JP6490419B2 - Regulatory T cell differentiation inducer - Google Patents

Description

  The present invention relates to a regulatory T cell differentiation inducer and its use for immunosuppression, GVHD, autoimmune diseases, and prevention and / or treatment of graft rejection.

  Graft-versus-host disease (GVHD) is caused by, after bone marrow transplantation, blood transfusion, etc., leukocytes from the donor identify the recipient's body tissue as “others” and attack by the immune response. GVHD is a disease that occurs at a relatively high frequency. For example, chronic GVHD is said to develop in about 40 to 50% of patients who have undergone bone marrow transplantation. Autoimmune diseases, on the other hand, are caused by the immune system reacting excessively against and attacking self tissue. These diseases are common in that the body tissues are attacked by the immune response, and immunosuppressants and steroids are usually used for the treatment. However, the conventional treatment methods still have room for improvement from the viewpoints of effects and side effects.

  Regulatory T cells are T cells that are responsible for suppression of immune responses, and are known to be involved in the development of GVHD and autoimmune diseases (Non-Patent Documents 1 to 8). Furthermore, it has been reported variously that these diseases can be treated by increasing the regulatory T cells (Non-Patent Documents 5 to 8).

  GM-CSF is known to control differentiation induction of macrophages, neutrophils, granulocytes, eosinophils and the like. Moreover, since it also promotes differentiation induction of antigen-presenting dendritic cells, it has been reported to be used for immunostimulation.

Ann N Y Acad Sci. 2008 December; 1150: 300-310.doi: 10.1196 / annals.1447.046. Immunological Reviews 2006 Vol. 212: 256-271. J Neuroimmunol. 2007 November; 191 (1-2): 51-60. The Journal of Experimental Medicine Vol. 201, No. 5, March 7, 2005 723-735. BLOOD, 18 JUNE 2009, VOLUME 113, NUMBER 25, 6277-6287. Journal of Autoimmunity 44 (2013) 13-20. The Journal of Experimental Medicine Vol. 206 No. 4, 2009, 751-760. BLOOD, 1 SEPTEMBER 2008 _ VOLUME 112, NUMBER 5, 2129-2138.

  An object of the present invention is to provide a regulatory T cell differentiation inducer. Furthermore, another object is to provide a differentiation inducer capable of specifically inducing differentiation of regulatory T cells.

  As a result of intensive studies to solve the above problems, the present inventors have found that a complex of GM-CSF and a GM-CSF antibody can specifically induce regulatory T cells. That is, this invention includes the following aspects.

  Item 1. Regulatory T cell differentiation inducer containing GM-CSF and GM-CSF antibody.

  Item 2. Item 2. The differentiation inducer according to Item 1, comprising a complex of GM-CSF and a GM-CSF antibody.

Item 3. Item 3. The differentiation inducing agent according to Item 1 or 2, wherein the regulatory T cell is FOXP3 ⁺ .

  Item 4. Item 4. The differentiation inducer according to any one of Items 1 to 3, wherein the GM-CSF antibody is a monoclonal antibody.

  Item 5. Item 5. The differentiation inducer according to any one of Items 1 to 4, which is used for immunosuppression.

  Item 6. Item 5. The differentiation inducer according to any one of Items 1 to 4, which is used for prevention and / or treatment of at least one disease selected from the group consisting of autoimmune diseases and graft-versus-host disease (GVHD).

  According to the present invention, a regulatory T cell differentiation inducer can be provided. According to this regulatory T cell differentiation inducer, it is possible to specifically induce regulatory T cells without inducing differentiation of conventional T cells that act on immune activation. Since regulatory T cells act on immunosuppression, autoimmune diseases, GVHD, graft rejection, and the like can be prevented and / or treated with the differentiation inducer.

The result (the ratio and number of regulatory T cells) of Test Example 1 is shown. The result (the ratio and number of conventional T cells) of Test Example 1 is shown. The result (ratio and number of dendritic cells) of Test Example 1 is shown. The result (The ratio and number of an immunosuppression dendritic cell) of the test example 1 is shown. The result of Experiment 1 (ratio and number of immune activated dendritic cells) is shown. Results of Test Example 2 (GVHD score after bone marrow transplantation)

  The present invention relates to a regulatory T cell differentiation inducer (hereinafter also referred to as “differentiation inducer of the present invention”) containing GM-CSF and a GM-CSF antibody. This will be described below.

1. GM-CSF
GM-CSF is an abbreviation of granulocyte monocyte colony stimulating factor (G ranulocyte M acrophage C olony- S timulating F actor), is a type of cytokine. As GM-CSF, GM-CSF derived from various mammals such as humans, monkeys, mice, rats, dogs, cats and rabbits can be employed. When a regulatory T cell differentiation inducer is administered to a living body, GM-CSF is preferably derived from the species to be administered.

  Specifically, for example, human GM-CSF includes a protein consisting of the amino acid sequence represented by SEQ ID NO: 1 (NCBI Reference Sequence: NP_000749.2), and mouse GM-CSF includes the amino acid represented by SEQ ID NO: 2. A protein consisting of a sequence (NCBI Reference Sequence: NP_034099.2) is mentioned.

  Moreover, it is preferable that GM-CSF lacks the signal peptide of the N terminal side. For example, in the case of human GM-CSF, the signal peptide is a peptide consisting of amino acids 1 to 17 of SEQ ID NO: 1, and in the case of mouse GM-CSF, it is a peptide consisting of amino acids 1 to 17 of SEQ ID NO: 2. . Examples of GM-CSF from which the signal peptide is deleted include human GM-CSF having a protein consisting of the amino acid sequence shown in SEQ ID NO: 3, and mouse GM-CSF consisting of the amino acid sequence shown in SEQ ID NO: 4. Examples include proteins.

As GM-CSF,
Preferably, the protein described in (a) below and the protein described in (b) below:
(A) a protein comprising the amino acid sequence represented by any one of SEQ ID NOs: 1 to 4,
(B) at least one selected from the group consisting of an amino acid sequence having 85% or more identity with the amino acid sequence shown in any of SEQ ID NOs: 1 to 4 and having a regulatory T cell differentiation-inducing activity Species are mentioned.

  “Identity” of amino acid sequences refers to the degree of amino acid sequences of two or more comparable amino acid sequences with respect to each other. Therefore, the higher the identity of two amino acid sequences, the higher the identity or similarity of those sequences. The level of amino acid sequence identity is determined, for example, using FASTA, a sequence analysis tool, using default parameters. Or, Karlin and Altschul by the algorithm BLAST (KarlinS, Altschul SF "Methods for assessing the statisticalsignificance of molecular sequence features by using general scoringschemes" Proc.Natl Acad Sci USA.87:. 2264-2268 (1990), KarlinS, Altschul SF. “Applications and statics for multiple high-scoring segments in molecular sequences.” Natl Acad Sci USA. 90: 5873-7 (1993). )). A program called BLASTX based on such a BLAST algorithm has been developed. Specific methods of these analysis methods are known, and the website of the National Center of Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov/) may be referred to.

  In the above (b), the identity is preferably 90% or more, more preferably 95% or more, and still more preferably 98% or more.

  The presence or absence of regulatory T cell differentiation-inducing activity can be determined according to the method described in Test Example 1 described later.

As an example of the protein described in the above (b), for example, (b ′) one or more amino acids are substituted, deleted, added, or inserted into the amino acid sequence shown in any one of SEQ ID NOs: 1 to 4. And a protein having a regulatory T cell differentiation-inducing activity.
In said (b '), a plurality is 2-20 pieces, for example, Preferably it is 2-10 pieces, More preferably, it is 2-5 pieces, More preferably, it is 2-3 pieces.

  In the proteins described in (b) and (b ′) above, the mutation is preferably a conservative substitution. A conservative substitution means that an amino acid residue is replaced with an amino acid residue having a similar side chain.

  For example, substitution with amino acid residues having basic side chains such as lysine, arginine, and histidine is a conservative substitution technique. In addition, amino acid residues having acidic side chains such as aspartic acid and glutamic acid; amino acid residues having non-charged polar side chains such as glycine, asparagine, glutamine, serine, threonine, tyrosine, and cysteine; alanine, valine, leucine, isoleucine, Amino acid residues with non-polar side chains such as proline, phenylalanine, methionine, and tryptophan; amino acid residues with β-branched side chains such as threonine, valine, and isoleucine, and aromatic side chains such as tyrosine, phenylalanine, tryptophan, and histidine Similarly, substitutions between amino acid residues are conservative substitutions.

  The method for obtaining GM-CSF is not particularly limited, and a known method may be adopted. For example, a recombinant protein may be prepared based on the above sequence, or a commercially available product may be used.

2. GM-CSF antibody The GM-CSF antibody is an antibody that recognizes GM-CSF described in the above "1. GM-CSF". Such an antibody may be a polyclonal antibody or a monoclonal antibody, and an antiserum containing these antibodies is also included in the antibody of the present invention, and can preferably be a monoclonal antibody.

  The method for obtaining the GM-CSF antibody is not particularly limited, and those skilled in the art can easily obtain the antibody of the present invention by adopting a known method based on the above-mentioned GM-CSF. Or what is marketed may be used.

3. Active ingredient The differentiation-inducing agent of the present invention contains the above-mentioned GM-CSF and GM-CSF antibodies as active ingredients.

  The differentiation inducer of the present invention preferably contains a complex of GM-CSF and GM-CSF antibody from the viewpoint of regulatory T cell differentiation inducing ability. The method for forming this complex is not particularly limited. For example, the complex can be easily formed by mixing GM-CSF and GM-CSF antibody under conditions where a normal antigen-antibody reaction occurs. In addition, even if a complex is not formed at the time of formulation (for example, a state where GM-CSF dry powder and GM-CSF antibody dry powder are mixed), the complex can be easily formed by mixing with a solvent at the time of administration. Can be formed.

  The content of GM-CSF in the differentiation inducer of the present invention is not particularly limited as long as differentiation induction of regulatory T cells is possible. The content of GM-CSF in the differentiation inducer of the present invention can be, for example, 0.000001% by weight or more, preferably 0.00001 to 10% by weight, more preferably 0.0001 to 1% by weight.

  The content of the GM-CSF antibody in the differentiation inducer of the present invention is not particularly limited as long as differentiation of regulatory T cells can be induced. The content of GM-CSF in the differentiation inducer of the present invention can be, for example, 0.000005% by weight or more, preferably 0.00005 to 20% by weight, more preferably 0.0005 to 5% by weight.

  The content ratio of the GM-CSF and GM-CSF antibody in the differentiation inducer of the present invention is not particularly limited as long as differentiation induction of regulatory T cells is possible. For example, with respect to 1 mol of GM-CSF, GM-CSF antibody is, for example, 0.01 to 10 mol, preferably 0.1 to 5 mol, more preferably 0.3 to 2 mol, and still more preferably 0.3 to It can be 1 mole.

4). Other components, dosage form, administration method The differentiation-inducing agent of the present invention may consist only of GM-CSF and GM-CSF antibody, or a pharmaceutically acceptable additive (hereinafter referred to as “a pharmaceutically acceptable additive”). , Or simply “additive”).

  Examples of the additive include a base, a carrier, a solvent, a dispersant, an emulsifier, a buffer, a stabilizer, an excipient, a binder, a disintegrant, a lubricant, a thickener, a moisturizer, a colorant, a fragrance, And chelating agents. When the opening agent of the present invention contains an additive, the differentiation inducer of the present invention can be produced by using the additive according to a conventional method according to the dosage form.

  The differentiation-inducing agent of the present invention can be in any dosage form such as tablets, pills, powders, solutions, injections, suspensions, emulsions, powders, granules, capsules and the like.

  When the differentiation-inducing agent of the present invention is administered to a living body, the administration route is not particularly limited. For example, enteral administration such as oral administration, tube feeding, enema administration; parenteral administration such as intravenous administration, transarterial administration, intramuscular administration, intracardiac administration, subcutaneous administration, intradermal administration, intraperitoneal administration, etc. Can be adopted. Among these, parenteral administration is preferable from the viewpoint that the effects of the present invention can be more reliably exhibited.

  When administering the differentiation-inducing agent of the present invention to a living body, the daily dose is determined according to the patient's condition and doctor's judgment, etc., and is not particularly limited. For example, for an adult weighing 60 kg, In terms of the active ingredient (GM-CSF and GM-CSF antibody), 1 to 100 mg can be administered. Administration may be divided into a plurality of times (for example, 2 to 5 times) per day. When administered multiple times, the dose per time can be determined so that the dose per day is the above amount.

5. Use The active ingredient of the present invention (GM-CSF and GM-CSF antibody) specifically induces differentiation and / or differentiation of regulatory T cells without inducing and / or proliferating T cells involved in immune activation. Can be propagated.

  Since regulatory T cells are cells that control immune responses, the differentiation-inducing agents of the present invention that can induce and / or proliferate them are immunosuppressants, autoimmune diseases, GVHD, graft rejection It can be used as a preventive and / or therapeutic agent. In addition, various reports have been reported that the induction and / or proliferation of regulatory T cells is useful for the prevention and treatment of various autoimmune diseases, GVHD, and the like (Non-Patent Documents 5 to 8).

  The autoimmune disease to be prevented and / or treated is not particularly limited. For example, systemic lupus erythematosus, scleroderma, polyarteritis, myasthenia gravis, multiple sclerosis, autoimmune thyroiditis, type 1 diabetes Rheumatoid arthritis, Sjogren's syndrome, dermatomyositis, polymyositis, ANCA-related vasculitis, Takayasu disease, Behcet's disease, adult still disease, relapsing polychondritis, IgA vasculitis, IgG4-related disease, mixed connective tissue disease, Rheumatoid polymyalgia, antiphospholipid antibody syndrome, ankylosing spondylitis, Kawasaki disease, Crohn's disease, ulcerative colitis, psoriasis vulgaris, pemphigoid, atopic dermatitis, bronchial asthma, primary bile Examples include cirrhosis, primary sclerosing cholangitis, idiopathic interstitial pneumonia.

  EXAMPLES The present invention will be described in detail below based on examples, but the present invention is not limited to these examples.

Test Example 1 Analysis of the effect on the number of regulatory T cells According to Table 1, each test solution (Comparative Examples 1-2 and Examples 1-3) was prepared. In the test solutions of Examples 1 to 3, GM-CSF and GM-CSF antibody are expected to form a complex. 200 μL of each test solution was intraperitoneally administered to mice (C57BL / 6 8-12 weeks old). This administration was performed for 3 consecutive days. The number of mice administered (n number) was “4” in Comparative Examples 1 and 2 and Example 1, and “6” in Examples 2 and 3.

  Four days after the first administration day, spleens were collected from each mouse. Cells were extracted from the spleen according to a conventional method. The obtained cells were immunostained according to a conventional method using labeled antibodies (CD4 antibody, CD8 antibody, CD11b antibody, CD11c antibody, CD25 antibody, FOXP3 antibody, and MHCclass II Iab antibody). The ratio of each cell (Table 2) in the obtained spleen cells and the number per spleen were determined by FACS (BD FACSCantoII) analysis. From the obtained ratio, the ratio of the cells 4 and 5 to the cells 3 was also determined.

  The results are shown in FIGS. In each figure, the tip of the ridge extending upward from the column indicates the 100th percentile value, the top of the column indicates the 75th percentile, the horizontal line in the column indicates the 50th percentile (median), and the bottom of the column indicates 25 This indicates the percentile value, and the tip of the ridge extending downward from the column indicates the 0th percentile value. In each figure, * indicates that there was a significant difference from Comparative Example 2 (P value obtained by two-sided student t-test was less than 0.05), and NS was not significant. It shows that. 1 shows the result of cell 1, FIG. 2 shows the result of cell 2, FIG. 3 shows the result of cell 3, FIG. 4 shows the result of cell 4, and FIG. In each figure, the left side shows the ratio in spleen cells (FIGS. 4 and 5 show the ratio in dendritic cells), and the right side shows the number per spleen.

  As shown in FIG. 1, when the test solutions of Examples 1 to 3 were administered, the ratio and number of regulatory T cells were significantly increased. On the other hand, as shown in FIG. 2, even when the test solutions of Examples 1 to 3 were administered, the ratio and number of conventional T cells, that is, T cells thought to act on immune activation, did not increase. . These results suggest that administration of a complex of GM-CSF and GM-CSF antibody can specifically induce differentiation of regulatory T cells.

  In addition, as shown in FIGS. 3 and 4, when the test solutions of Examples 1 to 3 are administered, the entire dendritic cells and immunosuppressed dendritic cells considered to be involved in induction of differentiation of regulatory T cells. The proportion and number of the increase increased. On the other hand, even when the test solutions of Examples 1 to 3 were administered, the ratio and number of immune activated T cells did not increase. The proportion of immune activated T cells tended to decrease rather. From these facts, administration of a complex of GM-CSF and a GM-CSF antibody first induces specific differentiation of immunosuppressed dendritic cells, and then differentiates regulatory T cells by the dendritic cells. It was suggested that it was induced.

Test Example 2 Analysis of effects on GVHD Bone marrow transplantation (allogeneic or allogeneic transplantation) was performed on mice (BALB / c 8-10 weeks old). For allogeneic transplanted mice, each test solution prepared in Test Example 1 (Comparative Examples 1-2 and 1 μL of Example 1) was injected into the peritoneal cavity on the 14th, 15th, and 16th days after transplantation. For each bone marrow transplanted mouse, from the date of transplantation to the 50th day after transplantation, the presence or absence of skin damage with hair loss and its area, and skin diseases (skin damage or skin desquamation) in the ears, tail, and hands The presence or absence was evaluated, and a GVHD score was calculated based on the evaluation result, and the GVHD score was “0” when there was no skin damage accompanied with hair loss, “1” when there was less than 1 cm ² , "2" if the skin injury 2 cm ² is present, the case where the skin damage is there more than 2 cm ² was evaluated as "3", further ear, tail, or when there is a skin disease in hand, to each part Every time there is a skin disease, .3 "was calculated by adding (minimum = 0, maximum value = 3.9).

  The average value of the GVHD score is shown in FIG. In FIG. 6, the horizontal axis indicates the number of days after bone marrow transplantation (BMT), “GM-CSF IC” indicates the group administered with the test solution of Example 1, and “GM-CSF” indicates the test solution of Comparative Example 2. “PBS” indicates the group to which the test solution of Comparative Example 1 was administered, and “Syngeneic” indicates the allograft group.

  As shown in FIG. 6, when the test solution of Example 1 was administered, the GVHD score was suppressed lower than when the test solution of Control (Comparative Examples 1 and 2) was administered. This suggested that administration of a complex of GM-CSF and a GM-CSF antibody is useful for the prevention and / or treatment of GVHD.

Claims (6)

Regulatory T cell differentiation inducer containing GM-CSF and GM-CSF antibody. The differentiation inducer according to claim 1, comprising a complex of GM-CSF and a GM-CSF antibody. The differentiation inducer according to claim 1 or 2, wherein the regulatory T cells are FOXP3 ⁺ . The differentiation inducer according to any one of claims 1 to 3, wherein the GM-CSF antibody is a monoclonal antibody. The differentiation inducer according to any one of claims 1 to 4, which is used for immunosuppression. It is for prevention and / or treatment of at least one disease selected from the group consisting of autoimmune disease, graft-versus-host disease (GVHD), and graft rejection. Differentiation inducer.

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