JP2016011272A - Immune response controlling agent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a nucleic acid which can activate or inhibit in vivo immune responses, particularly DC immune response-related functions.SOLUTION: The invention relates to an immune response controlling agent containing as an active ingredient at least one nucleic acid selected from the group consisting of a microRNA represented in tables 1-7, a microRNA complementary to the microRNA, an inhibitory nucleic acid to the microRNA and the complementary microRNA, and functional equivalents thereof. According to the invention, an immune response of an organism or a DC separated from the organism can be controlled by using a nucleic acid, and the prevention, improvement, or treatment of diseases caused by a decreased immune function or diseases caused by an abnormal enhancement of the immune function becomes possible.

Description

  The present invention relates to an immune response control agent comprising, as an active ingredient, a nucleic acid capable of controlling an immune response, particularly an immune response function of dendritic cells.

  Dendritic cells (hereinafter referred to as DC) play a very important role in the immune system by bridging innate and acquired immunity. In particular, DC is a cell that produces cytokines such as IFN-α and IFN-β that control immune-related cells, and has a function of presenting a foreign antigen to CD4 positive T cells or CD8 positive T cells, ie, antigen presenting cells. As having an important role in the immune system. For example, DC decomposes an antigen incorporated therein and presents the fragment to naive CD4 positive CD cells or CD8 positive T cells via MHC class I molecules or MHC class II molecules expressed on its surface. Inducing differentiation into effector T cells such as antigen-specific killer T cells and helper T cells, and further inducing differentiation of antibody-producing cells through the activation of helper T cells. It is involved in the immune response of the body by promoting production. DC also produces interleukin (IL) -12 and the like that act to induce differentiation into effector T cells.

  Originally, it is preferable that the antigen presentation by DC and the subsequent immune response are more activated against foreign cells to be excluded or abnormal cells such as cancer cells. On the other hand, when the immune response via DC becomes hypersensitive or excessive, an immune response to a foreign substance that should be tolerated is induced more than necessary, and allergic diseases may be induced or autoimmune diseases may be caused. is there. Therefore, a technique capable of appropriately regulating an immune response via a DC function, specifically, activating or suppressing a function related to a DC immune response according to a situation relates to the immune system. It can be useful in the treatment of various diseases.

  Known substances that can activate DC include IFN-α and IFN-β known as type 1-cytokines, and immune adjuvants that act via a toll-like receptor (TLR). Moreover, IL-10, IL-6, prostaglandin E2, etc. are known as a substance which can suppress the function of DC. In addition, since IL-17 has a function of inducing production of IL-6, IL-17 is considered to be one of the substances that can suppress the function of DC.

  IL-6 acts on DC to suppress the expression of MHC class II molecules and CD86 to reduce the antigen presentation ability, and to induce differentiation from naive T cells into helper T cells, which are effector T cells. Also suppresses the production of IL-12. In recent years, it has been reported that IL-6 production is induced in a cancer microenvironment, and as one of the causes that the therapeutic effect of immunotherapy on cancer cells is not sufficiently exhibited, Inhibition of DC function by expression of IL-6 has been pointed out (Non-patent Document 1).

  On the other hand, in connection with various diseases, particularly cancer, minute RNA having a specific base sequence (micro RNA, hereinafter referred to as miR) has attracted attention. miR is a short single-stranded RNA consisting of 20 to 25 bases, and is known to be involved in gene expression and transcriptional regulation by interacting with mRNA having a complementary sequence thereto. miR is also attracting attention as a biomarker for cancer, and its application to the suitability of cancer treatment methods and the determination of therapeutic effects has been studied.

  However, notable results have not yet been reported for miRs that control immune responses, in particular, act on DCs to activate or suppress their functions in immune responses via DCs.

Narita et al., J Immunol. 2013, 190, 812-820.

  An object of this invention is to provide the nucleic acid which can activate or suppress the function regarding the immune response of a biological body, especially the immune response of DC.

  The present inventors increase or decrease the expression level of a specific miR in DC cultured in a medium containing IL-6, IL-17, LPS or poly I: C, and immunize by utilizing these. The inventors found that it is possible to control the response, and completed the following inventions.

(1) At least one selected from the group consisting of miRs shown in Tables 1 to 7, miRs complementary to the miRs, inhibitory nucleic acids for the miRs and the complementary miRs, and functional equivalents thereof. An immune response control agent comprising the nucleic acid of 1 as an active ingredient.

(2) The immune response control agent according to (1) for controlling the antigen response function of dendritic cells.
(3) The immune response regulator according to (2), wherein the antigen response function of dendritic cells is a cytokine production function.
(4) A medicament comprising the immune response regulator according to any one of (1) to (3).

  ADVANTAGE OF THE INVENTION According to this invention, the immune response of DC isolate | separated from the living body or the living body using specific nucleic acid can be controlled, and the prevention, improvement, or treatment of the disease by the immune function fall or the abnormal increase of immune function Can be performed.

It is a graph showing the fluctuation | variation of the relative expression level by IL-6 stimulation of hsa-miR-4667-3p. It is a graph showing the fluctuation | variation of the relative expression level by IL-17 stimulation of hsa-miR-3934-5p. It is a graph showing the fluctuation | variation of the relative expression level by IL-17 stimulation of hsa-miR-6842-5p. It is a graph showing the fluctuation | variation of the relative expression level by IL-6 stimulation of hsa-miR-3646. It is a graph showing the fluctuation | variation of the relative expression level by IL-6 stimulation of hsa-miR-3126-5p. It is a graph showing the fluctuation | variation of the relative expression level by IL-17 stimulation of hsa-miR-5585-3p. It is the graph showing the fluctuation | variation of the relative expression level by LPS stimulation of hsa-miR-3646. It is the graph showing the fluctuation | variation of the relative expression level by LPS stimulation of hsa-miR-4667-3p. It is a graph showing the fluctuation | variation of the relative expression level by LPS stimulation of hsa-miR-20b-5p. It is a graph showing the fluctuation | variation of the relative expression level by LPS stimulation of hsa-miR-20b-3p. It is a graph showing the fluctuation | variation of the relative expression level by LPS stimulation of hsa-miR-3126-5p. It is a graph showing the fluctuation | variation of the relative expression level by LPS stimulation of hsa-miR-4650-5p. It is a graph showing the fluctuation | variation of the relative expression level by LPS stimulation of hsa-miR-101-5p. It is a graph showing the fluctuation | variation of the relative expression level by LPS stimulation of hsa-miR-224-5p. It is a graph showing the fluctuation | variation of the relative expression level by poly I: C stimulation of hsa-miR-101-3p. It is the graph showing the fluctuation | variation of the relative expression level by poly I: C stimulation of hsa-miR-511-5p. It is the graph showing the fluctuation | variation of the relative expression level by poly I: C stimulation of hsa-miR-511-3p. It is the graph showing the fluctuation | variation of the relative expression level by poly I: C stimulation of hsa-miR-20b-5p. It is the graph showing the fluctuation | variation of the relative expression level by poly I: C stimulation of hsa-miR-4650-5p. It is a graph showing the change of the gene expression level of IFN- (beta) and IL-12p35 after poly I: C stimulation in DC which introduce | transduced hsa-miR-3646. In the figure, the left is IFN-β and the right is IL-12p35. It is a graph showing the change of the gene expression level of IFN- (beta) and IL-12p35 after LPS stimulation in DC which introduce | transduced hsa-miR-3646. In the figure, the left is IFN-β and the right is IL-12p35. It is a graph showing the change of the gene expression level of IFN- (beta) and IL-12p35 after poly I: C stimulation in DC which introduce | transduced the inhibitory nucleic acid with respect to hsa-miR-3646. In the figure, the left is IFN-β and the right is IL-12p35. It is a graph showing the change of the gene expression level of IFN- (beta) and IL-12p35 after poly I: C stimulation in DC which introduce | transduced hsa-miR-3934-5p. In the figure, the left is IFN-β and the right is IL-12p35. It is a graph showing the change of the gene expression level of IFN- (beta) and IL-12p35 after LPS stimulation in DC which introduce | transduced hsa-miR-3934-5p. In the figure, the left is IFN-β and the right is IL-12p35. It is a graph showing the change of the gene expression level of IFN- (beta) and IL-12p35 after poly I: C stimulation in DC which introduce | transduced the inhibitory nucleic acid with respect to hsa-miR-3934-5p. In the figure, the left is IFN-β and the right is IL-12p35. It is a graph showing the change of the gene expression level of IFN- (beta) and IL-12p35 after LPS stimulation in DC which introduce | transduced the inhibitory nucleic acid with respect to hsa-miR-3934-5p. In the figure, the left is IFN-β and the right is IL-12p35. It is a graph showing the change of the gene expression level of IFN- (beta) after LPS stimulation in DC which introduce | transduced hsa-miR-1266-5p. It is a graph which shows the fixed_quantity | quantitative_assay result of IFN-gamma and IL-12 in the culture supernatant which culture | cultivated DC which introduce | transduced hsa-miR-224-5p in the culture medium containing a cancer antigen peptide. In the figure, the left is IFN-γ and the right is IL-12. It is a graph which shows the fixed_quantity | quantitative_assay result of IFN-gamma in the culture supernatant which culture | cultivated DC which introduce | transduced the inhibitory nucleic acid with respect to hsa-miR-224-5p- with the culture medium containing a cancer antigen peptide. It is a graph which shows the fixed_quantity | quantitative_assay result of IFN-gamma and IL-12 in the culture supernatant which culture | cultivated DC which introduce | transduced hsa-miR-224-3p in the culture medium containing a cancer antigen peptide. In the figure, the left is IFN-γ and the right is IL-12. It is a graph which shows the fixed_quantity | quantitative_assay result of IFN-gamma and IL-12 in the culture supernatant which culture | cultivated DC which introduce | transduced hsa-miR-20b-5p with the culture medium containing a cancer antigen peptide. In the figure, the left is IFN-γ and the right is IL-12.

  Throughout this specification, miR IDs and accession numbers are those specified in miRBase (http://www.mirbase.org/), which is a database that collects miR sequence information, etc. All the base sequences of miR are also registered in the database.

  The immune response control in the present invention means to suppress or activate an immune response. Suppressing the immune response means inhibiting, stopping, reducing or reducing the immune response to the antigen, that is, the progression of a biological defense reaction that eliminates or renders the antigen harmless. Inhibiting, reducing or reducing the expression or production of cytokines, such as IFN-β or IL-12, which inhibit, stop, reduce or reduce the ability to present, or promote differentiation induction or activation of T cells .

  In addition, activating an immune response means inducing, promoting or enhancing an immune response to an antigen, that is, progression of a host defense reaction that eliminates or renders the antigen harmless. Induces, promotes or enhances, or induces, promotes or enhances the expression or production of cytokines that promote T cell differentiation or activation, such as IFN-β or IL-12.

  The relative expression level mentioned in the present specification means a value obtained by correcting the expression level of miR measured using a DNA microarray by a comparative analysis method. Examples of such a method include a global normalization method, a quantile method, a lowess method, and a 75 percentile method. These methods are premised on the principle that there is no difference in the expression level when the expression data of a plurality of genes is collected as a lump for any sample and is taken as a gene expression data group. In this specification, a median of expression levels of all genes mounted on a DNA microarray is obtained, and calculated by a global normalization method (for example, Japanese Patent Application Laid-Open No. 2014-007995) that corrects the median to 25. The explanation will be made by adopting the relative expression level.

  The present invention is at least one selected from the group consisting of miRs shown in Tables 1 to 7, miRs complementary to the miRs, inhibitory nucleic acids for the miRs and the complementary miRs, and functional equivalents thereof. The present invention relates to an immune response control agent comprising the above nucleic acid as an active ingredient.

  The miRs shown in Tables 1 and 2 are DNA microarrays when compared to DCs (controls) cultured in a medium not containing these cytokines inside DCs cultured in a medium containing IL-6 or IL-17. It is miR in which the relative expression level calculated | required by the global normalization method using is reduced significantly (less than 1/2). The variation ratio in the table represents the reduction rate of each miR when the relative expression level determined by the global normalization method using the miR DNA microarray in the control is 1.

  The miRs shown in Table 3 and Table 4 used a DNA microarray when compared with DC cultured in a medium not containing these cytokines inside DC cultured in a medium containing IL-6 or IL-17. It is miR which the relative expression level calculated | required by the global normalization method rises significantly (2 times or more). The variation ratio in the table represents the rate of increase of each miR when the relative expression level determined by the global normalization method using the miR DNA microarray in the control is 1.

  The miRs shown in Tables 5 and 6 are cultured in a medium not containing these stimulating substances inside DC cultured in a medium containing lipopolysaccharide (LPS) or poly I: C, which are immunological stimulating substances. MiR in which the relative expression level determined by the quantitative PCR method increases when compared with the DC.

  The miRs shown in Table 7 are quantitative PCR when compared to DCs cultured in a medium containing poly I: C, an immunological stimulant, in DCs cultured in a medium not containing these stimulants. It is miR which the relative expression level calculated | required by the method reduces.

  IL-6 suppresses the expression of MHC class II molecules and CD86 against DC to reduce the antigen presentation ability, and has the ability to induce differentiation from naive T cells to helper T cells, which are effector T cells. It also suppresses the production of -12. That is, IL-6 is a cytokine that suppresses the immune response mediated by DC. IL-17 has a function of inducing the production of such IL-6, and as a result, is a cytokine that suppresses DC-mediated immune responses like IL-6. LPS is a substance that activates an immune response as in the case of infection of bacteria and the like, and poly I: C is a substance that activates an immune response as in the case of viral infection.

  MiRs shown in Tables 1 to 7 are expressed in DCs when stimulated by IL-6, LPS, or the like, and target various genes that suppress or activate immune responses and express their functions. It is thought that is adjusted. Such a target gene is assumed to have various functions such as inducing, promoting or enhancing, or inhibiting or suppressing an immune response via DC.

  Therefore, it is expected that an immune response via a living body, particularly DC, can be suppressed or activated by appropriately selecting miRs shown in Tables 1 to 7. In this sense, miR shown in Tables 1 to 7 can be used as an immune response control agent.

  The effect of the immune response control agent of the present invention may be confirmed by directly administering miR to a living body to confirm that an immune response is induced or suppressed. For example, miR that is an immune response control agent is applied to DC. Introduced and confirmed that expression of IFN-β or IL-12 involved in activation of immune response or IL-10 involved in suppression of immune response is induced, promoted, enhanced, inhibited or suppressed May be.

  In addition, i) miR complementary to miR shown in Tables 1 to 7, ie, miR comprising a base sequence complementary to the base sequence of miR, and ii) being placed under the control of an appropriate expression promoter, Table 1 -Nucleic acid capable of transcription-inducing RNA having the same base sequence as miR shown in Table 7; and iii) Inhibitory nucleic acid capable of inhibiting the function of miR shown in Table 1 to Table 7, for example, Table 1 Nucleic acids that can substantially inhibit the functions of miR shown in Tables 1 to 7 by having a base sequence that can be appropriately designed based on the base sequence of miR shown in Table 7; An RNA interference-inducing nucleic acid such as siRNA or shRNA capable of recognizing the miR base sequence shown in Table 7 and inhibiting its function, or placed under the control of an appropriate expression promoter Thus, RNA that can substantially inhibit the miR functions shown in Tables 1 to 7 or RNA that is RNA interference-inducing nucleic acid can be used as an immune response control agent in the present invention. It is expected to be possible. The miR and the miR complementary thereto are distinguished from each other by being expressed as 5p and 3p in the miR ID, such as hsa-miR-4709-5p and hsa-miR-4709-3p.

  Techniques for designing and producing inhibitory nucleic acids are widely known to those skilled in the art, and there are many companies that perform production from such design on consignment. For example, such consignment production can be performed using SIGMA-ALDRICH (http: // www. .Qiagen.com / products / catalog / assay-technologies / mirna / miscrypt-mirna-mimics), Applied Bioscience / http / c / id2 / AppID. ) Etc. That. Inhibitory nucleic acids can be obtained by providing such a trustee with a base sequence identified from the ID or accession number in each table described in this specification.

  Furthermore, a nucleic acid in which a part of the base sequence of the nucleic acid containing miR shown in Tables 1 to 7 is modified to improve stability against degradation by nuclease is also included in the immune response control agent of the present invention. Examples of the modification for improving stability against nuclease degradation include 2'O-methylation, 2'-Fation, 4'-thiolation and the like.

  In addition, chimeric RNAs in which a part of ribonucleotides in the RNA containing miR shown in Tables 1 to 7 is replaced with corresponding deoxyribonucleotides or nucleotide analogs are also included in the immune response control agent of the present invention. The Nucleotide analogs include, for example, 5-position modified uridine or cytidine, such as 5- (2-amino) propyluridine, 5-bromouridine, etc .; 8-position modified adenosine or guanosine, such as 8-bromoguanosine; Mention may be made, for example, of 7-deaza-adenosine; O- or N-alkylated nucleotides, such as N6-methyladenosine.

  Hereinafter, in the present invention, nucleic acids capable of transcription-inducing RNA having the same base sequence as miR shown in Tables 1 to 7 by being placed under the control of an appropriate expression promoter are shown in Tables 1 to 7. The nucleic acid functionally equivalent to the miRs shown in Tables 1 to 7 or the functions of miRs are obtained by modifying a part of the base sequence of miRs to improve the stability against degradation by nucleases and their chimeric RNAs. It shall be expressed as a physical equivalent.

  Similarly, nucleic acids capable of transcription-inducing RNAs having the same base sequence as the miR inhibitory nucleic acids shown in Tables 1 to 7 by being placed under the control of an appropriate expression promoter, Tables 1 to 7 Inhibitory nucleic acids and functions of miRs shown in Tables 1 to 7 and nucleic acids in which a part of the base sequence of the shown miR inhibitory nucleic acids is modified to improve the stability against degradation by nucleases and these chimeric RNAs It is expressed as a functionally equivalent nucleic acid or a functional equivalent of an inhibitory nucleic acid.

  One aspect of the immune response control agent of the present invention is an immunosuppressive agent containing miR that can suppress an immune response or a functional equivalent thereof as an active ingredient.

  For example, as shown in the Examples below, by introducing hsa-miR3646 whose expression level is increased by IL-6 shown in Table 3 into DC, the expression of IFN-β gene and IL-12p35 gene in DC is changed. Can be reduced. Therefore, hsa-miR-3646 can be used as an immunosuppressant.

  Another embodiment of the immune response regulator of the present invention is an immune activator comprising, as an active ingredient, miR that can activate an immune response or a functional equivalent thereof.

  For example, the expression of the IFN-β gene and the IL-12p35 gene in DC can be enhanced by introducing into the DC hsa-miR-3934-5p whose relative expression level is decreased by IL-17 stimulation. Therefore, hsa-miR-3934-5p can be used as an immune activator.

  Another aspect of the present invention is an immunosuppressive agent comprising an inhibitory nucleic acid against miR that can activate an immune response or a functional equivalent thereof as an active ingredient.

  For example, the expression of the IFN-β gene and the IL-12p35 gene in DC can be reduced by introducing into the DC an inhibitory nucleic acid against hsa-miR-3934-5p, which is the immunoactivator described above. . Therefore, an inhibitory nucleic acid against hsa-miR-3934-5p can be used as an immunosuppressant.

  A further aspect of the present invention is an immunostimulatory agent comprising an inhibitory nucleic acid against miR or a functional equivalent thereof capable of suppressing an immune response as an active ingredient.

  For example, the expression of the IFN-β gene and the IL-12p35 gene in DC can be enhanced by introducing into the DC an inhibitory nucleic acid against hsa-miR-3646, which is the immunosuppressant described above. Accordingly, inhibitory nucleic acids against hsa-miR-3646 can be used as immunostimulatory agents.

  In the present invention, miRs that can suppress or activate immunity are appropriately selected from the miRs shown in Tables 1 to 7, and these or functional equivalents thereof are immunosuppressive agents or immunoactivators. Can be used as Moreover, by appropriately selecting miRs that can suppress or activate immunity from the miRs shown in Tables 1 to 7, inhibitory nucleic acids or functional equivalents thereof can be used as an immunoactivator or immunity. It can be used as an inhibitor.

  Preferred immunoactivators or immunosuppressants in the present invention are miRs having a variation ratio of 0.4 or less shown in Tables 1 and 2, miRs having a variation ratio of 3 or more shown in Tables 3 and 4, and those Are functional equivalents thereof, inhibitory nucleic acids thereto, or functional equivalents thereof. More preferred immune activators or immunosuppressants in the present invention are miRs having a variation ratio of 0.3 or less shown in Tables 1 and 2, miRs having a variation ratio of 4 or more shown in Tables 3 and 4, Their functional equivalents, inhibitory nucleic acids for them or their functional equivalents.

  Nucleic acids that are immune response regulators of the present invention can be artificially synthesized using chemical synthesis techniques. As a method for chemically synthesizing nucleic acid, a method for synthesizing a non-natural base, or a method for synthesizing a nucleic acid containing the same, a method known or well known to those skilled in the art can be employed. Moreover, you may manufacture the immune response control agent of this invention by using apparatuses, such as what is called a DNA synthesizer.

  The nucleic acid that is the immune response control agent of the present invention may be directly administered to a living body. Moreover, you may introduce | transduce into DC isolate | separated from the biological body by a suitable method. In addition, the nucleic acid that is the immune response control agent of the present invention may be directly introduced into DC, or DC in the form of an expression vector functionally incorporated so that a desired RNA is appropriately induced in DC. May be introduced. The expression vector may further contain any functional base sequence that regulates transcriptional expression, for example, a promoter sequence such as a Pol III promoter, an operator sequence, an enhancer, and the like. These functional base sequences can be operably linked to the nucleic acid. Nucleic acid constructs such as expression vectors containing the nucleic acids of the invention are also within the scope of the invention. When two or more nucleic acids are used in the present invention, these nucleic acids may be incorporated into a single expression vector or separately into two or more vectors.

  The nucleic acid of the present invention can be obtained by any known cell introduction method such as calcium phosphate method, lipofection method, ultrasonic introduction method, electroporation method, particle gun method, virus vector (for example, adenovirus vector or retrovirus vector). It can be introduced into the DC by using a method to be used, a microinjection method or the like.

  Examples of genetic engineering methods for constructing an expression vector and introducing it into DC include methods known or well known to those skilled in the art, such as “Molecular Cloning: A Laboratory Manual 2nd edition” by Sambrook et al. (1989, Cold Spring Harbor). (Laboratory Press, Cold Spring Harbor, NY) and other techniques described in textbooks or handbooks in this field and widely used by those skilled in the art. In addition, when using a commercially available kit or reagent, it is preferable to follow the protocol and / or parameters determined by the manufacturer of the kit or reagent.

  The present invention also provides a method of controlling an immune response of a mammal, specifically, a method of controlling an immune response of a mammal by administering an effective amount of the immune response control agent of the present invention to the mammal, Or the method of controlling the immune response of a mammal by administering DC derived from the mammal cultured in presence of the immune response control agent of this invention is included.

  Examples of “mammals” used herein include humans, cows, horses, dogs, cats and the like, but the method of the present invention is particularly intended for humans.

  As used herein, an “effective amount” means an amount of an immune response control agent that is effective to activate or suppress a mammalian immune response. The effective amount is appropriately adjusted according to the type of disease, the severity of symptoms, the patient and other medical factors.

  One of the preferred embodiments of the treatment method of the present invention is to administer DC derived from a human cancer patient cultured in the presence of the immune response control agent of the present invention to the cancer patient, A method of activating a patient's immune response. Such DC culture is preferably performed using a medium containing an appropriate cancer antigen or cancer vaccine. It is also possible to activate the patient's immune response to infections caused by bacteria, fungi, parasites, viruses and other foreign organisms by similar methods.

  Another preferred embodiment of the treatment method of the present invention is a method of administering the immune response control agent of the present invention to a human patient to treat hepatitis, allergic disease or autoimmune disease in the patient. Diseases targeted by the treatment method of the present embodiment include hepatitis; allergic diseases such as hay fever, atopy, severe drug eruption; rheumatism, systemic lupus erythematosus (SLE), multiple sclerosis, transplantation disease, Crohn's disease, ulcer And autoimmune diseases such as ulcerative colitis and Sjogren's syndrome.

  The immune response control agent of the present invention is preferably used after forming or formulating a pharmaceutical composition with pharmaceutically acceptable excipients, carriers and other components. In particular, the use of excipients suitable for the preparation of nucleic acid preparations is preferred. The immune response control agent in the form of such a pharmaceutical composition or formulation is also an embodiment of the immune response control agent of the present invention.

  Pharmaceutically acceptable ingredients are well known to those skilled in the art, and those skilled in the art can use the ingredients from the ingredients described in the 16th revised Japanese Pharmacopoeia and other standards within the scope of their normal performance. Can be selected and used as appropriate. In addition, it is preferable to use various components used in preparations containing RNA interference-inducing nucleic acids and the like. Depending on the disease to be treated, the immune response control agent of the present invention may be used in combination with other drugs.

  The pharmaceutical composition containing the immune response control agent of the present invention is usually used in the form of parenteral preparations such as injections and drops. Examples of carriers that can be used in parenteral preparations include aqueous carriers that are usually used in preparations, such as isotonic solutions containing physiological saline, glucose, D-sorbitol, and the like. The pharmaceutical composition containing the immune response regulator of the present invention may further contain pharmaceutically acceptable buffers, stabilizers, preservatives and other components.

  In addition, the pharmaceutical composition containing the immune response control agent of the present invention can be encapsulated and / or fixed in an appropriate DDS such as polymer micelles, liposomes, emulsions, microspheres, and nanospheres.

  The administration method of the pharmaceutical composition containing the immune response control agent of the present invention is not particularly limited, but when it is a parenteral preparation, for example, intravascular administration (preferably intravenous administration), intraperitoneal administration, intestinal administration, Examples thereof include local administration in or near the tumor. In one of the preferred embodiments, the pharmaceutical composition comprising the immune response control agent of the present invention is administered to a subject by intravenous administration or local administration into or near a tumor.

When the expression of the nucleic acid of the present invention is induced in a mammalian body using a viral vector, the dose range is, for example, 1 × 10 ³ to 1 × 10 ¹⁴ , preferably 1 × 10 6 per human subject. ⁵ to 1 × 10 ¹² , more preferably 1 × 10 ⁶ to 1 × 10 ¹¹ , most preferably 1 × 10 ⁷ to 1 × 10 ¹⁰ plaque forming units (pf).

  The invention will now be described in more detail by way of non-limiting examples, but the invention is limited to the specific methodologies, protocols, cell lines, animal species and genera, constructs and reagents described herein. However, those skilled in the art can easily understand that these can be appropriately changed.

<Experimental example 1>
1) Preparation of mRNA of IL-6 or IL-17-stimulated DC Human peripheral blood mononuclear cells (2 × 10 ⁶ cells) collected from three healthy human blood were treated with 1 mL of AIM-V (Life) (Technologies) and incubated for 1 hour to allow the cells to adhere to the bottom surface. The adherent cells were cultured in the presence of 0.05 mg / mL IL-4 and GM-CSF for 7 days to obtain human monocyte-derived dendritic cells (Monocyte-derived Dendritic Cell, MoDC).

On day 7 of culture, human IL-6 (20 μg / mL) or human IL-17 (100 μg / mL) was added to the medium, and the culture was further continued for 8 hours. Thereafter, the cells were collected using Trypsin-EDTA solution, stained with anti-CD11c antibody and 7-AAD, and then CD11c positive 7-ADD negative cells were isolated using FACS Aria. Of these cells, 5 × 10 ⁶ cells were suspended in 350 μL of ISOGEN II, and then the cells were derived from three humans that had been stimulated with IL-6 and from three humans that had been stimulated with IL-17. After the cell suspensions were mixed together, mRNA was recovered. Also, mRNA was recovered from MoDC to which IL-6 and IL-17 were not added, and used as a control.

2) Microarray analysis The mRNA fraction collected from the IL-6-stimulated or IL-17-stimulated MoDC and control MoDC in 1) was commissioned to Toray Industries, Inc. to perform microarray analysis.

  From the analysis results of the microarray, miRs having a decrease rate by IL-6 stimulation of ½ or less and a value of unstimulated global normalization of 10 or more compared with the control were selected and summarized in Table 1. The graph which represented the fluctuation | variation by IL-6 irritation | stimulation of hsa-miR-4667-3p among Table 1 is shown in FIG.

  Similarly, miRs having a decrease rate by IL-17 stimulation of ½ or less and a non-stimulated global normalization value of 10 or more as compared with the control were selected and summarized in Table 2. The graph which represented the fluctuation | variation by IL-17 irritation | stimulation of hsa-miR-3934-5p and hsa-miR-6842-5p among Table 2 is shown in FIG.2 and FIG.3, respectively.

  In addition, miRs with a rate of increase by IL-6 stimulation of 2 times that of controls and a global normalization value of 10 or more after stimulation were selected and summarized in Table 3. The graph which represented the fluctuation | variation by IL-6 stimulation of hsa-miR-3646 and hsa-miR-3126-5p among Table 3 is shown in FIG.4 and FIG.5, respectively.

  Furthermore, miRs with a rate of increase by IL-17 stimulation of 2 times that of the control and a global normalization value of 10 or more after stimulation were selected and summarized in Table 4. FIG. 6 shows a graph showing the fluctuation of hsa-miR-5585-3p due to IL-17 stimulation in Table 4.

<Experimental example 2>
1) Preparation of mRNA of LPS or poly I: C-stimulated DCs To MoDC on the seventh day of culture prepared in the same manner as in 1) of Experimental Example 1, LPS (0.5 μg / mL) or poly I: C (10 μg / ML) was added to the medium and the cultivation was continued for another 8 hours. Thereafter, the same treatment as 1) of Experimental Example 1 was performed, and mRNA was recovered from each of the LPS-stimulated or poly I: C-stimulated MoDC and the control MoDC.

2) Quantitative PCR analysis Quantitative PCR analysis was performed on the mRNA fraction collected from the LPS-stimulated or poly-I: C-stimulated MoDC and the control MoDC of 1) using a synthetic primer that detects the target miR. It was.

  From the analysis results of quantitative PCR, miRs whose expression level increased by LPS stimulation compared with the control were selected and summarized in Table 5. Furthermore, miRs whose expression levels were increased by poly I: C stimulation compared to controls were selected and summarized in Table 6.

  Among Table 5, hsa-miR-3646, hsa-miR-4667-3p, hsa-miR-20b-5p, hsa-miR-20b-3p, hsa-miR-3126-5p, hsa-miR-4650-5p , Hsa-miR-101-5p and hsa-miR-224-5p are shown in FIGS.

  Moreover, the graph showing the fluctuation | variation by poly I: C irritation | stimulation of hsa-miR-101-3p, hsa-miR-511-5p, hsa-miR-511-3p, and hsa-miR-20b-5p among Table 6. Are shown in FIGS.

  In addition, miRs whose expression levels were reduced by poly I: C stimulation compared to controls were selected and summarized in Table 7.

  Of Table 7, FIG. 19 shows a graph showing fluctuations caused by stimulation with hsa-miR-4650-5p.

<Example 1>
Table 3 (miR whose expression level is increased by IL-6 stimulation) hsa-miR-3646 and its inhibitory nucleic acid (hsa-miR-3646-I) were obtained from SIGMA-ALDRICH (http: //www.sigmaaldrich. com / life-science / functional-genomics-and-rnai / mirna / microna-mimics.html and chemically synthesized respectively.

Using 1 ml of MoDC (1 × 10 ⁶ / well) prepared in 1) of Experimental Example 1 using lipofectamine® (life Technologies), according to the manufacturer's protocol, hsa-miR-3646 or hsa-miR -3646-I (30 pmol / well) was added respectively.

  After overnight culture of MoDC, LPS was added at 0.5 μg / mL or poly I: C was added at 10 μg / mL, and further cultured for 3 hours. After culturing, 350 mL of ISOGEN II is added to suspend the cells, and 140 mL of UltraPure DNase / RNase-Free Distilled Water (UPW, Life Technologies) is added and mixed, wait for 5 to 15 minutes, and 12,000 × g. Centrifuge for 15 minutes. Collect 350 μL of the centrifuged supernatant in a new tube, add 350 μL of isopropanol, mix by inverting, leave it for 10 minutes, and then centrifuge at 12,000 × g for 10 minutes. Further, the centrifugation supernatant was discarded, 500 μL of 75% ethanol was added, and the mixture was centrifuged at 8,000 × g for 2 to 3 minutes. The pellet obtained by repeating this operation twice was melted with 16 mL of UPW to obtain an mRNA fraction.

  Based on the recovered mRNA fraction, a reverse transcription reaction using SuperScript III Reverse transcriptases and Okigo dT was performed to prepare cDNA. Using this as a template, gene expression levels of IFN-β and IL-12p35 were analyzed by real-time quantitative PCR using Light cycler 2.0 (Roche) using TaqMan Universal PCR Master Mix (Roche). The expression level of each was corrected by the DDCt method (DCt = DCtssample-DCreference) based on the amount of β-actin mRNA, and the control value in each experiment was set to 1. The results are shown in FIGS.

  As shown in FIGS. 20 and 21, the expression of the IFN-β gene and IL-12p35 gene was suppressed by introducing hsa-miR3646 into MoDC that received LPS stimulation or poly I: C stimulation. Therefore, some of miRs whose expression levels are increased by IL-6 stimulation, such as hsa-miR-3646, are immunosuppressive agents that can inhibit the production of cytokines and suppress immune functions against DC. It was confirmed that it can be used as.

  On the other hand, as shown in FIG. 22, the expression of the IFN-β gene and the IL-12p35 gene was enhanced by introducing hsa-miR-3646-I into MoDC subjected to poly I: C stimulation. From this, an inhibitory nucleic acid against a part of miR whose expression level is increased by IL-6 stimulation such as hsa-miR-3646 enhances cytokine production against DC and activates immune function. It was confirmed that it can be used as an immune activator capable of

<Example 2>
The expression levels of hsa-miR-3646 and hsa-miR-3646-I of Example 1 are reduced by IL-17 stimulation, hsa-miR3934-5p and hsa-miR-3934-5p- The same experiment as in Example 1 was performed in place of I (both requested to SIGMA-ALDRICH for synthesis). The results are shown in FIGS.

  As shown in FIGS. 23 and 24, by introducing hsa-miR3934-5p into MoDC that receives LPS stimulation or poly I: C stimulation, the expression of IFN-β gene and IL-12p35 gene is enhanced. It was. From this, a part of miR whose expression level is decreased by IL-17 stimulation, such as hsa-miR-3934-5p, can enhance the production of cytokines against DC and activate the immune function. It was confirmed that it can be used as an immune activator.

On the other hand, as shown in FIG. 25 and FIG. 26, by introducing hsa-miR-3934-5p-I into MoDC which received LPS stimulation or poly I: C stimulation, IFN-β gene and IL-12p35 gene All expression was inhibited. Therefore, an inhibitory nucleic acid against a part of miR whose expression level is reduced by IL-17 stimulation, such as hsa-miR-3934-5p, inhibits cytokine production against DC and suppresses immune function. It was confirmed that it can be used as an immunosuppressive agent.
<Example 3>
Replacing hsa-miR-3646 and hsa-miR-3646-I of Example 1 with hsa-miR-1266-5p (requested to be synthesized by SIGMA-ALDRICH) whose expression level is increased by LPS stimulation, and stimulating DC The same experiment as in Example 1 was conducted except that only LPS was used, and the change in the expression level of IFN-β was measured. The result is shown in FIG.

  As shown in FIG. 27, the expression of the IFN-β gene was suppressed by introducing hsa-miR-1266-5p into MoDC that had been subjected to LPS stimulation. Therefore, some of miRs whose expression level is increased by LPS stimulation, such as hsa-miR-1266-5p, can inhibit the production of cytokines against DCs and suppress the immune function. It was confirmed that it can be used as.

<Example 4>
Table 5 (miR whose expression level is increased by LPS stimulation) hsa-miR-224-5p and its inhibitory nucleic acid (hsa-miR-224-5p-I) were chemically synthesized respectively (both were converted to SIGMA-ALDRICH). Request composition).

For MoDC (5 × 10 ³ cells / well) prepared in 1) of Experimental Example 1, using lipofectamine® (life Technologies), according to the manufacturer's protocol, hsa-miR-224-5p or hsa -MiR-224-5p-I (30 pmol / well) was added respectively.

After overnight culture of MoDC, CD4 positive T cells (5 × 10 ⁴ cells / well) induced with a peptide derived from the cancer antigen STEAP protein were added, and in the presence of STEAP peptide (2 μM / well), an additional 24 Incubate for hours. Culture supernatants were collected 24 hours after stimulation and human IFN-γ BD OptEIA set and human IL-12 BD OptEIA set (both BD Bioscience pharmingen) were used to quantify IFN-γ and IL-12 by ELISA. went. The results of setting the control value in each experiment to 1 are shown in FIGS.

  As shown in FIG. 28, by introducing hsa-miR-224-5p into MoDC, production of IFN-γ and IL-12 was both suppressed. Therefore, some miRs whose expression level is increased by LPS stimulation such as hsa-miR-224-5p inhibits the production of cytokines from T cells that have received antigen presentation by DCs and DCs, and immune function It was confirmed that the present invention can be used as an immunosuppressive agent capable of suppressing.

  On the other hand, as shown in FIG. 29, the production of IFN-γ was enhanced by introducing hsa-miR-224-5p-I into MoDC. Therefore, an inhibitory nucleic acid such as hsa-miR-224-5p, which inhibits a part of miR whose expression level is increased by stimulation with LPS, is stimulated by antigen-specific T cells activated by antigen presentation by DC. It was confirmed that the present invention can be used as an immune activator that can enhance the production of cytokines from the received DC and activate the immune function.

<Example 5>
Hsa-miR-224-5p of Example 4 is expressed in hsa-miR-224-3p or hsa-miR-20b-5p whose expression level is increased by LPS stimulation or poly I: C stimulation (both synthesized to SIGMA-ALDRICH) In the same manner as in Example 4. The results are shown in FIGS.

  As shown in FIGS. 30 and 31, by introducing hsa-miR-224-3p or hsa-miR-20b-5p into MoDC, production of IFN-γ and IL-12 was both suppressed. From this, a part of miR whose expression level is increased by LPS stimulation or poly I: C stimulation, such as hsa-miR-224-3p or hsa-miR-20b-5p, has been shown to be antigen-presented by DC. It was confirmed that the present invention can be used as an immunosuppressive agent that can suppress the production of cytokines from cells and DCs and suppress the immune function.

The present invention relates to an immune response control agent capable of activating or suppressing the immune response function of a living body or a DC isolated from the living body, and the invention relates to a disease caused by a decrease in immune function or a disease caused by abnormal enhancement of immune function. It can be used as a drug or medicine for prevention, improvement or treatment.

Claims (4)

At least selected from the group consisting of the microRNAs shown in Tables 1 to 7, microRNAs complementary to the microRNAs, inhibitory nucleic acids to the microRNAs and the complementary microRNAs, and functional equivalents thereof An immune response control agent comprising one or more nucleic acids as an active ingredient.
  The immune response control agent according to claim 1 for controlling an antigen response function of a dendritic cell.   The immune response control agent according to claim 2, wherein the antigen response function of the dendritic cell is a cytokine production function. The pharmaceutical containing the immune response control agent in any one of Claims 1-3.