 23 to 28, wherein a Th1-type immune response is stimulated.   The method according to any one of claims 23 to 28, wherein a Th2-type immune response is suppressed.   29. A method according to any one of claims 23 to 28, wherein the microcarrier is biologically denatureable.   The method according to any one of claims 23 to 28, wherein the microcarrier is biologically non-denaturing. [Administering to said individual an effective amount of a composition comprising an immunoregulatory immunoregulatory polynucleotide / microcarrier (IMP / MC) complex, said complex comprising an oligonucleotide bound to a microcarrier (MC) Wherein the oligonucleotide consists of the sequence 5′-TCG X ₁ X ₂ X ₃ X ₄ -3 ′, where X ₁ , X ₂ , X ₃ , X ₄ are nucleotides, and an effective amount is IFN in said individual -enough to increase γ],
A method of increasing interferon gamma (IFN-γ) in said individual.   The oligonucleotide according to claim 42, wherein the oligonucleotide consists of a sequence selected from the group consisting of 5'-TCGAAAA-3 ', 5'-TCGCCCC-3', 5'-TCGGGGG-3 ', 5'-TCGTTTT-3'. Method. It becomes oligonucleotide of SEQ 5'-TCGTCGX ₁ -3 ', where X ₁ is a nucleotide method of claim 42, wherein.   43. The method of claim 42, wherein the oligonucleotide consists of the sequence 5'-TCGTCGA-3 '. [Administering to said individual an effective amount of a composition comprising an immunomodulatory polynucleotide / microcarrier (IMP / MC) complex, wherein said complex comprises an oligonucleotide bound to a microcarrier (MC), wherein The oligonucleotide consists of the sequence 5′-X ₁ TCGX ₂ X ₃ X ₄ -3 ′, where X ₁ , X ₂ , X ₃ , X ₄ are nucleotides, and an effective amount is IFN-γ in said individual Enough to increase],
A method of increasing interferon gamma (IFN-γ) in said individual. [Administering to said individual an effective amount of a composition comprising an immunomodulatory polynucleotide / microcarrier (IMP / MC) complex, wherein said complex comprises an oligonucleotide bound to a microcarrier (MC), wherein The oligonucleotide consists of the sequence 5′-X ₁ X ₂ TCGX ₃ X ₄ -3 ′, where X ₁ , X ₂ , X ₃ , X ₄ are nucleotides, and an effective amount is IFN-γ in said individual Enough to increase],
A method of increasing interferon gamma (IFN-γ) in said individual.   48. The method according to any one of claims 42 to 47, wherein the microcarrier is a solid phase microcarrier.   48. The method according to any one of claims 42 to 47, wherein the microcarrier is a liquid phase microcarrier.   48. The method of any one of claims 42 to 47, wherein the oligonucleotide is covalently bound to the microcarrier.   48. The method of any one of claims 42 to 47, wherein the oligonucleotide is non-covalently bound to the microcarrier.   48. A method according to any one of claims 42 to 47, wherein the complex is free of antigen.   48. The method according to any one of claims 42 to 47, wherein the complex further comprises an antigen.   54. The method of claim 53, wherein the antigen is an allergen.   48. The method of any one of claims 42 to 47, wherein the oligonucleotide comprises a phosphate backbone modification.   57. The method according to claim 56, wherein the modification of the phosphate skeleton is phosphorothioate.   57. The method according to any one of claims 42 to 56, wherein the microcarrier is biologically denatureable.   57. The method according to any one of claims 42 to 56, wherein the microcarrier is biologically non-denaturing. [Administering to said individual an effective amount of a composition comprising an immunomodulatory polynucleotide / microcarrier (IMP / MC) complex, wherein said complex comprises an oligonucleotide bound to a microcarrier (MC), wherein The oligonucleotide consists of the sequence 5′-TCGX ₁ X ₂ X ₃ X ₄ -3 ′, where X ₁ , X ₂ , X ₃ , X ₄ are nucleotides, and an effective amount is defined as IFN-γ in said individual Enough to increase],
A method of increasing interferon alpha (IFN-α) in said individual comprising:   The oligonucleotide according to claim 59, wherein the oligonucleotide consists of a sequence selected from the group consisting of 5'-TCGAAAA-3 ', 5'-TCGCCCC-3', 5'-TCGGGGG-3 ', 5'-TCGTTTT-3'. Method. It becomes oligonucleotide of SEQ 5'-TCGTCGX ₁ -3 ', where X ₁ is a nucleotide, The method of claim 59.   62. The method of claim 61, wherein the oligonucleotide consists of the sequence 5'-TCGTCGA-3 '. [Administering to said individual an effective amount of a composition comprising an immunomodulatory polynucleotide / microcarrier (IMP / MC) complex, wherein said complex comprises an oligonucleotide bound to a microcarrier (MC), wherein The oligonucleotide consists of the sequence 5′-X ₁ TCGX ₂ X ₃ X ₄ -3 ′, where X ₁ , X ₂ , X ₃ , X ₄ are nucleotides, and an effective amount is defined as IFN-α in the individual. Enough to increase],
A method of increasing interferon alpha (IFN-α) in said individual comprising: [Administering to said individual an effective amount of a composition comprising an immunomodulatory polynucleotide / microcarrier (IMP / MC) complex, wherein said complex comprises an oligonucleotide bound to a microcarrier (MC), wherein The oligonucleotide consists of the sequence 5′-X ₁ X ₂ TCGX ₃ X ₄ -3 ′, where X ₁ , X ₂ , X ₃ , X ₄ are nucleotides, and an effective amount is defined as IFN-α in said individual. Enough to increase],
A method of increasing interferon alpha (IFN-α) in said individual comprising:   65. The method according to any one of claims 59 to 64, wherein the individual has a viral infection.   The method according to any one of claims 59 to 64, wherein the microcarrier is a solid phase microcarrier.   The method according to any one of claims 59 to 64, wherein the microcarrier is a liquid phase microcarrier.   65. The method according to any one of claims 59 to 64, wherein the oligonucleotide is covalently bound to the microcarrier.   65. The method according to any one of claims 59 to 64, wherein the oligonucleotide is non-covalently bound to the microcarrier.   65. The method according to any one of claims 59 to 64, wherein the complex is free of an antigen.   The method according to any one of claims 59 to 64, wherein the complex further comprises an antigen.   72. The method of claim 71, wherein the antigen is a viral antigen.   65. The method according to any one of claims 59 to 64, wherein the oligonucleotide comprises a phosphate backbone modification.   74. The method of claim 73, wherein the phosphate backbone modification is phosphorothioate.   65. The method according to any one of claims 59 to 64, wherein the microcarrier is biologically denatureable.   The method according to any one of claims 59 to 64, wherein the microcarrier is biologically non-denaturing. [Administering to said individual an effective amount of a composition comprising an immunomodulatory polynucleotide / microcarrier (IMP / MC) complex, wherein said complex comprises an oligonucleotide bound to a microcarrier (MC), wherein The oligonucleotide consists of the sequence 5′-TCGX ₁ X ₂ X ₃ X ₄ -3 ′, where X ₁ , X ₂ , X ₃ , X ₄ are nucleotides, and an effective amount reduces the level of IgE in said individual Is enough to reduce],
A method of reducing the level of IgE in said individual.   The oligonucleotide according to claim 77, wherein the oligonucleotide consists of a sequence selected from the group consisting of 5'-TCGAAAA-3 ', 5'-TCGCCCC-3', 5'-TCGGGGG-3 ', 5'-TCGTTTT-3'. Method. Oligonucleotide consists sequence 5'-TCGTCGX ₁ -3 ', where X ₁ is a nucleotide, The method of claim 77.   80. The method of claim 79, wherein the oligonucleotide consists of the sequence 5'-TCGTCGA-3 '. [Administering to said individual an effective amount of a composition comprising an immunomodulatory polynucleotide / microcarrier (IMP / MC) complex, wherein said complex comprises an oligonucleotide bound to a microcarrier (MC), wherein The oligonucleotide consists of the sequence 5′-X ₁ TCGX ₂ X ₃ X ₄ -3 ′, where X ₁ , X ₂ , X ₃ , X ₄ are nucleotides, and an effective amount reduces the level of IgE in said individual Enough to reduce],
A method of reducing the level of IgE in said individual. [Administering to said individual an effective amount of a composition comprising an immunomodulatory polynucleotide / microcarrier (IMP / MC) complex, wherein said complex comprises an oligonucleotide bound to a microcarrier (MC), wherein The oligonucleotide consists of the sequence 5′-X ₁ X ₂ TCGX ₃ X ₄ -3 ′, where X ₁ , X ₂ , X ₃ , X ₄ are nucleotides, and an effective amount reduces the level of IgE in said individual Enough to reduce],
A method of reducing the level of IgE in said individual.   83. The method according to any one of claims 77 to 82, wherein the microcarrier is a solid phase microcarrier.   83. The method according to any one of claims 77 to 82, wherein the microcarrier is a liquid phase microcarrier.   87. The method according to any one of claims 77 to 82, wherein the oligonucleotide is covalently bound to the microcarrier.   83. The method according to any one of claims 77 to 82, wherein the oligonucleotide is non-covalently bound to the microcarrier.   83. A method according to any one of claims 77 to 82, wherein the complex is free of antigen.   83. The method according to any one of claims 77 to 82, wherein the complex further comprises an antigen.   90. The method of claim 88, wherein the antigen is an allergen.   84. The method of any one of claims 77 to 82, wherein the oligonucleotide comprises a phosphate backbone modification.   92. The method of claim 90, wherein the phosphate backbone modification is phosphorothioate.   92. A method according to any one of claims 77 to 91, wherein the microcarrier is biologically denatureable.   92. The method according to any one of claims 77 to 91, wherein the microcarrier is biologically non-denaturing.   The method according to any one of claims 59 to 64, wherein the composition further comprises an antigen.   95. The method of claim 94, wherein the antigen is a viral antigen.   The method according to any one of claims 22 to 28, 42 to 47, and 77 to 82, wherein the composition further comprises an antigen.   99. The method of claim 96, wherein the antigen is an allergen. [Immunoregulatory polynucleotide / microcarrier (IMP / MC) complex comprises an oligonucleotide attached to a microcarrier (MC), where the oligonucleotide has the sequence 5′-TCG X ₁ X ₂ X ₃ X ₄ -3 ', Where X ₁ , X ₂ , X ₃ , X ₄ are nucleotides],
Including kit.   The oligonucleotide of claim 98, wherein the oligonucleotide consists of a sequence selected from the group consisting of 5'-TCGAAAA-3 ', 5'-TCGCCCC-3', 5'-TCGGGGG-3 ', 5'-TCGTTTT-3'. kit. Oligonucleotide consists sequence 5'-TCGTCGX ₁ -3 ', where X ₁ is a nucleotide, claim 98 kit according.   101. The method of claim 100, wherein the oligonucleotide consists of the sequence 5'-TCGTCGA-3 '. [Immunoregulatory polynucleotide / microcarrier (IMP / MC) complex comprises an oligonucleotide bound to a microcarrier (MC), where the oligonucleotide is of the sequence 5'-X ₁ TCGX ₂ X ₃ X ₄ -3 ' Where X ₁ , X ₂ , X ₃ , X ₄ are nucleotides],
Including kit. [Immunoregulatory polynucleotide / microcarrier (IMP / MC) complex comprises an oligonucleotide attached to a microcarrier (MC), where the oligonucleotide is of the sequence 5'-X ₁ X ₂ TCGX ₃ X ₄ -3 ' Where X ₁ , X ₂ , X ₃ , X ₄ are nucleotides],
Including kit.   104. The kit according to any one of claims 98 to 103, wherein the oligonucleotide is covalently bound to the microcarrier.   104. The kit according to any one of claims 98 to 103, wherein the oligonucleotide is non-covalently bound to the microcarrier.   104. The kit according to any one of claims 98 to 103, wherein the microcarrier is a liquid phase microcarrier.   104. The kit according to any one of claims 98 to 103, wherein the microcarrier is a solid phase microcarrier.   104. The kit according to any one of claims 98 to 103, wherein the microcarrier has a size smaller than 10 μm.   109. The kit according to claim 108, wherein the microcarrier has a size of 25 nm to 5 μm.   110. The kit according to claim 109, wherein the microcarrier has a size of 1.0 μm to 2.05 μm.   111. The kit according to claim 110, wherein the microcarrier has a size of 1.4 μm.   104. The kit according to any one of claims 98 to 103, wherein the microcarrier is a cation.   104. The kit according to any one of claims 98 to 103, wherein the complex is free of an antigen.   104. The method according to any one of claims 98 to 103, wherein the complex further comprises an antigen.   115. The method of claim 114, wherein the antigen is an allergen.   104. The kit according to any one of claims 98 to 103, wherein the oligonucleotide comprises a phosphate backbone modification.   117. The kit according to claim 116, wherein the phosphate skeleton modification is phosphorothioate.   104. The kit according to any one of claims 98 to 103, further comprising an antigen.   119. The kit according to claim 118, wherein the antigen is an allergen.   119. The kit according to claim 118, wherein the antigen is a viral antigen.   121. The kit according to any one of claims 98 to 120, wherein the microcarrier is biologically denatureable.   121. The kit according to any one of claims 98 to 120, wherein the microcarrier is biologically non-denaturing.   A composition comprising the IMP / MC complex of any one of claims 1 to 6 and a pharmaceutically acceptable excipient.   124. The composition of claim 123, wherein the composition is free of antigen.   124. The composition of claim 123, wherein the composition further comprises an antigen.   126. A composition according to claim 125, wherein the antigen is an allergen.   126. The composition according to claim 125, wherein the antigen is a viral antigen.