JP2007151536A - C-class oligonucleotide analog with enhanced immunostimulatory potency - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an immunostimulatory nucleic acid, to provide a composition thereof and to provide a method for using the immunostimulatory nucleic acid. <P>SOLUTION: An immunostimulatory CpG-containing oligonucleotide and oligonucleotide analog have a skeleton modified so as to contain a nuclease-resistant internucleotide linkage and a nuclease-sensitive internucleotide linkage. The immunostimulatory CpG-containing oligonucleotide and oligonucleotide analog have a secondary structure in which an invert repeat is not a strict Watson-Crick palindrome but can be interrupted by an intervening sequence or nucleotide analog at the molecule 3'-terminal or the vicinity thereof. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

(Field of Invention)
The present invention relates generally to immunostimulatory nucleic acids, compositions thereof and methods of using the immunostimulatory nucleic acids.

(Background of the Invention)
Bacterial DNA has an immunostimulatory effect that activates B cells and natural killer cells, whereas vertebrate DNA does not have that effect. Non-patent document 1; Non-patent document 2; Non-patent document 3; and Non-patent document 4 (Review Krieg, 1998) and Non-patent document 5. Currently, these immunostimulatory effects of bacterial DNA are the result of the presence of unmethylated CpG dinucleotides (CpG motifs), particularly in the base situation, although this unmethylated CpG dinucleotide is common in bacterial DNA. It has been found that vertebrate DNA is methylated and scarce. Non-patent document 6; Non-patent document 7.

  The immunostimulatory effect of bacterial DNA can be mimicked with synthetic oligodeoxynucleotides (ODN) containing these CpG motifs. Such CpG ODNs have a highly stimulatory effect on human and mouse leukocytes, B cell proliferation; cytokine and immunoglobulin secretion; natural killer (NK) cell lytic activity and interferon gamma (IFN-γ) ) Secretion; and other antigen presentations that express dendritic cells (DCs) and costimulatory molecules and secrete cytokines, particularly Thl-like cytokines that are important in promoting the development of Thl-like T cell responses. Induces cell activation. These immunostimulatory effects of the native phosphodiester backbone CpG ODN are dramatic when the CpG motif is methylated and changed to GpC or otherwise eliminated or altered. It is very CpG specific in that Non-patent document 6; Non-patent document 8.

In early studies, the immunostimulatory CpG motif was thought to follow the formula purine-purine-CpG-pyrimidine-pyrimidine. Non-patent document 6; Non-patent document 9; Non-patent document 10; However, it is now clear that mouse lymphocytes respond very well to phosphodiester CpG motifs that do not follow this “formula” (Non-Patent Document 12), and the same is true for human B cells and dendritic cells. (Non-patent document 8; Non-patent document 13).
Tokunaga T et al., Jpn J Cancer Res (1988) 79: 682-6 Tokunaga T et al., JCI (1984) 72: 955-62. Messina JP et al., J Immunol (1991) 147: 1759-64. C. A. Stein and A.M. M.M. Krieg, (eds.), Applied Oligonucleotide Technology, John Wiley and Sons, Inc. , New York, NY, pp. 431-448 Krieg AM, Annu Rev Immunol (2002) 20: 709-60 Krieg AM et al., Nature (1995) 374: 546-9. Krieg AM, Biochim Biophys Acta (1999) 1489: 107-16 Hartmann G et al., Proc Natl. Acad Sci USA (1999) 96: 9305-10 Pisetsky DS, J Immunol (1996) 156: 421-3. Hacker H et al., EMBO J (1998) 17: 6230-40 Lipford GB et al., Trends Microbiol (1998) 6: 496-500. Yi AK et al., J Immunol (1998) 160: 5898-906. Liang H et al., J Clin Invest (1996) 98: 1119-29.

の免疫刺激性核酸分子を含む組成物であって、ここで、
Ｚ_１、Ｚ_２、およびＺ_３の各々は、独立して、０〜１２ヌクレオチド長のあらゆる配列であって、該配列は、必要に応じて、非ヌクレオチドリンカーもしくは無塩基性ｄＳｐａｃｅｒを含み；
Ｘ_１およびＸ_２の各々は、独立して、ヌクレオチドであって、該ヌクレオチドは、チミン、ウラシル、アデニン、もしくは５−置換ウラシルを含むヌクレオチドであり；
Ｙ_１およびＹ_２の各々は、独立して、シトシン（Ｃ）もしくは改変シトシンであり；
Ｒ_１およびＲ_２の各々は、独立して、グアニン（Ｇ）もしくは改変グアニンであり；
ＮおよびＮ’の各々は、独立して、０〜１２ヌクレオチド長のあらゆる配列であって、該配列は、必要に応じて、非ヌクレオチドリンカーもしくは無塩基性ｄＳｐａｃｅｒを含み；
Ｓ_１は、非ヌクレオチドリンカー、無塩基性リンカー（ｄＳｐａｃｅｒ）、トリエチレングリコールユニットもしくはヘキサエチレングリコールユニットであって、２’５’−ヌクレオシド間結合、５’５’−ヌクレオシド間結合、３’３’−ヌクレオシド間結合、２’２’−ヌクレオシド間結合、もしくは２’３’−ヌクレオシド間結合を必要に応じて提供し；
Ｓ_２は、１〜１０ヌクレオチド長のあらゆる非パリンドローム配列もしくは非ヌクレオチドリンカー、無塩基性リンカー（ｄＳｐａｃｅｒ）、トリエチレングリコールユニットもしくはヘキサエチレングリコールユニットであり；
Ｎ_１，Ｎ_２，．．．．Ｎ_ｎ、およびＮ_１＃，Ｎ_２＃，．．．Ｎ_ｎ＃の各々は、任意のヌクレオチドもしくは改変ヌクレオチドであって、ここでＮ_１は、Ｎ_１＃と塩基対を形成し、Ｎ_２は、Ｎ_２＃と塩基対を形成し、．．．そしてＮ_ｎは、Ｎ_ｎ＃と塩基対を形成し；
ｋは、０〜５までの整数であり；
ｎは、２〜１６までの整数であり；
ｐは、１〜６までの整数であり；そして
ｑは、０〜１０までの整数であり、そしてここで
（Ｎ_ｎ）．．．（Ｎ_２）（Ｎ_１）Ｓ_２（Ｎ_１＃）（Ｎ_２＃）．．．（Ｎ_ｎ＃）が１０〜４２ヌクレオチド長である場合、Ｓ_２は、４〜１０ヌクレオチド長であり、かつＳ_２は、非ヌクレオチドリンカー、無塩基性リンカー（ｄＳｐａｃｅｒ）、トリエチレングリコールユニットもしくはヘキサエチレングリコールユニットを含み、かつ／または（Ｎ_ｎ）．．．（Ｎ_２）（Ｎ_１）Ｓ_２（Ｎ_１＃）（Ｎ_２＃）．．．（Ｎ_ｎ＃）は、２／３未満のＧＣ含量を有する、
組成物。
（項目２）
Ｎ_１，Ｎ_２，．．．．Ｎ_ｎおよびＮ_１＃，Ｎ_２＃，．．．Ｎ_ｎ＃の各々は、Ｃ、Ｇ、もしくはこれらの改変体から選択され、そしてＣはＧと塩基対を形成する、項目１に記載の免疫刺激性核酸分子。
（項目３）
Ｎ_１，Ｎ_２，．．．．Ｎ_ｎおよびＮ_１＃，Ｎ_２＃，．．．Ｎ_ｎ＃の各々は、Ｔ、Ａ、もしくはこれらの改変体から選択され、そしてＴはＡと塩基対を形成する、項目１に記載の免疫刺激性核酸分子。
（項目４）
Ｎ_１，Ｎ_２，．．．．Ｎ_ｎおよびＮ_１＃，Ｎ_２＃，．．．Ｎ_ｎ＃の各々は、Ｃ、Ｔ、Ａ、Ｇ、もしくはこれらの改変体から選択され、そしてＣはＧと塩基対を形成し、ＴはＧと塩基対を形成し、ＡはＴと塩基対を形成し、そしてＡはＧと塩基対を形成する、項目１に記載の免疫刺激性核酸分子。
（項目５）
Ｎ_１，Ｎ_２，．．．．Ｎ_ｎおよびＮ_１＃，Ｎ_２＃，．．．Ｎ_ｎ＃の各々は、ワトソン−クリック塩基対を形成する、非改変ヌクレオチドもしくは改変ヌクレオチドから選択される、項目１に記載の免疫刺激性核酸分子。
（項目６）
Ｎ_１，Ｎ_２，．．．．Ｎ_ｎおよびＮ_１＃，Ｎ_２＃，．．．Ｎ_ｎ＃の各々のうちの少なくとも１つは、ワトソン−クリック塩基対を形成する、非改変ヌクレオチドもしくは改変ヌクレオチドから選択される、項目１に記載の免疫刺激性核酸分子。
（項目７）
少なくとも１つのホスホジエステル結合で部分的に安定化した骨格をさらに含む、項目１に記載の免疫刺激性核酸分子。
（項目８）
少なくとも１つの安定化したヌクレオチド間結合を有する骨格をさらに含む、項目１に記載の免疫刺激性核酸分子。
（項目９）
前記オリゴヌクレオチドのヌクレオチド間結合は、全てホスホロチオエート結合である、項目１に記載の免疫刺激性核酸分子。
（項目１０）
Ｙ_１Ｒ_１もしくはＹ_２Ｒ_２のうちの少なくとも１つを結合するホスホジエステル結合で部分的に安定化した骨格をさらに含む、項目１に記載の免疫刺激性核酸分子。
（項目１１）
Ｙ_１はＣである、項目１に記載の免疫刺激性核酸分子。
（項目１２）
Ｒ_１はＧである、項目１に記載の免疫刺激性核酸分子。
（項目１３）
Ｙ_１はＣであり、Ｒ_１はＧである、項目１に記載の免疫刺激性核酸分子。
（項目１４）
Ｘ_１もしくはＸ_２は、Ｔである、項目１に記載の免疫刺激性核酸分子。
（項目１５）
Ｘ_１はＴであり、Ｘ_２はＴであり、Ｙ_１はＣであり、Ｒ_１はＧであり、ｋは１である、項目１に記載の免疫刺激性核酸分子。
（項目１６）
Ｘ_１はＴであり、Ｘ_２はＴであり、Ｙ_１はＣであり、Ｒ_１はＧであり、ｋは１であり、ｐは１であり、ＮおよびＮ’ならびにＺ_３は、各々、ヌクレオチドを含まず、Ｚ_２はＴＴＴＴもしくはｄ（ＵＵＵＵ）である、項目１に記載の免疫刺激性核酸分子。
（項目１７）
Ｓ_２は、非ヌクレオチドリンカーである、項目１に記載の免疫刺激性核酸分子。
（項目１８）
Ｓ_２は、少なくとも１つの無塩基性ｄＳｐａｃｅｒ残基を含む、項目１に記載の免疫刺激性核酸分子。
（項目１９）
前記オリゴヌクレオチドは、少なくとも１つの分枝状非ヌクレオシド結合を含む、項目１に記載の免疫刺激性核酸分子。
（項目２０）
少なくとも１つの二重ユニット、少なくとも１つの三重ユニット、または少なくとも１つの二重ユニットと少なくとも１つの三重ユニットをさらに含む、項目１に記載の免疫刺激性核酸分子。
（項目２１）
Ｓ_１は二重ユニットまたは三重ユニットである、項目１に記載の免疫刺激性核酸分子。
（項目２２）
前記オリゴヌクレオチドは、少なくとも１つの２’５’−ヌクレオシド間結合、５’５’−ヌクレオシド間結合、３’３’−ヌクレオシド間結合、２’２’−ヌクレオシド間結合、または２’３’−ヌクレオシド間結合を含む、項目１に記載の免疫刺激性核酸分子。
（項目２３）
式ＩＩＩ：
（Ｚ’）_ｍＺ_３（Ｓ_３） （式ＩＩＩ）
の免疫刺激性核酸分子であって、ここで
Ｚ’は、以下：

であり；
Ｚ_１、Ｚ_２、およびＺ_３の各々は、独立して、０〜１２ヌクレオチド長のあらゆる配列であり、該配列は、必要に応じて、非ヌクレオチドリンカーまたは無塩基性ｄＳｐａｃｅｒを含み；
Ｘ_１およびＸ_２の各々は、独立して、チミン、ウラシル、アデニン、または５−置換ウラシルを含むヌクレオチドであり；
Ｙ_１およびＹ_２の各々は、独立して、シトシンまたは改変シトシンであり；
Ｒ_１およびＲ_２の各々は、独立して、グアニンまたは改変グアニンであり；
ＮおよびＮ’の各々は、独立して、０〜１２ヌクレオチド長のあらゆる配列であり、該配列は、必要に応じて、非ヌクレオチドリンカーまたは無塩基性ｄＳｐａｃｅｒを含み；
Ｓ_１は非ヌクレオチドリンカー、無塩基性リンカー（ｄＳｐａｃｅｒ）、トリエチレングリコールユニットまたはヘキサエチレングリコールユニットであって、必要に応じて、２’５’−ヌクレオシド間結合、５’５’−ヌクレオシド間結合、３’３’−ヌクレオシド間結合、２’２’−ヌクレオシド間結合、または２’３’−ヌクレオシド間結合を提供し；
Ｓ_２は、１〜１０ヌクレオチド長のあらゆる非パリンドローム配列または非ヌクレオチドリンカー、無塩基性リンカー（ｄＳｐａｃｅｒ）、トリエチレングリコールユニットまたはヘキサエチレングリコールユニットであり；
Ｓ_３は、直接的または間接的な２’５’−ヌクレオシド間結合、５’５’−ヌクレオシド間結合、３’３’−ヌクレオシド間結合、２’２’−ヌクレオシド間結合、または２’３’−ヌクレオシド間結合、または非ヌクレオチドリンカーであり、該非ヌクレオチドリンカーは、無塩基性リンカー（ｄＳｐａｃｅｒ）、トリエチレングリコールユニット、またはヘキサエチレングリコールユニットを含み、該無塩基性リンカーまたは該ユニットは、ｍ配列部分の２’５’−結合、５’５’−結合、３’３’−結合、２’２’−結合、または２’３’−結合を促進し；
Ｎ_１，Ｎ_２，．．．Ｎ_ｎおよびＮ_１＃，Ｎ_２＃，．．．Ｎ_ｎ＃の各々は、任意のヌクレオチドまたは改変ヌクレオチドであって、ここでＮ_１は、Ｎ_１＃と塩基対を形成し、Ｎ_２は、Ｎ_２＃と塩基対を形成し、Ｎ_３は、Ｎ_３＃と塩基対を形成し、．．．そしてＮ_ｎは、Ｎ_ｎ＃と塩基対を形成し；
ｋは、０〜５の整数であり；
ｍは、２〜１０の整数であり；
ｎは、２〜１６の整数であり；
ｐは、１〜６の整数であり；そして
ｑは、０〜１０の整数である、
免疫刺激性核酸分子。
（項目２４）

は、非パリンドローム配列である、項目１または項目２３に記載の免疫刺激性核酸分子を含む組成物。
（項目２５）

は、

であり、ＬはｄＳｐａｃｅｒである、項目１または項目２３に記載の免疫刺激性核酸分子を含む組成物。
（項目２６）

は、パリンドローム配列である、項目１または項目２３に記載の免疫刺激性核酸分子を含む組成物。
（項目２７）

は、

である、項目１または項目２３に記載の免疫刺激性核酸分子。
（項目２８）

は、

である、項目１または項目２３に記載の免疫刺激性核酸分子。
（項目２９）

は、以下の配列

を含み、ここでＬは、ｄＳｐａｃｅｒである、項目１に記載の免疫刺激性核酸分子。
（項目３０）

は、以下の配列

を含む、項目１に記載の免疫刺激性核酸分子。
（項目３１）
以下の配列：

を含む、項目１に記載の免疫刺激性核酸分子。
（項目３２）
以下の配列：

を含み、ここでＬはｄＳｐａｃｅｒである、項目１に記載の免疫刺激性核酸分子。
（項目３３）
以下の配列：

を含む、項目１に記載の免疫刺激性核酸分子。
（項目３４）
以下の配列：

を含む、項目１に記載の免疫刺激性核酸分子。
（項目３５）
以下：

から選択される配列を含み、ここでＬはｄＳｐａｃｅｒであり、

はホスホロチオエートであり、

はホスホジエステルである、項目１に記載の免疫刺激性核酸分子。
（項目３６）
以下：

から選択される配列を含み、ここで、

はホスホロチオエートであり、

はホスホジエステルである、項目１に記載の免疫刺激性核酸分子。
（項目３７）
以下：

から選択される配列を含み、ここで、

はホスホロチオエートであり、

はホスホジエステルである、項目１に記載の免疫刺激性核酸分子。
（項目３８）
以下：

から選択される配列を含み、ここで、

はホスホロチオエートであり、

はホスホジエステルである、項目１に記載の免疫刺激性核酸分子。
（項目３９）
以下の配列：

を含み、ここで、

はホスホロチオエートであり、

はホスホジエステルである、項目１に記載の免疫刺激性核酸分子。
（項目４０）
以下：

から選択される配列を含み、ここで、

はホスホロチオエートであり、

はホスホジエステルである、項目１に記載の免疫刺激性核酸分子。
（項目４１）
以下の配列：

を含み、ここで、

はホスホロチオエートであり、

はホスホジエステルである、項目１に記載の免疫刺激性核酸分子。
（項目４２）
前記オリゴヌクレオチド中の少なくとも１つのヌクレオチドは、置換プリンもしくは改変プリンまたは置換ピリミジンもしくは改変ピリミジンである、項目１に記載の免疫刺激性核酸分子。
（項目４３）
前記置換ピリミジンは、Ｃ５−置換ピリミジンもしくはＣ６−置換ピリミジンである、項目４２に記載の免疫刺激性核酸分子。
（項目４４）
前記置換プリンは、Ｃ８−置換プリンもしくはＣ７−置換プリンである、項目４２に記載の免疫刺激性核酸分子。
（項目４５）
前記置換プリンもしくは改変プリンまたは置換ピリミジンもしくは改変ピリミジンは、５−置換シトシン、６−置換シトシン、Ｎ４−置換シトシン、５−アザ−シトシン、２−メルカプト−シトシン、イソシトシン、プソイド−イソシトシン、縮合環系を有するシトシンアナログ、およびウラシル誘導体、チミン誘導体、７−デアザグアニン、７−デアザ−７−置換グアニン、７−デアザ−８−置換グアニン、７−デアザ−８−アザグアニン、ヒポキサンチン、Ｎ２−置換グアニン、５−アミノ−３−メチル−３Ｈ，６Ｈ−チアゾロ［４，５−ｄ］ピリミジン−２，７−ジオン、２，６−ジアミノプリン、２−アミノプリン、プリン、インドール、置換アデニン、８−置換グアニン、ならびに６−チオグアニンからなる群より選択される、項目４２に記載の免疫刺激性核酸分子。
（項目４６）
前記置換プリンもしくは改変プリンまたは置換ピリミジンもしくは改変ピリミジンは、５−メチル−シトシン、５−フルオロ−シトシン、５−クロロ−シトシン、５−ブロモ−シトシン、５−ヨード−シトシン、５−ヒドロキシ−シトシン、６−ヒドロキシ−シトシン、５−ヒドロキシメチル−シトシン、５−ジフルオロメチル−シトシン、ならびに非置換型もしくは置換型の５−アルキニル−シトシン、Ｎ４−エチル−シトシン、Ｎ，Ｎ’−プロピレンシトシン、フェノキサジン、５−フルオロ−ウラシル、５−ブロモ−ウラシル、５−ブロモビニル−ウラシル、４−チオ−ウラシル、５−ヒドロキシ−ウラシル、５−プロピニル−ウラシル、２−チオチミン、４−チオチミン、６−置換チミン、７−デアザ−７−（Ｃ２−Ｃ６）アルキニルグアニン、Ｎ２−メチル−グアニン、Ｎ６−メチル−アデニン、８−オキソ−アデニン、８−ヒドロキシグアニン、および８−ブロモグアニンからなる群より選択される、項目４２に記載の免疫刺激性核酸分子。
（項目４７）
前記置換プリンもしくは改変プリンまたは置換ピリミジンもしくは改変ピリミジンは、ユニバーサル塩基（ｕｎｉｖｅｒｓａｌ ｂａｓｅ）、芳香族環系、および水素原子（ｄＳｐａｃｅｒ）からなる群より選択される、項目４２に記載の免疫刺激性核酸分子。
（項目４８）
前記置換プリンもしくは改変プリンまたは置換ピリミジンもしくは改変ピリミジンは、４−メチル−インドール、５−ニトロ−インドール、３−ニトロピロール、Ｐ−塩基、およびＫ−塩基、ベンズイミダゾール、ジクロロ−ベンズイミダゾール、１−メチル−１Ｈ−［１，２，４］トリアゾール−３−カルボン酸アミド、フルオロベンゼン、およびジフルオロベンゼンからなる群より選択される、項目４２に記載の免疫刺激性核酸分子。
（項目４９）
Ｎ、Ｓ、Ｘ、またはＺのいずれかは、Ｃ６−Ｃ３０アルキル鎖、胆汁酸、コール酸、タウロコール酸、デオキシコール酸、コレステロール、オレイルリトコール酸、オレオイルコラン酸、糖脂質、リン脂質、スフィンゴ脂質、イソプレノイド、ステロイド、ビタミン類、ビタミンＥ、飽和脂肪酸、不飽和脂肪酸、脂肪酸エステル、トリグリセリド、ピレン、ポルフィリン、テキサフィリン、アダマンタン、アクリジン、ビオチン、クマリン、フルオレセイン、ローダミン、テキサスレッド、ジゴキシゲニン、ジメトキシトリチル、ｔ−ブチルジメチルシリル、ｔ−ブチルジフェニルシリル、シアニン色素類、シアニン色素Ｃｙ３、シアニン色素Ｃｙ５７６、Ｈｏｅｃｈｓｔ ３３２５８色素、ソラレン、およびイブプロフェンからなる群より選択される残基によって置換されている、項目１に記載の免疫刺激性核酸分子。
（項目５０）
（ａ）（ＴＣＧ）_ｎＮおよびＲＤＣＧＹ_１Ｙ_２Ｎから選択される免疫刺激性モチーフで始まる５’末端であって、ここでＴはチミンであり、Ｃは非メチル化シトシンであり、Ｇはグアニンであり、Ｒはプリンであり、ＤはＣでなく、Ｙ_１およびＹ_２の各々は、独立してピリミジンであり、ｎは１〜４の整数であり、Ｎは、０〜１２塩基長のあらゆる配列である、５’末端；
（ｂ）ヘアピン構造もしくはステム−ループ構造を形成することができる逆方向反復で終結する３’末端であって、該構造は、
２〜６個の連続する塩基対長のＧＣ−リッチのステム、および
少なくとも１つのマッチしない塩基またはミスマッチ塩基
を含む、３’末端；ならびに
（ｃ）少なくとも１つのホスホジエステル５’−ＣｐＧ−３’結合を含む、複数の安定化された骨格、
を含む、免疫刺激性核酸分子。
（項目５１）
前記ＧＣ−リッチステムは、２個の連続する塩基対長である、項目５０に記載の免疫刺激性核酸分子。
（項目５２）
前記ＧＣ−リッチステムは、３個の連続する塩基対長である、項目５０に記載の免疫刺激性核酸分子。
（項目５３）
前記ＧＣ−リッチステムは、４個の連続する塩基対長である、項目５０に記載の免疫刺激性核酸分子。
（項目５４）
前記ＧＣ−リッチステムは、５個の連続する塩基対長である、項目５０に記載の免疫刺激性核酸分子。
（項目５５）
前記ＧＣ−リッチステムは、６個の連続する塩基対長である、項目５０に記載の免疫刺激性核酸分子。
（項目５６）
前記ＧＣ−リッチステムは、少なくとも２個のＧ−Ｃ塩基対を含む、項目５０に記載の免疫刺激性核酸分子。
（項目５７）
前記ＧＣ−リッチステムは、少なくとも３個のＧ−Ｃ塩基対を含む、項目５０に記載の免疫刺激性核酸分子。
（項目５８）
前記少なくとも１つのマッチしない塩基またはミスマッチ塩基は、Ｔである、項目５０〜５７のいずれか１項に記載の免疫刺激性核酸分子。
（項目５９）
少なくとも１つのホスホジエステル５’−ＣｐＧ−３’結合を含む前記部分的に安定化された骨格は、複数のホスホロチオエートヌクレオチド間結合をさらに含む、項目５０に記載の免疫刺激性核酸分子。
（項目６０）
前記５’末端は、

として提供される配列を有する、項目５０に記載の免疫刺激性核酸分子。
（項目６１）
前記逆方向反復で終結する３’末端は、

として提供される配列を有する、項目５０に記載の免疫刺激性核酸分子。
（項目６２）
前記逆方向反復で終結する３’末端は、

として提供される塩基配列を有する、項目５０に記載の免疫刺激性核酸分子。
（項目６３）

として提供される塩基配列を有する、免疫刺激性核酸。
（項目６４）

として提供される塩基配列を有する、免疫刺激性核酸。
（項目６５）

として提供される配列を有し、ここで、

はホスホロチオエートヌクレオチド間結合であり、

はホスホジエステルヌクレオチド間結合である、免疫刺激性核酸。
（項目６６）

として提供される配列を有し、ここで、

はホスホロチオエートヌクレオチド間結合であり、

はホスホジエステルヌクレオチド間結合である、免疫刺激性核酸。
（項目６７）

として提供される配列を有し、ここで、

はホスホロチオエートヌクレオチド間結合であり、

はホスホジエステルヌクレオチド間結合である、免疫刺激性核酸。
（項目６８）

として提供される配列を有し、ここで、

はホスホロチオエートヌクレオチド間結合であり、

はホスホジエステルヌクレオチド間結合である、免疫刺激性核酸。
（項目６９）
項目１〜６８のいずれか１項に記載の免疫刺激性核酸分子および抗原を含む、ワクチン。
（項目７０）
項目１〜６８のいずれか１項に記載の免疫刺激性核酸分子および薬学的に受容可能なキャリアを含む、薬学的組成物。
（項目７１）
Ｉ型インターフェロン（ＩＦＮ）発現を誘導するための方法であって、該方法は、
Ｉ型ＩＦＮを発現することができる細胞と、項目１〜６８のいずれか１項に記載の免疫刺激性核酸とを、Ｉ型ＩＦＮの発現を誘導するに有効な量で接触させる工程、
を包含する、方法。
（項目７２）
前記Ｉ型ＩＦＮは、インターフェロンα（ＩＦＮ−α）である、項目７１に記載の方法。
（項目７３）
γインターフェロン（ＩＦＮ−γ）発現を誘導するための方法であって、該方法は、
ＩＦＮ−γを発現することができる細胞と、項目１〜６８のいずれか１項に記載の免疫刺激性核酸とを、ＩＦＮ−γの発現を誘導するに有効な量で接触させる工程、
を包含する、方法。
（項目７４）
ナチュラルキラー（ＮＫ）細胞を活性化するための方法であって、該方法は、
ＮＫ細胞と、項目１〜６８のいずれか１項に記載の免疫刺激性核酸とを、該ＮＫ細胞を活性化させるに有効な量で接触させる工程、
を包含する、方法。
（項目７５）
感染を処置するための方法であって、該方法は、
感染を有するか、または感染を発生させる危険性がある被験体に、該感染を処置または予防するに有効な量の、項目１〜６８のいずれか１項に記載の免疫刺激性核酸を投与する工程、
を包含する、方法。
（項目７６）
前記被験体は、ウイルス感染、細菌感染、真菌感染、または寄生生物感染から選択される感染を有するかまたは該感染を発生させる危険性がある、項目７５に記載の方法。
（項目７７）
前記被験体は、Ｂ型肝炎ウイルス（ＨＢＶ）、Ｃ型肝炎ウイルス（ＨＣＶ）、サイトメガロウイルス（ＣＭＶ）、エプスタイン−バーウイルス（ＥＢＶ）、パピローマウイルス、ヒト免疫不全ウイルス（ＨＩＶ）、または単純疱疹ウイルス（ＨＳＶ）から選択されるウイルスによるウイルス感染を有するかまたは該ウイルス感染を発生させる危険性がある、項目７５に記載の方法。
（項目７８）
前記被験体は、Ｌｅｉｓｈｍａｎｉａ、Ｌｉｓｔｅｒｉａ、またはＡｎｔｈｒａｘから選択される細菌種による細菌感染を有するかまたは該細菌感染を発生させる危険性がある、項目７５に記載の方法。
（項目７９）
アレルギー状態を処置するための方法であって、該方法は、
アレルギー状態を有するかまたは該状態を発生させる危険性がある被験体に、該アレルギー状態を処置または予防するに有効な量の、項目１〜６８のいずれか１項に記載の免疫刺激性核酸を投与する工程、
を包含する、方法。
（項目８０）
前記アレルギー状態は、アレルギー性喘息である、項目７９に記載の方法。
（項目８１）
癌を処置するための方法であって、該方法は、
癌を有するかまたは癌を発生させる危険性のある被験体に、該癌を処置または予防するに有効な量の、項目１〜６８のいずれか１項に記載の免疫刺激性核酸を投与する工程、を包含する、方法。
（項目８２）
前記癌は、基底細胞癌、胆管癌、膀胱癌、骨癌、脳および中枢神経系の癌、乳癌、子宮頚癌、絨毛癌、結腸および直腸癌、結合組織癌、消化器系の癌、子宮内膜癌、食道癌、眼の癌、頭部および頸部の癌、胃癌、上皮内新生物、腎臓癌、咽頭癌、白血病、肝癌、肺癌、ホジキンリンパ腫および非ホジキンリンパ腫を含むリンパ腫、黒色腫、骨髄腫、神経芽腫、口腔癌、卵巣癌、膵臓癌、前立腺癌、網膜芽腫、横紋筋芽細胞腫、直腸癌、腎臓癌、呼吸器系の癌、肉腫、皮膚癌、胃癌、精巣癌、甲状腺癌、子宮癌、泌尿器系の癌、または他の癌腫および肉腫から選択される、項目８１に記載の方法。
（項目８３）
前記癌は、インターフェロンα（ＩＦＮ−α）での処置に感受性の癌である、項目８１に記載の方法。
（項目８４）
前記ＩＦＮ−αでの処置に感受性の癌は、ヘアリーセル白血病、慢性骨髄性白血病、皮膚Ｔ細胞白血病、多発性骨髄腫、濾胞性リンパ腫、悪性黒色腫、扁平上皮癌、ＡＩＤＳ関連カポージ肉腫、腎細胞癌、前立腺癌腫、子宮頚部形成異常、または結腸癌腫から選択される、項目８３に記載の方法。
（項目８５）
感染の処置において使用するための医薬の製造のための、項目１〜６８のいずれか１項に記載の免疫刺激性核酸の使用。
（項目８６）
アレルギー状態の処置において使用するための医薬の製造のための、項目１〜６８のいずれか１項に記載の免疫刺激性核酸の使用。
（項目８７）
アレルギー性喘息の処置において使用するための医薬の製造のための、項目１〜６８のいずれか１項に記載の免疫刺激性核酸の使用。
（項目８８）
癌の処置において使用するための医薬の製造のための、項目１〜６８のいずれか１項に記載の免疫刺激性核酸の使用。 For example, the present invention provides the following.
(Item 1)
The following formula I:

A composition comprising the immunostimulatory nucleic acid molecule of
Each of Z ₁ , Z ₂ , and Z ₃ is independently any sequence 0-12 nucleotides in length, optionally comprising a non-nucleotide linker or an abasic dSpacer;
Each of X ₁ and X ₂ is independently a nucleotide, the nucleotide comprising a thymine, uracil, adenine, or 5-substituted uracil;
Each of Y ₁ and Y ₂ is independently cytosine (C) or a modified cytosine;
Each of R ₁ and R ₂ is independently guanine (G) or modified guanine;
Each of N and N ′ is independently any sequence 0-12 nucleotides in length, optionally comprising a non-nucleotide linker or an abasic dSpacer;
S ₁ is a non-nucleotide linker, abasic linker (dSpacer), a triethylene glycol unit or a hexaethylene glycol unit, and includes a 2′5′-nucleoside bond, a 5′5′-internucleoside bond, and a 3′3. Optionally providing a '-internucleoside bond, a 2'2'-internucleoside bond, or a 2'3'-internucleoside bond;
S ₂ is any non-palindromic sequence or a non-nucleotide linker of 1-10 nucleotides in length, abasic linkers (dSpacer), be triethylene glycol units or hexaethylene glycol units;
N ₁ , N ₂ ,. . . . N _n , and N _{1 #} , N _{2 #,.} . . Each of N _{n #} is any nucleotide or modified nucleotide, where N ₁ forms a base pair with N _{1 #} , N ₂ forms a base pair with N _{2 #} ,. . . And N _n forms a base pair with N _{n #} ;
k is an integer from 0 to 5;
n is an integer from 2 to 16;
p is an integer from 1 to 6; and q is an integer from 0 to 10, where (N _n ). . . (N ₂ ) (N ₁ ) S ₂ (N _{1 #} ) (N _{2 #} ). . . When (N _{n #} ) is 10 to 42 nucleotides long, S ₂ is 4 to 10 nucleotides long and S ₂ is a non-nucleotide linker, abasic linker (dSpacer), triethylene glycol unit or hexa Contain ethylene glycol units and / or (N _n ). . . (N ₂ ) (N ₁ ) S ₂ (N _{1 #} ) (N _{2 #} ). . . (N _{n #} ) has a GC content of less than 2/3,
Composition.
(Item 2)
N ₁ , N ₂ ,. . . . N _n and N _{1 #} , N _{2 #,.} . . The immunostimulatory nucleic acid molecule of item 1, wherein each of N _{n #} is selected from C, G, or variants thereof, and C forms a base pair with G.
(Item 3)
N ₁ , N ₂ ,. . . . N _n and N _{1 #} , N _{2 #,.} . . 2. The immunostimulatory nucleic acid molecule of item 1, wherein each of N _{n #} is selected from T, A, or a variant thereof, and T forms a base pair with A.
(Item 4)
N ₁ , N ₂ ,. . . . N _n and N _{1 #} , N _{2 #,.} . . Each of N _{n #} is selected from C, T, A, G, or variants thereof, and C forms a base pair with G, T forms a base pair with G, and A forms a base with T The immunostimulatory nucleic acid molecule of item 1, wherein the immunostimulatory nucleic acid molecule forms a pair and A forms a base pair with G.
(Item 5)
N ₁ , N ₂ ,. . . . N _n and N _{1 #} , N _{2 #,.} . . 2. The immunostimulatory nucleic acid molecule of item 1, wherein each of N _{n #} is selected from unmodified nucleotides or modified nucleotides that form Watson-Crick base pairs.
(Item 6)
N ₁ , N ₂ ,. . . . N _n and N _{1 #} , N _{2 #,.} . . 2. The immunostimulatory nucleic acid molecule of item 1, wherein at least one of each of N _{n #} is selected from unmodified nucleotides or modified nucleotides that form a Watson-Crick base pair.
(Item 7)
The immunostimulatory nucleic acid molecule of item 1, further comprising a backbone partially stabilized with at least one phosphodiester bond.
(Item 8)
The immunostimulatory nucleic acid molecule of item 1, further comprising a backbone having at least one stabilized internucleotide linkage.
(Item 9)
The immunostimulatory nucleic acid molecule according to item 1, wherein all the internucleotide bonds of the oligonucleotide are phosphorothioate bonds.
(Item 10)
The immunostimulatory nucleic acid molecule of item 1, further comprising a backbone partially stabilized with a phosphodiester bond that binds at least one of Y ₁ R ₁ or Y ₂ R ₂ .
(Item 11)
The immunostimulatory nucleic acid molecule according to item 1, wherein Y ₁ is C.
(Item 12)
The immunostimulatory nucleic acid molecule according to item 1, wherein R ₁ is G.
(Item 13)
The immunostimulatory nucleic acid molecule of item 1, wherein Y ₁ is C and R ₁ is G.
(Item 14)
The immunostimulatory nucleic acid molecule according to item 1, wherein X ₁ or X ₂ is T.
(Item 15)
The immunostimulatory nucleic acid molecule according to item 1, wherein X ₁ is T, X ₂ is T, Y ₁ is C, R ₁ is G, and k is 1.
(Item 16)
X ₁ is T, X ₂ is T, Y ₁ is C, R ₁ is G, k is 1, p is 1, N and N ′ and Z ₃ are each not contain nucleotides, _{Z 2} is TTTT or d (UUUU), immunostimulatory nucleic acid molecule of claim 1.
(Item 17)
_2. The immunostimulatory nucleic acid molecule of item 1, wherein S2 is a non-nucleotide linker.
(Item 18)
_2. The immunostimulatory nucleic acid molecule of item 1, wherein S2 comprises at least one abasic dSpacer residue.
(Item 19)
The immunostimulatory nucleic acid molecule of item 1, wherein the oligonucleotide comprises at least one branched non-nucleoside linkage.
(Item 20)
2. The immunostimulatory nucleic acid molecule of item 1, further comprising at least one duplex unit, at least one triple unit, or at least one duplex unit and at least one triple unit.
(Item 21)
S ₁ is a double unit or triple unit, immunostimulatory nucleic acid molecule of claim 1.
(Item 22)
The oligonucleotide comprises at least one 2'5'-internucleoside bond, 5'5'-internucleoside bond, 3'3'-internucleoside bond, 2'2'-internucleoside bond, or 2'3'-. The immunostimulatory nucleic acid molecule of item 1, comprising an internucleoside linkage.
(Item 23)
Formula III:
(Z ′) _m Z ₃ (S ₃ ) (Formula III)
An immunostimulatory nucleic acid molecule, wherein Z ′ is:

Is;
Each of Z ₁ , Z ₂ , and Z ₃ is independently any sequence 0-12 nucleotides in length, optionally comprising a non-nucleotide linker or an abasic dSpacer;
Each of X ₁ and X ₂ is independently a nucleotide comprising thymine, uracil, adenine, or 5-substituted uracil;
Each of Y ₁ and Y ₂ is independently cytosine or modified cytosine;
Each of R ₁ and R ₂ is independently guanine or modified guanine;
Each of N and N ′ is independently any sequence 0-12 nucleotides in length, optionally comprising a non-nucleotide linker or an abasic dSpacer;
S ₁ is a non-nucleotide linker, an abasic linker (dSpacer), a triethylene glycol unit or a hexaethylene glycol unit, and if necessary, a 2′5′-nucleoside bond, a 5′5′-internucleoside bond Providing a 3'3'-internucleoside bond, a 2'2'-internucleoside bond, or a 2'3'-internucleoside bond;
S ₂ is any non-palindromic or non-nucleotide linker of 1-10 nucleotides in length, abasic linkers (dSpacer), be triethylene glycol units or hexaethylene glycol units;
S ₃ is a direct or indirect 2′5′-internucleoside bond, 5′5′-internucleoside bond, 3′3′-internucleoside bond, 2′2′-internucleoside bond, or 2′3. A '-nucleoside linkage, or a non-nucleotide linker, wherein the non-nucleotide linker comprises an abasic linker (dSpacer), a triethylene glycol unit, or a hexaethylene glycol unit, wherein the abasic linker or unit is m Promotes 2′5′-bonding, 5′5′-bonding, 3′3′-bonding, 2′2′-bonding, or 2′3′-binding of sequence portions;
N ₁ , N ₂ ,. . . N _n and N _{1 #} , N _{2 #,.} . . Each of N _{n #} is any nucleotide or modified nucleotide, where N ₁ forms a base pair with N _{1 #} , N ₂ forms a base pair with N _{2 #,} and N ₃ , N _{3 #} and base pair. . . And N _n forms a base pair with N _{n #} ;
k is an integer from 0 to 5;
m is an integer from 2 to 10;
n is an integer from 2 to 16;
p is an integer from 1 to 6; and q is an integer from 0 to 10,
Immunostimulatory nucleic acid molecule.
(Item 24)

24. A composition comprising the immunostimulatory nucleic acid molecule of item 1 or item 23, which is a non-palindromic sequence.
(Item 25)

Is

24. The composition comprising an immunostimulatory nucleic acid molecule of item 1 or item 23, wherein L is dSpacer.
(Item 26)

24. A composition comprising an immunostimulatory nucleic acid molecule according to item 1 or item 23, which is a palindromic sequence.
(Item 27)

Is

24. The immunostimulatory nucleic acid molecule according to item 1 or item 23, wherein
(Item 28)

Is

24. The immunostimulatory nucleic acid molecule according to item 1 or item 23, wherein
(Item 29)

Is the following array

The immunostimulatory nucleic acid molecule of item 1, wherein L is dSpacer.
(Item 30)

Is the following array

The immunostimulatory nucleic acid molecule according to item 1, comprising:
(Item 31)
The following sequence:

The immunostimulatory nucleic acid molecule according to item 1, comprising:
(Item 32)
The following sequence:

The immunostimulatory nucleic acid molecule of item 1, wherein L is dSpacer.
(Item 33)
The following sequence:

The immunostimulatory nucleic acid molecule according to item 1, comprising:
(Item 34)
The following sequence:

The immunostimulatory nucleic acid molecule according to item 1, comprising:
(Item 35)
Less than:

Wherein L is dSpacer, and

Is phosphorothioate;

The immunostimulatory nucleic acid molecule of item 1, wherein is a phosphodiester.
(Item 36)
Less than:

Comprising a sequence selected from

Is phosphorothioate;

The immunostimulatory nucleic acid molecule of item 1, wherein is a phosphodiester.
(Item 37)
Less than:

Comprising a sequence selected from

Is phosphorothioate;

The immunostimulatory nucleic acid molecule of item 1, wherein is a phosphodiester.
(Item 38)
Less than:

Comprising a sequence selected from

Is phosphorothioate;

The immunostimulatory nucleic acid molecule of item 1, wherein is a phosphodiester.
(Item 39)
The following sequence:

Where, where

Is phosphorothioate;

The immunostimulatory nucleic acid molecule of item 1, wherein is a phosphodiester.
(Item 40)
Less than:

Comprising a sequence selected from

Is phosphorothioate;

The immunostimulatory nucleic acid molecule of item 1, wherein is a phosphodiester.
(Item 41)
The following sequence:

Where, where

Is phosphorothioate;

The immunostimulatory nucleic acid molecule of item 1, wherein is a phosphodiester.
(Item 42)
The immunostimulatory nucleic acid molecule according to item 1, wherein at least one nucleotide in the oligonucleotide is a substituted purine or modified purine or a substituted pyrimidine or modified pyrimidine.
(Item 43)
43. The immunostimulatory nucleic acid molecule according to item 42, wherein the substituted pyrimidine is a C5-substituted pyrimidine or a C6-substituted pyrimidine.
(Item 44)
43. The immunostimulatory nucleic acid molecule according to item 42, wherein the substituted purine is C8-substituted purine or C7-substituted purine.
(Item 45)
The substituted purine or modified purine or substituted pyrimidine or modified pyrimidine is a 5-substituted cytosine, 6-substituted cytosine, N4-substituted cytosine, 5-aza-cytosine, 2-mercapto-cytosine, isocytosine, pseudo-isocytosine, condensed ring system And uracil derivatives, thymine derivatives, 7-deazaguanine, 7-deaza-7-substituted guanine, 7-deaza-8-substituted guanine, 7-deaza-8-azaguanine, hypoxanthine, N2-substituted guanine, 5-amino-3-methyl-3H, 6H-thiazolo [4,5-d] pyrimidine-2,7-dione, 2,6-diaminopurine, 2-aminopurine, purine, indole, substituted adenine, 8-substituted A term selected from the group consisting of guanine, and 6-thioguanine Immunostimulatory nucleic acid molecule according to 42.
(Item 46)
Said substituted or modified purines or substituted pyrimidines or modified pyrimidines are 5-methyl-cytosine, 5-fluoro-cytosine, 5-chloro-cytosine, 5-bromo-cytosine, 5-iodo-cytosine, 5-hydroxy-cytosine, 6-hydroxy-cytosine, 5-hydroxymethyl-cytosine, 5-difluoromethyl-cytosine, and unsubstituted or substituted 5-alkynyl-cytosine, N4-ethyl-cytosine, N, N′-propylene cytosine, phenoxazine 5-fluoro-uracil, 5-bromo-uracil, 5-bromovinyl-uracil, 4-thio-uracil, 5-hydroxy-uracil, 5-propynyl-uracil, 2-thiothymine, 4-thiothymine, 6-substituted thymine, 7-deaza-7- (C2-C6) alkynyl 43. The immunostimulatory nucleic acid molecule of item 42, selected from the group consisting of guanine, N2-methyl-guanine, N6-methyl-adenine, 8-oxo-adenine, 8-hydroxyguanine, and 8-bromoguanine.
(Item 47)
43. The immunostimulatory nucleic acid molecule according to item 42, wherein the substituted purine or modified purine or substituted pyrimidine or modified pyrimidine is selected from the group consisting of a universal base, an aromatic ring system, and a hydrogen atom (dSpacer). .
(Item 48)
Said substituted or modified purines or substituted pyrimidines or modified pyrimidines are 4-methyl-indole, 5-nitro-indole, 3-nitropyrrole, P-base, and K-base, benzimidazole, dichloro-benzimidazole, 1- 45. The immunostimulatory nucleic acid molecule of item 42, selected from the group consisting of methyl-1H- [1,2,4] triazole-3-carboxylic acid amide, fluorobenzene, and difluorobenzene.
(Item 49)
Any of N, S, X, or Z is a C6-C30 alkyl chain, bile acid, cholic acid, taurocholic acid, deoxycholic acid, cholesterol, oleyl lithocholic acid, oleoylcholanic acid, glycolipid, phospholipid, sphingo Lipid, isoprenoid, steroid, vitamins, vitamin E, saturated fatty acid, unsaturated fatty acid, fatty acid ester, triglyceride, pyrene, porphyrin, texaphyrin, adamantane, acridine, biotin, coumarin, fluorescein, rhodamine, Texas red, digoxigenin, dimethoxytrityl, Selected from the group consisting of t-butyldimethylsilyl, t-butyldiphenylsilyl, cyanine dyes, cyanine dye Cy3, cyanine dye Cy576, Hoechst 33258 dye, psoralen, and ibuprofen 2. The immunostimulatory nucleic acid molecule according to item 1, which is substituted by a selected residue.
(Item 50)
(A) (TCG) 5 ′ end beginning with an immunostimulatory motif selected from _n N and RDCGY ₁ Y ₂ N, where T is thymine, C is unmethylated cytosine, and G is Guanine, R is purine, D is not C, each of Y ₁ and Y ₂ is independently a pyrimidine, n is an integer from 1 to 4, and N is 0 to 12 bases in length The 5 ′ end of any sequence of
(B) a 3 ′ end that terminates in an inverted repeat capable of forming a hairpin structure or a stem-loop structure,
A 3 ′ end comprising 2 to 6 consecutive base-paired GC-rich stems and at least one unmatched or mismatched base; and (c) at least one phosphodiester 5′-CpG-3 ′ Multiple stabilized skeletons, including bonds,
An immunostimulatory nucleic acid molecule comprising:
(Item 51)
51. The immunostimulatory nucleic acid molecule of item 50, wherein the GC-rich stem is two consecutive base pair lengths.
(Item 52)
51. The immunostimulatory nucleic acid molecule of item 50, wherein the GC-rich stem is 3 consecutive base pairs long.
(Item 53)
51. The immunostimulatory nucleic acid molecule of item 50, wherein the GC-rich stem is 4 consecutive base pairs long.
(Item 54)
51. The immunostimulatory nucleic acid molecule of item 50, wherein the GC-rich stem is 5 consecutive base pairs long.
(Item 55)
51. The immunostimulatory nucleic acid molecule of item 50, wherein the GC-rich stem is 6 consecutive base pairs long.
(Item 56)
51. The immunostimulatory nucleic acid molecule of item 50, wherein the GC-rich stem comprises at least two GC base pairs.
(Item 57)
51. The immunostimulatory nucleic acid molecule of item 50, wherein the GC-rich stem comprises at least 3 GC base pairs.
(Item 58)
58. The immunostimulatory nucleic acid molecule of any one of items 50 to 57, wherein the at least one unmatched base or mismatched base is T.
(Item 59)
51. The immunostimulatory nucleic acid molecule of item 50, wherein the partially stabilized backbone comprising at least one phosphodiester 5'-CpG-3 'linkage further comprises a plurality of phosphorothioate internucleotide linkages.
(Item 60)
The 5 ′ end is

51. The immunostimulatory nucleic acid molecule of item 50, having the sequence provided as
(Item 61)
The 3 ′ end terminating in the inverted repeat is

51. The immunostimulatory nucleic acid molecule of item 50, having the sequence provided as
(Item 62)
The 3 ′ end terminating in the inverted repeat is

51. The immunostimulatory nucleic acid molecule according to item 50, having a base sequence provided as
(Item 63)

An immunostimulatory nucleic acid having the base sequence provided as
(Item 64)

An immunostimulatory nucleic acid having the base sequence provided as
(Item 65)

Having the sequence provided as

Is a phosphorothioate internucleotide linkage;

Is an immunostimulatory nucleic acid which is a phosphodiester internucleotide linkage.
(Item 66)

Having the sequence provided as

Is a phosphorothioate internucleotide linkage;

Is an immunostimulatory nucleic acid which is a phosphodiester internucleotide linkage.
(Item 67)

Having the sequence provided as

Is a phosphorothioate internucleotide linkage;

Is an immunostimulatory nucleic acid which is a phosphodiester internucleotide linkage.
(Item 68)

Having the sequence provided as

Is a phosphorothioate internucleotide linkage;

Is an immunostimulatory nucleic acid which is a phosphodiester internucleotide linkage.
(Item 69)
70. A vaccine comprising the immunostimulatory nucleic acid molecule according to any one of items 1 to 68 and an antigen.
(Item 70)
70. A pharmaceutical composition comprising the immunostimulatory nucleic acid molecule of any one of items 1-68 and a pharmaceutically acceptable carrier.
(Item 71)
A method for inducing type I interferon (IFN) expression comprising:
Contacting a cell capable of expressing type I IFN with the immunostimulatory nucleic acid according to any one of items 1 to 68 in an amount effective to induce expression of type I IFN;
Including the method.
(Item 72)
72. The method of item 71, wherein the type I IFN is interferon α (IFN-α).
(Item 73)
A method for inducing gamma interferon (IFN-γ) expression comprising:
Contacting a cell capable of expressing IFN-γ with the immunostimulatory nucleic acid according to any one of Items 1 to 68 in an amount effective to induce expression of IFN-γ,
Including the method.
(Item 74)
A method for activating natural killer (NK) cells comprising:
Contacting the NK cell with the immunostimulatory nucleic acid according to any one of items 1 to 68 in an amount effective to activate the NK cell;
Including the method.
(Item 75)
A method for treating an infection comprising:
70. An immunostimulatory nucleic acid according to any one of items 1 to 68 is administered to a subject having or at risk of developing an infection in an amount effective to treat or prevent the infection. Process,
Including the method.
(Item 76)
76. The method of item 75, wherein the subject has or is at risk of developing an infection selected from a viral infection, a bacterial infection, a fungal infection, or a parasitic infection.
(Item 77)
The subject may be hepatitis B virus (HBV), hepatitis C virus (HCV), cytomegalovirus (CMV), Epstein-Barr virus (EBV), papilloma virus, human immunodeficiency virus (HIV), or herpes simplex. 76. The method of item 75, wherein the method has or is at risk of causing a viral infection with a virus selected from viruses (HSV).
(Item 78)
76. The method of item 75, wherein the subject has or is at risk of developing a bacterial infection with a bacterial species selected from Leishmania, Listeria, or Anthrax.
(Item 79)
A method for treating an allergic condition, the method comprising:
69. An immunostimulatory nucleic acid according to any one of items 1 to 68 in an amount effective to treat or prevent the allergic condition in a subject who has or is at risk of developing the allergic condition. Administering,
Including the method.
(Item 80)
80. The method of item 79, wherein the allergic condition is allergic asthma.
(Item 81)
A method for treating cancer comprising the steps of:
70. A step of administering an immunostimulatory nucleic acid according to any one of items 1 to 68 to a subject who has cancer or is at risk of developing cancer, in an amount effective to treat or prevent the cancer. Including.
(Item 82)
The cancers include basal cell cancer, bile duct cancer, bladder cancer, bone cancer, brain and central nervous system cancer, breast cancer, cervical cancer, choriocarcinoma, colon and rectal cancer, connective tissue cancer, cancer of the digestive system, uterus Endometrial cancer, esophageal cancer, eye cancer, head and neck cancer, stomach cancer, intraepithelial neoplasia, kidney cancer, pharyngeal cancer, leukemia, liver cancer, lung cancer, lymphoma including Hodgkin lymphoma and non-Hodgkin lymphoma, melanoma , Myeloma, neuroblastoma, oral cancer, ovarian cancer, pancreatic cancer, prostate cancer, retinoblastoma, rhabdomyoblastoma, rectal cancer, kidney cancer, respiratory cancer, sarcoma, skin cancer, stomach cancer, 82. The method of item 81, selected from testicular cancer, thyroid cancer, uterine cancer, urinary cancer, or other carcinomas and sarcomas.
(Item 83)
82. The method of item 81, wherein the cancer is a cancer sensitive to treatment with interferon α (IFN-α).
(Item 84)
Cancers sensitive to treatment with IFN-α include hairy cell leukemia, chronic myelogenous leukemia, cutaneous T cell leukemia, multiple myeloma, follicular lymphoma, malignant melanoma, squamous cell carcinoma, AIDS-related Kaposi's sarcoma, kidney 84. The method of item 83, wherein the method is selected from cell carcinoma, prostate carcinoma, cervical dysplasia, or colon carcinoma.
(Item 85)
69. Use of an immunostimulatory nucleic acid according to any one of items 1 to 68 for the manufacture of a medicament for use in the treatment of an infection.
(Item 86)
69. Use of an immunostimulatory nucleic acid according to any one of items 1 to 68 for the manufacture of a medicament for use in the treatment of an allergic condition.
(Item 87)
69. Use of an immunostimulatory nucleic acid according to any one of items 1 to 68 for the manufacture of a medicament for use in the treatment of allergic asthma.
(Item 88)
69. Use of an immunostimulatory nucleic acid according to any one of items 1 to 68 for the manufacture of a medicament for use in the treatment of cancer.

(Summary of the Invention)
The present invention relates in part to immunostimulatory CpG-containing oligonucleotides and oligonucleotide analogs having secondary structures with inverted repeats at or near the 3 ′ end of the molecule. This secondary structure is responsible for the formation of duplex or higher order structures under certain conditions. As an important feature of the oligonucleotides and oligonucleotide analogs of the present invention, this inverted repeat is not a strict Watson-Crick palindrome, but can intervene between intervening sequences or nucleotide analogs. As another feature of the oligonucleotides and oligonucleotide analogs of the present invention, the backbone has been modified to include strategically located nuclease resistant and nuclease sensitive internucleotide linkages, thereby making them both active. Convenient and may reduce potential toxicity. Furthermore, it can be seen that these oligonucleotides and oligonucleotide analogs exert both A-class and B-class immunostimulatory activity and are therefore classified as novel C-class immunostimulatory nucleic acid molecules.

  The present invention relates to CpG-containing immunostimulatory oligonucleotides and oligonucleotide analogs, including incomplete palindrome at or near the 3 ′ end of the molecule, their preparation and their biological activity. Based on the discovery by the present invention that it has certain advantages in both. Specifically, the C-class oligonucleotides and oligonucleotide analogs of the present invention are characteristically monomers in solution. It is believed that these same nucleic acid molecules form an intramolecular duplex structure in vitro, which can stabilize these molecules against nuclease digestion. These same nucleic acid molecules can form intramolecular duplexes, and possibly higher order structures within the environment of the endosomal compartment, where these molecules exert their biological activity. Conceivable.

  In one aspect, the present invention provides the following formula I:

の免疫刺激性核酸分子を含む組成物を提供し、
ここでＺ_１、Ｚ_２、およびＺ_３の各々は、独立して、０〜１２ヌクレオチド長のあらゆる配列であって、この配列は、必要に応じて、非ヌクレオチドリンカーもしくは無塩基性ｄＳｐａｃｅｒを含み；Ｘ_１およびＸ_２の各々は、独立して、ヌクレオチドであって、該ヌクレオチドは、チミン、ウラシル、アデニン、もしくは５−置換ウラシルを含み；Ｙ_１およびＹ_２の各々は、独立して、シトシン（Ｃ）もしくは改変シトシンであり；Ｒ_１およびＲ_２の各々は、独立して、グアニン（Ｇ）もしくは改変グアニンであり；ＮおよびＮ’の各々は、独立して、０〜１２ヌクレオチド長のあらゆる配列であって、この配列は、必要に応じて、非ヌクレオチドリンカーもしくは無塩基性ｄＳｐａｃｅｒを含み；Ｓ_１は、非ヌクレオチドリンカー、無塩基性リンカー（ｄＳｐａｃｅｒ）、トリエチレングリコールユニットもしくはヘキサエチレングリコールユニットであって、２’５’−ヌクレオシド間結合、５’５’−ヌクレオシド間結合、３’３’−ヌクレオシド間結合、２’２’−ヌクレオシド間結合、もしくは２’３’−ヌクレオシド間結合を必要に応じて提供し；Ｓ_２は、１〜１０ヌクレオチド長のあらゆる非パリンドローム配列もしくは非ヌクレオチドリンカー、無塩基性リンカー（ｄＳｐａｃｅｒ）、トリエチレングリコールユニットもしくはヘキサエチレングリコールユニットであり；Ｎ_１，Ｎ_２，．．．．Ｎ_ｎ、およびＮ_１＃，Ｎ_２＃，．．．Ｎ_ｎ＃の各々は、任意のヌクレオチドもしくは改変ヌクレオチドであって、ここでＮ_１は、Ｎ_１＃と塩基対を形成し、Ｎ_２は、Ｎ_２＃と塩基対を形成し、．．．そしてＮ_ｎは、Ｎ_ｎ＃と塩基対を形成し；ｋは、０〜５までの整数であり；ｎは、２〜１６までの整数であり；ｐは、１〜６までの整数であり；そしてｑは、０〜１０までの整数であり、そしてここで（Ｎ_ｎ）．．．（Ｎ_２）（Ｎ_１）Ｓ_２（Ｎ_１＃）（Ｎ_２＃）．．．（Ｎ_ｎ＃）が１０〜４２ヌクレオチド長である場合、Ｓ_２は、４〜１０ヌクレオチド長であり、かつＳ_２は、非ヌクレオチドリンカー、無塩基性リンカー（ｄＳｐａｃｅｒ）、トリエチレングリコールユニットもしくはヘキサエチレングリコールユニットを含み、かつ／または（Ｎ_ｎ）．．．（Ｎ_２）（Ｎ_１）Ｓ_２（Ｎ_１＃）（Ｎ_２＃）．．．（Ｎ_ｎ＃）は、２／３未満のＧＣ含量を有する。

Providing a composition comprising the immunostimulatory nucleic acid molecule of
Where each of Z ₁ , Z ₂ , and Z ₃ is independently any sequence 0-12 nucleotides in length, optionally comprising a non-nucleotide linker or an abasic dSpacer. Each of X ₁ and X ₂ is independently a nucleotide, the nucleotide comprising thymine, uracil, adenine, or 5-substituted uracil; each of Y ₁ and Y ₂ is independently Each of R ₁ and R ₂ is independently guanine (G) or a modified guanine; each of N and N ′ is independently 0-12 nucleotides in length Wherein the sequence optionally includes a non-nucleotide linker or abasic dSpacer; S ₁ is a non-nucleotide linker An abasic linker (dSpacer), a triethylene glycol unit or a hexaethylene glycol unit, comprising a 2′5′-nucleoside bond, a 5′5′-nucleoside bond, a 3′3′-nucleoside bond, 2 '2'internucleoside linkage, or 2'3' internucleoside linkage as necessary to provide; S ₂ is any non-palindromic sequence or a non-nucleotidic linker of 1-10 nucleotides in length, abasic linkers ( dSpacer), a triethylene glycol unit or a hexaethylene glycol unit; N ₁ , N ₂ ,. . . . N _n , and N _{1 #} , N _{2 #,.} . . Each of N _{n #} is any nucleotide or modified nucleotide, where N ₁ forms a base pair with N _{1 #} , N ₂ forms a base pair with N _{2 #} ,. . . And N _n forms a base pair with N _{n #} ; k is an integer from 0 to 5; n is an integer from 2 to 16; p is an integer from 1 to 6 And q is an integer from 0 to 10 and where (N _n ). . . (N ₂ ) (N ₁ ) S ₂ (N _{1 #} ) (N _{2 #} ). . . When (N _{n #} ) is 10 to 42 nucleotides long, S ₂ is 4 to 10 nucleotides long and S ₂ is a non-nucleotide linker, abasic linker (dSpacer), triethylene glycol unit or hexa Contain ethylene glycol units and / or (N _n ). . . (N ₂ ) (N ₁ ) S ₂ (N _{1 #} ) (N _{2 #} ). . . (N _{n #} ) has a GC content of less than 2/3.

In one embodiment, N ₁ , N ₂ ,. . . . N _n and N _{1 #} , N _{2 #} . . . Each of N _{n #} is selected from C, G, or a variant thereof, where C forms a base pair with G.

In one embodiment, N ₁ , N ₂ ,. . . . N _n and N _{1 #} , N _{2 #,.} . . Each of N _{n #} is selected from T, A, or a variant thereof, and T forms a base pair with A.

  In these and other embodiments, each of C, G, A, and T is a deoxynucleotide and may refer to the corresponding bases cytosine, guanine, adenine, and thymine.

In one embodiment, N ₁ , N ₂ ,. . . . N _n and N _{1 #} , N _{2 #,.} . . Each of N _{n #} is selected from C, T, A, G, or variants thereof, and C forms a base pair with G, T forms a base pair with G, and A forms a base with T Forms a pair, and A forms a base pair with G.

In one embodiment, N ₁ , N ₂ ,. . . N _n and N _{1 #} , N _{2 #,.} . . Each of N _{n #} is selected from unmodified or modified nucleotides forming Watson-Crick base pairs, ie, each base pair (N ₁ -N _{1 #} , N ₂ -N _{2 #} ,... N _n- Nn _# ) is a Watson-Crick base pair.

In one embodiment, N ₁ , N ₂ ,. . . . N _n and N _{1 #} , N _{2 #,.} . . At least one of each of N _{n #} is selected from unmodified nucleotides or modified nucleotides that form Watson-Crick base pairs, ie, at least one base pair (N ₁ -N _{1 #} , N ₂ − _{n 2 #, ... n n -N} n #) is a non-Watson - is Crick base pairs.

  In one embodiment, the immunostimulatory nucleic acid molecule comprises a backbone that is partially stabilized with at least one phosphodiester bond.

  In one embodiment, the immunostimulatory nucleic acid molecule comprises a backbone having at least one stabilized internucleotide linkage.

  In one embodiment, the internucleotide linkages of the oligonucleotide are all phosphorothioate linkages.

In one embodiment, the immunostimulatory nucleic acid molecule comprises a backbone that is partially stabilized with a phosphodiester bond that binds at least one of Y ₁ R ₁ or Y ₂ R ₂ .

In one embodiment, Y ₁ is C.

In one embodiment, R ₁ is G.

In one embodiment, Y ₁ is C and R ₁ is G.

In one embodiment, X ₁ or X ₂ is T.

In one embodiment, X ₁ is T, X ₂ is T, Y ₁ is C, R ₁ is G, and k is 1.

In one embodiment, X ₁ is T, X ₂ is T, Y ₁ is C, R ₁ is G, k is 1, p is 1, N and N ′ and Z ₃ each contains no nucleotides and Z ₂ is TTTT or d (UUUU), where d (UUUU) represents dUdUdUdU (ie, (deoxyU) ₄ ).

In one embodiment, S ₂ is a non-nucleotide linker.

In one embodiment, _{S 2} contains at least one abasic dSpacer residue.

  In one embodiment, the oligonucleotide comprises at least one branched non-nucleoside linkage.

  In one embodiment, the immunostimulatory nucleic acid molecule comprises at least one duplex unit, at least one triple unit, or at least one duplex unit and at least one triple unit.

In one embodiment, S ₁ is a double unit or a triple unit.

  In one embodiment, the oligonucleotide comprises at least one 2'5'-internucleoside linkage, 5'5'-internucleoside linkage, 3'3'-internucleoside linkage, 2'2'-internucleoside linkage, or Contains a 2'3'-internucleoside linkage.

  In one embodiment, the immunostimulatory nucleic acid molecule of formula I is not an antisense nucleic acid.

  In one aspect, the present invention provides a compound of formula II:

の免疫刺激性核酸分子を提供し、ここでＺ_１、Ｚ_２、およびＺ_３の各々は、独立して、０〜１２個のヌクレオチド長のあらゆる配列であり、この配列は、必要に応じて、非ヌクレオチドリンカーまたは無塩基性ｄＳｐａｃｅｒを含み；Ｘ_１およびＸ_２の各々は、独立して、チミン、ウラシル、アデニン、または５−置換ウラシルを含み；Ｙ_１およびＹ_２の各々は、独立して、シトシン（Ｃ）または改変シトシンであり；Ｒ_１およびＲ_２の各々は、独立して、グアニン（Ｇ）または改変グアニンであり；Ｎは、０〜１２個のヌクレオチド長のあらゆる配列であり、この配列は、必要に応じて、非ヌクレオチドリンカーまたは無塩基性ｄＳｐａｃｅｒを含み；Ｓ_１は非ヌクレオチドリンカー、無塩基性リンカー（ｄＳｐａｃｅｒ）、トリエチレングリコールユニットまたはヘキサエチレングリコールユニットであって、必要に応じて、２’５’−ヌクレオシド間結合、５’５’−ヌクレオシド間結合、３’３’−ヌクレオシド間結合、２’２’−ヌクレオシド間結合、または２’３’−ヌクレオシド間結合を提供し；Ｓ_２は、１〜１０個のヌクレオチド長のあらゆる非パリンドローム配列または非ヌクレオチドリンカー、無塩基性リンカー（ｄＳｐａｃｅｒ）、トリエチレングリコールユニットまたはヘキサエチレングリコールユニットであり；Ｎ_１，Ｎ_２，．．．．Ｎ_ｎ−１，Ｎ_ｎ、およびＮ_１＃，Ｎ_２＃，．．．Ｎ_ｎ−１＃，Ｎ_ｎ＃の各々は、任意のヌクレオチドもしくは改変ヌクレオチドであって、ここでＮ_１は、Ｎ_１＃と塩基対を形成し、Ｎ_２は、Ｎ_２＃と塩基対を形成し、．．．Ｎ_ｎ−１は、Ｎ_ｎ−１＃と塩基対を形成し、そしてＮ_ｎは、Ｎ_ｎ＃と塩基対を形成し；ｋは、０〜５の整数であり；ｎは、２〜１６の整数であり；ｐは、１〜６の整数であり；そしてｑは、０〜１０の整数であり；そしてここで（Ｎ_ｎ）．．．（Ｎ_２）（Ｎ_１）Ｓ_２（Ｎ_１＃）（Ｎ_２＃）．．．（Ｎ_ｎ＃）が１０〜４２ヌクレオチド長である場合、Ｓ_２は、４〜１０ヌクレオチド長であり、かつＳ_２は、非ヌクレオチドリンカー、無塩基性リンカー（ｄＳｐａｃｅｒ）、トリエチレングリコールユニットもしくはヘキサエチレングリコールユニットを含み、かつ／または（Ｎ_ｎ）．．．（Ｎ_２）（Ｎ_１）Ｓ_２（Ｎ_１＃）（Ｎ_２＃）．．．（Ｎ_ｎ＃）は、２／３未満のＧＣ含量を有する。

Wherein each of Z ₁ , Z ₂ , and Z ₃ is independently any sequence 0-12 nucleotides in length, wherein the sequence is optionally Each of X ₁ and X ₂ independently includes thymine, uracil, adenine, or 5-substituted uracil; each of Y ₁ and Y ₂ is independently Each of R ₁ and R ₂ is independently guanine (G) or a modified guanine; N is any sequence 0-12 nucleotides in length the sequence may optionally include a non-nucleotide linker or abasic dSpacer; _{S 1} is non-nucleotide linker, an abasic linker (dSpacer), tri Tylene glycol unit or hexaethylene glycol unit, optionally 2'5'-internucleoside bond, 5'5'-internucleoside bond, 3'3'-internucleoside bond, 2'2'-nucleoside providing between bond, or 2'3' internucleoside linkage; _{S 2} is 1 to 10 nucleotides long any non palindromic or non-nucleotide linker, an abasic linker (dSpacer), triethylene glycol units Or a hexaethylene glycol unit; N ₁ , N ₂ ,. . . . N _n-1 , N _n , and N _{1 #} , N _{2 #,.} . . Each of N _{n-1 #} and N _{n #} is any nucleotide or modified nucleotide, where N ₁ forms a base pair with N _{1 #} and N ₂ forms a base pair with N _{2 #} Forming,. . . N _{n-1 forms} a _{N n-1 #} base pairs, and _{N n} form a _{N n #} base pair; k is an integer from 0 to 5; n is 2 to 16 P is an integer from 1 to 6; and q is an integer from 0 to 10; and where (N _n ). . . (N ₂ ) (N ₁ ) S ₂ (N _{1 #} ) (N _{2 #} ). . . When (N _{n #} ) is 10 to 42 nucleotides long, S ₂ is 4 to 10 nucleotides long and S ₂ is a non-nucleotide linker, abasic linker (dSpacer), triethylene glycol unit or hexa Contain ethylene glycol units and / or (N _n ). . . (N ₂ ) (N ₁ ) S ₂ (N _{1 #} ) (N _{2 #} ). . . (N _{n #} ) has a GC content of less than 2/3.

In one embodiment, Z ₁ (N _n ) (N _n-1 ) is TYR, where Y is cytosine or modified cytosine and R is guanine or modified guanine.

In one embodiment, N ₁ , N ₂ ,. . . . N _n-1 , N _n , and N _{1 #} , N _{2 #,.} . . Each of N _{n-1 #} , N _{n #} is selected from C, G, or a modification thereof, where C forms a base pair with G.

In one embodiment, N ₁ , N ₂ ,. . . . N _n-1 , N _n , and N _{1 #} , N _{2 #,.} . . Each of N _{n-1 #} and N _{n #} is selected from T, A or a modification thereof, where T forms a base pair with A.

  In these and other embodiments, each of C, G, A, and T is a deoxynucleotide and may refer to the corresponding bases cytosine, guanine, adenine, and thymine.

In one embodiment, N ₁ , N ₂ ,. . . . N _n-1 , N _n , and N _{1 #} , N _{2 #,.} . . Each of N _{n-1 #} , N _{n #} is selected from C, T, A, G, or variants thereof, and C forms a base pair with G and T forms a base pair with G , A forms a base pair with T, and A forms a base pair with G.

In one embodiment, N ₁ , N ₂ ,. . . . N _n−1 , N _n and N _{1 #} , N _{2 #,.} . . Each of N _{n-1 #} , N _{n #} is selected from unmodified nucleotides or modified nucleotides that form Watson-Crick base pairs, ie, each base pair (N ₁ -N _{1 #} , N ₂ -N _{2 #} ... _{n n -N} n _#) is Watson - is Crick base pairs.

In one embodiment, N ₁ , N ₂ ,. . . . N _n−1 , N _n and N _{1 #} , N _{2 #,.} . . At least one of each of N _{n-1 #} , N _{n #} is selected from unmodified nucleotides or modified nucleotides that form non-Watson-Crick base pairs, ie, at least one base pair N ₁ -N _{1 #} , N ₂ -N _{2 #} . . . N _n -N _{n #} is a non-Watson-Crick base pair.

  In one embodiment, the immunostimulatory nucleic acid molecule comprises a backbone that is partially stabilized with at least one phosphodiester bond.

  In one embodiment, the immunostimulatory nucleic acid molecule comprises a backbone having at least one stabilized internucleotide linkage.

  In one embodiment, the internucleotide linkages of the oligonucleotide are all phosphorothioate linkages.

In one embodiment, the immunostimulatory nucleic acid molecule comprises a backbone that is partially stabilized with a phosphodiester bond that binds at least one of Y ₁ R ₁ or Y ₂ R ₂ .

In one embodiment, Y ₁ is C.

In one embodiment, R ₁ is G.

In one embodiment, Y ₁ is C and R ₁ is G.

In one embodiment, X ₁ or X ₂ is T.

In one embodiment, X ₁ is T, X ₂ is T, Y ₁ is C, R ₁ is G, and k is 1.

In one embodiment, X ₁ is T, X ₂ is T, Y ₁ is C, R ₁ is G, k is 1, p is 1, N and N ′ and Z ₃ each contains no nucleotides and Z ₂ is TTTT or d (UUUU), where d (UUUU) represents (deoxy U) ₄ .

In one embodiment, S ₂ is a non-nucleotide linker.

In one embodiment, _{S 2} contains at least one abasic dSpacer residue.

  In one embodiment, the oligonucleotide comprises at least one branched non-nucleoside linkage.

  In one embodiment, the immunostimulatory nucleic acid molecule comprises at least one duplex unit, at least one triple unit, or at least one duplex unit and at least one triple unit.

In one embodiment, S ₁ is a double unit or a triple unit.

  In one embodiment, the oligonucleotide comprises at least one 2'5'-internucleoside linkage, 5'5'-internucleoside linkage, 3'3'-internucleoside linkage, 2'2'-internucleoside linkage, or Contains a 2'3'-internucleoside linkage.

  In one embodiment, the immunostimulatory nucleic acid molecule of formula I is not an antisense nucleic acid.

In one aspect, the present invention provides a compound of formula III:
(Z ′) _m Z ₃ (S ₃ ) (Formula III)
Of the immunostimulatory nucleic acid molecule, wherein Z ′ is:

であり；Ｚ_１、Ｚ_２、およびＺ_３の各々は、独立して、０〜１２ヌクレオチド長のあらゆる配列であり、この配列は、必要に応じて、非ヌクレオチドリンカーまたは無塩基性ｄＳｐａｃｅｒを含み；Ｘ_１およびＸ_２の各々は、独立して、チミン、ウラシル、アデニン、または５−置換ウラシルを含み；Ｙ_１およびＹ_２の各々は、独立して、シトシンまたは改変シトシンであり；Ｒ_１およびＲ_２の各々は、独立して、グアニンまたは改変グアニンであり；ＮおよびＮ’の各々は、独立して、０〜１２ヌクレオチド長のあらゆる配列であり、この配列は、必要に応じて、非ヌクレオチドリンカーまたは無塩基性ｄＳｐａｃｅｒを含み；Ｓ_１は非ヌクレオチドリンカー、無塩基性リンカー（ｄＳｐａｃｅｒ）、トリエチレングリコールユニットまたはヘキサエチレングリコールユニットであって、必要に応じて、２’５’−ヌクレオシド間結合、５’５’−ヌクレオシド間結合、３’３’−ヌクレオシド間結合、２’２’−ヌクレオシド間結合、または２’３’−ヌクレオシド間結合を提供し；Ｓ_２は、１〜１０ヌクレオチド長のあらゆる非パリンドローム配列または非ヌクレオチドリンカー、無塩基性リンカー（ｄＳｐａｃｅｒ）、トリエチレングリコールユニットまたはヘキサエチレングリコールユニットであり；Ｓ_３は、直接的または間接的な２’５’−ヌクレオシド間結合、５’５’−ヌクレオシド間結合、３’３’−ヌクレオシド間結合、２’２’−ヌクレオシド間結合、または２’３’−ヌクレオシド間結合、または非ヌクレオチドリンカーであり、この非ヌクレオチドリンカーは、無塩基性リンカー（ｄＳｐａｃｅｒ）、トリエチレングリコールユニット、またはヘキサエチレングリコールユニットを含み、該無塩基性リンカーまたは該ユニットは、ｍ配列部分の２’５’−結合、５’５’−結合、３’３’−結合、２’２’−結合、または２’３’−結合を促進し；Ｎ_１，Ｎ_２，．．．Ｎ_ｎおよびＮ_１＃，Ｎ_２＃，．．．Ｎ_ｎ＃の各々は、任意のヌクレオチドまたは改変ヌクレオチドであって、ここでＮ_１は、Ｎ_１＃と塩基対を形成し、Ｎ_２は、Ｎ_２＃と塩基対を形成し、Ｎ_３は、Ｎ_３＃と塩基対を形成し、．．．そしてＮ_ｎは、Ｎ_ｎ＃と塩基対を形成し；ｋは、０〜５までの整数であり；ｍは、２〜１０までの整数であり；ｎは、２〜１６までの整数であり；ｐは、１〜６までの整数であり；そしてｑは、０〜１０までの整数である。

Each of Z ₁ , Z ₂ , and Z ₃ is independently any sequence 0-12 nucleotides in length, which sequence optionally includes a non-nucleotide linker or an abasic dSpacer ; each of _{X 1} and _{X 2} are independently thymine, uracil, adenine or 5-substituted uracil comprises; each of _{Y 1} and _{Y 2} are independently cytosine or a modified cytosine; _{R 1} And each of R ₂ is independently guanine or modified guanine; each of N and N ′ is independently any sequence 0-12 nucleotides in length, which sequence may optionally be A non-nucleotide linker or abasic dSpacer; S ₁ is a non-nucleotide linker, abasic linker (dSpacer), triethylene glycol unit Or a hexaethylene glycol unit, if necessary, 2′5′-nucleoside bond, 5′5′-internucleoside bond, 3′3′-internucleoside bond, 2′2′-internucleoside bond or providing 2'3' internucleoside linkage; _{S 2} is any non-palindromic or non-nucleotide linker of 1-10 nucleotides in length, abasic linkers (dSpacer), triethylene glycol units or hexaethylene glycol S ₃ is a direct or indirect 2'5'-internucleoside bond, 5'5'-internucleoside bond, 3'3'-internucleoside bond, 2'2'-internucleoside bond, Or a 2′3′-nucleoside linkage, or a non-nucleotide linker, which is an abasic phosphorous A dSpacer, a triethylene glycol unit, or a hexaethylene glycol unit, wherein the abasic linker or unit is a 2′5′-link, 5′5′-link, 3′3 ′ - binding, it promotes 2'2'- bond, or 2'3'_{binding; N 1,} N _2,. . . N _n and N _{1 #} , N _{2 #,.} . . Each of N _{n #} is any nucleotide or modified nucleotide, where N ₁ forms a base pair with N _{1 #} , N ₂ forms a base pair with N _{2 #,} and N ₃ , N _{3 #} and base pair. . . And N _n forms a base pair with N _{n #} ; k is an integer from 0 to 5; m is an integer from 2 to 10; n is an integer from 2 to 16 P is an integer from 1 to 6; and q is an integer from 0 to 10.

  In certain embodiments,

は、非パリンドローム配列である。

Is a non-palindromic sequence.

  In certain embodiments,

は、

Is

であり、ＬはｄＳｐａｃｅｒである。

And L is dSpacer.

  In certain embodiments,

は、パリンドローム配列である。

Is a palindromic sequence.

  In certain embodiments,

は、

Is

である。

It is.

  In certain embodiments,

は、

Is

である。

It is.

In one embodiment, (N _n ). . . (N ₂ ) (N ₁ ) S ₂ (N _{1 #} ) (N _{2 #} ). . . (N _{n #} ) Z ₃ has the following sequence:

を含み、ここでＬは、ｄＳｐａｃｅｒである。

Where L is dSpacer.

In one embodiment, (N _n ). . . (N ₂ ) (N ₁ ) S ₂ (N _{1 #} ) (N _{2 #} ). . . (N _{n #} ) is the following sequence:

を含む。

including.

In one embodiment, the immunostimulatory nucleic acid is
The following sequence:

を含む。

including.

  In one embodiment, the immunostimulatory nucleic acid has the following sequence:

を含み、ここでＬはｄＳｐａｃｅｒである。

Where L is dSpacer.

  In one embodiment, the immunostimulatory nucleic acid has the following sequence:

を含む。

including.

  In one embodiment, the immunostimulatory nucleic acid has the following sequence:

を含む。

including.

  In one embodiment, the immunostimulatory nucleic acid is:

から選択される配列を含み、ここでＬはｄＳｐａｃｅｒであり、

Wherein L is dSpacer, and

はホスホロチオエートであり、

Is phosphorothioate;

はホスホジエステルである。

Is a phosphodiester.

  In one embodiment, the immunostimulatory nucleic acid is:

から選択される配列を含み、ここで、

Comprising a sequence selected from

はホスホロチオエートであり、

Is phosphorothioate;

はホスホジエステルである。

Is a phosphodiester.

  In one embodiment, the immunostimulatory nucleic acid is:

から選択される配列を含み、ここで、

Comprising a sequence selected from

はホスホロチオエートであり、

Is phosphorothioate;

はホスホジエステルである。

Is a phosphodiester.

  In one embodiment, the immunostimulatory nucleic acid is:

から選択される配列を含み、ここで、

Comprising a sequence selected from

はホスホロチオエートであり、

Is phosphorothioate;

はホスホジエステルである。

Is a phosphodiester.

  In one embodiment, the immunostimulatory nucleic acid has the following sequence:

を含み、ここで、

Where, where

はホスホロチオエートであり、

Is phosphorothioate;

はホスホジエステルである。

Is a phosphodiester.

  In one embodiment, the immunostimulatory nucleic acid is:

から選択される配列を含み、ここで、

Comprising a sequence selected from

はホスホロチオエートであり、

Is phosphorothioate;

はホスホジエステルである。

Is a phosphodiester.

  In one embodiment, the immunostimulatory nucleic acid has the following sequence:

を含み、ここで、

Where, where

はホスホロチオエートであり、

Is phosphorothioate;

はホスホジエステルである。

Is a phosphodiester.

  In one embodiment, the immunostimulatory nucleic acid has the following sequence:

を含み、ここで、

Where, where

はホスホロチオエートであり、

Is phosphorothioate;

はホスホジエステルである。

Is a phosphodiester.

  In one embodiment, at least one nucleotide in the oligonucleotide is a substituted or modified purine or a substituted or modified pyrimidine.

  In one embodiment, the substituted pyrimidine is a C5-substituted pyrimidine or a C6-substituted pyrimidine.

  In one embodiment, the substituted purine is a C8-substituted purine or a C7-substituted purine.

In one embodiment, the substituted purine or modified purine or substituted pyrimidine or modified pyrimidine is a 5-substituted cytosine, 6-substituted cytosine, N4-substituted cytosine, 5-aza-cytosine, 2-mercapto-cytosine, isocytosine, pseudo- Isocytosine, cytosine analogs with fused ring systems, and uracil derivatives, thymine derivatives, 7-deazaguanine, 7-deaza-7-substituted guanine, 7-deaza-8-substituted guanine, 7-deaza-8-azaguanine, hypoxanthine, N2-substituted guanine, 5-amino-3-methyl-3H, 6H-thiazolo [4,5-d] pyrimidine-2,7-dione, 2,6-diaminopurine, 2-aminopurine, purine, indole, substituted Consists of adenine, 8-substituted guanine, and 6-thioguanine More is selected.

  In one embodiment, the substituted purine or modified purine or substituted pyrimidine or modified pyrimidine is 5-methyl-cytosine, 5-fluoro-cytosine, 5-chloro-cytosine, 5-bromo-cytosine, 5-iodo-cytosine, 5 Hydroxy-cytosine, 6-hydroxy-cytosine, 5-hydroxymethyl-cytosine, 5-difluoromethyl-cytosine, and unsubstituted or substituted 5-alkynyl-cytosine, N4-ethyl-cytosine, N, N'- Propylene cytosine, phenoxazine, 5-fluoro-uracil, 5-bromo-uracil, 5-bromovinyl-uracil, 4-thio-uracil, 5-hydroxy-uracil, 5-propynyl-uracil, 2-thiothymine, 4-thiothymine, 6-substituted thymine, 7-deaza-7- ( 2-C6) alkynyl guanine, N2- methyl - guanine, N6-methyl - adenine, 8-oxo - adenine, are selected from the group consisting of 8-hydroxy guanine, and 8-bromo-guanine.

  In one embodiment, the substituted purine or modified purine or substituted pyrimidine or modified pyrimidine is selected from the group consisting of a universal base, an aromatic ring system, and a hydrogen atom (dSpacer).

  In one embodiment, the substituted purine or modified purine or substituted pyrimidine or modified pyrimidine is 4-methyl-indole, 5-nitro-indole, 3-nitropyrrole, P-base, and K-base, benzimidazole, dichloro- Selected from the group consisting of benzimidazole, 1-methyl-1H- [1,2,4] triazole-3-carboxylic acid amide, fluorobenzene, and difluorobenzene.

  In one embodiment, any of N, S, X, or Z is a C6-C30 alkyl chain, bile acid, cholic acid, taurocholic acid, deoxycholic acid, cholesterol, oleyl lithocholic acid, oleoyl cholonic acid. acid), glycolipid, phospholipid, sphingolipid, isoprenoid, steroid, vitamins, vitamin E, saturated fatty acid, unsaturated fatty acid, fatty acid ester, triglyceride, pyrene, porphyrin, texaphyrin, adamantane, acridine, biotin, coumarin, fluorescein, Rhodamine, Texas red, digoxigenin, dimethoxytrityl, t-butyldimethylsilyl, t-butyldiphenylsilyl, cyanine dyes, cyanine dye Cy3, cyanine dye Cy576, Hoechst Substituted by a residue selected from the group consisting of 33258 dye, psoralen, and ibuprofen.

In one aspect, the present invention is the 5 ′ end beginning with an immunostimulatory motif selected from the following (a) to (c): (a) (TCG) _n N and RDCGY ₁ Y ₂ N, wherein And T is thymine, C is unmethylated cytosine, G is guanine, R is purine, D is not C, and each of Y ₁ and Y ₂ is independently a pyrimidine; n is an integer from 1 to 4 and N is any sequence 0-12 bases in length, 5 ′ end; (b) terminates in inverted repeats capable of forming a hairpin or stem-loop structure A 3 ′ end, the structure comprising 2 to 6 consecutive base pair long GC-rich stems and at least one unmatched or mismatched base; and (c)
Immunostimulatory nucleic acid molecules comprising a plurality of stabilized scaffolds comprising at least one phosphodiester 5′-CpG-3 ′ linkage are provided.

  In one embodiment, the GC-rich stem is two consecutive base pair lengths.

  In one embodiment, the GC-rich stem is 3 consecutive base pair lengths.

  In one embodiment, the GC-rich stem is 4 consecutive base pair lengths.

  In one embodiment, the GC-rich stem is 5 consecutive base pairs long.

  In one embodiment, the GC-rich stem is 6 consecutive base pair lengths.

  In one embodiment, the GC-rich stem comprises at least 2 GC base pairs.

  In one embodiment, the GC-rich stem comprises at least 3 GC base pairs.

  In certain embodiments, the at least one unmatched base or mismatched base is T.

  In one embodiment, the partially stabilized backbone comprising at least one phosphodiester 5'-CpG-3 'linkage further comprises a plurality of phosphorothioate internucleotide linkages.

  In one embodiment, the 5 'end is

として提供される配列を有する。

As provided.

  In one embodiment, the 3 'end terminating in the inverted repeat is

として提供される塩基配列を有する。

As a base sequence.

  In one embodiment, the 3 'end terminating in the inverted repeat is

として提供される塩基配列を有する。

As a base sequence.

  In one aspect, the present invention provides:

として提供される塩基配列を有する、免疫刺激性核酸を提供する。

An immunostimulatory nucleic acid having the base sequence provided as

  In one aspect, the present invention provides:

として提供される塩基配列を有する、免疫刺激性核酸を提供する。

An immunostimulatory nucleic acid having the base sequence provided as

  In one aspect, the present invention provides:

として提供される塩基配列を有する免疫刺激性核酸を提供し、ここで、

An immunostimulatory nucleic acid having a base sequence provided as

はホスホロチオエートヌクレオチド間結合であり、

Is a phosphorothioate internucleotide linkage;

はホスホジエステルヌクレオチド間結合である。

Is a phosphodiester internucleotide linkage.

  In one aspect, the present invention provides:

として提供される塩基配列を有する免疫刺激性核酸を提供し、ここで、

An immunostimulatory nucleic acid having a base sequence provided as

はホスホロチオエートヌクレオチド間結合であり、

Is a phosphorothioate internucleotide linkage;

はホスホジエステルヌクレオチド間結合である。

Is a phosphodiester internucleotide linkage.

  In one aspect, the present invention provides:

として提供される塩基配列を有する免疫刺激性核酸を提供し、ここで、

An immunostimulatory nucleic acid having a base sequence provided as

はホスホロチオエートヌクレオチド間結合であり、

Is a phosphorothioate internucleotide linkage;

はホスホジエステルヌクレオチド間結合である。

Is a phosphodiester internucleotide linkage.

  In one aspect, the present invention provides:

として提供される塩基配列を有する免疫刺激性核酸を提供し、ここで、

An immunostimulatory nucleic acid having a base sequence provided as

はホスホロチオエートヌクレオチド間結合であり、

Is a phosphorothioate internucleotide linkage;

はホスホジエステルヌクレオチド間結合である。

Is a phosphodiester internucleotide linkage.

  In one aspect, the present invention provides a vaccine comprising the immunostimulatory nucleic acid molecule of the present invention and an antigen.

  In one aspect, the present invention provides a pharmaceutical composition comprising an immunostimulatory nucleic acid molecule of the present invention and a pharmaceutically acceptable carrier.

  In one aspect, the present invention provides a method for inducing type I interferon (IFN) expression. A method according to this aspect of the invention comprises contacting a cell capable of expressing type I IFN with an immunostimulatory nucleic acid of the invention in an amount effective to induce expression of type I IFN. .

  In one embodiment, the type I IFN is interferon α (IFN-α).

  In one embodiment, the type I IFN is interferon β (IFN-β).

In one aspect, the present invention provides a method for inducing gamma interferon (IFN-γ) expression. A method according to this aspect of the invention comprises contacting a cell capable of expressing IFN-γ with an immunostimulatory nucleic acid of the invention in an amount effective to induce expression of IFN-γ. .

  In one aspect, the present invention provides a method for activating natural killer (NK) cells. The method according to this aspect of the invention includes contacting NK cells with the immunostimulatory nucleic acid of the invention in an amount effective to activate the NK cells.

  In one aspect, the present invention provides a method for treating an infection. The method according to this aspect of the invention administers to a subject having or at risk of developing an infection an amount of an immunostimulatory nucleic acid of the invention effective to treat or prevent this infection. Process.

  In one embodiment, the subject has or is at risk of developing an infection selected from a viral infection, a bacterial infection, a fungal infection, or a parasitic infection.

  In one embodiment, the subject is hepatitis B virus (HBV), hepatitis C virus (HCV), cytomegalovirus (CMV), Epstein-Barr virus (EBV), papilloma virus, human immunodeficiency virus (HIV). ), Or at risk of having or developing a viral infection with a virus selected from herpes simplex virus (HSV).

  In one embodiment, the subject has or is at risk of developing a bacterial infection with a bacterial species selected from Leishmania, Listeria, or Anthrax.

  In one aspect, the present invention provides a method for treating an allergic condition. A method according to this aspect of the invention provides a subject having an allergic condition or at risk of developing this condition with an amount of an immunostimulatory nucleic acid of the invention effective to treat or prevent this allergic condition. Administering.

  In one embodiment, the allergic condition is allergic asthma.

  In one aspect, the present invention provides a method for treating cancer. The method according to this aspect of the invention comprises the step of administering to a subject having or at risk of developing cancer an amount of an immunostimulatory nucleic acid of the invention effective to treat or prevent the cancer. Is included.

  In one embodiment, the cancer is basal cell cancer, cholangiocarcinoma, bladder cancer, bone cancer, brain and central nervous system cancer, breast cancer, cervical cancer, choriocarcinoma, colon and rectal cancer, connective tissue cancer, digestive organs System cancer, endometrial cancer, esophageal cancer, eye cancer, head and neck cancer, gastric cancer, intraepithelial neoplasia, kidney cancer, pharyngeal cancer, leukemia, liver cancer, lung cancer, Hodgkin lymphoma and non-Hodgkin lymphoma Including lymphoma, melanoma, myeloma, neuroblastoma, oral cancer, ovarian cancer, pancreatic cancer, prostate cancer, retinoblastoma, rhabdomyoblastoma, rectal cancer, kidney cancer, respiratory cancer, sarcoma, Selected from skin cancer, stomach cancer, testicular cancer, thyroid cancer, uterine cancer, urinary cancer, or other carcinomas and sarcomas.

  In one embodiment, the cancer is a cancer that is sensitive to treatment with interferon alpha (IFN-alpha).

  In one embodiment, the cancer sensitive to treatment with IFN-α is hairy cell leukemia, chronic myelogenous leukemia, cutaneous T cell leukemia, multiple myeloma, follicular lymphoma, malignant melanoma, squamous cell carcinoma, AIDS Selected from related Kaposi's sarcoma, renal cell carcinoma, prostate carcinoma, cervical dysplasia, or colon carcinoma.

  In one aspect, the present invention provides the use of an immunostimulatory nucleic acid of the invention for the manufacture of a medicament for use in the treatment of an infection.

  In one aspect, the present invention provides the use of an immunostimulatory nucleic acid of the present invention for the manufacture of a medicament for use in the treatment of allergic conditions.

  In one aspect, the present invention provides the use of an immunostimulatory nucleic acid of the present invention for the manufacture of a medicament for use in the treatment of allergic asthma.

  In one aspect, the present invention provides the use of an immunostimulatory nucleic acid of the present invention for the manufacture of a medicament for use in the treatment of cancer.

  Each of the limitations of the invention can encompass various embodiments of the invention. Thus, it is understood that each of the limitations of the invention including any one element or combination of elements can be included in each aspect of the invention.

。

.

(Detailed description of the invention)
The present invention, in one aspect, includes the finding that certain subclasses of CpG immunostimulatory oligonucleotides having a defined secondary structure are highly effective in mediating immunostimulatory effects. These CpG nucleic acids serve to stimulate the immune system to treat cancer, infectious diseases, allergies, asthma and other disorders, and to protect against opportunistic infections after cancer chemotherapy Therefore, it is useful therapeutically and prophylactically. The strong, balanced, cellular and humoral immune responses resulting from CpG stimulation reflect the body's own innate defense system against pathogenic and cancerous cell invasion.

  The sequences of the present invention share some structural similarity with a class of CpG oligonucleotides, referred to as C class or combination motif CpG oligonucleotides. See published PCT international patent application WO 03/015711 pamphlet. Similar to the previously described C class oligonucleotides, the C class CpG oligonucleotides of the present invention have defined 5 'and 3' motifs as part of the molecule. These previously described C class oligonucleotides have traditional “stimulatory” CpG sequences (generally located at or near the 5 ′ or 3 ′ end of the molecule) and “GC-rich palindrome "With both motifs (generally located at or near the other end of the molecule). These combinatorial motif nucleic acids have the effects associated with the traditional “B class” CpG ODN, which is a potent inducer of B cell activation and dendritic cell activation, and more recently described classes. Related immunostimulatory nucleic acids ("Class A" CpG ODN; a strong inducer of IFN-α and NK cell activation, but relatively inducible for B cell and DC activation) It has an immune stimulating effect that fits somewhere in between.

  The novel C-class CpG oligonucleotides of the present invention are also structurally different from previously described C-class CpG oligonucleotides. Compared to previously described C class CpG oligonucleotides, the immunostimulatory nucleic acid molecules of the present invention are characterized by rather loose requirements with respect to the GC-rich palindrome at one end of the molecule. For example, previously described class C oligonucleotides include, in one embodiment, a strict or complete palindrome that is at least 10 nucleotides long and has a GC content of at least 2/3. In some embodiments, the palindrome of the previously described C class oligonucleotide may contain at most a minimal number of consecutive mismatched nucleotides.

  In contrast to previously described C class oligonucleotides, the C class oligonucleotide analogs of the present invention, in various embodiments, have less than 10 nucleotides; a GC content between 0 and less than 2/3. Various nucleotide analogs and substituents, including those lacking any nucleobases (dSpacers); extended intervening sequences comprising four or more consecutive nucleotides or nucleotide substituents that do not form Watson-Crick base pairs; and Features a palindromic motif that can have any combination of these. Further, in some embodiments, the 3 'portions of two or more molecules can be linked to each other via their 3' ends. This novel subclass of oligonucleotides that do not have a complete palindrome can still induce high levels of IFN production, similar to the previously described combinatorial motif CpG oligonucleotides, which are type I IFNs (eg, IFN- It has been discovered that α, IFN-β) and IFN-γ can be induced.

  “Palindrome” and equivalently “palindromic sequence”, as used herein, refers to its own fully opposite complement (ie, ABCDEEE'D'C'B'A '( Where A and A ′, B and B ′, C and C ′, D and D ′, and E and E ′ are normal Watson-Crick base pairs (ie, GC, AT, and A− U) refers to a nucleic acid sequence which is a base)). As used herein, “palindromic” excludes intervening sequences or intervening non-nucleotide structures that are not strictly involved in normal Watson-Crick base pairing.

  “Inverted repeat”, as used herein, is incomplete palindrome, ie, involved in normal Watson-Crick base pairing with nucleotides that can form normal Watson-Crick base pairs Not nucleotides, nucleotide analogs or other structures (eg, sequences such as ABCDE-S-E'D'C'B'A 'where A and A', B and B ', C and C', D And D ′, and E and E ′ are bases that can form normal Watson-Crick base pairs, and S is a non-palindromic sequence or non-nucleobase linker or abasic linker (dSpacer). A)) refers to the nucleic acid sequence present. In certain embodiments, nucleotides, nucleotide analogs or other structures that are not involved in normal Watson-Crick base pairing interfere with other complete palindromes. In certain embodiments, a nucleotide that is not involved in normal Watson-Crick base pairing may form a non-Watson-Crick base pair (eg, GT) with another nucleotide. As used herein, a non-Watson-Crick base pair is any base pair other than a Watson-Crick base pair, including Hoogsteen base pairs, and so-called wobble base pairs. However, it is not limited to these. In certain embodiments, nucleotides that are not involved in normal Watson-Crick base pairing do not match, and nucleotide bases or nucleotide base analogs to form Watson-Crick base pairs or non-Watson-Crick base pairs (eg, , DSpacer inverse G). In certain embodiments, a nucleotide that is not involved in base pairing can form a non-standard base pair with another nucleotide (eg, diaminopyridine can base pair with xanthosine).

In one embodiment, the 5 ′ end of the nucleic acid begins with an immunostimulatory motif selected from (TCG) _n N and RDCGY ₁ Y ₂ N. T is thymine, C is unmethylated cytosine, G is guanine, R is purine, D is not C, and each of Y ₁ and Y ₂ is independently a pyrimidine; n is an integer of 1 to 4 (including the end), and N is an arbitrary sequence having a length of 0 to 12 bases.

  The 3 'end of the nucleic acid terminates with an inverted repeat that can form a hairpin or stem-loop structure. The term “terminate” refers to a structure at or near the 3 ′ end. Thus, the end of the incomplete palindrome may be located at the actual 3 'end of the molecule, or the 3' end may contain one or more additional nucleotides that are not part of the inverted repeat structure. Preferably, the 3 'end of the molecule comprises 3 or fewer nucleotides that do not form part of the inverted structure.

  In one embodiment, “inverted repeats that can form a hairpin structure or stem-loop structure” as used herein is a GC-rich stem or 2-10 contiguous base pair length or Refers to the sequence of nucleotides forming the hairpin, which contains at least one unmatched or mismatched base. In individual embodiments, the GC-rich stem is 2, 3, 4, 5, 6, 7, 8, 9 or 10 consecutive base pair lengths. In some embodiments, the GC-rich stem comprises at least 2, 3 or 4 GC base pairs.

  In one embodiment, “inverted repeats that can form a hairpin or stem-loop structure” as used herein refers to an AT-rich stem or 2-10 consecutive base pair length or Refers to the sequence of nucleotides forming the hairpin, which contains at least one unmatched or mismatched base. In individual embodiments, the AT-rich stem is 2, 3, 4, 5, 6, 7, 8, 9 or 10 consecutive base pair lengths. In some embodiments, the AT-rich stem comprises at least 2, 3 or 4 AT base pairs.

  In some cases, at least one unmatched or mismatched base connects the ends of the stem or hairpin. This may allow the formation of secondary structures by providing a flexible point within the molecule where the stems base pair and form a hairpin. Alternatively, unmatched or mismatched bases can be inside the stem. Preferably, if the mismatched base is inside the stem, the stem is at least 3 base pairs long. An unmatched or mismatched base can be any nucleotide. In some embodiments, the unmatched or mismatched base is T. Unmatched nucleotides at the ends of duplexes are also known as overhanging nucleotides or dangling ends, which can significantly stabilize duplex formation or heparin formation. Freier SM et al. (1983) Effects of 3 'dangling end stacking on the stability of GGCC and CCGG double helices. Biochemistry 22: 6198-206.

  The nucleic acid also includes a partially stabilized backbone that includes at least one phosphodiester 5'-CpG-3 'linkage.

  In some cases, the duplex portion of the molecule may also include non-natural (non-standard) base pairs (eg, diaminopyridine paired with xanthosine). Lutz MJ et al. (1998) Recognition of a non-standard base pair by thermostable DNA polymers. Bioorg Med Chem Lett 8: 1149-52.

  The formula defines a subset of the class of CpG oligonucleotides that have demonstrated excellent immunostimulatory properties. In this formula, 5 'refers to the free 5' end of the oligonucleotide and 3 'refers to the free 3' end of the oligonucleotide.

  Oligonucleotides can have one or more accessible 5 'or 3' ends. In some embodiments, the 3 'end can be linked to another 3' end. Since the importance of the 5 'and 3' motifs has been discovered and described herein, it is also possible to create a modified oligonucleotide having two such 5 'or 3' ends. Is possible. This can be achieved, for example, by joining two oligonucleotides via 3'-3 'linkages to create an oligonucleotide with two accessible 5' ends. The 3'-3 'linkage or 5'-5' linkage can be a phosphodiester, phosphorothioate, or any other modified internucleoside bridge. Methods for achieving such binding are known in the art. For example, such linkages include Seliger H et al. (1991) Oligonucleotide analogues with terminal 3'-3'-and 5'-5'-internucleoside sequencial IDs and anti-lesions of N And Jiang Z et al (1999) Pseudo-cyclic oligonucleotides: in vitro and in vivo properties, Bioorg Med Chem 7: 2727-35.

In addition, 3′-3 ′ or 5′-5 ′ linked ODNs (the bond between the 3 ′ terminal nucleoside or the 5 ′ terminal nucleoside is not a phosphodiester, phosphorothioate, or other modified bridge) , Tri-, or tetraethylene glycol phosphate moieties (Durand M et al. (1992) Triple-helicon formation by one containing one (dA) 12 and two (dT) 12 sequences bridged
by two hexethylene chains, Biochemistry 31: 9197-204; US Pat. No. 5,658,738; and US Pat. No. 5,668,265). Alternatively, the non-nucleotide linker can be from ethanediol, propanediol, or an abasic deoxyribose (dSpacer) unit (Fontanel ML et al. (1994) Steady recognition by T4 polynucleotide kinase of non-nucleosidic acid 5 from oligonucleotides, Nucleic Acids Res 22: 2022-7) using standard phosphoramidite chemistry. This non-nucleotide linker may be incorporated one or more times, or combined with each other to allow any linking between the 3 ′ ends of two ODNs of any desired distance.

  “Non-nucleotide linker” as used herein refers to any linker element that is not a nucleotide or a polymer thereof (ie, a polynucleotide), where the nucleotide includes a purine nucleobase or a pyrimidine nucleic acid. Bases, and sugar phosphates. Thus, non-nucleotide linkers include abasic nucleotides (dSpacers), ie, nucleotide-like sugar phosphate units in which the nucleobase is replaced by a hydrogen atom. The non-nucleotide linker can be polyethylene glycol, including but not limited to triethylene glycol and hexaethylene glycol.

  In some embodiments, the oligonucleotide has the following structure:

のうちの１つを有する。

One of them.

  symbol

は、安定化されたヌクレオチド間結合の存在を表し、そして、

Represents the presence of a stabilized internucleotide linkage, and

は、ホスホジエステル結合の存在を表す。

Represents the presence of a phosphodiester bond.

  Immunostimulatory oligonucleotides generally have a length in the range of between 6 and 100 nucleotides. In some embodiments, the length is in the range of 6-40, 13-100, 13-40, 13-30, 15-50, or 15-30 nucleotides, or between Nucleotides in any integer range.

  The terms “nucleic acid” and “oligonucleotide” refer to a plurality of nucleotides (ie, sugars (eg, ribose or deoxyribose) linked to a phosphate group and an exchangeable organic base (such as substituted pyrimidines (eg, cytosine ( C), thymine (T) or uracil (U)) or a molecule containing a substituted purine (eg, either adenine (A) or guanine (G))) is used interchangeably. The As used herein, the terms “nucleic acid” and “oligonucleotide” refer to oligoribonucleotides and oligodeoxyribonucleotides. The terms “nucleic acid” and “oligonucleotide” also include polynucleotides (ie, polynucleotides minus a phosphate) and any other organic base-containing polymer. Nucleic acid molecules can be obtained from existing nucleic acid sources (eg, genomic DNA or cDNA), but are preferably synthetic (eg, produced by nucleic acid synthesis).

  The terms “nucleic acid” and “oligonucleotide” as used herein encompass the nucleic acid molecules and oligonucleotides of the invention, as well as the oligonucleotide analogs of the invention. The term “oligodeoxynucleotide”, and equivalently “ODN”, as used herein, encompasses unmodified oligodeoxynucleotides of the present invention as well as oligodeoxynucleotide analogs of the present invention.

  The terms “nucleic acid” and “oligonucleotide” also encompass nucleic acids or oligonucleotides in which, for example, bases and / or sugars are substituted or modified. For example, these include a nucleic acid having a skeleton sugar covalently bonded to a low molecular weight organic group other than a hydroxyl group at the 2 ′ position, and a skeleton covalently bonded to a low molecular weight organic group other than a phosphate group or a hydroxy group at the 5 ′ position. Examples include nucleic acids having sugars. Thus, the modified nucleic acid can comprise a 2'-O-alkylated ribose group. Furthermore, the modified nucleic acid may contain sugars such as arabinose or 2'-fluoroarabinose instead of ribose. Thus, nucleic acids can be heterogeneous in backbone composition, thereby including any possible combination of polymer units linked together as peptide-nucleic acids (having a peptide-like backbone with nucleobases). Other examples are described in more detail below.

  The immunostimulatory oligonucleotides of the present invention may include various chemical modifications and substitutions compared to natural RNA and DNA, which include phosphodiester internucleoside bridges, β-D-ribose units, and / or Contains natural nucleoside bases (adenine, guanine, cytosine, thymine, uracil). Examples of chemical modifications are known to those skilled in the art, see, for example, Uhlmann E et al. (1990) Chem Rev 90: 543; “Protocols for Oligonucleotides and Analogs” Synthesis and Properties & Sciences and Sciences. 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 17: 33. The oligonucleotides according to the invention may have one or more modifications, where each modification is a specific phosphodiester internucleoside bridge, and / or a specific β-D-ribose unit, and / or natural Located at a particular natural nucleoside base position compared to an oligonucleotide of the same sequence composed of DNA or RNA.

For example, the oligonucleotide can include one or more modifications, where each modification is independently selected from:
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) replacement of the sugar phosphate unit from the sugar phosphate backbone with another unit;
d) 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.

  Oligonucleotides can include modified internucleotide linkages, such as those described in a or b above. These modified bonds can be partially resistant to degradation (eg, stabilized). By “stabilized oligonucleotide molecule” is meant an oligonucleotide that is relatively resistant to in vivo degradation (eg, by exonuclease or endonuclease) resulting from such modifications. In some embodiments, an oligonucleotide having a phosphorothioate linkage provides maximum activity and can protect the oligonucleotide from degradation by intracellular exonucleases and endonucleases.

The phosphodiester internucleoside bridge located at the 3 'end and / or 5' end of the nucleoside can be replaced by a modified internucleoside bridge, wherein the modified internucleoside bridge is selected, for example, from : phosphorothioate, phosphorodithioate, ^NR 1 ^{R 2} - phosphoramidite, boranophosphate (boranophosphate), α- hydroxybenzyl phosphonate, 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 ) alkyl phosphonate, and / or (C ₆ -C ₁₂ ) aryl phosphonate bridge, (C ₇ -C ₁₂ ) -Α-Hydroxyme Til-aryl (eg as disclosed in WO 95/01363) (where (C ₆ -C ₁₂ ) aryl, (C ₆ -C ₂₀ ) aryl and (C ₆ -C ₁₄ ) aryl are optionally Accordingly substituted by halogen, alkyl, alkoxy, nitro, cyano, R ¹ and R ² , independently of one another, are 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 it, form a 5-6 membered heterocyclic ring, which is further O, S And N It may include heteroatoms from).

Replacement of phosphodiester bridges located at the 3 ′ and / or 5 ′ ends of nucleosides by dephosphorylation bridges (dephosphorylation bridges are described, for example, in Uhlmann E and Peyman A, “Methods in Molecular Biology”, Vol. 20, “ Protocols for Oligonucleotides
and Analogs ", S. et al. Edited by Agrawal, Humana Press, Totowa 1993, Chapter 16, pp. 355 ff). Here, the dephosphorization bridge is selected from, for example, a dephosphorization bridge form acetal, 3′-thioform acetal, methylhydroxylamine, oxime, methylenedimethyl-hydrazo, dimethylenesulfone, and / or a silyl group.

  Sugar phosphate units from the sugar phosphate backbone (ie, β-D-ribose and phosphodiester internucleoside bridges that together form the sugar phosphate unit) (ie, the sugar phosphate backbone is composed of sugar phosphate units Are replaced by another unit suitable for constructing, for example, “morpholino derivative” oligomers (eg, described in Stirchak EP et al. (1989) Nucleic Acids Res 17: 6129-41). Suitable for constructing a polyamide nucleic acid ("PNA"; described, for example, in Nielsen PE et al. (1994) Bioconjug Chem 5: 3-7); Can be replaced by another unit (ie, P By NA backbone (eg by 2-aminoethylglycine)). Oligonucleotides have other carbohydrate backbone modifications and substitutions (eg, peptide nucleic acids with phosphate groups (PHONA), locked nucleic acids (LNA), and oligonucleotides with backbone sections with alkyl or amino linkers) obtain. The alkyl linker can be branched or unbranched, substituted or unsubstituted, chirally pure, or a racemic mixture.

The β-ribose unit or β-D-2′-deoxyribose unit can be replaced by a modified sugar unit, for example, β-D-ribose, α-D-2′- Selected from deoxyribose, L-2′-deoxyribose, 2′-F-2′-deoxyribose, 2′-F-arabinose, 2′-O- (C ₁ -C ₆ ) alkyl-ribose, preferably _{, 2'-O- (C 1 ~C} 6) alkyl - ribose, 2'-O-methyl _{ribose, 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, beta-D-xylo - furanose, alpha-arabinofuranose, 2,4 Dideoxy-β-D-erythro-hexo-pyranose , And carbocyclic (e.g., as described in Froehler (1992) J Am Chem Soc 114: 8320), and / or open-chain sugar analogs (e.g., as described in Vandriessche et al. (1993) Tetrahedron 49: 7223), And / or bicyclic sugar analogs (for example, as described in Tarkov M et al. (1993) Helv Chim Acta 76: 481).

  In some embodiments, the sugar is 2'-O-methyl ribose, in particular one or both nucleosides are linked by phosphodiester or phosphodiester-like internucleoside linkages.

  Nucleic acids also include substituted purines and substituted pyrimidines, such as C-5 propyne pyrimidine modified bases and 7-deaza-7-substituted purine modified bases. Wagner RW et al. (1996) Nat Biotechnol 14: 840-4. Purines and pyrimidines include, but are not limited to, adenine, cytosine, guanine, thymine and uracil, and non-naturally occurring nucleobases, substituted and unsubstituted aromatic moieties.

Modified bases are chemically different from the naturally occurring bases typically found in DNA and RNA (eg, T, C, G, A and U), but with these naturally occurring bases. Any base that shares a basic chemical structure. The modified nucleoside base can be selected, for example, from: hypoxanthine, uracil, dihydrouracil, pseudouracil, 2-thiouracil, 4-thiouracil, 5-aminouracil, 5- (C ₁ -C ₆ ) -alkyl. uracil, 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-diamino - purine, 8 Azapurine, substituted 7-deazapurine (preferably 7-deaza-7-substituted purine and / or 7-deaza-8-substituted purine), 5-hydroxymethylcytosine, N4-alkylcytosine (eg, N4-ethylcytosine), 5-hydroxydeoxycytidine, 5-hydroxymethyldeoxycytidine, N4-alkyldeoxycytidine (eg N4-ethyldeoxycytidine), 6-thiodeoxyguanosine, and deoxyribonucleosides of nitropyrrole, C5-propylpyrimidine, and diaminopurine ( For example, 2,6-diaminopurine), inosine, 5-methylcytosine, 2-aminopurine, 2-amino-6-chloropurine, hypoxanthine, or other modifications of natural nucleoside bases. This list is meant to be exemplary and should not be intended to be limiting.

  In certain formulations described herein, modified bases can be incorporated. For example, cytosine can be replaced with modified cytosine. As used herein, a modified cytosine is a naturally occurring or non-naturally occurring pyrimidine base analog of cytosine that can replace this base without compromising the immunostimulatory activity of the oligonucleotide. is there. Modified cytosines include, but are not limited to, 5-substituted cytosines (eg, 5-methyl-cytosine, 5-fluoro-cytosine, 5-chloro-cytosine, 5-bromo-cytosine, 5- Iodo-cytosine, 5-hydroxy-cytosine, 5-hydroxymethyl-cytosine, 5-difluoromethyl-cytosine, and unsubstituted or substituted 5-alkynyl-cytosine), 6-substituted cytosines (eg, 6-hydroxy-cytosine) N4-substituted cytosine (eg, N4-ethyl-cytosine), 5-aza-cytosine, 2-mercapto-cytosine, isocytosine, pseudo-isocytosine, cytosine analogs having a condensed ring system (eg, N, N′-propylene cytosine) Or phenoxazine), and uracil and its induction (E.g., 5-fluoro - uracil, 5-bromo - uracil, 5-bromovinyl - uracil, 4-thio - uracil, 5-hydroxy - uracil, 5-propyl - uracil). Some preferred cytosines include 5-methyl-cytosine, 5-fluoro-cytosine, 5-hydroxy-cytosine, 5-hydroxymethyl-cytosine, and N4-ethyl-cytosine. In another embodiment of the invention, the cytosine base is replaced by a universal base (eg, 3-nitropyrrole, P-base), an aromatic ring system (eg, fluorobenzene or difluorobenzene), or a hydrogen atom (dSpacer). Is done.

Guanine can be replaced with a modified guanine base. Modified guanine, as used herein, is a naturally occurring or non-naturally occurring purine base of guanine that can replace this base without compromising the immunostimulatory activity of the oligonucleotide. It is analog. Modified guanines include, but are not limited to: 7-deazaguanine, 7-deaza-7 substituted guanine (eg, 7-deaza-7- (C ₂ -C ₆ ) alkynylguanine), 7 -Deaza-8 substituted guanine, hypoxanthine, N2-substituted guanine (eg N2-methyl-guanine), 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-methyl-adenine, 8-oxo-adenine), 8-substituted guanine, (eg, 8-hydroxy) Guanine and 8-bromoguanine), and 6-thioguanine. In another embodiment of the invention, the guanine base is a universal base (eg, 4-methyl-indole, 5-nitro-indole, and K-base), an aromatic ring system (eg, benzimidazole or dichloro-benzimidazole). , 1-methyl-1H- [1,2,4] triazole-3-carboxylic acid amide), or a hydrogen atom (dSpacer).

  In one embodiment, both C and G of the CG dinucleotide are an unmodified cytosine residue and an unmodified guanine residue, respectively. In one embodiment, the C of the CG dinucleotide is not methylated.

For use in the present invention, the oligonucleotides of the present invention can be synthesized using a number of procedures well known in the art, such as the β-cyanoethyl phosphoramidite method (Beaucage SL et al. (1981) Tetrahedron Lett 22: 1859); or nucleoside H-phosphonates. (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; Gaffney et al. (1988) Tetrahedron Lett 29: 2619-22)). These chemistries can be performed by a variety of automated nucleic acid synthesizers available on the market. These oligonucleotides are called synthetic oligonucleotides. An isolated oligonucleotide generally refers to an oligonucleotide that has been separated from components that are normally associated in nature. As an example, an isolated oligonucleotide can be an oligonucleotide separated from a cell, from the nucleus, from mitochondria, or from chromatin.

  Modified backbones such as phosphorothioates can be synthesized using automated techniques using either phosphoramidite or H-phosphonate chemistry. Aryl-phosphonates and alkyl-phosphonates can be made, for example, as described in US Pat. No. 4,469,863; and alkyl phosphotriesters (US Pat. No. 5,023,243 and European patent no. 092,574 can be prepared by automated solid phase synthesis using commercially available reagents). Methods for making other DNA backbone modifications and substitutions have been described (eg, Uhlmann E et al. (1990) Chem Rev 90: 544; Goodchild J (1990) Bioconjugate Chem 1: 165).

  Immunostimulatory oligonucleotides can also include one or more abnormal linkages between nucleotide or nucleoside analog moieties. A typical internucleoside linkage is a 3'5'-linkage. All other bonds (eg, 2′5′-bond, 5′5′-bond, 3′3′-bond, 2′2′-bond, and 2′3′-bond) are unusual internucleoside Considered a bond. Thereby, the 2'-5 'nomenclature is chosen according to the carbon atom of ribose. However, if a non-natural sugar moiety is used, such as a ring-expanded sugar analog (eg, hexanose, silohexene, or pyranose), or a bicyclic or tricyclic sugar analog, the nomenclature is Varies according to the nomenclature. In 3'-deoxy-β-D-ribopyranose analogs (also called β-DNA), mononucleosides are connected, for example, via 4'2'-linkages.

  If the nucleotide contains one 3'3'-link, the oligonucleotide analog usually has two unbound 5'-ends. Similarly, if a nucleotide contains one 5'5'-link, the oligonucleotide analog usually has two unbound 3'-ends. The accessibility of the unbound end of a nucleotide may be better accessible by its receptor. Both types of abnormal binding (3'3'- and 5'5'-) have been described by Ortigao JF et al. (1992) Antisense Res Dev 2: 129-46, where 3'3 ' -It was reported that oligonucleotides with bonds show enhanced stability against cleavage by nucleases.

  Different types of linkages can also be combined within one molecule, resulting in oligomeric branching. One part of the oligonucleotide is connected to the second oligonucleotide part at the 3'-end via a 3'3'-linkage and of the molecule at the 2'-end via a 2'3'-linkage. When connected to the third part, this results in a branched oligonucleotide having, for example, three 5′-ends (3′3′-branch, 2′3′-branch).

  In principle, binding between different parts of an oligonucleotide or between different oligonucleotides can occur through any part of the molecule as long as it does not negatively interfere with recognition by its receptor, respectively. Depending on the nature of the nucleic acid, conjugation may require a sugar moiety (Su), a heterocyclic nucleobase (Ba) or a phosphate backbone (Ph). Therefore, types of binding of Su-Su, Su-Ph, Su-Ba, Ba-Ba, Ba-Su, Ba-Ph, Ph-Ph, Ph-Su, and Ph-Ba are possible. If the oligonucleotide is further modified with certain non-nucleotide substituents, this linkage can also occur through the modified portion of the oligonucleotide. These modifications include modified nucleic acids (eg, PNA, LNA or morpholino oligonucleotide analogs).

The bond is preferably composed of C, H, N, O, S, B, P and halogen and contains 3 to 300 atoms. An example of having 3 atoms is, for example, an acetal linkage (ODN1-3′-O—CH ₂₎ connecting the 3′-hydroxy group of one nucleotide to the 3′-hydroxy group of a second oligonucleotide. -O-3'-ODN2; Froehler and Matteucci). An example of having about 300 atoms is PEG-40 (tetraconta polyethyleneglycol). Preferred linkages are phosphodiester, phosphorothioate, methylphosphonate, phosphoramidite, boranophosphonate, amide, ether, thioether, acetal, thioacetal, urea, thiourea, sulfonamide, Schiff base, and disulfide linkage. Another possibility is the use of a Sollink BioConjugation System (TriLink BioTechnologies, San Diego, CA).

  When the oligonucleotide is composed of two or more sequence parts, these parts may be the same or different. Thus, in an oligonucleotide having a 3′3′-linkage, the sequences are the same (eg, 5′-ODN1-3′3′-ODN1-5 ′) but different (eg, 5′-ODN1- 3′3′-ODN2-5 ′). Furthermore, the chemical modification of the various oligonucleotide moieties and the linker connecting them can be different. Since the incorporation of short oligonucleotides appears to be less efficient than the incorporation of long oligonucleotides, the joining of two or more short sequences results in improved immune stimulation. The length of the short oligonucleotide is preferably 2 to 20 nucleotides, more preferably 3 to 16 nucleotides, and most preferably 5 to 10 nucleotides. Preferably, they are linked with an oligonucleotide having two or more unbound 5'-ends.

  Oligonucleotide partial sequences can also be linked by non-nucleotide linkers, preferably in an abasic linker (dSpacer), a triethylene glycol unit, or a hexaethylene glycol unit. Other linkers include alkylamino linkers (eg, C3, C6, C12 amino linkers), and also alkyl thiol linkers (eg, C3 or C6 thiol linkers). Oligonucleotides can also be linked by aromatic residues, which can be further substituted with alkyl groups or substituted alkyl groups.

  Oligonucleotides can also include double or tripler units (Glen Research, Sterling, VA), particularly oligonucleotides with 3'3'-linkages. In one embodiment, the double unit is 1,3-bis- [5- (4,4′-dimethoxytrityloxy) pentylamido] propyl-2-[(2-cyanoethyl)-(N, N-diisopropyl). ] May be based on phosphoramidites. In one embodiment, the triple unit is tris-2,2,2- [3- (4,4′-dimethoxytrityloxy) propyloxymethyl] ethyl-[(2-cyanoethyl)-(N, N-diisopropyl). ]-Based on phosphoramidite incorporation. Branching of the oligonucleotide by multiple duplex, triple or other multiple units yields a dendrimer, which is a further embodiment of the invention. Oligonucleotides can also include linker units resulting from peptide modifying reagents, or oligonucleotide modifying reagents (Glen Research, Sterling, VA). Furthermore, the linkage may comprise one or more natural or non-natural amino acid residues connected by peptide (amide) linkages.

  Another possibility for linking oligonucleotides is via a heterocyclic base bridge (Verma S et al. (1998) Annu Rev Biochem 67: 99-134; pages 124). Yet another possibility is the linkage between the sugar moiety of one sequence portion and the heterocyclic base of another sequence portion (Iyer et al. (1999) Curr Opin Mol Therapeutics 1: 344-58; 352. page).

  Different oligonucleotides containing unusual linkages can be synthesized by established methods and linked together online during solid phase synthesis. Alternatively, they can be linked together following the synthesis of individual subsequences.

CpG phosphorothioate oligonucleotides with strong stimulatory activity in the mouse system tend to exhibit lower activity in human and other non-rodent immune cells. DNA containing these (TCG) _n N motifs or RDCGY ₁ Y ₂ N motifs strongly stimulated human peripheral blood to produce IFN-α.

  It has been discovered by the present invention that a subset of CpG immunostimulatory oligonucleotides has a dramatic immunostimulatory effect on human cells (eg, PBMC), which means that these CpG immunostimulatory oligonucleotides are Effective therapeutic agents for human vaccination, cancer immunotherapy, asthma immunotherapy, systemic enhancement of immune function, enhancement of hematopoietic recovery after irradiation or chemotherapy, and other immunomodulatory applications It is suggested that.

  As used herein, the term “treat, treat, or treat” when used with reference to disorders such as infectious diseases, cancer, allergies or asthma, tests for the onset of the disease. Prophylactic treatment to increase body resistance (eg, infection with a pathogen) or, in other words, reduce the likelihood that a subject will develop a disease (eg, to be infected with a pathogen); and Refers to treatment after a subject has developed a disease to fight the disease (eg, reduce or eliminate infection) or prevent the disease from getting worse.

  Accordingly, CpG immunostimulatory oligonucleotides are, in some aspects of the invention, suffering from allergies or asthma, infection with infectious organisms, or cancers for which specific cancer antigens have been identified, or these It is useful as a vaccine for the treatment of subjects at risk of developing. Thus, CpG immunostimulatory oligonucleotides can be administered to a subject in combination with an antigen or allergen for the treatment of infection, allergy, asthma or cancer. Alternatively and additionally, CpG immunostimulatory oligonucleotides can also be given alone without antigens or allergens, or administered with other therapeutic agents for protection against infection, allergy, or cancer. CpG immunostimulatory oligonucleotides can also be administered with other therapeutic agents. Repeated doses may allow longer periods of protection.

  As used herein, a “subject at risk” is any identifiable risk of exposure to a pathogen or allergen that causes infection, or cancer. A subject at risk of developing. For example, a subject at risk of developing an infection can be a subject planning to travel to an area where a particular type of infectious agent has been seen, or a lifestyle or medical procedure. A body fluid that may contain an infectious organism, or a subject that is directly exposed to that organism, or any subject living in an area where an infectious organism or allergen has been identified obtain. Subjects at risk of developing an infection also include the general public where medical institutions recommend vaccination with specific infectious organism antigens. If the antigen is an allergen and the subject develops an allergic reaction to that particular antigen and the subject can be exposed to the antigen, for example, during the pollen season, then the subject Risk of developing an allergic reaction. Subjects who are at risk of developing allergy or asthma have been identified as suffering from allergy or asthma but do not have an active disease during treatment with CpG immunostimulatory oligonucleotides Is mentioned. Subjects at risk of developing allergy or asthma also include subjects that are considered at risk of developing these diseases due to genetic or environmental factors.

  A subject at risk of developing cancer is a subject who is likely to develop cancer. These subjects include, for example, subjects with genetic abnormalities, existences that have been demonstrated to correlate with a high likelihood of developing cancer, cancers such as tobacco, asbestos or other chemical poisons And subjects that have been previously treated for cancer and in remission. If a subject at risk of developing a cancer is treated with a CpG immunostimulatory oligonucleotide and, optionally, an antigen specific for the type of cancer at which the subject is at risk of developing, this subject Can kill cancer cells when cancer develops. When a tumor begins to form in a subject, the subject develops an innate immune response, or a specific immune response against a tumor antigen.

  In addition to the use of CpG immunostimulatory oligonucleotides for prophylactic treatment, the present invention also provides the use of CpG immunostimulatory oligonucleotides for the treatment of a subject suffering from infection, allergy, asthma or cancer. Is included.

  A subject suffering from an infection is a subject that has been exposed to infectious pathogens and has acute or chronic detectable levels of pathogens in the body. CpG immunostimulatory oligonucleotides can be used with or without antigens or other therapeutic agents to reduce the level of infectious pathogens or to eradicate infectious pathogens Alternatively, it can elicit antigen-specific systemic or mucosal immune responses. As used herein, an “infectious disease” is a disease that results from the presence of exogenous microorganisms in the body. It is particularly important to develop effective vaccine strategies and treatments to protect the body's mucosal surface, the first site of pathogen entry.

  A subject suffering from an allergy is a subject who can develop an allergic reaction in response to an allergen. Allergy refers to acquired hypersensitivity to a substrate (allergen). Allergic conditions include, but are not limited to, eczema, allergic rhinitis or nasal cold, hay fever, conjunctivitis, bronchial asthma, allergic asthma, urticaria, food allergies, and other atopic conditions.

  Allergies are generally caused by the production of IgE antibodies against harmless allergens. Cytokines induced by systemic or mucosal administration of CpG immunostimulatory oligonucleotides are abundant in a class called Th1 (examples are IL-12, IP-10, IFN-α and IFN-γ), and These induce both humoral and cellular immune responses. Another major type of immune response associated with the production of IL-4 and IL-5 cytokines is called the Th2 immune response. In general, allergic diseases appear to be mediated by a Th2-type immune response. The immune response in a subject of the CpG immunostimulatory oligonucleotides described herein from a predominant Th2 (associated with the generation of IgE antibodies and allergies) to a balanced Th2 / Th1 response (protection against allergic reactions) Based on the ability to shift, an effective dose to induce an immune response of a CpG immunostimulatory oligonucleotide can be administered to a subject to treat asthma and allergies.

  Thus, CpG immunostimulatory oligonucleotides have significant therapeutic utility in the treatment of allergic conditions and asthma. Th2 cytokines (especially IL-4 and IL-5) are increased in the airways of asthmatic subjects. These cytokines promote important aspects of the inflammatory response of asthma, including IgE isotope switching, eosinophil chemotaxis and activation, and mast cell proliferation. Th1 cytokines (particularly IFN-γ and IL-12) can suppress the formation of Th2 clones and the production of Th2 cytokines. Asthma refers to a disorder of the respiratory system characterized by immunity, narrowing of the airways, and increased airway responsiveness to inhalants. Asthma is often, but not exclusively, associated with atopic or allergic symptoms. Thus, asthma includes allergic asthma and non-allergic asthma.

  A subject suffering from cancer is a subject having detectable cancerous cells. The cancer may be a malignant cancer or a non-malignant cancer. Cancer or tumor includes bile duct cancer; brain tumor; breast cancer; cervical cancer; choriocarcinoma; colon cancer; endometrial cancer; esophageal cancer; gastric cancer; intraepithelial neoplasia; lymphoma; liver cancer; Melanoma; neuroblastoma; oral cancer; ovarian cancer; pancreatic cancer; prostate cancer; rectal cancer; sarcoma; skin cancer; testicular cancer; thyroid cancer; and kidney cancer, and other carcinomas and others Include, but are not limited to, sarcomas. In one embodiment, the cancer is ciliary cell leukemia, chronic myelogenous leukemia, cutaneous T cell leukemia, multiple myeloma, follicular lymphoma, malignant melanoma, squamous cell carcinoma, renal cell carcinoma, prostate carcinoma, bladder cell Carcinoma or colon carcinoma.

  By subject is meant a human or vertebrate, including dogs, cats, horses, cows, pigs, sheep, goats, turkeys, chickens, primates (eg, monkeys), fish (aquatic species) (eg, , Salmon). Thus, this compound can be used to treat cancer and tumors, infections, and allergy / asthma in human and non-human subjects. Cancer is one of the leading causes of death of companion animals (eg, cats and dogs).

  In examples where CpG oligonucleotides are administered with an antigen, the subject can be exposed to the antigen. As used herein, the term “exposed to” refers to an active step of exposing a subject to an antigen, or passive exposure of a subject to an antigen in vivo. Point to either. Methods for active exposure of a subject to an antigen are well known in the art. In general, the antigen is administered directly to the subject by means such as intravenous, intramuscular, oral, transdermal, mucosal, intranasal, intratracheal, or subcutaneous administration. The antigen can be administered systemically or locally. Methods for administering antigens and CpG immunostimulatory oligonucleotides are described in more detail below. A subject is passively exposed to an antigen when the antigen becomes available for exposure to immune cells in the body. A subject can be passively exposed to an antigen, for example, by entry of a foreign pathogen into the body or by generating tumor cells that express the foreign antigen on its surface.

  The manner in which the subject is passively exposed to the antigen may depend, inter alia, on the timing of administration of the CpG immunostimulatory oligonucleotide. For example, in a subject at risk of developing cancer or an infectious disease, or an allergic or asthmatic response, the subject is at the highest risk (eg, during an allergic season or causing cancer) CpG immunostimulatory oligonucleotides can be administered periodically after exposure to the factors). In addition, CpG immunostimulatory oligonucleotides can be administered to travelers before they travel abroad that are at risk of being exposed to infectious agents. Similarly, CpG immunostimulatory oligonucleotides are soldiers at risk of being exposed to a biowarfare that induces a systemic or mucus immune response to the antigen when the subject is exposed to the antigen. Or it can be administered to the general public.

  As used herein, an antigen is a molecule that can elicit an immune response. Antigens include cells, cell extracts, proteins, polypeptides, peptides, polysaccharides, polysaccharide conjugates, polysaccharide peptidomimetics and non-peptide mimetics, and other molecules, small molecules, lipids, glycolipids, carbohydrates, viruses And viral extracts, as well as multicellular organisms such as parasites, and allergens. The term “antigen” broadly encompasses any type of molecule that is recognized as foreign by the host immune system. Antigens include, but are not limited to, cancer antigens, microbial antigens, and allergens.

As used herein, a cancer antigen is a peptide that binds to the surface of a tumor or cancer cell and can elicit an immune response when expressed on the surface of an antigen presenting cell in the context of an MHC molecule. Or a compound such as a protein. Cancer antigens are, for example, Cohen
PA et al. (1994) Cancer Res 54: 1055-8, preparing crude extracts of cancer cells, partially purifying antigens, recombinant techniques, or novel known antigens It can be prepared from cancer cells by any of the synthesis. Cancer antigens include, but are not limited to, recombinantly expressed antigens, immunogenic portions, or whole, or cancer cells. Such antigens can be isolated or prepared recombinantly or by any other means known in the art.

  As used herein, the terms “cancer antigen” and “tumor antigen” refer to an antigen that is differentially expressed by cancer cells and thereby can be utilized to target cancer cells. Used interchangeably. Cancer antigens are antigens that can potentially stimulate a tumor-specific immune response. Some of these antigens are not necessarily expressed, but are encoded by normal cells. These antigens are normally silent (ie not expressed) in normal cells, expressed only at specific stages of differentiation, and transiently expressed such as embryonic and fetal antigens Can be characterized as Other cancer antigens include mutant forms such as oncogenes (eg, activated ras oncogene), suppressor genes (eg, mutated but p53), fusion proteins resulting from internal deletions or chromosomal translocations. Encoded by cellular genes. Still other cancer antigens can be encoded by viral genes such as those carried on RNA and DNA tumor viruses.

  Microbial antigens as used herein are microbial antigens, which include but are not limited to viruses, bacteria, parasites and fungi. These antigens include intact microorganisms, as well as naturally occurring isolates and fragments or derivatives thereof, and also the same or similar to naturally occurring microorganisms and specific for that microorganism. Synthetic compounds that induce an immune response. A compound is similar to a naturally occurring microbial antigen when it induces an immune response (humoral and / or cellular) against the naturally occurring microbial antigen. Such antigens are routinely used in the art and are well known to those skilled in the art.

  Examples of viruses found in humans include, but are not limited to: retroviridae (eg, human immunodeficiency virus (eg, HIV-1 (HTLV-III, LAV or HTLV-III / LAV, or (Also referred to as HIV-III); and other isolates (eg, HIV-LP)); Picornaviridae (eg, poliovirus, hepatitis A virus; enterovirus, human coxsackie virus, renovirus, echovirus); calci Viridae (eg strain causing gastroenteritis); Togaviridae (eg equine encephalitis virus, rubella virus); Flavivirus (eg dengue virus, encephalitis virus, yellow fever virus); Coronaviridae (eg coronavirus) ); Ravine Ile Family (eg, vesicular stomatitis virus, rabies virus); Filoviridae (eg, Ebola virus); Paramyxoviridae (eg, parainfluenza virus, mumps virus, measles virus, RS virus); Ortho Myxoviridae (eg, influenza virus); Bunyaviridae (eg, HanTurn virus, Bunyavirus, flavovirus and Nairovirus); Arenaviridae (haemorrhagic fever virus); Reoviridae (eg, reovirus, Orbivirus) Virnaviridae; Hepadnaviridae (Hepatitis B virus); Parvoviridae (Parvovirus); Papovaviridae (Papillomavirus, Polyomavirus); Adenoviridae (most adenoviruses) Irs); herpesviridae (herpes simplex virus (HSV) 1 and herpes simplex virus 2, varicella-zoster virus, cytomegalovirus (CMV), herpes virus); poxviridae (decubitus virus, vaccinia virus, poxvirus) And Iridoviridae (e.g., African swine fever virus); and unclassified viruses (e.g., etiologic factor of hepatitis delta (considered to be a defective satellite of hepatitis B virus), etiological factor of hepatitis C; Norwalk virus and related viruses, and astrovirus).

  Both gram negative and gram positive bacteria function as antigens in vertebrates. Such Gram-positive bacteria include, but are not limited to, Pasteurella species, Staphylococci species, and Streptococcus species. Gram negative bacteria include, but are not limited to, Escherichia coli, Pseudomonas species, and Salmonella species. Specific examples of infectious bacteria include:

が挙げられるがこれらに限定されない。

However, it is not limited to these.

  Examples of fungi include:

が挙げられる。

Is mentioned.

  Other infectious organisms (ie, protists) include:

が挙げられる。血液由来および／または組織の寄生生物としては、以下：

Is mentioned. Blood-derived and / or tissue parasites include the following:

が挙げられる。

Is mentioned.

Other medically relevant microorganisms are extensively described in the literature. For example, C.I. G. A
See Thomas, Medical Microbiology, Bailier Tindall, Great Britain 1983, the entire contents of which are hereby incorporated by reference.

  Allergen refers to a substance (antigen) that can induce an allergic or asthmatic response in a subject susceptible to disease. The list of allergens is enormous and can include pollens, insect venoms, animal dander, dust, fungal spores and drugs (eg, penicillin). Examples of natural animal and plant allergens include the following species:

に特異的なタンパク質が挙げられるがこれらに限定されない。

But specific proteins are not limited to these.

  The antigen may be substantially purified. The term “substantially purified” as used herein refers to an antigen that is substantially free of other proteins, lipids, carbohydrates or other substances that are naturally associated with the antigen. That is, it refers to a polypeptide. One skilled in the art can purify polypeptide antigens using standard techniques for protein purification. A substantially pure polypeptide often obtains a single major band on a non-reducing polyacrylamide gel. For partially glycosylated polypeptides, or those with several initiation codons, there may be several bands on the non-reducing polyacrylamide gel, but these are characteristic patterns of the polypeptide. Form. The purity of the polypeptide antigen can also be determined by amino-terminal amino acid sequence analysis. Other types of antigens, such as polysaccharides, small molecules, mimetics, etc. are included in the present invention, which can be substantially pure, if desired.

  The oligonucleotides of the invention can be administered to a subject with an antibacterial agent. An antibacterial agent, as used herein, refers to a naturally occurring or synthetic compound that can kill or inhibit infectious microorganisms. The type of antibacterial agent useful in accordance with the present invention depends on the type of microorganism that the subject is infected with or is at risk of being infected. Antibacterial agents include, but are not limited to, anti-bacterial agents, antiviral agents, antifungal agents, and anti-parasitic agents. Phrases such as “anti-infective”, “anti-bacterial”, “anti-viral”, “anti-fungal”, “anti-parasitic” and “parasiticide” are well established to those skilled in the art. And has been defined in standard medical textbooks. Briefly, antibacterial agents kill or inhibit bacteria. Antibacterial agents include antibiotics, as well as other synthetic or natural compounds that have similar functions. Antibiotics are low molecular weight molecules produced as secondary metabolites by cells (eg, microorganisms). In general, antibiotics interfere with one or more bacteria or structures that are specific to the microorganism and are not present in the host cell. Antiviral agents may be isolated from natural sources or synthesized. Antiviral agents are useful for killing or suppressing viruses. Antifungal agents are used to treat superficial fungal infections as well as opportunistic and current systemic fungal infections. Antiparasitic agents kill or control parasites.

  Examples of anti-parasitic agents, also called insecticides useful for human administration, include, but are not limited to: albendazole, amphotericin B, benznidazole, bithionol, chloroquine HCl, chloroquine phosphate, Clindamycin, Dehydroemetine, Diethylcarbamazine, Diloxanide furoate, Efloornithine, Furazolidone, Glucocorticoid, Halofantrine, Iodoquinol, Ivermectin, Mebendazol, Mefloquine meme , Melarsoprol, metrifonate, metronidazole, nicrossamide, nifurtimox, oki Muniquine, paromomycin, pentamidine isethionate, piperazine, prodicantel, primaquine phosphate, proguanil, pyranterpamoate, pyrimethamine-sulfonamido, pyrimethamine-sulfidominxine-pyrimethaminexin HCl, quinine sulfate, quinidine gluconate, spiramycin, sodium stibogluconate (antimony sodium gluconate), suramin, tetracycline, doxycycline, thiabendazole, tinidazole, trimetropium-sulfamethoxazole, and tripalsamide (of these Some alone or in combination with others It is used in conjunction).

  Antibacterial agents kill bacteria or inhibit bacterial growth or function. A large class of antibacterial agents are antibiotics. Antibiotics that are effective in killing or controlling a wide range of bacteria are called broad spectrum antibiotics. Other types of antibiotics are primarily effective against gram positive bacteria or gram negative bacteria. These types of antibiotics are called narrow-range antibiotics. Other antibiotics that are effective against a single organism or disease and not against other types 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 inhibitors or nucleic acid function inhibitors, and competitive inhibitors.

An antiviral agent is a compound that prevents cell infection by the virus or prevents viral replication within the cell. There are significantly fewer antiviral agents than antibacterial agents. This is because the viral replication process is very closely related to DNA replication in the host, and non-specific antiviral agents are often toxic to the host. There are several stages during the viral infection process that can be blocked or inhibited by antiviral agents. These steps include viral attachment to host cells (immunoglobulin or binding peptide), viral decapping (eg, amantadine), viral mRNA synthesis or translation (eg, interferon), viral RNA or DNA replication ( For example, nucleoside analogs), maturation of novel viral proteins (eg, protease inhibitors), and viral budding and release.

  Nucleotide analogs are synthetic compounds that are similar to nucleotides but have incomplete or unusual deoxyribose or ribose groups. Once the nucleotide analogs enter the cell, they are phosphorylated to produce the thiophosphate form that competes with normal nucleotides for incorporation into the viral DNA or RNA. Once the thiophosphate form of the nucleotide analog is incorporated into the growing nucleic acid strand, this results in an irreversible association with the viral polymerase, thus terminating the linkage. Nucleotide analogs include acyclovir (used for the treatment of herpes simplex virus and varicella-zoster virus), ganciclovir (useful for the treatment of cytomegalovirus), idoxuridine, ribavirin (useful for the treatment of RS virus), dideoxyinosine , Dideoxycytidine, zidovudine (azidothymidine), imiquimod, and resiquimod, but are not limited thereto.

  Interferons are cytokines that are secreted by virus-infected cells and immune cells. Interferons function by binding to specific receptors on cells adjacent to infected cells and causing changes in the cells that protect them from infection by the virus. α-interferon and β-interferon also induce the expression of class I and class II MHC molecules on the surface of infected cells, resulting in increased antigen presentation for host immune cell recognition. α-interferon and β-interferon are available in recombinant form and are used to treat chronic hepatitis B and hepatitis C infections. At dosages effective against both antiviruses, interferon has severe side effects such as fever, fatigue and weight loss.

  Antiviral agents useful in the present invention include, but are not limited to: immunoglobulins, amantadine, interferons, nucleoside analogs, and protease inhibitors. Specific examples of antiviral agents include, but are not limited to: Acemannan; Acyclovir; Sodium Acyclovir; Adefovir; Alovudine; Alvircept Sudotox; Hydrochloric acid Amantadine; Aranotin; Arildone; Aterbidine mesylate; Abridine; Cidofovir; Cipamfylline; Citarabine didivirdinyl; Delavirdine; ; Edxuridine; Envira Enviroxine; Enviroxime; Famciclovir; Famotine Hydrochloride; Fiacitabine; Fialuridine; Phosalite; Fosalnet; Ganciclovir; ganciclovir sodium; idoxuridine; ketoxal; lamivudine; lobucavir; memotin hydrochloride; methisazone; nevirapine (Nevirapirvir; Rimantadine Hydrochloride; Saquinadil Mesilate; Somantazine Hydrochloride; Sorivudine; Statron; Stavudine; Tyrolone Hydrochloride; Trifluridine; Trivalvine Hydrochloride; Vidarabine Phosphate; Viroxime); Sarcitabine; Zidovudine; and Zinviroxime.

  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, basilungin / ECB. Other antifungal agents function by disrupting membrane integrity. These include imidazoles (e.g. clotrimazole, sertaconzole, fluconazole, itraconazole, ketoconazole, miconazole and voriconacol and FK463, amphotericin B, M Including but not limited to pradimicin, UK 292, butenafine and terbinafine Other antifungal agents function by destroying chitin (eg, chitinase), or Works by immunosuppression (501 cream).

  CpG immunostimulatory oligonucleotides can be combined with other therapeutic agents (eg, adjuvants) to enhance the immune response. The CpG immunostimulatory oligonucleotide and other therapeutic agent can be administered simultaneously or sequentially. When the other therapeutic agents are administered at the same time, the therapeutic agents can be administered in the same formulation or in separate formulations, but are administered at the same time. The other therapeutic agents are administered sequentially with each other and with the CpG immunostimulatory oligonucleotide when the administration of the other therapeutic agent and the CpG immunostimulatory oligonucleotide is separated in time. More specifically, the CpG immunostimulatory oligonucleotide may be administered before or after administration of (or exposure to) the at least one other therapeutic agent. The temporal separation between administrations of these compounds may be in minutes or longer. Other therapeutic agents include, but are not limited to, adjuvants, cytokines, antibodies, antigens and the like.

  The compositions of the invention can also be administered with non-nucleic acid adjuvants. A non-nucleic acid adjuvant is any molecule or compound other than the CpG immunostimulatory oligonucleotides described herein that can stimulate a humoral and / or cellular immune response. Non-nucleic acid adjuvants include, for example, adjuvants that produce a depot effect, immunostimulatory adjuvants, and adjuvants that produce a depot effect and stimulate the immune system.

  The CpG immunostimulatory oligonucleotides are also useful as mucosal adjuvants. It has been previously discovered that both systemic and mucosal immunity are induced by mucosal delivery of CpG nucleic acids. Thus, the oligonucleotide can be administered in combination with other mucosal adjuvants.

  The immune response may also include cytokines (Bueler and Mulligan, 1996; Chow et al., 1997; Geissler et al., 1997; Iwasaki et al., 1997; Kim et al., 1997) or costimulatory molecules (eg, B7 (Iwasaki et al., 1997; 1997) and CpG immunostimulatory oligonucleotides can be induced or enhanced by co-administration or co-linear expression The term “cytokine” acts as a humoral regulator at nanomolar or picomolar concentrations. And is used as a generic name for a diverse group of soluble proteins and soluble peptides that regulate the functional activity of individual cells and tissues under normal or physiological conditions. Directly mediates and regulates processes that occur in the extracellular environment Examples of cytokines include interleukin 1 (IL-1), IL-2, IL-3, IL-4, IL-5, IL-6 IL-7, IL-10, IL-12, IL-15, IL-18, granulocyte-macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), IFN-γ, IFN- Examples include, but are not limited to, α, IFN-β, tumor necrosis factor (TNF), TGF-β, Flt-3 ligand, and CD40 ligand In addition to cytokines, the CpG oligonucleotides are directed against specific cytokines. Antibodies (eg, anti-IL-10 and anti-TGF-β) and cyclooxygenase inhibitors (ie, COX-1 inhibitors) In combination with terpolymers and COX-2 inhibitors), it may be used.

  The oligonucleotides are also useful for changing the immune response from a Th2 immune response to a Th1 immune response. This results in the creation of a relatively balanced Th1 / Th2 environment. Altering an immune response from a Th2 immune response to a Th1 immune response can be accomplished by measuring the level of cytokines generated in response to the nucleic acid (eg, monocytes to generate Th1 cytokines (including IFN-α)) And by inducing other cells). Changing or rebalancing the immune response from a Th2 response to a Th1 response is particularly useful for the treatment of asthma. For example, an amount effective to treat asthma may be an amount useful for changing a Th2-type immune response associated with asthma to a Th1-type response or a balanced Th1 / Th2 environment. Th2 cytokines (particularly IL-4 and IL-5) are increased in the airways of asthmatic subjects. The CpG immunostimulatory oligonucleotides described herein cause an increase in Th1 cytokines that help re-equilibrate the immune system, thereby preventing or reducing the deleterious effects associated with prevailing Th2 immune responses.

  Altering an immune response from a Th2 immune response to a Th1 immune response can also be assessed by measuring the level of immunoglobulin of a particular isotype. For example, in mice, IgG2a is associated with a Th1 immune response and IgG1 and IgE are associated with a Th2 immune response.

  The CpG immunostimulatory oligonucleotide has a unique ability to promote cell survival, cell differentiation, cell activation, and maturation of dendritic cells, the CpG immunostimulatory oligonucleotide comprises an in vitro method involving dendritic cells, Useful for in vivo methods and ex vivo methods.

  CpG immunostimulatory oligonucleotides also increase natural killer cytolytic activity and antibody-dependent cellular cytotoxicity (ADCC). ADCC can be performed using CpG immunostimulatory oligonucleotides in combination with antibodies specific for cellular targets (eg, cancer cells). When the CpG immunostimulatory oligonucleotide is administered to a subject in combination with the antibody, the subject's immune system is induced to kill tumor cells. Antibodies useful in the ADCC procedure 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.

The CpG immunostimulatory oligonucleotide can also be administered in combination with an anti-cancer therapy. Anti-cancer treatments include cancer drugs, radiation procedures and surgical procedures. As used herein, “cancer drug” refers to an agent that is administered to a subject for the purpose of treating cancer. As used herein, “treating cancer” includes preventing the development of cancer, reducing the symptoms of cancer, and / or inhibiting the growth of established cancer. To do. In another aspect, the cancer drug is administered to a subject at risk of developing cancer for the purpose of reducing the risk of developing the cancer. Various types of medicaments for the treatment of cancer are described herein. For purposes herein, cancer drugs are classified as chemotherapeutic agents, immunotherapeutic agents, cancer vaccines, hormone therapy, and biological response modifiers.

  Furthermore, the methods of the invention are intended to encompass the use of more than one cancer drug in conjunction with the CpG immunostimulatory oligonucleotide. By way of example, where appropriate, the CpG immunostimulatory oligonucleotide can be administered with both a chemotherapeutic agent and an immunotherapeutic agent. Alternatively, the cancer drug is an immunotherapeutic agent and a cancer vaccine or chemotherapeutic agent administered to a subject for the purpose of treating a subject having cancer or a subject at risk of developing cancer. And a cancer vaccine, or a chemotherapeutic agent, an immunotherapeutic agent, and a cancer vaccine.

  The chemotherapeutic agents include methotrexate, vincristine, adriamycin, cisplatin, non-sugar-containing chloroethylnitrosoureas, 5-fluorouracil, mitomycin C, bleomycin, doxorubicin, dacarbazine, taxol, flagillin, megramin GLA, valrubicin, calmstein, and polyphe Luposan, MMI270, BAY 12-9566, RAS farnesyltransferase inhibitor, farnesyltransferase inhibitor, MMP, MTA / LY231514, LY264618 / lometexol, Gramolec, CI-994, TNP-470, Hicamtin / Topotecan, PKCC412, Pulse Podalno / PSC833 / Mitroxantrone, Metallette / Suramin Batimastat, E7070, BCH-4556, CS-682, 9-AC, AG3340, AG3433, Incel / VX-710, VX-853, ZD0101, ISI641, ODN698, TA2516 / malmistat, BB2516 / malmistat, CDP845, D2163, PD183805, DX8951f, remonal DP2202, FK317, picivanyl / OK-432, AD32 / valrubicin, metastron / strontium derivatives, temodar / temozolomide, ebasset / liposomal doxorubicin, utaxane / paclitaxel, taxol / paclitaxel , Cyclopax / oral paclitaxel, oral taxoid, SPU-07 7 / cisplatin, HMR 1275 / flavopiridol, CP-358 (774) / EGFR, CP-609 (754) / RAS oncogene inhibitor, BMS-182751 / oral platinum, UFT (tegaflu / uracil), ergamizole / levamisole, enil Uracil / 776C85 / 5FU enhancer, campto / levamisole, camptostar / irinotecan, tmodex / lalitrexed, leustatin / claribin, paxsex / paclitaxel, doxyl / liposomal doxorubicin, caelix / liposomal doxorubicin, fludarara / fludarabine / Epirubicin, Deposite (DepoCyt), ZD1839, LU79553 / Bis-naphthalimide, LU103793 / Dolastatin, Catechix / Liposomal doxorubicin, Gemzar / Gemcitabine, ZD0473 / Anormed, YM 116, Iodine seed, CDK4 inhibitor and CDK2 inhibitor, PARP inhibitor, D4809 / dexifosamide, Ifes / Menex / isofamide, Vumon / Teniposide, Paraplatin / carboplatin, plantinol / cisplatin, bepeside / etoposide, ZD9331, taxotere / docetaxel, prodrugs of guanine arabinoside, taxane analogs, nitrosoureas, alkylating agents (eg melferran and cyclophosphamide), amino Glutethimide, asparaginase, busulfan, carboplatin, chlorambucil, cytarabine HC , Dactinomycin, daunorubicin HCl, estramustine sodium phosphate, etoposide (VP16-213), floxlysine, fluorouracil (5-FU), flutamide, hydroxyurea (hydroxycarbamide), lomustine (CCNU), mechloretamine HCl (nitrogen) Mustard), mercaptopurine, mesna, mitoten (o. p'-DDD), mitoxantrone HCl, octretide, pricamycin, procarbazine HCl, streptozocin, tamoxifen citrate, thioguanine, thiotepa, vinblastine sulfate, amsacrine (m-AMSA), azacitidine, erythropoietin, hexamethylmelamine (HMM) , Interleukin 2, mitguazone (methyl-GAG; methylglyoxal bisguanylhydrazone; MGBG), pentostatin (2'deoxycoformycin), semustine (methyl-CCNU), teniposide (VM-26), and vindesine sulfate It can be selected from the group, but is not so limited.

  The above immunotherapeutic agents are Rituxan, Ributaxin, Herceptin, Quadramet, Panorex, IDEC-Y2B8, BEC2, C225, Oncolim, SMART M195, ATRAGEN, Ovalex, Bexar, LDP-03, iort6, MDX-210, MDX- 11, MDX-22, OV103, 3622W94, anti-VEGF, Zenapax, MDX-220, MDX-447, MELIMMUNE-2, MELIMMUNE-1, CEACIDE, pre-target, NovoMAb-G2, TNT, gliomab H, GNI-250, EMD -72000, Lymphoside, CMA676, Monofarm C, 4B5, ior egf. It can be selected from the group consisting of r3, ior c5, BABS, anti-FLK-2, MDX-260, ANA Ab, SMART 1D10 Ab, SMART ABL 364 Ab, and ImmuRAIT-CEA, but is not so limited.

  The above cancer vaccines are EGF, anti-idiotype cancer vaccine, Gp75 antigen, GMK melanoma vaccine, MGV ganglioside conjugate vaccine, Her2 / neu, Ovalex, M-Vax, O-Vax, L-Vax, STn-KHL teratope , BLP25 (MUC-1), liposomal idiotype vaccine, melacin, peptide antigen vaccine, toxin / antigen vaccine, MVA based vaccine, PACIS, BCG vaccine, TA-HPV, TA-CIN, DISC-virus, and ImmuCyst / TheraCys Can be selected from the group consisting of, but is not so limited.

  The use of CpG immunostimulatory oligonucleotides in combination with immunotherapeutic agents (eg, monoclonal antibodies) has resulted in numerous mechanisms (significant enhancement of ADCC (discussed above), NK cell activation, and IFN-α levels). It is possible to increase long-term survival. Such nucleic acids, when used in combination with monoclonal antibodies, serve to reduce the dose of antibody required to achieve biological results.

  The present invention also encompasses methods for using the CpG immunostimulatory oligonucleotides to induce non-antigen-specific innate immune activation and to induce broad spectrum resistance to infectious challenges. As used herein, the term “innate immune activation” refers to the activation of immune cells other than memory B cells, such as monocytes, neutrophils, macrophages, dendritic cells, NK cells, and It may include activation of other immune cells that may respond in an / or antigen-independent manner. Broad spectrum resistance to infectious challenges is induced because the immune cells are in an active form and are specifically stimulated to specific antigens. The cells need not be specifically stimulated against a particular antigen. This is particularly useful in biological warfare and other environments described above (eg, travelers).

  The CpG immunostimulatory oligonucleotide can be administered directly to the subject or can be administered in combination with a nucleic acid delivery complex. By nucleic acid delivery complex is meant a nucleic acid molecule that is associated (eg, ionic or covalent, or encapsulated therein) with a targeting means (eg, a molecule that produces high affinity binding to a target cell). 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). ) And nucleic acids associated therewith. Preferred complexes can be sufficiently stable to prevent significant uncoupling prior to internalization by the target cell. However, the complex may be cleavable under appropriate conditions in the cell such that the oligonucleotide is released in functional form.

  The CpG immunostimulatory oligonucleotide and / or antigen and / or other therapeutic agent can be administered alone (eg, in saline or buffer) or using any delivery vehicle known in the art Can be administered. For example, the following delivery vehicles have been described: Cochleate (Gould-Fogerite et al., 1994, 1996); Emulsome (Vancott et al., 1998; Lowell et al., 1997); ISCOM (Mowat et al., 1997); 1993; Carlsson et al., 1991; Hu et al., 1998; Morein et al., 1999); liposomes (Childers et al., 1999; Michalek et al., 1989, 1992; de Haan 1995a, 1995b); live bacterial vectors (eg, Salmonella, Escherichia coli) , Calmette-Guerin, Shigella, Lactobacillus) (Hone et al., 1996; Pouwels et al., 1998; Chat Liver 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 (Fyan et al., 1989); 1993; Kuklin et al., 1997; Sasaki et al., 1998; Okada et al., 1997; Ishii et al., 1997); polymers (eg, carboxymethylcellulose) Chitosan) (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); Sodium fluoride (Hashi) 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., 1998); 1999; Leibl et al., 1998). Other delivery vehicles are known in the art.

  The term “effective amount” generally refers to an amount necessary or sufficient to achieve a desired biological effect. For example, an effective amount of a CpG immunostimulatory oligonucleotide administered with an antigen to induce mucosal immunity is that amount necessary to cause the generation of IgA in response to the antigen when exposed to the antigen; On the other hand, the amount necessary to induce systemic immunity is the amount necessary to cause the generation of IgG in response to the antigen when exposed to the antigen. Select various active compounds and weighting factors (eg, potency, relative bioavailability, patient weight, severity of adverse side effects, and preferred mode of administration) in combination with the teaching provided herein By doing so, an effective prophylactic treatment regimen or an effective therapeutic treatment regimen can be planned that does not cause substantial toxicity but is completely effective for treating a particular subject. An effective amount for any particular application will depend on factors such as the disease or condition being treated, the particular CpG immunostimulatory oligonucleotide being administered, the size of the subject, or the severity of the disease or condition. Depending on it, it can change. One skilled in the art can empirically determine the effective amount of a particular CpG immunostimulatory oligonucleotide and / or antigen and / or other therapeutic agent without necessitating undue experimentation.

  The main dose of the compounds described herein for mucosal or topical delivery typically ranges from about 10 μg to 10 g for a single administration, depending on the application, It can be administered daily, weekly, or monthly and at other times in between or as needed. More typically, mucosal or topical doses range from about 1 mg to 500 mg per administration, most typically in the range of about 1 mg to 100 mg, with 2 to 4 administrations being Every few days or every few weeks. More typically, the dose of immunostimulant is in the range of 10 μg to 100 mg per administration, most typically in the range of 100 μg to 10 mg, administered daily or weekly. The dose of the compound described herein for parenteral delivery to induce an antigen-specific immune response (the compound is delivered with the antigen but not with another therapeutic agent) typically Is 5 to 10,000 times greater than the effective mucosal dose for vaccine adjuvant application or immunostimulant application, more typically 10 to 1,000 times greater, most typically 20 to 100 times greater. Twice as big. To induce an innate immune response, or to increase ADCC, or to induce an antigen-specific immune response when CpG immunostimulatory oligonucleotides are administered in combination with other therapeutic agents or in specialized delivery vehicles The amount of the compounds described herein for parenteral delivery ranges from about 100 μg to 10 g per administration, depending on the application, daily, weekly, or monthly , And other times in between or at other times as necessary. More typically, parenteral doses for these purposes are in the range of about 1 mg to 5 g per administration, most typically in the range of about 1 mg to 1 g, with 2 to 4 administrations. Are several days or weeks. However, in some embodiments, parenteral doses for these purposes can be used in a range of 5-fold to 10,000-fold greater than the representative doses described above.

  For any compound described herein, the therapeutically effective amount can be initially determined from animal models. Therapeutically effective doses are also those compounds known to show similar pharmacological activity for other CpG oligonucleotides tested in humans (human clinical trials are ongoing) (eg, LT antigen And other antigens for vaccination) can be determined from human data. Higher doses may be necessary for parenteral administration. The applied dose can be adjusted based on the relative bioavailability and potency of the administered compound. It is well within the ability of those skilled in the art to adjust the dosage to achieve maximum efficacy based on the methods described above and other methods well known in the art.

  The formulations of the present invention are administered in a pharmaceutically acceptable solution, which is conventionally used in pharmaceutically acceptable concentrations of salts, buffers, preservatives, compatible carriers, adjuvants, And may contain other therapeutic ingredients as needed.

  For therapeutic use, an effective amount of this CpG immunostimulatory oligonucleotide is administered to a subject by any mode that delivers the compound to the desired surface (eg, local surface, mucosal surface, systemic surface). obtain. Administering the pharmaceutical composition of the present invention may be accomplished by any means known to those skilled in the art. Preferred routes of administration include oral, parenteral, intramuscular, subcutaneous, intralesional, intratumoral, intranasal, sublingual, intratracheal, inhalation, intraocular, intravaginal, And rectal route, including but not limited to.

  For oral administration, the above compounds (ie, CpG immunostimulatory oligonucleotides, antigens, and other therapeutic agents) can be readily synthesized by combining the active compound with pharmaceutically acceptable carriers well known in the art. Can be prescribed. Such carriers can be used as tablets, pills, dragees, capsules, solutions, gels, syrups, slurries, suspensions, etc. for ingestion by a subject to which the compound of the invention is targeted. Allows to be prescribed. Pharmaceutical preparations for oral use include pulverizing the resulting mixture, if necessary, and adding appropriate auxiliaries, if desired, then treating the granule mixture to form a tablet core or dragee core. Can be obtained as a solid excipient. Suitable excipients are in particular fillers (for example sugars such as lactose, sucrose, mannitol or sorbitol); cellulose preparations (for example corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth gum, Methylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose) and / or polyvinylpyrrolidone (PVP)). If desired, disintegrating agents (eg, cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof (eg, sodium alginate)) can be added. If desired, the oral formulation can also be formulated in saline or buffer to neutralize internal acidity, or can be administered without any carrier.

  The dragee core is provided with a suitable coating. For this purpose, concentrated sugar solutions can be used, which if necessary include gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and / or titanium dioxide, lacquer solutions, and suitable Organic solvents or solvent mixtures can be included. Dyestuffs or pigments may be added to the tablets or dragee coatings to identify or characterize different active compound dose combinations.

  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 (eg, glycerol or sorbitol). This indented capsule is mixed with a filler (eg, lactose), a binder (eg, starch), and / or a lubricant (eg, talc or magnesium stearate), and optionally a stabilizer. Active ingredients may be included. In this soft capsule, the active compound can be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers can be added. Microspheres formulated for oral administration can also be used. Such microspheres are well defined in the art. All formulations for oral administration are in dosages suitable for such administration.

  For oral administration, the composition can take the form of tablets or lozenges formulated in conventional manner.

  The compounds can be administered by inhalation into the lung (particularly the bronchi, more particularly the deep alveoli) using standard inhalation devices. The compound is in the form of an aerosol spray presentation from a pressurized pack or nebulizer using a suitable propellant (eg, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas). Can be delivered. In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve to deliver a metered amount. An inhalation device can be used to deliver the compound to the subject. An inhalation device as described herein is any device for administering an aerosol (eg, a dry powder form of the above compound). Devices of this type are well known in the art and are described in detail (see, for example, Remington: The Science and Practice of Pharmacy, 19th Edition, 1995, Mac Publishing Company, Easton, Pennsylvania 92, 1676). Description). Many US patents (eg, US Pat. No. 6,116,237) also describe inhalation devices.

As used herein, “powder” refers to a composition composed of finely dispersed solid particles. Preferably, the compound floats relatively freely and can be dispersed in an inhalation device and then inhaled by a subject so that the compound reaches the lungs and allows penetration into the alveoli. To be. “Dry powder” refers to a powder composition having a moisture content that readily disperses in an inhalation device to form an aerosol. Its moisture content is generally less than about 10% by weight (% w), and in some embodiments, less than about 5%, preferably less than about 3% w. The powder can be formulated with a polymer or, if desired, with other materials (eg, liposomes, albumin, and / or other carriers).

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

  The compounds can be formulated for parenteral administration by infusion (eg, bolus infusion or continuous infusion) when it is desired to deliver systemically. Formulations for infusion can be presented in unit dosage form (eg, in ampoules or in multi-dose containers) with added preservatives. The composition can take the form of a suspension, solution, or emulsion in an oily or aqueous vehicle, and contains a formulation (eg, a suspension, stabilizer, and / or dispersant). obtain.

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

  Alternatively, the active compound can be in powder form for constitution prior to use with a suitable vehicle (eg, pyrogen-free sterile water).

  The compounds can also be formulated in rectal or vaginal compositions (eg, suppositories (eg, containing conventional suppository bases (eg, cocoa butter or other glycerides)) or retention enemas) .

  In addition to the formulations described above, the compounds can also be formulated as a depot preparation. Such long acting formulations may be prepared using suitable polymeric or hydrophobic materials (eg, as an acceptable emulsion in oil) or using ion exchange resins or poorly soluble derivatives ( For example, it may be formulated as a poorly soluble salt).

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

  Suitable liquid pharmaceutical preparation forms or solid pharmaceutical preparation forms are for example microencapsulated, encochrated, coated on microscopic gold particles, in liposomes Aqueous solution or saline solution for inhalation, contained in aerosols to be sprayed, contained in pellets for implantation into the skin, or dried on sharp objects that scratch the skin It is. The pharmaceutical composition may also be an extended release of the active compound in granules, powders, tablets, coated tablets, (micro) capsules, suppositories, syrups, emulsions, suspensions, creams, drops, or preparations In the preparation, excipients and additives and / or adjuvants (eg disintegrants, binders, coating agents, sweeteners, lubricants, flavoring agents, sweeteners, or solubilizers) This pharmaceutical composition is suitable for use in a variety of drug delivery systems, see Langer R (1990) for a brief review of methods for drug delivery. Science 249: 1527-33, which is incorporated herein by reference.

  The CpG immunostimulatory oligonucleotide and optionally other therapeutic agents and / or antigens can be administered per se (as is) or can be administered in the form of a pharmaceutically acceptable salt. When used in a drug, the salt should be pharmaceutically acceptable, but a non-pharmaceutically acceptable salt is conveniently used to prepare the pharmaceutically acceptable salt. Can be done. Such salts include, but are not limited to, salts prepared from the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, maleic acid, acetic acid, salicylic acid, p-toluenesulfone. Acids, tartaric acid, citric acid, methanesulfonic acid, formic acid, malonic acid, succinic acid, naphthalene-2-sulfonic acid, and benzenesulfonic acid. Such salts can also be prepared as alkali metal salts or alkaline earth metal salts (eg, sodium, potassium or calcium salts of carboxylic acid groups).

Suitable buffering agents include acetic acid and salts (1% w / v to 2% w / v); citric acid and salts (1% w / v to 3% w / v); boric acid and salts (0. 5% w / v to 2.5%
w / v); and phosphoric acid and salts (0.8% w / v to 2% w / v). Suitable preservatives include benzalkonium chloride (0.003% w / v to 0.03% w / v); chlorobutanol (0.3% w / v to 0.9% w / v); parabens (0.01% w / v to 0.25% w / v), and thimerosal (0.004% w / v to 0.02% w / v).

  A pharmaceutical composition of the invention comprises an effective amount of a CpG immunostimulatory oligonucleotide, and optionally an antigen and / or other agent, optionally in a pharmaceutically acceptable carrier, Including. The term “pharmaceutically acceptable carrier” refers to one or more compatible solid or liquid fillers, diluents, or encapsulating materials suitable for administration to humans or other vertebrates. means. The term “carrier” refers to a natural or synthetic, organic or inorganic component with which the active ingredient is combined to facilitate its application. The components of the pharmaceutical composition can also be mixed with the compounds of the present invention and with each other in a manner in which there are no interactions that substantially impair the desired pharmaceutical efficacy.

  The invention is further illustrated by the following examples. The following examples should not be construed as further limiting.

Example 1. C-class ODN analog induces IFN-α secretion and human TLR9 activity in vitro
In this series of experiments, the C-class ODN analogs of the present invention were stimulated with human TLR9 and NF-κB reporter constructs that stimulate human peripheral blood mononuclear cells (PBMC) against secreted IFN-α. Stablely transfected HEK293 cells were stimulated and tested in vitro for the ability to exhibit TLR9 signaling.

  ODN was purchased from Biospring (Frankfurt, Germany). The ODN was controlled for identity and purity by Coley Pharmaceutical GmbH (Langenfeld, Germany). ODN was diluted in phosphate buffered saline (Sigma, Germany) and stored at -20 ° C. All dilutions were performed using pyrogen-free reagents. The test ODN is

を含んだ。

Included.

は、ホスホロチオエート結合を示し、

Indicates a phosphorothioate bond;

は、ホスホジエステル結合を示す。ＣｐＧ ＯＤＮ２００６（ＴＣＧＴＣＧＴＴＴＴＧＴＣＧＴＴＴＴＧＴＣＧＴＴ、配列番号５６）を、ＴＬＲ９シグナル活性化についてのポジティブコントロールとして使用した。Ｃ−クラスＣｐＧ ＯＤＮ２４２９（ＴＣＧＴＣＧＴＴＴＴＣＧＧＣＧＧＣＣＧＣＣＧ、配列番号５３）を、ＩＦＮ−α誘導についてのポジティブコントロールとして使用した。非ＣｐＧ ＯＤＮ１９８２（ＴＣＣＡＧＧＡＣＴＴ
ＣＴＣＴＣＡＧＧＴＴ、配列番号１８）を、ネガティブコントロールとして使用した。

Represents a phosphodiester bond. CpG ODN2006 (TCGTCGTTTTGTCGTTTTGTCGTT, SEQ ID NO: 56) was used as a positive control for TLR9 signal activation. C-class CpG ODN2429 (TCGTCGTTTTCGGCCGCCGCCG, SEQ ID NO: 53) was used as a positive control for IFN-α induction. Non-CpG ODN 1982 (TCCAGGACTTT
CTCTCAGGGTT, SEQ ID NO: 18) was used as a negative control.

Peripheral blood buffy coat preparations derived from healthy male human donors and healthy female human donors
Obtained from Blood Bank of the University of Duesseldorf (Germany). From these, PBMCs were purified by centrifugation in Ficoll-Hypaque (Sigma). Purified PBMC were purified from 5% (v / v) heat-inactivated human AB serum (BioWhittaker) or 10% (v / v) heat-inactivated FCS, 2 mM L-glutamine (BioWhittaker), and 100 U / ml penicillin. And 100 μg / ml streptomycin (Invitrogen, Karlsruhe, Germany) and resuspended in RPMI 1640 culture medium (BioWhittaker, Belgium).

  Fresh PBMCs were seeded on 96-well round bottom plates and incubated for 48 hours in a 37 ° C. humidified incubator with the indicated concentrations of ODN. Culture supernatants were collected and frozen at -20 ° C until needed when not used immediately.

  The amount of IFN-α in the supernatant was assessed using an enzyme-linked immunosorbent assay (ELISA) that developed color using a commercially available antibody (Alexis GmbH, Gruenberg, Germany). ODN128, ODN331-ODN344, ODN611 and ODN620 were tested with PBMC from 4 different donors and ODN614 was tested with PBMC from 3 different donors.

  The stably transfected HEK293 cells used for the human TLR9 reporter gene assay expressed the human TLR9 receptor and the NF-κB reporter gene construct. The cells were incubated with ODN for 16 hours in a 37 ° C. humidified incubator. Each data point was done in triplicate. Cells were lysed and assayed for reporter gene activity. The stimulation index was calculated with respect to the reporter gene activity of the medium without the addition of ODN.

  (Results) Representative results are shown in FIGS. FIG. 1 shows that C-class ODN332, ODN333, and ODN334 induced large amounts of IFN-α (typically about 2000 pg / ml to 2500 pg / ml) when present at a concentration of 1 μM. The amount of IFN-α induced by these ODNs significantly exceeded the amount of IFN-α induced by the same concentration of ODN2006 or ODN1982. FIG. 1 also shows that C-class ODN332, ODN333, and ODN334 induced a significant amount of TLR9 signaling activity (typically at a stimulation index of about 15) when present at a concentration of 10 μM. Indicates. The amount of TLR9 signaling activity induced by these ODNs was approximately half of the TLR9 signaling activity induced by the same concentration of ODN2006. Essentially the same results were observed for ODN128 and ODN335-ODN344.

  FIG. 2 shows that C-class ODN611, ODN614, and ODN620 induced even higher amounts of IFN-α (typically about 3000 pg / ml to 4000 pg / ml) when present at a concentration of 1 μM. Show. The amount of IFN-α induced by these ODNs significantly exceeded the amount of IFN-α induced by the same concentration of ODN2006 or ODN1982. FIG. 2 also induces a significant amount of TLR9 signaling activity (typically at a stimulation index of about 10-20) when C-class ODN611, ODN614, and ODN620 are present at a concentration of 10 μM. Indicates that The amount of TLR9 signaling activity induced by these ODNs was approximately half of the TLR9 signaling activity induced by the same concentration of ODN2006.

  ODN611 (SEQ ID NO: 43), ODN614 (SEQ ID NO: 43), and ODN620 (SEQ ID NO: 43) were also compared to ODN2429 (one of the original palindrome-containing C class oligonucleotides described). These new C class ODNs showed higher activity in the human TLR9 assay, while the induction of IFN-α was similar to ODN2429 (FIG. 3).

  Taken together, the results of these experiments indicate that the C class ODN analogs of the present invention effectively induce IFN-α secretion and human TLR9 activity in vitro.

(Example 2)
(An additional C class ODN analog induces IFN-α secretion in vitro)
In this series of experiments, additional C class ODN analogs of the invention were tested in vitro for their ability to induce IFN-α secretion. In these experiments, C-class ODN analogs are characterized in part by the presence of AT-rich intermittent inverted repeats or by the presence of intermittent inverted repeats containing dSpacer residues at conventional nucleotide residue positions. It was.

  ODN was obtained as in Example 1. The test ODN is

を含んだ。

Included.

は、ホスホロチオエートヌクレオチド間結合を示し、

Indicates phosphorothioate internucleotide linkage;

は、ホスホジエステルヌクレオチド間結合を示し、Ｌは、ｄＳｐａｃｅｒを示す。

Indicates phosphodiester internucleotide linkage, and L indicates dSpacer.

  Human PBMC were obtained and processed in a manner similar to Example 1.

  The amount of IFN-α in the supernatant was assessed using an enzyme-linked immunosorbent assay (ELISA) in the same manner as in Example 1.

  Results C-class ODN645, ODN646, and ODN647, partially characterized by the presence of AT-rich intermittent inverted repeats, have moderate amounts of IFN-α (typical) when present at a concentration of 1 μM. About 1200 pg / ml to 1500 pg / ml). The amount of IFN-α induced by these ODNs significantly exceeded the amount of IFN-α induced by the same concentration of ODN2006 or ODN1982. C classes ODN649, ODN650, and ODN651, partially characterized by the presence of intermittent inverted repeats containing dSpacer residues, are found in large amounts of IFN-α (typically about 1% when present at a concentration of 1 μM). 2000 pg / ml to 2500 pg / ml). The amount of IFN-α induced by these ODNs significantly exceeded the amount of IFN-α induced by the same concentration of ODN2006 or ODN1982.

  In summary, the results of these experiments are partly characterized by the presence of AT-rich intermittent inverted repeats, or by the presence of intermittent inverted repeats containing dSpacer residues at conventional nucleotide residue positions. The C class ODN analogs of the present invention are shown to effectively induce IFN-α secretion in vitro.

(Example 3)
(Further C class ODN analogues induce IFN-α secretion and human TLR9 activity in vitro)
In this series of experiments, the C class ODN analogs of the present invention were used to stimulate HEK293 cells that stimulate human PBMC against secreted IFN-α or that are stably transfected with human TLR9 and NF-κB reporter constructs. Stimulated and tested in vitro for the ability to exhibit TLR9 signaling. The basic protocol is as described for Example 1, except that the test ODN is

を含んだ。

Included.

は、ホスホロチオエート結合を示し、

Indicates a phosphorothioate bond;

は、ホスホジエステル結合を示す。

Represents a phosphodiester bond.

Various concentrations of ODN were tested in the TLR9 reporter gene assay. EC50 (ODN concentration where the effect is 50% of the maximum effect) is expressed as SigmaPlot (Window
Version 8.0 was calculated using SigmaPlot 2002). Maximum stimulation index (max SI) was calculated as the quotient between the highest value of all concentrations tested for any ODN and vehicle control. The results are shown in FIG. It can be seen that a stimulation index in the range of 10-30 was common.

(Table 1)
(C class CpG ODN of the present invention activates human TLR9)

大量のＩＦＮ−αが、これらのＣクラスＣｐＧ ＯＤＮとともに４８時間インキュベーションした際に、ヒトＰＢＭＣによって分泌された。代表的なＩＦＮ−α量は、１μＭ以下の濃度のＯＤＮとともにインキュベートした後に、３０００ｐｇ／ｍｌ〜４０００ｐｇ／ｍｌの範囲にあった。これらの実験におけるＩＦＮ−αについての応答曲線は、図２において示される応答曲線と非常に類似した。

Large amounts of IFN-α were secreted by human PBMC when incubated with these C class CpG ODNs for 48 hours. Typical IFN-α amounts ranged from 3000 pg / ml to 4000 pg / ml after incubation with ODN at a concentration of 1 μM or less. The response curve for IFN-α in these experiments was very similar to the response curve shown in FIG.

Example 4
(C class ODN analogs induce antigen-specific immune responses in vivo)
In these series of experiments, the C class ODN of the present invention was tested in vivo in combination with vaccination of mice. The C class ODN of the present invention is at least equivalent to the B class CpG ODN2006 in antigen-specific total IgG and IgG2a (Th1-like IgG in mice) titers, and antigen-specific cytotoxic T lymphocyte (CTL) responses. It was observed to boost in the manner of

  ODN was obtained as in Example 1.

  Female BALB / c mice (6-8 weeks old) were used for all experiments. Animals were purchased from Charles River Canada (Quebec, Canada) and housed in small isolated incubators at the Ottawa Hospital Research Institute, Civic Site Animal Care Facility.

Naive BALB / c mouse splenocytes were used for all in vitro assays. The animals were anesthetized with isofluorane and the animals were euthanized by cervical dislocation. Spleens were removed under aseptic conditions and placed in phosphate buffered saline (PBS) + 0.2% bovine serum albumin (Sigma Chemical Company). The spleen was then homogenized and splenocytes were collected with 2% normal mouse serum (Cedarlane Laboratories, Ontario, Canada) and penicillin-streptomycin solution (final concentrations 1000 U / ml and 1 mg / ml, respectively; Sigma Chemical Company); Resuspended in RPMI 1640 tissue culture medium (Life Technologies, Grand Island, NY) supplemented with 5 × 10 ⁻⁵ M β-mercaptoethanol (Sigma Chemical Company).

  BALB / c mice (n = 10 / group) were treated with 1 μg hepatitis B surface antigen (HBsAg) subtype ad (International Enzymes, Calif.) Alone or 1 μg to 100 μg CpG ODN2006, CpG ODN608, CpG ODN618, Or immunized in combination with CpG ODN620. Blood was drawn from the animals and boosted 4 weeks after the first immunization. Two weeks after the boost, 5 animals from each group were euthanized and the spleen was removed for CTL assay.

  Antibodies specific for HBsAg (anti-HBs) (total IgG, IgG1, and IgG2a) were detected and quantified by endpoint dilution ELISA assay. This assay was performed in triplicate on samples from individual animals. The endpoint titer was defined as the highest plasma dilution that gave an absorbance value (OD450) greater than twice that of non-immune plasma at a cut-off value of 0.05. These were reported as group mean titers (GMT) ± SEM.

The CTL assay was performed according to a standard format. Briefly, treasures are removed 4 weeks after immunization, 10% fetal calf serum (Life Technologies, Grand Island, NY) and penicillin-streptomycin solution (final concentrations 1000 U / ml and 1 mg / ml, respectively; Sigma; , Irvine, UK) and 5 × 10 ⁻⁵ M β-mercaptoethanol (Sigma) supplemented with RPMI 1640 tissue culture medium (Life Technologies) (complete RPMI 1640) to homogenize to a single cell suspension. . HBsAg-specific lymphocytes in spleen cell suspension (3 × 10 ⁶ cells / ml) were restimulated for 5 days by incubating with a mouse cell line (p815-S) expressing HBsAg. Following restimulation, the ability of lymphocytes to kill cells expressing HBsAg was determined by using a ⁵¹ Cr release assay. The results are shown as% specific lysis at various effector / target (E: T) ratios.

(Results) Representative results are shown in FIGS. As shown in FIG. 4, the total IgG titer for ODN 2006 and the total IgG titer for ODN 620 are dose dependent, approximately 5 × 10 ³ and 6.5 × 10 ^{3 at} 1 μg ODN dose, respectively. I found out that The total IgG titer for ODN 2006 and ODN 620 was found to be about 1 × 10 ⁴ at a 1 μg ODN dose. As shown in FIG. 5, both ODN2006 and ODN620 significantly boosted IgG2a (Th1-like IgG in mice) compared to the non-CpG control ODN2137 (TGCTGCTTTTGTGCTTTTGTCTT; SEQ ID NO: 60). Specifically, the IgG2a titer for ODN2006, IgG2a titer for ODN620, and IgG2a titer for ODN2137, each administered at a dose of 10 μg, are about 3 × 10 ³ , 6 × 10 ³ , and It was 3 × 10 ² . As shown in FIG. 6, the CTL activity at an E: T ratio of 100: 1 for ODN2006 and ODN620 was significantly greater than the control at doses of 10 μg and 100 μg. For example, the% specific lysis for ODN 2006, the% specific lysis for ODN 260, and the% specific lysis for ODN 2137, each administered at a dose of 10 μg, were about 26%, 24%, and 12%.

  Taken together, the results of these experiments show that the C class ODN analogs of the present invention effectively induce an antigen-specific immune response in vivo.

(Example 5)
(C class ODN analogues improve survival and reduce tumor volume in vivo in tumor models)
In this series of experiments, the C class ODN analogs of the present invention were tested in vivo in a mouse neuroblastoma model. The C class ODN analogs of the present invention have been found to dramatically improve both overall survival and tumor burden.

  ODN was obtained as in Example 1.

  BALB / c mice were obtained as in Example 4.

BALB / c mice received 1 × 10 ⁶ mouse neuroblastoma (Neuro-2a) cells (eg, ATCC CCL-131, American Type Culture Collection, Manassas, Va.) On the left thigh on day 0 It was injected subcutaneously (sc). Mice were given daily subcutaneous injections of PBS, CpG 2006, CpG 620, or non-CpG control ODN2137 on days 10-25. % Survival and tumor volume were measured.

(Result) A typical result is shown in FIG. As shown in FIG. 7A, mice treated with 100 μg ODN620 had a 50% survival rate at 80 days. Compared to this, for ODN2006, ODN2137, or PBS, the corresponding survival rate was 0% to 20%. Furthermore, as shown in FIG. 7B, mice treated with 100 μg ODN620 had a tumor volume that peaked at about 1000 mm ³ on day 28 and decreased to 0 mm ^{3 by} day 38. In comparison, mice treated with ODN2137 or PBS had more rapid and monotonic tumor growth.

  Taken together, the results of these experiments show that the C class ODN analogs of the present invention effectively improve survival and effectively reduce tumor volume in vivo in tumor models.

(Example 6)
(Tissue metabolism and distribution of C class ODN analogs)
Adsorption studies / distribution studies using the C class ODN analogs of the present invention showed successful metabolism and clearance from organs such as kidney and liver after subcutaneous treatment of mice.

  Mice were divided into groups of 5 and each mouse received 250 μg of ODN by a single subcutaneous administration on day 0. At various time points, their organs (liver, kidney, and spleen) were removed and their oligonucleotides and their metabolites extracted for content quantification.

  Results ODNs of the present invention (eg, ODN611 and ODN620) showed less accumulation in organs compared to known ODNs (eg, ODN2429) that are completely phosphorothioate palindromic. For example, on day 3, kidney levels of ODN611, ODN620, and ODN2429 were about 70 ± 28 mg / kg, about 30 ± 18 mg / kg, and about 90 ± 10 mg / kg, respectively. Similarly, on the third day, liver levels of ODN611, ODN620, and ODN2429 were about 45 ± 15 mg / kg, about 28 ± 12 mg / kg, and about 150 ± 15 mg / kg, respectively.

  Taken together, these results indicate that the C-class ODN analogs of the present invention have smooth metabolism and clearance from organs where ODN can accumulate.

(Example 7)
(Physical characteristics of C class ODN analog)
In this set of experiments, ODN2429, ODN611, ODN620, ODN608

およびＯＤＮ６１８

And ODN618

を、サイズ排除クロマトグラフィー、キャピラリーゲル電気泳動（ＣＧＥ）、ＵＶ熱変性、および高速液体クロマトグラフィー（ＨＰＬＣ）を使用して、特徴付けた。ＯＤＮ６１１およびＯＤＮ６２０をサイズ排除クロマトグラフィー（ＰＢＳ中で２２５μＭ）にて調査する場合、ただ１つだけのピークが、各化合物について観察された。すなわち、各オリゴヌクレオチドは、モノマーのように溶出した。対照的に、ＯＤＮ６０８およびＯＤＮ６１８（各々は、パリンドローム配列を含む）をサイズ排除クロマトグラフィーによって調査した場合、各オリゴヌクレオチドについて２つのピークが観察された。これは、上記モノマーに加えて、分子間ダイマーの存在と一致した。しかし、ＵＶ熱変性研究は、ＯＤＮ６１１およびＯＤＮ６２０が、溶液中で二次構造を有すことを示した。これは、分子内ヘアピン構造と一致した。このヘアピン構造は、ＯＤＮ６１１の配列およびＯＤＮ６２０の配列中にある逆方向反復から生じると考えられる。一般的に言うと、これらの配列について、ＨＰＬＣおよびＣＧＥにおいては、ＯＤＮ２４２９およびＯＤＮ６０８と比較して鋭いピークが、観察された。

Were characterized using size exclusion chromatography, capillary gel electrophoresis (CGE), UV heat denaturation, and high performance liquid chromatography (HPLC). When examining ODN611 and ODN620 by size exclusion chromatography (225 μM in PBS), only one peak was observed for each compound. That is, each oligonucleotide eluted like a monomer. In contrast, when ODN608 and ODN618 (each containing palindromic sequences) were examined by size exclusion chromatography, two peaks were observed for each oligonucleotide. This was consistent with the presence of intermolecular dimers in addition to the above monomers. However, UV heat denaturation studies showed that ODN611 and ODN620 have secondary structure in solution. This was consistent with the intramolecular hairpin structure. This hairpin structure is thought to arise from inverted repeats in the ODN611 and ODN620 sequences. Generally speaking, sharp peaks were observed for these sequences in HPLC and CGE compared to ODN2429 and ODN608.

  Collectively, these results indicate that the C class ODN analogs of the present invention tend to form intramolecular secondary structures and do not form intermolecular complexes in vitro at the concentrations tested, but the palindrome-containing C Class ODN indicates that there is a tendency to assemble into a complex by breaking intermolecular interactions. However, in vivo, the ODN concentration achieved in the endosomal compartment is such that the ODN duplex or higher order complex (even the C class ODN analog duplex or higher order complex of the present invention). It may be high enough to support even

(Equivalent)
The above description is considered to be sufficient to enable one skilled in the art to practice the invention. Various modifications of the invention in addition to those shown and described herein will be apparent to those skilled in the art from the foregoing description and are within the scope of the appended claims. The advantages and objects of the invention are not necessarily encompassed by each embodiment of the invention.

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

The present invention may be more easily and completely understood when considered in conjunction with the accompanying drawings. The drawings are provided for purposes of illustration only and are not required to understand or practice the present invention.
FIG. 1 is a series of graphs illustrating the induction of IFN-α and TLR9 signaling by ODN332, ODN333, and ODN334. FIG. 2 is a series of graphs illustrating the induction of IFN-α and TLR9 signaling by ODN611, ODN614 and ODN620. FIG. 3 is a graph illustrating IFN-α production in peripheral blood mononuclear cells (PBMC) stimulated by a panel of oligonucleotides. FIG. 4 is a graph illustrating the group mean titer (GMT) of antigen-specific total IgG after immunization with 1 μg of hepatitis B surface antigen (HBsAg) with the indicated amount of ODN. FIG. 5 is a graph illustrating GMT of antigen-specific individual IgG isotypes after immunization with 1 μg HBsAg with the indicated amount of ODN. FIG. 6 is a graph illustrating the response of antigen-specific cytolytic T lymphocytes after immunization with 1 μg HBsAg with the indicated amount of ODN. FIG. 7A is a graph illustrating survival with different ODN treatments in a mouse neuroblastoma tumor model. FIG. 7B is a graph illustrating tumor volume with different ODN treatments in a mouse neuroblastoma tumor model.

Claims (28)

An immunostimulatory nucleic acid comprising TCGTCGTTTTACGGCGCCGTTGCCG (SEQ ID NO: 44). 2. The immunostimulatory nucleic acid of claim 1 consisting essentially of TCGTCGTTTTACGGCGGCCGTTGCCG (SEQ ID NO: 44). 2. The immunostimulatory nucleic acid of claim 1, wherein at least one internucleotide linkage is modified with phosphorothioate. 2. The immunostimulatory nucleic acid of claim 1, wherein each internucleotide linkage is modified with phosphorothioate. 2. The immunostimulatory nucleic acid of claim 1, wherein at least one nucleotide in the oligonucleotide is a substituted or modified purine or a substituted or modified pyrimidine. The immunostimulatory nucleic acid according to claim 5, wherein the substituted pyrimidine is a C5-substituted pyrimidine or a C6-substituted pyrimidine. The immunostimulatory nucleic acid according to claim 5, wherein the substituted purine is a C8-substituted purine or a C7-substituted purine. The substituted purine or modified purine or substituted pyrimidine or modified pyrimidine is a 5-substituted cytosine, 6-substituted cytosine, N4-substituted cytosine, 5-aza-cytosine, 2-mercapto-cytosine, isocytosine, pseudo-isocytosine, condensed ring system And uracil derivatives, thymine derivatives, 7-deazaguanine, 7-deaza-7-substituted guanine, 7-deaza-8-substituted guanine, 7-deaza-8-azaguanine, hypoxanthine, N2-substituted guanine, 5-amino-3-methyl-3H, 6H-thiazolo [4,5-d] pyrimidine-2,7-dione, 2,6-diaminopurine, 2-aminopurine, purine, indole, substituted adenine, 8-substituted Selected from the group consisting of guanine and 6-thioguanine, Immunostimulatory nucleic acid as claimed in claim 5. The substituted purine or modified purine or substituted pyrimidine or modified pyrimidine is 5-methyl-cytosine, 5-fluoro-cytosine, 5-chloro-cytosine, 5-bromo-cytosine, 5-iodo-cytosine, 5-hydroxy-cytosine; 6-hydroxy-cytosine, 5-hydroxymethyl-cytosine, 5-difluoromethyl-cytosine, and unsubstituted or substituted 5-alkynyl-cytosine, N4-ethyl-cytosine, N, N′-propylene cytosine, phenoxazine 5-fluoro-uracil, 5-bromo-uracil, 5-bromovinyl-uracil, 4-thio-uracil, 5-hydroxy-uracil, 5-propynyl-uracil, 2-thiothymine, 4-thiothymine, 6-substituted thymine, 7-deaza-7- (C2-C6) alkini Guanine, N2- methyl - guanine, N6-methyl - adenine, 8-oxo - adenine, are selected from the group consisting of 8-hydroxy guanine, and 8-bromo-guanine, immunostimulatory nucleic acid of claim 5. 6. The immunostimulatory nucleic acid of claim 5, wherein the substituted purine or modified purine or substituted pyrimidine or modified pyrimidine is selected from the group consisting of a universal base, an aromatic ring system, and a hydrogen atom (dSpacer). . The substituted purine or modified purine or substituted pyrimidine or modified pyrimidine is selected from 4-methyl-indole, 5-nitro-indole, 3-nitropyrrole, P-base, and K-base, benzimidazole, dichloro-benzimidazole, 1- 6. The immunostimulatory nucleic acid of claim 5 selected from the group consisting of methyl-1H- [1,2,4] triazole-3-carboxylic acid amide, fluorobenzene, and difluorobenzene. A vaccine comprising the immunostimulatory nucleic acid of any one of claims 1 to 11 and an antigen. A pharmaceutical composition comprising the immunostimulatory nucleic acid of any one of claims 1 to 11 and a pharmaceutically acceptable carrier. A pharmaceutical composition comprising the immunostimulatory nucleic acid according to any one of claims 1 to 11 and an anticancer drug. Use of an immunostimulatory nucleic acid according to any one of claims 1 to 11 for the manufacture of a medicament for use in the treatment of cancer. The cancer is basal cell cancer, cholangiocarcinoma, bladder cancer, bone cancer, brain and central nervous system cancer, breast cancer, cervical cancer, choriocarcinoma, colon and rectal cancer, connective tissue cancer, cancer of the digestive system, uterus Endometrial cancer, esophageal cancer, eye cancer, head and neck cancer, stomach cancer, intraepithelial neoplasia, kidney cancer, pharyngeal cancer, leukemia, liver cancer, lung cancer, lymphoma including Hodgkin lymphoma and non-Hodgkin lymphoma, melanoma , Myeloma, neuroblastoma, oral cancer, ovarian cancer, pancreatic cancer, prostate cancer, retinoblastoma, rhabdomyoblastoma, rectal cancer, kidney cancer, respiratory cancer, sarcoma, skin cancer, stomach cancer, 16. Use according to claim 15, selected from testicular cancer, thyroid cancer, uterine cancer, urinary cancer, or other carcinomas and sarcomas. 16. Use according to claim 15, wherein the cancer is a cancer sensitive to treatment with interferon alpha (IFN-α). Cancers sensitive to treatment with IFN-α are hairy cell leukemia, chronic myelogenous leukemia, cutaneous T cell leukemia, multiple myeloma, follicular lymphoma, malignant melanoma, squamous cell carcinoma, AIDS-related Kaposi's sarcoma, kidney 18. Use according to claim 17, selected from cell carcinoma, prostate carcinoma, cervical dysplasia, or colon carcinoma. 16. Use according to claim 15, wherein the drug is for use in combination with an anti-cancer treatment. 20. Use according to claim 19, wherein the anti-cancer treatment is radiation. 20. Use according to claim 19, wherein the anti-cancer treatment is a cancer drug. The use according to claim 21, wherein the cancer drug is a chemotherapeutic agent. The chemotherapeutic agent is methotrexate, vincristine, adriamycin, cisplatin, non-sugar-containing chloroethylnitrosoureas, 5-fluorouracil, mitomycin C, bleomycin, doxorubicin, dacarbazine, taxol, flagillin, megramin GLA, valrubicin, calmstein, and polyphe Luposan, MMI270, BAY 12-9566, RAS farnesyltransferase inhibitor, farnesyltransferase inhibitor, MMP, MTA / LY231514, LY264618 / lometexol, Gramolec, CI-994, TNP-470, Hicamtin / Topotecan, PKCC412, Pulse Podalno / PSC833 / Mitroxantrone, Metallette / Suramin Batimastat, E7070, BCH-4556, CS-682, 9-AC, AG3340, AG3433, Incel / VX-710, VX-853, ZD0101, ISI641, ODN698, TA2516 / malmistat, BB2516 / malmistat, CDP845, D2163, PD183805, DX8951f, remonal DP2202, FK317, picivanyl / OK-432, AD32 / valrubicin, metastron / strontium derivatives, temodar / temozolomide, ebasset / liposomal doxorubicin, utaxane / paclitaxel, taxol / paclitaxel , Cyclopax / oral paclitaxel, oral taxoid, SPU-07 7 / cisplatin, HMR 1275 / flavopiridol, CP-358 (774) / EGFR, CP-609 (754) / RAS oncogene inhibitor, BMS-182751 / oral platinum, UFT (tegaflu / uracil), ergamizole / levamisole, enil Uracil / 776C85 / 5FU enhancer, campto / levamisole, camptostar / irinotecan, tmodex / lalitrexed, leustatin / claribin, paxsex / paclitaxel, doxyl / liposomal doxorubicin, caelix / liposomal doxorubicin, fludarara / fludarabine / Epirubicin, Deposite (DepoCyt), ZD1839, LU79553 / Bis-naphthalimide, LU103793 / Dolastatin, Catechix / Liposomal doxorubicin, Gemzar / Gemcitabine, ZD0473 / Anormed, YM 116, Iodine seed, CDK4 inhibitor and CDK2 inhibitor, PARP inhibitor, D4809 / dexifosamide, Ifes / Menex / isofamide, Vumon / Teniposide, Paraplatin / carboplatin, plantinol / cisplatin, bepeside / etoposide, ZD9331, taxotere / docetaxel, prodrugs of guanine arabinoside, taxane analogs, nitrosoureas, alkylating agents (eg melferran and cyclophosphamide), amino Glutethimide, asparaginase, busulfan, carboplatin, chlorambucil, cytarabine HC , Dactinomycin, daunorubicin HCl, estramustine sodium phosphate, etoposide (VP16-213), floxlysine, fluorouracil (5-FU), flutamide, hydroxyurea (hydroxycarbamide), lomustine (CCNU), mechloretamine HCl (nitrogen) Mustard), mercaptopurine, mesna, mitoten (o. p'-DDD), mitoxantrone HCl, octretide, pricamycin, procarbazine HCl, streptozocin, tamoxifen citrate, thioguanine, thiotepa, vinblastine sulfate, amsacrine (m-AMSA), azacitidine, erythropoietin, hexamethylmelamine (HMM) , Interleukin 2, mitguazone (methyl-GAG; methylglyoxal bisguanylhydrazone; MGBG), pentostatin (2′deoxycoformycin), semustine (methyl-CCNU), teniposide (VM-26), or vindesine sulfate 23. Use according to claim 22. The use according to claim 21, wherein the cancer drug is an immunotherapeutic agent. The immunotherapeutic agent is Rituxan, Ributaxin, Herceptin, Quadramet, Panorex, IDEC-Y2B8, BEC2, C225, Oncolim, SMART M195, ATRAGEN, Ovalex, Bexar, LDP-03, iort6, MDX-210, MDX- 11, MDX-22, OV103, 3622W94, anti-VEGF, Zenapax, MDX-220, MDX-447, MELIMMUNE-2, MELIMMUNE-1, CEACIDE, pre-target, NovoMAb-G2, TNT, gliomab H, GNI-250, EMD -72000, Lymphoside, CMA676, Monofarm C, 4B5, ior egf. 25. Use according to claim 24, which is r3, ior c5, BABS, anti-FLK-2, MDX-260, ANA Ab, SMART 1D10 Ab, SMART ABL 364 Ab, or ImmuRAIT-CEA. The use according to claim 21, wherein the cancer drug is a cancer vaccine. The cancer vaccine is EGF, anti-idiotype cancer vaccine, Gp75 antigen, GMK melanoma vaccine, MGV ganglioside conjugate vaccine, Her2 / neu, Ovalex, M-Vax, O-Vax, L-Vax, STn-KHL teratope , BLP25 (MUC-1), liposomal idiotype vaccine, melacin, peptide antigen vaccine, toxin / antigen vaccine, MVA based vaccine, PACIS, BCG vaccine, TA-HPV, TA-CIN, DISC-virus, or ImmuCyst / TheraCys 27. Use according to claim 26, wherein 16. Use according to claim 15, wherein the drug is for use in combination with more than one anticancer therapy.

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