Immunostimulatory nucleic acid molecules for activating dendritic cells

Abstract

The present invention relates generally to methods and products for activating dendritic cells. In particular, the invention relates to oligonucleotides which have a specific sequence including at least one unmethylated CpG dinucleotide which are useful for activating dendritic cells. The methods are useful for in vitro, ex-vivo, and in vivo methods such as cancer immunotherapeutics, treatment of infectious disease and treatment of allergic disease.

We claim:  
     
         1 . A method for activating a dendritic cell, comprising: 
 contacting a dendritic cell with an isolated nucleic acid containing at least one unmethylated CpG dinucleotide wherein the nucleic acid is from about 8-80 bases in length in an amount effective to activate a dendritic cell.    
     
     
         2 . The method of  claim 1 , wherein the dendritic cell is an isolated dendritic cell.  
     
     
         3 . The method of  claim 1 , wherein the at least one unmethylated CpG dinucleotide has a formula: 
       5′N 1 X 1 CGX 2 N 2 3′ 
       wherein at least one nucleotide separates consecutive CpGs; X 1  is adenine, guanine, or thymine; X 2  is cytosine, adenine, or thymine; N is any nucleotide and N 1 +N 2  is from about 0-25 nucleotides.  
     
     
         4 . The method of  claim 2 , wherein the method is performed ex vivo.  
     
     
         5 . The method of  claim 1 , wherein the method is performed in vivo.  
     
     
         6 . The method of  claim 5 , wherein the isolated nucleic acid is administered to a human subject.  
     
     
         7 . The method of  claim 1 , further comprising contacting the dendritic cell with a cytokine selected from the group consisting of GM-CSF, IL-4, TNFαINF-γIL-6, Flt3 ligand, and IL-3.  
     
     
         8 . The method of  claim 4 , further comprising contacting the dendritic cell with an antigen prior to the isolated nucleic acid.  
     
     
         9 . The method of  claim 1 , wherein at least one nucleotide of the isolated nucleic acid has a phosphate backbone modification.  
     
     
         10 . The method of  claim 9 , wherein the phosphate backbone modification is a phosphorothioate or phosphorodithioate modification.  
     
     
         11 . The method of  claim 10 , wherein the phosphate backbone modification occurs at the 5′ end of the nucleic acid.  
     
     
         12 . The method of  claim 11 , wherein the phosphate backbone modification occurs at the first two internucleotide linkages of the 5′ end of the nucleic acid.  
     
     
         13 . The method of  claim 10 , wherein the phosphate backbone modification occurs at the 3′ end of the nucleic acid.  
     
     
         14 . The method of  claim 13 , wherein the phosphate backbone modification occurs at the last five internucleotide linkages of the 3′ end of the nucleic acid.  
     
     
         15 . The method of  claim 1 , wherein the at least one unmethylated CpG dinucleotide has a formula: 
       5′NX 1 X 2 CGX 3 X 4 N3′ 
       wherein at least one nucleotide separates consecutive CpGs; X 1 X 2  is selected from the group consisting of TpT, CpT, TpC, and ApT; X 3 X 4  is selected from the group consisting of GpT,GpA, ApA and ApT; N is any nucleotide and N 1 +N 2  is from about 0-25 nucleotides.  
     
     
         16 . The method of  claim 1 , wherein the isolated nucleic acid is selected from the group consisting of SEQ ID Nos. 97 and 98.  
     
     
         17 . A method for cancer immunotherapy, comprising: 
 administering an activated dendritic cell that expresses a specific cancer antigen to a subject having a cancer including the cancer antigen, wherein the activated dendritic cell is prepared by the method of  claim 1 .    
     
     
         18 . A method for treating an infectious disease, comprising: 
 administering an activated dendritic cell that expresses a specific microbial antigen to a subject having an infection with a microorganism including the microbial antigen, wherein the activated dendritic cell is prepared by the method of  claim 1 .    
     
     
         19 . A method for treating an allergy, comprising: 
 administering an activated dendritic cell that expresses a specific allergy causing antigen to a subject having an allergic reaction to the allergy causing antigen, wherein the activated dendritic cell is prepared by the method of  claim 1 .    
     
     
         20 . An isolated antigen-expressing dendritic cell population produced by the process of: 
 exposing an isolated dendritic cell to an antigen;    contacting the isolated dendritic cell with an isolated nucleic acid containing at least one unmethylated CpG dinucleotide wherein the isolated nucleic acid is from about 8-80 bases in length; and    allowing the isolated dendritic cell to process and express the antigen.    
     
     
         21 . The isolated antigen-expressing dendritic cell of  claim 20 , wherein the at least one unmethylated CpG dinucleotide has a formula: 
       5′N 1 X 1 CGX 2 N 2 3′ 
       wherein at least one nucleotide separates consecutive CpGs; X 1  is adenine, guanine, or thymine; X 2  is cytosine, adenine, or thymine; N is any nucleotide and N 1 +N 2  is from about 0-25 nucleotides.  
     
     
         22 . The isolated antigen-expressing dendritic cell of  claim 20 , wherein the isolated dendritic cell is contacted with a cytokine selected from the group consisting of GM-CSF, IL-4, TNFα, INF-γIL-6, Flt3 ligand, and IL-3.  
     
     
         23 . The isolated antigen-expressing dendritic cell of  claim 20 , wherein the dendritic cell is contacted with the antigen prior to the isolated nucleic acid.  
     
     
         24 . The isolated antigen-expressing dendritic cell of  claim 20 , wherein at least one nucleotide of the isolated nucleic acid has a phosphate backbone modification.  
     
     
         25 . The isolated antigen-expressing dendritic cell of  claim 20 , wherein the phosphate backbone modification is a phosphorothioate or phosphorodithioate modification.  
     
     
         26 . The isolated antigen-expressing dendritic cell of  claim 25 , wherein the phosphate backbone modification occurs at the 5′ end of the nucleic acid.  
     
     
         27 . The isolated antigen-expressing dendritic cell of  claim 26 , wherein the phosphate backbone modification occurs at the first two internucleotide linkages of the 5′ end of the nucleic acid.  
     
     
         28 . The isolated antigen-expressing dendritic cell of  claim 25 , wherein the phosphate backbone modification occurs at the 3′ end of the nucleic acid.  
     
     
         29 . The isolated antigen-expressing dendritic cell of  claim 28 , wherein the phosphate backbone modification occurs at the last five internucleotide linkages of the 3′ end of the nucleic acid.  
     
     
         30 . The isolated antigen-expressing dendritic cell of  claim 20 , wherein the at least one unmethylated CpG dinucleotide has a formula: 
       5′NX 1 X 2 CGX 3 X 4 N3′ 
       wherein at least one nucleotide separates consecutive CpGs; X 1 X 2  is selected from the group consisting of TpT, CpT, TpC, and ApT; X 3 X 4  is selected from the group consisting of GpT,GpA, ApA and ApT; N is any nucleotide and N 1 +N 2  is from about 0-25 nucleotides.  
     
     
         31 . A composition, comprising: 
 an effective amount for synergistically activating a dendritic cell of an isolated nucleic acid containing at least one unmethylated CpG dinucleotide wherein the nucleic acid is from about 8-80 bases in length; and    an effective amount for synergistically activating a dendritic cell of a cytokine selected from the group consisting of GM-CSF, IL-4, TNFαFlt3 ligand, and IL-3.    
     
     
         32 . The composition of  claim 31 , wherein the cytokine is GM-CSF.  
     
     
         33 . The composition of  claim 31 , further comprising an antigen.  
     
     
         34 . The composition of  claim 33 , wherein the antigen is selected from the group consisting of a cancer antigen, a microbial antigen, and an allergen.  
     
     
         35 . A screening assay for identifying compounds that are effective for preventing dendritic cell maturation, comprising: 
 contacting an immature dendritic cell with an isolated nucleic acid containing at least one unmethylated CpG dinucleotide wherein the nucleic acid is from about 8-80 bases in length;    exposing the dendritic cell to a putative drug; and    detecting the presence or absence of a maturation marker on the dendritic cell, wherein the absence of the maturation marker indicates that the putative drug is an effective compound for preventing dendritic cell maturation.    
     
     
         36 . The assay of  claim 34 , wherein the maturation marker is CD83.  
     
     
         37 . A method for generating a high yield of dendritic cells, comprising: 
 administering an isolated nucleic acid containing at least one unmethylated CpG dinucleotide wherein the nucleic acid is from about 8-80 bases in length in an amount effective for activating dendritic cells to a subject;    allowing the isolated nucleic acid to activate dendritic cells of the subject; and    isolating dendritic cells from the subject.    
     
     
         38 . A method for producing a CD40 expressing dendritic cell, comprising: 
 contacting a dendritic cell with an isolated nucleic acid containing at least one unmethylated CpG dinucleotide wherein the nucleic acid is from about 8-80 bases in length in an amount effective to produce a CD40 expressing dendritic cell.    
     
     
         39 . A method for causing maturation of a dendritic cell, comprising 
 contacting a dendritic cell with an isolated nucleic acid containing at least one unmethylated CpG dinucleotide wherein the nucleic acid is from about 8-80 bases in length in an amount effective to cause maturation of the dendritic cell.