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Sulpho-oligodeoxynucleotide with immune stimulation activity and uses thereof

An immunostimulatory activity, deoxynucleotide technology, applied in the direction of organic active ingredients, medical preparations containing active ingredients, organic chemistry, etc. Disclose issues such as nucleotides to achieve good immune stimulating activity, inhibit tumor growth, and enhance immune response.

Active Publication Date: 2009-07-29
许洪林
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, some CpG-ODNs and related applications are also disclosed in patent documents CN 1271733A, CN 1526718A, US 2005 / 0267057A1 or WO 99 / 56755, but in the above documents, there is no disclosure about having two or more than two copy of the nucleotide of the 5'-NTCGTT-3' motif (N does not represent A or G), nor does it disclose any information about the cross-immunity of thiooligonucleotides containing this motif to humans and mice Technical Implications for Stimulatory Activity

Method used

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  • Sulpho-oligodeoxynucleotide with immune stimulation activity and uses thereof
  • Sulpho-oligodeoxynucleotide with immune stimulation activity and uses thereof
  • Sulpho-oligodeoxynucleotide with immune stimulation activity and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Example 1. Human immunostimulatory activity of oligodeoxynucleotides with 5'-NTCGTPy-3' (wherein N represents A, G, C or T, Py represents pyrimidine C or T) motif

[0061] In order to elucidate the CpG motif for activating human immune cells, the inventors conducted mutation studies on the remaining four nucleotides in the CpG motif except for the core nucleotide CpG. To this end, the inventors designed a set of CpG-ODNs, as shown in Table 1, each sequence is 12 nucleotides in length and contains two identical copies of the CpG motif. These CpG-ODNs are used to stimulate human peripheral blood mononuclear cells (PBMC) cultured in vitro, and their activity on human immune cells can be evaluated by measuring the IgM content in the culture supernatant.

[0062] The CpG-ODN used in this example was designed by the present inventors, entrusted Shanghai Sangong Biotechnology Co., Ltd. to artificially synthesize it, and carried out full-chain thio modification, purified by pol...

Embodiment 2

[0068] Example 2. Immunostimulatory activity of oligodeoxynucleotides with 5'-NA / TCGTT-3' (wherein N represents A, G, C or T) motifs on mice

[0069] In order to elucidate the CpG motif that activates mouse immune cells, the inventors conducted mutation studies on the remaining four nucleotides in the CpG motif except the core nucleotide CpG. See Table 1 for the CpG-ODN sequences used in this example. Mouse spleen mononuclear cells (SMMC) were stimulated with these CpG-ODNs, and their activity on mouse immune cells could be evaluated by measuring the IgM content in the culture supernatant. The Balb / c mice used were female, 6-8 weeks old, purchased from the Experimental Animal Center of the Chinese Academy of Medical Sciences.

[0070] Take the spleen of Balb / c mice under sterile conditions, and prepare SMMC suspension with RPMI 1640 medium; after counting, use RPMI 1640 medium to adjust the cell concentration to 1×10 6 / ml, inoculated into 96-well cell plate, 200 μl per well...

Embodiment 3

[0073] Example 3. The immunostimulatory activity of oligodeoxynucleotides of the present invention on human immune cells

[0074] According to the CpG motifs found in Examples 1 and 2 that have cross-immunostimulatory activity to humans and mice, the inventors designed and synthesized a set of CpG-ODNs, as shown in Table 2, with a length of 15 to 35 nucleotides , respectively containing two or more than two copies of the 5'-TTCGTT-3' motif (numbered T1-T22) and the 5'-CTCGTT-3' motif (numbered C1 and C2).

[0075] The CpG-ODNs in Table 2 except for the control sequences T7C and 1826 are respectively quoted from the literature "Chinese Journal of Microbiology and Immunology" 2001, 21 (5), pages 471-475, "The Journal of Immunology" 1998, volume 160, Pages 870-876, all the other are designed by the inventor. The reference sequence used in this example and the sequence designed by the inventors were entrusted to Shanghai Sangon Biotechnology Co., Ltd. to synthesize, modify the wh...

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Abstract

The invention relates to a phosphothioate oligodeoxynucleotide with immunostimulation activity. A 5'-NTCGTT-3' primitive with two or more than two copies is arranged in the sequence thereof and the length thereof is of 15 to 35 nucleotide; wherein, CpG is unmethylated; and N does not represent A or G. The phosphothioate oligodeoxynucleotide has excellent immunostimulation activity both to a human body and a mouse immunocyte in vitro; and the immunostimulation activity in the human body thereof can be estimated according to the results of immunostimulation activity to the mouse. As a vaccine adjuvant, the phosphothioate oligodeoxynucleotide can remarkably enhance the immune response of the mouse to the hepatitis B vaccine of gene engineering, and can lead the immune response to lean to the direction of TH1, thus can be used for controlling and curing hypersusceptibility and infection. Meanwhile, the phosphothioate oligodeoxynucleotide of the invention also has excellent activity to inhibit the growth of tumors in the body of the mouse.

Description

technical field [0001] The invention relates to the field of biology, in particular to a thiooligo-deoxynucleotide with immunostimulatory activity and its application. Background technique [0002] In the 1980s, Japanese scholars discovered that the DNA component of Bacillus Calmette-Guerin (BCG) had anti-tumor activity in humans and animals. Later, scientists who studied antisense nucleic acid therapy discovered that artificially synthesized thiooligo-deoxynucleotides had non-toxic properties. Specific immunostimulatory activity. Krieg AM and others revealed the essence of these phenomena in "Nature" 1995, volume 374, pages 546-549, and found that the immunostimulatory active components in nucleic acids are some containing unmethylated 5'-CpG-3' dinuclear The hexanucleotide sequence of nucleotides is characterized by 5'-PuPuCpGPyPy-3' (Pu represents purine A or G; Py represents pyrimidine C or T), such as 5'-GACGTT-3', etc. Among them, the core nucleotides C and G are nec...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C07H21/04A61K31/7088A61P35/00A61P31/00A61P37/08C12N15/117
Inventor 许洪林
Owner 许洪林
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