JP2009510096A - Modulation of TLR-mediated immune responses using adapter oligonucleotides - Google Patents

Abstract

The present invention relates to the ability of certain oligonucleotides to modulate the profile of TLR ligands such as TLR7, TLR8, TLR9 ligands.

Description

  This application is a US patent from US Provisional Application No. 60 / 720,981 filed on Sep. 27, 2005, entitled “MODULATION OF TLR-MEDIATED IMMUNE RESPONSES USING ADAPTOR OLIGONUCLEOTIDES”, the entire contents of which are incorporated herein by reference. Claim for legal priority.

  The present invention relates to the modulation of TLR-mediated immune responses using specific nucleic acids.

  Reaction to certain motifs in bacterial DNA is an important function of innate immunity. Although bacterial DNA has long been known to exhibit mitogenic activity against mammalian B lymphocytes (B cells), mammalian DNA generally does not exhibit such properties. The discovery that this immune recognition is directed to specific DNA sequences centered on motifs containing unmethylated CpG dinucleotides has opened up the field for molecular immunological techniques. Krieg AM et al. (1995) Nature 374: 546-9. The so-called immunostimulatory action of CpG DNA can be reproduced using a synthetic oligodeoxynucleotide (ODN) containing CpG dinucleotides with respect to a preferred flanking sequence, the CpG motif. CpG-containing ODN (CpG-ODN) protects primary B cells from apoptosis, promotes entry into the cell cycle, and ramps the immune response to a Th1-type immune response, eg, interleukin 6 (IL-6), inter Various cell types of the immune system, including induction of leukin 12 (IL-12), gamma interferon (IFN-γ), activation of antigen-specific cytolytic T lymphocytes (CTL) and induction of IgG2a in mice It has been reported to have various effects.

  Recently, it was reported that the signal transduction by Toll-like receptor 9 (TLR9) is involved in the immunoregulatory action of CpG DNA. CpG DNA is taken up into the cell via a non-sequence-specific pathway and transported to the endosomal compartment where it interacts with TLR9 in a sequence-specific manner. The TLR9 signaling pathway induces a number of immune function related genes, particularly including NF-κB.

  TLRs are a large receptor family that recognizes unique molecular structures (pathogen-associated molecular patterns, or PAMPs) present in pathogens, and are also referred to as pattern recognition receptors (PRRs). Immune cells that express PRR are activated upon recognition of PAMP and induce the generation of an optimal adaptive immune response. There have been descriptions of PRRs consisting of 10 TLR subtypes, TLR1 to TLR10. Such TLRs include double-stranded RNA (TLR3), lipopolysaccharide (LPS) (TLR4), bacterial flagellin (TLR5), small molecule antiviral compounds (TLR7 and TLR8), and bacterial DNA or CpG ODN (TLR9). Has been described as being involved in recognition. (See review by Uhlmann et al. (2003) Curr Opin Drug Discov Dev 6: 204-17). Moreover, RNA molecules that interact with and signal through TLR7 and TLR8 have been identified. (See International Patent Application No. PCT / US03 / 10406). Such immunostimulatory RNA molecules are believed to have a base sequence that includes at least one guanine and at least one uracil. This immunostimulatory G · U abundant RNA does not require a CpG motif as described for TLR9. The corresponding class of RNA molecules found in nature is thought to exist in ribosomal RNA (rRNA), transfer RNA (tRNA), messenger RNA (mRNA) and viral RNA (vRNA).

Following the discovery of immunostimulatory CpG DNA, a number of reports have emerged describing short DNA sequences with immunostimulatory activity. It has long been known that poly G sequences are immunosuppressive. PCT Patent Application Publication No. WO 00/14217 contains an inhibitory motif N ₁ N ₂ GN ₃ G, and at least any _{two of} N ₁ , N ₂ and N ₃ are G (guanosine) Is described. Krieg and co-workers have described a group of inhibitory 15-mer ODNs that have triplicate or quadruplicate G and block apoptotic protection and cell cycle entry induced by activating ODNs. Lenert P et al. (2001) Antisense Nucleic Acid Drug Dev 11: 247-56; Stanz LL et al. (2002) Eur J Immunol 32: 1212-22; Lenert P et al. (2003) Antisense Nucleic Acid Drug Dev 13: 143-50. These immunosuppressive effects of ODN were reported to be specific for CpG-ODN and involve mechanisms other than mere competition for cell uptake. Stunz LL et al. (2002) Eur J Immunol 32: 1212-22. Independently, Klinman and his collaborators reported a single immunosuppressive ODN. Zeuner RA et al. (2002) Arthritis Rheum 46: 2219-24; Yamada H et al. (2002) J Immunol 169: 5590-4.

  The present invention provides methods and compositions for modulating TLR-mediated responses, including suppression of some responses, enhancement of other responses, or combinations thereof. The present invention is based in part on the finding that certain oligonucleotides can clearly alter the TLR signaling profile of TLR ligands. Thus, such “adapter” oligonucleotides can essentially convert a TLR7 ligand to a TLR8 ligand, according to one embodiment, and a TLR7 / 8 ligand to a TLR8 ligand, according to another embodiment.

  Thus, in one aspect, the invention comprises administering a TLR-7 / 8 ligand and an amount of an adapter oligonucleotide effective to stimulate a TLR8-mediated immune response to a subject in need thereof. Methods of stimulating a mediated immune response are provided.

  In another aspect, the invention includes administering to a subject experiencing a TLR7-mediated immune response an amount of an adapter oligonucleotide effective to redirect the TLR7-mediated immune response to a TLR8-mediated immune response. A method of redirecting a response to a TLR8-mediated immune response is provided.

  In yet another aspect, the present invention provides a composition comprising a TLR7 / 8 ligand and an adapter oligonucleotide.

  In one embodiment, the TLR7 / 8 ligand is a TLR7 ligand, such as a TLR7 specific ligand. The TLR7 ligand may be a guanosine analog such as, but not limited to, a C8 substituted guanosine. The TLR7 ligand may be 3M-001. The TLR7 ligand includes, but is not limited to, 6-amino-9-benzyl-2- (3-hydroxypropoxy) -9H-purin-8-ol and 6-amino-9-benzyl-2-butoxy-9H-purine. Adenosine compounds such as -8-ol may also be used. The TLR7 ligand may be 7-deazaguanosine.

  Examples of C8 substituted guanosines include 7-allyl-7,8-dihydro-8-oxoguanosine (loxoribine), 7-thia-8-oxoguanosine (immunosin, isatoribine, ANA245, 7-thia-8-oxo-7 , 8-dihydroguanosine, 5-amino-3- (β-D-ribofuranosyl) -3H, 6H-thiazole [4,5-d] pyrimidine-2,7-dione), 8-mercaptoguanosine, 8-bromoguanosine 8-methylguanosine, 8-oxo-7,8-dihydroguanosine, C8-arylamino-2′-deoxyguanosine, C8-propynylguanosine, C8 · N7 substituted guanine ribonucleoside, 7-methyl-8-oxoguanosine, 8-aminoguanosine, 8-hydroxy-2'-deoxyguanosine, 7-deaza-8 Conversion guanosine, and 8-hydroxy guanosine, and the like. In some embodiments, the C8-substituted guanosine is loxoribine, immunosin or 7-deazaguanosine.

  In another embodiment, the TLR7 / 8 ligand is a TLR8 ligand, including a TLR8 specific ligand. The TLR8 ligand may be 3M-002. In embodiments where the ligand is a TLR8 ligand, the TLR8-mediated immune response is at least 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, greater than the degree of immune response achieved in the absence of the adapter oligonucleotide, It can be enhanced 15-fold, 20-fold or more.

  In another embodiment, the TLR7 / 8 ligand is a TLR7 ligand and is a TLR8 ligand. An example of such a TLR7 / 8 ligand is imidazoquinoline. Examples of imidazoquinolines include imidazoquinoline amine, imidazopyridine amine, 6,7 condensed cycloalkylimidazopyridine amine, 1,2 bridged imidazoquinoline amine, R-848 (S-28463 or resiquimod), 4-amino-2ethoxy Methyl-α, α-dimethyl-1H-imidazo [4,5-c] quinolin-1-ethanol, 1- (2-methylpropyl) -1H-imidazo [4,5-c] quinolin-4-amine (R -837 or imiquimod), or S-27609. In some important embodiments, the imidazoquinoline is R848.

  The TLR7 / 8 ligand may be 3M-003. In embodiments of the ligand where the ligand is TLR7 and TLR8, the TLR8-mediated immune response is at least 2-fold, 3-fold, 4-fold, greater than the extent of the TLR8-mediated immune response achieved in the absence of the adapter oligonucleotide, It can be enhanced by 5 fold, 10 fold, 15 fold, 20 fold or more.

In one embodiment, the adapter oligonucleotide comprises formula 5′XN ₅ -X3 ′ or formula 5′XN ₄ 3 ′, wherein X can be any nucleotide and is present, N ₄ and N ₅ may be 4 or 5 T (thymidine), U (uracil), or A (adenine) such that each N in N ₄ or N ₅ is the same. To express. Depending on the embodiment, the oligonucleotide is 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or more, depending on the embodiment. More than nucleotides. In important embodiments, it includes at least one internucleotide phosphorothioate linkage (including up to the complete phosphorothioated backbone).

In a related embodiment, the adapter oligonucleotide comprises the formula 5′X _a -TTTTTT-X _b 3 ′, wherein X _a and X _b can independently be any nucleotides, and if present, It does not have to be. X _a and X _b may be one or more nucleotides (eg, 1-100 nucleotides). In one embodiment, the oligonucleotide comprises 6, 7 or more Ts. In one important embodiment, the adapter oligonucleotide is a thymidine (dT) homopolymer that is optionally 17 nucleotides in length.

In another embodiment, the adapter oligonucleotide comprises the formula 5′X _a -UUUUU-X _b 3 ′, wherein X _a and X _b can independently be any nucleotide, and if present, It does not have to be. X _a and X _b may be one or more nucleotides (eg, 1-100 nucleotides). In one embodiment, the oligonucleotide comprises six, seven, or more Us. In important embodiments, the oligonucleotide is a uracil (dU) homopolymer that is optionally 17 nucleotides in length.

In another embodiment, the adapter oligonucleotide comprises the formula 5′X _a -AAAAA-X _b 3 ′, wherein X _a and X _b can independently be any nucleotide, and if present, It does not have to be. X _a and X _b may be one or more nucleotides (eg, 1-100 nucleotides). In one embodiment, the oligonucleotide comprises six, seven, or more As. In important embodiments, the oligonucleotide is an adenine (dA) homopolymer that is optionally 17 nucleotides in length.

In yet another embodiment, the adapter oligonucleotide comprises the formula 5′C _n -T _m -C _p 3 ′, where n is an integer in the range of 0-100, p is an integer in the range of 0-100, m Is an integer in the range of 0-100. In one embodiment, the sum of n and p is such that the total C content of the oligonucleotide is 50%, less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, Less than or equal to the value of m so that it is less than 15%, less than 10%, less than 5%, or less. In some embodiments, n is in the range of 3-7, m is in the range of 2-10, and p is in the range of 4-8, provided that the above ratios (%) are satisfied. Examples include 5′C ₃ T ₁₀ C ₄ 3 ′ (SEQ ID NO: 1) and 5′C ₄ T ₈ C ₅ 3 ′ (SEQ ID NO: 2).

  The adapter oligonucleotide may contain a CpG motif or may lack such a motif. The motif may be an unmethylated CpG motif.

  In one embodiment, the TLR7 / 8 ligand and adapter oligonucleotide are administered separately to the subject. In these and other embodiments, the ligand and oligonucleotide may still be administered substantially simultaneously with each other. In yet another embodiment, the oligonucleotide and TLR7 / 8 ligand are conjugated to each other. In one embodiment, the adapter oligonucleotide is covalently linked to a TLR7 / 8 ligand.

  In one embodiment, the adapter oligonucleotide is DNA, while in another embodiment it is RNA. In one embodiment, the adapter oligonucleotide is not immunostimulatory when used alone. In one embodiment, the adapter oligonucleotide alone does not stimulate a TLR7 or TLR8 mediated immune response.

  In one embodiment, the composition further comprises an antigen and / or the method comprises administering the antigen to a subject.

  In one embodiment, the subject has an infection. In another embodiment, the subject has cancer. In yet another embodiment, the subject has allergy or asthma.

  The composition may be a pharmaceutical formulation further comprising a pharmaceutically acceptable carrier.

  In one aspect, the invention provides the use of a combination of a TLR ligand and an adapter oligonucleotide as described above for preparing a medicament for vaccinating a subject.

  In one aspect, the invention provides a method for preparing a vaccine. The method includes the step of bringing a combination of a TLR ligand and an adapter oligonucleotide as described above into intimate contact with an antigen and optionally a pharmaceutically acceptable carrier.

  In one aspect, the invention provides the use of a combination of a TLR ligand and adapter oligonucleotide as described above for the preparation of a medicament for treating an infectious disease in a subject.

  In one aspect, the invention provides a composition useful for the treatment of infectious diseases comprising a combination of a TLR ligand and an adapter oligonucleotide as described above and an antimicrobial agent or agent.

  In one aspect, the invention provides the use of a combination of a TLR ligand and adapter oligonucleotide as described above for the preparation of a medicament for treating a subject's cancer.

  In one aspect, the invention provides a composition useful for cancer treatment comprising a combination of a TLR ligand and an adapter oligonucleotide as described above, and a cancer drug.

  In one aspect, the invention provides the use of a combination of a TLR ligand and an adapter oligonucleotide as described above for the preparation of a medicament for treating an allergic condition in a subject.

  In one aspect, the present invention provides a composition useful for the treatment of allergic conditions comprising a combination of a TLR ligand and an adapter oligonucleotide as described above and an allergic agent.

  In one aspect, the invention provides the use of a combination of a TLR ligand and adapter oligonucleotide as described above for the preparation of a medicament for treating asthma in a subject.

  In one aspect, the present invention provides a composition useful for the treatment of asthma comprising a combination of a TLR ligand and an adapter oligonucleotide as described above and an asthma drug.

  In other aspects, the present invention provides screening methods for identifying TLR7 / 8 ligands and adapter oligonucleotides. Thus, in one aspect, the invention contacts a TLR8 expressing cell with a test ligand in the presence and absence of an adapter oligonucleotide and responds to the test ligand in the presence and absence of the adapter oligonucleotide. A method for identifying a TLR8 ligand comprising measuring a stimulatory amount of an expressed cell is provided. A TLR8 ligand is identified by an increase in the amount of stimulation in the presence of an adapter oligonucleotide. The increase in stimulation amount is preferably an increase over a non-zero stimulation amount in the absence of adapter oligonucleotide.

  In another aspect, the invention contacts TLR7-expressing cells and TLR8-expressing cells in the presence and absence of an adapter oligonucleotide with a test ligand, and the test ligand is present in the presence and absence of the adapter oligonucleotide. A method for identifying a TLR7 ligand comprising measuring the amount of stimulation of TLR7-expressing cells and TLR8-expressing cells in response to a TLR7 expressing cell. TLR7 ligands are identified by a decrease in the amount of stimulation of TLR7 expressing cells and an increase in the amount of stimulation of TLR8 expressing cells in the presence of adapter oligonucleotides. In this embodiment, the increase in stimulation amount is preferably an increase above the zero stimulation amount in the absence of the adapter oligonucleotide.

  In another aspect, the invention contacts a TLR8 expressing cell with a known TLR7 ligand in the presence and absence of a test adapter oligonucleotide, and TLR8 expression in response to the TLR7 ligand in the presence of the test adapter oligonucleotide. A method for identifying adapter oligonucleotides comprising measuring a stimulating amount of sex cells is provided. The stimulating amount is the amount in the absence of the test oligonucleotide, the amount in the absence of the TLR7 ligand and in the presence of the test oligonucleotide, and / or in the presence of the TLR7 ligand, a known adapter oligonucleotide. Can be compared. In one embodiment, the adapter oligonucleotide is identified by an increased amount of stimulation of TLR8 expressing cells in its presence compared to its absence provided that the adapter oligonucleotide itself is TLR8 mediated. It does not mediate the immune response (as determined by the control assay above).

  The ligand for TLR7 and TLR8, and the adapter oligonucleotide may be any of those described in the above aspects and embodiments. A TLR7 or TLR8 expressing cell may be a cell that naturally expresses TLR7 or TLR8, or may be a cell that has been modified to express TLR7 or TLR8 (eg, ectopic). The amount of stimulation is signal transduction via TLR7 or TLR8 and, but is not limited to, increased gene expression (eg when visualized using a reporter construct conjugated to a TLR7 or TLR8 downstream target promoter element), It can be measured by its downstream effects including increased growth factor (cytokine) expression, production or secretion.

  These and other aspects and embodiments of the invention are described in more detail herein.

  The present invention relates in part to the discovery that certain oligonucleotides can modulate TLR-mediated immune responses. As used herein, modulation of a TLR-mediated immune response refers to the ability to manipulate the signaling of one or more TLRs. For example, according to the present invention, the TLR profile of a particular TLR ligand can be altered such that in the presence of certain oligonucleotides such ligands signal via different TLRs. Another example shows that TLR ligands known to signal through multiple TLRs in the presence of certain oligonucleotides signal through only one TLR, often Such signaling is enhanced. In yet another example, the effect and efficacy of TLR signaling is significantly increased in the presence of certain oligonucleotides. Thus, the present invention allows selective suppression and / or enhancement of signaling by any one of TLR7, TLR8 and TLR9, or any combination thereof.

The present invention is directed to R against TLR8-expressing HEK293 cells when an imidazoquinoline derivative, resiquimod (R-848), which activates both TLR7 and TLR8 itself, is co-incubated with oligo (dT) ₁₇ homopolymer oligonucleotide (ODN6056). Some assumptions are made that the activity of -848 is increased while TLR7-mediated signaling is lost. Similarly, the combination of loxoribine, a guanosine analog that itself activates TLR7, with oligo (dT) ₁₇ ODN6056 induced TLR8-mediated signaling while TLR7-mediated signaling disappeared.

The cytokine profile induced by loxoribine in human immune cells was also altered by co-incubation with oligo (dT) ₁₇ . IL-12, TNF-α or IFN-γ was secreted in large amounts, but IFN-α production was stopped. While not intending to be bound by any particular mechanism, the observed changes in induced cytokines are attributed to the transition of activated cell types from plasma cell DCs with loxoribine alone to monocytes in combination with ODN, It is speculated that the sphere shows that it does not express functional TLR7.

  Thus, the present invention is useful for modulating a range of immune responses. The invention in certain embodiments is useful in suppressing TLR7 signaling (and associated TLR7-mediated immune response) while optionally inducing TLR8 signaling (and associated TLR8-mediated immune response). In another specific embodiment, the present invention results in the induction (including enhancement) of TLR8 signaling (and associated TLR8-mediated immune response). Depending on the nature of the adapter oligonucleotide, TLR9 signaling (and associated TLR9-mediated immune responses) can also be triggered simultaneously. For example, the use of adapter oligonucleotides containing a CpG motif in combination with a TLR7 ligand such as loxoribine will trigger TLR8 and TLR9 signaling (and their associated mediated immune responses).

  The present invention treats a series of pathologies that would be advantageous in suppressing and / or inducing (including enhancing) a TLR8-mediated immune response, optionally in the absence or presence of a TLR9-mediated immune response. Also useful.

  A series of medical conditions that may be advantageous in suppressing TLR7-mediated immune responses include, but are not limited to, autoimmune medical conditions, particularly those associated with infection with DNA viruses, such as lupus and rheumatoid arthritis. Suppression of a TLR7-mediated immune response that is accompanied by the induction of a TLR8-mediated immune response can be used when it is desirable to enhance a T cell response that is suppressed by regulatory T cells. Such medical conditions include, but are not limited to, several types of cancer and HCV, HBV and HIV infections. The combination of TLR7 suppression and TLR8 induction may be beneficial in the treatment of autoimmune diseases or when it is desirable to elicit an immune response but avoid TLR7 associated toxicity. Induction (including enhancement) of TLR8-mediated immune responses that are not involved in the modulation of TLR7-mediated immune responses is particularly useful for the treatment of cancer and infectious diseases with vaccine or non-vaccine methods.

  The present invention is directed to alterations in cytokine production or when a subject is refractory or refractory to the action of certain cytokines (eg, IFN-α sometimes found in the treatment of viral infections) It is also useful for the treatment of medical conditions that may be advantageous.

TLR
Toll-like receptors (TLRs) are a family of highly conserved polypeptides that play a critical role in mammalian innate immunity. To date, ten family members designated TLR1-TLR10 have been identified. The cytoplasmic domains of various TLRs are characterized by Toll-interleukin 1 (IL-1) receptor (TIR) domains. Medzitov R et al. (1998) Mol Cell 2: 253-8. Recognition of microbial invasion by TLRs triggers the activation of evolutionary conserved signaling cascades in Drosophila and mammals. A TIR domain-containing adapter protein, MyD88, has been reported to bind to TLR and recruit IL-1 receptor-related kinase (IRAK) and tumor necrosis factor (TNF) receptor-related factor 6 (TRAF6) to the TLR. The MyD88-dependent signaling pathway is the critical step in immune activation and production of inflammatory cytokines, the activity of NF-κB transcription factor and c-Jun NH _2- terminal kinase (Jnk) mitogen-activated protein kinase (MAPK) It is thought to cause For reviews, see Aderem A et al. (2000) Nature 406: 782-87.

  TLRs are thought to be differentially expressed in various tissues and on various immune cell types. For example, human TLR7 has been reported to be expressed in the placenta, lung, spleen, lymph nodes, tonsils, and on plasmaoid precursor dendritic cells (pDC). Chuang TH et al. (2000) Eur Cytokine Net 11: 372-8); Kadowaki N et al. (2001) J. Org. Exp Med 194: 863-9. Human TLR8 has been reported to be expressed in the lung, peripheral blood leukocytes (PBL), placenta, spleen, lymph nodes, and on monocytes. Kadowaki N et al. (2001) J. Org. Exp Med 194: 863-9; Chuang TH et al. (2000) Eur Cytokine Net 11: 372-8. Human TLR9 has been reported to be expressed in the spleen, lymph nodes, bone marrow, PBL, and on pDC and B cells. Kadowaki N et al. (2001) J. Org. Exp Med 194: 863-9; Bauer S et al. (2001) Proc Natl Acad Sci USA 98: 9237-42; Chuang TH et al. (2000) Eur Cytokine Net 11: 372-8.

  The nucleotide and amino acid sequences of human and mouse TLR7 are known. See, for example, GenBank accession numbers AF240467, AF245702, NM_016562, AF334492, NM_13311; and AAF60188, AAF78035, NP_057646, AAL73191 and AAL73192, the contents of each of which are incorporated herein by reference. Human TLR7 has been reported to be 1049 amino acids long. Mouse TLR7 has been reported to be 1050 amino acids long. A TLR7 polypeptide includes an extracellular domain with a leucine-rich repeat region, a transmembrane domain, and an intracellular domain that includes a TIR domain.

  The nucleotide and amino acid sequences of human and mouse TLR8 are known. For example, reference is made to GenBank accession numbers AF246971, AF245703, NM — 016706, XM — 045706, AY035890, NM — 1331212; Human TLR8 exists as at least two isoforms, one is reported to be 1041 amino acids long and the other 1059 amino acids long. Mouse TLR8 is 1032 amino acids long. A TLR8 polypeptide includes an extracellular domain with a rich leucine repeat region, a transmembrane domain, and an intracellular domain that includes a TIR domain.

  The nucleotide and amino acid sequences of human and mouse TLR9 are known. For example, GenBank accession numbers NM — 017442, AF259262, AB04180, AF245704, AB045181, AF348140, AF314224, NM — 031178; and NP — 059138, AAF72189, BAB19259, A2878037, A28780, A28780, A28780, A28780, A Please refer to. Human TLR9 exists as at least two isoforms, one being 1032 amino acids long and the other being 1055 amino acids long. Mouse TLR9 is 1032 amino acids long. A TLR9 polypeptide includes an extracellular domain with a leucine-rich leucine repeat region, a transmembrane domain, and an intracellular domain that includes a TIR domain.

TLR-mediated immune response As used herein, the term “TLR signaling” refers to any aspect of intracellular signaling associated with signaling through TLRs. As used herein, the term “TLR-mediated immune response” refers to an immune response associated with (eg, resulting from) TLR signaling.

  A TLR7-mediated immune response is a response associated with TLR7 signaling. TLR7-mediated immune responses are generally characterized by IFN-inducing cytokines such as IFN-α and IP-10, I-TAC. The amount of each cytokine IL-1α / β, IL-6, IL-8, MIP-1α / β and MIP-3α / β induced in a TLR7-mediated immune response is less than the amount induced in a TLR8-mediated immune response .

  A TLR8-mediated immune response is a response associated with TLR8 signaling. This response is associated with pro-inflammatory cytokines such as IFN-γ, IL-12p40 / 70, TNF-α, IL-1α / β, IL-6, IL-8, MIP-1α / β, MIP-3α / β. Characterized by induction.

  A TLR9-mediated immune response is a response associated with TLR9 signaling. This response is less than the amount achieved through a TLR8-mediated immune response, but is at least characterized by the production and / or secretion of IFN-γ and IL-12.

TLR7 / 8 ligand As used herein, a “TLR7 / 8 ligand” is any agent capable of increasing TLR7 and / or TLR8 signaling (ie, an agonist of TLR7 and / or TLR8). Is generically referred to. Thus, some TLR7 / 8 ligands (eg, TLR7-specific ligands) that elicit only TLR7 signaling when used in the absence of the adapter oligonucleotides of the invention (eg, TLR8-specific ligands). Ligands), and others induce both TLR7 and TLR8 signaling. According to the present invention, it has been found that the use of such ligands in combination with adapter oligonucleotides changes the TLR signaling profile of such ligands. In some examples, TLR7 signaling is suppressed and TLR8 signaling is induced. In other examples, TLR8 signaling is triggered (ie, increases beyond the amount of background) or enhanced (ie, increases above the amount of non-background), and in yet other embodiments, TLR8 , Both TLR9 signaling is induced or enhanced.

As used herein, the term “TLR7 ligand” refers to any agent that can increase TLR7 signaling (ie, an agonist of TLR7). In this regard, the amount of TLR7 signaling may be enhanced beyond the existing signaling amount or may be induced beyond the background signaling amount. TLR7 ligands include, but are not limited to, guanosine analogs such as C8 substituted guanosines, mixtures of ribonucleosides consisting essentially of G and U, guanosine ribonucleotides, and RNA or RNA-like molecules (PCT / US03 / 10406) Furthermore, adenosine compounds (for example, 6-amino-9-benzyl-2- (3-hydroxypropoxy) -9H-purin-8-ol (CL-029, Sumitomo), 6-amino-9-benzyl-2- Butoxy-9H-purin-8-ol (shown in FIG. 13D) and other related compounds such as those described in US Pat. No. 6,311,070 B1). TLR7 ligands are described in Gorden et al. J. et al. Immunol. 2005, 174: 1259-1268 (eg 3M-001, N- [4- (4-amino-2-ethyl-1H-imidazo [4,5-c] quinolin-1-yl) butyl -] _{methanesulfonamide, C 17 H 23 N 5 O} 2 S, mw361).

  As used herein, the term “guanosine analog” refers to a guanosine-like nucleotide (excluding guanosine) having a chemical modification involving either a guanine base, a guanosine nucleoside sugar, or both a guanine base and a guanosine nucleoside sugar. Specifically, guanosine analogs include, without limitation, 7-deazaguanosine.

  Guanosine analogues include 7-thia-8-oxoguanosine (immunosine), 8-mercaptoguanosine, 8-bromoguanosine, 8-methylguanosine, 8-oxo-7,8-dihydroguanosine, C8-arylamino-2 C8.N7 substituted guanine ribonucleosides such as '-deoxyguanosine, C8-propynylguanosine, 7-allyl-8-oxoguanosine (loxoribine) and 7-methyl-8-oxoguanosine, 8-aminoguanosine, 8-hydroxy-2' Further included are C8 substituted guanosines such as deoxyguanosine, 8-hydroxyguanosine, 7-deaza-8 substituted guanosine.

As used herein, the term “TLR8 ligand” refers to any agent that can increase TLR8 signaling (ie, an agonist of TLR8). In this regard, the amount of TLR8 signaling may be enhanced beyond the existing signaling amount or may be induced beyond the background signaling amount. TLR8 ligands include a mixture of ribonucleosides consisting essentially of G and U, guanosine ribonucleotides, and RNA or RNA-like molecules (PCT / US03 / 10406). Additional TLR8 ligands are described by Gorden et al. J. et al. Immunol. 2005,174: is also disclosed in 1259-1268 (e.g., 3M-002,2- propyl thiazolo [4,5-c] _{quinolin-4-amine, C 13 H 13 N 3 S} , mw243).

Some TLR7 / 8 ligands are both TLR7 and TLR8 ligands. Such ligands include imidazoquinolines, a mixture of ribonucleosides consisting essentially of G and U, guanosine ribonucleotides, and RNA or RNA-like molecules (PCT / US03 / 10406). Additional TLR7 / 8 ligands are described by Gorden et al. J. et al. Immunol. 2005, 174: 1259-1268 (eg 3M-003, 4-amino-2- (ethoxymethyl) -α, α-dimethyl-6,7,8,9-tetrahydro-1H-imidazo [4,5-c] quinolin-1-ethanol _{hydrate, C 17 H 26 N 4 O} 2, mw318).

  Imidazoquinolines induce the expression of several cytokines, including interferons (eg, IFN-α), TNF-α and some interleukins (eg, IL-1, IL-6, and IL-12). It is a possible immune response modifier. Imidazoquinolines can stimulate a Th1 immune response, as evidenced in part by their ability to induce increased amounts of IgG2a. It has been reported that imidazoquinoline agents can also suppress the production of Th2 cytokines such as IL-4, IL-5, and IL-13. Some of the cytokines induced by imidazoquinolines are produced by macrophages and dendritic cells. Several imidazoquinolines have been reported to increase NK cytolytic activity and stimulate B cell proliferation and differentiation, thereby inducing antibody production and secretion.

  As used herein, imidazoquinolines include imidazoquinoline amines, imidazopyridine amines, 6,7-fused cycloalkylimidazopyridine amines, and 1,2 bridged imidazoquinoline amines. These compounds are disclosed in U.S. Pat. Nos. 4,689,338, 4,929,624, 5,238,944, 5,266,575, 5,268,376, 5,346,905, 5,352,784, 5,389,640, 5,395,937, 5,549,936, 5,482,936. 5525612, 60399969 and 6110929. Specific species of imidazoquinolines include R-848 (S-28463), 4-amino-2ethoxymethyl-α, α-dimethyl-1H-imidazo [4,5-c] quinoline-1-ethanol, 1- ( 2-methylpropyl) -1H-imidazo [4,5-c] quinolin-4-amine (R837 or imiquimod), and S-27609. Imiquimod is currently used for topical treatment of vagina such as genitals and anal folds, and has also been tested for local treatment of basal cell carcinoma.

  As used herein, the term “TLR9 ligand” refers to any agent that can increase TLR9 signaling (ie, an agonist of TLR9). Specifically, TLR9 ligands include, without limitation, immunostimulatory nucleic acids, particularly immunostimulatory CpG nucleic acids.

  As used herein, the term “immunostimulatory CpG nucleic acid” refers to any CpG-containing nucleic acid that can activate immune cells. At least C of the CpG dinucleotide is usually but not necessarily unmethylated. Immunostimulatory CpG nucleic acids have been described in a number of issued and published patent applications, including US Pat. Nos. 6,194,388, 6,207,646, 6,218,371, 6,239,116, 6,339,068, 6,406,705 and 6,429,199. Has been.

  In some embodiments, the TLR ligand is a specific ligand. As used herein, a TLR7-specific ligand is a ligand that signals through TLR7 but not through TLR8 or TLR9 when used in the absence of the adapter oligonucleotide of the present invention. Similarly, a TLR8-specific ligand is a ligand that signals through TLR8 but not through TLR7 or TLR9 when used in the absence of an adapter oligonucleotide of the present invention. Preferably, the TLR7-specific ligand is signaled through TLR7 but not through other TLRs when used in the absence of the adapter oligonucleotide, and the TLR8-specific ligand is used in the absence of the adapter oligonucleotide. Then, the signal is transmitted through TLR8 but not through other TLRs.

  In some embodiments, the TLR ligand is not RNA.

  When TLR7 / 8 ligand is used with an adapter oligonucleotide, it can stimulate both TLR8 and TLR9 and cause downstream effects of both receptors simultaneously. For example, stimulation of TLR9 with the oligonucleotide can cause, for example, production of IFN-α, while stimulation of TLR8 with the oligonucleotide can cause production of, for example, IL-12, TNF-α, and IFN-γ. The ligand and oligonucleotide may be conjugated to each other or physically separated from each other.

Adapter Oligonucleotide According to the present invention, a TLR7 / 8 ligand is used in combination with an adapter oligonucleotide. An adapter oligonucleotide as used herein, when used with a TLR7 / 8 ligand, suppresses TLR7 signaling by the TLR7 ligand, induces TLR8 signaling by the TLR7 ligand, and / or both TLR7 and TLR8. An oligonucleotide that modulates the activity of the ligand by enhancing TLR8 signaling by the ligand being the ligand.

  As used herein, the term oligonucleotide is used interchangeably with the term nucleic acid. Preferably, the term excludes plasmids, vectors, and / or antisense nucleic acids. In some embodiments, the oligonucleotide may be immunostimulatory, while in other embodiments it may be non-immunostimulatory when used alone. The oligonucleotide may be of any length, but is preferably 7 or 8 to 100 nucleotides in length.

A vast class of adapter oligonucleotides includes the formula 5′XN ₄ —X3 ′ or 5′XN ₅ —X3 ′, where X can be any nucleotide and is present N ₄ and N ₅ may be 4 and 5 in series of T (thymidine), U (uracil) or A (adenine) so that N in N ₄ or N ₅ is the same. Represents. The oligonucleotide may be 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or more nucleotides in length. Preferably it comprises at least one internucleotide phosphorothioate linkage (up to a fully phosphorothioated backbone).

Thus, some classes of adapter oligonucleotides include the formula 5′X _a -TTTTTT-X _b 3 ′, where X _a and X _b can independently be any nucleotide, even if present You don't have to. X _a and X _b may represent one or more nucleotides (eg, 1-100 nucleotides). The oligonucleotide may have a length of 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or more in number of nucleotides. Good. The oligonucleotide may comprise 6, 7 or more Ts. Preferably, the adapter oligonucleotide is a dT homopolymer (ie, oligo dT as described herein in length). Even more preferably, the adapter oligonucleotide is a thymidine (dT) homopolymer of 17 nucleotides in length. Most preferably it comprises at least one internucleotide phosphorothioate linkage (up to a fully phosphorothioated backbone).

  The adapter oligonucleotide is 100% T, 99% T, 198T, 97% T, 96% T, 95% T, 94% T, 93% T, 92% T, 91% T, depending on the embodiment. 90% T, 85% T, 80% T, 75% T, 70% T, 65% T, 60% T, 55% T, 50% T, 45% T, or lower T May be.

Another class of adapter oligonucleotides includes the formula 5′X _a -UUUUU-X _b 3 ′, where X _a and X _b can independently be any nucleotide, and if present, It does not have to be. X _a and X _b may represent one or more nucleotides (eg, 1-100 nucleotides). The oligonucleotide may have a length of 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or more in number of nucleotides. Good. The oligonucleotide may comprise U, 6 or 7 or more Us. In important embodiments, the oligonucleotide is preferably a dU homopolymer that is 17 nucleotides in length and has at least one internucleotide phosphorothioate linkage (up to a fully phosphorothioated backbone).

  The adapter oligonucleotide may be 100% U, 99% U, 198U, 97% U, 96% U, 95% U, 94% U, 93% U, 92% U, 91% U, depending on the embodiment. 90% U, 85% U, 80% U, 75% U, 70% U, 65% U, 60% U, 55% U, 50% U, 45% U, or lower U May be.

Yet another class of adapter oligonucleotides includes the formula 5′X _a -AAAAA-X _b 3 ′, where X _a and X _b can independently be any nucleotide, You don't have to. X _a and X _b may represent one or more nucleotides (eg, 1-100 nucleotides). The oligonucleotide may have a length of 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or more in number of nucleotides. Good. The oligonucleotide may comprise six, seven, or more As. In important embodiments, the oligonucleotide is a dA homopolymer, preferably 17 nucleotides in length and having at least one internucleotide phosphorothioate linkage (up to a fully phosphorothioated backbone).

  The adapter oligonucleotide is 100% A, 99% A, 198A, 97% A, 96% A, 95% A, 94% A, 93% A, 92% A, 91% A, depending on the embodiment. 90% A, 85% A, 80% A, 75% A, 70% A, 65% A, 60% A, 55% A, 50% A, 45% A, or lower A May be.

Another class of adapter oligonucleotides includes the formula 5′C _n -T _m -C _p 3 ′, where n is an integer in the range of 0-100 (eg, 3-7), p is 0-100 (eg, An integer in the range of 4 to 8) and m is an integer in the range of 0 to 100 (for example, 2 to 10). Preferably, the sum of n and p has a C content of 50%, less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10% Is less than or equal to m so that it is less than, less than 5%, or less. In some embodiments, n is in the range of 3-7, m is in the range of 2-10, and p is in the range of 4-8, provided that the above ratios (%) are satisfied. Several of this equation are shown in FIG. Examples include 5′C ₃ T ₁₀ C ₄ 3 ′ (SEQ ID NO: 1), 5′C ₄ T ₈ C ₅ 3 ′ (SEQ ID NO: 2), 5′C ₅ T ₆ C ₆ 3 ′ (SEQ ID NO: 1). 6) 5′C ₆ T ₄ C ₇ 3 ′ (SEQ ID NO: 7) and 5′C ₇ T ₂ C ₈ 3 ′ (SEQ ID NO: 8).

  In some embodiments, the adapter oligonucleotide comprises an immunostimulatory CpG motif. The motif may be methylated or unmethylated. In the latter example, the entire oligonucleotide can be unmethylated, or a portion thereof can be unmethylated, but at least C of 5'CG3 'must be unmethylated. Unmethylated motifs and their effects on immunomodulation have been described in US patents such as US Pat. No. 6,194,388 B1, US Pat. No. 6,207,646 B1, US Pat. No. 6,239,116 B1, US Pat. No. 6,218,371 B1, and PCT / US98 / 03678, PCT / It has been extensively described in published patent applications such as US98 / 10408, PCT / US98 / 04703, PCT / US99 / 09863. The entire contents of each of these patents and patent applications are incorporated herein by reference.

  As noted above, the terms “oligonucleotide” and “nucleic acid” refer to multiple nucleotides (ie, phosphate groups and pyrimidines (eg, cytosine (C), thymidine (T) or uracil (U)), purines (adenine ( Used interchangeably to mean a sugar (eg, a molecule comprising ribose or deoxyribose) linked to an exchangeable organic base that is either A) or guanine (G)). Thus, this term encompasses both DNA and RNA oligonucleotides. The term is also intended to encompass polynucleosides (ie, polynucleotides minus phosphate) and any other organic base-containing polymer. Oligonucleotides can be obtained from existing nucleic acid sources (eg, genomic or cDNA), but are preferably synthetic (eg, produced by a nucleic acid synthesizer).

  The oligonucleotide may be either double-stranded or single-stranded. In certain embodiments, when the oligonucleotide is single stranded, it can form secondary and tertiary structures (eg, selected by folding or over, or along length). By hybridizing with itself in the segment). Therefore, even when the primary structure of such an oligonucleotide is single-stranded, the higher-order structure may be double-stranded or triple-stranded.

  The length of the oligonucleotide has a range, but the length is preferably 100 nucleotides (or bases) or less. The adapter oligonucleotide is preferably not a plasmid or vector. It is also preferred that there is no anti-antisense activity ability, or that the action that appears in accordance with the present invention is at least not related to anti-antisense activity.

  The oligonucleotide may have a modified backbone. For example, it may contain at least one internucleotide linkage that is not a phosphodiester linkage. Such a linking part may be a phosphorothioate linking part. In some embodiments, the oligonucleotide may have a chimeric backbone (ie, a backbone consisting of at least two internucleotide linkages).

  As used herein, the term “phosphorothioate backbone” refers to a stabilized sugar phosphate of an oligonucleotide in which the non-crosslinkable phosphate oxygen is replaced by sulfur at at least one internucleotide linkage. Refers to the ester skeleton. In one embodiment, the non-crosslinkable phosphate oxygen is replaced by sulfur at every and every internucleotide linkage.

Oligonucleotide Sources and Preparations Oligonucleotides of the invention comprise natural RNA and phosphodiester internucleoside bridges, β-D-ribose units, and / or natural nucleoside bases (adenine, guanine, cytosine, thymine, uracil) and A variety of chemical modifications and substitutions can be included as compared to DNA. Examples of chemical modifications are known to those skilled in the art and are described, for example, in Uhlmann E et al. (1990) Chem Rev 90: 543; “Protocols for Oligonucleotides and Analogs” Synthesis and Properties & Synthesis and Analytical Technologies, S .; Agrawal, Ed, Humana Press, Totowa, USA 1993; Crooke ST et al. (1996) Annu Rev Pharmacol Toxicol 36: 107-29 and Hunziker J et al. (1995) Mod Synth Methods 7: 331-417. Oligonucleotides according to the present invention are specific phosphodiester internucleoside bridges and / or specific β-D-ribose units and / or specific natural, compared to oligonucleotides of the same sequence composed of natural DNA or RNA. It may have one or more modifications, each located at a nucleoside base position.

For example, the oligonucleotide may comprise one or more modifications, each modification comprising:
a) replacement of the phosphodiester internucleoside bridge located at the 3 ′ and / or 5 ′ end of the nucleoside by a modified internucleoside bridge;
b) replacement of the phosphodiester bridge located at the 3 ′ and / or 5 ′ end of the nucleoside by dephosphorylation,
c) Substitution by another unit of sugar phosphate unit derived from sugar phosphate skeleton,
d) independently selected from the replacement of β-D-ribose units with modified sugar units, and e) replacement of natural nucleoside bases with modified nucleoside bases.

  A more detailed example for the chemical modification of an oligonucleotide is as follows.

  The oligonucleotide may comprise a modified internucleotide linkage such as those described in (a) or (b) above. Such internucleotide linkages can be partially resistant to degradation (eg, stabilized). “Stabilized oligonucleotide” shall mean an oligonucleotide that is relatively resistant to degradation in vivo (eg, by exo or endonuclease) as a result of such modifications. In some embodiments, an oligonucleotide having a phosphorothioate linkage exhibits the greatest activity and may protect the oligonucleotide from degradation by intracellular exo or endonucleases.

The phosphodiester internucleoside bridge located at the 3 ′ and / or 5 ′ end of the nucleoside can be replaced by a modified internucleoside bridge, such as phosphorothioate, phosphorodithioate, NR ¹ R ² -Phosphoramidate, boranophosphate, α-hydroxybenzylphosphonate, phosphate- (C ₁ -C ₂₁ ) -O-alkyl ester, phosphate-[(C ₆ -C ₁₂ ) aryl- (C ₁ -C ₂₁ ) -O- alkyl] _{ester, (C} 1 -C ₈₎ alkylphosphonate and / or _(C 6 _{-C 12)} aryl phosphonate _{bridge, (C 7} ~C 12) _-α- hydroxymethyl aryl (e.g., WO 95 (Disclosed in / 01363), wherein: C ₆ -C ₁₂ ) aryl, (C ₆ -C ₂₀ ) aryl and (C ₆ -C ₁₄ ) aryl may be substituted with halogen, alkyl, alkoxy, nitro, cyano, R ¹ and R ² are , Independently of one another, hydrogen, (C ₁ -C ₁₈ ) alkyl, (C ₆ -C ₂₀ ) aryl, (C ₆ -C ₁₄ ) aryl- (C ₁ -C ₈ ) alkyl, preferably hydrogen, (C ₁ ~ C ₈ ) alkyl, preferably (C ₁ -C ₄ ) alkyl, and / or methoxyethyl, or R ¹ and R ² together with the nitrogen atom bearing both, additionally from the group of O, S and N To form a 5-6 membered heterocycle which can contain additional heteroatoms.

  Phosphodiester bridges located at the 3 ′ and / or 5 ′ end of the nucleoside can be replaced by dephosphorylation bridges (for example, “Methods in Molecular Biology”, Vol. 20, “Protocols for Oligonucleotides”). and Analogs ", S. Agrawal, Ed, Humana Press, Totowa, 1993, Uhlmann E and Peyman A, Chapter 16, pp. 355ff), dephosphorylated bridges are, for example, formacetal groups, 3 ' -Dephosphorization of thioform acetal group, methylhydroxylamine group, oxime group, methylenedimethylhydrazo group, dimethylenesulfone group and / or silyl group It is selected from.

  A sugar phosphate unit derived from a sugar phosphate ester skeleton (ie, a sugar phosphate ester skeleton is composed of sugar phosphate ester units) (ie, a sugar phosphate ester unit is formed together) , Β-D-ribose and phosphodiester internucleoside bridges) can be replaced by another unit, for example a “morpholino derivative” oligomer (eg Stirchak EP et al. (1989) Nucleic Acids Res). 17: 6129-41), i.e. construction of alternatives having, for example, morpholino derivative units, or polyamide nucleic acids ("PNA", e.g. Nielsen PE et al. (1994) Bioconjug Chem 5: 3-7. That is, for example, PNA Having Rating Unit, for example, by 2-aminoethyl glycine it is suitable for the construction of alternatives. The oligonucleotide can be used for other modifications and alternatives of the sugar chain backbone, such as peptide nucleic acid having a phosphate group (PHONA), immobilized nucleic acid (LNA), and oligonucleotide having an alkyl linker or amino linker in the backbone. You may have. The alkyl linker may be branched or unbranched, substituted or unsubstituted, and pure chiral or racemic mixtures.

The β-ribose unit or β-D-2′-deoxyribose unit can be replaced by a modified sugar unit, such as β-D-ribose, α-D-2′-deoxyribose. L-2′-deoxyribose, 2′-F-2′-deoxyribose, 2′-F-arabinose, 2′-O— (C ₁ -C ₆ ) alkylribose, 2′-O-methylribose, _{2'-O- (C 2 ~C 6} ) _{alkenyl-ribose, 2 '- [O- (C} 1 ~C 6) alkyl -O- _(C 1 ~C ₆₎ alkyl] ribose, _2'-NH 2 -2 '-Deoxyribose, β-D-xylofuranose, α-arabinofuranose, 2,4-dideoxy-β-D-erythrohexopyranose, as well as carbocyclic saccharide analogs (eg, Froehler (1992) J Am Chem Soc 114: 8320), and / or open-chain saccharide analogs (eg, described in Vandendriesche et al. (1993) Tetrahedron 49: 7223), and / or bicyclic saccharide analogs (eg, Tarkov M et al. (1993) Helv Chim Acta 76: 481).

  In some embodiments, the modified sugar is 2 'modified ribose. In some embodiments, the sugar is 2'-O-methyl ribose, particularly for one or both nucleotides linked by a phosphodiester or phosphodiester-like internucleoside linkage.

  Nucleic acids also include substituted purines and pyrimidines such as C5 propyne pyrimidine and each base modified with 7-deaza-7 substituted purines. Wagner RW et al. (1996) Nat. Biotechnol 14: 840-4. Purines and pyrimidines include, but are not limited to, adenine, cytosine, guanine and thymine, and other natural and non-natural nucleobases, substituted and unsubstituted aromatic moieties.

A modified base is any base that is chemically different from the natural bases normally found in DNA and RNA, such as T, C, G, A, and U, but shares a basic chemical structure with these natural bases. . The modified nucleoside base is, for example, hypoxanthine, uracil, dihydrouracil, pseudouracil, 2-thiouracil, 4-thiouracil, 5-aminouracil, 5- (C ₁ -C ₆ ) alkyluracil, 5- (C ₂ -C ₆₎ alkenyl uracil, 5- _(C 2 -C ₆₎ alkynyl uracil, 5- (hydroxymethyl) uracil, 5-chloro-uracil, 5-fluorouracil, 5-bromouracil, 5-hydroxy cytosine, 5- _(C 1 ~ C ₆₎ alkyl cytosine, 5- _(C 2 ~C ₆₎ alkenyl cytosine, 5- _(C 2 ~C ₆₎ alkynyl cytosine, 5-chloro-cytosine, 5-fluorocytosine, 5-bromo-cytosine, ^{N 2} - dimethyl guanine 2,4-diaminopurine, 8-azapurine, substituted 7-deazapurine, preferred Or 7-deaza-7-substituted purine and / or 7-deaza-8-substituted purine, 5-hydroxymethylcytosine, N4-alkylcytosine such as N4-ethylcytosine, 5-hydroxydeoxycytidine, 5-hydroxymethyldeoxycytidine, N4 Alkyldeoxycytidines such as N4-ethyldeoxycytidine, 6-thiodeoxyguanosine, and deoxyribonucleosides of nitropyrrole, C5-propynylpyrimidine, and diaminopurines such as 2,6-diaminopurine, inosine, 5-methylcytosine, 2-amino It may be selected from purine, 2-amino-6-chloropurine, hypoxanthine, or other modifications of natural nucleoside bases. This list is intended to be illustrative and should not be construed as limiting.

  Modified bases may be incorporated into the specific formulas described herein. For example, a cytosine may be replaced with a modified cytosine. Modified cytosine as used herein is a natural or non-natural pyrimidine base analog of this base that can replace cytosine without compromising the immunostimulatory activity of the oligonucleotide. Modified cytosines include, but are not limited to, 5-substituted cytosines (eg, 5-methylcytosine, 5-fluorocytosine, 5-chlorocytosine, 5-bromocytosine, 5-iodocytosine, 5-hydroxycytosine, 5-hydroxymethyl Cytosine, 5-difluoromethylcytosine, and unsubstituted or substituted 5-alkynylcytosine), 6-substituted cytosine, N4-substituted cytosine (eg, N4-ethylcytosine), 5-azacytosine, 2-mercaptocytosine, isocytosine, pseudoisocytosine , Cytosine analogs having a fused ring system (eg, N, N′-propylene cytosine or phenoxazine), and uracil and its derivatives (eg, 5-fluorouracil, 5-bromouracil, 5-bromovinyluracil, 4-thiouracil) , 5- Mud carboxymethyl uracil, include 5-propynyl uracil) it is. Some preferred cytosines include 5-methylcytosine, 5-fluorocytosine, 5-hydroxycytosine, 5-hydroxymethylcytosine and N4-ethylcytosine. In another embodiment of the invention, the cytosine base is a universal base (eg, 3-nitropyrrole, P-base), an aromatic ring system (eg, fluorobenzene or difluorobenzene), or a hydrogen atom (d spacer). Has been replaced by

  As another example, guanine may be replaced with a modified guanine base. Modified guanine as used herein is a natural or unnatural pyrimidine base analog of this base that can replace guanine without compromising the immunostimulatory activity of the oligonucleotide. Modified guanines include, but are not limited to, 7-deazaguanine, 7-deaza-7 substituted guanine (such as 7-deaza-7- (C2-C6) alkynylguanine), 7-deaza-8 substituted guanine, hypoxanthine, N2 Substituted guanine (N2-methylguanine), 5-amino-3-methyl-3H, 6H-thiazolo [4,5-d] pyrimidine-2,7-dione, 2,6-diaminopurine, 2-aminopurine, purine , Indole, adenine, substituted adenine (eg, N6-methyladenine, 8-oxoadenine), 8-substituted guanine (eg, 8-hydroxyguanine, 8-bromoguanine), and 6-thioguanine. In another embodiment of the invention, the guanine base is a universal base (eg, 4-methylindole, 5-nitroindole, and K-base), an aromatic ring system (eg, benzimidazole or dichlorobenzimidazole, 1 -Methyl-1H- [1,2,4] triazole-3-carboxylic acid amide), or a hydrogen atom (d spacer).

  For use in the present invention, the oligonucleotides of the present invention can be synthesized using, for example, the β-cyanoethyl phosphoramidite method (Beaucage SL et al. (1981) Tetrahedron Lett 22: 1859), or the nucleoside H-phosphonate method (Garegg et al. (1986) Tetrahedron Lett 27: 4051-4; Froehler BC et al. (1986) Nucleic Acids Res 14: 5399-407; Garegg et al. (1986) Tetrahedron Lett 27: 4055-8; Any of a number of procedures well known in the art, including Tetrahedron Lett 29: 2619-22) It can be synthesized from scratch using. Such chemical operations can be performed by other types of commercially available automatic nucleic acid synthesizers. These oligonucleotides are referred to as synthetic oligonucleotides. An isolated oligonucleotide generally refers to an oligonucleotide that has been separated from naturally associated components. For example, an isolated oligonucleotide can be isolated from cells, nuclei, mitochondria, or chromatin.

  Modified backbones such as phosphorothioates can be synthesized using automated techniques using phosphoramidite chemistry or H-phosphonate chemistry. Aryl and alkyl phosphonates can be made, for example, as described in US Pat. No. 4,469,863, where alkyl phosphotriesters (with charged oxygen moieties described in US Pat. No. 5,023,243 and European Patent No. 092574). Can be prepared by automated solid phase synthesis using commercially available reagents. Methods for making other modifications and substitutions of the DNA backbone have been described (eg, Uhlmann E et al. (1990) Chem Rev 90: 544; Goodchild J (1990) Bioconjugate Chem 1: 165).

Isolation In certain embodiments, the TLR7 / 8 ligand and / or adapter oligonucleotide is isolated. An isolated ligand or oligonucleotide may be substantially pure or contain other substances normally found together in nature or in vivo systems to the extent practical and appropriate for the intended use. No ligand or oligonucleotide. In particular, the ligand or oligonucleotide is sufficiently pure and does not contain other biological components sufficiently to be useful, for example, in the manufacture of pharmaceutical formulations. Since an isolated ligand or oligonucleotide can be miscible with a pharmaceutically acceptable carrier in a pharmaceutical formulation, the ligand or oligonucleotide may represent only a small percentage of the formulation weight. Nonetheless, the ligand or oligonucleotide is isolated because it is substantially separated from materials that may be related in biological systems.

Conjugate As used herein, the term “conjugate” refers to any combination of two or more component parts linked directly or indirectly by any physicochemical interaction. In one embodiment, the conjugate is a combination of two or more component parts linked directly or indirectly by covalent bonds.

  In one aspect, the invention provides a composition comprising a conjugate of a TLR ligand and an adapter oligonucleotide. In one embodiment, the conjugate is made through a covalent bond. In one embodiment, the conjugate includes a linker.

  The conjugate can comprise one or more adapter oligonucleotides of the invention and one or more TLR7 / 8 ligands. The conjugate may alternatively or additionally include one or more other molecules including but not limited to antigens or other agents.

  FIGS. 13-15 illustrate many features of such conjugates, including attachment points in both ligands and oligonucleotides, and the optional use of linkers to facilitate such conjugation. .

  In one embodiment, the conjugate is a conjugate of an adapter oligonucleotide and certain TLR7 specific ligands such as, but not limited to, immunosyn and loxoribine. The adapter oligonucleotide may be, but is not limited to, a poly (dT) oligonucleotide having a length in the range of 8 to 20, preferably 10 to 20, more preferably 14 to 18 nucleotides. . In some embodiments, conjugates of adapter oligonucleotides with certain TLR7 / 8 ligands, such as imidazoquinolines, C8 substituted guanosines, in particular, are adapter oligonucleotides that are themselves immunostimulatory or TLR9. Are excluded when signaling via

FIGS. 13A-C illustrate the structures of immunosynthetic and its two monomeric variants that can be used in conjugation methods. The variant shown in FIG. 13B can be conjugated to the oligonucleotide at an internal position or at the 3 ′ end. There may be one or more of such variants attached to the oligonucleotide. The variant shown in FIG. 13C can be conjugated to an oligonucleotide at the 5 ′ end. FIG. 14 illustrates the conjugation of immunosine to an oligonucleotide using a linker. FIG. 15 illustrates the points of attachment in R-848, CL-029 and immunosine to which linkers and / or oligonucleotides can be conjugated. The conjugates of the present invention may comprise one, two, three, four, five, or more linkers, or may lack a linker. An example of a conjugate is poly (dT) ₁₄ -L ₂ -IM (wherein L represents a linker, IM represents an immunosine, and the subscript indicates the number of each unit in the conjugate) ( SEQ ID NO: 9). Another example, _IM-L 2 - is a _{(dT) 14} (SEQ ID NO: 10). The difference between the two examples is that the arrangement of the immunosyn and linker is at either the 3 'end or the 5' end.

  The linker may be attached to any reactive group on the oligonucleotide, including but not limited to a backbone phosphate group or a sugar hydroxyl group. For example, the linker may be incorporated via phosphodiester, phosphorothioate, methylphosphonate and / or amide linkages.

  A linker may be non-nucleotide in nature. Linkers include, for example, non-basic residues (d-spacers), oligoethylene glycols such as triethylene glycol (spacer 9) and hexaethylene glycol (spacer 18), or alkane diols such as butanediol. The linkers may be connected to each other by a phosphodiester or phosphorothioate bond. Other linkers are alkyl amino linkers such as C3, C6, C12 amino linkers, as are alkyl thiol linkers such as C3 or C6 thiol linkers. Heterogeneous linkers may be used in one conjugate.

  The oligonucleotides may be linked by aromatic residues that may be further substituted with alkyl groups or substituted alkyl groups. The oligonucleotide may contain double or triple units that allow conjugation of homogeneous or heterogeneous multiple ligands to the oligonucleotide. The oligonucleotide may contain a linker resulting from a peptide modifying agent or oligonucleotide modifying agent. Furthermore, it may contain one or more natural or non-natural amino acid residues connected by peptide (amide) linkages.

  Various oligonucleotides can be synthesized by established methods and linked together on the line during solid phase synthesis. Alternatively, after the individual units are synthesized, they may be linked together.

Immune Response In one embodiment, the TLR-mediated immune response is a Th1-like immune response. As used herein, the term “Th1-like” refers to having characteristics characteristic of a Th1 immune response. A Th1 immune response may include features of induction of certain cytokines such as IFN-γ, secretion of IgG2a immunoglobulin (in mice), and macrophage activation. The term “Th1-like” should be contrasted with the term “Th2-like”, which refers to having characteristics characteristic of a Th2 immune response, as discussed below.

  A Th1-like immune response is a species that is characteristically associated with a Th1 immune response, including IFN-α, IFN-β, IFN-γ, TNF-α, IL-12, IL-18, IP-10. Expression of any of the cytokines and chemokines, and any combination thereof. Th1 and Th2 immune responses are thought to be retroregulated. In some embodiments, a Th1-like immune response can include suppression of certain Th2-related cytokines, including IL-4, IL-5, IL-10 and IL-13. A Th1-like immune response may include the expression of certain antibody isotypes, including IgG2a (in mice), with or without suppression of certain Th2-related antibody isotypes, including IgE and IgG1 (in mice). it can. In one embodiment, the Th1-like immune response is a Th1 response.

  A Th2-like immune response is any of certain cytokines and chemokines, including IL-4, IL-5, IL-10, IL-13, and any of them characteristically associated with a Th2 immune response Expression of any combination of these can be included. In some embodiments, a Th2-like immune response can include suppression of certain Th1-related cytokines. A Th2-like immune response includes expression of certain antibody isotypes, including IgE and IgG1 (in mice), with or without suppression of certain Th1-related antibody isotypes, including IgG2a (in mice) Can do. In one embodiment, the Th2-like immune response is a Th2 response.

  Accordingly, in one embodiment, the present invention provides a method of eliciting a Th1-like immune response in a subject. Inducing a Th1-like immune response includes enhancing or enhancing a Th1-like immune response. The method comprises administering to the subject an effective amount of a TLR7 / 8 ligand and adapter oligonucleotide of the invention to elicit a Th1-like immune response in the subject.

  In one embodiment, the present invention provides a method of suppressing a Th2-like immune response in a subject. The method includes administering to the subject an effective amount of a TLR7 / 8 ligand and adapter oligonucleotide of the invention to suppress a Th2-like immune response in the subject. Such a method may find particular use in the treatment of subjects having or at risk for a condition characterized primarily by a Th2 characteristic immune response. Such medical conditions include, without limitation, allergies and asthma.

  In one embodiment, the immune response involves upregulation of cell surface markers of immune cell activation, such as CD25, CD80, CD86, CD154. Methods for measuring cell surface expression of such markers are well known in the art and include FACS analysis.

  In order to measure an immune response in a cell or cell population, in one embodiment, the cell or cell population expresses at least one of TLR7, TLR8 or TLR9, and in some cases preferably only one. The cell can naturally express TLR or can be manipulated to express TLR by introducing an appropriate expression vector for TLR into the cell. In one embodiment, the cell or cell population is obtained as peripheral blood mononuclear cells (PBMC). In one embodiment, the cell or cell population is obtained as a cell line that expresses a TLR. In one embodiment, the cell or cell population is obtained as a cell line transiently transfected with a TLR. In one embodiment, the cell or cell population is obtained as a cell line stably transfected with TLR.

  It may also be advantageous to introduce into the cell or cell population a reporter construct that is responsive (ie, regulated) to intracellular signaling by the TLR for use in measuring an immune response in the cell or cell population. . In one embodiment, such a reporter is a gene under the control of the NF-κB promoter. In one embodiment, the gene under the control of its promoter is luciferase, but other reporter genes can be used, including green fluorescent protein (GFP), β-galactosidase, alkaline phosphatase, and the like. Under appropriate conditions of activation, for example, a luciferase reporter construct is expressed and emits a detectable light signal that can be measured quantitatively using a luminometer. These and other reporter constructs are commercially available. In other embodiments, a TLR-mediated immune response can be measured by production (mRNA or protein) or secretion of cytokines such as IFN-α, TNF-α, IL-12, IFN-γ. In the Examples, this type of assay is shown. The present invention also envisages the use of a cell-free detection method for TLR activation.

  The present invention also encompasses antigen-specific immune responses (described in more detail herein) and non-specific innate immune activation, as well as methods for eliciting widespread resistance to infection attacks. As used herein, the term non-antigen-specific innate immune activation refers to non-B cells, NK cells, T cells, or other immune cells that can respond in an antigen-independent manner, or these cells Refers to the activation of immune cells comprising a combination of Widespread resistance to infectious attack is induced because the immune cells are in the active form and are primed to respond to any compound or microorganism that enters. The cells need not be individually primed against a particular antigen. This is particularly useful in biological warfare and other such situations such as travelers.

Subject As used herein, a subject refers to a human or non-human vertebrate. Non-human vertebrates include livestock animals, pets and laboratory animals. Non-human subjects specifically include non-human primates and rodents. Non-human subjects specifically include, without limitation, chickens, horses, cows, pigs, goats, dogs, cats, guinea pigs, hamsters, minks and rabbits.

  As used herein, a subject at risk of developing a condition is a subject that is apparently or suspected of being exposed to an agent known to cause or be associated with the condition, or A subject with a known or suspected predisposition to develop the condition (eg, a genetic marker for the condition or a family of the condition).

  In one aspect, the invention provides a method of treating a subject with immune system deficiency. The method according to this aspect of the invention includes the step of administering to the subject an effective amount of the composition of the invention to treat the subject. As used herein, immune system deficiency refers to an abnormal decline in the ability of the immune system to initiate an immune response to an antigen. In one embodiment, the immune system deficiency is that the subject's immune system is not functioning with normal adaptive capacity, or enhances the subject's immune response, for example, to remove the subject's tumor or cancer, or infection It is a disease or disorder that would be useful. As used herein, an immunodeficient subject refers to a subject that exhibits a reduced ability of the subject's immune system to initiate, for example, an immune response to an antigen. Subjects with immune system deficiencies include subjects with acquired immune system deficiencies as well as subjects with innate immune system deficiencies. Subjects with acquired immune system deficiencies include, but are not limited to, subjects with chronic inflammatory conditions, subjects with chronic kidney failure or renal failure, subjects with infections, subjects with cancer, subjects with immunosuppressive drugs, other Includes immunosuppressed subjects and malnourished subjects. In one embodiment, the subject has a suppressed CD4 + T cell population. In one embodiment, the subject has a human immunodeficiency virus (HIV) infection or has acquired immune deficiency syndrome (AIDS). Accordingly, the method according to this aspect of the invention provides a method of enhancing an immune response or increasing the ability to initiate an immune response in a subject in need of a more active immune response.

  As used herein, inhibiting shall mean reducing the result or effect compared to normal.

  As used herein, treatment in use with respect to a disease or condition refers to preventing or delaying the onset of the disease or condition, preventing or delaying its progression, stopping its progression, or eliminating it. As such, it is meant to intervene in such a disease or condition. For example, treatment of cancer includes prevention of cancer development (eg, from a precancerous condition), reduction of cancer symptoms, and / or inhibition of growth of established cancer.

Disease states The present invention contemplates treatment of disease states where suppression and / or induction of certain TLR-mediated immune responses may be beneficial. As used herein, a condition associated with TLR7-mediated immunostimulation or immune response is any disease in a subject in which immune activation occurs concomitantly with TLR7 signaling and such activation is detrimental. Refers to a disease or other medical condition. Such pathologies usually involve activation of TLR7 signaling in response to contact with a TLR7 ligand.

  As used herein, a condition associated with TLR8-mediated immunostimulation or immune response is any disease in a subject in which immune activation occurs concomitant with TLR8 signaling and such activation is detrimental. Refers to a disease or other medical condition. Such pathologies usually involve activation of TLR8 signaling in response to contact with a TLR8 ligand.

  As used herein, a condition associated with TLR9-mediated immunostimulation or an immune response is any disease in a subject in which immune activation occurs concomitant with TLR9 signaling and such activation is detrimental. Refers to a disease or other medical condition. Such pathologies usually involve activation of TLR9 signaling in response to contact with a TLR9 ligand.

Infectious Diseases The present invention can be used to treat medical conditions such as infectious diseases. Infectious disease refers to any condition in which there is an abnormal collection or population of living microorganisms extracellular or intracellular in a subject. Such microorganisms may be endogenous to the subject or exogenous to the subject. A subject with an infectious disease is a subject who has a disorder that occurs superficially, locally or systemically upon entry of an infectious microorganism into the subject, or an upregulated overgrowth of endogenous microorganisms naturally occurring in the subject It is. Infectious microorganisms may be viruses, bacteria, fungi or parasites as described above. Various microorganisms can cause infection, including microorganisms that are bacteria, microorganisms that are viruses, microorganisms that are fungi, and microorganisms that are parasites.

  Bacteria are unicellular organisms that grow asexually by bisection. Bacteria are classified and named based on morphology, staining reaction, nutrient and metabolic requirements, antigenic structure, chemical composition, and genetic homology. Bacteria can be classified into three groups, spheres (cocci), straight bars (gonococci), and curved or spiral bars (vibrio, campylobacter, spirillum and spirochete) based on morphological shape. Bacteria are also more commonly characterized into two groups of microorganisms, gram positive and gram negative, based on the staining reaction. Gram refers to a staining method commonly performed in a microbiology laboratory. Gram-positive microorganisms retain staining after the staining operation and appear dark purple. Gram-negative microorganisms do not retain staining and take up counterstaining and thus appear pink.

  Infectious bacteria include, but are not limited to, gram positive and gram negative bacteria. Gram positive bacteria include, but are not limited to, Pasteurella species, Staphylococcus species, Streptococcus species, and the like. Gram negative bacteria include, but are not limited to, E. coli, Pseudomonas species, Salmonella species, and the like. Specific examples of infectious bacteria include, but are not limited to, Helicobacter pylori, Borrelia burgdorferi, Legionella pneumophila, mycobacterial species (eg, Mycobacterium tuberculosis, Avian tuberculosis, M. intracellulare, S. aureus, Neisseria gonorrhoeae, Neisseria meningitidis, Listeria monocytogenes, Streptococcus pyogenes (Group A streptococci), Streptococcus agalactier (Group B streptococci), Streptococcus (Vilidans group) ), Streptococcus faecalis, Streptococcus bovis, Streptococcus (anaerobic species), Streptococcus pneumoniae, Pathogenic Campylobacter species, Enterococcus species, Haemophilus influenzae, Bacillus anthracis, Diphtheria, Corynebacterium species, Swine bacilli, Welch , Tetanus, Enterobacter aerogenes, Neisseria pneumoniae, Pasteurella Rutoshida, Bacteroides species, Fusobacterium Nukureatsumu, Streptomyces Bacillus Moniriforumisu, Treponema pallidum, Furanpejia-Treponema, Leptospira, Rickettsia, and the like Israel actinomycetes.

  Examples of bacteria include, but are not limited to, Pasteurella species, Staphylococcus species, Streptococcus species, Escherichia coli, Pseudomonas species, Salmonella species, and the like. Specific examples of infectious bacteria include, but are not limited to, Helicobacter pylori, Borrelia burgdorferi, Legionella pneumophila, mycobacterial species (eg, Mycobacterium tuberculosis, Avian tuberculosis, M. intracellulare, S. aureus, Neisseria gonorrhoeae, Neisseria meningitidis, Listeria monocytogenes, Streptococcus pyogenes (Group A streptococci), Streptococcus agalactier (Group B streptococci), Streptococcus (Vilidans group) ), Streptococcus faecalis, Streptococcus bovis, Streptococcus (anaerobic species), Streptococcus pneumoniae, Pathogenic Campylobacter species, Enterococcus species, Haemophilus influenzae, Bacillus anthracis, Diphtheria, Corynebacterium species, Swine bacilli, Welch , Tetanus, Enterobacter aerogenes, Neisseria pneumoniae, Pasteurella Rutoshida, Bacteroides species, Fusobacterium Nukureatsumu, Streptomyces Bacillus Moniriforumisu, Treponema pallidum, Furanpejia-Treponema, Leptospira, Rickettsia, and the like Israel actinomycetes.

  Viruses are small infectious agents that generally contain a nucleic acid core and a protein membrane, but are not independent organisms. Viruses can also take the form of infectious nucleic acids lacking protein. Viruses cannot survive in the absence of live cells capable of replicating therein. Viruses invade certain living cells by endocytosis or direct injection of DNA (phage), proliferate, and cause disease. The propagated virus is then released and can infect additional cells. Some viruses are DNA-containing viruses and others are RNA-containing viruses. In some embodiments, the present invention involves prions in disease progression, such as bovine spongiform encephalopathy (ie, mad cow disease, BSE), animal scrapie infection, or human Creutzfeldt-Jakob disease. It is also intended for the treatment of diseases.

  Viruses include, but are not limited to, enteroviruses (including but not limited to picornaviridae viruses such as poliovirus, coxsackie virus, echovirus), rotaviruses, adenoviruses, and hepatitis viruses. Specific examples of viruses found in humans include, but are not limited to, retroviridae (eg, HIV-1 (also referred to as HTLV-III, LAV or HTLV-III / LAV) or HIV-III, and HIV-LP). Human immunodeficiency virus such as other isolates), Picornaviridae (eg, poliovirus, hepatitis A virus, enterovirus, human coxsackie virus, rhinovirus, echovirus), calciviridae (eg cause gastroenteritis) Strain), Togaviridae (eg, equine encephalitis virus, rubella virus), Flaviviridae (eg, dengue virus, encephalitis virus, yellow fever virus), Coronaviridae (eg, coronavirus), Rhabdoviridae (eg, Bullous stomatitis virus , Rabies virus), filoviridae (eg Ebola virus), paramyxoviridae (eg parainfluenza virus, mumps virus, measles virus, respiratory syncytial virus), orthomyxoviridae (eg influenza virus), Bunyaviridae (eg, Hantavirus, Bunyavirus, Frevovirus and Nairovirus), Arenaviridae (eg, hemorrhagic fever virus), Reoviridae (eg, reovirus, Orbivirus and rotavirus), Birnaviridae Hepadnaviridae (eg, hepatitis B virus), Parvoviridae (eg parvovirus), Papovaviridae (papillomavirus, polyomavirus), Adenoviridae (most adenovirus), Rupesviridae (type 1 and type 2 herpes simplex virus (HSV), chicken pox virus, cytomegalovirus (CMV)), poxviridae (pox virus, vaccinia virus, poxvirus), iridoviridae (eg, African swine) Cholera virus), as well as unclassified viruses (eg, spongiform encephalopathy pathogens, hepatitis delta pathogens (considered as defective satellites of hepatitis B virus), non-A non-B hepatitis pathogens (class 1 = in vivo) Transmission, class 2 = parenteral transmission (ie, hepatitis C)), Norwalk and related viruses, astrovirus).

  Examples of viruses found in humans include, but are not limited to, retroviridae (such as HIV-1 (also referred to as HTLV-III, LAV or HTLV-III / LAV) or HIV-III, and HIV-LP). Human immunodeficiency virus such as other isolates), Picornaviridae (eg, poliovirus, hepatitis A virus, enterovirus, human coxsackie virus, rhinovirus, echovirus), calciviridae (eg, strain causing gastroenteritis) ), Togaviridae (eg equine encephalitis virus, rubella virus), Flaviviridae (eg dengue virus, encephalitis virus, yellow fever virus), Coronaviridae (eg coronavirus), Rhabdoviridae (eg blister) Sexual stomatitis virus, Canine virus), Filoviridae (eg, Ebola virus), Paramyxoviridae (eg, parainfluenza virus, mumps virus, measles virus, respiratory syncytial virus), Orthomyxoviridae (eg, influenza virus), Bunyaviridae (eg, Hantavirus, Bungavirus, Frevovirus and Nairovirus), Arenaviridae (eg, hemorrhagic fever virus), Reoviridae (eg, Reovirus, Orbivirus and Rotavirus), Bornaviridae, Hepadnaviridae (eg, hepatitis B virus), Parvoviridae (eg, parvovirus), Papovaviridae (papillomavirus, polyomavirus), Adenoviridae (most adenovirus), Hell Sviridae (type 1 and type 2 herpes simplex virus (HSV), varicella virus, cytomegalovirus (CMV), herpes virus), poxviridae (smallpox virus, vaccinia virus, poxvirus), and iridoviridae ( For example, African swine fever virus), as well as unclassified viruses such as hepatitis delta (considered as a defective satellite for hepatitis B virus), hepatitis C pathogens, Norwalk and related viruses, astrovirus) It is.

  Fungi are eukaryotes, only a small part of which cause infections in vertebrate mammals. Since fungi are eukaryotes, they differ considerably from prokaryotic bacteria in size, structural organization, life cycle and growth mechanism. Fungi are generally classified based on morphological characteristics, reproductive system and culture characteristics. Fungi cause various diseases such as respiratory allergy after inhalation of fungal antigens, fungal poisoning due to ingestion of toxic substances such as amanta phalloides toxin and farotoxin produced by poisonous mushrooms, aflatoxin produced by Aspergillus species There is a fear, but not all fungi cause infection.

  Infectious fungi can cause systemic or superficial infections. Primary systemic infections can occur in normal healthy subjects, and opportunistic infections are most frequently found in subjects with immune disorders. The most common fungal bodies that cause primary systemic infections include the genera Blastmyces, Coccidioides, and Histoplasma. Common fungi that cause opportunistic infections in immune disorders or immunosuppressed subjects include, but are not limited to, Candida albicans, Cryptococcus neoformans, and various Aspergillus species. A systemic fungal infection is an invasive infection of the viscera. The organism usually enters the body through the lungs, gastrointestinal tract or venous catheter. These types of infections can be caused by primary pathogenic fungi or opportunistic fungi.

  Superficial fungal infections involve the growth of fungi on the outer surface without invading internal tissues. Common superficial fungal infections include skin fungal infections involving the skin, hair or nails.

  Diseases associated with fungal infections include aspergillosis, blastomiasis, candidiasis, chromoblastosis, coccidioidomycosis, cryptococcosis, fungal infections of the eyes, hair, nails and skin, histoplasmosis, robotics Examples include mycosis, foot mycosis, otomycosis, paracoccidioidomycosis, disseminated penicillium marnefei, pheohyfomycosis, linospodium disease, sporotrichosis, and zygomycosis.

  Fungi include yeast and mold. Examples of fungi include, but are not limited to, Aspergillus fumigatus, Blastmyces dermatitis, Candida species including Candida albicans, Coccidioides imimitis, Cryptococcus neoformans, Histoplasma capsulatum, Pneumocystis carini, Examples include Rhizomucor species and Kumonosukabi species.

  A parasite is an organism that depends on other organisms to survive and therefore must invade or infect another organism to continue its life cycle. The infected organism, or host, provides both nutrients and habitat for the parasite. The term parasite encompasses, in the broadest sense, all infectious agents (i.e. bacteria, viruses, fungi, protozoa and helminths), but generally speaking the term includes protozoa, helminths and ectoparasite arthropods. Used only to refer to animals (eg, ticks, mites, etc.). Protozoa are unicellular organisms that can replicate both intracellularly and extracellularly, particularly in the blood, gastrointestinal tract, or extracellular matrix of tissues. Helminths are multicellular organisms that are almost always extracellular (the exception is Trichinella species). Helminths usually need to be excreted from the primary host and transmitted to the secondary host in order to replicate. In contrast to these classes described above, ectoparasitic arthropods form a parasitic relationship with the outer surface of the host organism.

  Parasites include intracellular parasites and obligate intracellular parasites. Examples of parasites include, but are not limited to, Plasmodium falciparum, oval malaria parasites, Plasmodium falciparum, Plasmodium falciparum, Plasmodium nouresi, Babesia microti, Babesia divergens, Cruz. Trypanosoma, Toxoplasma gondii, Trichinella spiralis, Forest type tropical Leishmania, Donovan Leishmania, Brazilian Leishmania, Tropical Leishmania, Gambia trypanosoma, Rhodesia trypanosoma .

  Other infectious organisms (ie, protists) include Plasmodium species such as Plasmodium falciparum, Plasmodium falciparum, Oval malaria parasite, Plasmodium falciparum, and Toxoplasma. Blood infectious and / or tissue parasites include Plasmodium species, Babesia microti, Babesia divergens, Trachoma chlamydia, Tropical leishmania, Leishmania, Brazil leishmania, Donovan leishmania, Gambia Trypanosoma and Rhodesia trypanosoma (African sleeping sickness), Cruise trypanosoma (Chagas disease), Toxoplasma and the like.

  Other medically relevant microorganisms have been extensively described in the literature, such as C.I., which is incorporated herein by reference in its entirety. G. See A Thomas, Medical Microbiology, Bailier Tindall, Great Britain 1983.

Cancer In one aspect, the invention provides a method of treating a subject having cancer.

  A subject with cancer is a subject with detectable cancer cells. The cancer may be malignant or non-malignant. “Cancer” as used herein refers to the uncontrolled growth of cells that interferes with the normal functioning of internal organs and systems. Cancer that migrates from the site of development and disseminates to critical organs can ultimately cause the subject to die due to reduced function of the affected organs. Hematopoietic cancer, such as leukemia, can overwhelm the normal hematopoietic compartment in a subject, thereby causing hematopoietic failure (in the form of anemia, thrombocytopenia, and neutropenia) and ultimately death. it can.

  A metastasis | transition part is a cancer cell area | region different from the primary tumor site | part which arises by seed | inoculation of the cancer cell from a primary tumor to another body part. Upon diagnosis of the primary tumor mass, the subject may be monitored for the presence of metastases. In addition to monitoring specific symptoms, metastatic sites may be detected by magnetic resonance imaging (MRI) scans, computed tomography (CT) scans, blood and platelet counts, liver function tests, chest x-rays, and bone scans, either alone or in combination. , Most often detected.

  Cancers include, but are not limited to, basal cell cancer, bile duct cancer, bladder cancer, bone cancer, brain and central nervous system (CNS) cancer, breast cancer, cervical cancer, choriocarcinoma, colorectal cancer, connective tissue cancer, Digestive system cancer, endometrial cancer, esophageal cancer, eye cancer, head and neck cancer, intraepithelial tumor, renal cancer, laryngeal cancer, leukemia, liver cancer, lung cancer (eg, small and non-small cells), Hodgkin And lymphoma including non-Hodgkin lymphoma, melanoma, myeloma, neuroblastoma, oral cancer (eg, lip, tongue, mouth, throat), ovarian cancer, pancreatic cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma, Examples include rectal cancer, respiratory cancer, sarcoma, skin cancer, gastric cancer, testicular cancer, thyroid cancer, uterine cancer, urinary cancer, and other carcinomas, adenocarcinoma, and sarcoma.

Autoimmune pathologies Conditions associated with innate or Th1-like immune responses include inflammation, acute and chronic allograft rejection, graft-versus-host disease (GvHD), certain autoimmune diseases, sepsis and the like. The present invention can be used for the treatment of such medical conditions in view of the selective suppression of TLR signaling that can be realized by the present invention.

  Autoimmune diseases can be generally classified as antibody mediated, T cell mediated, or a combination of antibody and T cell mediated. The adapter ODN and TLR ligand combinations of the present invention involve antibody-mediated or T-cell mediated immunity and include insulin-dependent (type I) diabetes, rheumatoid arthritis, multiple sclerosis, systemic lupus erythematosus (SLE), And is considered useful in the treatment of a wide variety of autoimmune diseases, including inflammatory bowel disease (ie, Crohn's disease and ulcerative colitis). Animal models for such autoimmune diseases are available and are useful for assessing the efficacy of the combinations of the invention in such diseases. Other autoimmune diseases include, but are not limited to, alopecia areata, acquired hemophilia, ankylosing spondylitis, antiphospholipid syndrome, autoimmune hepatitis, autoimmune hemolytic anemia, Behcet's syndrome, myocardium , Celiac disease dermatitis, chronic fatigue immune dysfunction syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy, Churg-Strauss syndrome, scar pemphigoid, CREST syndrome, cold agglutinin disease, discoid lupus , Essential mixed cryoglobulinemia, fibromyalgia, fibromyositis, Guillain-Barre syndrome, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura, IgA nephropathy, juvenile arthritis, lichen planus, myasthenia gravis Disease, nodular polyarteritis, polychondritis, polyendocrine syndrome, dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, Raynaud's phenomenon, lighter Syndrome, sarcoidosis, stiff-man syndrome, Takayasu arthritis, temporal arteritis / giant cell arteritis, uveitis, vasculitis and vitiligo.

  In some autoimmune diseases, antibodies against self antigens are often found. For example, for systemic lupus erythematosus, autoantibodies have been described against single and double stranded DNA or RNA. Vallin H et al. (1999) J Immunol 163: 6306-13; Hoet RM et al. (1999) J Immunol 163: 3304-12; ven Venrooij (1990) J Clin Invest 86: 2154-60. The amount of autoantibodies found in the serum of autoimmune patients has been found to correlate very often with the severity of the disease. For example, the autoantibody pattern that occurs in human SLE suggests that intact polymer particles, such as RNA-containing or DNA-containing complexes, can themselves be immunogenic and thus can generate anti-nucleic acid antibodies. Lotz M et al. (1992) Mol Biol Rep 16: 127; Mohan C et al. (1993) J Exp Med 177: 1367-81. For example, such DNA or RNA released from apoptotic cells, or DNA-containing or RNA-containing microorganisms present in the serum of autoimmune patients can cause inflammation that promotes autoimmune diseases. Fatenejad S (1994) J Immunol 152: 5523-31; Malmegrim KC et al. (2002) Isr Med Assoc J 4: 706-12; Newkirk MM et al. (2001) Arthritis Res 3: 253-8. Indeed, CpG-containing sequences could be identified from SLE sera eliciting an efficient immune response dominated by IFN-α secretion, which is thought to facilitate the development of autoimmune diseases. Magnusson M et al. (2001) Scand J Immunol 54: 543-50; Ronnblom L et al. (2001) J Exp Med 194: F59-63. In addition, epitopes for anti-RNA antibodies have been identified that are composed of G, U-rich sequences. Tsai DE et al. (1992) Proc Natl Acad Sci USA 89: 8864-8; Tsai DE et al. (1993) J Immunol 150: 1137-45.

Allergy “Allergic condition” or “allergy” refers to acquired hypersensitivity to a substance (allergen). “Subject having an allergic condition” shall refer to a subject who is currently experiencing or previously experienced an allergic reaction in response to an allergen. Allergic conditions include, but are not limited to, eczema, allergic rhinitis or coryza, hay fever, allergic conjunctivitis, bronchial asthma, urticaria (urticaria) and food allergies, atopic dermatitis Anaphylaxis, drug allergy, angioedema and the like.

  Allergy is a seizure pathology that usually accompanies the production of antibodies from IgE, a specific class of immunoglobulins against allergens. The development of an IgE-mediated response to common air allergens is also a factor that indicates a trend towards the development of asthma. When an allergen contacts specific IgE bound to an IgE Fc receptor (FcεR) on the surface of basophils (circulating in the blood) or mast cells (dispersed throughout the solid tissue), The cells are activated and produce and release mediators such as histamine, serotonin, lipid mediators.

  Allergic reactions occur when IgE-type tissue sensitizing immunoglobulins react with foreign allergens. The IgE antibody binds to mast cells and / or basophils, and these special cells, when stimulated and stimulated by an allergen bridging the end of the antibody molecule, are chemical mediators of allergic reactions. Releases (vasoactive amines). Histamine, platelet activating factor, arachidonic acid metabolites, and serotonin are among the best known mediators of allergic reactions in humans. Histamine and other vasoactive amines are normally stored in mast cells and basophils. Mast cells are dispersed throughout the animal tissue, and basophils circulate in the vasculature. These cells produce and store histamine within the cell unless that particular series of events with IgE binding occurs to trigger the release of histamine.

  The symptoms of an allergic reaction vary depending on the site in the body where IgE reacts with the allergen. If the reaction occurs along the respiratory epithelium, the symptoms are generally sneezing, coughing and asthmatic reactions. Abdominal pain and diarrhea are common if the interaction occurs in the digestive tract as in the case of food allergies. For example, systemic allergic reactions after bee sting and administration of penicillin to allergic subjects can be severe and often life-threatening.

  Allergy is associated at least in part with a Th2-type immune response characterized by the Th2 cytokines IL-4 and IL-5, and the switch of antibody isotypes to IgE. Since the Th1 and Th2 immune responses are retrogradely regulated with respect to each other, when the immune response is inclined toward the Th1-type immune response, the Th2-type immune response including allergies can be prevented or improved.

Asthma “Asthma” as used herein refers to a disorder of the respiratory system characterized by inflammation and stenosis of the airways and increased airway responsiveness to inhaled agents. Asthma is often, but not necessarily, associated with an atopic or allergic condition. Symptoms of asthma include wheezing resulting from airway obstruction, apnea, chest pain, and recurrent seizures of cough. Airway inflammation associated with asthma is associated with a number of physiology, including airway epithelial exposure, collagen deposition under the basement membrane, edema, mast cell activation, inflammatory cell infiltration including neutrophils, eosinophils and lymphocytes It can be detected by observing changes in the environment. As a result of airway inflammation, asthmatics often experience airway hyperresponsiveness, restricted airflow, respiratory problems, and disease chronification. Airflow limitation includes airway remodeling, a feature that causes acute bronchoconstriction, airway edema, mucus plug formation, and often airway obstruction. In some asthma cases, subbasement fibrosis may occur, resulting in persistent abnormal lung function.

  Studies over the last few years have shown that asthma appears to result from complex interactions between inflammatory cells, mediators, and other cells and tissues that are resident in the respiratory tract. Mast cells, eosinophils, epithelial cells, macrophages and activated T cells all play an important role in the inflammatory process associated with asthma. Djukanovic R et al. (1990) Am Rev Respir Dis 142: 434-457. It is believed that these cells can affect airway function through secretion of preformed and newly synthesized mediators that can act directly or indirectly on local tissues. It was also recognized that a subpopulation of T lymphocytes (Th2) plays an important role in regulating allergic inflammation in the respiratory tract by establishing selective cytokine release and disease chronogenesis. Robinson DS et al. (1992) N Engl J Med 326: 298-304.

  Asthma is a complex disorder that occurs at various stages of onset and can be classified as acute, subacute or chronic based on the degree of symptoms. An acute inflammatory response is associated with the initial recruitment of cells into the respiratory tract. The subacute inflammatory response involves cell mobilization as well as resident cell activation, resulting in a more sustained inflammatory pattern. Chronic inflammatory responses are characterized by sustained levels of cell damage and ongoing repair processes that can cause permanent abnormalities in the airways.

A “subject with asthma” is a subject with a respiratory disorder characterized by inflammation and stenosis of the respiratory tract and increased airway responsiveness to inhaled agents. Factors associated with initiation of asthma include, but are not limited to, allergens, cold, exercise, and viral infections, and SO _2.

  As described herein, asthma may be associated at least in part with a Th2-type immune response characterized by the Th2 cytokines IL-4 and IL-5, and the switch of antibody isotypes to IgE. Since the Th1 and Th2 immune responses are retrogradely regulated with respect to each other, when the immune response is inclined toward the Th1-type immune response, the Th2-type immune response including allergies can be prevented or improved.

  The combination of a TLR7 / 8 ligand and adapter oligonucleotide of the present invention is also useful for improving dendritic cell survival, differentiation, activation and maturation.

  In certain aspects, the present invention provides a method of enhancing epitope spreading. As used herein, “epitope diffusion” refers to an initial focal dominant epitope-specific immune response against a self or foreign protein from that protein (intramolecular diffusion) or other proteins ( Refers to diversification of epitope specificity to non-dominant and / or cryptic epitopes on intermolecular diffusion). Epitope spreading results in multiple epitope-specific immune responses.

Antimicrobial Agents The combination of TLR7 / 8 ligand and adapter oligonucleotide can be used with one or more antimicrobial agents. Antimicrobial agents or such agents include, but are not limited to, antibacterial agents, antiviral agents, antifungal agents, and antiparasitic agents. Phrases such as “anti-infective”, “antibiotic”, “anti-bacterial”, “anti-viral”, “anti-fungal”, “anti-parasitic”, “parasitic” etc. Has a well-established meaning and is defined in standard medical textbooks. Briefly, antibacterial agents kill or inhibit bacteria and include antibiotics as well as other synthetic or natural compounds that have similar functions. Antiviral agents can be isolated or synthesized from natural sources and are useful for killing or inhibiting viruses. Antifungal agents are used to treat superficial fungal infections, as well as opportunistic and primary systemic fungal infections. Antiparasitic agents kill or inhibit parasites. Many antibiotics are low molecular weight molecules produced as secondary metabolites by cells such as microorganisms. In general, antibiotics interfere with one or more functions or structures that are unique to the microorganism and that are not present in the host cell.

  One problem with anti-infective drugs is the side effects that occur in hosts that are treated with anti-infective agents. For example, many anti-infective agents can kill or inhibit a wide range of microorganisms and are not specific for a particular type of species. Treatment with these types of anti-infectives kills not only infectious microorganisms, but also the normal microflora that live in the host. Since the microbiota competes with and acts as a barrier to infectious pathogens, loss of the microbiota can cause complications and make the host susceptible to infection by other pathogens. Other side effects may occur as a result of the specific or non-specific action of these chemicals on non-microbial host cells or tissues.

  Another problem with widespread use of anti-infective agents is the development of antibiotic resistant strains of microorganisms. Vancomycin-resistant enterococci, penicillin-resistant pneumococci, multi-drug resistant Staphylococcus aureus and multi-drug resistant tuberculosis strains have already been developed and are becoming a major clinical problem. The widespread use of anti-infectives will likely produce many antibiotic-resistant bacterial strains. As a result, new anti-infective strategies will be needed to combat these microorganisms.

  Antibacterial antibiotics that are effective in killing or inhibiting a wide range of bacteria are termed broad spectrum antibiotics. Other types of antibacterial antibiotics are mainly effective against gram positive or gram negative group bacteria. These types of antibiotics are called narrow-range antibiotics. Other antibiotics that are effective against a single species of microorganism or disease and ineffective against other species of bacteria are termed limited antibiotics.

  Antibacterial agents are sometimes classified based on their primary mode of action. In general, antibacterial agents are cell wall synthesis inhibitors, cell membrane inhibitors, protein synthesis inhibitors, nucleic acid synthesis or function inhibitors, and competitive inhibitors. Cell wall synthesis inhibitors inhibit the process of cell wall synthesis, generally the step in the synthesis of bacterial peptidoglycan. Cell wall synthesis inhibitors include β-lactam antibiotics, natural penicillin, semi-synthetic penicillin, ampicillin, clavulanic acid, cephalosporin, bacitracin and the like.

  β-lactams are antibiotics that contain a four-membered β-lactam ring that inhibits the final step of peptidoglycan synthesis. β-lactam antibiotics can be synthesized or even natural. The β-lactam antibiotics produced by penicillium are natural penicillins such as penicillin G and penicillin V. These are produced by the fermentation of Penicillium chrysogenum. The natural penicillin has narrow activity and is generally effective against streptococci, bacilli and staphylococci. Other types of natural penicillins that are also effective against gram positive bacteria include penicillins F, X, K, and O.

  Semi-synthetic penicillin is generally a modified form of the molecule 6-aminopenicillanic acid produced by mold. 6-Aminopenicillanic acid can be modified by the addition of side chains that produce penicillins having a broader range of activity or other various advantageous properties than natural penicillins. Some semi-synthetic penicillins have broad activity against gram positive and gram negative bacteria but are inactivated by penicillinase. Such semi-synthetic penicillins include ampicillin, carbenicillin, oxacillin, azurocillin, mezlocillin, piperacillin and the like. Other types of semi-synthetic penicillins have a narrower activity against Gram-positive bacteria, but express the property that they are not inactivated by penicillinase. These include, for example, methicillin, dicloxacillin, nafcillin and the like. Some of the broad-range semi-synthetic penicillins can be used in combination with β-lactamase inhibitors such as clavulanic acid and sulbactam. β-lactamase inhibitors do not have antimicrobial activity but function to inhibit penicillinase and thus protect semisynthetic penicillin from degradation.

  Another type of β-lactam antibiotic is that cephalosporin. This class is sensitive to degradation by bacterial β-lactamases and is therefore not necessarily effective alone. However, cephalosporin is resistant to penicillinase. This class is effective against a variety of gram positive and gram negative bacteria. Cephalosporins include, but are not limited to, cephalotin, cefapirin, cephalexin, cefamandol, cefaclor, cefazolin, cefuroxykin, cefoxitin, cefotaxime, cefrosin, cefetamet, cefixime, ceftriaxone, cefoperazone, ceftazidine, etc. It is done.

  Bacitracin is another class of antibiotics that inhibit cell wall synthesis by inhibiting the release of subunits or peptidoglycans from molecules that send out muropeptide subunits outside the membrane. Although bacitracin is effective against gram-positive bacteria, its use is generally limited to topical administration due to its high toxicity.

  Carbapenem is another broad-band β-lactam antibiotic that can inhibit cell wall synthesis. Examples of carbapenem include, but are not limited to, imipenem. Monobactam is also a broad-spectrum β-lactam antibiotic and includes usetreonam. The antibiotic vancomycin produced by Streptomyces is also effective against gram-positive bacteria by inhibiting cell membrane synthesis.

  Another class of antibacterial agents are cell membrane inhibitor antibacterial agents. Such compounds disrupt the structure of the bacterial membrane or inhibit its function. One problem with cell membrane inhibitors, antibacterial agents, is that the phospholipids of bacterial and eukaryotic cell membranes are similar and can affect not only bacteria but also eukaryotic cells. Thus, such compounds are rarely specific enough to allow them to be used systemically and prevent the use of high doses for local administration.

  A clinically useful single cell membrane inhibitor is polymyxin. Polymyxins interfere with membrane function by binding to membrane phospholipids. Polymyxin is primarily effective against gram-negative bacteria and is generally used for severe Pseudomonas infections or Pseudomonas infections that are resistant to less toxic antibiotics. Severe side effects associated with systemic administration of this compound include damage to the kidneys and other organs.

  Other cell membrane inhibitors include amphotericin B and nystatin, which are antifungal agents used primarily for the treatment of systemic fungal infections and Candida yeast infections. Imidazole is another class of antibiotics that are cell membrane inhibitors. Imidazole is used as an antibacterial and antifungal agent and is used, for example, in the treatment of yeast infections, dermatophyte infections, and systemic fungal infections. Imidazole includes, but is not limited to, clotrimazole, miconazole, ketoconazole, itraconazole, fluconazole and the like.

  Many antibacterial agents are protein synthesis inhibitors. Such compounds prevent the synthesis of bacterial structural proteins and enzymes, thus causing bacterial cell growth or inhibition of function or cell death. In general, such compounds interfere with the transcription or translation process. Antibacterial agents that block transcription include, but are not limited to, rifampin and ethambutol. Rifampin, which inhibits the RNA polymerase enzyme, has a broad spectrum activity and is effective against Gram-positive and Gram-negative bacteria, and Mycobacterium tuberculosis. Ethambutol is effective against Mycobacterium tuberculosis.

  Antibacterial agents that block translation prevent translation of mRNA into protein by interfering with bacterial ribosomes. In general, this class of compounds includes, but is not limited to, tetracycline, chloramphenicol, macrolides (eg, erythromycin), aminoglycosides (eg, streptomycin), and the like.

  Aminoglycosides are produced by Streptomyces bacteria and are a class of antibiotics such as streptomycin, kanamycin, tobramycin, amikacin, gentamicin and the like. Aminoglycosides have been used against a wide range of bacterial infections caused by gram positive and gram negative bacteria. Streptomycin has been widely used as a primary drug in the treatment of tuberculosis. Gentamicin is used in particular in combination with tobramycin for many strains of gram positive and gram negative bacteria, including Pseudomonas infections. Kanamycin is used against many gram positive bacteria, including penicillin resistant staphylococci. One side effect of aminoglycosides that has clinically limited their use is that prolonged use at doses essential for efficacy has been shown to damage renal function and cause hearing nerve damage resulting in hearing loss. .

  Another type of translation-inhibiting antibacterial agent is tetracycline. Tetracyclines are a broad class and a class of antibiotics that are effective against a variety of gram positive and gram negative bacteria. Examples of tetracyclines include tetracycline, minocycline, doxycycline, chlortetracycline and the like. Although this class is important for the treatment of many types of bacteria, it is particularly important for the treatment of Lyme disease. Tetracyclines have been a problem because they have been abused and misused by the medical community due to their low toxicity and few direct side effects. For example, a wide range of resistance has been developed due to its abuse.

  Antibacterial agents, such as macrolides, reversibly bind to the 50S ribosomal subunit, inhibit protein elongation by peptidyl transferase, prevent release of empty tRNA from bacterial ribosomes, or both. Such compounds include erythromycin, roxithromycin, clarithromycin, oleandromycin, azithromycin and the like. Erythromycin is active against most Gram-positive bacteria, Neisseria, Legionella and Haemophilus, but is inactive against the Enterobacteriaceae family. Lincomycin and clindamycin, which block peptide bond formation during protein synthesis, are used against gram positive bacteria.

  Another type of translation inhibitor is chloramphenicol. Chloramphenicol binds to the 70S ribosome and inhibits the bacterial enzyme peptidyltransferase, thereby preventing the growth of polypeptide chains during protein synthesis. One severe side effect associated with chloramphenicol is aplastic anemia. Aplastic anemia develops in a small proportion (1/50000) of patients with a dose of chloramphenicol effective in treating bacteria. Chloramphenicol, once a well-prescribed antibiotic, is rarely used today as a result of death from aplastic anemia. Because of its effectiveness, it is still used in life-threatening situations (eg typhoid).

  Some antibacterial agents disrupt nucleic acid synthesis or function, eg, bind to DNA or RNA so that the message cannot be read. These include, but are not limited to, both the synthetic drugs quinolone and cotrimoxazole, and the natural or semi-synthetic drug rifamycin. Quinolone blocks bacterial DNA replication by inhibiting DNA gyrase, an enzyme that bacteria need to synthesize circular DNA. Such are widespread and examples include norfloxacin, ciprofloxacin, enoxacin, nalidixic acid, temafloxacin and the like. Nalidixic acid is an antibacterial agent that is essential for DNA replication, binds to DNA gyrase enzyme (topoisomerase) that relaxes and reforms the supercoil, and inhibits DNA gyrase activity. The main uses of nalidixic acid are Escherichia coli, Enterobacter aerogenes, K. et al. It is a treatment for lower UTI because it is effective against several gram-negative bacteria such as Pneumonier and Proteus. Cotrimoxazole is a combination of sulfamethoxazole and trimethoprim and blocks bacterial synthesis of folic acid necessary for the production of DNA nucleotides. Rifampicin is a rifamycin derivative that is active against gram-positive bacteria (M. tuberculosis and meningitis caused by Neisseria meningitidis) and some gram-negative bacteria. Rifampicin binds to the β subunit of the polymerase and prevents the addition of the first nucleotide required for activation of the polymerase, thereby preventing mRNA synthesis.

  Another class of antibacterial agents are compounds that function as competitive inhibitors of bacterial enzymes. Competitive inhibitors are almost all structurally similar to bacterial growth factors and compete for binding but do not exercise metabolic function within the cell. Such compounds include sulfonamides and chemically modified sulfanilamides with increased antibacterial activity and broadening. Sulfonamides (eg, gantricin and trimethoprim) are useful for the treatment of Streptococcus pneumoniae, beta-hemolytic streptococci and E. coli, for the treatment of uncomplicated UTI by E. coli and for the treatment of meningococcal meningitis. Have been used.

  An antiviral agent is a compound that prevents infection of a cell by a virus or replication of the virus in the cell. Antiviral drugs are far more effective than antibacterial drugs because the process of viral replication is so closely related to DNA replication in the host cell that nonspecific antiviral agents are often thought to be toxic to the host. Few. There are several stages in the process of viral infection that an antiviral agent can block or inhibit. These steps include viral binding to the host cell (immunoglobulin or binding peptide), viral unshelling (eg, amantadine), viral mRNA synthesis or translation (eg, interferon), viral RNA or DNA replication ( For example, nucleoside analogs), maturation of new viral proteins (eg, protease inhibitors), and viral budding and release.

  Another type of antiviral agent is a nucleoside analog. A nucleoside analog is a synthetic compound that is similar to a nucleoside but has an incomplete or unusual deoxyribose or ribose group. Once the nucleoside analog enters the cell, it is phosphorylated to produce the triphosphate form, which competes with normal nucleotides for incorporation into the viral DNA or RNA. Once the triphosphate form of the nucleoside analog is incorporated into a growing nucleic acid chain, it undergoes an irreversible bond with the viral polymerase, thus causing chain termination. Nucleoside analogs include, but are not limited to, acyclovir (used to treat herpes simplex and varicella viruses), ganciclovir (useful for the treatment of cytomegalovirus), idoxuridine, ribavirin (respiratory syncytial virus Useful for treatment), dideoxyinosine, dideoxycytosine, zidovudine (azidothymidine) and the like.

  Another class of antiviral agents includes cytokines such as interferons. Interferons are cytokines that are secreted not only by immune cells but also by virus-infected cells. Interferons function by binding to specific receptors on cells adjacent to infected cells and causing changes in the cells that protect themselves from infection by the virus. α and β-interferons induce the expression of class I and class II MHC molecules on the surface of infected cells, increasing the amount of antigen presentation for host immune cell recognition. α and β-interferon are available as recombinant forms and have been used to treat chronic hepatitis B and C infections. At doses effective for antiviral therapy, interferon exhibits severe side effects such as fever, malaise, and weight loss.

  Immunoglobulin therapy is used to prevent viral infections. Immunoglobulin therapy for viral infections binds to extracellular virions rather than antigen-specific and prevents the virions from binding to and entering cells susceptible to viral infection. It is different from bacterial infections because the therapy works. This therapy is useful for the prevention of viral infections for the period in which antibodies are present in the host. In general, there are two types of immunoglobulin therapy, normal immunoglobulin therapy and hyperimmunoglobulin therapy. Normal immunoglobulin therapy utilizes antibody products prepared and pooled from serum of normal blood donors. This pooled product contains low titers of antibodies against a wide range of human viruses, particularly in neonates, such as hepatitis A, parvovirus, enterovirus. Hyperimmunoglobulin therapy utilizes antibodies prepared from an individual's serum that contain high titers of antibodies against specific viruses. The antibody is then used against a specific virus. Examples of hyperimmunoglobulins include herpes zoster immunoglobulin (useful for the prevention of chickenpox in poorly immunized children and newborns), human rabies immunoglobulin (useful for prevention after virus exposure in subjects bitten by rabies animals), Hepatitis B immunoglobulin (useful for the prevention of hepatitis B virus, particularly in subjects exposed to the virus), as well as RSV immunoglobulin (useful for the treatment of respiratory syncytial virus infection).

  Antifungal agents are useful for the treatment and prevention of infectious fungi. Antifungal agents are sometimes classified by their mechanism of action. Some antifungal agents function as cell wall inhibitors by inhibiting glucose synthase. These include, but are not limited to Basiungin / ECB. Other antifungal agents function by destabilizing membrane integrity. These include, but are not limited to, imidazoles such as clotrimazole, sertaconzole, fluconazole, itraconazole, ketoconazole, miconazole, voriconacol, and FK463, amphotericin B, BAY 38-9502, MK991, prazimicin, UK292, butenafine, Examples include terbinafine. Other antifungal agents function by destroying chitin (eg, chitinase) or immunosuppression (501 cream).

  Parasiticides are agents that directly kill parasites. Such compounds are known in the art and are generally commercially available. Examples of parasiticides useful for human administration include, but are not limited to, albendazole, amphotericin B, benznidazole, bitionol, chloroquine HCl, chloroquine phosphate, clindamycin, dehydroemetine, diethylcarba Mazine, diloxanide furoate, eflornithine, furazolidanone, glucocorticoid, halofantrine, iodoquinol, ivermectin, mebendazole, mefloquine, meglumine antimonate, melarsoprolol, metrifonate, metronidazole, niclosamide, niflutimox, oxamuniquine , Paromomycin, pentamidine isethionate, piperazine, praziquantel, primaquine phosphate, proganneal, pyrantel pamoate, pyrimethamine sulfonamide, Rimethamine sulfadoxine, quinacrine HCl, quinine sulfate, quinidine gluconate, spiramycin, sodium stibogluconate (antimony sodium gluconate), suramin, tetracycline, doxycycline, thiabendazole, tinidazole, trimethoprim-sulfamethoxazole, tripalsamide Etc.

Anti-cancer therapy The combination of TLR7 / 8 ligand and adapter oligonucleotide of the present invention can be used with anti-cancer therapy.

  Anti-cancer therapies include cancer drugs, radiation and surgical procedures. As used herein, “cancer medicament” refers to an agent that is administered to a subject to treat cancer. A variety of medicaments for treating cancer are described herein. For purposes of this specification, cancer drugs are classified as chemotherapeutic agents, immunotherapeutic agents, cancer vaccines, hormonal therapeutic agents, and biological response modifiers.

  Chemotherapeutic agents include methotrexate, vincristine, adriamycin, cisplatin, sugar-free chloroethylnitrosourea, 5-fluorouracil, mitomycin C, bleomycin, doxorubicin, dacarbazine, taxol, furazirin, megramin GLA, valrubicin, carmustain and polyferposan, MMI270 , BAY12-9566, RAS farnesyltransferase inhibitor, farnesyltransferase inhibitor, MMP, MTA / LY231514, Alimta, LY264618 / rometexol, Gramorec, CI-994, TNP-470, Hicamtin / Topotecan, PKC412, Valspodartron / PSC8 / PSC Mitroxantrone, Metallet / Suramin, Bachimaster G, E7070, BCH-4556, CS-682, 9-AC, AG3340, AG3433, In-cell / VX-710, VX-853, ZD0101, ISI641, ODN698, TA2516 / Malmistat, BB2516 / Malmistat, CDP845, D2163, PD183805, DX8951f, remonal DP2202, FK317, picibanil / OK-432, AD32 / valrubicin, metastron / strontium derivatives, temodar / temozolomide, ebasset / liposomal doxorubicin, utaxane / paclitaxel, taxol / vinciflutaxel Cyclopax / oral paclitaxel, oral taxoid, SPU-077 / sysp Chin, HMR1275 / flavopiridol, CP-358 (774) / EGFR, CP-609 (754) / RAS oncogene inhibitor, BMS-182751 / oral platinum, UFT (tegafur / uracil), elgamisole / levamisole, eniluracil / 776C85 / 5FU enhancer, campto / levamisole, camptosar / irinotecan, tumodex / lalitrexed, leustatin / cladribine, paxsex / paclitaxel, doxyl / liposomal doxorubicin, caelix / liposomal doxorubicin, fludara / fludarubicin DepoCyt, ZD1839, LU79553 / Bisnaphthalimide, LU103793 / Dolstein, Cathetics / Liposomal doxo Rubicin, gemzar / gemcitabine, ZD0473 / anormed, YM116, iodine seed crystal, CDK4 and CDK2 inhibitor, PARP inhibitor, D4809 / dexifosamide, ifes / mesnex / ifosamide, bumon / teniposid, paraplatin / carboplatin, platinol / cisplatin, / Etoposide, ZD9331, taxotere / docetaxel, guanine arabinoside prodrug, taxane analog, nitrosourea, alkylating agents (melferran, cyclophosphamide, etc.), aminoglutethimide, asparaginase, busulfan, carboplatin, chloroumbucil Cytarabine HCl, dactinomycin, daunorubicin HCl, estramustine sodium phosphate, etoposide (VP16-21) ), Floxuridine, fluorouracil (5-FU), flutamide, hydroxyurea (hydroxycarbamide), ifosfamide, interferon α-2a, α-2b, leuprolide acetate (LHRH releasing factor analogue), lomustine (CCNU), mechlorethamine HCl (Nitrogen mustard), mercaptopurine, mesna, mitotane (o. p'-DDD), mitoxantrone HCl, octreotide, pricamycin, procarbazine HCl, streptozocin, tamoxifen citrate, thioguanine, thiotepa, vinblastine sulfate, amsacrine (m-AMSA), azacitidine, elsulopoietin, hexamethylmelamine ( HMM), interleukin 2, mitoguazone (methyl GAG, methylglyoxal bisguanylhydrazone, MGBG), pentostatin (2′deoxycoformycin), semustine (methyl CCNU), teniposide (VM-26), and vindesine sulfate The group can be selected from, but not limited to.

  The immunotherapeutic agents are 3622W94, 4B5, ANA Ab, anti-FLK-2, anti-VEGF, ATRAGEN, AVASTIN (Bevacizumab, Genentech), BABS, BEC2, BXXAR (Toshitsumabu, GlaxoSmithKline), C225, CAMPZmab, G , CEACID, CMA676, EMD-72000, ERBITUX (Cetuximab, ImClone Systems Inc.), Gliomab-H, GNI-250, HERCEPTIN (Trastuzumab, Genentech), IDEC-Y2B8, ImmuRAit-c, ImmuRAit-C. r3, ior t6, LDP-03, LymphoCide, MDX-11, MDX-22, MDX-210, MDX-220, MDX-260, MDX-447, MELIMMUNE-1, MELIMMUNE-2, Monopharm-C, NovoMAb-G2 , Oncolym, OV103, Ovarex, Panorex, Pretarget, Quadramet, Ributaxin, RITUXAN (Rituximab, Genentech), SMART 1D10 Ab, SMART ABL 364 Ab, TMA MZAP, TMA MZAP Not limited to this.

  Cancer vaccines include EGF, anti-idiotype cancer vaccine, Gp75 antigen, GMK melanoma vaccine, MGV ganglioside conjugate vaccine, Her2 / neu, Ovarex, M-Vax, O-Vax, L-Vax, STn-KHL therapepe, BLP25 ( Selected from the group consisting of MUC-1), liposomal idiotype vaccine, Melacine, peptide antigen vaccine, toxin / antigen vaccine, MVA vaccine, PACIS, BCG vaccine, TA-HPV, TA-CIN, DISC virus and ImmuCyst / TheraCys However, it is not limited to this.

Antiallergic Drug The TLR7 / 8 ligand and adapter oligonucleotide of the present invention can be used together with an antiallergic drug.

  Conventional methods of treating or preventing allergies involve the use of allergy medications and desensitization therapy. Some therapies that are being developed to treat or prevent allergies include the use of anti-IgE antibody neutralization. Antihistamines and other drugs that block the action of chemical mediators of allergic reactions help to control the severity of allergic symptoms, but do not prevent allergic reactions and have no effect on subsequent allergic responses. Desensitization therapy is performed by administering small doses of allergen, usually subcutaneously, to elicit an IgG type response to the allergen. The presence of IgG antibodies is believed to help neutralize mediator production resulting from the induction of IgE antibodies. To avoid severe induction of response, the subject is initially treated with a very small dose of allergen and gradually dosed. This type of therapy is dangerous because a compound that causes an allergic response is actually administered to the subject and an allergic reaction may occur.

  Allergic drugs include, but are not limited to, antihistamines, corticosteroids, prostaglandin inducers, and the like. Antihistamines are compounds that antagonize histamine released by mast cells or basophils. Such compounds are well known in the art and are widely used for allergy treatment. Antihistamines include, but are not limited to, acribastine, astemizole, azatazine, azelastine, betatastin, brompheniramine, buclidine, cetirizine, cetirizine analogs, chlorpheniramine, clemastine, CS560, cyproheptadine, desloratadine, dexchlorpheniramine, Examples include ebastine, epinastine, fexofenadine, HSR609, hydroxyzine, levocabastine, loratidine, methoscopolamine, mizolastine, norastemizol, phenindamine, promethazine, pyrilamine, terfenadine, and tranilast.

  Corticosteroids include, but are not limited to, methylprednisolone, prednisolone, prednisone, beclomethasone, budesonide, dexamethasone, flunisolide, fluticasone propionate, triamcinolone and the like. Dexamethasone is a corticosteroid with anti-inflammatory action, but it is highly absorbed and has long-term inhibitory side effects at effective doses, so it is not commonly used in the treatment of allergies or asthma in inhaled form. However, when administered in combination with the composition of the present invention, dexamethasone can be used according to the present invention to treat allergies or asthma because it can be administered at a low dose to suppress side effects. Among the side effects associated with corticosteroid use are cough, difficulty speaking, acne in the mouth (candidiasis), and at higher doses, adrenal suppression, impaired glucose tolerance, osteoporosis, sterile necrosis of the bone, cataract formation, growth Includes systemic effects such as suppression, hypertension, muscle weakness, skin thickness reduction, and easy contusion formation. Barnes & Peterson (1993) Am Rev Respir Dis 148: S1-S26 and Kamada AK et al (1996) Am J Respir Crit Care Med 153: 1739-48.

Anti-Asthma Drug The combination of TLR7 / 8 ligand and adapter oligonucleotide of the present invention can be used with an anti-asthma drug.

Drugs for treating asthma (i.e., anti-asthma drugs) are generally divided into two types: immediate relief drugs and long-term suppressive drugs. Asthmatic patients achieve and maintain sustained asthma suppression by taking long-term suppressive drugs daily. Long-term suppressive drugs include anti-inflammatory agents such as corticosteroids, cromolyn sodium, nedocromil, long-acting β ₂ agonists, long-acting bronchodilators such as methylxanthine, leukotriene modifiers, and the like. Immediate-effect alleviating drugs include short-acting β ₂ agonists, anticholinergic agents, systemic corticosteroids and the like. There are many side effects associated with each of these drugs, none of which can prevent or completely treat asthma alone or in combination.

  Asthma medicines include, but are not limited to, PDE-4 inhibitors, bronchodilators / β-2 agonists, K + channel openers, VLA-4 antagonists, neurokin antagonists, thromboxane A2 (TXA2) synthesis inhibitors, Xanthine, arachidonic acid antagonists, 5-lipoxygenase inhibitors, TXA2 receptor antagonists, TXA2 antagonists, 5-lipox-activating protein inhibitors, protease inhibitors and the like.

Bronchodilator / beta ₂ agonist is a compound of one class to cause bronchodilation or smooth muscle relaxation. The bronchodilator / beta ₂ agonists include, but are not limited to, salmeterol, salbutamol, albuterol, terbutaline, D2522 / formoterol, fenoterol, bitolterol, pyruvaldehyde Errol methylxanthines, and the like orciprenaline. Long acting β ₂ agonists and bronchodilators are compounds used for long-term prevention of symptoms in addition to anti-inflammatory therapy. Long acting β ₂ agonists include, but are not limited to, salmeterol, albuterol, and the like. Such compounds are commonly used with corticosteroids and are generally not used without any inflammatory therapy. Such have been associated with side effects such as tachycardia, skeletal muscle tremor, hypokalemia, and prolonged QTc interval when overdose.

For example, methylxanthine, including theophylline, has been used for long-term suppression and prevention of symptoms. Such compounds cause bronchodilation as a result of phosphodiesterase inhibition and possibly adenosine antagonism. Dose-related acute toxicity is a special problem with these types of compounds. As a result, serum concentrations must be routinely monitored to account for toxicity and the narrow therapeutic range arising from individual differences in metabolic clearance. Side effects include tachycardia, tachyarrhythmia, nausea and vomiting, central nervous system stimulation, headache, seizures, vomiting, hyperglycemia, hypokalemia. Short-acting β ₂ agonists include, but are not limited to, albuterol, vitorterol, pyrbuterol, terbutaline and the like. Some side effects associated with the administration of short-acting beta ₂ agonists, tachycardia, skeletal muscle tremor, hypokalemia, increased lactic acid, include headache and hyperglycemia.

  Cromolyn sodium and nedocromil are used as long-term suppressive drugs, primarily to prevent asthma symptoms resulting from exercise or allergic symptoms resulting from allergens. These compounds are believed to block early and late reactions to allergens by interfering with chloride channel function. They stabilize the mast cell membrane and inhibit the activation and release of mediators from inosineophil and epithelial cells. To maximize benefit, a dose period of 4-6 weeks is generally required.

  Anticholinergics are commonly used to relieve acute bronchospasm. Such compounds are believed to function by competitive inhibition of muscarinic cholinergic receptors. Anticholinergic agents include, but are not limited to, ipratropium bromide. These compounds only improve choline-mediated bronchospasm and do not alter the response to antigens at all. Side effects include dry mouth and respiratory secretions, increased wheezing in some individuals, and blurred eyes when sprayed into the eye.

  The compositions of the present invention can also be useful in the treatment of airway remodeling. Airway remodeling results from smooth muscle cell proliferation and / or submucosal thickening in the airway and ultimately restricts airflow as a result of airway narrowing. The composition of the present invention may prevent further airway remodeling and possibly even reduce the tissue gain resulting from the remodeling process.

Adjuvant The TLR7 / 8 ligand and adapter oligonucleotide of the present invention may be used in combination with other agents such as an adjuvant. Adjuvants as used herein are different from antigens, TLR7 / 8 ligands, and adapter oligonucleotides, and can stimulate immune cell activation in response to antigens, eg, humoral and / or cellular immune responses. Refers to the substance to be enhanced. Adjuvants promote the accumulation and / or activation of accessory cells to enhance the antigen-specific immune response. Adjuvants are used to increase the efficacy of vaccines used to elicit protective immunity against antigens, ie, antigen-containing compositions.

  Adjuvants generally include adjuvants that create a depot effect, immunostimulatory adjuvants, and adjuvants that create a depot effect and stimulate the immune system. As used herein, an adjuvant that creates a depot effect is an adjuvant that gradually releases the antigen in the body and thus prolongs the exposure of immune cells to the antigen. This class of adjuvants includes, but is not limited to, alum (eg, aluminum hydroxide, aluminum phosphate), mineral oil, non-mineral oil-in-water or oil-in-water emulsion, and Septan ISA series of Montanide adjuvants (eg, , Montanide ISA720, AirLiquid, Paris, France), MF-59 (Squalene-in-water emulsion stabilized with Span 85 and Tween 80, Chiron Corporation, Emeryville, Calif.), And PROVAX (stabilizing surfactant and micelle forming agent) Including oil-in-water emulsions, IDEC Pharmaceuticals Corporation, San Diego, California) Emulsion-based formulations.

  An immunostimulatory adjuvant is an adjuvant that causes activation of cells of the immune system. For example, it allows immune cells to produce and secrete cytokines. This class of adjuvant includes, but is not limited to, Q.I. saponins purified from saponaria bark, such as QS21 (glycolipids that elute in peak 21 in the HPLC fraction, Aqua Biopharmaceuticals, Inc., Worcester, Mass.), poly [di (carboxylatophenoxy) phosphazene (PCPP polymer) , Virus Research Institute, USA), monophosphoryl lipid A (MPL, Ribi ImmunoChem Research, Inc., Hamilton, Montana) derivatives of lipopolysaccharide, such as muramyl dipeptide (MDP, Ribi) and threonyl muramyl dipeptide (t -MDP, Ribi), OM-174 (lipid A-related glumisamine disaccharide, OM Pharma SA, Meyrin, Switzerland), and Lee Yumania elongation factor (purified Leishmania protein, Corixa Corporation, Seattle, Washington) are included. This class of adjuvants also includes CpG DNA.

  Adjuvants that create a depot effect and stimulate the immune system are compounds that have both of the functions specified above. This class of adjuvants includes, but is not limited to, ISCOMS (mixed saponins, immunostimulatory complexes that contain large lipids containing lipids and capable of retaining antigens, CSL, Melbourne, Australia), SB-AS2 (SmithKli Beeham adjuvant system No. 2 which is an oil-in-water emulsion containing MPL and QS21, SmithKline Beecham Biologicals [SBB], Ricusent, Belgium), SB-AS4 (Smith4ch system containing Alm and MPL) No., SBB, Belgium), nonionic block copolymers such as CRL1005 that form micelles (these are hydrophobic polyoxypropylenes with adjacent polyoxyethylene chains) Vaxcel, Inc. Norcross, Georgia, which contains a linear chain of Len, and Syntex Adjuvant Formulation (oil-in-water emulsions containing SAF, Tween 80 and non-ionic block copolymers, Syntex Chemicals, Inc., Boulder, Colorado) included.

  The adjuvant may be a lipopeptide such as Pam3Cys, a cationic polysaccharide such as chitosan, or a cationic peptide such as protamine.

Cytokines A combination of a TLR7 / 8 ligand and adapter oligonucleotide of the present invention may be used with a cytokine. Cytokines are soluble proteins and glycoproteins produced by many types of cells that mediate inflammatory and immune responses. Cytokines act both locally and systemically to mediate communication between cells of the immune system, mobilize cells and regulate their function and proliferation. Cytokine types include innate immunity mediators and regulators, adaptive immunity mediators and regulators, and hematopoietic activators. Some cytokines include interleukins (eg, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL- 10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, and interleukin 19-32 (IL-19-IL-32)) Chemokines (especially IP-10, RANTES, MIP-1α, MIP-1β, MIP-3α, MIP-1, MIP-2, MIP-3, MIP-4, eotaxin, I-TAC and BCA-1) , And type 1 interferons (eg, IFN-α and IFN-β), type 2 interferons (eg, IFN-γ), tumor necrosis factor α (TNF-α), transforming growth factor β (T F-beta), and GM-CSF, include other cytokines, including various colony stimulating factors (CSF) including G-CSF and M-CSF.

Antigen The combination of TLR7 / 8 ligand and adapter oligonucleotide of the present invention can optionally be used with an antigen in a vaccine formulation. As used herein, “antigen” refers to any molecule that can be recognized by a T cell antigen receptor or a B cell antigen receptor. This term broadly encompasses any type of molecule that the host immune system recognizes as foreign. In general, antigens include, but are not limited to, cells, cell extracts, proteins, polypeptides, peptides, polysaccharides, polysaccharide conjugates, peptidic and non-peptidomimetics of polysaccharides and other molecules, small molecules, Examples include lipids, lipoproteins, glycolipids, polysaccharides, carbohydrates, viruses and virus extracts, and multicellular organisms such as parasites, as well as allergens. For antigens that are proteins, polypeptides or peptides, such antigens can include nucleic acid molecules encoding such antigens. More specifically, antigens include, but are not limited to, cancer cells and cancer antigens including molecules expressed in or on cancer cells, microorganisms and microorganism antigens including molecules expressed in or on microorganisms As well as allergens. Accordingly, the present invention in certain embodiments provides vaccines for cancer, infectious agents and allergens.

  In various embodiments, the antigen is a microbial antigen, a cancer antigen or an allergen. A “microbial antigen” as used herein is an antigen of a microorganism, including but not limited to viruses, bacteria, parasites and fungi. Such antigens include not only naturally isolated antigens and fragments or derivatives thereof, but also intact microorganisms, and are identical or similar to natural microbial antigens, eliciting an immune response specific for that microorganism. Also includes synthetic compounds. If a compound elicits an immune response (humoral and / or cellular) against a natural microbial antigen, it is similar to a natural microbial antigen. Such antigens are routinely used in the art and are well known to those skilled in the art. The present invention is intended to encompass antigens from any of the infectious agents described herein.

  As used herein, the terms “cancer antigen” and “tumor antigen” are associated with tumors or cancer cells and expressed on the surface of antigen presenting cells involved in major histocompatibility complex (MHC) molecules. Are used interchangeably to refer to compounds such as peptides, proteins, lipoproteins or glycoproteins that can elicit an immune response. Cancer antigens are differentially expressed by cancer cells and can thus be utilized to target cancer cells. Cancer antigens are antigens that can potentially stimulate an apparent tumor-specific immune response. Some of these antigens are encoded by normal cells but are not always expressed. Such antigens are antigens that are normally silent (ie, not expressed) in normal cells, antigens that are expressed only at specific stages of differentiation, and transiently expressed antigens such as embryonic and fetal antigens. The characteristic can be expressed as Other cancer antigens are encoded by mutant cellular genes such as oncogenes (eg, activated ras oncogene), suppressor genes (eg, mutant p53), fusion proteins resulting from internal deletions or chromosomal translocations. Still other cancer antigens can be encoded by viral genes such as those carried on RNA and DNA tumor viruses.

  Cancer antigens are described, for example, in Cohen PA et al. (1994) Cancer Res 54: 1055-8 as described in Preparation of a crude extract of cancer cells, partial purification of the antigen by recombinant techniques, or de novo synthesis of a known antigen. Can do. A cancer antigen includes, but is not limited to, a recombinantly expressed antigen, an immunogenic portion thereof, or the entire tumor, cancer, cell. Such cancer antigens can be isolated or prepared by recombination or by any other means known in the art.

Examples of tumor antigens include MAGE, MART-1 / Melan-A, gp100, dipeptidyl peptidase IV (DPPIV), adenosine deaminase binding protein (ADAbp), cyclophilin b, colorectal associated antigen (CRC) C017-1A / GA733, carcinoembryonic antigen (CEA) and its immunogenic epitopes CAP-1 and CAP-2, etv6, aml1, prostate specific antigen (PSA) and its immunogenic epitopes PSA-1, PSA-2 and PSA- 3. Prostate-specific membrane antigen (PSMA), T cell receptor / CD3-ζ chain, MAGE family tumor antigen (eg, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6) , MAGE-A7, MAGE-A8, MAGE-A9, MA E-A10, MAGE-A11, MAGE-A12, MAGE-Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-B3), MAGE-Xp4 (MAGE-B4), MAGE-C1, MAGE-C2, MAGE-C3 , MAGE-C4, MAGE-C5), GAGE family tumor antigens (eg, GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE-8, GAGE) -9), BAGE, RAGE, LAGE-1, NAG, GnT-V, MUM-1, CDK4, tyrosinase, p53, MUC family, HER2 / neu, p21ras, RCAS1, α-fetoprotein, E-cadherin, α-catenin , Β-catenin and γ-catenin, p120ctn, gp1 ^{0 Pmel117, PRAME, NY-ESO} -1, cdc27, adenomatous poly Po cis protein (APC), fodrin, Connexin 37, Ig-idiotype, p15, gp75, GM2 and GD2 gangliosides, viral products such as human papilloma virus proteins, Smad family Tumor antigen, lmp-1, P1A, EBV-encoded nuclear antigen (EBNA) -1, brain glycogen phosphorylase, SSX-1, SSX-2 (HOM-MEL-40), SSX-1, SSX-4, SSX-5, Examples include SCP-1 and CT-7, c-erbB-2, and the like. This list is not intended to be limiting.

  An “allergen” as used herein is a molecule that can elicit an immune response characterized by the production of IgE. Allergens are also substances that can induce allergic or asthmatic responses in susceptible subjects. Thus, in the context of the present invention, the term allergen means a specific type of antigen capable of inducing an allergic response mediated by IgE antibodies.

  The list of allergies is enormous and includes pollen, insect venom, animal skin antigens, fungal spores and drugs (eg penicillin). Examples of natural animal and plant allergens include proteins specific for the following genera: Such genera include dogs (Canis familiaris), a single mite (eg, Dermatophagoides farinae), cats (Felis dometicia), ragweed (Ambrosia artemilum ceria), and durum (Lolium perum). (Alternaria alternata), alder, alnus galtinosa, birch (Betula verrucosa), quercus alba, olive (Olea europa), mugwort (Artemisia), In example, Plantago lanceolata), Hikagemizu (e.g., Parietaria officinalis and Parietaria Judaica), German cockroach (e.g., Blattella germanica), bees (e.g., Apis multiflorum), cypress (e.g., Cupressus sempervirens, Cupressus arizonica and Cupressus macrocarpa), juniper (e.g. , Juniperus sabinoides, Juniperus virginiana, Juniperus communis and Juniperus ashei), Kurobe (eg, Thuya orientalis), Hinoki ( For example, Chamecyparis obtusa), cockroaches (e.g., Periplaneta americana), Camellia (e.g., Agropyron repens), rye (e.g., Secreum cereal), wheat (e.g., Triticum aestima, e.g. Festuca elatior), Strawberry jumpweed (eg, Poa platensis and Poa compressa), Oats (eg, Avena sativa), Shiragaya (eg, Holcus lanatus), eg, Anthoxanthratum, eg. erum elatius), euglena (e.g., Agrostis alba), red squirrel (e.g., Phleum platenase), cedar reed (e.g., Phalaris arundinacea), scorpion (e.g., Pasalum notatum), sau (e.g., Sor, um) , Bromus inertis) and the like.

Antibodies and ADCC
The combination of TLR7 / 8 ligand and adapter oligonucleotide of the present invention also increases natural killer cytolytic activity and antibody dependent cellular cytotoxicity (ADCC). ADCC can be performed using a combination with an antibody specific for a cellular target and can induce the subject's immune system to kill the tumor cells. Antibodies useful for the progression of ADCC include antibodies that interact with cells in the body. Many such antibodies specific for cellular targets have been described in the art and many are commercially available. In one embodiment, the antibody is an IgG antibody.

  Therapeutic antibodies useful in the present invention can be specific for microbial antigens (eg, bacterial, viral, parasitic or fungal antigens), cancer or tumor associated antigens, and self antigens. A preferred antibody is an antibody that recognizes and binds to an antigen present on or within the cell. Examples of suitable antibodies include, but are not limited to, Rituxan ™ (Rituximab, anti-CD20 antibody), Herceptin (Trastuzumab), Quadramet, Panorex, IDEC-Y2B8, BEC2, C225, Oncolym, SMART M195, ATRAGEN, , Bexxar, LDP-03, iort6, MDX-210, MDX-11, MDX-22, OV103, 3622W94, anti-VEGF, Zenapax, MDX-220, MDX-447, MELIMMUNE-2, MELIMMUNE-1, CEACIDE, Pretarget , NovoMAb-G2, TNT, Gliomab-H, GNI-250, EMD-72000, LymphoCide, CMA676, Mono pharm-C, 4B5, ior egf. r3, ior c5, BABS, anti-FLK-2, MDX-260, ANA Ab, SMART 1D10 Ab, SMART ABL 364 Ab, CC49 (mAb B72.3), ImmuRAIT-CEA, anti-IL-4 antibody, anti-IL-5 Examples include antibodies, anti-IL-9 antibodies, anti-Ig antibodies, anti-IgE antibodies, serum-derived hepatitis B antibodies, and recombinant hepatitis B antibodies.

  Other antibodies that are also useful for the present invention include alemtuzumab (B-cell chronic lymphocyte leukemia), gemtuzumab ozogamicin (CD33 + acute myeloid leukemia), hP67.6 (CD33 + acute myeloid leukemia), Infliximab (inflammatory bowel disease and rheumatoid arthritis), etanercept (rheumatoid arthritis), tositumomab, MDX-210, oregovomab, anti-EGF receptor mAb, MDX-447, anti-tissue factor protein (TF) (Sunol), ior-c5, c5, edrecolomab, ibritumomab tiuxetan, anti-idiotype mAb mimetic of ganglioside GD3 epitope, anti-HLA-Dr10 mAb, anti-CD33 humanized mAb, anti-CD52 humAb, anti-CD1 mAb (iort6), MDX-22, celogobab Anti-17- A mAb, bevacizumab, daclizumab, anti-TAG-72 (MDX-220), high molecular weight proteoglycan anti-idiotype mAb mimetic (I-Mel-1), high molecular weight proteoglycan anti-idiotype mAb mimetic (I-Mel- 2), anti-CEA Ab, hmAbH11, anti-DNA or DNA-related protein (histone) mAb, Gliomab-H mAb, GNI-250 mAb, anti-CD22, CMA676), anti-idiotype human mAb against GD2 ganglioside, ior egf / r3, Anti-ior c2 glycoprotein mAb, ior c5, anti-FLK-2 / FLT-3 mAb, anti-GD-2 bispecific mAb, antinuclear autoantibody, anti-HLA-Dr Ab, anti-CEA mAb, palivizumab, bevacizumab, alemtuzu Bed, BLyS-mAb, anti-VEGF2, anti Trail receptor, B3 mAb, mAb BR96, breast cancer, and the like Abx-Cb1 mAb.

Immunoglobulin Therapy The agents of the present invention can also be used with normal and hyperimmunoglobulin therapy. Normal immunoglobulin therapy utilizes antibody products prepared and pooled from normal blood donor sera. This pooled product contains low titers of antibodies to a wide range of antigens such as antigens of infectious pathogens (eg, viruses such as bacteria, hepatitis A, parvovirus, enterovirus, fungi and parasites). Hyperimmunoglobulin therapy utilizes antibodies prepared from individual serum that contain high titers of antibodies against specific viruses. Examples of hyperimmunoglobulins include herpes zoster immunoglobulin (useful for the prevention of chickenpox in poorly immunized children and newborns), human rabies immunoglobulin (useful for prevention after virus exposure in subjects bitten by rabies animals), Hepatitis B immunoglobulins (especially useful for the prevention of hepatitis B virus in subjects exposed to the virus), as well as RSV immunoglobulins (useful for the treatment of respiratory syncytial virus infection).

Dosage regime and administration The ligand and oligonucleotide used in combination to achieve the desired effect can be formulated in separate compositions. For example, the adapter oligonucleotide and TLR7 / 8 ligand can be mixed together, administered to the subject substantially simultaneously as a combination, or contacted with a cell. In another example, the adapter oligonucleotide and TLR7 / 8 ligand can be administered to the subject at different times or contacted with cells. In yet another example, the adapter oligonucleotide and TLR7 / 8 ligand can be administered to different sites in the subject.

  As noted above, the term “effective amount” generally refers to an amount necessary or sufficient to achieve a desired biological effect. In combination with the teachings provided herein, a substantial selection of active compounds can be made by considering factors such as efficacy, bioavailability, patient weight, severity of adverse side effects, preferred mode of administration, etc. An effective prophylactic or therapeutic treatment regimen can be developed that does not cause toxicity but is completely effective for the treatment of a particular subject. The effective amount for any particular application can vary depending on such factors as the disease or condition being treated, the particular oligonucleotide being administered, the size of the subject, and the severity of the disease or condition. One of ordinary skill in the art can empirically determine the effective amount of a particular adapter oligonucleotide and TLR7 / 8 ligand and / or antigen and / or other therapeutic agent without necessitating undue experimentation.

  Subject dosages for systemic or local delivery of the compounds described herein typically range from about 10 ng to 10 mg per dose, and can be administered daily, weekly or monthly, depending on the application. And any amount of time between them or at another time as needed. More typically, systemic or local doses are typically in the range of about 1 μg to 1 mg, most typically in the range of about 10 μg to 100 μg per administration, with 2-4 administrations over several days Or at intervals of several weeks. Parenteral administration may require higher doses. However, in some embodiments, parenteral dosages for this may be used in the range of 5 to 10,000 times the typical dosages described above.

  For any compound described herein, the therapeutically effective dose can be initially determined from animal models. The applied dose can be adjusted based on the bioavailability and potency of the administered compound. Based on the methods described above and other methods well known in the art, it is well within the skill of the artisan to adjust the dosage to achieve the maximum effect.

Route of Administration For clinical use, the combination of a TLR7 / 8 ligand of the invention and an adapter oligonucleotide can be administered alone via any suitable route of administration that is effective in achieving the desired therapeutic result, Or it can be formulated as a delivery complex. Routes of administration include enteral and parenteral routes of administration. Examples of enteral routes of administration include oral, gastric, enteral and rectal. Non-limiting examples of parenteral routes of administration include intravenous, intramuscular, subcutaneous, intraperitoneal, intrathecal, topical injection, local, nasal, transmucosal and transpulmonary.

Delivery vehicles Adapter oligonucleotides and TLR7 / 8 ligands and / or antigens and / or other therapeutic agents can be used alone (eg, in saline solution or buffer) or any delivery vehicle known in the art. May be administered as well.

  For example, the combination of a TLR7 / 8 ligand and adapter oligonucleotide of the present invention may be administered directly to a patient or may be administered with a nucleic acid delivery complex. By nucleic acid delivery complex is meant a nucleic acid molecule that is bound (eg, ionically or covalently bound or encapsulated therein) to a target-inducing means (eg, a molecule that enhances affinity binding to a target cell). Shall. Examples of nucleic acid delivery complexes include sterols (eg, cholesterol), lipids (eg, cationic lipids, virosomes or liposomes), or target cell specific binding agents (eg, ligands recognized by target cell specific receptors). ). Preferred complexes may be stable in vivo sufficiently to prevent appreciable decoupling prior to uptake by target cells. However, the complex can be cleaved under suitable conditions in the cell so that the oligonucleotide is released in functional form.

  Other delivery vehicles described so far and which can be used according to the present invention include cochleates (Gould-Fogerite et al., 1994, 1996), emalsomes (Vancott et al., 1998, Lowell et al., 1997). ISCOM (Mowat et al., 1993, Carlsson et al., 1991, Hu et al., 1998, Morein et al., 1999), liposomes (Childers et al., 1999, Michaelk et al., 1989, 1992, de Haan, 1995) a, 1995 b), live bacterial vectors (eg, Salmonella, E. coli, Calmette Guerin, Shigella, Lactobacillus) (Hone et al., 1996, Pouwels et al., 1998, Chatfield et al., 1993, Stover et al. 1991, Nugent et al., 1998), live viral vectors (eg, vaccinia, adenovirus, herpes simplex) (Gallican et al., 1993, 1995, Moss et al., 1996, Nugent et al., 1998, Flexner et al., 1998 (Morrow et al., 1999), microspheres (Gupta et al., 1998, Jones et al., 1996, Malloy et al., 1994, Moore et al., 1995, O'Hagan et al., 1994, Eldridge et al., 1989) Nucleic acid vaccines (Fynan et al., 1993, Kuklin et al., 1997, Sasaki et al., 1998, Okada et al., 1997, Ishii et al., 1997), polymers (eg, carboxymethylcellulose, chito Sun) (Hamajima et al., 1998, Jabbal-Gill et al., 1998), polymer ring (Wyatt et al., 1998), proteosome (Vancott et al., 1998, Lowell et al., 1988, 1996, 1997), Foo Sodium hydroxide (Hashi et al., 1998), transgenic plants (Tacket et al., 1998, Mason et al., 1998, Haq et al., 1995), virosomes (Gluck et al., 1992, Mengiardi et al., 1995, Cryz et al., 1998), virus-like particles (Jiang et al., 1999, Leibl et al., 1998) and the like. Other delivery vehicles are known in the art.

Pharmaceutical Composition The combination of TLR7 / 8 ligand and adapter oligonucleotide of the present invention can be formulated as a pharmaceutical composition further comprising a pharmaceutically acceptable carrier. A pharmaceutically acceptable carrier means one or more compatible, solid or liquid fillers, diluents or encapsulating materials suitable for administration to humans and other vertebrates. The carrier is any natural or synthetic organic or inorganic component that, when mixed with the active ingredient (s), promotes the desired effect. The components of the pharmaceutical composition are mixed with one another so that no interaction occurs that substantially impairs the desired pharmaceutical efficiency.

  For oral administration, the compounds (ie, adapter oligonucleotides and TLR7 / 8 ligands, antigens and / or other therapeutic agents) are pharmaceutically acceptable carriers and active compound (s) well known in the art. Can be easily formulated. With such carriers, the compounds of the present invention can be formulated as tablets, pills, dragees, capsules, solutions, gels, syrups, slurries, suspensions, etc., for ingestion by the subject being treated. . Oral pharmaceutical preparations can be obtained as solid excipients, optionally by grinding the resulting mixture and processing the granule mixture to obtain tablets or dragee cores, if desired, after addition of suitable auxiliaries. . Suitable excipients are in particular sugars including lactose, sucrose, mannitol or sorbitol, such as corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth gum, methylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, etc. Cellulose products and / or fillers such as polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate. In some cases, oral formulations may be formulated in saline solution or buffer to neutralize internal acidic conditions, or may be administered without any carrier.

  A suitable coating is applied to the sugar-coated tablet core. For this purpose, concentrated sugar solutions can be used, which can optionally contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol and / or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragees for identification or to characterize different combinations of active compound doses.

  Pharmaceutical preparations that can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. Indented capsules can contain the active ingredients in a mixture with a filler such as lactose, a binder such as starch, and / or a lubricant such as talc or magnesium stearate, and optionally a stabilizer. For soft capsules, the activity may be dissolved or suspended in a suitable liquid, such as fatty oil, liquid paraffin, or liquid polyethylene glycol. In addition, stabilizers may be added. Microspheres formulated for oral administration may be used. Such microspheres have been well defined in the art. All formulations for oral administration should be in dosages suitable for such administration.

  For buccal administration, the composition may be in the form of conventionally formulated tablets or lozenges.

  The compound may be administered using standard inhalation devices to the lung airways, particularly the bronchi, and more particularly the deep alveoli. The compound may be in the form of an aerosol spray supplied from a pressurized container or nebulizer using a suitable high pressure gas such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, or other suitable gas. Can be delivered. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. An inhalation device may be used to deliver the compound to the subject. An inhalation device for use in the present invention is any device that administers an aerosol, such as a dry powder form of the compound. Such devices are well known in the art and are described in detail in Remington: The Science and Practice of Pharmacy, 19th Edition, 1995, Mac Publishing Company, Easton, Pennsylvania, pages 1676-1692, etc. Has been described so far. A number of US patents, such as US Pat. No. 6,116,237, also describe inhalation devices.

  “Powder” as used herein refers to a composition consisting of finely dispersed solid particles. Preferably, the compound is fairly free flowing and can be dispersed in an inhaler, after which it can reach the lungs and enter the alveoli by being inhaled by the subject. . “Dry powder” refers to a powder composition having a moisture content such that the particles can be easily dispersed in an inhaler to form an aerosol. The moisture content is generally less than about 10% by weight (% w) water, in some embodiments less than about 5% w, preferably less than about 3% w. The powder may be formulated with the polymer or optionally with other substances such as liposomes, albumin and / or other carriers.

  Aerosol doses and delivery systems are described, for example, in Gonda, I., in Critical Reviews in Therapeutic Drug Carrier Systems, 6: 273-313 (1990). “Aerosols for delivery of therapeutic and diagnostic agents to the respiratory tract”, and Aerosols in Medicine. Principles, Diagnostics and Therapy, Moren, et al. Eds. , Elsevier, Amsterdam, 1985, as described in Moren, “Aerosol dosage forms and formulations”, can be selected by one skilled in the art for specific therapeutic applications.

  Where it is desired to deliver the compound systemically, the compound may be formulated for parenteral administration by infusion, eg, bolus injection or continuous infusion. Injectable formulations may be presented in unit dosage form, for example, in ampoules or in multi-dose containers supplemented with preservatives. The compositions may take the form of suspensions, solutions, emulsions, etc. in oily or aqueous media and may contain compounding agents such as suspending, stabilizing and / or dispersing agents.

  Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water form. In addition, suspensions of the active compounds may be prepared as appropriate oily suspensions for injection. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate and triglycerides, or liposomes. Aqueous injection suspensions may contain sodium carboxymethylcellulose, sorbitol or dextran as substances that increase the viscosity of the suspension. Optionally, the suspension may contain suitable stabilizers or agents that increase the solubility of the compound to allow for the preparation of highly concentrated solutions.

  Alternatively, the active compound may be in powder form for constitution with a suitable medium, eg, pyrogen-free sterile water, before use.

  The compounds may be formulated in rectal or vaginal compositions such as suppositories or retention enemas, eg, containing conventional suppository bases such as cocoa butter or other glycerides.

  In addition to the preparations described so far, the compounds may be formulated as a depot preparation. Such long acting formulations may be formulated with suitable polymeric or hydrophobic substances (eg, as acceptable emulsions in oil), or with ion exchange resins, or as sparingly soluble derivatives, such as sparingly soluble salts. Also good.

  The pharmaceutical composition may comprise a suitable solid phase or gel phase carrier or excipient. Examples of such carriers or excipients include, but are not limited to, calcium carbonate, calcium phosphate, various sugars, starch, cellulose derivatives, gelatin, and polymers such as polyethylene glycol.

  Suitable solid or liquid dosage forms are, for example, aqueous solutions for inhalation or saline solutions, microencapsulated, cochlear, coated on gold microparticles, contained in liposomes Being sprayed, aerosolized, pellets for skin implantation, or dried on sharp objects for scraping into the skin. Such pharmaceutical compositions also include granules, powders, tablets, coated tablets, (micro) capsules, suppositories, syrups, emulsions, suspensions, creams, drops or sustained release formulations of the active compounds, at the time of their preparation. Excipients such as disintegrants, binders, coatings, swelling agents, lubricants, flavoring agents, sweeteners or solubilizers and additives and / or adjuvants are commonly used as described above. The pharmaceutical composition is suitable for use in a variety of drug delivery systems. For a brief review on drug delivery methods, see Langer R (1990) Science 249: 1527-33, which is incorporated herein by reference.

  The agents of the present invention, and optionally other therapeutic agents and / or antigens, may be administered per se (undiluted) or in the form of a pharmaceutically acceptable salt. When used in medicine, the salt should be pharmaceutically acceptable, but the pharmaceutically acceptable salt may be prepared by convenient use of pharmaceutically unacceptable salts. Such salts include, but are not limited to, the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, maleic acid, acetic acid, salicylic acid, p-toluenesulfonic acid, tartaric acid, citric acid, methanesulfone. Salts prepared from acids, formic acid, malonic acid, succinic acid, naphthalene-2-sulfonic acid, benzenesulfonic acid are included. Such salts can also be prepared as alkali metal salts or alkaline earth metal salts such as sodium, potassium and calcium salts of the carboxylic acid group.

  Suitable buffers include acetic acid and salt (1-2% w / v), citric acid and salt (1-3% w / v), boric acid and salt (0.5-2.5% w / v). ), Phosphoric acid and salts (0.8-2% w / v). Suitable preservatives include benzalkonium chloride (0.003-0.03% w / v), chlorobutanol (0.3-0.9% w / v), paraben (0.01-0.25). % W / v), thimerosal (0.004 to 0.02% w / v), and the like.

  The pharmaceutical composition of the present invention contains an effective amount of the agent of the present invention, optionally contained in a pharmaceutically acceptable carrier, and optionally an antigen and / or other therapeutic agent. The term pharmaceutically acceptable carrier means one or more compatible, solid or liquid fillers, diluents or encapsulating materials suitable for administration to humans or other vertebrates. The term carrier means any natural or synthetic organic or inorganic ingredient whose application is facilitated when mixed with the active ingredient. Each component of the pharmaceutical composition can also be miscible with the compound of the present invention so that there is no interaction that would substantially impair the desired pharmaceutical efficiency.

Screening Methods In yet another aspect, the present invention provides a method for identifying TLR7 / 8 ligands and / or adapter oligonucleotides. The steps of the method will vary depending on the agent identified (ie, ligand vs. oligonucleotide), whether the ligand is TLR7, TLR8, TLR7 / 8 ligand, what cell type is used for the method, etc. .

  Based on the teachings presented herein, TLR7 ligands are capable of stimulating only TLR7 signaling, ability to stimulate TLR8 signaling (compared to background levels) in the presence of adapter oligonucleotides, and / or adapters. Can be identified by inhibition of TLR7 stimulation in the presence of oligonucleotide. TLR8 ligands can be identified by enhanced TLR8 signaling (compared to higher levels than background) when used in combination with adapter oligonucleotides. Ligands that stimulate both TLRs 7 and 8 (when used alone) can be identified by any combination of these activities. Adapter oligonucleotides can be identified by their ability to inhibit TLR7 stimulation by a TLR7 ligand, induce TLR8 stimulation by a TLR7 ligand, and / or enhance TLR8 stimulation from a TLR8 ligand.

  Such methods can be performed either in vivo or in vitro. In vitro assays are recorded in this example. Suitable readings include, but are not limited to, IL-12, TNF-α and / or IFN-γ for TLR8 stimulation, and IFN-α for TLR7 stimulation. Alternatively, the assay may employ a reporter construct having a reporter gene linked to a transcriptional regulatory element (eg, NF-κB response element) that is responsive to TLR7 and / or TLR8 signaling. Such constructs are described in this example. The assay may measure transcriptional up or down regulation, translation up or down regulation, protein expression and / or secretion, and the like.

  For example, in a TLR8 ligand assay, a TLR8 expressing cell (or cell population) can be contacted with a test ligand in the presence and absence of an adapter oligonucleotide. The cell is preferably a cell that expresses TLR8 but does not express TLR7 or TLR9. TLR8 signaling is measured from cells responding to the test ligand in the presence and absence of oligonucleotides. A test ligand having a TLR8 signaling profile that is enhanced in the presence of the oligonucleotide as compared to the absence of the oligonucleotide is then identified as a TLR8 ligand. This assay may include analysis of TLR7 signaling using TLR7 expressing cells that do not express TLR8 or TLR9. This latter analysis can be used to rule out the possibility that the ligand is a TLR7 ligand that switches to TLR8 signaling in the presence of oligonucleotides.

  Results from a similar assay using known ligands are shown in FIG. Loxoribine and immunosin (TLR7 specific ligand) both stimulate TLR8 signaling in the presence of ODN6056. A hypothetical TLR7-specific ligand would be expected to have a qualitatively similar profile. TLR8 signaling by R848 (TLR7, TLR8 combined ligand) is enhanced in the presence of ODN6056. A hypothetical TLR8-specific ligand would be expected to have a qualitatively similar profile. Ribavirin, which is neither a TLR7 nor a TLR8 ligand, does not stimulate TLR8 signaling in the presence or absence of ODN 6056, indicating the specificity of this assay for TLR7 / 8 ligand.

  In adapter oligonucleotide assays, TLR8-expressing cells (or cell populations) can be contacted with a test oligonucleotide in the presence and absence of a TLR7, TLR8 or TLR7 / 8 ligand. If the ligand is a TLR7 ligand (when used alone), this assay can measure inhibition of TLR7 signaling and / or induction of TLR8 signaling when the oligonucleotide is used with the ligand. If the ligand is a TLR8 ligand (when used alone), this assay can measure the enhancement of TLR8 signaling compared to the effect of the ligand alone when the oligonucleotide is used with the ligand. . If the ligand stimulates both TLR7 and TLR8 (when used alone), this assay can measure one or more of the readings. The assay results can be compared to positive control assays (eg, assays using known adapter oligonucleotides) and the like. The method can also include an assay that determines whether the oligonucleotide is a TLR7 and / or TLR8 ligand itself.

  Such methods are weakly stimulating when used alone, but can be used to identify ligands with new and / or enhanced signaling profiles when used in combination with oligonucleotides. .

  The invention is further illustrated by the following examples, which should in no way be construed as further limiting.

Materials and Methods Oligonucleotides and Reagents All oligonucleotides were purchased from Biospring (Frankfurt, Germany) or provided by Coley Pharmaceutical GmbH (Langenfeld, Germany) and are controlled by Coley Pharmaceutical GmbH for identification and purity. The assay showed an endotoxin level (<0.1 EU / ml) below the detection limit (BioWhittaker, Verviers, Belgium). Oligonucleotides were suspended in sterile, endotoxin-free Tris-EDTA (Sigma, Daisenhofen, Germany) and stored and handled under aseptic conditions to prevent contamination of both microorganisms and endotoxins. All dilutions were made with endotoxin-free Tris-EDTA. Each sequence of the oligonucleotide is listed in Tables 1 and 2. Loxoribine (7-allyl-7,8-dihydro-8-oxoguanosine) (Sigma) was first dissolved in 1N NaOH, diluted in RPMI medium and the pH adjusted to 7.4 with 1N HCl (19). . R-848 (1- (2-hydroxy-2-methylopropyl) -2-methyl-1H-imidazo [4,5-c] quinolin-4-amine) is commercially available from GL Synthesis (Worthester, Massachusetts, USA). It was synthesized and dissolved in 10% DMSO. 7-deazaguanosine (ChemGenes, Wilmington, Mass., USA) was dissolved in 1M in 1N NaOH. Inosine (Sigma) was dissolved in H2O. All dilutions were made in Tris-EDTA without endotoxin.

TLR assay Stably transfected HEK293 cells expressing human TLR9, TLR8 or TLR7 have been described previously (16, 20). Briefly, HEK293 cells were transfected by electroporation with a vector expressing each human TLR and 6xNF-κB luciferase reporter plasmid. Stable transfectants (3 × 10 ⁴ cells / well) were incubated with loxoribine or R-848 in a humidified incubator at 37 ° C. for 16 hours in the absence or presence of ODN. Each data point was repeated three times. Cells were lysed and assayed for luciferase gene activity (using Perkin-Elmer, Zaventem, Belgium's BriteLite kit). The stimulation index was calculated based on the reporter gene activity of the medium without the addition of oligonucleotide.

Cell Purification Peripheral blood buffy coat preparations from healthy donors were obtained from the University of Dusseldorf (Germany) Blood Bank and PBMCs were purified by centrifugation on Ficoll-Hypaque (Sigma). 5% (v / v) heat-inactivated human AB serum (BioWhittaker) or 10% (v / v) heat-inactivated FCS, 2 mM L-glutamine, 100 U / ml penicillin, and 100 μg / ml at 37 ° C. in a humidified incubator Cells were cultured in RPMI 1640 medium supplemented with streptomycin (all from Sigma).

Cytokine detection and flow cytometry analysis PBMC were resuspended at a concentration of 5 × 10 ⁶ cells / ml and added to 96-well round bottom plates (250 μl / well). PBMCs were incubated with various concentrations of ODN and / or loxoribine and culture supernatants (SN) were collected after the indicated time points. If not used immediately, SN was stored at −20 ° C. until needed. Cytokine levels in SN are relative to commercially available ELISA kits (for BD Biosciences, Heidelberg, Germany), IFN-γ and TNF-α (from Diaclone, Besançon, France), or IFN-γ. Evaluation was performed using a homemade ELISA developed using commercially available antibodies (PBL, New Brunswick, New Jersey, USA).

For intracellular staining, incubate PBMC at 5 × 10 ⁶ cells / ml in 96-well round bottom plates (250 μl / well) with the indicated oligonucleotide amount and / or loxoribine concentration and add Brefeldin A solution (BD Biosciences) did. PBMC were incubated for 6 hours. For intracellular staining of IFN-γ, cells were incubated with oligonucleotide and / or loxoribine for 16 hours prior to the addition of Brefeldin A solution. Cells were harvested and intracellular staining was performed using Intraprep reagent (Beckman-Coulter, Neuss, Germany) according to the manufacturer's procedure. For the identification of monocytes (CD14 ⁺ ), B cells (CD19 ⁺ ), and NK cells (CD56 ⁺ , CD3 ⁻ ), the cells were stained with appropriate antibodies. Flow cytometry data was collected on a FACSCalibur and analyzed using the computer program CellQuest (both from BD Biosciences). All antibodies (mAb) for flow cytometry analysis were purchased from BD Biosciences except for CD11c from Diaclone, CD14 from Immunotech (Marseille, France) and CD123 from Miltenyi (Bergish Gladbach, Germany). Human monocytes were separated from total PBMC using a CD14 cell separation kit as described by the manufacturer (Miltenyi). To determine purity, cells were stained with mAbs against CD11c and CD14 and identified by flow cytometry. In all experiments, monocyte purity exceeded 95%. Purified monocytes (4 × 10 ⁶ cells / ml) were incubated for 24 hours with increasing concentrations of ODN. PDCs were enriched using a BDCA-4 pDC separation kit as described by the manufacturer (Miltenyi). PDC purification was confirmed by staining with mAbs against CD123 (Miltenyi), HLA-DR and CD11c (BD Biosciences). The purity was about 85%. Cells (5 × 10 ⁵ cells / ml, 250 μl / well) were cultured for 24 hours with or without oligonucleotide and loxoribine. IFN-α or IL-12p40 in SN was measured as described above.

Results Sequence Selective Enhancement of TLR8-Mediated Signaling by Co-Incubation with Homopolymeric Oligonucleotides HEK293 cells stably expressing hTLR8 and NF-κB luciferase reporter constructs were isolated from R-848 (1- (2-hydroxy-2- Methylopropyl) -2-methyl-1H-imidazo [4,5-c] quinolin-4amine) was incubated in the presence or absence of oligonucleotide. The effect of co-incubation with different oligonucleotides on TLR8-mediated NF-κB activation was then analyzed.

ODN 6056, an oligo (dT) ₁₇ homopolymer ODN with a phosphorothioate backbone, was confirmed to significantly increase the level of NF-κB activation stimulated by R-848 (FIG. 1B). ODN 6056 alone did not stimulate significant NF-κB activation (less than 2.5 times background) in TLR8 expressing cells at concentrations up to 25 μM (unpublished data). Not all tested oligonucleotides equally enhanced TLR8 signaling (eg, an unrelated heteropolymer (ODN 1982) did not significantly affect NF-κB activation by R-848). This effect appeared to be oligonucleotide dependent.

  Co-incubation of R-848 with ODN 6056 significantly increased not only the efficacy of TLR8 activation but also the efficacy. The EC50 of R-848 was significantly reduced as early as in the presence of 0.1 μM ODN6056 (average of three experiments 0.1 μM ODN6056: EC50 (R-848) = 4.9 μM; EC50 (R-848 )> 30 μM alone). However, as the concentration of ODN 6056 increased, the EC50 further decreased (1 μM ODN 6056: EC50 (R-848) = 1.4 μM; 5 μM ODN: EC50 (R-848) = 0.4 μM). This effect was specific to ODN 6056, as the decrease in EC50 was much smaller with ODN 1982 (5 μM 1982: EC50 (R-848) = 19.0 μM). In contrast to the stimulatory effect on TLR8, co-incubation of ODN 6056 (or ODN 1982) with TLR9 ligand CpG ODN2006 on hTLR9 expressing HEK293 cells did not affect CpG-mediated NF-κB stimulation (unpublished data).

Potential sequence requirements for the observed enhancement effect were investigated and several different phosphorothioate homopolymers were tested (Table 1). Oligo (dT) ₁₇ (ODN6056) had the strongest enhancement, followed by oligo (dU) ₁₇ (SEQ ID NO: 11) and oligo (dA) ₁₇ (SEQ ID NO: 12). Significantly small enhancement was detected for oligo (dC) ₁₇ (SEQ ID NO: 13) and randomized oligo (dN) ₁₅ . The heteropolymer ODN 1982 showed little enhancing activity, and oligo (dG) ₂₄ (SEQ ID NO: 14) did not enhance activity, but rather inhibited R-848-mediated NF-κB stimulation. The sequence of ODN6056 with a phosphorodiester backbone did not affect TLR8-mediated stimulation. The length of the oligonucleotide also seemed to have some influence. That is, oligo (dA) ₂₄ (SEQ ID NO: 15) had a smaller synergistic effect with R-848 than short-chain oligo (dA) ₁₇ (SEQ ID NO: 12).

Stimulation of TLR8-expressing cells with TLR7-specific ligand in the presence of homopolymer T oligonucleotide R-848 is a ligand for both hTLR7 and hTLR8 (16), but loxoribine (7-allyl-7,8-dihydro-8 oxoguanosine ) Was described as a TLR7 specific ligand (18). Concentrations up to 10 mM loxoribine did not activate NF-κB signaling in HEK293 cells transfected with either hTLR8 or hTLR9, but activated hTLR7-mediated signaling (FIG. 2A). Although TLR7 ligand, loxoribine and oligonucleotide co-incubated with hTLR8 expressing HEK293 cells activated NF-κB signaling (FIG. 2B). This concentration-dependent effect was observed exclusively in the presence of ODN 6056 and not in the presence of ODN 1982 (FIG. 2B). Similar observations were made for 7-deazaguanosine, another TLR7 ligand (18). 7-deazaguanosine alone was inactive on hTLR8 expressing cells at concentrations up to 5 mM in the absence of ODN6056, but stimulated TLR8-mediated NF-κB activation in the presence of ODN6056. Inosine, a compound that appears to non-specifically activate NF-κB signaling in HEK293 cells (unpublished data), was also co-incubated with ODN6056 on hTLR8 expressing HEK293 cells (FIG. 2C). NF-κB activation by inosine in the presence of ODN 6056 did not change at concentrations up to 10 mM compared to non-specific activation of inosine.

Similar effects have been observed for 6-amino-9-benzyl-2-butoxy-9H-purin-8-ol, an adenosine compound. When used alone, this compound is a TLR7 ligand but not a TLR8 ligand (FIGS. 9A and 9B). When used in the presence of poly (dT) ₁₇ adapter oligonucleotide, it can stimulate TLR8 signaling (FIG. 9B).

Sequence Independent Inhibition of TLR7 Signaling These data indicate that small molecule TLR7 ligands induce TLR8-dependent NF-κB signaling in the presence of certain oligonucleotides. Nevertheless, the effect was reversed in TLR7 expressing HEK293 cells. Oligonucleotides actually inhibited TLR7-mediated NF-κB activation by TLR7-specific and TLR7 / 8 ligands as shown in FIGS. 1A, 3 and 9A. This effect appeared to be non-sequence specific since all phosphorothioate oligonucleotides tested so far inhibited hTLR7 activation (unpublished data).

Enhancement of TLR8 ligand signaling using RNA adapter oligonucleotides Similar experiments were performed using R-848 and RNA adapter oligonucleotides as TLR7 / 8 ligands. FIG. 10 shows the effect of mixed sequence oligonucleotides and poly (rU) ₁₈ oligonucleotides on NF-κB stimulation in hTLR8-LUC-293 cells. The poly (rU) ₁₈ oligonucleotide has (rU ^* rU ^* rU ^* rU ^* rU ^* rU ^* rU ^* rU ^* rU ^* rU ^* rU ^* rU ^* rU ^* rU ^* rU ^* rU ^* rU ^* rU: SEQ ID NO: 5) When used alone, it signals through TLR8 as shown by the slight activation of NF-κB by this oligonucleotide (FIG. 10). The mixed sequence RNA oligonucleotide (rG ^* rC ^* rC ^* rA ^* rC ^* rC ^* rG ^* rA ^* rG ^* rC ^* rC ^* rG ^* rA ^* rA ^* rG ^* rG ^* rC ^* rA ^* rC ^* rC: SEQ ID NO: 16 ) Does not signal through TLR8 when used alone.

In FIG. 11, increasing amounts of hTLR8-LUC-293 cells R-848 and either (a) TE and 50 μg / ml DOTAP, or (b) 5 μM of the indicated ORN in the presence of 50 μg / ml DOTAP. Incubated in the presence of Stimulation of NF-κB was measured after 16 hours by determination of luciferase activity. The stimulation index was calculated based on the background in the presence of medium alone. As the figure shows, the effect of poly (rU) ₁₈ oligonucleotide on TLR8 signaling is dramatically enhanced when the oligonucleotide is combined with R-848 (FIG. 11). Mixed sequence RNA oligonucleotides that are not ligands for either TLR7 or TLR8 also enhance TLR8 signaling by R-848, as shown in FIG. This data indicates that RNA-based oligonucleotides can also function as adapters.

Co-incubation of loxoribine with oligo (dT) ₁₇ ODN alters the cytokine profile exhibited by human PBMC The effect of co-incubation of loxoribine with ODN 6056 and 1982 on human immune cells was examined. Incubation with loxoribine alone stimulated human PBMC to produce IFN-α (FIG. 4A). However, in the presence of increasing ODN concentration, loxoribine-induced IFN-α decreased to the background level. This effect was observed for both types of ODN, but ODN 6056 appeared to have a stronger inhibitory action.

  The production of other cytokines was also examined. Loxoribine alone induced only a small amount of IL-12p40 secretion from human PBMC (FIG. 4B). In contrast, in the presence of ODN 6056, IL-12p40 was produced in significant amounts, but the same concentration of ODN 1982 shared with loxoribine does not induce IL-12p40 beyond the background using medium alone. There wasn't. Similar results were obtained for IL-12p70 (unpublished data) and IFN-γ (FIG. 4C). TNF-α production was also stimulated when loxoribine and ODN 6056 were co-incubated. However, TNF-α production was also observed when ODN 1982 was co-incubated with loxoribine, much less than the amount obtained with co-incubation with ODN 6056. Neither ODN nor loxoribine alone induced a significant amount of TNF-α secretion (FIG. 4D).

IL-12p40 and TNF-α are produced by monocytes when co-stimulated with loxoribine and oligo (dT) ₁₇ ODN The combination of loxoribine and ODN activates TLR8-expressing HEK293 cells that suppress TLR7-mediated signaling To do. It was hypothesized that human TLR8 positive immune cells are the primary source of IL-12 and TNF-α, and TLR7 positive pDC is the primary source of IFN-α. Intracellular FACS staining revealed that the majority of CD14 + cells produced TNF-α when co-incubated with loxoribine and ODN6056 and human PBMC (FIG. 5A). Loxoribine, used alone or in combination with ODN 1982, produced few TNF positive monocytes. The ratio (%) of IL-12 positive monocytes was smaller than the corresponding ratio for TNF-α. Nevertheless, the synergistic effect of the combination of loxoribine and ODN6056 was clearly detectable for intracellular IL-12 (Fig. 5B). In any of these experiments, intracellular IL-12 or TNF-α could not be detected in CD19 + B cells upon stimulation with ODN and loxoribine (unpublished data). The cell source of IFN-γ was also examined. In combination with loxoribine and ODN, IL-12 was highly stimulated, and previous studies have shown that IL-12 induces IFN-γ secretion in NK cells (21-23). Therefore, IFN-γ production was evaluated in human NK cells (FIG. 6). When co-incubating loxoribine and ODN6056 with human PBMC, IFN-γ production could actually be observed in human NK cells, but as already shown in the IFN-γ protein ELISA of human PBMC supernatant, Stimulation alone did not induce a significant degree of IFN-γ production.

  To demonstrate direct TLR-mediated monocyte stimulation in response to loxoribine and ODN 6056, human PBMC monocytes were purified and incubated with loxoribine in the presence or absence of ODN 6056 or 1982. Isolated monocytes produced IL-12p40 (FIG. 7A) or TNF-α (unpublished data) only in response to both types of stimuli, loxoribine and ODN6056.

Incubation of human PBMC with small immunomodulatory compounds such as R-848 and loxoribine induced IFN-α production by pDC (FIGS. 7B and (24, 25)). Thus, if the inhibitory effect of ODN6056 on IFN-α production stimulated by loxoribine was observed, it could have been a direct result of inhibition of TLR7-mediated signaling in pDC. To investigate this possibility, human pDCs were isolated and stimulated with loxoribine in the presence of oligo (dT) ₁₇ homopolymer or heteropolymer ODN (FIG. 7B). Enriched human pDC produced large amounts of IFN-α when incubated with loxoribine alone. In contrast, IFN-α production was stopped when co-incubated with ODN 6056 or ODN 1982. The inhibitory effect of ODN 6056 appeared more powerful than that of ODN 1982 because the ODN concentration required to stop IFN-α production was lower.

  FIG. 8 shows data generated using TLR8-LUC-293 cells. A constant concentration of R-848 (50 μmol) and increasing amounts of the specified ODN were used. For normalization, the R-848 luciferase readout was set to 100%. The result is shown in the left panel. The right panel of FIG. 8 shows the effect of increasing T content in 1 μmol of 17-mer poly C oligonucleotide in the presence of increasing amounts of R-848. Its activity is enhanced with only two thymidines, and increases dramatically with more than 6 thymidines.

Discussion This data shows that the activity of hTLR8 can be modulated by the presence of certain phosphorothioate ODNs. HTLR8 is most closely related to hTLR7 (26), as reflected by the ability of the immunostimulatory small compound R-848 to activate both hTLR7 and hTLR8 (16). However, hTLR7 is much more sensitive to stimulation by R-848 than hTLR8 because the R-848 concentration required for TLR7 activation is lower. Co-incubation of R848 with phosphorothioate oligo (dT) ₁₇ increased the sensitivity of TLR8 to stimulation by R848, but inhibited TLR7 activation, but this result showed that the T homopolymer alone showed that these TLRs Contrary to the fact that none of them had a significant effect.

To determine whether the T homopolymer affects TLR activation by other stimuli, we investigated its effect on two TLR7 ligands, loxoribine and 7-deazaguanosine (18) that do not stimulate TLR8 It was. Surprisingly, the presence of oligo (dT) ₁₇ for both ligands causes a complete switch from TLR7-dependent to TLR8-dependent NF-κB activation, and TLR7-mediated signaling is oligo (dT) _17. It was suppressed by. It is conceivable that human TLR8 has a weak binding affinity for loxoribine that is probably not detectable in available biological assays. Because the presence of certain oligonucleotides can enhance the affinity of loxoribine for TLR8 by an unknown mechanism, loxoribine-mediated stimulation of TLR8 in stably transfected cells can be detected.

The possibility that oligonucleotides affect the overall signaling pathway or uptake mechanism cannot be ruled out. However, the fact that oligo (dT) ₁₇ showed a different mode of action on HEK293 cells transfected with hTLR7 (inhibition of NF-κB signaling), hTLR9 (no effect) or hTLR8 (strong enhancement of signaling) Strongly denies this possibility. The observed synergy was not limited to cells ectopically expressing TLR8. Human TLR7 is mainly expressed on pDC and B cells. Such cells do not express TLR8, whereas TLR8 is expressed in myeloid cells (27-30). Indeed, synergy of loxoribine and ODN on cytokine production (IL-12 and TNF-α) was measured in human face cells that endogenously express TLR8, such as monocytes. In contrast, the majority of loxoribine and R-848-induced IFN-α is produced by activation of TLR7 in human pDC (our data and (4, 24, 25)). These TLR7-stimulated activation of IFN-α production in pDC was completely suppressed by incubation with phosphorothioate ODN, consistent with the inhibitory effect seen in TLR7-transfected cells. In contrast to the sequence-selective stimulatory effects observed for TLR8, inhibition of TLR7-mediated signaling by phosphorothioate ODN in transfected cells or pDC did not have obvious sequence dependence. The combination of ODN and loxoribine caused not only switching from pDC-derived IFN-α to monocyte-derived IL-12 and TNF-α, but also secretion of IFN-γ from NK cells. Since human NK cells lack TLR7 and TLR8 expression (31), stimulation of NK cell-derived IFN-γ appears to be an IL-12-mediated indirect effect (32). In summary, a complete change in the profile of loxoribine-mediated immune effects was observed with the addition of certain oligonucleotides. Furthermore, these data indicate some sequence-specific effects, and phosphorothioate T-rich ODN is the most effective ODN that can be used in combination with TLR7-specific ligands to activate TLR8.

Several reports have shown that myeloid dendritic cells and monocytes express both TLR7 and TLR8 at the mRNA level (27-29). Data that actually shows the expression of TLR protein in these cells has not been obtained. Direct stimulation of monocytes with loxoribine was also not detected in these experiments. In contrast, only the combination of loxoribine and oligonucleotide induced the production of monocyte-derived cytokines. These results negate the functional expression of TLR7 in monocytes and indicate TLR8 as a receptor targeted by oligonucleotides and loxoribine. Furthermore, loxoribine alone did not induce significant amounts of IL-12, but R-848-induced IL-12 production in human PBMC has been reported (29), which stimulates both species TLRs. Consistent with the capabilities of R-848 (16). In cells that can express both TLR7 and TLR8, it is very difficult to clearly identify which TLR is activated by a compound that stimulates both types of receptors. Ito et al (33) reported that mDC produced IL-12 when stimulated with R-848, but not IFN-α. In contrast, pDC does not produce IL-12 when stimulated with R-848 or other TLR ligands, but does produce IFN-α (25), indicating that IL-12 in mDCs FIG. 6 shows TLR8-mediated induction and TLR7-mediated induction of IFN-α in pDC. This signal is insufficient to activate the proper pathway in pDC because even co-incubation of R-848 or loxoribine with oligo (dT) ₁₇ ODN did not cause IL-12 production by human pDC. I suggested that.

  It is attractive to assume a model where certain TLRs have regulatory sites (possibly allosteric) to which oligonucleotides can bind and function as effector molecules. In the case of TLR7, the phosphorothioate oligonucleotide appears to act as an antagonist. Thus, an oligonucleotide that binds to TLR7 may inhibit proper binding of TLR7 ligand to the binding pocket, or proper downstream signaling, for example by exacerbating proper conformational changes in its receptor. . On the other hand, T-rich oligonucleotide binding to TLR8 can function as an (allosteric) activator, and the amount of other small molecule ligands such as R-848 or loxoribine bound to the active TLR8 binding pocket. Allows for increased or downstream signaling. It is not currently possible to determine whether the binding domain is involved in two places, or whether binding of the small molecule ligand and effector ODN can occur at the same site. Allosteric enhancers are known for several families of receptors. For example, Knoflach et al. (34) reported the identification of two classes of small molecules that behave as allosteric enhancers for metabotropic glutamate receptors belonging to the G protein-coupled receptor family. Through detailed analysis of the receptor structure, the enhancer binding site was located within the transmembrane domain. Other studies have shown that small molecules can act as allosteric enhancers for muscarinic acetylcholine receptors (35). Certain 2-aminothiophenes stabilize the ligand-receptor-G protein ternary complex and increase allosteric enhancement of adenosine receptor A1 by increasing unoccupied receptor and G protein binding (38). Acts as an agent (36, 37). Interestingly, Gao et al. (39) found a series of imidazoquinoline derivatives that act as agonist allosteric enhancers that bind to the human A3 adenosine receptor. Recently, Rutz et al. (40) demonstrated direct blocking of CpG ODN binding to purified TLR9 protein by small molecules chloroquine and quinacrine using SPR biosensor analysis. These findings indicate that these molecules bind directly to the extracellular domain of TLR9 and act as antagonists of TLR9-mediated signaling. Thus, it is possible that the oligonucleotides and small molecules bind directly to TLRs 7 and 8. Binding studies with isolated proteins and each receptor ligand will provide further insight into the exact mechanism of action of the combination of small molecule TLR ligands and oligonucleotides on TLR7 or TLR8. These findings can have important implications for understanding TLR molecular signaling mechanisms, as well as for pharmaceutical research and drug development. According to the present invention, it was demonstrated that the signaling activities of two members of the TLR family, TLR8 and TLR7, can be manipulated by the presence or absence of certain oligonucleotides. Taken together, these results suggest new ways of modifying the immune response using loxoribine (or other small molecules) in combination with certain oligonucleotides. By combining these molecules, it is possible to alter its cytokine profile by redirecting the signaling activity of the TLR small molecule ligand to a different TLR. An altered or enhanced immune response will be beneficial in the treatment of various diseases.

ｈＴＬＲ８およびＮＦ−κＢレポーター構築体を発現するＨＥＫ２９３細胞を、表示したＯＤＮ５μＭの非存在下または存在下でＲ−８４８の５０μＭとインキュベートした。ＯＤＮ非存在下でのＲ−８４８によるＮＦ−κＢ刺激を１００％に設定し、それに従ってＲ８４８活性に対する効果を計算した。数値は２〜４回の実験の平均値（±ＳＤ）を表す。「Ｎ」はランダムな順序でＡ、Ｃ、ＧまたはＴを表す。「^＊」はホスホロチオエート骨格を表示し、「＿」はホスホジエステル骨格を表示する。

HEK293 cells expressing hTLR8 and NF-κB reporter constructs were incubated with 50 μM R-848 in the absence or presence of the indicated ODN 5 μM. NF-κB stimulation by R-848 in the absence of ODN was set at 100% and the effect on R848 activity was calculated accordingly. The numerical value represents an average value (± SD) of 2 to 4 experiments. “N” represents A, C, G or T in a random order. “ ^* ” ^Indicates a phosphorothioate skeleton, and “_” indicates a phosphodiester skeleton.

参考文献

References

Equivalents The foregoing specification is considered to be sufficient to enable one skilled in the art to practice the invention. Since the examples presented are intended to be merely illustrative of one aspect of the present invention, the present invention should not be limited in scope to the examples, and other functionally equivalent embodiments are possible. Within the scope of the present invention. Various modifications of the invention other than those shown and described herein will be apparent to those skilled in the art from the foregoing description, and will fall within the scope of the appended claims. The advantages of the invention are not necessarily encompassed by each embodiment of the invention.

  All references, patents and patent publications cited in this application are hereby incorporated by reference in their entirety.

1A-1B show the selective enhancement and inhibition of R-848 mediated activity against human TLR8 and TLR7 by ODN. FIG. 1A shows inhibition of R-848 activity against human TLR7 by ODN 6056 (SEQ ID NO: 3) and 1982 (SEQ ID NO: 4) (see Tables 1 and 2 for each sequence). HEK293 cells stably expressing TLR7 and NF-κB luciferase reporter constructs are incubated with 2 μM R-848 in the presence or absence of the indicated amounts of oligo (dT) 17 homopolymer ODN6056 or heteropolymer ODN1982. did. The activity of R-848 alone was set to 100%. All data points are assayed in triplicate and expressed as mean values (+/− SD). The result shown is one of three or more independent experiments. FIG. 1B shows sequence selective enhancement of R-848 activity against human TLR8 by oligo (dT) ₁₇ ODN. HEK293 cells stably expressing hTLR8 and NF-κB luciferase reporter constructs were increased in the absence of oligo (dT) ₁₇ homopolymer ODN6056 or heteropolymer ODN1982 or in the presence of 0.1, 1 and 5 μM. Incubated with a series of R-848s. Stimulation of NF-κB activation was calculated relative to the media background. All data points are assayed in triplicate and expressed as mean values (± SD). The result shown is one of four independent experiments. 2A-C show the altered target specificity of the TLR7 ligands loxoribine and 7-deazaguanosine as compared to oligo (dT) ₁₇ ODN. In FIG. 2A, HEK293 cells stably expressing hTLR7, hTLR8 or hTLR9 were incubated with increasing series of loxoribine. The activation of NF-κB was measured by an assay for luciferase activity after 16 hours, and was expressed as a stimulation factor relative to the medium background. In FIG. 2B, HEK293 cells expressing hTLR8 were incubated with increasing series of loxoribine in the absence of homopolymer ODN6056 or heteropolymer ODN1982 or in the presence of the indicated concentrations. Activation of NF-κB is shown as the induction factor relative to the medium background. In FIG. 2C, increasing series of 7-deazaguanosine (left panel) and inosine (right panel) were assayed on hTLR8 expressing HEK293 cells in the absence (filled circles) or presence (open circles) of 5 μM ODN6056, The activation of NF-κB relative to the medium background was calculated. All data points were assayed in triplicate. Data from one of three independent experiments are shown. FIGS. 3A-3B show that ODN 6056 inhibits TLR-mediated NF-κB activation in HEK293 cells. HEK293 cells stably expressing hTLR7 and NF-κB luciferase reporter constructs were incubated with 2.5 mM loxoribine (FIG. 3A) or 2 μM R-848 (FIG. 3B) in the presence or absence of the indicated concentrations of ODN6056 for 16 hours. Incubated. Stimulation of NF-κB activation was calculated relative to the media background. All data points are assayed in triplicate and expressed as mean values (± SD). The result shown is one of two independent experiments. 4A-4D show the redirection of loxoribine-induced cytokine production by specific ODNs. Human PBMC were incubated with 1 mM loxoribine in the absence of ODN 6056 and ODN 1982, or in the presence of their respective increasing series. Supernatants were collected 24 hours later and the amounts of IFN-α (FIG. 4A), IL-12p40 (FIG. 4B), IFN-γ (FIG. 4C) and TNF-α (FIG. 4D) were measured by ELISA. The numerical value represents an average value (± SEM) of three donors. Data are from a representative one of four experiments. FIGS. 5A-5F show IL-12p40 and TNF-α production upon stimulation of human monocytes when co-cultured with loxoribine and oligo (dT) ₁₇ ODN. Human PBMC were incubated with the indicated amounts of loxoribine and ODN. Intracellular staining was performed using anti-IL-12p40 / p70 (FIG. 5A) and anti-TNF-α (FIG. 5B) antibodies. Cells were stained simultaneously with anti-CD14 and anti-CD19 antibodies. CD14 positive cells were selected from the cells, and the ratio (%) of IL-12p40 / p70 positive or TNF-α positive cells was calculated. A result shows the average value (+/- SEM) of 3 donors. Figures 5C-5F display representative dot blots of flow cytometry. One of two independent experiments is shown. FIGS. 6A-6L show IFN-γ production upon stimulation of human NK cells when co-cultured with loxoribine and oligo (dT) ₁₇ ODN6056. Human PBMC were incubated with the indicated amounts of loxoribine and ODN. Intracellular staining was performed using an anti-IFN-γ antibody. Cells were stained with anti-CD56 and anti-CD3 antibodies. CD56 positive / CD3 negative cells (NK cells) were selected from the cells. After calculating the ratio (%) of IFN-γ positive cells, it is displayed in a dot blot of flow cytometry. Two of the five donors are shown. Figures 7A and 7B show the effect of co-incubation of loxoribine and ODN on cytokine production of isolated cell populations. In FIG. 7A, monocytes were separated from human PBMC using CD14 mAb (96% purity) and incubated with the indicated amount of loxoribine in the presence or absence of ODN. The amount of IL-12p40 was determined in the supernatant by ELISA. The data displays the individual results of the two donors (gray and black bars). One of three similar experiments is shown. In FIG. 7B, PDC was enriched (85% purity) from two donor (gray and black bars) human PBMC using BDCA-4 mAb and incubated for 24 hours with the indicated combination of loxoribine and ODN. IFN-α was detected in the supernatant by ELISA. Data from one experiment out of three similar experiments are shown. FIG. 8A shows the effect on hTLR8-LUC-293 cells incubated with 50 μM R-848 for 16 hours in the presence of the indicated amount of ODN. The amount of NF-κB stimulation was measured by luciferase activity assay. The activity of NF-κB induced by 50 μM R-848 alone was set to 100%. FIG. 8B shows hTLR8-LUC-293 cells incubated for 16 hours with increasing concentrations of R-848 in the presence of 1 μM of the indicated ODN. The amount of NF-κB stimulation was measured by luciferase activity assay. FIGS. 9A and 9B show TLR7 and TLR8 signaling using a combination of a TLR7 specific ligand, 6-amino-9-benzyl-2-butoxy-9H-purin-8-ol and ODN6056. FIG. 9A shows the effect of the combination on TLR7 signaling. FIG. 9B shows the effect of the combination on TLR8 signaling. FIG. 10 shows the effect of two RNAs on TLR8 signaling. Poly (rU) ₁₈ (SEQ ID NO: 5) stimulates TLR8 in the absence of TLR7 / 8 ligand. The mixed RNA oligo (SEQ ID NO: 16) does not stimulate TLR8 alone. FIG. 11 shows the effect of two RNAs on TLR8 signaling in the presence of TLR7 / 8 ligand. FIG. 12 shows the TLR8-mediated effects of ODN6056 on TLR7 ligands (loxoribine, immunosine), TLR7, a ligand that is a combined TLR8 ligand (R848), and a compound that is not a TLR7 / 8 ligand (ribavirin). Figures 13A-D show two immunosine variants / monomers (B and C) that can be conjugated to an oligonucleotide at the immunosyn (A), 3 'or internally (B), or 5' end (C), And the structure of 6-amino-9-benzyl-2-butoxy-9H-purin-8-ol (D). FIG. 14 shows the structure of an immunosine-oligonucleotide conjugate containing a linker. FIGS. 15A-C show the structures of R848 (A), CL-029 (B) and immunosine (C), and the points at which linkers and / or oligonucleotides can be attached.

<Sequence Listing>

Claims (42)

A method of stimulating a TLR8-mediated immune response comprising administering to a subject in need thereof a TLR7 / 8 ligand and an amount of an adapter oligonucleotide effective to stimulate the TLR8-mediated immune response. Redirecting a TLR7-mediated immune response to a TLR8-mediated immune response comprising administering to a subject experiencing a TLR7-mediated immune response an amount of an adapter oligonucleotide effective to redirect the TLR7-mediated immune response to a TLR8-mediated immune response Method.   The method according to claim 1 or 2, wherein the TLR7 / 8 ligand is a TLR7-specific ligand.   The method according to claim 1 or 2, wherein the TLR7-specific ligand is a C8-substituted guanosine.   C8-substituted guanosine is 7-allyl-7,8-dihydro-8-oxoguanosine (loxoribine), 7-thia-8-oxoguanosine (immunosine), 8-mercaptoguanosine, 8-bromoguanosine, 8-methylguanosine, 8-oxo-7,8-dihydroguanosine, C8-arylamino-2'-deoxyguanosine, C8-propynylguanosine, C8- and N7-substituted guanine ribonucleosides, 7-methyl-8-oxoguanosine, 8-aminoguanosine 5. The method of claim 4, which is 8-hydroxy-2′-deoxyguanosine, 7-deaza-8-substituted guanosine, and 8-hydroxyguanosine.   5. The method of claim 4, wherein the C8 substituted guanosine is loxoribine.   4. The method of claim 3, wherein the TLR7 specific ligand is 7-deazaguanosine.   TLR7 specific ligand is 6-amino-9-benzyl-2- (3-hydroxy-propoxy) -9H-purin-8-ol or 6-amino-9-benzyl-2-butoxy-9H-purin-8-ol The method of claim 3, wherein   The method according to claim 1 or 2, wherein the TLR7 / 8 ligand is a TLR8 specific ligand.   The method according to claim 1 or 2, wherein the TLR7 / 8 ligand is a TLR7 ligand and is a TLR8 ligand.   11. The method of claim 10, wherein the TLR7 / 8 ligand is imidazoquinoline.   Imidazoquinoline is imidazoquinolineamine, imidazopyridineamine, 6,7-fused cycloalkylimidazopyridineamine, 1,2 bridged imidazoquinolineamine, R-848 (S-28463 or resiquimod), 4-amino-2 Ethoxymethyl-α, α-dimethyl-1H-imidazo [4,5-c] quinolin-1-ethanol, 1- (2-methylpropyl) -1H-imidazo [4,5-c] quinolin-4-amine ( 12. The method of claim 11, wherein R-837 or imiquimod) or S-27609.   12. The method of claim 11, wherein the imidazoquinoline is R-848.   The method of claim 1 or 2, wherein the adapter oligonucleotide comprises an unmethylated CpG motif.   The method of claim 1 or 2, wherein the adapter oligonucleotide does not comprise unmethylated CpG.   3. The method of claim 1 or 2, wherein the adapter oligonucleotide comprises 5'N-TTTTTT-N3 ', where N is any nucleotide.   The method according to claim 1 or 2, wherein the adapter oligonucleotide comprises 5'N-TTTTTT-N3 ', where N is any nucleotide.   The method according to claim 1 or 2, wherein the adapter oligonucleotide is a dT homopolymer.   The method of claim 1 or 2, wherein the adapter oligonucleotide comprises a phosphorothioate backbone modification.   3. The method of claim 1 or 2, wherein the TLR7 / 8 ligand and adapter oligonucleotide are administered separately.   3. The method of claim 1 or 2, wherein the TLR7 / 8 ligand and adapter oligonucleotide are conjugated to each other.   The method according to claim 1 or 2, wherein the adapter oligonucleotide is DNA.   The method according to claim 1 or 2, wherein the adapter oligonucleotide is RNA.   3. The method of claim 1 or 2, further comprising administering an antigen to the subject.   3. The method of claim 1 or 2, wherein the subject has an infection.   3. The method of claim 1 or 2, wherein the subject has cancer.   3. The method of claim 1 or 2, wherein the subject has allergy or asthma. Selected from the group consisting of 6-amino-9-benzyl-2- (3-hydroxy-propoxy) -9H-purin-8-ol and 6-amino-9-benzyl-2-butoxy-9H-purin-8-ol A TLR7-specific ligand,
A composition comprising an adapter oligonucleotide. A composition comprising a TLR8-specific ligand and an adapter oligonucleotide.   30. The composition of claim 28 or 29, wherein the adapter oligonucleotide comprises an unmethylated CpG motif.   30. The composition of claim 28 or 29, wherein the adapter oligonucleotide does not comprise unmethylated CpG.   30. The composition of claim 28 or 29, wherein the adapter oligonucleotide comprises 5'N-TTTTTT-N3 ', where N is any nucleotide.   30. The composition of claim 28 or 29, wherein the adapter oligonucleotide comprises 5'N-TTTTTT-N3 ', where N is any nucleotide.   30. The composition of claim 28 or 29, wherein the adapter oligonucleotide is a dT homopolymer.   30. The composition of claim 28 or 29, wherein the adapter oligonucleotide comprises at least one internucleotide phosphorothioate linkage.   30. The composition of claim 28 or 29, wherein the adapter oligonucleotide is DNA.   30. The composition of claim 28 or 29, wherein the adapter oligonucleotide is RNA.   30. The composition of claim 28 or 29, wherein the adapter oligonucleotide and the TLR7-specific or TLR8-specific ligand are conjugated to each other.   30. The composition of claim 28 or 29, further comprising an antigen.   30. A composition according to claim 28 or 29 which is a pharmaceutical formulation. A method for identifying a TLR8 ligand comprising:
Contacting TLR8-expressing cells with a test ligand in the presence and absence of an adapter oligonucleotide, and measuring the amount of stimulation of TLR8-expressing cells in response to the test ligand in the presence and absence of an adapter oligonucleotide And wherein the TLR8 ligand is identified by an increased amount of stimulation in the presence of the adapter oligonucleotide. A method for identifying an adapter oligonucleotide comprising:
Contacting the TLR8-expressing cell with a TLR7 ligand in the presence and absence of the test adapter oligonucleotide, and the amount of stimulation of the TLR8-expressing cell in response to the TLR7 ligand in the presence and absence of the test adapter oligonucleotide. A method wherein the adapter oligonucleotide is identified by an increased amount of stimulation of TLR8-expressing cells in the presence of the adapter oligonucleotide.

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