JP2008000001A - Immune stimulating oligonucleotide and use in pharmaceutical - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain a novel immune stimulating oligonucleotide exhibiting an immune response inducing activity in human in which the activity exhibits cross-reactivity to a mouse and to provide a pharmaceutical use of the immune stimulating oligonucleotide. <P>SOLUTION: The oligonucleotide has a specific base sequence and has immune stimulating activity to a human and a mouse. The therapeutic agent comprises the same and is useful for treating, preventing or ameliorating diseases or disorders in which an immune system does not normally function or diseases or disorders in which enhancement of immune function is effective. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to immunostimulatory oligonucleotide sequences and therapeutic agents comprising the oligonucleotides.

  As immunomodulatory therapeutic agents for malignant tumors, infections, immunodeficiency diseases or autoimmune diseases, various bacterial preparations, various synthetic low molecular compounds such as polysaccharides and levamisole, or various cytokines such as interferon are used. Or have been researched. However, the effects of these therapeutic agents are not always sufficient, and all of them exhibit various side effects. Therefore, there is a need for therapeutic agents that are more effective and have fewer side effects.

  It is known that some polynucleotides have a biological response regulating action. A typical example is Poly (I: C) which is RNA. Poly (I: C) is a potent inducer of IFN production, and an activator of macrophages and an inducer of NK activity. Poly (I: C) has been studied for tumor treatment, but has not been put into practical use as a therapeutic drug to date due to side effects such as fever.

  It has also been shown that certain types of bacterial DNA stimulate immune responses. Bacterial DNA induces cytokine production by macrophages and natural killer (NK) cells. Cellular activation of bacterial DNA is said to be due to a short sequence centered on unmethylated CpG dinucleotide (hereinafter referred to as CpG). 3'-AACGCT-5 ', 3'-AGCGCT-5', 3'-GACGTC-5 'as the most immunostimulatory hexamers when tested for activity to enhance NK cell activity in mouse splenocytes Has been found (Non-Patent Document 1). In addition, studies measuring B cell activation show that the most stimulating hexameric sequences are 5'-purine, purine or pyrimidine, CpG, purine or pyrimidine, pyrimidine-3 'sequences. (Patent Document 1, Non-Patent Document 2).

  It has also been reported that bacterial DNA stimulates macrophages to induce IL-12 and TNF-α production. These cytokines are further known to induce IL-12 and IFN-γ production from splenocytes. In addition, in vitro treatment of splenocytes with either bacterial DNA or immunostimulatory oligonucleotides containing CpG has been reported to induce IL-6, IL-12, IFN-γ production (Non-Patent Documents). 3). These results indicate that CpG-containing sequences have the ability to induce Th1-type immune responses.

  Several types of CpG-containing immunostimulatory oligonucleotide sequences showing such properties, ie, enhanced NK cell activity or induction of Th1-type immune response, have already been found, and there are many reports. As an example, a sequence of polydeoxynucleotide having a palindromic structure containing a certain type of CpG has been found by Tokunaga et al., And this sequence has been reported to show an activity of inducing immune response in mice (Patent Literature). 2). Known CpG-containing sequences that induce human immune responses include D-Type immunostimulatory oligonucleotides (Patent Literature 3, Non-Patent Literature 4). D-Type immune oligonucleotides are known to induce IFN-γ production in human peripheral blood mononuclear cells and to activate human NK cells. Sequences and properties of other types of CpG ODN have also been reported (Non-patent Document 5).

  Studies aimed at enhancing the activity of oligonucleotides as described above have also been conducted. Tokunaga et al. Have found that induction of an immune response is enhanced by adding a repeating structure of deoxyguanylic acid (poly G sequence) to a sequence outside the palindrome containing CpG (Non-patent Document 6). In addition, naturally occurring phosphodiester nucleotides are susceptible to degradation by various nucleolytic activities in cells and in cell culture media. Therefore, stabilization by substituting a phosphodiester bond between nucleotides, which is an attack target of nucleolytic activity, and further study on increased activity as a result are being conducted. A frequently used substitution method is substitution with phosphorothioate. A study by Klinman et al. Shows that the induction of an immune response is enhanced by modifying the G sequence outside the palindrome of the immunostimulatory oligonucleotide by phosphotyrosoate modification (Non-patent Document 7).

  In recent studies, a receptor called Toll-like receptor (TLR) has been found. The role of the TLR family in mammals is thought to be involved in innate immune recognition as a pattern recognition receptor that recognizes common bacterial structures. The TLR family is also known to induce NF-κB activation and Type I interferon production. Bacterial DNA containing unmethylated CpG is specifically recognized by TLR9 and has been found in studies using TLR9 knockout mice to exhibit immunostimulatory activity as described above. (Patent Document 5, Non-Patent Document 8)

  Therapies using immunostimulatory oligonucleotides that show induction of Th1-type immune responses are being studied. For example, a method of administering a conjugate of an antigen and an immunostimulatory nucleotide, or a similar analogue has been found. Administration of the conjugate has been shown to enhance the Th1-type immune response against the same antigen while down-regulating the Th2-type immune response against a particular antigen. Such treatment is stated to be effective for diseases such as allergies, tumors, and autoimmune diseases. (Patent Document 4)

  Although the immunostimulatory oligonucleotides reported so far show an immune response inducing activity in either humans or mice, no species cross-reactivity is found and no activity is found at the same level in both. The low cross-reactivity to non-human species in sequences that exhibit immunostimulatory activity in humans is a major obstacle when developing immunostimulatory oligonucleotides as pharmaceuticals. That is, in non-clinical studies using animals of immunostimulatory oligonucleotide-containing pharmaceuticals that are essential before clinical trials in humans, it is difficult to confirm therapeutic effects and detect early toxic effects. Therefore, it is important and highly useful for immunostimulatory oligonucleotides to exhibit high immunostimulatory activity in humans and cross-reactivity to non-human species.

10-506265 JP 4-352724 WO00 / 61151 JP2002-34565 Special table 2002-517156 Yamamoto et al. J. Immunol. 1992 148: 4072-4076 Kurieg et al. Nature 374: 546-549 Klinman et al. Proc. Natl. Acad. Sci. 1996 93: 2879-2883 Klinman et al. J. Immunol. 2001 166: 2372-2377 Klinman et al. Immunological review 2004 199: 201-216 Tokunaga et al. J. Biochem. 116: 991-994 2001 J. Immunol. 166: 2372-2377 Hemmi et al. Nature 2000 408: 740-745

  The problem to be solved by the present invention is to provide a novel immunostimulatory oligonucleotide that exhibits an immune response-inducing activity in humans and whose activity is cross-reactive to mice, and a pharmaceutical use of this immunostimulatory oligonucleotide That is.

  As a result of intensive studies in order to solve this problem, the present inventors have found that the bases at the 5′- and 3′-ends of the palindrome composed of 6 bases including CpG, which Tokunaga et al. Among the oligonucleotides to which sequences have been added, an oligonucleotide that exhibits high immunostimulatory activity in humans and immunostimulatory activity in mice has been found, and the present invention has been completed. That is, the present invention includes a nucleotide sequence represented by SEQ ID NO: 1, or a nucleotide sequence in which 1 to 4 nucleotides are substituted, deleted and / or inserted in the nucleotide sequence, and An oligonucleotide having immunostimulatory activity is provided. The present invention also includes a base sequence represented by SEQ ID NO: 1, or a base sequence in which 1 to 4 bases are substituted, deleted and / or inserted in the base sequence, An oligonucleotide having immunostimulatory activity is provided. Furthermore, the present invention provides oligonucleotide derivatives having immunostimulatory activity against humans and mice, wherein all or part of the above-mentioned oligonucleotides of the present invention are modified in phosphate bond, ribose sugar part and / or base part. To do. Furthermore, the present invention provides an immunostimulatory agent containing the oligonucleotide or oligonucleotide derivative of the present invention as an active ingredient. Provided is an oligonucleotide delivery complex for use as a medicament, comprising the oligonucleotide or oligonucleotide derivative of the present invention. Further, the present invention enhances the immune function by treating a disease or disorder in which the immune system is not functioning normally, comprising the oligonucleotide or oligonucleotide derivative of the present invention or the oligonucleotide delivery substance of the present invention as an active ingredient. A therapeutic agent for use in treating, preventing or ameliorating a disease or disorder for which is effective. Furthermore, the present invention provides a vaccine comprising the oligonucleotide or oligonucleotide derivative of the present invention or the delivery substance of the present invention as an adjuvant.

  The immunostimulatory oligonucleotide provided by the present invention has a strong immunostimulatory activity in humans. Furthermore, the activity has crossing ability to animal species other than human, which is not found in known nucleotides. Such properties make it possible to accurately predict the effects of immunostimulatory oligonucleotides in humans using other animal species. Therefore, the immunostimulatory nucleotides provided by the present invention and their use are expected to be highly beneficial and useful in pharmaceutical and industrial applications.

  The immunostimulatory oligonucleotide of the present invention preferably comprises a nucleotide sequence represented by SEQ ID NO: 1 or 2 in the sequence listing and has immunostimulatory activity against humans and mice. As specifically shown in the following Examples, it was confirmed that the oligonucleotide consisting of the base sequence represented by SEQ ID NO: 1 or 2 has immunostimulatory activity against humans and mice.

  In general, in a nucleic acid having physiological activity, even if a small number of bases are substituted, deleted, and / or inserted, the physiological activity of the original nucleic acid may be maintained. It is widely known by contractors. Therefore, the nucleotide sequence represented by SEQ ID NO: 1 or 2 comprises a nucleotide sequence in which 1 to 4 bases are substituted, deleted and / or inserted, and is immunized against humans and mice. Oligonucleotides having stimulating activity are within the scope of the present invention. The number of bases for such substitution, deletion and / or insertion is preferably 2 or less, more preferably 1 or less, and most preferably 0. Further, the site of substitution, deletion and / or insertion is not limited as long as immunostimulatory activity against humans and mice is maintained, but the site is different between SEQ ID NO: 1 and SEQ ID NO: 2, The 6th to 11th positions from the 5 ′ side are usually preferred. The oligonucleotide of the present invention may consist of only the base sequence represented by SEQ ID NO: 1 or 2 or the modified sequence thereof described above, or may contain human and mouse as long as it contains the base sequence. As long as it has immunostimulatory activity, other base sequences may be further bound. The size of the oligonucleotide is not particularly limited as long as it has immunostimulatory activity against humans and mice, but is preferably 20 to 50 bases, more preferably 20 to 25 bases.

  The immunostimulatory oligonucleotides of the present invention have immunostimulatory activity in humans and mice. Peripheral blood mononuclear cells of human specimens having an average reactivity in humans, and cytokines from bone marrow-derived dendritic cells in mice In the human peripheral blood mononuclear cells, it shows an effect of inducing the production of interferon-γ (hereinafter referred to as IFN-γ) of 20 ng / ml or more by treatment with an immunostimulatory oligonucleotide of 10 μM or less. This refers to induction of 40 ng / ml or more of interleukin-12p40 (hereinafter IL-12p40) by immunostimulatory oligonucleotide treatment of 1 μM or less. Crossing in humans and mice indicates that the ratio of the minimum dose that shows immunostimulatory activity in humans and mice is 10 or less.

In vitro induction evaluation test of interferon-γ and α from human peripheral blood mononuclear cells (PBMC) as a means of confirming the presence or absence of induction of cytokine production in human peripheral blood mononuclear cells by the immunostimulatory oligonucleotide of the present invention Is mentioned. That is, PBMC is isolated from human blood by performing density gradient centrifugation using Histopaque 1077 at 2000 rpm for 25 minutes at room temperature. After preparing the isolated PBMC in RPMI 1640 medium containing 10% FCS to contain 4.0 x 10 ⁶ cells per ml, seed 4.0 x 10 ⁵ cells per well in a U-bottom 96-well microplate, At the same time, CpG-ODN is stimulated in the presence for 24 hours for 7 days, and the culture supernatant is recovered. Using the collected culture supernatant, the amount of IFNα produced after completion of stimulation for 24 hours and the amount of IFNγ produced after completion of stimulation for 7 days are respectively determined by ELISA.

In vitro induction evaluation test of cytokine (IL-12p40) of immunostimulatory oligonucleotide in mouse dendritic cells as means for confirming the presence or absence of induction of cytokine production in mouse bone marrow-derived dendritic cells by the immunostimulatory oligonucleotide of the present invention Is mentioned. Specifically, bone marrow cells are obtained from femur and tibia of male 10-25 week old CL57BL / 6N mice and cultured in RPMI1640 medium containing 10% FCS containing 10 ng / ml murine GM-CSF. By exchanging the medium every two days, the cells are cultured for 6 days while removing floating cells mainly consisting of granulocytes, and differentiated from dendritic progenitor cells to immature dendritic cells (imDC). After culturing for 6 days, imDCs are collected by pipetting, and 2.0 × 10 ⁵ cells per well are seeded in a flat-bottom 96-well plate (IWAKI) and stimulated with CpG-ODN for 2 days. After stimulation, the amount of IL-12p40, a cytokine derived from the Th1 reaction, released into the culture supernatant is quantified by ELISA.

  The present invention also includes immunostimulatory oligonucleotides in which the phosphate bond, ribose sugar part and base part of all or part of the above sequence are modified. In a preferred embodiment, the modification is phosphorothioate type oligonucleotideization. In a more preferred embodiment, phosphorothioate modification is performed between some or all consecutive G-sequence nucleotide residues at the 5′- and 3′-ends.

  The immunostimulatory oligonucleotides of the present invention can be synthesized by conventional techniques and nucleic acid synthesizers. These synthetic methods include, but are not necessarily limited to, enzymatic methods, chemical methods, and degradation of sequences longer than the sequences of the present invention. Modified oligonucleotides are also synthesized in the prior art. For example, a phosphorothioate-modified oligonucleotide can be obtained by treating oligonucleotide phosphoramidate with sulfur, but is not necessarily limited thereto. These oligonucleotide synthesis techniques and modification techniques are also used in patent documents and non-patent documents cited in the present specification, and are other known techniques for which many reports have been confirmed.

  The above-described immunostimulatory oligonucleotide of the present invention or the derivative thereof has a use as an immunostimulant. Alternatively, the immunostimulatory oligonucleotide of the present invention described above or the derivative thereof described above may be in the form of an oligonucleotide delivery complex intended for use as a medicament. Delivery complexes include, but are not necessarily limited to, mixtures and conjugates of the immunostimulatory oligonucleotide or derivative thereof with other substances, liposomes and nanospheres incorporating the immunostimulatory oligonucleotide. is not. In a preferred embodiment, the delivery complex is a liposome embedded with an oligonucleotide or derivative thereof. Embedding refers to binding of the liposome to the lipid membrane surface, incorporation into the lipid membrane, or incorporation of the liposome into the lumen.

  The immunostimulant is a therapeutic agent for use in treating, preventing or ameliorating a disease or disorder in which the immune system is not functioning normally or a disease or disorder in which enhancement of immune function is effective Can be used as Diseases or disorders in which the immune system is not functioning normally include allergies and malignant tumors, and are not necessarily limited thereto, but in a preferred embodiment, the disease or disorder is an allergic disease. . An allergic disease is caused by an antigenic substance derived from pollen, mites, house dust, etc., and is a disease whose symptoms are inflammation such as rhinitis, conjunctivitis, dermatitis, asthma, etc. The allergic disease is pollen allergy. The pollen allergy refers to an allergy that specifically uses a protein derived from cedar pollen as an antigen, but other pollen-derived substances such as ragweed and cypress may be the antigen.

  When used as such a therapeutic agent, the administration route is not particularly limited, but subcutaneous injection, intradermal injection, intravenous injection, intramuscular injection, injection into affected tissue, nasal administration, ophthalmic administration, etc. Administration, pulmonary administration, transdermal administration and the like are preferred. The dosage is appropriately selected according to the patient's condition, therapeutic purpose, administration route, etc., but is usually about 0.1 pM to 10 μM, preferably about 1 pM to 1 μM as the amount of oligonucleotide per day for adults. In addition, the oligonucleotide or a derivative thereof is usually prepared by a well-known preparation method for preparing a dosage form to be adopted.

  The present invention also has the use of the immunostimulatory oligonucleotides or delivery complexes described above as vaccine adjuvants. Diseases that can be used for vaccines are infectious diseases. Infectious diseases are diseases that develop due to viruses, bacteria, fungi, protozoa, and the like. The dose for use as an adjuvant is also appropriately selected depending on the purpose of administration, the route of administration, etc., but it may usually be the same dose as described above.

The comparative examples and examples will be described in detail below. The sequence abbreviations and characteristics of the immunostimulatory oligonucleotides used in the following description are described in the sequence listing.
Comparative Example 1: Cytokine-inducing effect of known immune oligonucleotides in mice and humans For known immunostimulatory oligonucleotides (Table 1), cytokine-inducing effects in mice and humans, i.e. cross-reactivity, were examined.

  Treatment of human PBMC with 10 μM D19N, 3 and 10 μM D19N + GS confirmed the induction of IFN-γ, among which the sequence D19N + GS modified with phosphorothioate modification of the terminal G sequence was active at low doses. Confirmed to have. Moreover, the induction | guidance | derivation of the cytokine by the treatment of other well-known sequences (D19GS, M-CpGS, M-non-CpGS) was hardly seen. (Figure 1)

  When each immunostimulatory oligonucleotide was treated with BMDC in mice, the production of IL-12p40 was significantly induced by the treatment with M-CpGS, a known sequence having immunostimulatory activity in mice, and the production was as low as 1 μM. Also confirmed. Other immunostimulatory oligonucleotides induce IFN-γ production with 10 μM treatment, but the production is low and little production with 1 μM treatment is observed. Therefore, in the known sequence, the activity to induce IFN-γ production in human and mouse was not crossed. (Figure 2)

  From these test results, it was proved that the known sequences D19N and D19N + GS have activity in humans, but their activity in mice is low and the immunostimulatory cross-reactivity in mice and humans is low. . Also, such properties are not improved by phosphorylation of the entire sequence or terminal G sequence. Therefore, it was confirmed that the enhancement of the crossing property cannot be achieved by simply adding the above-described modification to the sequence exhibiting immunomodulating activity.

Example 1: Palindromic Sequence Immunostimulatory Oligonucleotides Containing Phosphorothioate-Modified G Sequences at Both Ends Induced Cytokine Production in Human PBMC Palindromic sequence shown by Tokunaga et al. And 15), a immunity-inducing oligonucleotide obtained by adding a nucleotide sequence containing a phosphorothioate-modified G sequence to both ends, mod1 (SEQ ID NO: 1), mod2 (SEQ ID NO: 2), mod9 (SEQ ID NO: 3) ), And mod12 (SEQ ID NO: 4). Then, screening of the cytokine production induction activity in human PBMC of these sequences was performed.

  When human PBMC were treated with D19N + GS, mod1, 2, 9 and 12, respectively, the induction of IFN-γ production by 3μM treatment was higher in the mod1 and 2 sequences compared to D19N + GS. Confirmed (FIG. 3).

  When each of the above oligonucleotides was treated in mouse BMDC, it was confirmed that in 1 μM treatment of mod1 and 2, IL-12p40 production-inducing activity was higher compared to D19N + GS, and the activity increased further at 5 μM ( FIG. 4).

  From these results, it was confirmed that the oligonucleotide sequences mod1 and 2 have human and mouse cross-activity. Also, mod 9 and 12 had weak activity in humans and mice. These results indicated that adding a nucleotide sequence containing a G sequence to the end of the Tokunaga et al. Sequence does not necessarily lead to increased activity in humans and mice. Therefore, it was confirmed that human and mouse cross-activity is a property unique to the mod1 and mod2 sequences.

Example 2: Induction activity of IFN-α production in human PBMCs of D19N-GS, mod1 and mod2
D19N-GS, mod1 and mod2 were screened for inducing activity of IFN-α production in human PBMC.

  When these oligonucleotides (3 μM) were treated with PBMC, D19N-GS and mod1 yielded about 3 ng / ml. On the other hand, when mod2 was treated, a production amount of 25 ng / ml or more was confirmed, and IFN-α production from PBMC was significantly induced as compared with D19N-GS and mod1 (FIG. 5).

It is a figure which shows the result of the production amount of IFN-γ in human PBMC treated with a known immunostimulatory oligonucleotide. It is a figure which shows the result of IL-12p40 production amount in the mouse | mouth BMDC which processed the known immunostimulatory oligonucleotide. It is a figure which shows the result of the amount of IFN-gamma production in human PBMC which treated this invention and the known immunostimulatory oligonucleotide. It is a figure which shows the result of IL-12p40 production amount in mouse | mouth BMDC which treated this invention and the well-known immunostimulatory oligonucleotide. It is a figure which shows the result of the amount of IFN- (alpha) production in human PBMC which treated this invention and the known immunostimulatory oligonucleotide.

Claims (16)

  Contains the nucleotide sequence shown in SEQ ID NO: 1 or a nucleotide sequence in which 1 to 4 nucleotides are substituted, deleted and / or inserted in the nucleotide sequence, and has immunostimulatory activity against humans and mice , Oligonucleotides.   The oligonucleotide according to claim 1, comprising the base sequence represented by SEQ ID NO: 1.   The oligonucleotide according to claim 2, which is a 20-base oligonucleotide consisting of the base sequence shown in SEQ ID NO: 1.   Contains the nucleotide sequence shown in SEQ ID NO: 2 or a nucleotide sequence in which 1 to 4 nucleotides are substituted, deleted and / or inserted in the nucleotide sequence, and has immunostimulatory activity against humans and mice , Oligonucleotides.   The oligonucleotide according to claim 4, comprising the base sequence represented by SEQ ID NO: 2.   The oligonucleotide according to claim 5, which is a 20-base oligonucleotide consisting of the base sequence shown in SEQ ID NO: 2.   An oligonucleotide having immunostimulatory activity against humans and mice, wherein all or part of the phosphate bond, ribose sugar part and / or base part of the oligonucleotide according to any one of claims 1 to 6 is modified. Derivative.   The oligonucleotide derivative according to claim 7, wherein the modification is phosphorothioate-type oligonucleotide formation in which an oxygen atom of a phosphate group of an oligonucleotide having a phosphodiester bond is substituted with a sulfur atom.   The oligonucleotide derivative according to claim 8, wherein the nucleotide residues in the 5'- and 3'-terminal consecutive G sequences are phosphorothioate-modified.   The immunostimulant which contains the oligonucleotide or oligonucleotide derivative of any one of Claim 1 thru | or 9 as an active ingredient.   An oligonucleotide delivery complex comprising the oligonucleotide or oligonucleotide derivative according to any one of claims 1 to 9 and used as a medicament.   The oligonucleotide delivery complex according to claim 11, wherein the complex is a liposome embedded with an oligonucleotide.   A disease or disorder in which the immune system does not function normally, comprising the oligonucleotide or oligonucleotide derivative according to any one of claims 1 to 9 or the oligonucleotide delivery substance according to claim 11 or 12 as an active ingredient, or A therapeutic agent for use in treating, preventing or ameliorating a disease or disorder for which it is effective to enhance immune function.   The therapeutic agent according to claim 13, wherein the disease or disorder is an allergic disease.   The therapeutic agent according to claim 14, wherein the allergic disease is pollen allergy. A vaccine comprising the oligonucleotide or oligonucleotide derivative according to any one of claims 1 to 9 or the oligonucleotide delivery substance according to claim 11 or 12 as an adjuvant.

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