WO2010050584A1 - ヘルパーt細胞の選択的機能制御法 - Google Patents
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- A01K2267/035—Animal model for multifactorial diseases
- A01K2267/0368—Animal model for inflammation
Definitions
- the present invention relates to a pharmaceutical composition having an immunosuppressive activity, an antiasthmatic activity and / or an antiallergic activity, comprising a helper T cell selective activity inhibitor.
- the present invention also relates to a method for screening a substance exhibiting immunosuppressive activity, anti-asthma activity and / or anti-allergic activity, including a step of measuring the ability to selectively inhibit helper T cell activity.
- T cells are immune cells that occupy 70-80% of peripheral blood lymphocytes, and are widely distributed in the spleen and systemic lymph nodes, contributing to biological defense.
- a T cell expresses a T cell antigen receptor (T ⁇ cell receptor; TCR) on its surface and plays a central role in the acquired immune system.
- T cell activation is triggered by TCR on the T cell membrane recognizing the major histocompatibility complex (MHC) + antigen expressed by antigen presenting cells.
- MHC major histocompatibility complex
- One T cell can exert a specific immune response against any antigen by expressing only one type of antigen-specific TCR.
- T cells express CD4 or CD8 molecules on the cell surface, and are distinguished as CD4 positive T cells and CD8 positive T cell subsets, respectively, and form completely different cell populations with different immune functions.
- the CD4-positive T cell subset is called a helper T cell, and has a function of inducing functional expression of other T cells or differentiating B cells into antibody-producing cells.
- the CD8 positive T cell subset is called a killer T cell, has a function of destroying virus-infected cells, cancer cells and the like, and is also involved in a rejection reaction that becomes a problem in organ transplantation.
- Helper T cells are further functionally divided into four subgroups (Th1, Th2, Th17, and Treg cells). Th1 cells are effector cells differentiated mainly in the presence of IL-12, and are involved in autoimmune diseases and delayed allergy by mainly producing IFN- ⁇ to induce cellular immunity.
- Th2 cells are effector cells differentiated mainly in the presence of IL-4, and are involved in immediate allergy by mainly producing IL-4 and inducing humoral immunity.
- Th17 cells are a recently identified subgroup that is differentiated in the presence of IL-6 and TGF- ⁇ to produce IL-17 and is functionally thought to be involved in autoimmune diseases.
- Treg cells are CD25-positive CD4-positive T cells called regulatory T cells, and contribute to maintaining the homeostasis of the immune system by controlling the immune response.
- T cells play an important role in all pathologies involving immunity such as infections, tumors, organ transplants, and allergies.
- CD4 positive helper T cells play a central role in biological defense such as controlling other immune cells, while excessive helper T cell immune responses also cause autoimmune diseases and allergic diseases. That is, appropriate and appropriate activation of helper T cells is essential for maintaining the homeostasis of the immune system.
- Immunosuppressive agents are frequently used as a conventional method for controlling T cell immunity. However, it is a problem that the effect of the immunosuppressive agent is non-specific to the T cell subset. Cyclosporine and FK506 are known as typical immunosuppressive agents, and these suppress the activation of all T cells. Steroidal drugs are also frequently used for the treatment of autoimmune diseases and allergic diseases, but they have a problem that they act non-specifically and have strong side effects. Thus, a method for selectively controlling the function of helper T cells has not yet been established.
- Ganglioside is a group of glycosphingolipids having sialic acid, and all endogenous glycosphingolipids are biosynthesized from ceramide through a series of enzymatic reactions (FIG. 1).
- GM3 is a starting molecule for all gangliosides and is synthesized from lactosylceramide by GM3 biosynthetic enzyme (sialic acid transferase I; SAT-I).
- SAT-I GM3 biosynthetic enzyme
- Gangliosides are expressed in all mammalian cells, and together with cholesterol and sphingomyelin constitute a microdomain on the cell membrane called raft. Recent research has revealed that rafts function as an important field for transmitting information from receptors into cells (Non-patent Document 1).
- rafts provide an indispensable field for intracellular signal transduction via TCR, and when the raft structure is destroyed using an agent that removes cholesterol from the cell membrane, TCR-mediated signal transduction is suppressed.
- the cells are not activated (Non-patent Document 2).
- Non-patent Document 3 the functional role of gangliosides in T cell activation has been verified by the addition of glycosphingolipid biosynthetic enzyme inhibitors or exogenous gangliosides.
- Various effects were recognized for each ganglioside added (Non-patent Document 3).
- no studies have analyzed the function of gangliosides for TCR-mediated activation in each T cell subset in vivo. K Simons and D Toomre.
- helper T cells Some pathological conditions involving immune reactions such as infection, tumor, organ transplant rejection, allergy, etc. are caused by abnormal function or hyperfunction of helper T cells.
- helper T cells killer T cells Controlling the entire immune system such as immunodeficiency causes an immune deficiency or an immune enhancement state. From such a viewpoint, a method capable of selectively controlling the function of helper T cells is desired.
- the present inventors diligently tried to solve the above-mentioned problems, and produced a mouse (SAT-I KO) deficient in a gene involved in ganglioside biosynthesis (ganglioside GM3 synthase gene).
- ganglioside GM3 synthase gene The functional role of gangliosides in activating T cell subsets at the level was analyzed.
- helper T cells by T cell receptor (TCR) stimulation can be selectively suppressed while maintaining the function of killer T cells. Furthermore, it was confirmed that inflammatory infiltration can be reduced in an individual by suppressing proliferation of this helper T cell selectively.
- TCR T cell receptor
- the present invention includes controlling the growth of glycosphingolipids in helper T cells and selectively suppressing the proliferation of helper T cells during an immune response and mitigating or suppressing the excessive immune response resulting from this proliferation suppression. And the like, ie, screening methods for substances exhibiting immunosuppressive activity, anti-asthma action and / or anti-allergic action.
- the present invention relates to the following pharmaceutical compositions, screening methods, therapeutic methods and the like: (1) A pharmaceutical composition having an immunosuppressive activity, an antiasthma activity and / or an antiallergic activity, comprising a helper T cell selective activity inhibitor. (2) The composition according to (1), wherein the helper T cell selective activity inhibitor is a substance that selectively suppresses proliferation of helper T cells. (3) The composition according to (2), wherein the helper T cell selective activity inhibitor is a substance that selectively suppresses the production of glycosphingolipids in helper T cells. (4) The composition according to (3), wherein the glycosphingolipid is ganglioside. (4a) The composition according to (3), wherein the ganglioside is GM3.
- helper T cell selective activity inhibitor comprises an antibody against GM3.
- the helper T cell selective activity inhibitor is a GM3 synthase inhibitor.
- the helper T cell selective activity inhibitor is an antisense nucleic acid, ribozyme or RNAi RNA that inhibits the expression of GM3 synthase, according to any one of (1) to (4) Composition.
- a screening method for a substance exhibiting immunosuppressive action, anti-asthma action and / or anti-allergic action which comprises contacting a test substance with helper T cells and measuring cell proliferation in the helper T cells.
- a screening method for a substance exhibiting immunosuppressive action, anti-asthma action and / or anti-allergic action wherein a test substance is brought into contact with a helper T cell and the production amount of glycosphingolipid in the helper T cell is measured.
- the method according to (10), wherein the glycosphingolipid is ganglioside.
- 11a The method according to (11), wherein the ganglioside is GM3.
- a screening method for a substance exhibiting immunosuppressive action, anti-asthmatic action and / or anti-allergic action wherein a test substance is brought into contact with a helper T cell and the expression level of GM3 synthase in the helper T cell is measured.
- the method according to (13), wherein the non-human animal is a mouse.
- helper T cell selective activity inhibitor suppresses the production amount of glycosphingolipid expressed in helper T cells.
- the glycosphingolipid is ganglioside.
- the ganglioside is GM3.
- the helper T cell selective activity inhibitor is a GM3 synthase inhibitor or a substance that inhibits the expression of GM3 synthase.
- the helper T cell selective activity inhibitor is an antisense nucleic acid, dsRNA, or ribozyme that inhibits the expression of GM3 synthase.
- a method for selectively controlling the immune function of a helper T cell by controlling the production of glycosphingolipid in the helper T cell comprising: (i) contacting a helper T cell with a glucosylceramide synthase inhibitor D-PDMP and ganglioside GM3; (ii) a step of contacting a helper T cell with a glucosylceramide synthase inhibitor D-PDMP, ganglioside GM3 and a test substance, and (iii) comparing the cell proliferation of (i) and (ii) above, A screening method comprising a step of determining a test substance that suppresses T cell proliferation.
- a method for screening a helper T cell selective activity inhibitor comprising: (i) contacting a helper T cell with a test substance; (ii) GM3, GM2, GM1, GD1a and GT1a (a-series), GD3, GD2, GD1b, GT1b and GQ1b (b-series), and GT3, GT2, GT1c, GQ1c and GP1c ( c-series), a step of measuring at least one ganglioside amount selected from the group consisting of: and (iii) selecting a test substance that reduces at least one ganglioside amount.
- test substance in which the above cells do not proliferate in the presence of a combination of an anti-CD3 antibody and an anti-CD28 antibody, a superantigen or a syngeneic heterologous antigen subset, and the above cells proliferate in the presence of a combination of Ionomycin and PMA.
- the pharmaceutical composition of the present invention can selectively control the function of helper T cells while maintaining the function of killer T cells, it can provide a therapeutic method as a new immunoregulatory method clinically. .
- the pharmaceutical composition of the present invention or the substance obtained by the screening method of the present invention is considered to be useful for the purpose of selectively inhibiting the growth of helper T cells, for example, in cell therapy.
- FIG. 1 shows the ganglioside biosynthesis pathway.
- FIG. 2 shows a method for producing a GM3 synthase gene-deficient mouse (SAT-I KO).
- FIG. 3 shows reduction of allergic airway inflammation after ovalbumin (OVA) inhalation in SAT-I gene-deficient mice.
- FIG. 4 shows a diagram of mucosal staining by PAS of the airway after inhalation of ovalbumin (OVA) in the SAT-I gene-deficient mouse of FIG.
- FIG. 5 shows a decrease in serum IgE level after inhalation of ovalbumin (OVA) in SAT-I gene-deficient mice.
- FIG. 1 shows the ganglioside biosynthesis pathway.
- FIG. 2 shows a method for producing a GM3 synthase gene-deficient mouse (SAT-I KO).
- FIG. 3 shows reduction of allergic airway inflammation after ovalbumin (OVA) inhalation in SAT-I gene-deficient mice.
- FIG. 6 shows the decrease in serum IL-4 concentration and IL-5 concentration after ovalbumin (OVA) inhalation in SAT-I gene-deficient mice.
- FIG. 7 shows selective suppression of TCR-dependent proliferative response of CD4-positive T cells in SAT-I gene-deficient mice.
- FIG. 8 shows that only SAT-I KO mouse-derived CD4 + T cells lack antigen-specific responses.
- FIG. 9 shows that only CD4 + T cells derived from SAT-I KO mice lack superantigen-specific responses.
- FIG. 10 shows that only the SAT-I ⁇ KO mouse-derived CD4 + T cells lack alloantigen-specific responses.
- FIG. 11 shows the recovery effect of GM3 addition on the functional decline of CD4-positive T cells in SAT-I gene-deficient mice.
- FIG. 12 shows the recovery effect of SAT-I gene transfer on the reduced function of CD4-positive T cells in SAT-I gene-deficient mice.
- composition and therapeutic method of the present invention provides a pharmaceutical composition having immunosuppressive activity, anti-asthmatic activity and / or anti-allergic activity, comprising a helper T cell selective activity inhibitor.
- helper T cell selective activity inhibitor used in the present invention is a selective bioactive function by helper T cells (including Th1 cells, Th2 cells, Th17 cells and Treg cells which are four subgroups). And it is a substance that suppresses specifically. That is, for example, a substance that acts on helper T cells but has little or no harmful effect on killer T cells. More specifically, the “helper T cell selective activity inhibitor” used in the present invention is determined by the method described in the immunological protocol, Hiromitsu Nakauchi / Ed., September 2004, Yodosha).
- the growth inhibitory activity of helper T cells at the time of stimulation to be measured is 10% or more, preferably 20% or more, more preferably 40% or more, more preferably 60% or more, and further preferably 80% or more. It is a substance having a growth inhibitory activity of killer T cells at the time of stimulation of 20% or less, more preferably 10% or less, and still more preferably 5% or less.
- helper T cell selective activity evaluation is made by the production amount of IL-4, IL-5 and IFN- ⁇ , cytokines selectively produced from helper T cells by activation. There is a way to do it. Furthermore, it can be evaluated by the expression level of a gene or protein of a transcription factor (T-bet, GATA-3) whose expression increases with activation of helper T cells.
- T-bet a transcription factor
- helper T cell selective activity inhibitor preferably used in the present invention is a substance that selectively suppresses the proliferation of helper T cells, and selectively suppresses the production of glycosphingolipids in the helper T cells. It is a substance.
- typical immune responses induced in the living body by the physiologically active function of helper T cells include, for example, autoimmune diseases and delayed allergy by producing IFN- ⁇ to induce cellular immunity. It is involved in immediate allergy by inducing humoral immunity by producing IL-4, functionally involved in autoimmune disease by producing IL-17, immune response (Shu ⁇ L and Paul WE CD4T cells: fates, functions, and faults. Blood, 112, 1557-1569, 2008).
- immunosuppressive activity means an activity that suppresses, reduces, alleviates, sedates, improves, etc. an excessive immune response in a living body (for example, Gummert JF et al. Newer Immunosuppressive Drugs: A Review. J Am Soc Neprol 10, 1366-1380, ⁇ ⁇ ⁇ 1999).
- immunosuppressive agents such as cyclosporine.
- anti-asthma activity means treating and / or preventing symptoms of asthma. Asthma is caused by exacerbation of airway inflammation caused by allergens, bacterial or viral infections, etc., resulting in increased airway hypersensitivity and reversible airway narrowing, causing symptoms such as paroxysmal wheezing and coughing It is a respiratory disease.
- bronchodilators beta receptor agonists, xanthine derivatives, anticholinergic agents, corticosteroids (such as beclomethasone propionate as an inhaled steroid), and antiallergic agents (as histamine release inhibitors)
- bronchodilators beta receptor agonists, xanthine derivatives, anticholinergic agents
- corticosteroids such as beclomethasone propionate as an inhaled steroid
- antiallergic agents as histamine release inhibitors
- Cromoglycic acid and histamine receptor antagonists include terfenadine, thromboxane A2 receptor antagonists, leukotriene antagonists, etc.) (for example, asthma prophylaxis / management guidelines 2006; see the Japanese Society of Allergology supervision).
- antiallergic activity means treatment and / or prevention of allergic symptoms.
- the immune reaction is an essential physiological function for the living body that works to eliminate foreign substances (antigens). Allergy means that this immune reaction reacts excessively with a specific antigen.
- the cause of allergy is unknown, but it is thought to be caused by extraneous factors such as excessive exposure to the antigen, inherited intrinsic factors (eg Asthma Prevention and Management Guidelines ⁇ ⁇ 2006; supervised by the Japanese Society of Allergology) Can be referred to).
- “selectively inhibiting the proliferation of helper T cells” means selectively and specifically inhibiting only the proliferation of helper T cells among somatic cells.
- Examples of the selective proliferation of helper T cells include cell proliferation by receiving proliferation stimulation via CD4 specific to helper T cells.
- Examples of other helper T cell-specific proliferation stimuli include MHC class II molecule-dependent antigen and the same molecule-specific antibody.
- Glycosphingolipid refers to a lipid that contains sugar, fatty acid, and long-chain base sphingosine in the molecule.
- Ganglioside is a general term for the glycosphingolipid family containing sialic acid, and is a molecule in which a sugar chain containing sialic acid is covalently bound to a lipid called ceramide.
- ceramide a lipid in which a sugar chain containing sialic acid is covalently bound to a lipid called ceramide.
- GM3 is the first ganglioside molecule in its biosynthetic pathway (see FIG. 1). That is, all endogenous ganglio gangliosides are biosynthesized by a series of enzymatic reactions starting with ceramide and starting with GM3 synthase.
- GM3 is a molecule that is the starting point of all gangliosides and is synthesized from lactosylceramide by GM3 biosynthetic enzyme (SAT-I).
- SAT-I GM3 biosynthetic enzyme
- the sugar chain moiety is sequentially synthesized from a glycosyltransferase using a sugar nucleotide as a donor in the Golgi lumen in the cell.
- GM3 is generated from Gal-Glc-Cer by GM3 biosynthetic enzyme (SAT-I), and the a-series (GM3, Gangliosides of GM2, GM1, GD1a and GT1a), b-series (GD3, GD2, GD1b, GT1b and GQ1b) and c-series (GT3, GT2, GT1c, GQ1c and GP1c) are produced. From this figure, it is understood that the generation of these ganglioside molecules depends on GM3 synthase.
- SAT-I GM3 biosynthetic enzyme
- the helper T cell selective activity inhibitor may specifically inhibit the function of GM3 with an antibody against GM3.
- antibodies include known monoclonal antibodies having specificity for GM3 (Kotani, M., et al.,: Biochem. Biophys. Acta, 1117, 97-103 (1992)). .
- Such antibodies can inhibit the action of GM3 at the cell membrane in helper T cells.
- antibodies include monoclonal antibodies, polyclonal antibodies, anti-idiotype antibodies, antibody fragments (eg, Fab and F (ab ′) 2, Fv variable regions, or complementarity determining regions). Understood.
- an antibody is specific for GM3 or GM3 synthase if it binds with a Ka of 10-7 M or higher, preferably 10-8 M or higher.
- the affinity of a monoclonal antibody can be readily determined by one skilled in the art (see Scatchard, Ann. N.Y. Acad. Sci. 51: 660-672, 1949).
- the helper T cell selective activity inhibitor may be a compound that suppresses GM3 biosynthesis.
- the GM3 synthase (SAT-I) in the present specification is the amino acid sequence of SEQ ID NO: 2 or 4, or 1 to several (1-9, 1-8, 1-7, 1-6) of these sequences. 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1) refers to a protein containing an amino acid sequence having a deletion, substitution, insertion and / or addition mutation of amino acids.
- glucosylceramide synthase acts on ceramide (Cer) to synthesize glucosylceramide (GlcCer), and then lactosylceramide synthase (LacCer) Generate.
- GM3 synthase recognizes this LacCer as a substrate and synthesizes GM3. That is to say, inhibition of enzyme reaction by substrate analogs of glucosylceramide synthase, lactosylceramide synthase and GM3 synthase, and those capable of reducing the amount of GM3 by binding to these enzymes.
- the GM3 synthase inhibitor of the present invention includes glucosylceramide biosynthesis inhibitor D-PDMP and its analogs (Inokuchi, J., and Radin, N. Preparation of active isomer of 1-phenyl-2-decanoylamino-3- morpholino-1-propanol. J. Lipid Res. 28, 565-571, 1987; Radin NS., et al. Metabolic effects of inhiboting glucosylceramide synthesis with PDMP and other substances. Adv. Lipid. Res. 26, 183-213, 1993; Lee. L., et al. Iproved inhibitors of glucosylceramide synthase., J. Biol. Chem.
- the helper T cell selective activity inhibitor may be a substance that inhibits the expression of GM3 synthase.
- the cDNA of GM3 synthase (SAT-I) in this specification is represented by SEQ ID NO: 1 or 3, for example.
- substances that inhibit the expression of GM3 synthase include antisense nucleic acids, ribozymes, dsRNA having RNAi effect, and the like.
- nucleic acid is used interchangeably with “polynucleotide”, “gene” or “nucleic acid molecule” and is intended to be a polymer of nucleotides.
- base sequence is used interchangeably with “nucleic acid sequence” or “nucleotide sequence” and refers to the sequence of deoxyribonucleotides (abbreviated A, G, C, and T). As shown.
- polynucleotide containing the base sequence of SEQ ID NO: 1 or a fragment thereof intends the polynucleotide containing the sequence represented by each deoxynucleotide A, G, C and / or T of SEQ ID NO: 1 or a fragment thereof. Is done.
- the nucleic acid according to the present invention may exist in the form of RNA (for example, mRNA) or in the form of DNA (for example, cDNA or genomic DNA).
- DNA can be double-stranded or single-stranded.
- Single-stranded DNA or RNA can be the coding strand (also known as the sense strand) or it can be the non-coding strand (also known as the antisense strand).
- “substances that inhibit the expression of GM3 synthase” include suppression of transcription of the GM3 synthase gene and suppression of protein translation. Also included is a decrease in expression as well as complete cessation of DNA expression.
- One embodiment of the “substance inhibiting the expression of GM3 synthase” is a nucleic acid encoding an antisense strand complementary to the GM3 synthase gene.
- Antisense technology is known as a method for suppressing the expression of a specific endogenous gene, and is described in various literatures (for example, Hirashima and Inoue: Acupuncture Chemistry Laboratory 2 Nucleic acid IV gene replication and expression (Japan (See Biochemical Society, Tokyo Chemical Co., Ltd.) pp.319-347, 1993).
- An antisense nucleic acid can be prepared, for example, by the phosphorothioate method (Stein, Nucleic Acids Res., 16: 3209-3221, 1988) based on the sequence information of the cDNA described in SEQ ID NO: 1 or 3. Is possible.
- the prepared nucleic acid can be used in a known method, which can be directly applied to cells, and can be transformed into a suitable expression by incorporating it into a vector equipped with a known expression system. it can.
- the sequence of the antisense nucleic acid is preferably a sequence complementary to the transcription product of the endogenous gene possessed by the cell to be transformed, but may not be completely complementary as long as the gene expression can be effectively inhibited. .
- the transcribed RNA preferably has a complementarity of 90% or more (eg, 95%, 96%, 97%, 98%, 99% or more) to the transcript of the target gene.
- the length of the antisense nucleic acid is at least 15 bases, preferably 100 bases or more, more preferably 500 bases or more. is there.
- the length of the antisense nucleic acid used is shorter than 5 kb, preferably shorter than 2.5 kb.
- suppression of endogenous GM3 synthase gene expression can also be carried out using DNA encoding a ribozyme.
- a ribozyme is an RNA molecule having catalytic activity, which inhibits the function of the gene by cleaving the target DNA transcript.
- Various known literatures can also be referred to for the design of ribozymes (for example, FEBS Lett. 228: 228, 1988; FEBS Lett. 239: 285, 1988; Nucl. Acids. Res. 17: 7059, 1989; Nature 323). : 349, 1986; Nucl. Acids. Res. 19: 6751, 1991; Protein Eng 3: 733, 1990; Nucl. Acids Res.
- polynucleotide encoding RNA that suppresses DNA expression by co-suppression refers to a nucleotide that inhibits the function of the target DNA by “co-suppression”.
- suppression of endogenous gene expression in the present invention can also be achieved by transforming a desired cell with a gene having a dominant negative trait of the target gene.
- a gene having a dominant negative trait refers to a gene having a function of eliminating or reducing the activity of an endogenous wild type gene inherent in a desired cell by expressing the gene.
- RNA that suppresses DNA expression by the RNAi effect.
- RNAi refers to a phenomenon in which, when a double-stranded RNA having the same or similar sequence as a target gene sequence is introduced into a cell, expression of the introduced foreign gene and target endogenous gene are both suppressed.
- Examples of RNA used herein include double-stranded RNA that causes RNA interference of 21 to 25 bases in length, such as dsRNA (double strand RNA), siRNA (small interfering RNA), or shRNA (short hairpin RNA). .
- RNA can be locally delivered to a desired site by a delivery system such as a liposome, and can also be locally expressed using a vector capable of producing the double-stranded RNA.
- dsRNA, siRNA or shRNA double-stranded RNA
- Methods for preparing and using such double-stranded RNA are known from many literatures (Japanese translations of PCT publication No. 2002-516062; U.S. Publication No. 2002 / 086356A; Nature Genetics , 24 (2), 180-183, 2000 Feb .; Genesis, 26 (4), 240-244, 2000 April; Nature, 407: 6802, 319-20, 2002 Sep. 21; Genes & Dev., Vol. 16, (8), 948-958, 2002 Apr.15; Proc Natl.
- a pharmaceutical composition having immunosuppressive activity, anti-asthma activity and / or anti-allergic activity contains a substance having an inhibitory action on helper T cell selective activity, and includes autoimmune diseases, asthma, allergies, infectious diseases, It can be used for treatment or prevention of transplant rejection (eg, kidney transplantation, heart transplantation, bone marrow transplantation) reaction of tumors and organs.
- the pharmaceutical composition having an inhibitory action on the helper T cell selective activity of the present invention is preferably transplant rejection of an organ or the like, autoimmune disease, asthma, infection, allergy, tumor, more preferably autoimmune disease, asthma, It is used for treatment or prevention of infectious diseases, allergies, etc., and particularly preferably used for treatment or prevention of asthma and / or allergic diseases.
- the pharmaceutical composition of the present invention when used, for example, oral, intravenous, oral mucosa, rectal, vaginal, transdermal, nasal or inhalation can be used, but oral administration is preferable.
- the active ingredients of the pharmaceutical composition of the present invention may be blended singly or in combination.
- a pharmaceutically acceptable carrier or a pharmaceutical additive may be blended with the active ingredient and provided in the form of a pharmaceutical preparation. it can.
- the active ingredient of the present invention can be contained, for example, in the preparation in an amount of 0.1 to 99.9% by weight.
- Examples of pharmaceutically acceptable carriers or additives include excipients, disintegrants, disintegration aids, binders, lubricants, coating agents, dyes, diluents, solubilizers, solubilizers, isotonic agents. Agents, pH adjusters, stabilizers and the like can be used.
- preparations suitable for oral administration include powders, tablets, capsules, fine granules, granules, liquids or syrups.
- various excipients such as microcrystalline cellulose, sodium citrate, calcium carbonate, dipotassium phosphate, glycine are added to starch, preferably corn, potato or tapioca starch, and alginic acid and certain species. It can be used with various disintegrants such as silicate double salts and granulating binders such as polyvinylpyrrolidone, sucrose, gelatin, gum arabic.
- lubricants such as magnesium stearate, sodium lauryl sulfate, and talc are often very effective for tablet formation.
- the same kind of solid composition can also be used by filling gelatin capsules.
- suitable substances in this connection include lactose or lactose as well as high molecular weight polyethylene glycols.
- the active ingredient is used in combination with various sweeteners or flavors, colorants or dyes, and if necessary, an emulsifier and / or suspending agent is also used.
- an emulsifier and / or suspending agent is also used.
- preparations suitable for parenteral administration include injections and suppositories.
- a solution in which the active ingredient of the present invention is dissolved in either sesame oil or peanut oil or dissolved in an aqueous propylene glycol solution can be used.
- the aqueous solution should be appropriately buffered as necessary (preferably pH 8 or more), and the liquid diluent must first be made isotonic.
- Such aqueous solutions are suitable for intravenous injection and oily solutions are suitable for intra-articular, intramuscular and subcutaneous injection. All these solutions can be prepared aseptically by standard pharmaceutical techniques well known to those skilled in the art.
- the active ingredient of the present invention can also be administered locally such as on the skin. In this case, topical administration in the form of creams, jellies, pastes, ointments is desirable according to standard pharmaceutical practice.
- the dose of the pharmaceutical composition of the present invention is not particularly limited, and an appropriate dose is selected according to various conditions such as the type of disease, the age and symptoms of the patient, the route of administration, the purpose of treatment, the presence or absence of a concomitant drug, etc. Is possible.
- the dosage of the pharmaceutical composition of the present invention is, for example, 1 to 5000 mg, preferably 10 to 1000 mg per day for an adult (for example, body weight 60 kg). These daily doses may be administered in two to four divided doses.
- the screening method of the present invention further comprises a helper T cell selective activity inhibitor, ie, an immunosuppressive activity, an anti-asthma effect and / or an anti-allergic effect, which comprises a step of measuring the ability to suppress the selective activity of helper T cells.
- a helper T cell selective activity inhibitor ie, an immunosuppressive activity, an anti-asthma effect and / or an anti-allergic effect.
- Such screening methods include methods performed in vitro and / or in vivo.
- Examples of the screening method of the present invention include a method in which a test substance is contacted with a helper T cell in vitro, and selective cell proliferation in the helper T cell is measured.
- Test substances include peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, plasma and the like.
- the helper T cell may be a cell isolated from a living body or a cell established.
- examples of the direct measurement method include a method of visual measurement using a hemocytometer, and a method of using equipment such as a Coulter counter and a flow cytometer.
- Examples of the indirect measurement method include a method using a commercially available reagent / kit such as an XTT assay kit (for example, a kit manufactured by R & D " systems " MN " USA).
- a known method such as trypan blue staining for excluding dead cells can be used in combination as necessary.
- the proliferation-suppressing activity of helper T cells upon stimulation measured by the method described in the above-mentioned immunological protocol is 10% or more, Preferably, it is 20% or more, more preferably 40% or more, more preferably 60% or more, and further preferably 80% or more, and the growth inhibitory activity of the killer T cell under the same conditions as a control is 20% or less, More preferably, it is 10% or less, More preferably, it is 5% or less.
- Examples of the screening method of the present invention include a method in which a test substance is contacted with a helper T cell in vitro, and the amount of selective glycosphingolipid production in the helper T cell is measured.
- the glycosphingolipid to be measured is preferably ganglioside, and more preferably GM3.
- Known methods for purification and quantitative detection of glycolipids include, for example, Macher BA and Klock JC (J. Biol. Chem. 255, 2092-2096, 1980) and LedeenLet al. (J. Neurochem. 21 , 829-839, 1973).
- quantitative detection using an antibody that specifically acts on gangliosides such as GM3 is also possible.
- the suppression of GM3 production in helper T cells upon stimulation measured by the method described in the aforementioned immunological protocol, Hiromitsu Nakauchi / hen, Yodosha, is 10% or more, Preferably it is 20% or more, More preferably, it is 40% or more, More preferably, it is 60% or more, More preferably, it is 80% or more.
- Examples of the screening method of the present invention include an assay method in which a helper T cell is contacted with a test substance in vitro to examine the ganglioside composition in the helper T cell.
- GM3, GM2, GM1, GD1a and GT1a (a-series), GD3, GD2, GD1b, GT1b and GQ1b (b-series), and GT3, GT2, GT1c, GQ1c and GP1c (c-series)
- a test substance that significantly reduces at least one ganglioside selected from the group consisting of:
- “significantly reduce” means at least 10% or more, preferably 20% or more, more preferably 40% or more, more preferably 60% or more, and even more preferably 80% or more compared to the control. , A reduction of 90% or more.
- Examples of the screening method of the present invention include a method in which a test substance is contacted with a helper T cell in vivo, and the expression level of selective GM3 synthase in the helper T cell is measured.
- Examples of a method for measuring the expression level of GM3 synthase include a method using hybridization using a sequence of GM3 synthase. For example, Northern blot, a method using a probe immobilized on a carrier, a gene chip Examples of the method used include quantitative PCR.
- suppression of GM3 synthase expression in helper T cells upon stimulation measured by the method described in the aforementioned immunological protocol, Keiko Nakauchi / hen, Yodosha, is 10% Above, preferably 20% or more, more preferably 40% or more, more preferably 60% or more, and further preferably 80% or more, 90% or more.
- the test substance in the screening method of the present specification includes a polynucleotide that specifically inhibits the expression of GM3 synthase.
- GM3 synthase expression-inhibiting polynucleotide examples include the aforementioned antisense nucleic acid, ribozyme, and double-stranded RNA.
- These GM3 synthase expression-inhibiting polynucleotides can be prepared by known methods. Furthermore, these nucleic acids may be subjected to various chemical modifications.
- a cell derived from a genetically modified animal or a genetically modified cell in which a reporter gene is linked downstream of the promoter of GM3 synthase is contacted with the test substance in vivo, and the cell A method of measuring the expression level of the reporter gene in Various cells can be used as the genetically modified cells used in this method, preferably T cells, and more preferably helper T cells.
- the gene sequence 5 ′ upstream of the GM3 synthase gene is described in Kim JW., Et al., Gene 273, 163-171, 2001, and the promoter is Kim SW., Et al., Biochim. Biophys.
- the protein encoded by the reporter gene used preferably includes various known proteins such as firefly luciferase, Renilla luciferase, green fluorescent protein (GFP), ⁇ -galactosidase, alkaline phosphatase and the like. These reporter proteins can be detected using known detection methods or kits. In such a screening method, for example, suppression of reporter protein expression upon stimulation with a test substance is 10% or more, preferably 20% or more, more preferably 40% or more, 60% or more, more preferably 80% or more, 90% or more.
- helper T cell selectivity of the test substance selected in the screening method of the present invention various known T cell receptor stimulations, for example, combinations of anti-CD3 antibody and anti-CD28 antibody, superantigen, or alloantigen ( This can be confirmed by cell proliferation of helper T cells in the presence of allogeneic antigen).
- the stimulation not involving the T cell receptor as a control include a combination of Ionomycin and PMA. By this stimulation, both helper T cells and killer T cells can be proliferated. See Immunological Protocol, Keigo Nakauchi / Ed., Yodosha.
- the screening method of the present invention can further include a step of administering a test substance confirmed to have a cell proliferation activity of helper T cells to a non-human animal exhibiting an immune disease, asthma and / or allergic reaction.
- the test substance can be modified so as to specifically act on helper T cells in the living body, or a known modification such as using a delivery system specific to helper T cells can be added. .
- the screening method of the present invention can be detected by, for example, the amount of cytokine produced by helper T cells.
- cytokines that produce helper T include IL-2, IL-4, IL-5, IL-6, and IFN- ⁇ that activate B cells, such as IL-4, IL-12, and IFN- that activate macrophages.
- IL-3, IL-5, GM-CSF, etc. that activate eosinophils, such as ⁇ , TNF- ⁇ , etc.
- Examples of methods for detecting these cytokines include, but are not limited to, a method for measuring proliferation of these cytokine-dependent cells, a method for confirming physiological activity such as antibody production, and a method using an antibody against each cytokine. .
- the reduction of specific cytokine production by helper T cells upon stimulation measured by the method described in the aforementioned immunological protocol, Keiko Nakauchi / hen, Yodosha, is 10% Or more, preferably 20% or more, more preferably 40% or more, more preferably 60% or more, still more preferably 80% or more, 90% or more, and the production of a specific cytokine in a killer cell under the same conditions as a control
- the suppression is 20% or less, more preferably 10% or less, and still more preferably 5% or less.
- non-human animals examples include monkeys, chimpanzees, dogs, cats, guinea pigs, rats, mice, pigs, sheep, horses, and other animals generally used in pharmaceutical tests.
- Preferred are monkeys, chimpanzees, rats and mice, and more preferred are mice and rats.
- the screening method of the present invention can further include a step of measuring the number of infiltrating cells such as macrophages, eosinophils, neutrophils, lymphocytes, mast cells and the like in inflammatory sites, for example, in vivo. That is, for example, when a test substance is administered to a subject having an inflammatory site, all or any one or more of macrophages, eosinophils, neutrophils, lymphocytes, mast cells, etc. in the inflammatory site are present. If significantly reduced, the test substance can be assumed to be therapeutically effective. In the present specification, “significantly reduced” means at least 10% or more, preferably 20% or more, more preferably 40% or more, more preferably 60% or more, and even more preferably 80% or more compared to the control. 90% or more and 95% or more.
- the screening method of the present invention can further include a step of measuring the IgE concentration in serum during allergic response, for example, in vivo. For example, when administering a test substance to a subject having an inflammatory site, if the blood IgE concentration of the subject is significantly reduced, it can be estimated that the test substance is therapeutically effective.
- the present invention provides a method for treating immune diseases, asthma and / or allergy, wherein the immune function of helper T cells is selectively controlled by administering an effective amount of a helper T cell selective activity inhibitor to a patient.
- a helper T cell selective activity inhibitor include the above-mentioned pharmaceutical composition or the test substance confirmed by the screening method.
- the helper T cell selective activity inhibitor is, as described above, a substance that selectively suppresses the proliferation of the helper T cell, or a substance that suppresses the production amount of glycosphingolipid expressed in the helper T cell.
- Substances that suppress the production of gangliosides expressed in helper T cells, substances that suppress the production of GM3 expressed in helper T cells, GM3 synthase inhibitors, substances that inhibit the expression of GM3 synthase, etc. are administered, the dosage can be appropriately adjusted according to the individual case in consideration of symptoms, age of the administration subject, sex, and the like. The dosage and the number of administrations can be appropriately adjusted after the doctor considers the subject's age, medical history, drugs currently used, and the like.
- SAT-I KO mice are a-series (GM3, GM2, GM1, GD1a and GT1a), b-series (GD3, GD2, GD1b, GT1b and GQ1b) and c-series (GT3, GT2, GT1c, GQ1c).
- GP1c are completely deficient in the ganglioside (see FIG. 1).
- helper T cells was selectively suppressed in SAT-I KO mice, and the function of killer T cells was normal.
- T cells was recovered by exogenously supplementing gangliosides lacking in T cells of SAT-I KO mice or by reintroducing the SAT-I gene.
- FIG. 2A and FIG. 2B show a schematic diagram of the targeting vector used for the production of SAT-I KO mice and confirmation of gene deletion in KO mice, respectively.
- FIG. 2A shows the targeting vector at the top, the wild type gene at the second, and the mutant gene at the third.
- determination of SAT-I genotype by PCR is shown.
- 5′-GGAATCCATCCCTTTTCTCACAGAG-3 SEQ ID NO: 5′-TGAACTCACTTGGCATTGCTGG-3 ′ (SEQ ID NO: 6) were used as primers.
- FIG. 2 (c) shows the results of TLC analysis of brain ganglioside.
- wild-type mice (+ / +) and heterozygous mice (+/-) GM1, GD1a, GD1b, GT1b, etc. were expressed, but in knockout mice (-/-), all of these disappeared and were compensated.
- GM1b and GD1a were expressed (see FIG. 1).
- helper T Since infiltration of eosinophils into allergic regions depends on the activation of helper T cells (see Asthma Prevention and Management Guidelines 2006; supervised by the Japanese Society of Allergology), helper T in vivo in SAT-I KO mice It may be due to the lack of cellular activity.
- IL-4 / IL-5 production ability of SAT-I KO mice Induction of asthma / allergic reaction and production of antigen-specific IgE antibody include IL-4 and IL-5 produced from activated helper T cells. Asthma / allergy model by OVA inhalation after OVA sensitization prepared by the same method as above, because it depends on so-called type 2 helper T cytokine (see Allergy Disease Diagnosis / Treatment Guidelines 2007; Japan Allergy Society) Serum IL-4 and IL-5 concentrations were measured by ELISA. Serum was collected from the same individual at the 5th day after inhalation of antigen at the time of evaluation of asthma / allergic reaction shown in FIG.
- SAT-I in helper T cells or controlling the function of ganglioside, by suppressing the function of helper T cells and suppressing infiltration by eosinophils and lymphocytes, Resistance to immune responses, asthma, allergic reactions, etc. can be provided to the subject.
- FIG. 12 shows the cell proliferation response when the SAT-I gene is reintroduced into helper and killer T cells of SAT-I KO mice.
- SAT-I gene or LacZ gene is introduced into each cell via adenovirus or untreated After (-), cells were stimulated by the same method as in FIG. 1 to analyze cell proliferation (Miyake S. et al., Efficient generation of recombinant adenoviruses using adenovirus DNA-terminal protein complex and a cosmid bearing the full-length virus genome. Proc.
- D-PDMP D-threo-1-phenyl-2-decanoylamiono-3-morpholino-1-prppanol ⁇ HCl, product name D-threo-PDMP, manufacturer: Matreya, Inc. Catalog Number: # 1756
- D-PPMP D-threo-1-phenyl-2-hexadecanoylamiono-3-morpholino-1-prppanol ⁇ HCl, Manufacturer: Matreya, Inc.
- 14662 AMP-DNM N- (5-adamantane-1-yl-methoxy) -pentyl-1-deoxynojirimycin, Diabetes 56, 1341-1349, 2007
- GlcCer glucosylceramide
- GM3 should be added to the culture solution after treating all T cells with a GlcCer synthase inhibitor or simultaneously with the treatment. Then, confirm the recovery of the function (proliferative ability) of CD4 positive T cells. At this time, the GlcCer synthase inhibitor also suppresses the function of CD8 + T cells, but the addition of GM3 does not restore the proliferation ability of CD8 + T cells.
- ⁇ Reference Example 2 Procedure for constructing screening system 2 (in vivo system) Control (wild type) mice (+ / +) and SAT-I KO mice (-/-) were sensitized twice by OVA with intraperitoneal administration, and several days later Inhalation of OVA induces asthma and allergic reactions.
- Glucosylceramide synthase inhibitor eg, D-PDMP
- Tween 80 5% Tween 80 at a concentration such as 10 mg / ml, and under conditions such that CD4 positive T cells proliferate only when GM3 is used, for example, 75 Intraperitoneally administer mg / kg twice a day for 6 consecutive days after inhalation of OVA.
- Asthma and allergic reactions were evaluated using the SAT-I KO mouse, as described above. Measurement of the number of inflammatory cells and the amount of cytokines contained in the lung lavage fluid, airway mucus (principal component) staining of lung tissue, serum It can be performed by the above-mentioned various methods such as measurement of the amount of anti-OVA-specific IgE in the medium.
- the present invention is an unprecedented breakthrough that enables selective function control of helper T cells. Based on this invention, development of a novel treatment method targeting infectious diseases, tumors, organ transplant rejection, allergies and the like can be expected.
Abstract
Description
K Simons and D Toomre. Lipid raft and signal transduction. Nat. Rev. Mol. Cell Biol. 1, 31-39, 2000 P.W. Janes, S.C. Ley, A.I. Magee and P.S. Kabouridis. The role of lipid rafts in T cell receptor signaling. Semin. Immunol. 12, 23-34, 2000 M Potapenko, G.V. Shurin and J de Leon. Gangliosides as immunomodulator. Adv. Exp. Med. Biol.601, 195-203, 2007
(1) ヘルパーT細胞選択的活性抑制剤を含有する、免疫抑制活性、抗喘息活性及び/又は抗アレルギー活性を有する医薬組成物。
(2) 前記ヘルパーT細胞選択的活性抑制剤が、ヘルパーT細胞の増殖を選択的に抑制する物質である、(1)に記載の組成物。
(3) 前記ヘルパーT細胞選択的活性抑制剤が、ヘルパーT細胞におけるスフィンゴ糖脂質の産生を選択的に抑制する物質である、(2)に記載の組成物。
(4) 前記スフィンゴ糖脂質がガングリオシドである、(3)に記載の組成物。
(4a) 前記ガングリオシドがGM3である、(3)に記載の組成物。
(5) 前記ヘルパーT細胞選択的活性抑制剤がGM3に対する抗体を含む、(4)に記載の組成物。
(6) 前記ヘルパーT細胞選択的活性抑制剤が、GM3合成酵素阻害剤である、(1)~(4)のいずれかに記載の組成物。
(7) 前記ヘルパーT細胞選択的活性抑制剤が、GM3合成酵素の発現を阻害する、アンチセンス核酸、リボザイムまたはRNAi効果を有するRNAである、(1)~(4)のいずれかに記載の組成物。
(8) ヘルパーT細胞の選択的活性抑制能を測定する工程を含む、免疫抑制活性、抗喘息作用及び/又は抗アレルギー作用を示す物質のスクリーニング方法。
(9) 被験物質を、ヘルパーT細胞に接触させ、ヘルパーT細胞における細胞増殖を測定する、免疫抑制作用、抗喘息作用及び/又は抗アレルギー作用を示す物質のスクリーニング方法。
(10) 被験物質を、ヘルパーT細胞に接触させ、ヘルパーT細胞におけるスフィンゴ糖脂質の産生量を測定する、免疫抑制作用、抗喘息作用及び/又は抗アレルギー作用を示す物質のスクリーニング方法。
(11) 前記スフィンゴ糖脂質がガングリオシドである、(10)に記載の方法。
(11a) 前記ガングリオシドがGM3である、(11)に記載の方法。
(12) 被験物質を、ヘルパーT細胞に接触させ、ヘルパーT細胞におけるGM3合成酵素の発現量を測定する、免疫抑制作用、抗喘息作用及び/又は抗アレルギー作用を示す物質のスクリーニング方法。
(13) ヘルパーT細胞の細胞増殖活性を有することが確認された被験物質を、免疫疾患、喘息及び/又はアレルギー反応を示す非ヒト動物に投与する工程を含む、(8)~(12)のいずれかに記載の方法。
(14) 前記非ヒト動物がマウスである、(13)に記載の方法。
(15) 前記非ヒト動物の炎症部位における好酸球及び/又はリンパ球の浸潤細胞数を対照と比較する工程をさらに含む、(13)に記載の方法。
(16) ヘルパーT細胞選択的活性抑制剤の有効量を患者に投与することによりヘルパーT細胞の免疫機能を選択的に制御する、免疫疾患、喘息及び/又はアレルギーの治療方法。
(17) 前記ヘルパーT細胞選択的活性抑制剤が、ヘルパーT細胞の増殖を抑制する、(16)に記載の方法。
(17a) 前記非ヒト動物において、血清中IgE濃度、IL-4濃度およびIL-5濃度のうちの少なくとも1つを低減させる被験物質を決定する工程を含む、(16)に記載のスクリーニング方法。
(18) 前記ヘルパーT細胞選択的活性抑制剤が、ヘルパーT細胞に発現するスフィンゴ糖脂質の産生量を抑制する、(16)に記載の方法。
(19) 前記スフィンゴ糖脂質がガングリオシドである、(18)に記載の方法。
(19a) 前記ガングリオシドがGM3である、(18)に記載の組成物。
(20) 前記ヘルパーT細胞選択的活性抑制剤が、GM3合成酵素阻害剤又はGM3合成酵素の発現を阻害する物質である、(16)に記載の方法。
(20a) 前記ヘルパーT細胞選択的活性抑制剤が、GM3合成酵素の発現を阻害するアンチセンス核酸、dsRNAまたはリボザイムである、(20)に記載の組成物。
(21)ヘルパーT細胞におけるスフィンゴ糖脂質の産生を制御することによりヘルパーT細胞の免疫機能を選択的に制御する方法。
(22) ヘルパーT細胞選択的活性抑制剤のスクリーニング方法であって、以下:
(i) ヘルパーT細胞と、グルコシルセラミド合成酵素阻害剤D-PDMPおよびガングリオシドGM3とを接触させる工程、
(ii) ヘルパーT細胞と、グルコシルセラミド合成酵素阻害剤D-PDMP、ガングリオシドGM3および被験物質とを接触させる工程、ならびに
(iii)上記(i)と(ii)との細胞増殖を比較し、ヘルパーT細胞の増殖を抑制する被験物質を決定する工程
を含む、スクリーニング方法。
(23) (v) キラーT細胞と上記被験物質とを、キラーT細胞が増殖できる条件下で接触させる工程、および
(vi) キラーT細胞の細胞増殖には影響しない被験物質を決定する工程、
をさらに含む、(22)に記載のスクリーニング方法。
(24) ヘルパーT細胞選択的活性抑制剤のスクリーニング方法であって、以下:
(i) ヘルパーT細胞と、被験物質としてGM3合成酵素発現阻害ポリヌクレオチドとを接触させる工程、および
(ii) ヘルパーT細胞を増殖させない被験物質を決定する工程、
を含む、スクリーニング方法。
(25) ヘルパーT細胞選択的活性抑制剤のスクリーニング方法であって、以下:
(i) SAT-I遺伝子のプロモーターの下流にレポーター遺伝子を発現するよう組み換えられた哺乳動物細胞を作製する工程、
(ii)上記遺伝子組み換え細胞が増殖する条件下で、その細胞と被験物質とを接触させる工程、および
(iii)レポーター遺伝子の発現量を低減させる被験物質を決定する工程
を含む、スクリーニング方法。
(26) ヘルパーT細胞選択的活性抑制剤のスクリーニング方法であって、以下:
(i) ヘルパーT細胞と、被験物質とを接触させる工程、
(ii) 上記細胞の細胞膜における、GM3、GM2、GM1、GD1aおよびGT1a(a-シリーズ)、GD3、GD2、GD1b、GT1bおよびGQ1b(b-シリーズ)、ならびにGT3、GT2、GT1c、GQ1cおよびGP1c(c-シリーズ)からなる群より選択される少なくとも1つのガングリオシド量を測定する工程、ならびに
(iii)少なくとも1つのガングリオシド量を低減させる被験物質を選択する工程
を含む、スクリーニング方法。
(27) 抗CD3抗体と抗CD28抗体との組合せ、スーパー抗原または同系異種抗原サブセットの共存下では上記細胞が増殖せず、IonomycinとPMAとの組合せの共存下では上記細胞が増殖する被験物質を決定する工程をさらに含む、上記(22)~(26)のいずれか1項に記載のスクリーニング方法。
本発明は、ヘルパーT細胞選択的活性抑制剤を含有する、免疫抑制活性、抗喘息活性及び/又は抗アレルギー活性を有する医薬組成物を提供する。
本発明はさらに、ヘルパーT細胞の選択的活性抑制能を測定する工程を含む、ヘルパーT細胞選択的活性抑制剤、すなわち免疫抑制活性、抗喘息作用及び/又は抗アレルギー作用を示す物質のスクリーニング方法を提供する。このようなスクリーニング方法としては、インビトロ及び/又はインビボで実施する方法が挙げられる。
ガングリオシドのin vivoにおける免疫機能における役割を解析するために、卵白アルブミン(OVA)に対する喘息・アレルギー反応モデルを用いて、GM3合成酵素遺伝子欠損マウス(SAT-I KOマウス)の喘息・アレルギー反応を検討した。その結果、アレルギー性免疫応答および気道炎症の惹起にはGM3および関連ガングリオシドの関与が証明され、ガングリオシドはヘルパーT細胞の機能に必須であることが強く示唆された。
SAT-I KOマウスの作製に用いたターゲティングベクターの模式図およびKOマウスにおける遺伝子欠損の確認をそれぞれ図2Aおよび図2Bに示す。図2Aの一番上にtargetting vector、2番目に野生型の遺伝子、3番目に変異体の遺伝子を示す。また、図2Bでは、PCRによるSAT-I ジェノタイプの決定を示す。野生型のSAT-I遺伝子アレル(エキソン2)の同定には、5’-GGAATCCATCCCTTTTCTCACAGAG-3(配列番号:5)と 5’-TGAACTCACTTGGCATTGCTGG-3’(配列番号:6)をプライマーとして用いた。SAT-I ノックアウトの確認には、挿入されたネオマイシン耐性遺伝子を、5’-GGAATCCATCCCTTTTCTCACAGAG-3’(配列番号:7) と 5’-TGAACTCACTTGGCATTGCTGG-3’(配列番号:8)のプライマーをもちいた。図2 (c)において、脳ガングリオシドのTLC分析の結果を示す。野生型マウス(+/+)およびヘテロマウス(+/-)では、GM1, GD1a, GD1b, GT1bなどが発現していたが、ノックアウトマウス(-/-)では、これらが全て消失し、代償的にGM1bおよびGD1aが発現していた(図1を参照)。
SAT-I KOマウスにおける、卵白アルブミン(OVA)感作後のOVA吸入による喘息・アレル
ギー反応を検証した。コントロール(野生型)マウス(+/+)およびSAT-I KOマウス(-/-)に腹腔内投与にてOVAで感作し、12日後にOVAを吸入させることによって喘息・アレルギー反応を惹起させた。喘息・アレルギー反応の評価は、抗原吸入後5日目に肺洗浄液を採取してそこに含まれる炎症細胞数を計測することにより行った(図3)。その結果、SAT-I KOマウスでは、気道への炎症細胞数がコントロールマウスと比較して約50%以下に減少しており、なかでもリンパ球および好酸球の浸潤が約30%までに減少していた(*P<0.05)。さらに抗原吸入後5日目において、コントロール(野生型)マウス(+/+)およびSAT-I KOマウス(-/-)の気道粘膜をPAS染色により染色した。染色の結果、SAT-I KOマウスの気道粘膜は、コントロールマウスよりも明確に減少していた(図4)。以上より、SAT-I KOマウスでは喘息・アレルギー反応に対して抵抗性であることが判明した。アレルギー局所への好酸球の浸潤はヘルパーT細胞の活性化に依存することから(喘息予防・管理ガイドライン 2006; 日本アレルギー学会監修を参照のこと)、SAT-I KOマウスではin vivoにおいてヘルパーT細胞の活性が欠如していることが考えられる。
そこで、上記の方法により作成したOVA感作後のOVA吸入による喘息・アレルギーモデルにおける血清中のOVA特異的IgE抗体をELISAにて測定した。血清は、図3に示した喘息・アレルギー反応の評価時に同じ個体から採取した。その結果、SAT-I KOマウスでは、OVA特異的IgE抗体量がコントロールマウスに比較して約50%以下まで低下していた(**P<0.01)(図5)。このことから、SAT-I KOマウスにおける喘息・アレルギー反応の低下(図3)と対応して抗原特異的IgE抗体産生の低下が見られた。このことは、抗体産生におけるIgEクラススイッチの機能低下も考えられる。
喘息・アレルギー反応の惹起や抗原特異的IgE抗体の産生は、活性化ヘルパーT細胞から産生されるIL-4、IL-5などのいわゆる2型ヘルパーTサイトカインに依存することから(アレルギー疾患 診断・治療ガイドライン 2007; 日本アレルギー学会作成を参照のこと)、上記と同様の方法により作成したOVA感作後のOVA吸入による喘息・アレルギーモデルにおける血清中のIL-4およびIL-5濃度をELISAにて測定した。血清は、図3に示した喘息・アレルギー反応の評価時に同じ個体から抗原吸入後5日目に採取した。その結果、SAT-I KOマウスでは、OVA特異的な血清中IL-4濃度およびIL-5濃度が、それぞれコントロールマウスに比較してそれぞれ約10%、約30%まで低下していた(*P<0.05)(図6)。一方、IFN-γは有意な差が見られなかった。この結果からも、SAT-I KOマウスにおける喘息・アレルギー反応の低下(図3)と対応して、抗原特異的なIL-4産生低下およびIL-5産生低下が見られた。
6.1.SAT-I KOマウス由来T細胞のTCR媒介性増殖応答について
次に、喘息・アレルギーモデルにおいて、SAT-I KOマウスは気道炎症反応、ならびに血清IgE量、IL-4量およびIL-5量が低下する機序の一連の検討を行った。T細胞抗原受容体(TCR)に対する抗体にて刺激したときの細胞増殖応答を測定したコントロールおよびSAT-I KOのT細胞をCD4陽性とCD8陽性T細胞とに分離した後、各々の細胞に無刺激、抗CD3抗体+抗CD28抗体刺激あるいはPMA+Ionomycin刺激を行い、72時間後、XTTアッセイキット(R & D systems MN USA社製、カタログ番号4891-025-K)により細胞増殖を検出した。抗CD3抗体+抗CD28抗体によりTCRと共刺激分子CD28に刺激を与えることが公知である(免疫学的プロトコール 中内 啓光 /編 羊土社)。また、ホルボールエステルでありプロテインキナーゼCの活性化剤であるPMA(例えば、Sigama-Aldrich Japan 社、カタログ番号P1585より入手可能である)、およびカルシウムイオノフォアであり細胞内カルシウム濃度を上昇させる薬剤であるIonomycin(例えば、Sigama-Aldrich Japan 社、カタログ番号I0634より入手可能である)のこれら両者は、同時使用によってTCRをバイパスした刺激を細胞に与えて細胞を増殖させる強制的な活性化剤であることが公知である(Davis L., and Lipsky PE., Signals involved in T cell activation. II. Distinct roles of intact accessory cells, phorbol esters, and interleukin 1 in activation and cell cycle progression of resting T lymphocytes. J. Immunol.136, 3558-3596, 1986)。これらの結果、SAT-I KOマウスのCD4陽性T細胞はTCR依存的な増殖応答がコントロールと比較して約30%に低下していた(図7左)。PMA+ionomycinの刺激ではコントロールと同程度の増殖応答を示したことから、SAT-I KOマウスにおいてTCR媒介性の細胞内情報伝達が欠損していることが分かる。一方、CD8陽性T細胞では、TCR媒介性の増殖応答はSAT-I KOマウスの方が強い傾向が認められた(図7右)。
野生型およびSAT-I KOマウスの足底にハプテンとしてトリニトロフェノール(TNP)結合させたOVA (TNP-OVA(200 μg))を皮下注射にて免疫した。8日後、免疫マウスより脾臓を摘出しCD4+T細胞, CD8+T細胞をそれぞれ精製した。このCD4+T細胞, CD8+T細胞に対して抗原提示細胞+OVA抗原にて72時間再刺激を行うことによって、in vitroにおける抗原特異的なT細胞の増殖反応を検討した(図8)。その結果、SAT-I KOマウス由来のCD4+T細胞でのみ、抗原特異的反応が欠如していることが明らかとなった。
そこで、SAT-I KOマウスにおけるヘルパーT細胞の選択的機能低下がスーパー抗原によっても認められるか否かを検討した。スーパー抗原としてTCR Vb8に結合するSEB (streptococcul enterotoxin B)を用い、これを野生型およびSAT-I KOマウスの脾臓から精製したCD4+T細胞およびCD8+T細胞にそれぞれ添加した後、72時間後のT細胞の増殖反応を検討した(図9)。その結果、SAT-I KOマウス由来のCD4+T細胞でのみ、スーパー抗原特異的反応が欠如していることが明らかとった。
次に、アロ抗原(同種異系抗原)に対するT細胞免疫応答を検討するため混合リンパ球試験(MLR: mixed lymphocyte reaction)を実施した(図10)。Responder細胞として野生型およびSAT-I KOマウス(ともにC57BL/6系統)の脾臓から精製したCD4+T細胞およびCD8+T細胞を用い、Stimulator細胞としてBalb/c系統マウスの脾臓細胞を採取してマイトマイシン処理にて不活性化したものを準備した。Responder(3 x105個)とStimulator(6 x105個)を混合培養し、72時間後の増殖反応およびサイトカイン産生を検定した。その結果、SAT-I KOマウス由来のCD4+T細胞でのみ、アロ抗原特異的反応が欠如していることが明らかとなった(図10)。
以上より、SAT-I KOマウスでは、ヘルパーT細胞の機能が選択的に低下していること、および対照的にキラーT細胞の免疫機能は正常であることを実証した。すなわち、ガングリオシドはT細胞サブセットのなかでヘルパーT細胞による免疫機能に対して不可欠な要素であることを示した。
7.1.GM3添加による回復
SAT-I KOマウスのヘルパーT細胞に対してそれが欠如するガングリオシドGM3を外来性に補充したときの細胞増殖応答を測定した。コントロールおよびSAT-I KOマウスのT細胞をCD4陽性とCD8陽性T細胞とに分離した後、各々の細胞に未処理(-)あるいはGM3処理(+)を1時間行い、続いて、図1と同じ方法で細胞を刺激して細胞増殖を解析した(図11)。この結果、SAT-I KOマウスのCD4陽性T細胞におけるTCR媒介性の増殖応答の低下は、GM3の添加によって完全に回復した。
図12にSAT-I KOマウスのヘルパーおよびキラーT細胞にSAT-I遺伝子を再導入したときの細胞増殖応答を示す。コントロールおよびSAT-I遺伝子欠損マウスのT細胞をCD4陽性とCD8陽性T細胞とに分離した後、各々の細胞にアデノウイルスを介してSAT-I遺伝子あるいはLacZ遺伝子(実験コントロール)を導入あるいは未処理(-)した後、続いて、図1と同じ方法で細胞を刺激して細胞増殖を解析した(Miyake S. et al., Efficient generation of recombinant adenoviruses using adenovirus DNA-terminal protein complex and a cosmid bearing the full-length virus genome. Proc. Natl. Acad. Sci. USA 93, 1320-1324, 1996)。その結果、 SAT-I KOマウスのCD4陽性T細胞におけるTCR媒介性の増殖応答の低下は、SAT-I遺伝子の再導入によってのみ完全に回復した。また、CD8陽性T細胞にSAT-I遺伝子を導入した場合には、全く変化は認められなかった。
以上の結果より、SAT-I KOマウスでは、ヘルパーT細胞が選択的に機能不全に陥っており、SAT-I活性の遺伝子導入による回復またはSAT-I生成物であるGM3の補充によって、ヘルパーT細胞の抗原特異的細胞増殖が可能であった。したがって、これらヘルパー細胞特異的な機能不全が、SAT-IによるGM3合成活性単独の欠失によるものであることを確認した。
8.1.目的
前述のように、SAT-I遺伝子ノックアウトマウスは、a-シリーズ、b-シリーズおよびc-シリーズのガングリオシを完全に欠損している(図1)。その結果として、ヘルパーT細胞特異的にTCR媒介性増殖応答が低下し、種々の免疫応答の低下が認められた。現在のところ、このSAT-I合成酵素、またはGM3合成経路に着目してたヘルパーT細胞特異的な免疫抑制物質は報告されていない。そこで、本明細書中に記載されるようなSAT-I活性が欠失または低下した実験系を利用することにより、過剰な免疫応答、喘息、アレルギー反応などに対して有効な薬理物質の同定できる、以下のスクリーニング方法を構築した。
スフィンゴ糖脂質生合成阻害剤D-PDMP (D-threo-1-phenyl-2-decanoylamiono-3-morpholino-1-prppanol・HCl,製品名 D-threo-PDMP, メーカー:Matreya,Inc. カタログ番号:#1756)、D-PPMP (D-threo-1-phenyl-2-hexadecanoylamiono-3-morpholino-1-prppanol・HCl, メーカー:Matreya, Inc. カタログ番号:#1865)、D-PBPP (D-threo-1-phenyl-2-benzyloxycarbonylamino-3-pyrrolidinoamino-1-prppanol・HCl: Jimbo et al., J. Biochem. 127 485-491, 2000)、D-threo-3',4'-Ethylenedioxy-P4 (D-threo-3', 4'-Ethylenedioxy 1-phenyl-2-palmitoylamino-3-pyrrolidino-1-propanol, 文献名;Lee, L., et al. J Biol Chem. 274, 14662-14669, 1999)などの類縁化合物や別の基本骨格を有するAMP-DNM (N-(5-adamantane-1-yl-methoxy)-pentyl-1-deoxynojirimycin, Diabetes 56, 1341-1349, 2007)は、スフィンゴ糖脂質生合成の中でグルコシルセラミド(GlcCer)合成酵素を選択的に阻害し、すべてのガングリオシドの生成を抑制する(図1左上を参照のこと)。従って、D-PDMPなどのGlcCer合成酵素阻害剤で処理した場合、CD4陽性T細胞およびCD8陽性T細胞のどちらの機能も抑制することなる(Nagafuku,M., et al., J. Biol. Chem. 278, 51920-51927, 2003. ; Blank N. et al., Biochem. Pharmacol. 126-135, 2005)。そこで、CD4陽性T細胞の機能を選択に抑制する物質をスクリーニングできることを明確にするために、GlcCer合成酵素阻害剤で全T細胞を処理後、または処理と同時にGM3を培養液中に添加することで、CD4陽性T細胞の機能(増殖能)の回復を確認する。この時、GlcCer合成酵素阻害剤はCD8陽性T細胞の機能も抑制するが、GM3を加えてもCD8陽性T細胞の増殖能は回復しないことになる。
スクリーニング系1(インビトロ系)の構築
実験手順
野生型マウスのリンパ節細胞よりCD4T陽性細胞を精製し、抗体96穴プレートに2x10^5個の細胞を播種し、無刺激、抗CD3抗体+抗CD28抗体刺激あるいはPMA+ Ionomycin刺激を行う。刺激と同時にグルコシルセラミド(GlcCer)合成酵素阻害剤(例えば、D-PDMP(10μM))およびGM3(2μg/ml)について両方あるいは片方の添加あるいは両方無添加を行う。約72時間後、細胞の増殖応答をXTTアッセイにて評価する。この実験系において、糖脂質を枯渇した際、CD4T細胞の活性化に伴う細胞増殖が抑制されるが、GM3を共存させることで、D-PDMPによる増殖抑制効果が解除されることになるので、ヘルパーT細胞の活性を抑制する有効な化合物のスクリーニング法が確立される。
スクリーニング系2(インビボ系)の構築
実験手順
コントロール(野生型)マウス(+/+)およびSAT-I KOマウス(-/-)を腹腔内投与にてOVAで2回感作し、数日後にOVAを吸入させることによって喘息・アレルギー反応を惹起さる。グルコシルセラミド合成酵素阻害剤(例えば、D-PDMP)を5% Tween 80に10 mg/mlなどの濃度で溶解し、GM3を併用する場合のみCD4陽性T細胞が増殖するような条件で、例えば75 mg/kg を一日2回をOVAを吸入前から継続して6日間連続して腹腔内投与する。喘息・アレルギー反応の評価は、上記SAT-I KOマウスを用いて行ったように、肺洗浄液に含まれる炎症細胞数およびサイトカイン量の計測と肺組織の気道粘液(喀痰の主成分)染色、血清中の抗OVA特異的IgE量測定など、上記の種々の方法により行うことができる。
Claims (21)
- ヘルパーT細胞選択的活性抑制剤を含有する、免疫抑制活性、抗喘息活性及び/又は抗アレルギー活性を有する医薬組成物。
- 前記ヘルパーT細胞選択的活性抑制剤が、ヘルパーT細胞の増殖を選択的に抑制する物質である、請求項1に記載の組成物。
- 前記ヘルパーT細胞選択的活性抑制剤が、ヘルパーT細胞におけるスフィンゴ糖脂質の産生を選択的に抑制する物質である、請求項2に記載の組成物。
- 前記スフィンゴ糖脂質がガングリオシドである、請求項3に記載の組成物。
- 前記ヘルパーT細胞選択的活性抑制剤がGM3に対する抗体を含む、請求項4に記載の組成物。
- 前記ヘルパーT細胞選択的活性抑制剤が、GM3合成酵素阻害剤である、請求項1~4のいずれかに記載の組成物。
- 前記ヘルパーT細胞選択的活性抑制剤が、GM3合成酵素の発現を阻害する、アンチセンス核酸、リボザイムまたはRNAi効果を有するRNAである、請求項1~4のいずれかに記載の組成物。
- ヘルパーT細胞の選択的活性抑制能を測定する工程を含む、免疫抑制活性、抗喘息作用及び/又は抗アレルギー作用を示す物質のスクリーニング方法。
- 被験物質を、ヘルパーT細胞に接触させ、ヘルパーT細胞における細胞増殖を測定する、免疫抑制作用、抗喘息作用及び/又は抗アレルギー作用を示す物質のスクリーニング方法。
- 被験物質を、ヘルパーT細胞に接触させ、ヘルパーT細胞におけるスフィンゴ糖脂質の産生量を測定する、免疫抑制作用、抗喘息作用及び/又は抗アレルギー作用を示す物質のスクリーニング方法。
- 前記スフィンゴ糖脂質がガングリオシドである、請求項10に記載の方法。
- 被験物質を、ヘルパーT細胞に接触させ、ヘルパーT細胞におけるGM3合成酵素の発現量を測定する、免疫抑制作用、抗喘息作用及び/又は抗アレルギー作用を示す物質のスクリーニング方法。
- ヘルパーT細胞の細胞増殖活性を有することが確認された被験物質を、免疫疾患、喘息及び/又はアレルギー反応を示す非ヒト動物に投与する工程を含む、請求項8~12のいずれかに記載の方法。
- 前記非ヒト動物がマウスである、請求項13に記載の方法。
- 前記非ヒト動物の炎症部位におけるマクロファージ、好中球、好酸球及び/又はリンパ球の浸潤細胞数を対照と比較する工程をさらに含む、請求項13に記載の方法。
- ヘルパーT細胞選択的活性抑制剤の有効量を患者に投与することによりヘルパーT細胞の免疫機能を選択的に制御する、免疫疾患、喘息及び/又はアレルギーの治療方法。
- 前記ヘルパーT細胞選択的活性抑制剤が、ヘルパーT細胞の増殖を抑制する、請求項16に記載の方法。
- 前記ヘルパーT細胞選択的活性抑制剤が、ヘルパーT細胞に発現するスフィンゴ糖脂質の産生量を抑制する、請求項16に記載の方法。
- 前記スフィンゴ糖脂質がガングリオシドである、請求項18に記載の方法。
- 前記ヘルパーT細胞選択的活性抑制剤が、GM3合成酵素阻害剤又はGM3合成酵素の発現を阻害する物質である、請求項16に記載の方法。
- ヘルパーT細胞におけるスフィンゴ糖脂質の産生を制御することによりヘルパーT細胞の免疫機能を選択的に制御する方法。
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US13/126,140 US8691514B2 (en) | 2008-10-31 | 2009-10-30 | Method for selective control of helper T cell function |
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WO2011162115A1 (ja) * | 2010-06-21 | 2011-12-29 | 国立大学法人東京工業大学 | 膵臓β細胞増殖因子を利用した医薬組成物 |
WO2017094705A1 (ja) * | 2015-11-30 | 2017-06-08 | 公益財団法人野口研究所 | Gm3促進炎症抑制剤及び炎症性サイトカイン産生抑制剤 |
WO2020085414A1 (ja) * | 2018-10-25 | 2020-04-30 | 学校法人 麻布獣医学園 | グルコシルセラミド合成酵素遺伝子欠損t細胞の使用およびその治療的利用 |
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EP3600967B1 (en) | 2017-03-30 | 2021-01-20 | Volvo Truck Corporation | Mirror assembly |
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Cited By (6)
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WO2011162115A1 (ja) * | 2010-06-21 | 2011-12-29 | 国立大学法人東京工業大学 | 膵臓β細胞増殖因子を利用した医薬組成物 |
JPWO2011162115A1 (ja) * | 2010-06-21 | 2013-08-19 | 国立大学法人東京工業大学 | 膵臓β細胞増殖因子を利用した医薬組成物 |
WO2017094705A1 (ja) * | 2015-11-30 | 2017-06-08 | 公益財団法人野口研究所 | Gm3促進炎症抑制剤及び炎症性サイトカイン産生抑制剤 |
US10849926B2 (en) | 2015-11-30 | 2020-12-01 | The Noguchi Institute | GM3-promoted inflammation inhibitor and inflammatory cytokine production inhibitor |
WO2020085414A1 (ja) * | 2018-10-25 | 2020-04-30 | 学校法人 麻布獣医学園 | グルコシルセラミド合成酵素遺伝子欠損t細胞の使用およびその治療的利用 |
CN112912503A (zh) * | 2018-10-25 | 2021-06-04 | 学校法人麻布兽医学园 | 葡萄糖神经酰胺合成酶基因缺失t细胞的用途和其治疗应用 |
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CA2741664C (en) | 2014-12-23 |
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