The present invention relates to a modified CpG oligodeoxynucleotide (ODN) which is prepared by coupling a consecutive sequence of deoxyribothymine (dT) to the 3'-terminus of CpG ODN having immunoregularory function, thereby improving immunoactivity of splenocytes, macrophages and peripheral mononuclear cells, and therefore, can be effectively used as a vaccine adjuvant for preventing and treating hepatitis B or an anti-cancer agent. Since the phosphorothioate CpG ODN having the consecutive sequence of dT at its 3'-terminus shows high activity inducing Th-1 immune response and does not elicit in vivo toxicity with guaranteeing its safety, it can be effectively used as a vaccine adjuvant.

Description of the Invention

FIELD OF THE INVENTION

The present invention relates to a modified CpG oligodeoxynucleotide (ODN) with improved immunoregulatory functions. In particular, the present invention relates to the modified CpG ODN which is prepared by coupling a consecutive sequence of deoxyribothymine (dT) to the 3'-terminus of CpG ODN having immunoregularory function, leading to improved immunoactivity of splenocytes, macrophages and peripheral mononuclear cells, and therefore, can be effectively used as a vaccine adjuvant for preventing and treating hepatitis B or an anti-cancer agent.

BACKGROUND OF THE INVENTION

Vertebrate animals can suppress expression of CpG dinucleotide in their genomic DNA sequence or have a cytosine residue of the expressed CpG dinucleotide methylated (Krieg, Ann. Rev. Immunol., 2002, 20, 709; McClelland & Ivarie, Nucleic Acids Res. 1982, 23, 78). Meanwhile, a microbial CpG dinucleotide is expressed at a normal rate and not methylated, and therefore it enables to detect microbial infection in vertebrates using the difference in the levels of the expressed CpG dinucleotide between vertebrate animals and microorganisms.

A microbial genomic DNA is recognized by dendritic cells or B cells expressing the TLR9 via toll-like receptor 9 (TLR9), a pattern recognition receptor, and eventually activates an innate immune system of a host cell. The innate immune system is endowed with a mechanism for removing cancer cells as well as a self defense mechanism against microbial or parasitic infections, and thus it is expected to develop a carcinostatic immunological adjuvant capable of inducing anti-cancer activity of an immune system by properly modifying the CpG ODN.

Studies for activating immune system using the CpG ODN have been actively progressed for the past few decades. The CpG ODN contains at least four bases at both 5'- and 3'-termini with reference to the CpG dinucleotide as the center, and immunoactivity of the CpG ODN is characterized by the base sequence. The CpG ODN is subdivided into two groups, K type and D type. K type ODN stimulates myeloid lineage cells and B cells thus resulting in their proliferation or secretion of immunoglobulin M or IL-6 (Klinman et al., Microbes Infect., 2002, 897-901). On the other hand, D type ODN activates monocytes to be differentiated into dendritic cells or stimulates natural killer cells to secrete IL-6 (Klinman et al., Eur. J. Immunol., 2002, 32, 2617-22; Gursel et al., J. Leukoc. Biol., 71, 813-20, 2002). Further, the D type ODN activates B220.sup.+ dendritic cells to produce IFN-.alpha. while TLR9.sup.+ B220.sup.+ dendritic cells release IL-12 in response to D type ODN. These results suggest that there might be several pathways to improve immune activity of the CpG ODN by stimulating specific immunocytes (Hemmi et al., J. Immunol., 2003, 170, 3059-3064; Kerkman et al., J. Immunol., 2003, 170, 4465-4474).

Various types of CpG ODNs have been designed to redirect the pathologic condition such as infection, autoimmune disease and cancer. Of these, the strategy toward the development of immunotherapeutic CpG ODN against cancer can rely upon effector cells mainly stimulated by CpG ODN. To augment cell-mediated immunity using CpG ODN, the following two methods are commonly exploited.

The first method is to augment a local or systemic immunity via an activation of naive or professional dendritic cells. Cancer cells down-regulate their antigen presentation capabilities to escape an immune surveillance system of a host cell, thereby enabling to survive in the host cell taking advantage of the fact that host cytotoxic T cells are unable to recognize them. To solve this problem, there has been developed a method that immunizes a host with a strong immunogenic peptide in a cancer antigen together with the CpG ODN as an immune adjuvant. By this method, the dendritic cells with high antigen presentation activity can uptake the peptide of cancer antigen and when activated by the CpG ODN, leading to activating cytotoxic T cells. The activated cytotoxic T cells can then effectively eliminate the cancer cells. It has been reported that this method can kill RMA from a mouse (Stern et al., J. Immunol., 2002, 168, 6099-6105). IFN-.gamma. plays an important role in these immune responses. Although this is not done through activation of dendritic cells, it still enables to activate anti-cancer immunity same as in the mechanism of dendritic cells by rendering CpG ODN 2006 to directly work on B cells as well as to increase the expression of a costimulator which can induce an interaction between B and T cells (Jahrsdorfer et al., J. Leukoc. Biol., 2001, 69, 81-88).

The other method is to augment innate immunity via activation of natural killer cells. It is possible to activate cytotoxic T cells by activating dendritic cells by introducing a cancer antigen from the outside, but it is essential to present the cancer antigen on the surface of MHC class I molecule for eliciting cytotoxicity from cytotoxic T cells. However, in many cases the level of presenting caner antigens on the surface of cancer cells is too low to elicit cytotoxicity from cytotoxic T cells, and therefore the method for removing cancer cells by activating the dendritic cells often becomes ineffective. To overcome this limitation, it has been suggested to activate natural killer cells which exert cytotoxicity regardless of cancer antigen presentation on the surface of a cancer cell. Further, the activated natural killer cells activate monocytes or macrophages, leading to activation of antigen-independent anti-cancer immune system. It has been reported that CpG ODN 1584 administration in vivo blocks the metastasis of NK sensitive B16.F1 melanoma whereas CpG ODN 1826 injection effectively rejects NK resistant EL4 lymphoma in an in vivo_mouse tumor model using the above method (Ballas et al., J. Immunol., 2001, 167, 4878-4886). Further, when ODN 1826 was directly injected into a tumorigenic lesion after induction of C26 colon carcinoma mass in BALB/C mice, the size of the tumor was markedly decreased. This data shows that peritumoral CpG ODN monotherapy elicits a strong CD8 T cell response and innate effector mechanisms that seem to act in concert to overcome unresponsiveness of the immune system toward a growing tumor. (Heckelsmiller et al., J. Immunol., 2002, 169, 3892-3899). In contrast, it is difficult to eliminate the murine 38C13 B cell lymphoma in vivo by means of CpG ODN monotherapy. However, if a monoclonal antibody specific to 38C13 lymphoma antigen is treated with the CpG ODN, the activated natural killer cell is capable of efficiently removing 38C13 lymphoma by exerting antibody-dependent cell cytotoxicity (Woodridge et al., Blood, 1997, 89, 2994-2998).

Meanwhile, humoral immunity against cancer can be induced by using an antigen or an equivalent thereof together with the CpG ODN as an immunostimulant. Trastuzumab and rituximab have been known as commercial monoclonal antibodies specific to HER-2 protein over-expressing cancer cell and non-Hodgkins B cell lymphoma, respectively. Recently, the combination therapy with the CpG ODN plus tumor antigen specific antibody demonstrated the potent tumor rejection preclinically and is now entering into clinical trials. (Jahrsdorfer et al., Sem. Oncol., 2003, 30, 476-482).

The CpG ODN has strong innate immunoactivity due to the nucleotide sequences containing CpG dinucleotides present at 5'- and 3'-termini. However, since the CpG ODN itself is of a wild-type structure having no difference from a structure of in vivo DNA molecule, it does not show any cytotoxicity. However, there is a disadvantage in the CpG ODN that it is easily degraded in vivo due to its wild-type structure, consequently leading to reducing half-life of immunoactivity. Although it is possible to increase a daily dosage of CpG ODN to overcome this drawback, it is not economical. It has been reported that when a phosphodiester bond known as a backbone of DNA molecule is changed into a phosphorothioate bond during the synthesis of CpG ODN, immunoactivity of the CpG ODN is amplified about 10 to 100-folds (Sester et al., J. Immunol., 2000, 165, 4165-4173). However, this method has the problems of bringing about cytotoxic effects to immune cells as well as changes in immunoactivity of the CpG due to a modification in backbone. Therefore, it is unclear whether this method would be optimal to structural modification of CpG ODNs.

The CpG ODN shows species-specificity and there has been no report that the CpG ODN shows a high immunoactivity in humans comparable to that it has shown in experimental animals. Further, since the action mechanism of CpG ODN is still unknown, it is hard to develop potent immunotherapeutic CpG ODN to target human immune cells. Consequently, there is very rare CpG ODN for a clinical trial. These limitations necessitate the development of CpG ODN either with superior immune-stimulating activity or lower side effects comparing to the existent CpG ODN.

The present inventors devised the simple method to solve the above problems. That is, a modified CpG ODN was prepared by coupling a consecutive sequence of deoxyribothymine (dT) to the 3'-terminus of CpG ODN, thus improving immunoactivity of splenocytes, macrophages and peripheral mononuclear cells, and therefore, can be effectively used as a vaccine adjuvant for preventing and treating hepatitis B or an anti-cancer agent.

SUMMARY OF THE INVENTION

Accordingly, an object of the present invention is to provide a modified CpG ODN having an improved immunoregulatory function.

Another object of the present invention is to provide a vaccine adjuvant or an anti-cancer agent comprising a modified CpG ODN as an effective ingredient.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a modified CpG ODN showing immunoactivity and containing a CpG motif, wherein a consecutive sequence of dT is coupled to the 3'-terminus of ODN.

Further, the present invention provides a use of the modified CpG ODN as a vaccine adjuvant or an anti-cancer agent.

Hereinafter, the present invention is described in detail.

The present invention relates to the modified CpG ODN which is prepared by coupling a consecutive sequence of dT to the 3'-terminus of CpG ODN having immunoregularory function, leading to improve the immunoactivity of splenocytes, macrophages and peripheral blood mononuclear cells, and therefore, can be effectively used as a vaccine adjuvant for preventing and treating hepatitis B or an anti-cancer agent.

Hereinafter, the term "ODN" means an oligodeoxynucleotide having 11 to 26 nucleotides in length which is capable of activating an immune response. In particular, the term "CpG ODN" means an oligodeoxynucleotide having a CpG motif (5'-purine purine CpG pyrimidine pyrimidine-3') which shows immunoregularory function. The term "a consecutive sequence of dT" means a form of successively coupling at least 4 deoxyribothymines to the CpG ODN by a phosphodiester bond.

The CpG ODNs used in the present invention are artificially synthesized by MetaBion (Germany), wherein all phosphodiester bonds are replaced by phosphorothioate bonds. After the synthesized CpG ODNs are subjected to HPLC (high-pressure liquid chromatography) and NAP purification procedures to maintain high purity, they are provided in a freeze-dried state. The nucleotide sequences of the CpG ODNs used in the present invention are shown in Table 1 (see Original Patent).

When a backbone of the ODN is formed by a natural phosphodiester bond, it can easily degraded by an attack of in vivo nuclease. In order for the CpG ODN with anti-cancer immunoactivity to exhibit a proper immunological effect, it is essential to increase its dose or modify its structure to avoid the above-mentioned attack by a nuclease. The CpG ODN of the present invention has a structure where its backbone is formed by a phosphorothioate bond, thus avoiding nuclease's attack while extending its in vivo half-life. However, a safety problem, when CpG ODN is administered in vivo, may be occurred in a non-specific immune response caused by the phosphorothioate bond itself. Thus, the present inventors examined the safety of the phosphorothioate bond by administering 50 .mu.g of the CpG ODN having the phosphorothioate bond, pre-treated with aluminum hydroxide, along with other antigen such as gDE2t derived from CHO cell into a mouse. The result showed that there was no abnormal finding such as granuloma and necrosis at the injection site of the mouse, thus proving the safety of CpG ODN having the phosphorothioate bond.

The CpG ODN of the present invention activates myeloid lineaged cells to secrete proinflammatory and Th1 type cytokines, and eventually augments anti-cancer immunity. CpG ODN candidates were selected by primary screening using myeloid cell lines such as U-937, RAW264.7 and THP-1. Since the above cell lines may elicit a different immune response from primary cell lines to be targeted by the CpG ODN of the present invention, the inventors of the present invention examined the impact of the CpG ODN of the present invention on fresh murine splenocytes and peritoneal macrophages. The result showed that the CpG ODN significantly enhanced the levels of TNF-.alpha., IL-6, IL-12 and IFN-.gamma. mRNA expression and upregulated expression of MHC class II and B7.2 proteins on the surface of macrophages. These results showed that the CpG ODN of the present invention has a superior anti-cancer immunoactivity as compared with the activity of control natural killer cells present in mouse splenocytes and human peripheral blood mononuclear cells.

Accordingly, the CpG ODN of the present invention can be effectively used as a vaccine adjuvant, in particular, for preventing and treating hepatitis B. Here, it is preferable to administer an antigen for inducing an immune response and the CpG ODN twice or three times at intervals of from 2 to 4 week with a dose of ranging from 50 to 5,000 .mu.g/kg body weight, respectively. Further, the ratio of the CpG ODN to the antigen is preferable to be in the range of from 5:1 to 4:1. The antigen may be immunized alone or together with aluminum hydroxide as a pharmaceutically acceptable vaccine adjuvant, and can be administered via an intramuscular injection or a subcutaneous injection.
 

Claim 1 of 7 Claims

1. A structurally modified oligodeoxynucleotide (ODN) consisting of the nucleotide sequence of SEQ ID NO: 5 having immunoactivity and a CpG motif, wherein the ODN has a consecutive sequence of 4 deoxyribothymines (dT) at the 3'-terminus of ODN, which is modified by changing the phosphodiester bonds into phosphorothioate bonds.
 

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