WO2011071059A1 - Inhibiteur d'activation des lymphocytes t, composition pharmaceutique contenant celui-ci, et procédé de criblage pour une substance inhibant l'activation des lymphocytes t - Google Patents

Inhibiteur d'activation des lymphocytes t, composition pharmaceutique contenant celui-ci, et procédé de criblage pour une substance inhibant l'activation des lymphocytes t Download PDF

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WO2011071059A1
WO2011071059A1 PCT/JP2010/071965 JP2010071965W WO2011071059A1 WO 2011071059 A1 WO2011071059 A1 WO 2011071059A1 JP 2010071965 W JP2010071965 W JP 2010071965W WO 2011071059 A1 WO2011071059 A1 WO 2011071059A1
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rgm
cell activation
rgma
cells
antibody
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PCT/JP2010/071965
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Japanese (ja)
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山下　俊英
武一 久保
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国立大学法人大阪大学
国立大学法人千葉大学
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First worldwide family litigation filed litigation Critical https://patents.darts-ip.com/?family=44145603&utm_source=google_patent&utm_medium=platform_link&utm_campaign=public_patent_search&patent=WO2011071059(A1) "Global patent litigation dataset” by Darts-ip is licensed under a Creative Commons Attribution 4.0 International License.
Priority to KR1020127017515A priority Critical patent/KR20120120193A/ko
Priority to US13/514,915 priority patent/US9334323B2/en
Priority to KR1020217023891A priority patent/KR102425676B1/ko
Priority to KR1020177024100A priority patent/KR20170102378A/ko
Priority to JP2011545222A priority patent/JP5785873B2/ja
Application filed by 国立大学法人大阪大学, 国立大学法人千葉大学 filed Critical 国立大学法人大阪大学
Priority to KR1020217005837A priority patent/KR102284780B1/ko
Priority to CN2010800633729A priority patent/CN102811739A/zh
Priority to EP10835979.5A priority patent/EP2510948B1/fr
Priority to ES10835979.5T priority patent/ES2555060T3/es
Priority to KR1020197027496A priority patent/KR102223464B1/ko
Publication of WO2011071059A1 publication Critical patent/WO2011071059A1/fr
Priority to US15/149,314 priority patent/US9751938B2/en

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Definitions

  • the present invention relates to a T cell activation inhibitor, a pharmaceutical composition containing the same, and a method for screening a T cell activation inhibitor, and in particular, inhibits T cell activation containing an RGM inhibitor as an active ingredient. And a method for screening a T cell activation inhibitor comprising a pharmaceutical composition for preventing or treating a disease caused by T cell activation containing the T cell activation inhibitor, and a step of bringing the test substance into contact with RGM It is.
  • RGM (repulsive guidance molecule) is a membrane protein initially identified as a visual axon guidance molecule (see Non-Patent Document 1).
  • the RGM family includes three types of members called RGMa, RGMb, and RGMc, and at least RGMa and RGMb are known to work in the same signal transduction mechanism (see Non-Patent Document 2).
  • RGM has functions such as axonal guidance and lamina formation in xenopaths and chicken embryos, and control of cranial neural tube closure in mouse embryos (Non-Patent Document 3). reference).
  • Patent Document 1 discloses an axonal regeneration promoter containing an anti-RGM neutralizing antibody as an active ingredient.
  • Multiple Sclerosis is a visual, motor, and sensory decline because myelin (myelin sheath) that covers nerve fibers in the brain and spinal cord is inflamed and demyelinating, and nerve information cannot be transmitted well. It is a disease that causes various symptoms such as balance disorder, a chronic disease whose cause has not been clarified yet and cannot be completely cured by modern medicine. Although it is recognized as one of the autoimmune diseases, details of the onset mechanism have not been elucidated. For example, Non-Patent Document 5 reports that CD4 + T cells immunologically attack myelin and oligodendrocytes in the white matter of the brain and spinal cord.
  • the present invention finds a novel function of RGM, and provides a novel T cell activation inhibitor, a pharmaceutical composition for preventing or treating a disease caused by T cell activation, and a screening method for a T cell activation inhibitor. For the purpose.
  • a T cell activation inhibitor comprising an RGM inhibitor as an active ingredient.
  • a pharmaceutical composition for preventing or treating a disease caused by T cell activation comprising the T cell activation inhibitor according to any one of [1] to [3].
  • [5] The pharmaceutical composition according to the above [4], wherein the disease caused by T cell activation is an autoimmune disease.
  • the pharmaceutical composition according to the above [5], wherein the autoimmune disease is multiple sclerosis.
  • a step of bringing a test substance into contact with RGM, a step of measuring the RGM activity level, a step of comparing the activity level with an activity level of RGM not in contact with the test substance, and an RGM activity level A method for screening a T cell activation inhibitor, which comprises the step of selecting a test substance that reduces the amount of the test substance.
  • a method for inhibiting T cell activation comprising a step of bringing an RGM inhibitor into contact with an RGM-expressing cell.
  • a novel T cell activation inhibitor can be provided.
  • the T cell activation inhibitor is useful for preventing or treating diseases caused by T cell activation, such as autoimmune diseases.
  • the T cell activation inhibitor obtained by the screening method of the present invention is a candidate for an active ingredient of a prophylactic or therapeutic agent for diseases caused by T cell activation.
  • (A) is a figure which shows the clinical symptom incidence rate of EAE in each group
  • (B) is a figure which shows the average value of the EAE onset day in each group
  • (C) is an EAE score of each mouse
  • (D) is a figure which shows the average value of the EAE score cumulative value of each mouse
  • (A) and (b) are HE-stained histological images of the cervical spinal cord of a multiple sclerosis model mouse administered with a control antibody
  • (c) and (d) are multiple sclerosis models administered with an anti-RGMa neutralizing antibody. It is a HE-stained tissue image of the mouse cervical spinal cord.
  • FIG. Antigen-specific and non-specific T cell activation of spleen cells prepared from multiple sclerosis model mice administered with control antibody and multiple sclerosis model mice administered with anti-RGMa neutralizing antibody was examined.
  • A is the cell growth
  • (b) is the secretion amount of IL-2
  • (c) is the secretion amount of IFN- ⁇
  • (d) is the secretion amount of IL-17.
  • the figure which shows the result of having digitized the clinical symptom of EAE in the multiple sclerosis model mouse which administered the anti-RGMa neutralizing antibody and the multiple sclerosis model mouse which administered the control antibody, and observed the change over time. is there.
  • MOG stimulation was performed on BMDCs with RGMa gene knockdown or BMDCs without RGMa gene knockdown, these BMDCs were transplanted into recipient mice, and the clinical symptoms of EAE in the recipient mice were quantified by score, It is a figure which shows the result of having observed the change with time.
  • the present inventors found for the first time that RGM is expressed in bone marrow-derived dendritic cells (BMDCs), and that RGM receptor is expressed in CD4 + T cells and CD11b + macrophages, and RGM is CD4 + T. It has been found that Rap1 is activated by binding to RGMa receptors of cells and CD11b + macrophages, and as a result, cell adhesion activity of CD4 + T cells and CD11b + macrophages is enhanced. In addition, in the experiment using the multiple sclerosis model mouse, the present inventors administered the anti-RGM neutralizing antibody to the multiple sclerosis model mouse (experimental) induced by myelin oligodendrocyte glycoprotein (MOG).
  • MOG myelin oligodendrocyte glycoprotein
  • EAE autoimmune encephalomyelitis
  • T cell activation inhibitor The present invention provides a T cell activation inhibitor containing an RGM inhibitor as an active ingredient.
  • the RGM inhibitor which is an active ingredient of the T cell activation inhibitor of the present invention may be either a substance that inhibits RGM activity (RGM activity) or a substance that inhibits RGM expression.
  • Examples of substances that inhibit RGM activity include low molecular weight compounds that inhibit RGM activity, and anti-RGM neutralizing antibodies.
  • RGM activity indicators include RGM binding activity to the RGM receptor (see (1-2) in Example 1), Rap1 activation inducing activity (see (1-3) in Example 1), Examples include an activity for enhancing the adhesion of T cells to ICAM (see (1-4) in Example 1) and the like, and an RGM inhibitory substance that inhibits (reduces) RGM activity when evaluated using these indicators. Called.
  • Examples of low molecular weight compounds that inhibit RGM activity include Y27632 (M.32Uehata, et al. Calcium sensitization of smooth muscle mediated by a Rho-associated protein kinase in hypertension. Nature 389, 990, which is known as a Rho kinase inhibitor. (1997)).
  • the anti-RGM neutralizing antibody may be an antibody that binds to RGM and inhibits its activity, and examples thereof include an anti-RGM antibody that binds to RGM and prevents RGM from binding to the RGM receptor.
  • An anti-RGM neutralizing antibody can be prepared by a known method using RGM or a fragment thereof as an immunogen. Whether the obtained antibody is a neutralizing antibody can be confirmed using the above-mentioned indicator of RGM activity.
  • RGM include, but are not limited to, human RGMa consisting of the amino acid sequence shown in SEQ ID NO: 2, rat RGMa consisting of the amino acid sequence shown in SEQ ID NO: 4, and the like.
  • RGMs derived from various organisms can be suitably used as the immunogen. These amino acid sequences can be easily obtained from a known database (Protein
  • the anti-RGM neutralizing antibody may be a polyclonal antibody or a monoclonal antibody. Further, it may be a complete antibody molecule or an antibody fragment (for example, Fab, F (ab ′) 2 , Fab ′, Fv, scFv, etc.) that can specifically bind to an antigen.
  • a polyclonal antibody can be prepared and obtained, for example, as follows. That is, a mammal (mouse, rat, rabbit, goat, horse, etc.) is prepared by dissolving an antigen (RGM or a fragment thereof) in PBS and mixing an appropriate amount of a normal adjuvant (for example, Freund's complete adjuvant) if necessary. Immunize.
  • the immunization method is not particularly limited, for example, a method of subcutaneous injection or intraperitoneal injection once or a plurality of times at appropriate intervals is preferable.
  • blood can be collected from the immunized animal, serum can be separated, and the polyclonal antibody fraction can be purified.
  • a monoclonal antibody can be obtained by fusing immune cells (eg, spleen cells) obtained from the immunized mammal with myeloma cells to obtain a hybridoma, and collecting the antibody from the hybridoma culture.
  • an antibody gene can be cloned from a hybridoma, incorporated into an appropriate vector, introduced into a host cell, and a recombinant monoclonal antibody can be produced using gene recombination techniques. Furthermore, it can also be prepared using a phage display method.
  • the anti-RGM neutralizing antibody is preferably a human chimeric antibody or a humanized antibody.
  • a human chimeric antibody refers to an antibody comprising a heavy chain variable region and a light chain variable region of an antibody derived from a non-human animal, and a heavy chain constant region and a light chain constant region of a human antibody.
  • a humanized antibody is obtained by grafting a CDR (complementarity determining region) of an antibody derived from a non-human animal into a CDR of a human antibody, and is also referred to as a CDR-grafted antibody, a reconstituted antibody, or the like.
  • the FR (framework region) of the humanized antibody is selected so that CDR forms a favorable antigen-binding site.
  • amino acid sequence of the FR in the variable region of the antibody may be substituted so that the CDR of the humanized antibody forms an appropriate antigen-binding site.
  • the amino acid sequence of the constant region of a human antibody can be obtained from a known database (Protein Data Bank etc.).
  • RGM gene siRNA short interfering RNA
  • shRNA short hairpin RNA
  • antisense oligonucleotides examples include RGM gene siRNA (short interfering RNA), shRNA (short hairpin RNA), and antisense oligonucleotides.
  • RGM gene examples include, but are not limited to, a human RGMa gene consisting of the base sequence shown in SEQ ID NO: 1, a rat RGMa gene consisting of the base sequence shown in SEQ ID NO: 3, and the like.
  • the base sequences of RGM genes derived from various organisms can be easily obtained from known databases (GenBank etc.).
  • siRNA is a short double-stranded RNA that can suppress the expression of a target gene (in the present invention, an RGM gene).
  • the base sequence and length are not particularly limited as long as they function as siRNA, but are preferably less than about 30 bases, more preferably about 19 to 27 bases, and further preferably about 21 to 25 bases.
  • shRNA is a single-stranded RNA that contains a partially palindromic base sequence, so that it has a double-stranded structure in the molecule and a short hairpin structure having a protruding portion at the 3 ′ end. It refers to the above molecules. After such shRNA is introduced into the cell, it is degraded into a length of about 20 bases (typically, for example, 21 bases, 22 bases, 23 bases) in the cell and becomes a target in the same manner as siRNA.
  • siRNA and shRNA may be in any form as long as it can suppress the expression of the RGM gene.
  • siRNA or shRNA can be artificially chemically synthesized.
  • antisense and sense RNA can be synthesized in vitro from template DNA using T7 RNA polymerase and T7 promoter.
  • the antisense oligonucleotide may be any nucleotide that is complementary to or hybridizes to a continuous 5 to 100 nucleotide sequence in the DNA sequence of the RGM gene, and may be either DNA or RNA. Moreover, it may be modified as long as the function is not hindered.
  • Antisense oligonucleotides can be synthesized by conventional methods, for example, can be easily synthesized by a commercially available DNA synthesizer.
  • the RGM inhibitor can inhibit T cell activation by contacting with an RGM-expressing cell (a cell expressing RGM or a cell having RGM-expressing ability). Therefore, a method for inhibiting T cell activation, comprising the step of bringing an RGM inhibitor into contact with an RGM-expressing cell is also included in the present invention.
  • the present invention provides a pharmaceutical composition for preventing or treating a disease caused by T cell activation, comprising the above T cell activation inhibitor of the present invention.
  • the pharmaceutical composition of the present invention is a pharmaceutical composition for preventing or treating a disease caused by T cell activation containing an RGM inhibitor as an active ingredient.
  • Examples of diseases caused by T cell activation include autoimmune diseases and cerebrovascular disorders.
  • Examples of autoimmune diseases include cellular autoimmune diseases, rheumatism, multiple sclerosis, neuroautoimmune diseases (Guillain Barre syndrome, neuro-Behcet's disease, etc.), pernicious anemia, type I (insulin-dependent) diabetes, systemic lupus erythematosus (SLE), inflammatory bowel disease (IBD), Sjogren's syndrome, atopic dermatitis, Goodbascher syndrome, Graves' disease, autoimmune hemolytic anemia, autoimmune thrombocytopenic purpura, glomerulonephritis, severe muscle Examples include asthenia and Hashimoto's disease.
  • Cerebrovascular disorders include cerebral infarction, cerebral hemorrhage, subarachnoid hemorrhage and the like.
  • the pharmaceutical composition of the present invention is preferably used for prevention or treatment of autoimmune diseases, and particularly preferably used for prevention or treatment of multiple sclerosis.
  • the pharmaceutical composition of the present invention can be formulated by appropriately blending a pharmaceutically acceptable carrier or additive with an RGM inhibitor as an active ingredient.
  • oral preparations such as tablets, coated tablets, pills, powders, granules, capsules, solutions, suspensions, emulsions; parenterals such as injections, infusions, suppositories, ointments, patches, etc. can do.
  • What is necessary is just to set suitably about the mixture ratio of a carrier or an additive based on the range normally employ
  • Carriers or additives that can be blended are not particularly limited.
  • various carriers such as water, physiological saline, other aqueous solvents, aqueous or oily bases; excipients, binders, pH adjusters, disintegrants, absorption
  • Various additives such as an accelerator, a lubricant, a colorant, a corrigent, and a fragrance are included.
  • RGM inhibitor is an anti-RGM neutralizing antibody
  • a parenteral route of administration such as intravenous, intramuscular, intradermal, intraperitoneal, It is preferably administered subcutaneously or locally.
  • An injection or infusion containing an anti-RGM neutralizing antibody can be used as a solution, suspension or emulsion.
  • the solvent include distilled water for injection, physiological saline, glucose solution and isotonic solution (for example, solutions of sodium chloride, potassium chloride, glycerin, mannitol, sorbitol, boric acid, borax, propylene glycol, etc.) Can be used.
  • this injection or infusion may contain stabilizers, solubilizers, suspending agents, emulsifiers, soothing agents, buffers, preservatives, preservatives, pH adjusters and the like.
  • a stabilizer for example, albumin, globulin, gelatin, mannitol, glucose, dextran, ethylene glycol, propylene glycol, ascorbic acid, sodium bisulfite, sodium thiosulfate, EDTA sodium, sodium citrate, dibutylhydroxytoluene, etc. should be used. Can do.
  • solubilizers include alcohols (eg, ethanol), polyalcohols (eg, propylene glycol, polyethylene glycol, etc.), nonionic surfactants (eg, polysorbate 80 (registered trademark), HCO-50, etc.) Etc.
  • alcohols eg, ethanol
  • polyalcohols eg, propylene glycol, polyethylene glycol, etc.
  • nonionic surfactants eg, polysorbate 80 (registered trademark), HCO-50, etc.
  • Etc. can be used.
  • the suspending agent for example, glyceryl monostearate, aluminum monostearate, methyl cellulose, carboxymethyl cellulose, hydroxymethyl cellulose, sodium lauryl sulfate and the like can be used.
  • emulsifier for example, gum arabic, sodium alginate, tragacanth and the like can be used.
  • the soothing agent for example, benzyl alcohol, chlorobutanol, sorbitol and the like can be used.
  • the buffer for example, phosphate buffer, acetate buffer, borate buffer, carbonate buffer, citrate buffer, Tris buffer, and the like can be used.
  • Preservatives include, for example, methyl paraoxybenzoate, ethyl paraoxybenzoate, propyl paraoxybenzoate, butyl paraoxybenzoate, chlorobutanol, benzyl alcohol, benzalkonium chloride, sodium dehydroacetate, sodium edetate, boric acid, boron Sand or the like can be used.
  • preservative for example, benzalkonium chloride, paraoxybenzoic acid, chlorobutanol and the like can be used.
  • pH adjuster for example, hydrochloric acid, sodium hydroxide, phosphoric acid, acetic acid and the like can be used.
  • the RGM inhibitor is a nucleic acid (siRNA, shRNA, antisense oligonucleotide, etc.)
  • it can be administered in the form of a non-viral vector or a viral vector.
  • a method for introducing nucleic acid molecules using liposomes liposome method, HVJ-liposome method, cationic liposome method, lipofection method, lipofectamine method, etc.
  • microinjection method gene gun (Gene Gun )
  • a method of transferring a nucleic acid molecule into a cell together with a carrier (metal particle) can be used.
  • a viral vector such as a recombinant adenovirus or a retrovirus
  • DNA expressing siRNA or shRNA is added to DNA viruses or RNA viruses such as detoxified retrovirus, adenovirus, adeno-associated virus, herpes virus, vaccinia virus, pox virus, poliovirus, Sindbis virus, Sendai virus, SV40, etc.
  • a gene can be introduced into a cell or tissue by introducing and infecting the cell or tissue with this recombinant virus.
  • the preparation thus obtained can be administered, for example, to humans and other mammals (eg, rats, mice, rabbits, sheep, pigs, cattle, cats, dogs, monkeys, etc.) by administering an effective amount thereof.
  • Diseases caused by T cell activation can be prevented or treated.
  • the dose is appropriately determined in consideration of the purpose, the severity of the disease, the patient's age, weight, sex, medical history, type of active ingredient, and the like.
  • the active ingredient is an anti-RGM neutralizing antibody
  • the total daily dose may be a single dose or divided doses.
  • the screening method of the present invention includes a step of bringing a test substance into contact with RGM, a step of measuring the activity level of RGM that has contacted the test substance, and a step of comparing the measured activity level with the activity level of RGM that has not been in contact with the test substance Any method that includes a step of selecting a test substance that reduces the activity level of RGM may be used.
  • a T cell activation inhibitor can be screened simply and efficiently.
  • the contact between the test substance and the RGM can be performed, for example, by adding the test substance to the RGM solution and dissolving or suspending it.
  • the RGM a commercially available recombinant RGM or a recombinant RGM manufactured in-house by a conventional method can be used.
  • the contact time and the contact temperature are not particularly limited and may be appropriately selected.
  • RGM activity levels include RGM binding activity to the RGM receptor (see (1-2) in Example 1), Rap1 activation inducing activity (see (1-3) in Example 1), It can be measured by using, as an index, adhesion enhancing activity to ICAM (see (1-4) in Example 1).
  • the test substance is a T cell activation inhibitor by comparing the measured activity level with the activity level of RGM not in contact with the test substance. If the activity level of the RGM contacted with the test substance is low compared to the activity level of the RGM not contacted with the test substance, the test substance is judged to be a T cell activation inhibitor and is selected. That's fine.
  • the substance is determined to be a T cell activation inhibitor when the RGM activity level contacted with the test substance is 50% or less, more preferably 25% or less.
  • the T cell activation inhibitory substance obtained by the screening method of the present invention is useful as an active ingredient candidate substance for a prophylactic or therapeutic drug for diseases caused by T cell activation such as autoimmune diseases. In particular, it is extremely useful as an active ingredient candidate substance for a prophylactic or therapeutic drug for multiple sclerosis.
  • Example 1 Expression of RGMa and RGMa receptor in immune cells
  • (1-1) Expression of RGMa in bone marrow-derived dendritic cells (BMDCs) C57BL / 6 mice according to the method of Lutz et al. (Lutz, MB, et al. J. Immunol. Methods 223, 77-92 (1999).) Bone marrow cells were collected from (8 to 10 weeks old) and cultured in a medium containing GM-CSF (20 ng / mL, Sigma-Aldrich) to obtain BMDCs. In the experiment, BMDCs 6 days after the start of culture were used.
  • GM-CSF 20 ng / mL, Sigma-Aldrich
  • RGMa mRNA in BMDCs was measured by real-time quantitative PCR. That is, LPS (Sigma Aldrich) was added to BMDCs 6 days after the start of culture at 0, 0.01, 0.1 or 1 ⁇ g / mL and cultured for 24 hours to collect activated BMDCs. Total RNA was extracted from BMDCs using RNeasy Kit (Qiagen), and cDNA was obtained using reverse transcriptase (GE Healthcare). TaqMan real-time PCR (ABI Prism 7500 Sequence Detection System) was performed according to the manufacturer's protocol using the obtained cDNA as a template to quantitatively analyze RGMa mRNA expression. Specific primers and probes were purchased from Applied Biosystems.
  • BMDCs (2 ⁇ 10 6 cells) 24 hours after addition of LPS were converted into 2 ⁇ sample buffer (250 mM Tris-HCl, 4% SDS, 20% glycerol, 0.02% bromophenol blue, and 10% ⁇ -mercaptoethanol). ) And after boiling for 5 minutes, an equal amount of each sample was subjected to 15% SDS-PAGE under reducing conditions. After electrophoresis, the protein was transferred to a PVDF membrane (Immobilon-P, Millipore).
  • FIG. 1 The results of real-time PCR are shown in FIG. 1, and the results of Western blotting are shown in FIG.
  • FIG. 1 it was shown that the expression of RGMa mRNA in BMDCs was enhanced by stimulation with LPS.
  • FIG. 2 35 kDa and 50 kDa bands to which the anti-RGMa antibody binds existed, and it was shown that the intracellular amount increased by LPS stimulation.
  • the 50 kDa band corresponds to the full length of RGMa
  • 35 kDa is a C-terminal fragment of RGMa and is presumed to be a mature form of RGMa (Mueller, BK, Yamashita, T., Schaffar, G. & Mueller, R.losPhilos. Trans R. Soc. Lond. B Biol. Sci. 361, 1513-1529 (2006)). From these results, it was revealed that RGMa expression is induced in activated BMDCs.
  • RGMa-Fc C-terminal fragment of human RGMa
  • CD4 + T cells and CD11b + macrophages were isolated by positive sorting using anti-CD4 magnetic beads and anti-CD11b magnetic beads (Miltenyi Biotec), respectively.
  • the active Rap1 was measured.
  • Rap1 activation assay kit (Upstate Biotech) was used, and splenocytes before isolating CD4 + T cells and CD11b + macrophages were used as controls.
  • FIG. 4 (A) The result of Western blotting is shown in FIG. 4 (A), and a graph showing relative Rap1 activity is shown in FIG. 4 (B).
  • FIG. 4B the graph of relative activity values is represented by the mean and standard error of 3 to 4 independent experiments, and “*” represents P ⁇ 0.05 in Student's t-test.
  • the active Rap1 Pull down Rap1 in the figure
  • FIG. 4 (B) it was revealed that Rap1 of spleen cells, CD4 + T cells and CD11b + macrophages were significantly activated by RGMa stimulation for 5 minutes. . From these results, it was suggested that RGMa expressed in dendritic cells may activate intracellular Rap1 through RGMa receptors on the surface of CD4 + T cells and CD11b + macrophages.
  • the plate was washed with PBS and blocked with RPMI 1640 (Invitrogen) containing 2% BSA at 37 ° C. for 1 hour to prevent non-specific binding.
  • the plate coated with ICAM-1 / Fc was used for evaluation of CD4 + T cells, and the plate coated with fibronectin was used for evaluation of splenocytes.
  • Freshly prepared spleen cells or CD4 + T cells were added with 2.5 ⁇ M BCECF-AM (2′7′-bis- (2-carboxyethyl) -5- (and-6) -carboxyfluorescein, acetoxymethyl ester, Calbiochem) Labeled at 37 ° C. for 30 minutes, followed by washing with RPMI 1640 containing 0.5% BSA. 5 ⁇ 10 5 cells were added to the precoated plate and incubated for 1 hour at 37 ° C. in RPMI 1640 with 0.5% BSA with or without 2 ⁇ g / mL recombinant RGMa. Non-adherent cells were removed by washing 3 times with warm RPMI 1640 containing 0.5% BSA.
  • Adhesion was quantified using Spectra MAX (Molecular Devices) at an excitation wavelength of 485 nm and a fluorescence wavelength of 538 nm. The adhesion rate was expressed as a percentage of the total number of cells seeded in one well.
  • FIG. 5 (A) The results of splenocytes are shown in FIG. 5 (A), and the results of CD4 + T cells are shown in FIG. 5 (B).
  • the adhesion graph is expressed as the mean and standard error of three independent experiments, where “*” and “**” are P ⁇ 0.05 and P ⁇ 0.01, respectively, in Student's t-test. Represents.
  • FIG. 5 (A) it was shown that the splenocytes stimulated with RGMa had a significantly higher adhesion rate to fibronectin than splenocytes not stimulated with RGMa.
  • FIG. 5 (A) it was shown that the splenocytes stimulated with RGMa had a significantly higher adhesion rate to fibronectin than splenocytes not stimulated with RGMa.
  • FIG. 5 (A) it was shown that the splenocytes stimulated with RGMa had a significantly higher adhesion rate to fibronectin than splenocytes not stimulated with
  • CD4 + T cells stimulated with RGMa have a significantly higher adhesion rate to ICAM-1 than CD4 + T cells not stimulated with RGMa. It was shown to be. From these results, it was clarified that RGMa activates Rap1 to enhance the adhesion activity of splenocytes and CD4 + T cells.
  • Example 2 Examination of effect of anti-RGMa neutralizing antibody in multiple sclerosis model mouse
  • (2-1) Generation of multiple sclerosis model mice As a model animal that is widely accepted as a research model for clinical and pathological features of multiple sclerosis, myelin oligodendrocyte glycoprotein (hereinafter “myelin oligodendrocyte glycoprotein”) Experimental autoimmune encephalomyelitis (hereinafter referred to as “EAE”) mice induced by “MOG”) were used.
  • myelin oligodendrocyte glycoprotein hereinafter "myelin oligodendrocyte glycoprotein”
  • EAE Experimental autoimmune encephalomyelitis mice induced by “MOG” mice were used.
  • EAE consists of 100 ⁇ L of PBS containing 100 ⁇ g of (MOG) 35-55 peptide (MEVGWYRSPFSRVVHLYRNGK (SEQ ID NO: 5), Greiner Bio-one), and 100 ⁇ L of Freund's complete adjuvant containing 500 ⁇ g of Mycobacterium tuberculosis (H37Ra, Difco).
  • 200 ⁇ L of the emulsion was administered subcutaneously to the flank of C57BL / 6 mice (8-10 weeks old).
  • 200 ng of pertussis toxin (List Biological Laboratories) was intravenously administered after MOG administration and 48 hours later.
  • mice were euthanized, the spinal cord was removed, paraformaldehyde-fixed and paraffin-embedded sections were prepared and subjected to histological evaluation. Sections were stained with hematoxylin and eosin (HE) to assess inflammation. From the cervical spine to the thoracic spinal cord, 20-30 sections per mouse were observed, and a semi-quantitative inflammation histological evaluation (inflammatory index) was blindly performed based on the following criteria. The anti-RGMa neutralizing antibody administration group was evaluated for 5 mice, and the control antibody administration group was evaluated for 6 mice.
  • HE hematoxylin and eosin
  • the change in EAE score during the observation period is shown in FIG.
  • the EAE score was expressed as an average value and standard error.
  • the EAE score of the anti-RGMa neutralizing antibody administration group after 14 days was significantly lower than that of the control antibody administration group.
  • P ⁇ 0.05 was observed on the 14th day, and P ⁇ 0.01 after the 15th day, and a statistically significant difference was observed between the two groups.
  • FIG. 7 (A) shows the incidence of clinical symptoms of EAE in each group
  • FIG. 7 (B) shows the average value of EAE onset days in each group
  • FIG. 7 (C) shows the maximum score of each mouse in each group. The average value was shown
  • FIG. 7D shows the average score accumulated value of each mouse in each group.
  • “ns” indicates that there is no significant difference in Student's t test
  • “*” and “**” indicate that P ⁇ 0.05 and P ⁇ 0.01, respectively, in Student's t test.
  • FIG. 7 (A) all the control antibody administration groups (100%) developed clinical symptoms of EAE, whereas 44% of mice in the anti-RGMa neutralizing antibody administration group had clinical symptoms of EAE.
  • FIG. (A) and (b) are HE-stained tissue images of the control antibody administration group
  • FIG. (c) and (d) are HE-stained tissue images of the anti-RGMa neutralizing antibody administration group
  • the scale bar indicates 250 ⁇ m.
  • FIGS. 8 (a) and (b) inflammatory mononuclear cells were widely present in the bone marrow of the control antibody administration group, indicating a wide range of inflammatory lesions.
  • FIGS. 8 (c) and (d) it was shown that the bone marrow of the anti-RGMa neutralizing antibody administration group had significantly fewer inflammatory mononuclear cells and reduced inflammatory lesions.
  • FIG. 9 The evaluation results of the inflammation index are shown in FIG. In the figure, “*” indicates that P ⁇ 0.05 in Student's t-test. As is clear from FIG. 9, the inflammation index of the anti-RGMa neutralizing antibody administration group was significantly lower than that of the control antibody administration group. As described above, the results of experiments using multiple sclerosis model mice revealed that administration of an anti-RGMa neutralizing antibody alleviated both clinical symptoms and histological lesions in EAE.
  • Example 3 Examination of T cell activation in spleen cells of multiple sclerosis model mice administered with anti-RGMa neutralizing antibody
  • Removing the spleen from the multiple sclerosis model mice terminated clinical symptoms observed in Example 2 (EAE mouse) 21 days after MOG administration
  • Splenocytes were isolated, 5 ⁇ 10 5 cells / well in 96-well plates And then cultured for 3 days to analyze cell proliferation.
  • As the medium RPMI1640 medium containing glutamine, sodium pyruvate, penicillin, streptomycin, 2-ME, and 10% heat-inactivated FBS was used.
  • the splenocytes were seeded at 2 ⁇ 10 6 cells / well in a 24-well plate, and 20 ⁇ g / mL MOG peptide or 5 ⁇ g Cultured in the presence or absence of / mL anti-CD3 monoclonal antibody.
  • the culture supernatant 24 hours after the start of the culture was used for the measurement of IL-2, and the culture supernatant 72 hours after the start of the culture was used for the measurement of other cytokines. Cytokine was measured using an ELISA kit (Invitrogen) as described in the instructions.
  • FIG. (A) is a measurement result of cell proliferation
  • (b) is a measurement result of IL-2
  • (c) is a measurement result of IFN- ⁇
  • (d) is a measurement result of IL-17.
  • FIG. 10 (a) cell proliferation of splenocytes obtained from mice in the anti-RGMa neutralizing antibody administration group is due to either antigen-specific stimulation with MOG peptide or non-specific stimulation with anti-CD3 monoclonal antibody.
  • relapsing multiple sclerosis model mouse Recurrent EAE model by administering myelin proteolipid protein (hereinafter referred to as “PLP”) to SJL / J mice instead of MOG Mice were made.
  • PLP myelin proteolipid protein
  • recurrent EAE is 100 ⁇ L of PBS containing 100 ⁇ g of (PLP) 139-151 peptide (HSLGKWLGHPDKF (SEQ ID NO: 6), Greiner Bio-one), and Freund's complete adjuvant containing 500 ⁇ g of Mycobacterium tuberculosis (H37Ra, Difco).
  • Example 5 Examination of the effect of RGMa gene knockdown on EAE clinical symptoms in mice transplanted with BMDCs stimulated with MOG]
  • 5-1) Preparation of RGMa gene knockdown BMDCs
  • Mouse RGMa siRNA (Stealth RNAi (trade name), Invitrogen) having the following base sequence was purchased and used.
  • Non-targeting dsRNA (Invitrogen) was used as a control siRNA.
  • Sense strand 5'-AAAGAGGCCGCAGUGAGUGUAGUUG-3 '(SEQ ID NO: 7)
  • Antisense strand 5'-CAACUACACUCACUGCGGCCUCUUU-3 '(SEQ ID NO: 8)
  • BMDCs 7 days after the start of culture were used (see Example 1). 1 ⁇ 10 6 BMDCs were suspended in 100 ⁇ L of a nucleic acid introduction solution containing 500 pmol of mouse RGMa siRNA or control siRNA. The sample was transferred to a cuvette and siRNA was introduced into the cells using a Nucleofector® device (Lonza) according to the manufacturer's instructions. After siRNA introduction, the cells were cultured in RPMI 1640 medium supplemented with glutamine, sodium pyruvate, penicillin, streptomycin, 2-ME, 10% heat-inactivated FBS. The expression of RGMa in BMDCs into which mouse RGMa siRNA was introduced was significantly reduced.
  • the RGMa siRNA group (5 mice) and the control siRNA group (5 mice) were observed from cell transplantation (day 0) to the 21st day, and the clinical symptoms of EAE are shown in Example 2 above. Evaluation was based on the criteria described. Changes in the EAE score during the observation period are shown in FIG. The EAE score was expressed as an average value and standard error. In the figure, “*” and “**” represent P ⁇ 0.05 and P ⁇ 0.01, respectively, in Student's t test. As is clear from FIG. 12, the EAE score of the RGMa siRNA group was significantly lower than the EAE score of the control siRNA group. From this result, it was revealed that RGMa siRNA is effective for the prevention or treatment of multiple sclerosis.
  • Example 6 Examination of effect of anti-RGMa neutralizing antibody on clinical symptoms of EAE in mice transplanted with CD4 + T cells subjected to MOG stimulation]
  • (6-1) Preparation of donor mouse CD4 + T cells
  • Anti-RGMa neutralizing antibody (see Non-Patent Document 3) or control antibody (rabbit IgG, sigma Aldrich) was administered intraperitoneally (400 ⁇ g each) ( ⁇ 2 days). Two days later (day 0), the anti-RGMa neutralizing antibody or the control antibody is administered for the second time, and (MOG) 100 ⁇ L of PBS containing 35-55 peptide and 500 ⁇ g of Mycobacterium tuberculosis (H37Ra, Difco) are included.
  • mice were euthanized, the spleen and draining lymph nodes were removed, cells were isolated, and a cell suspension was prepared.
  • To restimulate CD4 + T cells cells (1 ⁇ 10 6 cells / mL) were cultured for 3 days in medium containing 40 ⁇ g / mL (MOG) 35-55 peptide. After completion of the culture, cells were collected and separated CD4 + T cells with CD4 + T cells isolation kit (Miltenyi Biotec).
  • the EAE score was expressed as an average value and standard error.
  • “*” and “**” represent P ⁇ 0.05 and P ⁇ 0.01, respectively, in Student's t test.
  • the EAE score of the anti-RGMa neutralizing antibody group was significantly lower than the EAE score of the control antibody group. From these results, the anti-RGMa neutralizing antibody-based multiple sclerosis prevention or treatment effect is based on the fact that the anti-RGMa neutralizing antibody suppresses antigen-specific T cell activation and autoreactive T cell differentiation. Became clear.
  • the present invention has extremely high utility value in the pharmaceutical industry.

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Abstract

La présente invention concerne un inhibiteur d'activation des lymphocytes T qui contient une substance inhibitrice de RGM telle qu'un anticorps neutralisant anti-RGM. L'inhibiteur d'activation des lymphocytes T est utile en tant que composition pharmaceutique pour la prévention ou le traitement de maladies auto-immunes y compris la sclérose en plaques et d'autres maladies causées par l'activation de lymphocytes T. De plus, le criblage d'une substance inhibitrice d'activation des lymphocytes T peut être effectué en mettant des substances à soumettre à essai en contact avec un RGM et en sélectionnant une substance qui réduit le taux d'activité du RGM.
PCT/JP2010/071965 2009-12-09 2010-12-08 Inhibiteur d'activation des lymphocytes t, composition pharmaceutique contenant celui-ci, et procédé de criblage pour une substance inhibant l'activation des lymphocytes t WO2011071059A1 (fr)

Priority Applications (11)

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KR1020197027496A KR102223464B1 (ko) 2009-12-09 2010-12-08 Ｔ 세포 활성화 저해제, 이것을 함유하는 의약 조성물 및 ｔ 세포 활성화 저해 물질의 스크리닝 방법
US13/514,915 US9334323B2 (en) 2009-12-09 2010-12-08 Method of reducing recurrence of multiple sclerosis symptoms in a mammal by administering an anti-repulsive guidance molecule neutralizing antibody
KR1020217023891A KR102425676B1 (ko) 2009-12-09 2010-12-08 Ｔ 세포 활성화 저해제, 이것을 함유하는 의약 조성물 및 ｔ 세포 활성화 저해 물질의 스크리닝 방법
KR1020177024100A KR20170102378A (ko) 2009-12-09 2010-12-08 Ｔ 세포 활성화 저해제, 이것을 함유하는 의약 조성물 및 ｔ 세포 활성화 저해 물질의 스크리닝 방법
JP2011545222A JP5785873B2 (ja) 2009-12-09 2010-12-08 Ｔ細胞活性化阻害剤、これを含有する医薬組成物およびｔ細胞活性化阻害物質のスクリーニング方法
KR1020127017515A KR20120120193A (ko) 2009-12-09 2010-12-08 Ｔ 세포 활성화 저해제, 이것을 함유하는 의약 조성물 및 ｔ 세포 활성화 저해 물질의 스크리닝 방법
KR1020217005837A KR102284780B1 (ko) 2009-12-09 2010-12-08 Ｔ 세포 활성화 저해제, 이것을 함유하는 의약 조성물 및 ｔ 세포 활성화 저해 물질의 스크리닝 방법
CN2010800633729A CN102811739A (zh) 2009-12-09 2010-12-08 T细胞活化抑制剂、含有其的药物组合物以及抑制t细胞活化的物质的筛选方法
EP10835979.5A EP2510948B1 (fr) 2009-12-09 2010-12-08 Inhibiteur d'activation des lymphocytes t, composition pharmaceutique contenant celui-ci, et procédé de criblage pour une substance inhibant l'activation des lymphocytes t
ES10835979.5T ES2555060T3 (es) 2009-12-09 2010-12-08 Inhibidor de la activación de linfocitos T, composición farmacéutica que lo contiene y método de cribado de sustancias inhibidoras de la activación de linfocitos T
US15/149,314 US9751938B2 (en) 2009-12-09 2016-05-09 Method of inhibiting activation of a T cell by administering an anti-RGM antibody or an RGM siRNA

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US8962803B2 (en) 2008-02-29 2015-02-24 AbbVie Deutschland GmbH & Co. KG Antibodies against the RGM A protein and uses thereof
US9605069B2 (en) 2008-02-29 2017-03-28 AbbVie Deutschland GmbH & Co. KG Antibodies against the RGM a protein and uses thereof
US9175075B2 (en) 2009-12-08 2015-11-03 AbbVie Deutschland GmbH & Co. KG Methods of treating retinal nerve fiber layer degeneration with monoclonal antibodies against a retinal guidance molecule (RGM) protein
US9102722B2 (en) 2012-01-27 2015-08-11 AbbVie Deutschland GmbH & Co. KG Composition and method for the diagnosis and treatment of diseases associated with neurite degeneration
US9365643B2 (en) 2012-01-27 2016-06-14 AbbVie Deutschland GmbH & Co. KG Antibodies that bind to repulsive guidance molecule A (RGMA)
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CN113267630A (zh) * 2014-09-10 2021-08-17 艾伯维德国有限责任两合公司 基于RGMa片段的诊断测定
JP2017526930A (ja) * 2014-09-10 2017-09-14 アッヴィ・ドイチュラント・ゲー・エム・ベー・ハー・ウント・コー・カー・ゲー ＲＧＭａ断片ベース診断アッセイ
AU2016253886B2 (en) * 2015-04-28 2019-05-16 Mitsubishi Tanabe Pharma Corporation RGMa binding protein and use thereof
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KR20190124350A (ko) 2015-04-28 2019-11-04 미쓰비시 타나베 파마 코퍼레이션 RGMa 결합 단백질 및 그 사용
RU2705304C2 (ru) * 2015-04-28 2019-11-06 Мицубиси Танабе Фарма Корпорейшн Связывающий rgma белок и его использование
EP3584260A1 (fr) 2015-04-28 2019-12-25 Mitsubishi Tanabe Pharma Corporation Protéine de liaison à rgma et son utilisation
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US10287346B2 (en) 2015-04-28 2019-05-14 Mitsubishi Tanabe Pharma Corporation RGMa binding protein and use thereof
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JP2023030057A (ja) * 2015-04-28 2023-03-07 田辺三菱製薬株式会社 ＲＧＭａ結合タンパク質及びその使用
JP7307446B2 (ja) 2015-04-28 2023-07-12 田辺三菱製薬株式会社 ＲＧＭａ結合タンパク質及びその使用
RU2809500C2 (ru) * 2015-04-28 2023-12-13 Мицубиси Танабе Фарма Корпорейшн СВЯЗЫВАЮЩИЙ RGMa БЕЛОК И ЕГО ИСПОЛЬЗОВАНИЕ
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KR20190110633A (ko) 2019-09-30
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EP2510948B1 (fr) 2015-09-09
EP2510948A1 (fr) 2012-10-17
JPWO2011071059A1 (ja) 2013-04-22
KR20120120193A (ko) 2012-11-01
US9334323B2 (en) 2016-05-10
KR102284780B1 (ko) 2021-08-02
JP5785873B2 (ja) 2015-09-30
ES2555060T3 (es) 2015-12-28
KR102223464B1 (ko) 2021-03-05
US20160244517A1 (en) 2016-08-25
US20120328633A1 (en) 2012-12-27
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