The content of the following submission on ASCII text file is incorporated herein by reference in its entirety: a computer readable form (CRF) of the Sequence Listing (file name: 377882003304SUBSEQLIST.TXT, date recorded: Jan. 7, 2020, size: 64 KB).

TECHNICAL FIELD

The present invention relates to immunomodulatory polynucleotides. It also relates to the administration of the polynucleotides to modulate an immune response.

BACKGROUND OF THE INVENTION

The type of immune response generated to infection or other antigenic challenge can generally be distinguished by the subset of T helper (Th) cells involved in the response. The Th1 subset is responsible for classical cell-mediated functions such as delayed-type hypersensitivity and activation of cytotoxic T lymphocytes (CTLs), whereas the Th2 subset functions more effectively as a helper for B-cell activation. The type of immune response to an antigen is generally influenced by the cytokines produced by the cells responding to the antigen. Differences in the cytokines secreted by Th1 and Th2 cells are believed to reflect different biological functions of these two subsets. See, for example, Romagnani (2000)Ann. Allergy Asthma Immunol.85:9-18.

The Th1 subset may be particularly suited to respond to viral infections, intracellular pathogens, and tumor cells because it secretes IL-2 and IFN-γ, which activate CTLs. The Th2 subset may be more suited to respond to free-living bacteria and helminthic parasites and may mediate allergic reactions, since IL-4 and IL-5 are known to induce IgE production and eosinophil activation, respectively. In general, Th1 and Th2 cells secrete distinct patterns of cytokines and so one type of response can moderate the activity of the other type of response. A shift in the Th1/Th2 balance can result in an allergic response, for example, or, alternatively, in an increased CTL response.

For many infectious diseases, such as tuberculosis and malaria, Th2-type responses are of little protective value against infection. Proposed vaccines using small peptides derived from the target antigen and other currently used antigenic agents that avoid use of potentially infective intact viral particles, do not always elicit the immune response necessary to achieve a therapeutic effect. Protein-based vaccines typically induce Th2-type immune responses, characterized by high titers of neutralizing antibodies but without significant cell-mediated immunity.

Moreover, some types of antibody responses are inappropriate in certain indications, most notably in allergy where an IgE antibody response can result in anaphylactic shock. Generally, allergic responses also involve Th2-type immune responses. Allergic responses, including those of allergic asthma, are characterized by an early phase response, which occurs within seconds to minutes of allergen exposure and is characterized by cellular degranulation, and a late phase response, which occurs 4 to 24 hours later and is characterized by infiltration of eosinophils into the site of allergen exposure. Specifically, during the early phase of the allergic response, allergen cross-links IgE antibodies on basophils and mast cells, which in turn triggers degranulation and the subsequent release of histamine and other mediators of inflammation from mast cells and basophils. During the late phase response, eosinophils infiltrate into the site of allergen exposure (where tissue damage and dysfunction result).

Antigen immunotherapy for allergic disorders involves the subcutaneous injection of small, but gradually increasing amounts, of antigen. Such immunization treatments present the risk of inducing IgE-mediated anaphylaxis and do not efficiently address the cytokine-mediated events of the allergic late phase response. Thus far, this approach has yielded only limited success.

Administration of certain DNA sequences, generally known as immunostimulatory sequences, induces an immune response with a Th1-type bias as indicated by secretion of Th1-associated cytokines. 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 injected intradermally withEscherichia coli(E. coli) β-galactosidase (β-Gal) in saline or in the adjuvant alum responded by producing specific IgG1 and IgE antibodies, and CD4+cells that secreted IL-4 and IL-5, but not IFN-γ, demonstrating that the T cells were predominantly of the Th2 subset. However, mice injected intradermally (or with a tyne skin scratch applicator) with plasmid DNA (in saline) encoding β-Gal and containing an immunostimulatory sequence responded by producing IgG2a antibodies and CD4+cells that secreted IFN-γ, but not IL-4 and IL-5, demonstrating that the T cells were predominantly of the Th1 subset. Moreover, specific IgE production by the plasmid DNA-injected mice was reduced 66-75%. Raz et al. (1996)Proc. Natl. Acad. Sci. USA93:5141-5145. In general, the response to naked DNA immunization is characterized by production of IL-2, TNFα and IFN-γ by antigen-stimulated CD4+T cells, which is indicative of a Th1-type response. This is particularly important in treatment of allergy and asthma as shown by the decreased IgE production. The ability of immunostimulatory polynucleotides to stimulate a Th1-type immune response has been demonstrated with bacterial antigens, viral antigens and with allergens (see, for example, WO 98/55495).

References describing immunostimulatory activity of polynucleotides include: Krug et al. (2001)Eur. J. Immunol.31:3026; Bauer et al. (2001)J. Immunol.166:5000; Klinman et al. (1999)Vaccine17:19; Jahn-Schmid et al. (1999)J. Allergy Clin. Immunol.104:1015; Tighe et al. (2000)Eur. J. Immunol.30:1939; Shirota et al. (2000)J. Immunol.164:5575; Klinman et al. (1999)Infect. Immun.67:5658; Sur et al. (1999)J. Immunol.162:6284; Magone et al. (2000)Eur. J. Immunol.30:1841; Kawarada et al. (2001)J. Immunol.167:5247; Kranzer et al. (2000)Immunology99:170; Krug et al. (2001)Eur. J. Immunol.31:2154; Hartmann et al. (2000)J. Immunol.164:944; Bauer et al. (1999)Immunology97:699; Fujieda et al. (2000)Am. J. Respir. Crit. Care Med.162:232; Krieg (2002)Annu. Rev. Immunol.20:709; Verthelyi et al. (2002)J. Immunol.168:1659; Hornung et al. (2002)J. Immunol.168:4531; Yamamoto et al. (2000)Springer Semin. Immunopathol.22:35; Lee et al. (2000)J. Immunol.165:3631; Gursel et al. (2002)J. Leukoc. Biol.71:813; Gursel et al. (2002)Eur. J. Immunol.32:2617; Broide et al. (2001)J. Clin. Immunol.21:175; Zhu et al. (2001)Immunology103:226; Klinman et al. (2002)Microbes Infect.4:897; Hartmann et al. (2000)J. Immunol.164:1617; Krieg (1999)Biochim. Biophys. Acta1489:107; Dalpke et al. (2002)Immunology106:102; Yu et al. (2002)Biochem. Biophys. Res. Commun.297:83; Hafner et al. (2001)Cancer Res.61:5523; Zwaveling et al. (2002)J. Immunol.169:350; Davis et al. (2000)Vaccine18:1920; Gierynska et al. (2002)J. Virol.76:6568; Lipford et al. (2000)J. Immunol.165:1228; Freidag et al. (2000)Infect. Immun.68:2948; Dieudonne et al. (2001)J. Allergy Clin. Immunol.107:S233.

Other references describing immunostimulatory sequences include: Krieg et al. (1989)J. Immunol.143:2448-2451; Tokunaga et al. (1992)Microbiol. Immunol.36:55-66; Kataoka et al. (1992)Jpn. J. Cancer Res.83:244-247; Yamamoto et al. (1992)J. Immunol.148:4072-4076; Mojcik et al. (1993)Clin. Immuno. and Immunopathol.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)Jpn. J. Cancer Res.85:775-779; Raz et al. (1994)Proc. Natl. Acad. Sci. USA91:9519-9523; Kimura et al. (1994)J. Biochem. (Tokyo) 116:991-994; Krieg et al. (1995)Nature374:546-549; Pisetsky et al. (1995)Ann. N.Y. Acad. Sci.772:152-163; Pisetsky (1996a)J. Immunol.156:421-423; Pisetsky (1996b)Immunity5:303-310; 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)Science273:352-354; Stacey et al. (1996)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.84:185-193; 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. USA94:10833-10837; Macfarlane et al. (1997)Immunology91:586-593; Schwartz et al. (1997)J. Clin. Invest.100:68-73; Stein et al. (1997)Antisense Technology, Ch. 11 pp. 241-264, C. Lichtenstein and W. Nellen, Eds., IRL Press; Wooldridge et al. (1997)Blood89:2994-2998; 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 Antisense Oligonucleotide TechnologyCh. 24, pp. 431-448, C. A. Stein and A. M. 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. USA95:12631-12636; Spiegelberg et al. (1998)Allergy53(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 Development8:351-356; McCluskie et al. (1998)J. Immunol.161(9):4463-4466; Gramzinski et al. (1998)Mol. Med.4:109-118; Liu et al. (1998)Blood92:3730-3736; Moldoveanu et al. (1998)Vaccine16: 1216-1224; Brazolot Milan et al. (1998)Proc. Natl. Acad. Sci. USA95:15553-15558; 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 98/37919; WO 98/40100; WO 98/52581; WO 98/55495; WO 98/55609 and WO 99/11275. See also Elkins 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 and U.S. Pat. Nos. 5,663,153, 5,723,335 and 5,849,719. See also Liang et al. (1996)J. Clin. Invest.98:1119-1129; Bohle et al. (1999)Eur. J. Immunol.29:2344-2353 and WO 99/56755. See also WO 99/61056; WO 00/06588; WO 00/16804; WO 00/21556; WO 00/54803; WO 00/61151; WO 00/67023; WO 00/67787 and U.S. Pat. No. 6,090,791. See also Manzel et al. (1999)Antisense Nucl. Acid Drug Dev.9:459-464; Verthelyi et al. (2001)J. Immunol.166:2372-2377; WO 01/15726; WO 01/12223; WO 01/22972; WO 01/22990; WO 01/35991; WO 01/51500; WO 01/54720; U.S. Pat. Nos. 6,174,872, 6,194,388, 6,207,646, 6,214,806, 6,218,371, 6,239,116. See also, WO 01/12804; WO 01/45750; WO 01/55341; WO 01/55370; WO 01/62207; WO 01/68077; WO 01/68078; WO 01/68103; WO 01/68116; WO 01/68117; WO 01/68143; WO 01/68144; WO 01/72123; WO 01/76642; WO 01/83503; WO 01/93902; WO 02/026757; WO 02/052002; WO 02/069369; WO 02/074922; U.S. Pat. Nos. 6,339,068, 6,406,705, 6,426,334, 6,426,336, 6,429,199, 6,476,000.

Immunomodulatory polynucleotides generally include a CG sequence. Nucleotides flanking the CG of an IMP also appear to play a role in the immunomodulatory activity of the polynucleotide. There remains a need for continued identification of immunomodulatory polynucleotides.

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

DISCLOSURE OF THE INVENTION

The invention relates to immunomodulatory polynucleotides (IMP) and methods for modulating immune responses in individuals using these polynucleotides, particularly humans.

In one aspect, the invention provides immunomodulatory polynucleotides. In certain embodiments, the invention includes compositions which comprise any of the immunomodulatory polynucleotides described herein. The compositions may also include, for example, a pharmaceutically acceptable excipient or any of a number of other components, such as an antigen.

In one aspect, the immunomodulatory polynucleotide of the invention comprises (a) a palindromic sequence comprising at least two CG dinucleotides, wherein the CG dinucleotides are separated by 0, 1, 2, 3, 4 or 5 bases and wherein the palindromic sequence is at least 8 bases in length; and (b) a (TCG)y, wherein y is 1 or 2, wherein the 5′ T of the (TCG), is positioned 0, 1, 2 or 3 bases from the 5′ end of the polynucleotide and wherein the (TCG), is separated from the 5′ end of the palindromic sequence by 0, 1, or 2 bases. In some immunomodulatory polynucleotides of the invention, whether described in this paragraph or elsewhere in this application, the palindromic sequence has a base composition of less than two-thirds G's and C's. In some embodiments, the palindromic sequence has a base composition of greater than one-third A's and T's.

In another aspect, the immunomodulatory polynucleotide of the invention comprises (a) a palindromic sequence comprising at least two CG dinucleotides, wherein the CG dinucleotides are separated by 0, 1, 2, 3, 4 or 5 bases and wherein the palindromic sequence is at least 8 bases in length; and (b) a (TCG), sequence, wherein y is 1 or 2, wherein the 5′ T of the (TCG), sequence is positioned 0, 1, 2 or 3 bases from the 5′ end of the polynucleotide, and further wherein the palindromic sequence of (a) includes all or part of the (TCG)ysequence and wherein a CG of the (TCG), sequence may be one of the CG dinucleotides of the palindromic sequence of (a).

In another aspect, the immunomodulatory polynucleotide of the invention comprises (a) 5′-Nx(TCG(Nq))yNw(X1X2CGX2′X1′(CG)p)z(SEQ ID NO: 156) wherein N are nucleosides, x=0-3, y=1-4, w=−2, −1, 0, 1 or 2, p=0 or 1, q=0, 1 or 2, and z=1-20, X1and X1′ are self-complementary nucleosides, X2and X2′ are self-complementary nucleosides, and wherein the 5′ T of the (TCG(Nq))ysequence is 0-3 bases from the 5′ end of the polynucleotide; and (b) a palindromic sequence at least 8 bases in length wherein the palindromic sequence comprises the first (X1X2CGX2′X1′) of the (X1X2CGX2′X1′(CG)p)zsequences. In some embodiments, X1and X2are each either A or T.

In another aspect, the immunomodulatory polynucleotide of the invention comprises (a) 5′-Nx(TCG(Nq))yNw(X1X2CGX3X3′CGX2′X1′(CG)p)z(SEQ ID NO: 159) wherein N are nucleosides, x=0-3, y=1-4, w=−2, −1, 0, 1 or 2, p=0 or 1, q=0, 1 or 2, and z=1-20, X1and X1′ are self-complementary nucleosides, X2and X2′ are self-complementary nucleosides, X3and X3′ are self-complementary nucleosides and wherein the 5′ T of the (TCG(Nq))ysequence is 0-3 bases from the 5′ end of the polynucleotide; and (b) a palindromic sequence at least 10 bases in length wherein the palindromic sequence comprises the first (X1X2CGX3X3′CGX2′X1′) (SEQ ID NO:216) of the (X1X2CGX3X3′CGX2′X1′(CG)p)z(SEQ ID NO:217) sequences. In some embodiments, when p=1, X1, X2, and X3are each either A or T. In some embodiments, when p=0, at least two of X1, X2, and X3are either A or T.

In another aspect, the immunomodulatory polynucleotide of the invention comprises (a) 5′-Nx(TCG(Nq))yNw(X1X2X3X4X5CGX5′X4′X3′X2′X1′(CG)p)z(SEQ ID NO: 160) wherein N are nucleosides, x=0-3, y=1-4, w=−3, −2, −1, 0, 1 or 2, p=0 or 1, q=0, 1 or 2, and z=1-20, X1and X1′ are self-complementary nucleosides, X2and X2′ are self-complementary nucleosides, X3and X3′ are self-complementary nucleosides, X4and X4′ are self-complementary nucleosides, X5and X5′ are self-complementary nucleosides, and wherein the 5′ T of the (TCG(Nq))ysequence is 0-3 bases from the 5′ end of the polynucleotide; and (b) a palindromic sequence at least 12 bases in length wherein the palindromic sequence comprises the first (X1X2X3X4X5CGX5′X4′X3′X2′X1′) (SEQ ID NO:218) of the (X1X2X3X4X5CGX5′X4′X3′X2′X1′(CG)p)z(SEQ ID NO:219) sequences. In some embodiments, at least three of X1, X2, X3, X4, and X5are either A or T.

In another aspect, the immunomodulatory polynucleotide of the invention comprises (a) 5′-Nx(TCG(Nq))yNw(CGX1X1′CG(CG)p)z(SEQ ID NO:161) wherein N are nucleosides, x=0-3, y=1-4, w=−2, 0, 1 or 2, p=0 or 1, q=0, 1 or 2, and z=1-20, wherein X1and X1′ are self-complementary nucleosides and wherein the 5′ T of the (TCG(Nq))ysequence is 0-3 bases from the 5′ end of the polynucleotide; and (b) a palindromic sequence at least 8 bases in length wherein the palindromic sequence comprises the first (CGX1X1′CG) of the (CGX1X1′CG(CG)p)zsequences.

In another aspect, the immunomodulatory polynucleotide of the invention comprises (a) 5′-Nx(TCG(Nq))yNw(X1CGCGX1′(CG)p)z(SEQ ID NO: 162) wherein N are nucleosides, x=0-3, y=1-4, w=−1, 0, 1 or 2, p=0 or 1, q=0, 1 or 2, and z=1-20, X1and X1′ are self-complementary nucleosides and wherein the 5′ T of the (TCG(Nq))ysequence is 0-3 bases from the 5′ end of the polynucleotide; and (b) a palindromic sequence at least 8 bases in length wherein the palindromic sequence comprises the first (X1CGCGX1′) of the (X1CGCGX1′(CG)p)zsequences.

In another aspect, the immunomodulatory polynucleotide of the invention comprises (a) 5′-Nx(TCG(Nq))yNw(X1X2CGCGX2′X1′(CG)p)z(SEQ ID NO: 163) wherein N are nucleosides, x=0-3, y=1-4, w=−2, −1, 0, 1 or 2, p=0 or 1, q=0, 1 or 2, and z=1-20, X1and X1′ are self-complementary nucleosides, X2and X2′ are self-complementary nucleosides, and wherein the 5′ T of the (TCG(Nq))ysequence is 0-3 bases from the 5′ end of the polynucleotide; and (b) a palindromic sequence at least 8 bases in length wherein the palindromic sequence comprises the first (X1X2CGCGX2′X1′) of the (X1X2CGCGX2′X1′(CG)p)z(SEQ ID NO:220) sequences. In some embodiments, X1and X2are each either A or T.

In another aspect, the immunomodulatory polynucleotide of the invention comprises (a) 5′-Nx(TCG(Nq))yNw(X1X2X3CGCGX3′X2′X1′(CG)p)z(SEQ ID NO: 164) wherein N are nucleosides, x=0-3, y=1-4, w=−3, −2, −1, 0, 1 or 2, p=0 or 1, q=0, 1 or 2, and z=1-20, X1and X1′ are self-complementary nucleosides, X2and X2′ are self-complementary nucleosides, X3and X3′ are self-complementary nucleosides, and wherein the 5′ T of the (TCG(Nq))ysequence is 0-3 bases from the 5′ end of the polynucleotide; and (b) a palindromic sequence at least 10 bases in length wherein the palindromic sequence comprises the first (X1X2X3CGCGX3′X2′X1′) (SEQ ID NO:221) of the (X1X2X3CGCGX3′X2′X1′(CG)p)z(SEQ ID NO:222) sequences. In some embodiments, when p=1, X1, X2, and X3are each either A or T. In some embodiments, when p=0, at least two of X1, X2, and X3are either A or T.

In another aspect, the immunomodulatory polynucleotide of the invention comprises a) 5′-Nx(TCG(Nq))yNw(CGX1X2X2′X1′CG(CG)p)z(SEQ ID NO: 165) wherein N are nucleosides, x=0-3, y=1-4, w=−2, 0, 1 or 2, p=0 or 1, q=0, 1 or 2, and z=1-20, X1and X1′ are self-complementary nucleosides, X2and X2′ are self-complementary nucleosides, and wherein the 5′ T of the (TCG(Nq))ysequence is 0-3 bases from the 5′ end of the polynucleotide; and (b) a palindromic sequence at least 8 bases in length wherein the palindromic sequence comprises the first (CGX1X2X2′X1′CG) of the (CGX1X2X2′X1′CG(CG)p)z(SEQ ID NO:223) sequences. In some embodiments, X1and X2are each either A or T.

In another aspect, the invention provides methods of modulating an immune response in an individual, comprising administering to an individual an immunomodulatory polynucleotide of the invention in an amount sufficient to modulate an immune response in said individual. Immunomodulation according to the methods of the invention may be practiced on individuals including those suffering from a disorder associated with a Th2-type immune response (e.g., allergies, allergy-induced asthma, or atopic dermatitis), individuals receiving vaccines such as therapeutic vaccines (e.g., vaccines comprising an allergy epitope, a mycobacterial epitope, or a tumor associated epitope) or prophylactic vaccines, individuals with cancer and individuals having an infectious disease.

In another aspect, the invention provides methods of increasing interferon-gamma (IFN-γ) in an individual, comprising administering an effective amount of an immunomodulatory polynucleotide of the invention to said individual. Administration of an immunomodulatory polynucleotide in accordance with the invention increases IFN-γ in the individual.

In another aspect, the invention provides methods of increasing interferon-alpha (IFN-α) in an individual, comprising administering an effective amount of an immunomodulatory polynucleotide of the invention to said individual. Administration of an immunomodulatory polynucleotide in accordance with the invention increases IFN-α in the individual.

In another aspect, the invention provides methods of ameliorating one or more symptoms of an infectious disease, comprising administering an effective amount of an immunomodulatory polynucleotide of the invention to an individual having an infectious disease. Administration of an immunomodulatory polynucleotide in accordance with the invention ameliorates one or more symptoms of the infectious disease.

In another aspect, the invention provides methods of ameliorating one or more symptoms of an IgE-related disorder, comprising administering an effective amount of an immunomodulatory polynucleotide of the invention to an individual having an IgE-related disorder. Administration of an immunomodulatory polynucleotide in accordance with the invention ameliorates one or more symptoms of the IgE-related disorder.

The invention further relates to kits, preferably for carrying out the methods of the invention. The kits of the invention generally comprise an immunomodulatory polynucleotide of the invention (generally in a suitable container), and may further include instructions for use of the immunomodulatory polynucleotide in immunomodulation of an individual.

