WO2015147335A1 - Diagnostic et traitement du paludisme cérébral - Google Patents

Diagnostic et traitement du paludisme cérébral Download PDF

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WO2015147335A1
WO2015147335A1 PCT/JP2015/060403 JP2015060403W WO2015147335A1 WO 2015147335 A1 WO2015147335 A1 WO 2015147335A1 JP 2015060403 W JP2015060403 W JP 2015060403W WO 2015147335 A1 WO2015147335 A1 WO 2015147335A1
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ccl21
cxcr3
ccr7
antibody
factor
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ジェヴァイア チョバン，
芳親 吉岡
石井　健
大貴 青枝
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国立大学法人大阪大学
国立研究開発法人医薬基盤・健康・栄養研究所
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Priority to JP2016510589A priority Critical patent/JP6618191B2/ja
Publication of WO2015147335A1 publication Critical patent/WO2015147335A1/fr

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    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/05—Detecting, measuring or recording for diagnosis by means of electric currents or magnetic fields; Measuring using microwaves or radio waves 
    • A61B5/055—Detecting, measuring or recording for diagnosis by means of electric currents or magnetic fields; Measuring using microwaves or radio waves  involving electronic [EMR] or nuclear [NMR] magnetic resonance, e.g. magnetic resonance imaging
    • A—HUMAN NECESSITIES
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    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/40—Detecting, measuring or recording for evaluating the nervous system
    • A61B5/4005—Detecting, measuring or recording for evaluating the nervous system for evaluating the sensory system
    • A61B5/4011—Evaluating olfaction, i.e. sense of smell
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    • C07—ORGANIC CHEMISTRY
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    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • C07K14/7158—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons for chemokines
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
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    • A61B2576/00—Medical imaging apparatus involving image processing or analysis
    • A61B2576/02—Medical imaging apparatus involving image processing or analysis specially adapted for a particular organ or body part
    • A61B2576/026—Medical imaging apparatus involving image processing or analysis specially adapted for a particular organ or body part for the brain
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/40—Detecting, measuring or recording for evaluating the nervous system
    • A61B5/4058—Detecting, measuring or recording for evaluating the nervous system for evaluating the central nervous system
    • A61B5/4064—Evaluating the brain
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    • G16H—HEALTHCARE INFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR THE HANDLING OR PROCESSING OF MEDICAL OR HEALTHCARE DATA
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    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
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    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
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    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A90/00—Technologies having an indirect contribution to adaptation to climate change
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Definitions

  • the present invention relates to the diagnosis and treatment of cerebral malaria (sometimes referred to herein as “CM”).
  • CM cerebral malaria
  • CM Cerebral malaria
  • Non-Patent Document 1 Idro, R. et al. (2010) .Pediatr Res 68, 267-274.
  • Non-patent document 2 Taylor, TE et al. (2004). Nat Med 10, 143-145.
  • Non-Patent Document 4 Langhorne, J. et al. (2011). Malar J 10, 23.
  • the brain forms a blood-brain barrier (BBB) in which dense endothelial cells are firmly anchored in specific blood vessels, and is a privileged site that prevents the invasion of exogenous pathogens. It is thought to result from destruction and subsequent inflammatory responses and cellular accumulation in the brain.
  • BBB blood-brain barrier
  • Non-patent Document 5 Investigationa, FG et al. (2010). InfectImmun 78, 4033-4039.
  • Patent Literature 6 Haque, A. et al. (2011) .J Immunol 186,6148-6156.;
  • Non-patent Literature 7 Howland, SW et al.
  • Non-patent document 8 Lundie, R.J. et al. (2008).
  • Non-patent document 9 McQuillan, JA et al. (2011) .Int J Parasitol 41,155-163.
  • Non-patent document 10 Renia, L. et al. (2012). 193-201 .
  • Non-Patent Document 11 Yui, K. (2013) .EMBO Mol Med. (Online release on June 5, 2013, doi: 10.1002 / emmm.201302849)).
  • Non-Patent Document 12 Tamura
  • BBB dysfunction is a major feature of ECM, and systemic and local events contribute to this
  • Non-Patent Document 13 Combes, V. et al. (2012). Trends Parasitol 28, 311-319.
  • Non-Patent Document 14 Nacer, A. et al. (2012). PLoS Pathog 8, e1002982.).
  • the inventors have used a combination of ultra-high magnetic field 11.7T MRI and multiphoton (MP) microscopy, a powerful imaging technique, to pathophysiological mechanisms and immunology of mouse and non-human primate CM.
  • MP multiphoton
  • the spatiotemporal control of the mechanical mechanism was investigated.
  • PbA mouse CM model
  • P.A The basic mechanism by which BBB was destroyed and tolerated during systemic infection with coatneyi (monkey CM model) was elucidated.
  • OLF olfactory bulb
  • the inventors are a region in which the olfactory bulb (OLF) is physically damaged by the Plasmodium parasite and functionally impaired (loss of olfaction) in the mouse model and monkey model of CM (olfaction). It was revealed that high fever occurred.
  • Two powerful imaging technologies ultra-high magnetic field MRI and in vivo multiphoton microscopy, show that OLF undergoes parasite accumulation and cell blockage, followed by microbleeding due to palisade microcapillary structures. Visualized. Parasites that circulate in the OLF blood vessels release CCL21 that can be detected by OLF glomeruli and perivascular astrocytes at an early stage of infection and can attract CD11c + CD8 T cells via CXCR3.
  • the present invention also provides the following.
  • MRI nuclear magnetic resonance imaging
  • a method of diagnosing cerebral malaria comprising measuring the state of a subject's olfactory bulb (OLF).
  • a device for detecting or diagnosing cerebral malaria including means for measuring the state of the olfactory bulb.
  • the means is a magnetic resonance imaging (MRI) means.
  • the MRI is an MRI of 7T or more.
  • a preventive or therapeutic agent for cerebral malaria comprising an inhibitor of at least one factor of the CCL21-CXCR3-CCR7 signaling system.
  • the inhibitor of at least one factor of the CCL21-CXCR3-CCR7 signaling system includes an inhibitor of at least one factor selected from the group consisting of CCL21, CXCR3 and CCR7, and nucleic acids encoding them.
  • Item 15 The preventive or therapeutic agent for cerebral malaria according to Item 14.
  • the inhibitor is a low molecular weight compound, an antibody or a fragment or functional equivalent thereof, siRNA, shRNA, antisense nucleic acid, aptamer, pharmaceutically acceptable salt or solvate thereof, or pharmaceutical thereof Item 16.
  • the preventive or therapeutic agent for cerebral malaria according to claim 1. (18)
  • the inhibitor is any one of items 14 to 17, comprising at least two antibodies selected from the group consisting of an anti-CCL21 antibody, an anti-CXCR3 antibody, and an anti-CCR7 antibody, or a fragment or functional equivalent thereof
  • the inhibitor according to item 20 further comprising the feature of any one or more of items 15 to 19.
  • a method for preventing or treating brain malaria in a subject comprising administering an effective amount of an inhibitor of at least one factor of the CCL21-CXCR3-CCR7 signaling system to the subject in need thereof.
  • a marker for cerebral malaria comprising at least one factor of the CCL21-CXCR3-CCR7 signaling system.
  • At least one factor of the CCL21-CXCR3-CCR7 signaling system is at least one selected from the group consisting of CCL21, CXCR3, CCR7, and nucleic acids encoding them, their expression products and their derivatives.
  • At least one factor of the CCL21-CXCR3-CCR7 signaling system is at least selected from the group consisting of (1) IP-10 (CXCR3 ligand), CXCL11 (I-TAC), and CXCL9 (MIG)
  • IP-10 CXCR3 ligand
  • CXCL11 I-TAC
  • MIG CXCL9
  • One inflammatory cytokine and at least one selected from the group consisting of nucleic acids encoding them, their expression products and their derivatives
  • CCL21 and / or CCL19 comprising at least one factor selected from the group consisting of: CD8 ⁇ dendritic cells and other dendritic cells; and (4) CD8 T cells and other T cells.
  • Item 25 The marker for brain malaria according to item 23 or 24.
  • the at least one factor of the CCL21-CXCR3-CCR7 signaling system is a nucleic acid sequence represented by SEQ ID NO: 1, 3 or 5, or a fragment or variant thereof, or an amino acid sequence represented by SEQ ID NO: 2, 4 or 6 Alternatively, the marker according to any one of items 23 to 28, comprising a fragment or a variant thereof.
  • Detection agent or diagnostic agent (2) The detection agent or diagnostic agent according to item 31, wherein the detection agent is an antibody, a fragment or functional equivalent thereof, or a nucleic acid.
  • the expression of any of at least one factor of the CCL21-CXCR3-CCR7 signaling system of the target sample is increased compared to that of a normal sample, the target sample is diagnosed as having brain malaria 36.
  • the present invention provides a diagnosis and treatment technique for cerebral malaria that has not existed previously.
  • FIG. 1 shows that an ultrahigh magnetic field MRI is applied to the olfactory bulb. It shows a mode that it identifies as a place easy to be attacked by berghei ANKA parasite.
  • C57BL / 6 mice were infected with 10 6 PbA parasites.
  • B A diffusion-weighted image of the coronal section of the mouse head on day 6.
  • a square and an arrow surrounded by a gray dotted line indicate the location of HE staining in (C).
  • a and B images are representative of at least 5 animals.
  • C The histology of the coronal section of OLF on day 6 after infection shows low density areas corresponding to several bleeding sites with HE staining (scale bar is 1 mm). ONL (olfactory nerve layer), GL (glomerular layer), MCL (mitral cell layer), GCL (granular cell layer).
  • D IHC of the OLF cross section on the 6th day. Red blood cells were stained with TER119 (red) and GFP-parasite (green). Nuclei were visualized with DAPI (blue). The scale bar is 100 ⁇ m.
  • FIG. 1E to 1F show RI images of a living body and mouse brain fixed with paraformaldehyde.
  • E Naive mouse head in vivo and paraformaldehyde (PFA) fixed 11.7T MRI imaging (coronal cross section).
  • F shows coronal and sagittal sections of an initial 11.7 T MRI image of PbA-infected mice.
  • FIG. 2 shows in vivo multiphoton imaging of OLF palisade microvessels during infection.
  • A Schematic diagram of MP microscope observation through the thin skull of the back OLF is shown. The depth of the image is about 150 ⁇ m from the buffy coat surface, and capillaries (red, less than 5 ⁇ m in diameter) in the GL are visualized.
  • Red indicates a blood vessel labeled with red TRITC-dextran
  • green (green arrow) indicates a PbA parasite expressing GFP
  • blue indicates CD8 T cells (anti-CD8 antibody, white arrow).
  • the red area in the circle indicates the area that has already bleeded
  • the yellow arrow indicates the horned blood vessel where new bleeding will occur in the future
  • the white arrow in the right image already indicates that new bleeding has already occurred.
  • An expanded blood vessel is shown to indicate that FIG. 3 shows how olfactory loss and fever occur in mouse brain malaria.
  • C57BL / 6 mice were infected with 10 6 PbA parasites.
  • A To assess OLF function, mice were subjected to food burying studies at indicated time points after infection.
  • FIGS. 3 (H)-(I) show the lack of involvement of the OLF olfactory bulb during PyL and in Rag2 ⁇ / ⁇ mice with PbA infection.
  • (H) shows an 11.7T MRI image of the C58B / 6 mouse head on the fifth day of infection with PyL.
  • (I) shows an 11.7T MRI image of the Rag2 ⁇ / ⁇ mouse head 6 days after PbA infection (FLASH T2 * signal, coronal section).
  • a circle surrounded by a dotted line corresponds to the OLF olfactory bulb.
  • FIG. 4 shows how chemokine CCL21 activity occurs in the olfactory bulb during PbA infection.
  • D WT and Ccr7 ⁇ / ⁇ mice were infected and subjected to food embedding tests to measure OLF function.
  • AB Flow cytometric analysis of whole brain (A) and spleen (B) cells 6 days after infection.
  • the numbers in the inset show the percentage of CD8 T cells and their CD11c expression, and the right figure of A shows the absolute number of CD11c + CD8 T cells in the brain (4-8 mice in each group).
  • C The activation state of CD11 + CD8 T cells in the spleens of WT (Ccr7 +/ ⁇ ) and Ccr7 ⁇ / ⁇ mice was determined by CD44 surface staining.
  • D Intracellular staining of IFN- ⁇ secreting CD11c + CD8 T cells in the spleen of WT mice 6 days after infection.
  • E Ccr7 ⁇ / ⁇ mice were adoptively immunized with concentrated CD8 ⁇ DC from infected Rag2 ⁇ / ⁇ spleen, infected with PbA, and survival was monitored.
  • Ccr7 ⁇ / ⁇ mice adoptively immunized with Ccr7 ⁇ / ⁇ vs. CD8 ⁇ DC by Log-rank (Mantel-Cox) test, *** p 0.00055.
  • the number of CD11c + CD8 T cells accumulated in the brains of Ccr7 ⁇ / ⁇ mice was determined (mean ⁇ SD of 3 mice in each group). ** p ⁇ 0.01 in Student Ccr7 +/ ⁇ mice vs.
  • FIGS. 5 (F)-(G) show the absence of thermal involvement during the PbA-infected day 5 cytokine storm and non-lethal PyNL infection.
  • Fig. 2 shows fever in yoelii NL infected C57B / 6 mice. Average heat measurements were calculated every 3 hours. The data presented is representative of at least 3 infected mice per group.
  • FIGS. 5 (H)-(I) show the absence of signs in Ccr7 ⁇ / ⁇ mice 6 days after infection.
  • H shows MRI images of coronal sections from naive and infected Ccr7 ⁇ / ⁇ mice 6 days after infection.
  • I shows evaluation by Evans blue staining of BBB leakage in Ccr7 ⁇ / ⁇ mice on day 6 and day 8 after PbA infection.
  • FIG. 6 shows that CCL21 expressed in the OLF can be involved in the migration of CD11c + CD8 T cells into the OLF, consistent with astrocyte activation.
  • A Co-localization with CCL21 stained astrocyte marker GFAP. OLF sections from PbA-infected WT mice 6 days after infection were stained with GFAP (green) and CCL21 (red) antibodies. Nuclei were visualized with DAPI (blue). The image is representative of at least 3 different animals. The scale bar is 50 ⁇ m.
  • B Shows how the end feet of astrocytes wrap blood vessels in the GL.
  • OLF GL sections from naive and infected WT mice were stained with PECAM (green) and GFAP (red) antibodies. Nuclei were visualized with DAPI (blue). Arrows indicate intact blood vessels (naive mice) and broken blood vessels (infected mice). The scale bar is 10 ⁇ m.
  • FIGS. 6 (E), (F) and (G) show that CD8 ⁇ dendritic cells (DC) are required for CD11c + CD8 T cell activation and cerebral malaria pathogenesis.
  • (F) Flow cytometric analysis of whole brain and splenic CD8 T cells 6 days after infection.
  • FIGS. 6 (H) and (I) show flow cytometric analysis 5 days after infection of Rag2 ⁇ / ⁇ mouse spleen CD8 ⁇ DC.
  • the upper (green) rectangular region (CD8 ⁇ + DC) highly expresses CDR7, while the lower (black) rectangular region (CD8 ⁇ - DC) expresses low levels of CCD7.
  • FIG. 7 shows that the olfactory bulb can be a “gateway” for the accumulation of Plasmodium-infected erythrocytes into the brain.
  • Some of the activated CD8 T cells become an effector phenotype by expressing CD11c and CXCR3 (2).
  • the products of these activated immune cells, infected red blood cells (iRBCs) or parasites are sensed by the astrocytes near the blood vessels in the OLF glomerulus and their surrounding end feet (3), thereby Probably induces gradual CCL21 secretion from astrocytes (4) and is partly involved in opening the BBB entrance to primed immune cells.
  • Activated CD8 T cells migrate specifically to OLF, where CCL21 and various other chemokines are secreted (5, 6), leading to fever and bleeding in OLF (7).
  • brain malaria is the most lethal complication of Plasmodium falciparum (P. falciparum) malaria, and is caused by the destruction of the blood brain barrier (BBB). It is said that neurological symptoms such as decreased consciousness and language tangles occur, and if it progresses, it falls into a coma and is said to die.
  • CM cerebral malaria
  • PbA berghei ANKA parasite mouse model
  • the term “olfactory bulb (sometimes abbreviated as OLF)” is used in the same meaning as is commonly used in the art, and refers to a bulge of the front end of the olfactory lobe, which is located at the front end of the telencephalon and is spherical. Has a protruding structure. Inside, there are the main olfactory bulb and the accessory olfactory bulb showing a layer structure, and it is said that the olfactory nerve from the olfactory organ, which is a chemosensory receptor, and the vomeronasal nerve from the vomeronasal organs are all over. It is said to play an important role in olfactory information processing.
  • OLF olfactory bulb
  • the state of the olfactory bulb includes the appearance, the structure or the functional state of the olfactory bulb. More specifically, the state of the olfactory bulb can be determined by an olfactory sensation abnormality, a cell test, a diagnostic image of the olfactory bulb, and the like, but is not limited thereto.
  • ultra-high magnetic field nuclear magnetic resonance imaging refers to MRI at a high magnetic field exceeding the magnetic field of 3 Tesla currently used for clinical use, for example, about 4 Tesla or more, About 5 Tesla or more, about 6 Tesla or more, about 7 Tesla or more. In the embodiment, 11.7 Tesla is used, and the state of the olfactory bulb can be determined.
  • spot in a diagnostic image means that a spotted object is seen in a densely packed image in an olfactory bulb found in a normal state. Dysfunction is presumed.
  • cell examination has the same meaning as commonly used in the art, and includes staining such as staining (for example, HE staining) and immunohistochemistry.
  • CCL21-CXCR3-CCR7 signal transduction system or “CCL21-CXCR3-CCR7 axis” refers to the signal transduction pathway of CCL21, CXCR3 and CCR7, and refers to the onset mechanism of brain malaria. Say. The schematic diagram is shown in FIG.
  • Factors of “CCL21-CXCR3-CCR7 signaling system” include CCL21, CXCR3 and CCR7, IP-10 (CXCR3 ligand), CXCL11 (I-TAC), CXCL9 (MIG), interleukin (IL) -6, At least one inflammatory cytokine selected from the group consisting of IL-12p40, interferon (IFN) - ⁇ , KC, MCP-1 (CCR2 ligand), MIP-1 ⁇ , MIP-1 ⁇ , and RANTES, CD8 ⁇ dendritic cells ( DC), CD11c + CD8 T cells, etc. can also be considered as this factor.
  • immune cells are activated in the periphery such as blood and spleen.
  • CD8 ⁇ + dendritic cells activated by infection in the spleen acquire the ability to cross-prime antigen to CD8 T cells via CCR7 expression.
  • Some activated CD8 T cells become effector phenotypes by expressing CD11c and / or CXCR3.
  • the products of these activated immune cells, infected red blood cells (iRBCs) or parasites are sensed by the astrocytes near the blood vessels in the OLF's glomeruli and their surrounding end feet, thereby causing the astrocytes It is thought to induce gradual CCL21 secretion from and open the BBB entry and exit for primed immune cells.
  • Activated CD8 T cells migrate specifically to OLF, where CCL21 is secreted, which is thought to lead to fever and bleeding in the OLF.
  • CCL21 is an endogenous ligand of CCR7 and is found in high endothelial venules (specialized blood vessels through which lymphocytes leave the blood and enter lymph nodes) It is a chemokine ligand and a member of the CC subfamily. It is also called 6Ckine, CKb9, ECL, SCYA21, SLC, TCA4.
  • the nucleic acid sequence and amino acid sequence of human CCL21 are disclosed in NCBI accession numbers NM_002989 (SEQ ID NO: 1) and NP_002980 (SEQ ID NO: 2), respectively, and the mouse nucleic acid sequence and amino acid sequence are disclosed in NM_011124 and NP_035254, respectively.
  • CCL 21 can be identified by an accession number with OMIM: 602737.
  • CCL21 is not only a protein having an amino acid sequence described in a specific sequence number or accession number (or a nucleic acid encoding it), but also functionally active. A derivative thereof, or a functionally active fragment thereof, or a homologue thereof, or a variant encoded by a nucleic acid that hybridizes under high or low stringency conditions to a nucleic acid encoding this protein. , Understood to mean.
  • a given protein or nucleic acid name not only refers to a protein or nucleic acid as shown in the sequence listing, but also a functionally active derivative, or a functionally active fragment thereof, or a homologue thereof, or a high stringency. It also refers to a variant encoded by a nucleic acid that hybridizes to a nucleic acid encoding the protein under conditions of genency or low stringency, preferably under the conditions described above.
  • a “derivative” or “analog of a component protein” or “variant” preferably includes, but is not intended to be limited, a region that is substantially homologous to the component protein.
  • Such molecules in various embodiments, at least 30% when compared to sequences aligned over amino acid sequences of the same size or aligned by computer homology programs known in the art. , 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% identical or nucleic acid encoding such a molecule is moderately stringent under stringent conditions Can hybridize to sequences encoding component proteins under mild or non-stringent conditionsThis is a product of a naturally occurring protein modified by amino acid substitutions, deletions and additions, respectively, and derivatives thereof that do not necessarily exhibit the same degree of biological function of the naturally occurring protein. means. For example, the biological function of such proteins can be examined by suitable and available in vitro assays described herein or known in the art.
  • CCL21-CXCR3-CCR7 signaling factor of the present invention for example, CCL21 protein
  • these mammals are also of the present invention. It is understood that it falls within the scope.
  • a CCL21-CXCR3-CCR7 signaling factor fragment (for example, CCL21) is a polypeptide containing any region of a CCL21-CXCR3-CCR7 signaling factor in the case of a protein or nucleic acid. As long as the object of the present invention can be achieved, it does not have to have the biological function of a natural CCL21-CXCR3-CCR7 signaling system factor.
  • a typical nucleotide sequence of CCL21 is: (A) a polynucleotide having the base sequence set forth in SEQ ID NO: 1 or a fragment sequence thereof; (B) a polynucleotide encoding a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 2 or a fragment thereof; (C) a variant polypeptide or fragment thereof in which one or more amino acids have one mutation selected from the group consisting of substitution, addition and deletion in the amino acid sequence set forth in SEQ ID NO: 2, A polynucleotide that encodes a variant polypeptide having functional activity; (D) a polynucleotide which is a splice variant or allelic variant of the base sequence set forth in SEQ ID NO: 1 or a fragment thereof; (E) a polynucleotide encoding a species homologue of the polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 2 or a fragment thereof; (F) a poly
  • amino acid sequence of CCL21 As the amino acid sequence of CCL21, (A) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2 or a fragment thereof; (B) a polypeptide having one mutation in which one or more amino acids are selected from the group consisting of substitution, addition and deletion in the amino acid sequence set forth in SEQ ID NO: 2 and having biological activity; (C) a polypeptide encoded by a splice variant or allelic variant of the base sequence set forth in SEQ ID NO: 1; (D) a polypeptide that is a species homologue of the amino acid sequence set forth in SEQ ID NO: 2; or (e) an amino acid sequence that is at least 70% identical to any one polypeptide of (a) to (d) And a polypeptide having biological activity.
  • the biological activity typically means the activity of CCL21.
  • CXCR3 has the same meaning as commonly used in the art, and is one of the CXC chemokine receptor families that are G protein-coupled receptors.
  • G protein-coupled receptor 9 GPR9
  • CD183 CD182
  • CKR-L2 CD182
  • CMKAR3 CMKAR3
  • IP10-R Mig-R
  • CXCR3-A and CXCR3-B There are two variants, and there are two types: CXCR3-A and CXCR3-B.
  • CXCR3-A is supposed to bind to CXCL9 (MIG), CXCL10 (IP-10) and CXCL11 (I-TAC), and CXCR3-B is CXCL9 (MIG), CXCL10 (IP-10) and CXCL11 (I -TAC) in addition to CXCL4.
  • the nucleic acid sequence and amino acid sequence of human CXCR3 are disclosed in NCBI accession numbers NM_001142797 (SEQ ID NO: 3) and NP_001136269 (SEQ ID NO: 4), respectively, and the mouse nucleic acid sequence and amino acid sequence are disclosed in NM_009910 and NP_034040, respectively. This information is also incorporated herein.
  • CXCR3 can be identified by an accession number with OMIM: 300574.
  • “CXCR3” is not only a protein having an amino acid sequence described in a specific sequence number or accession number (or a nucleic acid encoding it), but also functionally active. A derivative thereof, or a functionally active fragment thereof, or a homologue thereof, or a variant encoded by a nucleic acid that hybridizes under high or low stringency conditions to a nucleic acid encoding this protein. , Understood to mean.
  • a typical nucleotide sequence of CXCR3 is: (A) a polynucleotide having the base sequence set forth in SEQ ID NO: 3 or a fragment sequence thereof; (B) a polynucleotide encoding a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 4 or a fragment thereof; (C) a variant polypeptide or fragment thereof in which one or more amino acids have one mutation selected from the group consisting of substitution, addition and deletion in the amino acid sequence set forth in SEQ ID NO: 4, A polynucleotide that encodes a variant polypeptide having functional activity; (D) a polynucleotide which is a splice variant or allelic variant of the base sequence set forth in SEQ ID NO: 3 or a fragment thereof; (E) a polynucleotide encoding a species homologue of the polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 4 or a fragment thereof; (F) a
  • amino acid sequence of CXCR3 As the amino acid sequence of CXCR3, (A) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 4 or a fragment thereof; (B) a polypeptide having one mutation in which one or more amino acids are selected from the group consisting of substitution, addition and deletion in the amino acid sequence of SEQ ID NO: 4 and having biological activity; (C) a polypeptide encoded by a splice variant or allelic variant of the base sequence set forth in SEQ ID NO: 3; (D) a polypeptide that is a species homologue of the amino acid sequence set forth in SEQ ID NO: 4; or (e) an amino acid sequence that is at least 70% identical to any one polypeptide of (a) to (d) And a polypeptide having biological activity.
  • the biological activity typically refers to the activity of CXCR3.
  • CCR7 has the same meaning as commonly used in the field, is an abbreviation for chemokine (C-C motif) receptor 7, and is one of chemokine receptors.
  • CCL21 which is found in high endothelial venules, blood vessels specialized to allow lymphocytes to leave the blood and enter lymph nodes.
  • CCL19 which is said to be present in the T cell region of lymph nodes (site where T cells are separated from B cells and gather).
  • CCR7 The nucleic acid sequence and amino acid sequence of human CCR7 are disclosed in NCBI accession numbers NM_001838 (SEQ ID NO: 5) and NP_001829 (SEQ ID NO: 6), respectively, and the mouse nucleic acid sequence and amino acid sequence are disclosed in NM_007719 and NP_031745, respectively. This information is also incorporated herein.
  • CCR7 can be identified by an accession number with OMIM: 60000242.
  • “CCR7” is not only a protein having an amino acid sequence described in a specific sequence number or accession number (or a nucleic acid encoding it), but also functionally active.
  • a representative nucleotide sequence of CCR7 is: (A) a polynucleotide having the base sequence set forth in SEQ ID NO: 5 or a fragment sequence thereof; (B) a polynucleotide encoding a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 6 or a fragment thereof; (C) a variant polypeptide or fragment thereof in which one or more amino acids have one mutation selected from the group consisting of substitution, addition and deletion in the amino acid sequence set forth in SEQ ID NO: 6, A polynucleotide that encodes a variant polypeptide having functional activity; (D) a polynucleotide which is a splice variant or allelic variant of the base sequence set forth in SEQ ID NO: 5 or a fragment thereof; (E) a polynucleotide encoding a species homologue of the polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 6 or a fragment thereof; (F) a poly
  • amino acid sequence of CCR7 As the amino acid sequence of CCR7, (A) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 6 or a fragment thereof; (B) a polypeptide having one mutation in which one or more amino acids are selected from the group consisting of substitution, addition and deletion in the amino acid sequence set forth in SEQ ID NO: 6 and having biological activity; (C) a polypeptide encoded by a splice variant or allelic variant of the base sequence set forth in SEQ ID NO: 5; (D) a polypeptide that is a species homologue of the amino acid sequence set forth in SEQ ID NO: 6; or (e) an amino acid sequence that is at least 70% identical to any one polypeptide of (a) to (d) And a polypeptide having biological activity.
  • the biological activity typically refers to the activity possessed by CCR7.
  • CCL21-CXCR3-CCR7 signal transduction system factors such as the following: (1) At least one inflammatory cytokine selected from the group consisting of IP-10 (CXCR3 ligand), CXCL11 (I-TAC), and CXCL9 (MIG), and a nucleic acid encoding them, its expression product and its origin At least one selected from the group consisting of: (2) CCL21 and / or CCL19 (3) CD8 ⁇ dendritic cells and other dendritic cells; and (4) CD8 T cells and other T cells.
  • IP-10 CXCR3 ligand
  • I-TAC CXCL11
  • MIG CXCL9
  • a substance that binds to a factor of CCL21-CXCR3-CCR7 signaling system or “an interacting molecule of a factor of CCL21-CXCR3-CCR7 signaling system” at least temporarily refers to CCL21, CXCR3, CCR7. It is a molecule or substance that binds to a factor of the CCL21-CXCR3-CCR7 signaling system, such as, and is preferably capable of indicating that it is bound (eg, labeled or labelable).
  • Substances that bind to CCL21-CXCR3-CCR7 signaling system factors are inhibitors of CCL21-CXCR3-CCR7 signaling system factors (eg, CCL21, CXCR3, CCR7, etc.).
  • Examples thereof include antibodies, antisense oligonucleotides, siRNA, low molecular weight molecules (LMW), binding peptides, aptamers, ribozymes, and peptidomimetics, etc., and CCL21-CXCR3 -Activity of factors of CCL21-CXCR3-CCR7 signaling system (for example, CCL21, CXCR3, CCR7, etc.) directed against CCR7 signaling system factors (for example, CCL21, CXCR3, CCR7, etc.)
  • CCL21-CXCR3-CCR7 signaling system factors for example, CCL21, CXCR3, CCR7, etc.
  • a binding protein or binding peptide that is directed to a position, and a nucleic acid directed to a gene (nucleic acid) of a CCL21-CXCR3-CCR7 signaling system factor (eg, CCL21, CXCR3, CCR7, etc.) included.
  • Nucleic acids for CCL21-CXCR3-CCR7 signaling system factors include, for example, CCL21 gene expression or CCL21-CXCR3-CCR7 signaling system factors (eg, CCL21, CXCR3, CCR7, etc.)
  • CCL21 gene expression or CCL21-CXCR3-CCR7 signaling system factors eg, CCL21, CXCR3, CCR7, etc.
  • CCL21, CXCR3, CCR7, etc. refers to double-stranded or single-stranded DNA or RNA, or a modification or derivative thereof, including, without limitation, antisense nucleic acids, aptamers, siRNA (small interfering RNA) and ribozymes.
  • binding protein or “binding peptide” for a CCL21-CXCR3-CCR7 signal transduction factor (eg, CCL21, CXCR3, CCR7, etc.) refers to a CCL21-CXCR3-CCR7 signal transduction factor.
  • Polyclonal or monoclonal antibodies, antibody fragments and protein backbones that are directed against the type of protein or peptide that binds to and that are directed against factors of the CCL21-CXCR3-CCR7 signaling system (eg, CCL21, CXCR3, CCR7, etc.) Including but not limited to.
  • protein protein
  • polypeptide oligopeptide
  • peptide refers to a polymer of amino acids having an arbitrary length.
  • This polymer may be linear, branched, or cyclic.
  • the amino acid may be natural or non-natural and may be a modified amino acid.
  • the term can also encompass one assembled into a complex of multiple polypeptide chains.
  • the term also encompasses natural or artificially modified amino acid polymers. Such modifications include, for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation or any other manipulation or modification (eg, conjugation with a labeling component).
  • amino acid may be natural or non-natural as long as it satisfies the object of the present invention.
  • polynucleotide As used herein, “polynucleotide”, “oligonucleotide”, and “nucleic acid” are used interchangeably herein and refer to a nucleotide polymer of any length. The term also includes “oligonucleotide derivatives” or “polynucleotide derivatives”. “Oligonucleotide derivatives” or “polynucleotide derivatives” refer to oligonucleotides or polynucleotides that include derivatives of nucleotides or that have unusual linkages between nucleotides, and are used interchangeably.
  • oligonucleotide examples include, for example, 2′-0-methyl-ribonucleotide, an oligonucleotide derivative in which a phosphodiester bond in an oligonucleotide is converted to a phosphorothioate bond, and a phosphodiester bond in an oligonucleotide.
  • oligonucleotide derivative in which ribose and phosphodiester bond in oligonucleotide are converted to peptide nucleic acid bond
  • uracil in oligonucleotide is C ⁇
  • Oligonucleotide derivatives substituted with 5-propynyluracil oligonucleotide derivatives wherein uracil in oligonucleotide is substituted with C-5 thiazole uracil
  • cytosine in oligonucleotide is C-5 propynylcytosine
  • oligonucleotide derivatives in which the cytosine in the oligonucleotide is substituted with phenoxazine-modified cytosine
  • oligonucleotide derivatives in which the ribose in DNA is substituted with 2'-0-
  • a particular nucleic acid sequence may also be conservatively modified (eg, degenerate codon substitutes) and complementary sequences, as well as those explicitly indicated. Is contemplated. Specifically, a degenerate codon substitute creates a sequence in which the third position of one or more selected (or all) codons is replaced with a mixed base and / or deoxyinosine residue. (Batzeret al., Nucleic Acid Res. 19: 5081 (1991); Ohtsuka et al., J. Biol. Chem. 260: 2605-2608 (1985); Rossolinite al., Mol. Cell. Probes). 8: 91-98 (1994)).
  • nucleic acid is also used interchangeably with gene, cDNA, mRNA, oligonucleotide, and polynucleotide.
  • nucleotide may be natural or non-natural.
  • gene refers to a factor that defines a genetic trait
  • gene may refer to “polynucleotide”, “oligonucleotide”, and “nucleic acid”.
  • homology of a gene refers to the degree of identity of two or more gene sequences to each other, and generally “having homology” means that the degree of identity or similarity is high. Say. Therefore, the higher the homology between two genes, the higher the sequence identity or similarity. Whether two genes have homology can be examined by direct sequence comparison or, in the case of nucleic acids, hybridization methods under stringent conditions. When directly comparing two gene sequences, the DNA sequence between the gene sequences is typically at least 50% identical, preferably at least 70% identical, more preferably at least 80%, 90% , 95%, 96%, 97%, 98% or 99% are identical, the genes are homologous.
  • a “homolog” or “homologous gene product” is a protein in another species, preferably a mammal, that performs the same biological function as the protein component of the complex further described herein. Means. Such homologues may also be referred to as “ortholog gene products”. It will be understood that such homologues, homologous gene products, orthologous gene products and the like can be used as long as they meet the objectives of the present invention.
  • Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides may also be referred to by a generally recognized one letter code.
  • BLAST is a sequence analysis tool.
  • the identity search can be performed using, for example, NCBI BLAST 2.2.28 (issued 2013.4.2).
  • the identity value usually refers to a value when the BLAST is used and aligned under default conditions. However, if a higher value is obtained by changing the parameter, the highest value is the identity value. When identity is evaluated in a plurality of areas, the highest value among them is set as the identity value. Similarity is a numerical value calculated for similar amino acids in addition to identity.
  • “several” may be, for example, 10, 8, 6, 5, 4, 3, or 2, or may be less than any of these values.
  • Polypeptides that have undergone deletion, addition, insertion, or substitution with other amino acids of one or several amino acid residues are known to maintain their biological activity (Market al., Proc Natl. Acad Sci US A. 1984 Sep; 81 (18): 5562-5666., Zoller et al., Nucleic Acids Res. 1982 Oct 25; 10 (20): 6487-6500., Wang et al., Science. 1984. Jun 29; 224 (4656): 1431-1433.).
  • Antibodies with deletions and the like can be prepared by, for example, site-specific mutagenesis, random mutagenesis, or biopanning using an antibody phage library.
  • site-specific mutagenesis method for example, KOD-Plus-Mutageness Kit (TOYOBO CO., LTD.) Can be used. It is possible to select an antibody having the same activity as that of the wild type from mutant antibodies into which deletions have been introduced by performing various characterizations such as FACS analysis and ELISA.
  • “90% or more” may be, for example, 90, 95, 96, 97, 98, 99, or 100% or more, and is within the range of any two of them. Also good.
  • the above-mentioned “homology” may be calculated according to a method known in the art, based on the ratio of the number of amino acids homologous in the amino acid sequence between two or more amino acids. Before calculating the ratio, the amino acid sequences of the group of amino acid sequences to be compared are aligned, and a gap is introduced into a part of the amino acid sequence when necessary to maximize the ratio of the same amino acids.
  • polynucleotide hybridizing under stringent conditions refers to well-known conditions commonly used in the art. Such a polynucleotide can be obtained by using a colony hybridization method, a plaque hybridization method, a Southern blot hybridization method or the like using a polynucleotide selected from among the polynucleotides of the present invention as a probe. Specifically, hybridization was performed at 65 ° C. in the presence of 0.7 to 1.0 M NaCl using a filter on which DNA derived from colonies or plaques was immobilized, and then a 0.1 to 2-fold concentration was obtained. It means a polynucleotide that can be identified by washing a filter under conditions of 65 ° C.
  • SSC serum-sodium citrate
  • composition of 1-fold concentration of SSC solution is 150 mM sodium chloride, 15 mM sodium citrate
  • stringent conditions for example, the following conditions can be adopted.
  • Use low ionic strength and high temperature for washing eg, 0.015 M sodium chloride / 0.0015 M sodium citrate / 0.1% sodium dodecyl sulfate at 50 ° C.
  • Use denaturing agents such as formamide during hybridization (eg, at 42 ° C., 50% (v / v) formamide and 0.1% bovine serum albumin / 0.1% Ficoll / 0.1% polyvinylpyrrolidone / 50 mM PH 6.5 sodium phosphate buffer and 750 mM sodium chloride, 75 mM sodium citrate), or (3) 20% formamide, 5 ⁇ SSC, 50 mM sodium phosphate (pH 7.6), 5 ⁇ Denhardt's solution, 10 37% in a solution containing dextran sulfate and 20 mg / ml denatured sheared salmon sperm DNA Incubate overnight at 0 ° C, then wash the filter with 1X SSC at about 37-50 ° C.
  • the formamide concentration may be 50% or more.
  • the cleaning time may be 5, 15, 30, 60, or 120 minutes, or longer.
  • a plurality of factors such as temperature and salt concentration are conceivable.
  • “highly stringent conditions” are 0.0015 M sodium chloride, 0.0015 M sodium citrate, 65-68 ° C., or 0.015 M sodium chloride, 0.0015 M sodium citrate, and 50% formamide, 42 ° C. Hybridization, Molecular Cloning 2nd ed.
  • sequence containing only the A sequence or only the T sequence is preferably excluded from the sequences that hybridize under stringent conditions.
  • Moderate stringent conditions can be readily determined by those skilled in the art based on, for example, the length of the DNA, Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd edition, Vol.
  • a polypeptide used in the present invention is encoded by a nucleic acid molecule that hybridizes under highly or moderately stringent conditions to a nucleic acid molecule encoding a polypeptide specifically described in the present invention. are also included.
  • a “purified” substance or biological factor refers to a substance from which at least a part of the factor naturally associated with the substance or biological factor has been removed. .
  • the purity of a biological agent in a purified biological agent is higher (ie, enriched) than the state in which the biological agent is normally present.
  • the term “purified” as used herein is preferably at least 75% by weight, more preferably at least 85% by weight, even more preferably at least 95% by weight, and most preferably at least 98% by weight, Means the presence of the same type of biological agent.
  • the substance or biological agent used in the present invention is preferably a “purified” substance.
  • an “isolated” substance or biological agent is substantially free of the factors that naturally accompany the substance or biological agent. Say things.
  • the term “isolated” as used herein does not necessarily have to be expressed in purity, as it will vary depending on its purpose, but is preferably at least 75% by weight, more preferably if necessary. Means that there is at least 85%, more preferably at least 95%, and most preferably at least 98% by weight of the same type of biological agent.
  • the materials used in the present invention are preferably “isolated” materials or biological agents.
  • a “corresponding” amino acid or nucleic acid or moiety refers to a predetermined amino acid or polypeptide in a reference polypeptide or polynucleotide for comparison in a polypeptide molecule or polynucleotide molecule (eg, CCL21, CXCR3, CCR7).
  • an antisense molecule can be a similar part in an ortholog corresponding to a particular part of the antisense molecule.
  • Corresponding amino acids are, for example, cysteinylated, glutathione, SS bond formation, oxidation (eg, oxidation of methionine side chain), formylation, acetylation, phosphorylation, glycosylation, myristylation, etc. Of amino acids.
  • the corresponding amino acid can be an amino acid responsible for dimerization.
  • Such “corresponding” amino acids or nucleic acids may be regions or domains spanning a range. Thus, in such cases, it is referred to herein as a “corresponding” region or domain. Such corresponding regions or domains are useful when designing complex molecules in the present invention.
  • a “corresponding” gene eg, a polynucleotide sequence or molecule
  • a gene for example, a polynucleotide sequence or a molecule
  • a gene corresponding to a gene can be an ortholog of that gene. Therefore, human CCL21, CXCR3, and CCR7 can find the corresponding CCL21, CXCR3, and CCR7, respectively, in other animals (particularly mammals).
  • Such corresponding genes can be identified using techniques well known in the art.
  • a corresponding gene in an animal is a reference gene of the corresponding gene (for example, CCL21, CXCR3, CCR7, etc.) is SEQ ID NO: 1, 3, 5 or SEQ ID NO: 2, 4 , 6 etc. as a query sequence and can be found by searching a database containing the animal's sequence.
  • fragment refers to a polypeptide or polynucleotide having a sequence length of 1 to n ⁇ 1 with respect to a full-length polypeptide or polynucleotide (length is n).
  • the length of the fragment can be appropriately changed according to the purpose.
  • the lower limit of the length is 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50 and more amino acids, and lengths expressed in integers not specifically listed here (eg 11 etc.) are also suitable as lower limits obtain.
  • examples include 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 75, 100 and more nucleotides.
  • Non-integer lengths may also be appropriate as a lower limit.
  • such a fragment falls within the scope of the present invention as long as the full-length fragment functions as a marker or target molecule as long as the fragment itself also functions as a marker or target molecule. Is understood.
  • the term “activity” refers herein to the function of a molecule in the broadest sense. Activity is not intended to be limiting, but generally includes the biological function, biochemical function, physical function or chemical function of a molecule. Activity activates, promotes, stabilizes, inhibits, suppresses, or destabilizes, for example, enzyme activity, the ability to interact with other molecules, and the function of other molecules Ability, stability, and ability to localize to specific intracellular locations. Where applicable, the term also relates to the function of the protein complex in the broadest sense.
  • biological function refers to a specific function that a gene, nucleic acid molecule or polypeptide may have in vivo when referring to a gene or a nucleic acid molecule or polypeptide related thereto.
  • the antibody include, but are not limited to, generation of specific antibodies, enzyme activity, and imparting resistance.
  • biological activity refers to activity that a certain factor (eg, polynucleotide, protein, etc.) may have in vivo, and exhibits various functions (eg, transcription promoting activity). For example, an activity in which another molecule is activated or inactivated by interaction with one molecule.
  • the biological activity can be the binding between the two molecules and the resulting biological change, and for example, one molecule was precipitated using an antibody Sometimes two molecules are considered bound when other molecules co-precipitate. Therefore, seeing such coprecipitation is one of the judgment methods.
  • a factor is an enzyme
  • the biological activity includes the enzyme activity.
  • an agent is a ligand
  • the ligand includes binding to the corresponding receptor.
  • Such biological activity can be measured by techniques well known in the art.
  • “activity” indicates or reveals binding (either direct or indirect); affects the response (ie, has a measurable effect in response to some exposure or stimulus), refers to various measurable indicators, such as the affinity of a compound that directly binds to a polypeptide or polynucleotide of the invention, or the amount of protein upstream or downstream after some stimulation or event or other A measure of similar function.
  • expression of a gene, polynucleotide, polypeptide or the like means that the gene or the like undergoes a certain action in vivo to take another form.
  • a gene, a polynucleotide or the like is transcribed and translated into a polypeptide form.
  • transcription and production of mRNA are also an aspect of expression.
  • expression product includes such a polypeptide or protein, or mRNA. More preferably, such polypeptide forms may be post-translationally processed. For example, the expression levels of CCL21, CXCR3, CCR7, etc. can be determined by any method.
  • the amount of mRNA such as CCL21, CXCR3, and CCR7 by evaluating the amount of mRNA such as CCL21, CXCR3, and CCR7, the amount of protein such as CCL21, CXCR3, and CCR7, and the biological activity of the protein such as CCL21, CXCR3, and CCR7, CCL21, CXCR3,
  • the expression level of CCR7 etc. can be known. Such measurements can be used in companion diagnostics.
  • the amount of mRNA or protein such as CCL21, CXCR3, CCR7 and the like can be determined by a method as described in detail elsewhere in this specification or other methods known in the art.
  • the “functional equivalent” refers to any target having the same target function but a different structure from the original target entity. Therefore, “CCL21, CXCR3, CCR7, etc.” or a functional equivalent of an antibody thereof is not CCL21, CXCR3, CCR7, etc. or the antibody itself, but CCL21, CXCR3, CCR7 etc. or a variant or variant of the antibody ( (For example, amino acid sequence variants) having the biological action of CCL21, CXCR3, CCR7, etc. or an antibody thereof, and CCL21, CXCR3, CCR7, etc. Those that can be changed to CCL21, CXCR3, CCR7, etc.
  • CCL21, CXCR3, CCR7, etc. or the antibody itself, or CCL21, CXCR3, CCR7, etc., or a variant or modification of the antibody thereof Body Vectors containing nucleic acids, and the nucleic acids, including cells, etc. are understood to be included.
  • CCL21, CXCR3, CCR7 or the like or a functional equivalent thereof can be used in the same manner as CCL21, CXCR3, CCR7 or the like or an antibody thereof, even if not specifically mentioned. Functional equivalents can be found by searching a database or the like.
  • search refers to finding another nucleobase sequence having a specific function and / or property using a nucleobase sequence electronically or biologically or by other methods.
  • Electronic searches include BLAST (Altschul et al., J. Mol. Biol. 215: 403-410 (1990)), FASTA (Pearson & Lipman, Proc. Natl. Acad. Sci., USA 85: 2444-2448 ( 1988), Smith and Waterman method (Smith and Waterman, J. Mol. Biol. 147: 195-197 (1981)), and Needleman and Wunsch method (Needleman and Wunsch, J. Mol. Biol. 48: 443-453 (1970)). ) And the like, but is not limited thereto.
  • Bio searches include stringent hybridization, macroarrays with genomic DNA affixed to nylon membranes, microarrays affixed to glass plates (microarray assays), PCR and in situ hybridization. It is not limited. In the present specification, it is intended that the gene used in the present invention should include a corresponding gene identified by such an electronic search or biological search.
  • an amino acid sequence having one or more amino acid insertions, substitutions or deletions, or those added to one or both ends can be used.
  • “insertion, substitution or deletion of one or a plurality of amino acids, or addition to one or both ends thereof in an amino acid sequence” means a well-known technical method such as site-directed mutagenesis. It means that a modification has been made by substitution of a plurality of amino acids to the extent that can occur naturally by a method or by natural mutation.
  • the modified amino acid sequence has, for example, 1 to 30, preferably 1 to 20, more preferably 1 to 9, further preferably 1 to 5, particularly preferably 1 to 2 amino acid insertions, substitutions, or deletions. Lost or added to one or both ends thereof.
  • the modified amino acid sequence preferably has one or more (preferably 1 or several or 1, 2, 3, or 4) conservative substitutions in the amino acid sequence such as CCL21, CXCR3, CCR7, etc.
  • the amino acid sequence may have.
  • conservative substitution means substitution of one or more amino acid residues with another chemically similar amino acid residue so as not to substantially alter the function of the protein. For example, when a certain hydrophobic residue is substituted by another hydrophobic residue, a certain polar residue is substituted by another polar residue having the same charge, and the like. Functionally similar amino acids that can make such substitutions are known in the art for each amino acid.
  • non-polar (hydrophobic) amino acids such as alanine, valine, isoleucine, leucine, proline, tryptophan, phenylalanine, and methionine.
  • polar (neutral) amino acids include glycine, serine, threonine, tyrosine, glutamine, asparagine, and cysteine.
  • positively charged (basic) amino acids include arginine, histidine, and lysine.
  • negatively charged (acidic) amino acids include aspartic acid and glutamic acid.
  • inhibitor refers to a substance or factor that inhibits the biological action of a target entity (for example, receptor or cell) of the receptor or cell, and is also referred to as a suppressor or the like.
  • the inhibitor of CCL21, CXCR3, CCR7 etc. of the present invention may temporarily or permanently reduce or eliminate the function of the target CCL21, CXCR3, CCR7 etc. or cells expressing CCL21, CXCR3, CCR7 etc. It is a possible factor.
  • examples of such factors include, but are not limited to, antibodies, antigen-binding fragments thereof, derivatives thereof, functional equivalents, antisense, and nucleic acid forms such as RNAi factors such as siRNA.
  • the term “agonist” refers to a substance that expresses or enhances the biological action of a target entity (eg, receptor).
  • a target entity eg, receptor
  • synthetic antagonists and modified ones can be mentioned.
  • an antagonist can be included in the concept of an inhibitor (suppressor) or an inhibitory (suppressing) factor because it suppresses or inhibits a physiological phenomenon. Accordingly, as used herein, an antagonist is used interchangeably with “inhibitor”.
  • anti-CCL21 antibody, anti-CXCR3 antibody, anti-CCR7 antibody and the like includes an antibody having binding properties to CCL21, CXCR3, CCR7 and the like.
  • the production method of the anti-CCL21 antibody, anti-CXCR3 antibody, anti-CCR7 antibody and the like is not particularly limited.
  • the “functional equivalent” of “an antibody against CCL21, CXCR3, CCR7, etc. (anti-CCL21 antibody, anti-CXCR3 antibody, anti-CCR7 antibody, etc.) or fragment thereof” is, for example, CCL21, CXCR3,
  • binding activity such as CCR7, if necessary, antibody itself and its fragment itself, chimeric antibody, humanized antibody, multifunctional antibody, bispecific or oligospecific antibody, single chain antibody , ScFV, diabody, sc (Fv) 2 (single chain (Fv) 2 ), scFv-Fc and the like are also encompassed.
  • the anti-CCL21 antibody, anti-CXCR3 antibody, anti-CCR7 antibody and the like according to one embodiment of the present invention are specifically specific to specific epitopes such as CCL21, CXCR3, and CCR7 from the viewpoint of particularly strongly suppressing the growth of malignant tumors.
  • Anti-CCL21 antibody, anti-CXCR3 antibody, anti-CCR7 antibody and the like that bind are preferable.
  • the anti-CCL21 antibody, anti-CXCR3 antibody, anti-CCR7 antibody and the like may be monoclonal antibodies. If it is a monoclonal antibody, it can be made to act on CCL21, CXCR3, CCR7 etc. efficiently compared with a polyclonal antibody. From the viewpoint of efficiently producing anti-CCL21 monoclonal antibody, anti-CXCR3 monoclonal antibody, anti-CCR7 monoclonal antibody and the like, it is preferable to immunize chickens with CCL21, CXCR3, CCR7 and the like.
  • the antibody class such as anti-CCL21 antibody, anti-CXCR3 antibody, and anti-CCR7 antibody according to an embodiment of the present invention is not particularly limited, but may be, for example, IgM, IgD, IgG, IgA, IgE, or IgY.
  • the anti-CCL21 antibody, anti-CXCR3 antibody, anti-CCR7 antibody and the like may be an antibody fragment having an antigen-binding activity (hereinafter also referred to as “antigen-binding fragment”).
  • antigen-binding fragment an antibody fragment having an antigen-binding activity
  • the anti-CCL21 antibody, anti-CXCR3 antibody, anti-CCR7 antibody and the like may be a fusion protein.
  • This fusion protein may be a polypeptide or oligopeptide bound to the N- or C-terminus of anti-CCL21 antibody, anti-CXCR3 antibody, anti-CCR7 antibody or the like.
  • the oligopeptide may be a His tag.
  • the fusion protein may be a fusion of a mouse, human, or chicken antibody partial sequence.
  • Such fusion proteins are also included in one form of the anti-CCL21 antibody, anti-CXCR3 antibody, anti-CCR7 antibody and the like according to this embodiment.
  • Anti-CCL21 antibody, anti-CXCR3 antibody, anti-CCR7 antibody and the like include, for example, purified CCL21, CXCR3, CCR7, etc., expressing cells such as CCL21, CXCR3, CCR7, etc., or CCL21, CXCR3, CCR7, etc. It may be an antibody obtained through a step of immunizing an organism with the containing lipid membrane. From the viewpoint of enhancing the therapeutic effect on positive malignant tumors such as CCL21, CXCR3, and CCR7, it is preferable to use cells expressing CCL21, CXCR3, and CCR7 for immunization.
  • Anti-CCL21 antibody, anti-CXCR3 antibody, anti-CCR7 antibody and the like are purified CCL21, CXCR3, CCR7, etc., expressed cells such as CCL21, CXCR3, CCR7, etc. or lipid membranes containing CCL21, CXCR3, CCR7, etc. It may be an antibody having a CDR set of an antibody obtained through a step of immunizing an organism. From the viewpoint of enhancing the therapeutic effect on positive malignant tumors such as CCL21, CXCR3, and CCR7, it is preferable to use cells expressing CCL21, CXCR3, and CCR7 for immunization.
  • a CDR set is a set of heavy chain CDRs 1, 2, and 3 and light chain CDRs 1, 2, and 3.
  • “expressing cells such as CCL21, CXCR3, CCR7” are expressed by, for example, introducing CCL21, CXCR3, CCR7, etc. into a cell and then expressing CCL21, CXCR3, CCR7, etc. May be obtained.
  • CCL21, CXCR3, CCR7 and the like include fragments such as CCL21, CXCR3 and CCR7.
  • “CCL21, CXCR3, CCR7, etc.-containing lipid membrane” may be obtained, for example, by mixing CCL21, CXCR3, CCR7, etc. with a lipid bilayer membrane.
  • CCL21, CXCR3, CCR7 and the like include fragments such as CCL21, CXCR3 and CCR7.
  • the anti-CCL21 antibody, anti-CXCR3 antibody, anti-CCR7 antibody and the like include a step of immunizing a chicken with an antigen from the viewpoint of enhancing the therapeutic effect on positive malignant tumors such as CCL21, CXCR3, and CCR7 An antibody obtained via the antibody or an antibody having a CDR set of the antibody is preferable.
  • the anti-CCL21 antibody, anti-CXCR3 antibody, anti-CCR7 antibody and the like may have any binding force as long as the object is achieved, for example, at least 1.0 ⁇ 10 6.
  • 2.0 ⁇ 10 6 or more, 5.0 ⁇ 10 6 or more, 1.0 ⁇ 10 7 or more can be mentioned, but it is not limited to these, and usually the KD value is 1.0 ⁇ 10 7. It may be the above.
  • the anti-CCL21 antibody, anti-CXCR3 antibody, anti-CCR7 antibody and the like may have ADCC or CDC activity.
  • the anti-CCL21 antibody, anti-CXCR3 antibody, anti-CCR7 antibody and the like may be antibodies that bind to wild-type or mutant types such as CCL21, CXCR3, and CCR7. Variants include those resulting from differences in DNA sequences between individuals.
  • the amino acid sequence of wild-type or mutant CCL21, CXCR3, CCR7, etc. is preferably 80% or more, more preferably 90% or more, more preferably 95% with respect to the amino acid sequence shown in SEQ ID NOs: 2, 4, 6, etc. As described above, the homology is particularly preferably 98% or more.
  • antibody includes a molecule or population thereof that can specifically bind to a particular epitope on an antigen.
  • the antibody may be a polyclonal antibody or a monoclonal antibody.
  • the antibody can exist in various forms, for example, full-length antibody (an antibody having Fab region and Fc region), Fv antibody, Fab antibody, F (ab ′) 2 antibody, Fab ′ antibody, diabody, single Chain antibodies (for example, scFv), dsFv, multivalent specific antibodies (for example, bivalent specific antibodies), peptides or polypeptides having antigen binding properties, chimeric antibodies (for example, mouse-human chimeric antibodies, chicken-human chimeric antibodies, etc.) ), One or more forms selected from the group consisting of mouse antibodies, chicken antibodies, humanized antibodies, human antibodies, or their equivalents (or equivalents).
  • the antibody includes an antibody modified product or an antibody unmodified product.
  • an antibody and various molecules such as polyethylene glycol may be bound.
  • the modified antibody can be obtained by chemically modifying the antibody using a known technique.
  • such antibodies may be covalently linked or recombinantly fused to enzymes such as alkaline phosphatase, horseradish peroxidase, alpha galactosidase, and the like.
  • the anti-CCL21 antibody, anti-CXCR3 antibody, anti-CCR7 antibody, etc. used in the present invention may be bound to proteins such as CCL21, CXCR3, CCR7, etc., and their origin, type, shape, etc. are not questioned.
  • known antibodies such as non-human animal antibodies (eg, mouse antibodies, rat antibodies, camel antibodies), human antibodies, chimeric antibodies, and humanized antibodies can be used.
  • monoclonal or polyclonal antibodies can be used as antibodies, but monoclonal antibodies are preferred.
  • the binding of the antibody to a protein such as CCL21, CXCR3, CCR7 is preferably specific binding.
  • the antibody includes an antibody modified product or an antibody unmodified product.
  • an antibody and various molecules such as polyethylene glycol may be bound.
  • the modified antibody can be obtained by chemically modifying the antibody using a known technique.
  • a “polyclonal antibody” refers to, for example, mammals (eg, rats, mice, rabbits, cows, monkeys, etc.), birds, etc. in order to induce the production of polyclonal antibodies specific to the antigen. It can be generated by administering an immunogen containing the antigen of interest. Administration of the immunogen may involve infusion of one or more immunizing agents and, if desired, an adjuvant.
  • An adjuvant may be used to increase the immune response and may include Freund's adjuvant (complete or incomplete), mineral gel (such as aluminum hydroxide), or a surfactant (such as lysolecithin). .
  • Immunization protocols are known in the art and may be performed by any method that elicits an immune response depending on the host organism chosen (Protein Experiment Handbook, Yodosha (2003): 86-91). .).
  • a “monoclonal antibody” is an antibody in which the individual antibodies constituting the population substantially correspond to a single epitope, except for antibodies having mutations that can naturally occur in small amounts. Including the case of Alternatively, the individual antibodies that make up the population may be antibodies that are substantially identical except for antibodies that have mutations that can occur naturally in small amounts. Monoclonal antibodies are highly specific and differ from normal polyclonal antibodies, which typically include different antibodies corresponding to different epitopes. In addition to its specificity, monoclonal antibodies are useful in that they can be synthesized from hybridoma cultures that are not contaminated by other immunoglobulins.
  • the form “monoclonal” may be characterized as being derived from a substantially homogeneous population of antibodies, but does not mean that the antibodies must be produced in any particular way.
  • the monoclonal antibody may be prepared by a method similar to the hybridoma method described in “Kohler G, Milstein C., Nature. 1975 Aug7; 256 (5517): 495-497.”.
  • monoclonal antibodies may be produced by methods similar to recombinant methods such as those described in US Pat. No. 4,816,567.
  • the monoclonal antibody may be “Clacksonet al., Nature. 1991 Aug 15; 352 (6336): 624-628.” Or “Markset al., JMol Biol.
  • any technique known in the art can be used.
  • the construction of a typical mass production system of antibodies and the production of antibodies can be exemplified as follows. That is, CHO cells are transfected with an H chain antibody expression vector and an L chain antibody expression vector, cultured using selection reagents G418 and Zeocin, and cloned by limiting dilution. After cloning, clones that stably express the antibody are selected by ELISA. The selected CHO cells are expanded and cultured, and the culture supernatant containing the antibody is collected. The antibody can be purified from the collected culture supernatant by Protein A or Protein G purification.
  • the “Fv antibody” is an antibody containing an antigen recognition site. This region contains a dimer of one heavy chain variable domain and one light chain variable domain by non-covalent bonds. In this configuration, the three CDRs of each variable domain can interact to form an antigen binding site on the surface of the VH-VL dimer.
  • the “Fab antibody” refers to, for example, a fragment obtained by treating an antibody containing a Fab region and an Fc region with proteolytic enzyme papain, about half of the N-terminal side of the H chain and the entire L chain. Is an antibody bound through some disulfide bonds.
  • the Fab can be obtained, for example, by treating the anti-CCL21 antibody, anti-CXCR3 antibody, anti-CCR7 antibody or the like according to the embodiment of the present invention containing the Fab region and the Fc region with the proteolytic enzyme papain.
  • F (ab ′) 2 antibody refers to, for example, 2 sites corresponding to Fab in a fragment obtained by treating an antibody containing a Fab region and an Fc region with proteolytic enzyme pepsin.
  • One antibody. F (ab ′) 2 can be obtained, for example, by treating the anti-CCL21 antibody, anti-CXCR3 antibody, anti-CCR7 antibody, etc. according to the embodiment of the present invention containing the Fab region and the Fc region with the proteolytic enzyme pepsin. . Further, for example, it can be prepared by linking the following Fab ′ with a thioether bond or a disulfide bond.
  • Fab ′ antibody is, for example, an antibody obtained by cleaving a disulfide bond in the hinge region of F (ab ′) 2 .
  • F (ab ′) 2 can be obtained by treating with a reducing agent dithiothreitol.
  • the “scFv antibody” is an antibody in which VH and VL are linked via an appropriate peptide linker.
  • scFv antibody obtains cDNA encoding VH and VL such as anti-CCL21 antibody, anti-CXCR3 antibody, and anti-CCR7 antibody according to the embodiment of the present invention, and constructs a polynucleotide encoding VH-peptide linker-VL Then, the polynucleotide can be incorporated into a vector and produced using expression cells.
  • “diabody” is an antibody having a bivalent antigen binding activity.
  • the bivalent antigen binding activity can be the same, or one can be a different antigen binding activity.
  • Diabody for example, constructs a polynucleotide encoding scFv so that the length of the amino acid sequence of the peptide linker is 8 residues or less, incorporates the obtained polynucleotide into a vector, and produces it using cells for expression. it can.
  • “dsFv” is an antibody in which a polypeptide in which a cysteine residue is introduced into VH and VL is bound via a disulfide bond between the cysteine residues.
  • the position to be introduced into the cysteine residue can be selected based on the three-dimensional structure prediction of the antibody according to the method shown by Reiter et al. (Reiter et al., Protein Eng. it can.
  • an “antigen-binding peptide or polypeptide” is an antibody comprising antibody VH, VL, or CDR1, 2, or 3 thereof. Peptides containing multiple CDRs can be linked directly or via a suitable peptide linker.
  • Fv antibody Fab antibody, F (ab ′) 2 antibody, Fab ′ antibody, scFv antibody, diabody, dsFv antibody, peptide or polypeptide having antigen binding properties (hereinafter also referred to as “Fv antibody etc.”)
  • Production method is not particularly limited.
  • DNA encoding a region such as an Fv antibody in the anti-CCL21 antibody, anti-CXCR3 antibody, anti-CCR7 antibody and the like can be incorporated into an expression vector and produced using an expression cell.
  • the antigen-binding fragment according to one embodiment of the present invention may be one or more of the above Fv antibodies.
  • a “chimeric antibody” is, for example, a variable region of an antibody between heterologous organisms and a constant region of the antibody, and can be constructed by a gene recombination technique.
  • the mouse-human chimeric antibody can be prepared by the method described in, for example, “Roguska et al., ProcNatl Acad Sci US A. 1994 Feb 1:91 (3): 969-973”.
  • the basic method for making a mouse-human chimeric antibody is, for example, encoding the mouse leader and variable region sequences present in the cloned cDNA, and the human antibody constant region already present in the expression vector of a mammalian cell.
  • the mouse leader sequence and variable region sequence present in the cloned cDNA may be linked to a sequence encoding a human antibody constant region and then linked to a mammalian cell expression vector.
  • Human antibody constant region fragments can be of any human antibody heavy chain constant region and human antibody light chain constant region, eg, for human heavy chain, C ⁇ 1, C ⁇ 2, C ⁇ 3 or C ⁇ 4, For the light chain, C ⁇ or C ⁇ can be mentioned, respectively.
  • a “humanized antibody” has, for example, one or more CDRs derived from a non-human species, a framework region (FR) derived from a human immunoglobulin, and a constant region derived from a human immunoglobulin. And an antibody that binds to the desired antigen.
  • Antibody humanization can be performed using various techniques known in the art (Almagroet al., FRONt Biosci. 2008 Jan 1; 13: 1619-1633.). For example, CDR grafting (Ozaki et al., Blood. 1999 Jun 1; 93 (11): 3922-3930.), Re-surfacing (Roguska et al., Proc Natl Acad Sci US A.
  • FR shuffle Damschroder et al., Mol Immunol. 2007 Apr; 44 (11): 3049-3060.Epub 2007 Jan 22.
  • amino acid residues of the human FR region may be replaced with corresponding residues from the CDR donor antibody. This FR substitution can be performed by methods well known in the art (Riechmann et al., Nature. 1988 Mar 24; 332 (6162): 323-327.).
  • FR residues important for antigen binding may be identified by modeling the interaction of CDR and FR residues.
  • an abnormal FR residue at a particular position may be identified by sequence comparison.
  • a “human antibody” is derived from a gene encoding human immunoglobulin, for example, the variable region and the constant region of the heavy chain and the region including the variable region and the constant region of the light chain that constitute the antibody.
  • the main production methods include a transgenic mouse method for producing human antibodies, a phage display method, and the like.
  • human antibodies having various antigen-binding abilities can be produced instead of the mouse antibody.
  • a human monoclonal antibody can be obtained by a conventional hybridoma method.
  • phage display method a foreign gene is fused to the N-terminal side of the coat protein (g3p, g10p, etc.) of filamentous phages such as M13 and T7, which are typically E. coli viruses. It is a system that expresses as protein. For example, it can be prepared by the method described in “Vaughanet al., Nat Biotechnol. 1996 Mar; 14 (3): 309-314.”.
  • the antibody may be any antibody by CDR-grafting (Ozaki et al., Blood. 1999 Jun 1; 93 (11): 3922-3930.), And the anti-CCL21 antibody, anti-CXCR3 antibody, anti-CXCR3 antibody, You may produce by grafting heavy chain CDR or light chain CDR, such as CCR7 antibody.
  • a heavy chain of a DNA encoding a heavy chain CDR or a light chain CDR such as an anti-CCL21 antibody, an anti-CXCR3 antibody, or an anti-CCR7 antibody according to an embodiment of the present invention, and an antibody derived from a known human or non-human organism
  • a DNA encoding a region excluding the CDR or light chain CDR can be obtained by ligation to a vector according to a method known in the art and then expression using a known cell.
  • a method known in the art for example, the amino acid residue of the antibody is randomly mutated to react with the reactive antigen.
  • the region excluding the heavy chain CDR or the light chain CDR may be optimized using a method for screening a high one or a phage display method or the like.
  • FR shuffle Damschroderet al., Mol Immunol.
  • the FR region may be optimized using JP-A-2006-241026, or Footet et al., J Mol Biol. 1992 Mar 20; 224 (2): 487-499.).
  • the “heavy chain” is typically the main component of a full-length antibody.
  • the heavy chain is usually linked to the light chain by disulfide bonds and non-covalent bonds.
  • the domain on the N-terminal side of the heavy chain has a region called a variable region (VH) in which the amino acid sequence is not constant even with antibodies of the same type and the same class.
  • VH variable region
  • a “CDR (complementarity determining region)” is a region in an antibody that is actually in contact with an antigen to form a binding site.
  • the CDRs are located on the Fv (variable region: including heavy chain variable region (VH) and light chain variable region (VL)) of the antibody.
  • VH heavy chain variable region
  • VL light chain variable region
  • CDR1, CDR2, and CDR3 consisting of about 5 to 30 amino acid residues.
  • CDRs of heavy chains contribute to the binding of antibodies to antigens.
  • CDR3 contributes most to the binding of an antibody to an antigen.
  • FR regions other than CDRs are called framework regions (FR) and are composed of FR1, FR2, FR3, and FR4 and are relatively well conserved among antibodies (Kabatet al., “Sequence of Proteins of Immunological Interest” USDept. .Health and Human Services, 1983.). That is, it can be said that the factor characterizing the reactivity of the antibody is in the CDR, particularly in the heavy chain CDR.
  • Kabat Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD. (1991)
  • Chothia Chothia et al., J. Mol. Biol., 1987; 196: 901-917
  • the Kabat definition is adopted as a preferred example, but is not necessarily limited thereto. In some cases, it may be determined in consideration of both the Kabat definition and the Chothia definition.
  • an overlapping part of CDRs according to each definition or a part including both CDRs according to each definition can also be a CDR.
  • a CDR As a specific example of such a method, Martin et al. (Proc. Natl. Acad. Sci. 86: 9268-9272).
  • Such CDR information can be used to produce variants that can be used in the present invention.
  • one or several eg, 2, 3, 4, 5, 6, 7, 8, 9, 10 in the framework of the original antibody).
  • Substitutions, additions or deletions, but the CDRs can be produced without mutations.
  • antigen refers to any substrate that can be specifically bound by an antibody molecule.
  • immunogen refers to an antigen capable of initiating lymphocyte activation that produces an antigen-specific immune response.
  • epitope or “antigenic determinant” refers to a site in an antigen molecule to which an antibody or lymphocyte receptor binds. Methods for determining epitopes are well known in the art, and such epitopes can be determined by those skilled in the art using such well known techniques once the primary sequence of the nucleic acid or amino acid is provided. It will be understood that the antibodies of the present invention can be used in the same manner even if they have the same epitope, even antibodies having other sequences.
  • the antibody used in the present invention may be a polyclonal antibody or a monoclonal antibody.
  • “means” refers to any tool that can achieve a certain purpose (for example, detection, diagnosis, treatment).
  • a certain purpose for example, detection, diagnosis, treatment.
  • “means for selectively recognizing” A means by which one object can be recognized differently from another.
  • the term “marker (substance or gene)” refers to whether or not there is a certain state (for example, the level of disease state, disorder state, or malignant state of cerebral malaria, or its risk). This refers to the substance that will be tracked. Such markers can include genes, gene products, metabolites, enzymes, and the like. In the present invention, detection, diagnosis, preliminary detection, prediction or pre-diagnosis for a certain state (for example, a disease state such as cerebral malaria) is a drug, agent, factor or means specific for the marker associated with the state. Or a composition, kit or system containing them.
  • expression product also referred to as gene product refers to a protein or mRNA encoded by a gene. In the present specification, it has been found that gene products (CCL21, CXCR3, CCR7, etc.) that have not been shown to be related to cerebral malaria can be used as indicators of cerebral malaria.
  • the “subject (person)” refers to a subject to be diagnosed or detected or treated according to the present invention (for example, an organism such as a human or a cell, blood, serum, etc. removed from the organism). .
  • sample refers to any substance obtained from a subject or the like, and includes, for example, serum. Those skilled in the art can appropriately select a preferable sample based on the description of the present specification.
  • drug drug
  • drug may also be a substance or other element (eg energy such as light, radioactivity, heat, electricity).
  • Such substances include, for example, proteins, polypeptides, oligopeptides, peptides, polynucleotides, oligonucleotides, nucleotides, nucleic acids (eg, DNA such as cDNA, genomic DNA, RNA such as mRNA), poly Saccharides, oligosaccharides, lipids, small organic molecules (for example, hormones, ligands, signaling substances, small organic molecules, molecules synthesized by combinatorial chemistry, small molecules that can be used as pharmaceuticals (for example, small molecule ligands, etc.)) , These complex molecules are included, but not limited thereto.
  • a polynucleotide having a certain sequence homology to the sequence of the polynucleotide (for example, 70% or more sequence identity) and complementarity examples include, but are not limited to, a polypeptide such as a transcription factor that binds to the promoter region.
  • Factors specific for a polypeptide typically include an antibody specifically directed against the polypeptide or a derivative or analog thereof (eg, a single chain antibody), and the polypeptide is a receptor.
  • specific ligands or receptors in the case of ligands, and substrates thereof when the polypeptide is an enzyme include, but are not limited to.
  • diagnosis identifies various parameters related to a disease, disorder, condition (eg, cerebral malaria), etc. in a subject, and determines the current state or future of such a disease, disorder, or condition. That means.
  • conditions within the body can be examined, and such information can be used to formulate a disease, disorder, condition, treatment to be administered or prevention in a subject.
  • various parameters such as methods can be selected.
  • diagnosis in a narrow sense means diagnosis of the current state, but in a broad sense includes “early diagnosis”, “predictive diagnosis”, “preliminary diagnosis”, and the like.
  • the diagnostic method of the present invention is industrially useful because, in principle, the diagnostic method of the present invention can be used from the body and can be performed away from the hands of medical personnel such as doctors.
  • diagnosis, prior diagnosis or diagnosis may be referred to as “support”.
  • detection agent or “test agent (agent)” refers to any agent that can detect or inspect a target object in a broad sense.
  • diagnostic agent refers to any agent that can diagnose a target condition (for example, a disease such as cerebral malaria) in a broad sense.
  • treatment refers to prevention of worsening of a disease or disorder when it becomes such a condition or disease (for example, cerebral malaria). Preferably, it refers to reduction, more preferably elimination, and includes the ability to exert a symptom-improving effect or a preventive effect on one or more symptoms associated with a patient's disease or disease. Diagnosing in advance and performing appropriate treatment is referred to as “companion treatment”, and the diagnostic agent therefor is sometimes referred to as “companion diagnostic agent”.
  • the term “therapeutic agent (agent)” broadly refers to any drug capable of treating a target condition (for example, a disease such as cerebral malaria), and an inhibitor (for example, provided by the present invention) Antibody).
  • the “therapeutic agent” may be a pharmaceutical composition comprising an active ingredient and one or more pharmacologically acceptable carriers.
  • the pharmaceutical composition can be produced by any method known in the technical field of pharmaceutics, for example, by mixing the active ingredient and the carrier.
  • the form of use of the therapeutic agent is not limited as long as it is a substance used for treatment, and it may be an active ingredient alone or a mixture of an active ingredient and an arbitrary ingredient.
  • the shape of the carrier is not particularly limited, and may be, for example, a solid or a liquid (for example, a buffer solution).
  • prevention means that a certain disease or disorder (for example, cerebral malaria) is prevented from becoming such a state before it becomes such a state. Diagnosis can be performed using the drug of the present invention, and for example, cerebral malaria can be prevented or a preventive measure can be taken using the drug of the present invention as necessary.
  • a certain disease or disorder for example, cerebral malaria
  • prophylactic agent refers to any agent that can prevent a target condition (for example, a disease such as cerebral malaria) in a broad sense.
  • interaction refers to two substances. Force (for example, intermolecular force (van der Waals force), hydrogen bond, hydrophobic interaction between one substance and the other substance. Etc.). Usually, two interacting substances are in an associated or bound state. The detection, inspection and diagnosis of the present invention can be realized by utilizing such interaction.
  • bond means a physical or chemical interaction between two substances or a combination thereof. Bonds include ionic bonds, non-ionic bonds, hydrogen bonds, van der Waals bonds, hydrophobic interactions, and the like.
  • a physical interaction (binding) can be direct or indirect, where indirect is through or due to the effect of another protein or compound. Direct binding refers to an interaction that does not occur through or due to the effects of another protein or compound and does not involve other substantial chemical intermediates.
  • a “factor” (or drug, detection agent, etc.) that interacts (or binds) “specifically” to a biological agent such as a polynucleotide or a polypeptide is defined as that
  • the affinity for a biological agent such as a nucleotide or polypeptide thereof is typically equal or greater than the affinity for other unrelated (especially less than 30% identity) polynucleotides or polypeptides. Includes those that are high or preferably significantly (eg, statistically significant). Such affinity can be measured, for example, by hybridization assays, binding assays, and the like.
  • a first substance or factor interacts (or binds) “specifically” to a second substance or factor means that the first substance or factor has a relationship to the second substance or factor. Interact (or bind) with a higher affinity than a substance or factor other than the second substance or factor (especially other substances or factors present in the sample containing the second substance or factor) That means.
  • Specific interactions (or bindings) for substances or factors include, for example, hybridization in nucleic acids, antigen-antibody reactions in proteins, enzyme-substrate reactions, nucleic acid and protein reactions, protein-lipid interactions, nucleic acid-lipids Examples include, but are not limited to, interactions.
  • the first substance or factor “specifically interacts” with the second substance or factor means that the first substance or factor has the second substance Or having at least a part of complementarity to the factor.
  • both substances or factors are proteins
  • the fact that the first substance or factor interacts (or binds) “specifically” to the second substance or factor is, for example, by antigen-antibody reaction Examples include, but are not limited to, interactions by receptor-ligand reactions, enzyme-substrate interactions, and the like.
  • the first substance or factor interacts (or binds) “specifically” to the second substance or factor by means of an antibody and its antigen Interaction (or binding) between is included.
  • an object in a sample can be detected or quantified.
  • detection or “quantification” of polynucleotide or polypeptide expression includes mRNA measurement and immunological measurement methods, including, for example, binding or interaction with a detection agent, test agent or diagnostic agent. It can be achieved using any suitable method.
  • molecular biological measurement methods include Northern blotting, dot blotting, and PCR.
  • immunological measurement methods include ELISA using a microtiter plate, RIA, fluorescent antibody method, luminescence immunoassay (LIA), immunoprecipitation (IP), immunodiffusion method (SRID), immunization. Examples are turbidimetry (TIA), Western blotting, immunohistochemical staining, and the like.
  • Examples of the quantitative method include an ELISA method and an RIA method. It can also be performed by a gene analysis method using an array (eg, DNA array, protein array).
  • the DNA array is widely outlined in (edited by Shujunsha, separate volume of cell engineering "DNA microarray and latest PCR method”).
  • Examples of gene expression analysis methods include, but are not limited to, RT-PCR, RACE method, SSCP method, immunoprecipitation method, two-hybrid system, in vitro translation and the like.
  • expression level refers to the amount of polypeptide or mRNA expressed in a target cell, tissue or the like. Such expression level is evaluated by any appropriate method including immunoassay methods such as ELISA method, RIA method, fluorescent antibody method, Western blot method, and immunohistochemical staining method using the antibody of the present invention. The amount of expression of the polypeptide of the present invention at the protein level, or the polypeptide used in the present invention evaluated by any suitable method including molecular biological measurement methods such as Northern blotting, dot blotting, and PCR. The expression level of the peptide at the mRNA level can be mentioned.
  • “Change in expression level” means expression at the protein level or mRNA level of the polypeptide used in the present invention evaluated by any appropriate method including the above immunological measurement method or molecular biological measurement method. Means that the amount increases or decreases. By measuring the expression level of a certain marker, various detection or diagnosis based on the marker can be performed.
  • “decrease” or “suppression” or synonyms for activity, expression products (eg, proteins, transcripts (RNA, etc.)) or a synonym is a decrease in the quantity, quality or effect of a particular activity, transcript or protein. Or activity to decrease. When “disappears” of the decrease, it means that the activity, the expression product, etc. are below the detection limit, and in particular, may be “disappear”. As used herein, “disappearance” is encompassed by “decrease” or “suppression”.
  • an expression product eg, a protein, transcript (such as RNA)
  • a synonym thereof refers to a quantity, quality or effect of a particular activity, transcript or protein.
  • nucleic acid primer refers to a substance necessary for the initiation of a reaction of a polymer compound to be synthesized in a polymer synthase reaction.
  • a nucleic acid molecule for example, DNA or RNA
  • the primer can be used as a marker detection means.
  • the term “probe” refers to a substance that serves as a search means used in biological experiments such as screening in vitro and / or in vivo.
  • a nucleic acid molecule containing a specific base sequence or a specific Examples include, but are not limited to, peptides containing amino acid sequences, specific antibodies or fragments thereof.
  • the probe is used as a means for marker detection, inspection or diagnosis.
  • the “label” refers to a presence (for example, a substance, energy, electromagnetic wave, etc.) for distinguishing a target molecule or substance from others.
  • a labeling method include RI (radioisotope) method, fluorescence method, biotin method, chemiluminescence method and the like.
  • the labeling is performed with fluorescent substances having different fluorescence emission maximum wavelengths. The difference in the maximum fluorescence emission wavelength is preferably 10 nm or more.
  • Alexa TM Fluor is preferred as the fluorescent substance.
  • Alexa TM Fluor is a water-soluble fluorescent dye obtained by modifying coumarin, rhodamine, fluorescein, cyanine, etc., and is a series corresponding to a wide range of fluorescent wavelengths. Compared with other applicable fluorescent dyes, Stable, bright and low pH sensitive. Examples of combinations of fluorescent dyes having a fluorescence maximum wavelength of 10 nm or more include a combination of Alexa TM 555 and Alexa TM 633, a combination of Alexa TM 488 and Alexa TM 555, and the like. Any nucleic acid can be used as long as it can bind to its base moiety.
  • cyanine dyes eg, CyDye TM series Cy3, Cy5, etc.
  • rhodamine 6G reagent 2-acetylaminofluorene ( AAF), AAIF (iodine derivative of AAF) and the like
  • the fluorescent substance having a difference in fluorescence emission maximum wavelength of 10 nm or more include a combination of Cy5 and rhodamine 6G reagent, a combination of Cy3 and fluorescein, a combination of rhodamine 6G reagent and fluorescein, and the like.
  • the target object by using such a label, the target object can be modified so that it can be detected by the detection means used. Such modifications are known in the art, and those skilled in the art can appropriately carry out such methods depending on the label and the target object.
  • a “tag” is a substance for sorting molecules by a specific recognition mechanism such as a receptor-ligand, more specifically, a binding for binding a specific substance.
  • a substance that plays the role of a partner for example, having a relationship such as biotin-avidin, biotin-streptavidin
  • label can be included in the category of “label”.
  • a specific substance to which a tag is bound can be selected by bringing the substrate to which the binding partner of the tag sequence is bound into contact.
  • tags or labels are well known in the art.
  • Representative tag sequences include, but are not limited to, myc tag, His tag, HA, Avi tag and the like.
  • Such a tag may be bound to the marker of the present invention, a marker detection agent, a test agent, or a diagnostic agent (which may be a primer or a probe).
  • in vivo refers to the inside of a living body. In a particular context, “in vivo” refers to the location where a target substance is to be placed.
  • in vitro refers to a state in which a part of a living body is excised or released “outside the living body” (for example, in a test tube) for various research purposes. A term that contrasts with in vivo.
  • ex vivo refers to a series of operations ex vivo when a certain treatment is performed outside the body but is intended to be returned to the body afterwards. Also in the present invention, an embodiment in which cells in the living body are treated with the drug of the present invention and returned to the patient can be envisaged.
  • the “kit” is a unit provided with a portion to be provided (eg, a test agent, a diagnostic agent, a therapeutic agent, an antibody, a label, an instruction, etc.) usually divided into two or more compartments.
  • a portion to be provided eg, a test agent, a diagnostic agent, a therapeutic agent, an antibody, a label, an instruction, etc.
  • This kit form is preferred when it is intended to provide a composition that should not be provided in admixture for stability or the like, but preferably used in admixture immediately before use.
  • Such kits preferably include instructions or instructions that describe how to use the provided parts (eg, test agents, diagnostic agents, therapeutic agents, or how the reagents should be processed).
  • the kit when the kit is used as a reagent kit, the kit usually contains instructions including usage of test agents, diagnostic agents, therapeutic agents, antibodies, etc. Is included.
  • the “instruction sheet” describes the method for using the present invention for a doctor or other user.
  • This instruction manual includes a word indicating that the detection method of the present invention, how to use a diagnostic agent, or administration of a medicine or the like is given.
  • the instructions may include a word indicating that the administration site is oral or esophageal administration (for example, by injection).
  • This instruction is prepared in accordance with the format prescribed by the national supervisory authority (for example, the Ministry of Health, Labor and Welfare in Japan and the Food and Drug Administration (FDA) in the United States, etc. in the United States) where the present invention is implemented, and is approved by the supervisory authority. It is clearly stated that it has been received.
  • the instruction sheet is a so-called package insert and is usually provided as a paper medium, but is not limited thereto.
  • the instruction sheet may be in a form such as an electronic medium (for example, a home page or an e-mail provided on the Internet). Can be provided.
  • the present invention provides a method using the state of a subject's olfactory bulb (OLF) as an indicator of brain malaria.
  • the present invention provides a method for diagnosing cerebral malaria, comprising the step of determining the state of a subject's olfactory bulb (OLF).
  • OVF olfactory bulb
  • an abnormality of the subject's olfactory bulb is an indicator that the subject suffers from cerebral malaria.
  • Such a diagnosis should be noted as enabling an early diagnosis of cerebral malaria because it can be found before other symptoms of cerebral malaria occur. That is, the abnormality of the subject's olfactory bulb can be said to be an early indicator of cerebral malaria.
  • the determination of the state of the olfactory bulb can be performed by various techniques, and early administration of cerebral malaria and administration of a preventive or therapeutic agent for cerebral malaria such as those provided for the first time by the present invention, It is also noted that it enables full-scale treatment of malaria.
  • the state of the subject's olfactory bulb (OLF) determined by the present invention can include, but is not limited to, olfactory abnormalities, cytological examination, and diagnostic images of the olfactory bulb. Therefore, the diagnostic method of the present invention includes a step of performing at least one selected from olfactory abnormalities, cytological examinations, diagnostic images of the olfactory bulb, and the like, and the result determines the state of the olfactory bulb (OLF) of the subject. Can be used as an indicator of early stage of cerebral malaria.
  • olfaction includes reactions to foods, reactions to fragrances, (please list other possible tests), etc.
  • olfactory measurement methods include standard olfactory tests for various odorous substances, alinamine, etc. Can be tested by a venous olfactory test, an ASTM syringe method, an odorless chamber method, a centmeter method, a saline balance method, a three-point comparison odor bag method, and the like.
  • the diagnostic image of the olfactory bulb may be obtained by nuclear magnetic resonance imaging (MRI), but is not limited thereto.
  • the “ultra-high magnetic field” nuclear magnetic resonance imaging (MRI) used by the present invention includes a magnetic field of 3 Tesla (T) currently used for clinical use or a high magnetic field exceeding it, for example, at least 3 Tesla or more.
  • MRI at least 4 Tesla, at least 5 Tesla, at least 6 Tesla, at least 7 Tesla, at least 8 Tesla, at least 9 Tesla, at least 10 Tesla, at least 11 Tesla, at least 12 Tesla be able to.
  • 11.7 Tesla is used, and the state of the olfactory bulb can be determined.
  • the observation of spots in the diagnostic image is an indicator of cerebral malaria.
  • a spot indicates that there is an abnormality in the olfactory bulb, and it has been clarified in the present invention that it is an index of brain malaria infection.
  • the state of the olfactory bulb may be determined by cytological examination, and such determination is examined by staining (for example, hematoxylin and eosin (HE) staining) or immunohistochemistry.
  • staining for example, hematoxylin and eosin (HE) staining
  • immunohistochemistry for example, immunohistochemistry.
  • the present invention provides an apparatus for detecting or diagnosing cerebral malaria, including means for measuring the state of the olfactory bulb. It will be understood that such an apparatus may be any apparatus that implements the above method.
  • the means used in the apparatus of the present invention is a magnetic resonance imaging (MRI) means.
  • the MRI used in the present invention is an ultra high magnetic field MRI.
  • Ordinary MRI did not capture abnormalities in the olfactory bulb, but in the present invention, by using MRI with an ultrahigh magnetic field, the relationship between abnormalities in the olfactory bulb and brain malaria could be elucidated for the first time. Therefore, it can be said that the present invention is remarkable in that MRI using an ultrahigh magnetic field is used.
  • the present invention relates to a prophylactic or therapeutic agent for cerebral malaria, a prophylactic or therapeutic agent, a prophylactic or therapeutic pharmaceutical agent or an inhibitor comprising an inhibitor of at least one factor of the CCL21-CXCR3-CCR7 signaling system.
  • a prophylactic or therapeutic agent for cerebral malaria a prophylactic or therapeutic agent, a prophylactic or therapeutic pharmaceutical agent or an inhibitor comprising an inhibitor of at least one factor of the CCL21-CXCR3-CCR7 signaling system.
  • pharmaceutical compositions (these terms are used interchangeably herein unless otherwise indicated) and refer to the same subject.
  • the present invention provides an inhibitor of at least one factor of the CCL21-CXCR3-CCR7 signaling system for the prevention or treatment of cerebral malaria. Brain malaria can be treated or prevented using this therapeutic or prophylactic agent or a specific therapeutic or prophylactic inhibitor.
  • this prophylactic or therapeutic agent is superior in that it provides a prophylactic and therapeutic agent for the first time for cerebral malaria, for which there has been no treatment / prevention method so far, and since an antibody is used when used in a preferred embodiment, Excellent from the viewpoint of safety.
  • the inhibitor of at least one factor of the CCL21-CXCR3-CCR7 signaling system used by the present invention is at least one selected from the group consisting of CCL21, CXCR3 and CCR7, and nucleic acids encoding them. Inhibitor of one factor.
  • the inhibitor of the present invention is a small molecule compound, antibody or fragment or functional equivalent thereof, siRNA, shRNA, antisense nucleic acid, aptamer, pharmaceutically acceptable salt or solvate thereof. Or at least one solvate of a pharmaceutically acceptable salt thereof. Two or more of these may be used.
  • the inhibitor used in the present invention comprises at least one antibody selected from the group consisting of anti-CCL21 antibody, anti-CXCR3 antibody, and anti-CCR7 antibody, or a fragment or functional equivalent thereof.
  • the inhibitor used in the present invention comprises at least two antibodies selected from the group consisting of anti-CCL21 antibody, anti-CXCR3 antibody and anti-CCR7 antibody, or fragments or functional equivalents thereof. .
  • the inhibitor used in the present invention comprises three antibodies, or a fragment or functional equivalent thereof, an anti-CCL21 antibody, an anti-CXCR3 antibody and an anti-CCR7 antibody.
  • An anti-CCL21 antibody, anti-CXCR3 antibody, anti-CCR7 antibody, etc. comprise a set of amino acid sequences of heavy chain CDRs 1, 2, and 3 and light chain CDRs 1, 2, and 3, At least one, preferably two, three, four, five, six, seven, or all frameworks of chain FR1, 2, 3, 4, light chain FR1, 2, 3, and 4 Can be the same or substantially the same as any of those specified or the same except for conservative substitutions.
  • One or more antibodies may be used.
  • Another embodiment of the present invention provides an anti-CCL21 antibody, an anti-CXCR3 antibody, an anti-CCR7 antibody, etc., comprising at least one of the amino acid sequence sets of heavy chains FR1, 2, 3, and 4 listed above. It is.
  • the anti-CCL21 antibody, anti-CXCR3 antibody, anti-CCR7 antibody and the like may be in the form of scFv, in which case the linker between the heavy chain and the light chain is a heavy chain and a light chain. It may have an amino acid sequence between As long as the anti-CCL21 antibody, anti-CXCR3 antibody, anti-CCR7 antibody, etc. have the desired effect, (i) one or several nucleotide sequences are deleted or substituted in the above amino acid sequences.
  • An inserted or added amino acid sequence (ii) an amino acid sequence having 90% or more homology to the above amino acid sequence, and (iii) a base complementary to the base sequence encoding the above amino acid sequence It may be one or more amino acid sequences selected from the group consisting of an amino acid sequence encoded by a polynucleotide that specifically hybridizes under stringent conditions to a polynucleotide comprising the sequence.
  • a transformant can be prepared by introducing a polynucleotide or vector encoding an anti-CCL21 antibody, anti-CXCR3 antibody, anti-CCR7 antibody or the like according to an embodiment of the present invention into a cell.
  • an anti-CCL21 antibody, an anti-CXCR3 antibody, an anti-CCR7 antibody and the like according to an embodiment of the present invention can be produced.
  • the transformant may be a cell of a human or a mammal other than a human (eg, rat, mouse, guinea pig, rabbit, cow, monkey, etc.). Examples of mammalian cells include Chinese hamster ovary cells (CHO cells), monkey cells COS-7, and the like. Alternatively, the transformant may be Escherichia genus, yeast or the like.
  • vectors examples include plasmids derived from E. coli (eg, pET-Blue), plasmids derived from Bacillus subtilis (eg, pUB110), yeast-derived plasmids (eg, pSH19), and animal cell expression plasmids (eg, pA1-11, pcDNA3.1-). V5 / His-TOPO), bacteriophages such as ⁇ phage, virus-derived vectors, and the like can be used. These vectors may contain components necessary for protein expression, such as a promoter, origin of replication, or antibiotic resistance gene.
  • the vector may be an expression vector.
  • a method for introducing the polynucleotide or vector into the cell for example, calcium phosphate method, lipofection method, electroporation method, adenovirus method, retrovirus method, microinjection, etc. can be used (Revised 4th edition) New Genetic Engineering Handbook, Yodosha (2003): 152-179.).
  • a production method using antibody cells for example, the method described in “Protein Experiment Handbook, Yodosha (2003): 128-142.” Can be used.
  • the inhibitor of the CCL21-CXCR3-CCR7 signaling system factor is an antibody or a fragment or functional equivalent thereof.
  • the antibody is an antibody or an antigen-binding fragment thereof containing an arbitrary sequence including a CDR of the full-length sequence, or an antibody or an antigen-binding fragment thereof containing a variable region of a specific sequence, one in its framework region, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 17, or 20 or more substitutions, impossible, or Or an antibody or antigen-binding fragment thereof containing the deletion.
  • an antibody or fragment or functional equivalent thereof preferably has an inhibitory activity downstream of the CCL21-CXCR3-CCR7 signaling system.
  • nucleic acid can be selected as an inhibitor of the nucleic acid form of the present invention using antisense activity as an index.
  • antisense activity refers to an activity capable of specifically suppressing or reducing the expression of a target gene. More specifically, depending on a certain nucleotide sequence introduced into the cell, an activity that can reduce the protein expression level by specifically reducing the mRNA level of a gene having a nucleotide sequence region complementary to that sequence.
  • a technique there are a method of directly introducing an RNA molecule complementary to mRNA produced from a target gene into a cell, and a method of introducing a construction vector capable of expressing RNA complementary to a target gene in the cell. Broadly divided.
  • Antisense activity is usually achieved by a nucleic acid sequence of at least 8 consecutive nucleotides that is complementary to the nucleic acid sequence of the gene of interest.
  • a nucleic acid sequence is preferably at least 9 contiguous nucleotides long, more preferably 10 contiguous nucleotides long, even more preferably 11 contiguous nucleotides long, 12 contiguous nucleotides long, 13 19 contiguous nucleotide lengths, 14 contiguous nucleotide lengths, 15 contiguous nucleotide lengths, 16 contiguous nucleotide lengths, 17 contiguous nucleotide lengths, 18 contiguous nucleotide lengths, 19 contiguous lengths Nucleotide length, 20 consecutive nucleotide lengths, 21 consecutive nucleotide lengths, 22 consecutive nucleotide lengths, 23 consecutive nucleotide lengths, 24 consecutive nucleotide lengths, 25 consecutive nucleotide lengths Of 40 consecutive
  • nucleic acid sequences include nucleic acid sequences that are at least 70% homologous, more preferably at least 80% homologous, more preferably 90% homologous, 95% homologous to the sequences described above.
  • antisense activity is preferably complementary to a sequence at the 5 'end of the nucleic acid sequence of the gene of interest.
  • antisense nucleic acid sequences also include those having one, several or one or more nucleotide substitutions, additions and / or deletions relative to the sequences described above. Therefore, in the present specification, antisense activity includes, but is not limited to, a decrease in gene expression level.
  • RNA interference RNA interference
  • RNA interference or “RNAi” is an abbreviation for RNA interference, and is an organism generally known in the art that inhibits or down-regulates gene expression in cells mediated by factors that cause RNAi. Process. For example, a phenomenon in which homologous mRNA is specifically decomposed by introducing a factor causing RNAi such as double-stranded RNA (also referred to as dsRNA) into a cell, and the synthesis of a gene product is suppressed, and a technique used therefor Say.
  • RNAi can also be used synonymously with “factor causing RNAi”, “factor causing RNAi”, “RNAi factor” and the like in some cases.
  • RNAi see, for example, Zamore and Haley, 2005, Science, 309, 1519-1524; Vahn and Martinsensen, 2005, Science, 309, 1525-1526; Zamore et al. 2000, Cell, 101, 25-33; Bass, 2001, Nature, 411, 428-429; Elbashirital.
  • RNAi is synonymous with other terms used to describe sequence-specific RNA interference such as post-transcriptional gene silencing, translational inhibition, transcriptional inhibition, and epigenetics. Understood.
  • the “factor causing RNAi” may be any as long as it causes “RNAi”.
  • the “factor causing RNAi” includes “small interfering nucleic acid”, “siNA”, “small interfering RNA”, “siRNA”, “small interfering nucleic acid molecule”, “small interfering oligonucleotide molecule”. ”Or“ chemically modified small interfering nucleic acid molecules ”and the like, these terms inhibit or down-regulate gene expression or viral replication by mediating RNA interference“ RNAi ”or gene silencing in a sequence-specific manner. Refers to any nucleic acid molecule that can. These terms may also represent individual nucleic acid molecules, a plurality of such nucleic acid molecules, or a pool of such nucleic acid molecules. These molecules can be double stranded nucleic acid molecules comprising self-complementary sense and antisense regions.
  • siRNA typically used in the present invention is a double-stranded RNA having a short length, usually about 20 bases (eg, typically about 21 to 23 bases) or less. .
  • Such siRNA can be used for treatment, prevention, prognosis, etc. of a disease because it suppresses gene expression by expressing in a cell and suppresses expression of a pathogenic gene targeted by the siRNA.
  • the siRNA used in the present invention may take any form as long as it can cause RNAi.
  • the antisense region in an agent that causes RNAi such as siRNA, includes a nucleotide sequence that is complementary to a nucleotide sequence in a target nucleic acid molecule or a part thereof, and a nucleotide sequence corresponding to the target nucleic acid sequence or a part thereof
  • a sense region having These molecules can be assembled from two separate oligonucleotides, one strand is the sense strand and the other is the antisense strand.
  • the antisense strand and the sense strand are self-complementary (i.e., each strand has a nucleotide sequence in the other strand such that the antisense strand and the sense strand form a double-stranded or double-stranded structure).
  • Complementary nucleotide sequences are included, where, for example, the double-stranded region is from about 15 to about 30, for example, about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25.
  • the antisense strand comprises a nucleotide sequence that is complementary to a nucleotide sequence in the target nucleic acid molecule or a portion thereof
  • the sense strand includes a nucleotide sequence corresponding to the target nucleic acid sequence or portion thereof (eg, about 15 to about 25 or more nucleotides of the molecule are present in the target nucleic acid or portion thereof.
  • these molecules are assembled from a single oligonucleotide, and the self-complementary sense and antisense regions of these molecules are linked by a nucleic acid linker or a non-nucleic acid linker.
  • These molecules can be polynucleotides having a double-stranded, asymmetric duplex, hairpin or asymmetric hairpin secondary structure comprising self-complementary sense and antisense regions, where the antisense region is A nucleotide sequence that is complementary to a nucleotide sequence in a separate target nucleic acid molecule, or a portion thereof, and a sense region that has a nucleotide sequence corresponding to the target nucleic acid sequence, or a portion thereof, the molecule comprising two or more A ring having a loop structure and a stem comprising a self-complementary sense region and an antisense region
  • the antisense region may comprise a nucleotide sequence that is complementary to a nucleotide sequence in a target nucleic acid molecule or a portion thereof, and a nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof.
  • the circular polynucleotide can be processed in vivo or in vitro to produce an active molecule that can mediate RNAi, these factors being present in the nucleotide sequence or part thereof in the target nucleic acid molecule.
  • Single-stranded polynucleotides having nucleotide sequences that are complementary can also be included (eg, these factors do not require a nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof to be present in these factors).
  • Single-stranded polynucleotides are 5 ′ phosphates (eg, Martinez et al . , 2002, Cell. 110, 563-574 and Schwartz et al.
  • inhibitors of the present invention such as CCL21, CXCR3, CCR7 comprise separate sense and antisense sequences or regions.
  • the sense and antisense regions are covalently linked by nucleotide or non-nucleotide linker molecules known in the art, or ionic interactions, hydrogen bonds, van der Waals interactions, hydrophobic interactions and / or Alternately non-covalently linked by stacking interactions.
  • the inhibitor of the present invention comprises a nucleotide sequence that is complementary to the nucleotide sequence of the target gene.
  • an inhibitor such as CCL21, CXCR3, CCR7 of the present invention interacts with the nucleotide sequence of the target gene so as to inhibit the expression of the target gene.
  • inhibitors such as CCL21, CXCR3, and CCR7 are not necessarily limited to molecules containing only RNA, but also include chemically modified nucleotides and non-nucleotides.
  • the invention when the invention is a small interfering nucleic acid molecule, it may be devoid of 2'hydroxy (2'-OH) containing nucleotides.
  • the invention can be a small interfering nucleic acid that does not require the presence of a nucleotide having a 2 'hydroxyl group to mediate RNAi. Therefore, when the present invention is a small interfering nucleic acid molecule, ribonucleotides (for example, nucleotides having a 2'-OH group) may not be contained.
  • a linked linker comprising one or more nucleotides having a 2′-OH group, or It may have other bonds or associated groups, moieties or chains.
  • an agent that inhibits CCL21, CXCR3, CCR7, etc. of the present invention may comprise ribonucleotides at about 5, 10, 20, 30, 40 or 50% of the nucleotide positions.
  • inhibitors such as CCL21, CXCR3, CCR7 are nucleic acid molecules that can mediate sequence-specific RNAi, such as small interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA), It may be a short hairpin RNA (shRNA), a small interfering oligonucleotide, a small interfering nucleic acid, a small interfering modified oligonucleotide, a chemically modified siRNA, or a post-transcriptional gene silencing RNA (ptgsRNA).
  • siRNA small interfering RNA
  • dsRNA double-stranded RNA
  • miRNA microRNA
  • ptgsRNA post-transcriptional gene silencing RNA
  • factors that cause RNAi include, for example, a sequence having at least about 70% homology to a portion of the nucleic acid sequence of a target gene or a sequence that hybridizes under stringent conditions. Examples include, but are not limited to, RNA comprising a double-stranded portion of nucleotide length or a variant thereof.
  • the factor preferably includes a 3 'protruding end, and more preferably, the 3' protruding end may be DNA having a length of 2 nucleotides or more (for example, DNA having a length of 2 to 4 nucleotides).
  • RNAi used in the present invention include, but are not limited to, a pair of short reverse complementary sequences (for example, 15 bp or more, for example, 24 bp).
  • RNAi RNAi to work
  • a relatively long (eg, 40 base pairs or more) RNA helicase
  • Dicer an RNaseIII-like nuclease having a domain excises the molecule from the 3 ′ end by about 20 base pairs to produce a short dsRNA (also called siRNA).
  • siRNA is an abbreviation for short interfering RNA, which is artificially chemically synthesized, biochemically synthesized, synthesized in an organism, or about 40
  • a protein specific to the siRNA binds to form RISC (RNA-induced-silencing-complex). This complex recognizes and binds to mRNA having the same sequence as siRNA, and cleaves mRNA at the center of siRNA by RNaseIII-like enzyme activity.
  • siRNA sequence and the mRNA sequence cleaved as a target is preferably 100% identical.
  • the cleavage activity by RNAi is not completely lost, but a partial activity remains.
  • the mutation of the base at the center of siRNA has a large effect, and the cleavage activity of mRNA by RNAi is extremely reduced.
  • siRNA itself can be used as a factor that causes RNAi, and a factor that generates siRNA (for example, a dsRNA typically having about 40 bases or more) can be used as such a factor. it can.
  • siRNA is synthesized by dsRNA in addition to the pathway described above, where the antisense strand of siRNA binds to mRNA and acts as a primer for RNA-dependent RNA polymerase (RdRP). It is also contemplated that this dsRNA becomes Dicer's substrate again, generating new siRNA and amplifying the action.
  • RdRP RNA-dependent RNA polymerase
  • siRNA itself and factors that produce siRNA are also useful.
  • 35 dsRNA molecules almost completely degrade the mRNA in a cell having 1,000 copies or more, so it is understood that siRNA itself and factors that generate siRNA are useful. Is done.
  • the factor causing RNAi of the present invention may be a short hairpin structure (shRNA; short hairpin RNA) having a protruding portion at the 3 'end.
  • shRNA short hairpin structure
  • shRNA is a single-stranded RNA that includes a partially palindromic base sequence, and thus has a double-stranded structure within the molecule, resulting in a hairpin-like structure.
  • shRNA is artificially chemically synthesized.
  • such shRNA can be generated by synthesizing RNA in vitro with T7 RNA polymerase, which has a hairpin structure DNA in which the DNA sequences of the sense strand and antisense strand are ligated in the reverse direction.
  • shRNAs are approximately 20 bases in length (typically 21 bases, 22 bases, 23 bases, etc.) in length after being introduced into the cell. It should be understood that it is degraded to cause RNAi as well as siRNA and has the therapeutic effect of the present invention. It should be understood that such effects are exerted in a wide range of organisms such as insects, plants, animals (including mammals). Thus, since shRNA causes RNAi similarly to siRNA, it can be used as an active ingredient of the present invention.
  • the shRNA can also preferably have a 3 'overhang.
  • the length of the double-stranded part is not particularly limited, but may preferably be about 10 nucleotides or more, more preferably about 20 nucleotides or more.
  • the 3 'protruding end may be preferably DNA, more preferably DNA having a length of at least 2 nucleotides, and further preferably DNA having a length of 2 to 4 nucleotides.
  • the factor causing RNAi used in the present invention can be either artificially synthesized (for example, chemical or biochemical) or naturally occurring, and the effect of the present invention can be achieved between the two. There is no essential difference. Those chemically synthesized are preferably purified by liquid chromatography or the like.
  • RNAi used in the present invention can also be synthesized in vitro.
  • antisense and sense RNAs are synthesized from template DNA using T7 RNA polymerase and T7 promoter. When these are annealed in vitro and then introduced into cells, RNAi is caused through the mechanism described above, and the effects of the present invention are achieved.
  • RNA can be introduced into cells by any appropriate method such as the calcium phosphate method.
  • Factors causing RNAi of the present invention also include factors such as single strands that can hybridize to mRNA, or all similar nucleic acid analogs thereof. Such factors are also useful in the present invention.
  • One embodiment of the present invention is a therapeutic agent for CCL21, CXCR3, CCR7 and other positive brain malaria comprising an RNAi molecule against CCL21, CXCR3, CCR7 or the like, or a polynucleotide encoding the RNAi molecule. If this RNAi molecule or a polynucleotide encoding the RNAi molecule is used, the growth of positive brain malaria cells such as CCL21, CXCR3, and CCR7 can be suppressed.
  • the “polynucleotide” may be a polymer compound having 10 or more nucleotides and in which nucleotides are linearly polymerized.
  • the “RNAi molecule” is an RNA strand having an RNAi action, and examples thereof include siRNA, shRNA, miRNA, and a small RNA having an RNAi action.
  • RNAi is a function of a target gene or mRNA or the like by one or more of siRNA, shRNA, miRNA, short or long single or double stranded RNA, or a modification thereof. Including the phenomenon that is suppressed or silenced.
  • siDirect2.0 (Naito et al., BMC Bioinformatics. 2009 Nov 30; 10: 392.) Can be used for designing RNAi molecules. Further, it may be entrusted to a trust company (for example, Takara Bio Inc.). The RNAi action can be confirmed by quantifying the RNA strand expression level by real-time RT-PCR. Alternatively, it can also be performed by methods such as analysis of RNA strand expression level by Northern blot, analysis of protein amount by Western blot, and observation of phenotype. Moreover, the plasmid which produces siRNA or shRNA with respect to a specific gene can be purchased from a trust company (for example, Takara Bio Inc. etc.), for example.
  • siRNA includes an RNA strand capable of inducing RNAi.
  • the duplex of siRNA can be divided into a guide strand and a passenger strand, and the guide strand is incorporated into RISC.
  • the guide strand incorporated into the RISC is used to recognize the target RNA.
  • artificially prepared materials are mainly used, but those existing endogenously in the living body are also known.
  • the guide strand may be composed of RNA having 15 or more bases. If it is 15 bases or more, the possibility of being able to bind to the target polynucleotide with high accuracy increases.
  • the guide strand may be composed of RNA of 40 bases or less. If it is 40 bases or less, the risk that disadvantageous phenomena, such as an interferon response, will arise becomes lower.
  • shRNA includes a single-stranded RNA strand capable of inducing RNAi and forming a hairpin-like folded structure (hairpin-like structure).
  • shRNA is cleaved by Dicer in the cell, and siRNA is excised. It is known that target RNA is cleaved by this siRNA.
  • the shRNA may be composed of 35 or more nucleotides. If it is 35 or more, the possibility that a hairpin-like structure peculiar to shRNA can be formed with high accuracy increases.
  • the shRNA may be composed of RNA having 100 bases or less. If it is 100 bases or less, the risk that disadvantageous phenomena such as an interferon response occur will be reduced.
  • the length of shRNA is not necessarily 100 bases or less. However, it is thought that it can function as shRNA.
  • miRNA includes an RNA strand having a function similar to that of siRNA, and is known to suppress or degrade the translation of a target RNA strand.
  • the difference between miRNA and siRNA generally lies in the production pathway and detailed mechanism.
  • small RNA refers to a relatively small RNA strand, and examples thereof include siRNA, shRNA, miRNA, antisense RNA, and 1 or double-stranded small RNA.
  • the RNAi molecule may contain an overhang consisting of 1 to 5 bases at the 5 'end or 3' end. In this case, it is considered that the efficiency of RNAi increases. This number may be, for example, 5, 4, 3, 2, or 1 base, and may be within the range of any two of them. When the RNAi molecule is double stranded, mismatch RNA may exist between the RNA strands. The number may be, for example, 1, 2, 3, 4, 5, or 10 or less, and may be in the range of any two of them.
  • the RNAi molecule may contain a hairpin loop. The number of bases of the hairpin loop may be, for example, 10, 8, 6, 5, 4, or 3 bases, and any two values thereof. It may be within the range.
  • each base sequence may be deleted, substituted, inserted, or added.
  • the notation of each base sequence is the 5 'end on the left side and the 3' end on the right side.
  • the length of the RNAi molecule may be, for example, 15, 18, 20, 25, 30, 40, 50, 60, 80, 100, 200, or 400 bases, and is within the range of any two of them. It may be. This number is preferably 15 or more or 100 or less from the viewpoint of enhancing the therapeutic effect on positive malignant tumors such as CCL21, CXCR3, and CCR7.
  • the “RNA strand” includes those in which RNA or an equivalent thereof is constituted in a combined form.
  • the “DNA strand” includes those in which a plurality of DNAs or their equivalents are combined.
  • the RNA strand or DNA strand includes an RNA strand or a DNA strand in the form of a single strand or a plurality of strands (for example, a double strand).
  • the RNA strand or DNA strand may be bound to a cell uptake promoting substance (for example, PEG or a derivative thereof), a label tag (for example, a fluorescent label tag), or a linker (for example, a nucleotide linker).
  • RNA strands or DNA strands can be synthesized using a nucleic acid synthesizer. In addition, it can also be purchased from a trust company (for example, Invitrogen). In vivo RNA or DNA strands may form salts or solvates. In addition, the RNA strand or DNA strand in the living body may be chemically modified.
  • the term RNA strand or DNA strand includes, for example, an RNA strand or DNA strand that forms a salt or solvate, or an RNA strand or DNA strand that has undergone chemical modification.
  • the RNA strand or DNA strand may be an RNA strand analog or a DNA strand analog.
  • the RNAi molecule preferably contains a base sequence complementary to a part of the base sequence of mRNA such as CCL21, CXCR3, CCR7 and the like from the viewpoint of stably exhibiting RNAi action.
  • the “part” may be, for example, 5, 10, 15, 18, 20, 22, 24, 26, 28, 30, 35, 40, or 50 bases or more, and any two values thereof. It may be within the range.
  • “inhibiting protein expression” includes, for example, inhibiting a transcription mechanism from a gene to mRNA, or inhibiting a translation mechanism from mRNA to protein. It also includes reducing the amount of protein as a result, eg, by inducing degradation of a gene, mRNA, or protein.
  • “inhibiting the function of a protein” includes causing a structural change in the protein and reducing the activity of the protein. In addition, for example, it includes a decrease in the amount of mRNA or protein produced as a result of inhibiting gene expression.
  • the “state in which expression is inhibited” includes a state in which the expression level is significantly reduced as compared with the normal state.
  • the expression level may be based on the amount of mRNA or the amount of protein.
  • “significantly” may include, for example, when statistical significance is evaluated using Student's t-test (one-sided or two-sided) and p ⁇ 0.05. . Or the state in which the difference has arisen substantially is included.
  • “the state in which the function is inhibited” includes a state in which the activity is significantly decreased as compared with that in the normal state.
  • the present invention provides a subject in need of an effective amount of an inhibitor of at least one factor of CCL21-CXCR3-CCR7 signaling system or a composition or medicament (therapeutic or prophylactic agent) containing the same.
  • a method for preventing or treating cerebral malaria in said subject comprising administering. So far, it has not been known that the CCL21-CXCR3-CCR7 signaling system can be applied to the prevention or treatment of cerebral malaria, and the effective amount has not been known.
  • an effective amount can be selected by those skilled in the art depending on the inhibitor to be used based on the knowledge obtained in the present specification.
  • the administration route of the therapeutic agent is preferably an effective route for treatment, and may be, for example, intravenous, subcutaneous, intramuscular, intraperitoneal, or oral administration.
  • the administration form may be, for example, an injection, capsule, tablet, granule or the like.
  • Aqueous solutions for injection may be stored, for example, in vials or stainless steel containers.
  • the aqueous solution for injection may contain, for example, physiological saline, sugar (for example, trehalose), NaCl, or NaOH.
  • the therapeutic agent may contain, for example, a buffer (for example, phosphate buffer), a stabilizer and the like.
  • compositions, medicaments, therapeutic agents, prophylactic agents, etc. of the present invention comprise a therapeutically effective amount of a therapeutic agent or active ingredient, and a pharmaceutically acceptable carrier or excipient.
  • pharmaceutically acceptable refers to a licensed or otherwise recognized pharmacopoeia of a government for use in animals, and more particularly in humans, by a government supervisory authority. It means that it is enumerated.
  • carrier refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic agent is administered.
  • Such carriers can be sterile liquids, such as water and oils, including but not limited to those of petroleum, animal, vegetable or synthetic origin, including but not limited to peanut oil, soybean oil, minerals Oil, sesame oil, etc. are included.
  • Water is a preferred carrier when the drug is administered orally.
  • Saline and aqueous dextrose are preferred carriers when the pharmaceutical composition is administered intravenously.
  • saline solutions and aqueous dextrose and glycerol solutions are used as liquid carriers for injectable solutions.
  • Suitable excipients include light anhydrous silicic acid, crystalline cellulose, mannitol, starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, chloride Sodium, nonfat dry milk, glycerol, propylene, glycol, water, ethanol, carmellose calcium, carmellose sodium, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylacetal diethylaminoacetate, polyvinylpyrrolidone, gelatin, medium chain fatty acid triglyceride, polyoxyethylene hardening Castor oil 60, sucrose, carboxymethylcellulose, corn starch, inorganic salts and the like are included.
  • compositions can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents, if desired.
  • These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. It is also possible to formulate the composition as a suppository, with traditional binders and carriers such as triglycerides. Oral formulations may also include standard carriers such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate. Examples of suitable carriers are E.I. W. Martin, Remington's Pharmaceutical Sciences (Mark Publishing Company, Easton, USA).
  • compositions contain a therapeutically effective amount of the therapeutic agent, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient.
  • the formulation must be suitable for the mode of administration.
  • surfactants, excipients, coloring agents, flavoring agents, preservatives, stabilizers, buffering agents, suspending agents, tonicity agents, binders, disintegrating agents, lubricants, fluidity Accelerators, flavoring agents and the like may be included.
  • the “salt” includes, for example, an anion salt formed with any acidic (eg, carboxyl) group or a cation salt formed with any basic (eg, amino) group.
  • Salts include inorganic salts or organic salts, for example, Berge et al. , J .; Pharm. Sci. , 1977, 66, 1-19. Examples thereof include metal salts, ammonium salts, salts with organic bases, salts with inorganic acids, salts with organic acids, and the like.
  • a “solvate” is a compound formed by a solute and a solvent. Solvates are described in, for example, J. Honiget al.
  • the solvent is water, the solvate formed is a hydrate. This solvent is preferably one that does not interfere with the biological activity of the solute. Examples of such preferred solvents include, but are not limited to, water or various buffers.
  • “chemical modification” includes, for example, modification with PEG or a derivative thereof, fluorescein modification, biotin modification, or the like.
  • various delivery systems are known, and such systems can be used to administer the therapeutic agent of the present invention to an appropriate site (eg, esophagus).
  • Such systems include, for example, encapsulation in liposomes, microparticles, and microcapsules: the use of recombinant cells capable of expressing therapeutic agents (eg, polypeptides), receptor-mediated endocytosis Use; such as the construction of therapeutic nucleic acids as part of a retroviral vector or other vector.
  • Introduction methods include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes.
  • an inhaler or nebulizer can be used with an aerosolizing agent and can be administered with other biologically active agents. Administration can be systemic or local.
  • the present invention when used in the brain (eg, olfactory bulb) region, it can be further administered by any suitable route, such as by direct injection into the brain (eg, olfactory bulb).
  • the nucleic acid is administered in vivo by constructing the nucleic acid as part of a suitable nucleic acid expression vector and administering the nucleic acid so that it is intracellular. It is also possible to facilitate the expression of the protein, for example by the use of retroviral vectors or by direct injection or by the use of microparticle guns or the nucleic acids with lipids, cell surface receptors or transfection agents. This can be done by coating or by administering a nucleic acid linked to a tag sequence known to enter the nucleus. Alternatively, nucleic acid therapeutics can be introduced into cells and incorporated by homologous recombination into host cell DNA for expression.
  • the composition can be formulated as a pharmaceutical composition adapted for human administration according to known methods. Such compositions can be administered by injection. Typically, compositions for injection administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition can also include a solubilizing agent and a local anesthetic such as lidocaine to ease pain at the site of the injection. In general, the ingredients are supplied separately or mixed together in a unit dosage form, for example in a sealed container such as an ampoule or sachet indicating the amount of active agent, lyophilized powder or water-free concentration Can be supplied as a product.
  • a sealed container such as an ampoule or sachet indicating the amount of active agent, lyophilized powder or water-free concentration Can be supplied as a product.
  • composition is to be administered by infusion
  • it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline.
  • an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.
  • compositions, medicament, therapeutic agent, and preventive agent of the present invention in a neutral form, salt form, or other prodrug (for example, ester).
  • pharmaceutically acceptable salts include those formed with free carboxyl groups derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine And those formed with free amine groups such as those derived from, and those derived from sodium, potassium, ammonium, calcium, ferric hydroxide, and the like.
  • the amount of the therapeutic agent of the invention effective for the treatment of a particular disorder or condition can vary depending on the nature of the disorder or condition, but can be determined by those skilled in the art by standard clinical techniques based on the description herein. Furthermore, in some cases, in vitro assays can be used to help identify optimal dosage ranges.
  • the exact dose to be used in the formulation can also vary depending on the route of administration and the severity of the disease or disorder and should be determined according to the judgment of the attending physician and the circumstances of each patient. However, the dose is not particularly limited, and may be, for example, 0.001, 1, 5, 10, 15, 100, or 1000 mg / kg body weight per dose, and within the range of any two of these values There may be.
  • the dosing interval is not particularly limited.
  • the dose, administration interval, and administration method may be appropriately selected depending on the age, weight, symptoms, target organ, etc. of the patient.
  • the therapeutic agent preferably contains a therapeutically effective amount or an effective amount of an active ingredient that exhibits a desired action. If the malignant tumor marker is significantly decreased after administration, it may be determined that there is a therapeutic effect. Effective doses can be extrapolated from dose-response curves derived from in vitro or animal model test systems.
  • a “patient” or “subject” is a human or non-human mammal (eg, mouse, guinea pig, hamster, rat, mouse, rabbit, pig, sheep, goat, cow, horse, One or more of a cat, dog, marmoset, monkey, or chimpanzee).
  • the patient or subject may be a patient or subject that has been determined or diagnosed as having abnormal expression of CCL21, CXCR3, CCR7, etc.
  • the determination or diagnosis is preferably performed by detecting the protein level of CCL21, CXCR3, CCR7 or the like.
  • the pharmaceutical composition or therapeutic agent or prophylactic agent of the present invention can be provided as a kit.
  • the present invention provides a drug pack or kit comprising one or more containers filled with one or more components of the composition or medicament of the present invention.
  • a drug pack or kit comprising one or more containers filled with one or more components of the composition or medicament of the present invention.
  • associated with such containers manufactured, used or sold for human administration by a government agency in a manner prescribed by the government agency that regulates the manufacture, use or sale of a pharmaceutical or biological product. It is also possible to indicate information indicating authorization.
  • the kit of the present invention can also contain an expression vector encoding a protein for use as a composition, therapeutic agent, prophylactic agent or medicament of the present invention, and the protein is biologically expressed after being expressed. Can also be reconstituted to form a complex that is active. Such a kit preferably also contains the necessary buffers and reagents. In some cases, such containers may be accompanied by instructions for use of the kit (package insert) and / or in a form prescribed by a government agency that regulates the manufacture, use or sale of a pharmaceutical or biological product. It is also possible to provide information indicating the approval of manufacture, use or sale for human administration by a government agency.
  • a pharmaceutical composition comprising a nucleic acid of the invention can be administered via liposomes, microparticles, or microcapsules. In various embodiments of the present invention, it may be useful to achieve sustained release of nucleic acids using such compositions.
  • the present invention provides a marker for cerebral malaria comprising at least one factor of the CCL21-CXCR3-CCR7 signaling system.
  • the present invention provides a method for identifying or diagnosing cerebral malaria using a substance that binds to any of at least one factor of the CCL21-CXCR3-CCR7 signaling system or an expression product thereof.
  • the expression of any of at least one factor of the CCL21-CXCR3-CCR7 signaling system of the target sample is increased compared to that of the normal sample, the target sample is diagnosed as having brain malaria. .
  • the method of the present invention includes the step of examining the expression of any of at least one factor of the CCL21-CXCR3-CCR7 signaling system in the subject specimen, and the result may be used as an index of cerebral malaria disease. it can.
  • This CCL21-CXCR3-CCR7 signal transduction system refers to the signal transduction pathway of CCL21, CXCR3 and CCR7, and refers to the onset mechanism of brain malaria.
  • the schematic diagram is shown in FIG.
  • the factor of “CCL21-CXCR3-CCR7 signaling system” is at least selected from the group consisting of CCL21, CXCR3 and CCR7, IP-10 (CXCR3 ligand), CXCL11 (I-TAC), and CXCL9 (MIG).
  • CD8 ⁇ dendritic cells (DC) and other dendritic cells, CD11c + CD8 T cells (CD8 T cells in humans) and other T cells should also be considered as this factor Can do.
  • At least one factor of the CCL21-CXCR3-CCR7 signaling system used in the present invention is derived from CCL21, CXCR3, CCR7, and nucleic acids encoding them, their expression products and their derivatives. At least one selected from the group consisting of: Regarding each factor of CCL21, CXCR3, and CCR7, the association with brain malaria is not known, and the present invention shows that this Yuna pathway and each factor can be used as an index for diagnosis or detection of brain malaria. Offer for the first time.
  • At least one factor of the CCL21-CXCR3-CCR7 signaling system used in the present invention is a group consisting of CCL21, CXCR3, CCR7, and nucleic acids encoding them, their expression products and their derivatives.
  • At least one inflammatory cytokine selected from the group consisting of IP-10 (CXCR3 ligand), CXCL11 (I-TAC), and CXCL9 (MIG), and a nucleic acid encoding them, its expression product and its origin At least one selected from the group consisting of: (2) CCL21 and / or CCL19 (3) CD8 ⁇ dendritic cells and other dendritic cells; and (4) at least one factor selected from the group consisting of CD11c + CD8 T cells (CD8 T cells in the case of humans) and other T cells.
  • IP-10 CXCR3 ligand
  • CXCL11 I-TAC
  • MIG CXCL9
  • the detection or diagnosis of the present invention comprises the following: (A) Increased expression of CCL21 and / or CCL19 in the olfactory bulb; (B) Increased expression of IFN- ⁇ in the cortex; (C) that the CD8 ⁇ dendritic cells are observed to be primed in a CCR7-dependent manner; and / or (D) that CD11c + CD8T cells are observed in the brain or spleen.
  • A Increased expression of CCL21 and / or CCL19 in the olfactory bulb
  • B Increased expression of IFN- ⁇ in the cortex
  • C that the CD8 ⁇ dendritic cells are observed to be primed in a CCR7-dependent manner
  • D that CD11c + CD8T cells are observed in the brain or spleen.
  • CD11c + CD8T cells are observed in the brain or spleen.
  • the brain malaria is Produced by a human malaria parasite corresponding to a malaria parasite selected from the group consisting of falciparum.
  • brain malaria targeted by the present invention includes rodents or primates.
  • the present invention provides that at least one factor of the CCL21-CXCR3-CCR7 signaling system used by the present invention is the nucleic acid sequence described in SEQ ID NOs: 1, 3, 5, etc., or a fragment thereof or an equivalent thereof Alternatively, the amino acid sequence described in SEQ ID NOs: 2, 4, 6, etc. or a fragment thereof or an equivalent thereof is included.
  • the marker of the invention is (1) a CCL21 protein or a nucleic acid encoding it; (2) CXCR3 or a nucleic acid encoding the same; and / or (3) a CCR7 protein or a nucleic acid encoding the same.
  • the present invention binds to at least one factor of CCL21-CXCR3-CCR7 signaling system, and at least one selected from the group consisting of nucleic acids encoding them, their expression products and their derivatives.
  • the present invention provides a detection agent or a diagnostic agent for cerebral malaria containing a substance. It was first found in the present invention that the CCL21-CXCR3-CCR7 signaling system is related to cerebral malaria, and detection, examination, or diagnosis using this was also realized for the first time by the present invention.
  • brain malaria is detected by detecting the expression of a CCL21-CXCR3-CCR7 signaling system factor in a subject subject or a sample derived therefrom (eg, a cell sample, cerebral spinal fluid, serum, etc.). Can be detected or selected. Therefore, it is understood that an agent for treating cerebral malaria can be detected and screened using the ability of the marker of the present invention to reduce, suppress, increase or activate as an index.
  • the present invention provides a detection agent, a test agent, or a diagnostic agent for identifying cerebral malaria, including a substance that binds to or interacts with a CCL21-CXCR3-CCR7 signaling factor.
  • a detection agent e.g., a test agent, or a diagnostic agent for identifying cerebral malaria
  • the substance binding is preferably specific.
  • any substance may be used as long as it can bind to or interact with a factor of the CCL21-CXCR3-CCR7 signaling system.
  • antibodies of these factors or fragments or functional equivalents thereof, or nucleic acids encoding these factors in particular nucleic acid primers or CCL21-CXCR3- capable of amplifying factors of the CCL21-CXCR3-CCR7 signaling system Examples include, but are not limited to, probes that can bind to or interact with factors of the CCR7 signaling system.
  • the detection agent, test agent or diagnostic agent of the present invention can be used as a detection kit, test kit or diagnostic kit.
  • the detection agent, test agent or diagnostic agent of the present invention is a complex in which another substance (for example, a label or the like) is bound to a moiety (for example, an antibody or the like) that enables detection, inspection or diagnosis.
  • a moiety for example, an antibody or the like
  • it may be a complex molecule.
  • “complex” or “complex molecule” means any construct comprising two or more moieties.
  • the other part may be a polypeptide or other substance (eg, sugar, lipid, nucleic acid, other hydrocarbon, etc.).
  • two or more parts constituting the complex may be bonded by a covalent bond, or bonded by other bonds (for example, hydrogen bond, ionic bond, hydrophobic interaction, van der Waals force, etc.). May be.
  • bonds for example, hydrogen bond, ionic bond, hydrophobic interaction, van der Waals force, etc.
  • the “complex” includes a molecule formed by linking a plurality of molecules such as a polypeptide, a polynucleotide, a lipid, a sugar, and a small molecule.
  • the detection agent, test agent or diagnostic agent of the present invention can take the form of probes and primers.
  • the probes and primers of the present invention can specifically hybridize with CCL21-CXCR3-CCR7 signaling system factors such as CCL21, CXCR3, CCR7.
  • expression of CCL21-CXCR3-CCR7 signaling factors such as CCL21, CXCR3, CCR7 is an indicator of brain malaria and is useful as an indicator.
  • the probes and primers according to the invention can be used to identify brain malaria.
  • the probe and primer of the present invention only need to be able to detect the expression of a CCL21-CXCR3-CCR7 signaling factor such as CCL21, CXCR3, CCR7, etc. It refers to a polymer composed of bases or base pairs such as nucleic acids (RNA). Double stranded cDNA is also known to be available for tissue in situ hybridization, and probes and primers of the invention include such double stranded cDNA. As a particularly preferred probe and primer for detecting RNA in tissue, an RNA probe (riboprobe) can be mentioned.
  • RNA probe riboprobe
  • the present invention can take the form of a primer.
  • nucleic acid molecules that are usually used as primers include those having a nucleic acid sequence of at least 8 consecutive nucleotides that is complementary to the nucleic acid sequence of the target gene (eg, SEQ ID NO: 1).
  • Such a nucleic acid sequence is preferably at least 12 contiguous nucleotides long, at least 9 contiguous nucleotides, more preferably at least 10 contiguous nucleotides, and even more preferably at least 11 contiguous nucleotides.
  • Nucleic acid sequences used as probes are nucleic acid sequences that are at least 70% homologous, more preferably at least 80% homologous, more preferably at least 90% homologous, at least 95% homologous to the sequences described above. Is included.
  • a sequence suitable as a primer may vary depending on the nature of the sequence intended for synthesis (amplification), but those skilled in the art can appropriately design a primer according to the intended sequence. Such primer design is well known in the art, and may be performed manually or using a computer program (eg, LASERGENE, PrimerSelect, DNAStar).
  • the primer according to the present invention can also be used as a primer set composed of two or more of the primers.
  • the primer and primer set according to the present invention are known to detect a target gene using a nucleic acid amplification method such as a PCR method, an RT-PCR method, a real-time PCR method, an in situ PCR method, or a LAMP method. In the method, it can utilize as a primer and a primer set according to a conventional method.
  • the primer set according to the present invention can be selected so that the nucleotide sequence of a target protein such as a CCL21-CXCR3-CCR7 signaling factor such as CCL21, CXCR3, CCR7 can be amplified by a nucleic acid amplification method such as a PCR method.
  • Nucleic acid amplification methods are well known, and selection of primer pairs in nucleic acid amplification methods is obvious to those skilled in the art.
  • one of two primers is paired with the plus strand of the double-stranded DNA of the target protein such as a CCL21-CXCR3-CCR7 signaling factor such as CCL21, CXCR3, CCR7, etc.
  • the primer can be selected such that the other primer is paired with the minus strand of the double-stranded DNA, and the other primer is paired with the extended strand extended by one primer.
  • LAMP method WO00 / 28082
  • the primer of the present invention can be chemically synthesized based on the nucleotide sequence disclosed herein.
  • the present invention may take the form of a “probe”.
  • nucleic acid molecules that are usually used as probes include those having a nucleic acid sequence of at least 8 consecutive nucleotides that is homologous or complementary to the nucleic acid sequence of the target gene (eg, SEQ ID NO: 1).
  • Such a nucleic acid sequence is preferably at least 12 contiguous nucleotides long, at least 9 contiguous nucleotides, more preferably at least 10 contiguous nucleotides, and even more preferably at least 11 contiguous nucleotides.
  • Nucleic acid sequences used as probes are nucleic acid sequences that are at least 70% homologous, more preferably at least 80% homologous, more preferably at least 90% homologous, at least 95% homologous to the sequences described above. Is included.
  • the detection agent of the present invention may be labeled.
  • the detection agent, test agent or diagnostic agent of the present invention may have a tag attached thereto.
  • the label or tag used in the present invention can take any of the forms described herein.
  • the present invention is a method for using a factor of CCL21-CXCR3-CCR7 signaling system such as CCL21, CXCR3, CCR7 as an index for identifying cerebral malaria, or detecting, examining or diagnosing cerebral malaria.
  • a factor of CCL21-CXCR3-CCR7 signaling system such as CCL21, CXCR3, CCR7 as an index for identifying cerebral malaria, or detecting, examining or diagnosing cerebral malaria.
  • a CCL21-CXCR3-CCR7 signaling factor such as CCL21, CXCR3, CCR7 as an index for identifying brain malaria, for example, CCL21, CXCR3, CCR7, etc.
  • the CCL21-CXCR3-CCR7 signal transduction factor expression product eg, protein or mRNA
  • a detection agent, a test agent or a diagnostic agent containing a substance that binds to an expression product of a factor of CCL21-CXCR3-CCR7 signaling system such as CCL21, CXCR3, CCR7, etc. it can.
  • Such a detection agent, test agent or diagnostic agent is described in the present specification, and a person skilled in the art performs the method of the present invention using a technique known in the art as necessary based on the description. It is understood that you can.
  • CCL21-CXCR3-CCR7 signaling such as CCL21, CXCR3, CCR7, etc., which is a target object in the sample is brought into contact with the target sample of the detection agent, test agent or diagnostic agent of the present invention.
  • contact means that a plurality of substances are arranged so as to cause interaction or binding between the plurality of substances.
  • a polypeptide or polynucleotide can be achieved either in direct or indirect physical proximity to a marker of the invention or a sample containing it.
  • the polypeptide or polynucleotide can be present in many buffers, salts, solutions, and the like.
  • Contact includes placing the compound in, for example, a beaker, microtiter plate, cell culture flask or microarray (eg, gene chip) containing a polypeptide encoding a nucleic acid molecule or fragment thereof.
  • a specific method for detecting an expression product of a factor of CCL21-CXCR3-CCR7 signaling system such as CCL21, CXCR3, CCR7, such as a protein or mRNA is the method of detecting CCL21, CXCR3, CCR7, etc. in a sample (eg, serum).
  • the method is not particularly limited as long as it can detect an expression product of a factor of the CCL21-CXCR3-CCR7 signaling system, for example, protein or mRNA, and examples thereof include a hybridization method, a nucleic acid amplification method, and an antigen-antibody reaction method.
  • the sample used may be any sample that is considered to contain an expression product.
  • serum can be used.
  • Serum can be obtained by a conventional method.
  • the detection, test or diagnosis according to the invention comprises the hybridization of a probe according to the invention with a nucleic acid sample (such as mRNA or complementary DNA (cDNA) transcribed therefrom), ie a hybridization complex,
  • a nucleic acid sample such as mRNA or complementary DNA (cDNA) transcribed therefrom
  • cDNA complementary DNA
  • the expression of CCL21-CXCR3-CCR7 signaling factor such as CCL21, CXCR3, CCR7, etc. in the cell sample can be detected.
  • Detection of an expression product of a factor of CCL21-CXCR3-CCR7 signaling system such as CCL21, CXCR3, CCR7, etc. using a hybridization method is performed by, for example: (a) a polynucleotide derived from a test sample, and the present invention And (b) detecting the hybridization complex.
  • step (a) mRNA prepared from the target test sample or complementary DNA (cDNA) transcribed from the mRNA can be brought into contact with the probe as a polynucleotide derived from the test cell sample.
  • the probe can be labeled and used.
  • radioactive activity for example, 32 P, 14 C, and 35 S
  • fluorescence for example, FITC, europium
  • enzymatic reaction such as chemical color development (for example, peroxidase, alkaline phosphatase), etc.
  • chemical color development for example, peroxidase, alkaline phosphatase
  • the detection of the hybridization product can be performed using a well-known method such as Northern hybridization, Southern hybridization, colony hybridization and the like.
  • the sample in which the hybridization complex is detected indicates that the tissue of the subject expresses CCL21-CXCR3-CCR7 signaling factor such as CCL21, CXCR3, CCR7, etc. It can be determined that the possibility of brain malaria is high.
  • a nucleic acid sample (mRNA or its transcription product) is amplified by a nucleic acid amplification method using the primer or primer set according to the present invention, and the amplified product is detected.
  • the expression of CCL21-CXCR3-CCR7 signaling factor such as CCL21, CXCR3, CCR7, etc. in the sample can be detected, examined or diagnosed using this.
  • CCL21-CXCR3-CCR7 signal transduction system factors such as CCL21, CXCR3, CCR7 using nucleic acid amplification method, for example, (i) Primer or primer according to the present invention using a polynucleotide derived from a test sample as a template Performing a nucleic acid amplification method using the set; and (ii) detecting a formed amplification product.
  • mRNA prepared from the target test sample or complementary DNA (cDNA) transcribed from the mRNA can be used as a template.
  • Detection of the amplification product can be performed using a nucleic acid amplification method such as a PCR method, an RT-PCR method, a real-time PCR method, or a LAMP method.
  • the detection of the amplification product in this sample indicates that the subject's tissue expresses CCL21-CXCR3-CCR7 signaling factor such as CCL21, CXCR3, CCR7, etc., so the subject from which the sample is derived It can be determined that the possibility of brain malaria is high.
  • the CCL21-CXCR3-CCR7 signaling system such as CCL21, CXCR3, CCR7, etc. in the sample is detected by contacting the antibody according to the present invention with the sample and detecting an antigen-antibody reaction.
  • the expression of the factor can be detected, examined or diagnosed using it.
  • Detection of expression of factors of CCL21-CXCR3-CCR7 signaling system such as CCL21, CXCR3, CCR7 using antigen-antibody reaction is performed by, for example, (I) contacting a protein derived from a test cell sample with the antibody according to the present invention. And (II) a step of measuring an antigen-antibody complex.
  • Methods for detecting antigen-antibody reactions are well known to those skilled in the art.
  • CCL21-CXCR3-CCR7 signaling factors such as CCL21, CXCR3, CCR7, etc. in serum can be detected by immunological methods.
  • Immunological methods include cell tissue samples that have been appropriately treated, such as cell separation and extraction procedures, immunohistochemical staining, enzyme immunoassay, western blotting, agglutination, competition, etc.
  • a known method such as a method or a sandwich method can be applied.
  • the immunohistochemical staining method can be performed by, for example, a direct method using a labeled antibody, an indirect method using a labeled antibody against the antibody, or the like.
  • the labeling agent known labeling substances such as fluorescent substances, radioactive substances, enzymes, metals, and dyes can be used.
  • the sample in which the antigen-antibody complex is detected includes cells expressing CCL21-CXCR3-CCR7 signaling factor such as CCL21, CXCR3, CCR7, etc. It can be determined that the property is high.
  • cerebral malaria can be diagnosed with higher accuracy by performing the above-described steps twice or more.
  • CCL21-CXCR3-CCR7 signaling factors such as CCL21, CXCR3, CCR7 (for example, SCC or CEA which are known markers) or these factors are used in combination
  • CCL21, CXCR3, CCR7 for example, SCC or CEA which are known markers
  • the preparation procedure of the diagnostic agent and the like of the present invention as a medicine is known in the art, and is described in, for example, the Japanese Pharmacopeia, the US Pharmacopeia, and the pharmacopoeia of other countries. Accordingly, those skilled in the art can determine the embodiment, such as the amount to be used, without undue experimentation as described herein.
  • the concentration of the marker can be measured by mass spectrometry.
  • mass spectrometry As ionization methods in this case, both matrix-assisted laser ionization (matrix-assisted laser desorption / ionization, MALDI) and electrospray ionization (electrospray ionization, ESI) can be applied, but the production of multivalent ions is small. MALDI is preferred.
  • MALDI-TOF-MS combined with a time-of-flight mass spectrometer (TOF)
  • the concentration of the marker can be measured more accurately.
  • MS / MS using two mass spectrometers the marker concentration can be measured more accurately.
  • the test material When measuring the concentration of the marker by electrophoresis, for example, the test material is subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) to separate the target marker, and the gel is stained with an appropriate dye or fluorescent substance. What is necessary is just to measure the density and fluorescence intensity of the band corresponding to the target marker.
  • SDS-PAGE SDS-polyacrylamide gel electrophoresis
  • Two-dimensional electrophoresis combined with isoelectric focusing (IEF) can also be used when marker separation is insufficient by SDS-PAGE alone.
  • Western blotting can be performed to measure the amount of marker on the membrane.
  • a method by liquid high performance chromatography can be used. That is, the concentration of the marker in the sample can be measured by subjecting the sample to HPLC to separate the target marker and measuring the peak area of the chromatogram.
  • the present invention provides a kit for detection, inspection and / or diagnosis for carrying out the method for detection, inspection and / or diagnosis according to the present invention.
  • This kit contains the detection agent, test agent and / or diagnostic agent of the present invention.
  • any embodiment described in this specification can be used alone or in combination.
  • the detection kit according to the present invention includes a detection kit for performing the detection of the embodiment according to the present invention, specifically, CCL21-CXCR3-CCR7 signal such as CCL21, CXCR3, CCR7, etc.
  • a kit for detecting expression of a factor of a transmission system which includes at least a probe according to the present invention. This probe may be labeled.
  • This detection kit detects the expression of a CCL21-CXCR3-CCR7 signaling factor such as CCL21, CXCR3, CCR7, etc. by a hybridization method.
  • the detection method of the first aspect optionally further comprises various reagents for carrying out the hybridization method, such as substrate compounds, hybridization buffers, instructions, and / or instruments used for detection of the label. Can be included.
  • the detection kit of this embodiment provides a marker gene for brain malaria other than factors of CCL21-CXCR3-CCR7 signaling system such as CCL21, CXCR3, CCR7 (for example, SCC, CEA) in order to perform highly accurate detection.
  • Etc. may further comprise a probe, a primer, a primer set, or an antibody capable of detecting the expression. These probes, primers, primer sets, or antibodies may be labeled.
  • This detection kit expresses marker genes of brain malaria other than factors of CCL21-CXCR3-CCR7 signal transduction system such as CCL21, CXCR3, CCR7, etc. by any of hybridization, nucleic acid amplification, and antigen-antibody reaction methods Is further detected.
  • the detection kit according to the present invention includes a detection kit for performing the detection according to another embodiment of the present invention, specifically, CCL21-CXCR3 such as CCL21, CXCR3, CCR7, etc. -A kit for detecting the expression of a factor of the CCR7 signaling system, which includes at least a primer according to the present invention or a primer set according to the present invention.
  • This detection kit detects the expression of CCL21-CXCR3-CCR7 signaling factors such as CCL21, CXCR3, CCR7, etc. by nucleic acid amplification.
  • the detection method of the second aspect includes various reagents for carrying out the nucleic acid amplification method, for example, a buffer, an internal standard indicating that PCR can proceed normally, instructions, and / or instruments, if desired. Can further be included.
  • the detection kit of this embodiment according to the present invention can detect the expression of a marker gene of cerebral malaria other than factors of CCL21-CXCR3-CCR7 signaling system such as CCL21, CXCR3, CCR7, etc. in order to perform highly accurate detection.
  • a probe, primer, primer set, or antibody may further be included. These probes, primers, primer sets, or antibodies may be labeled.
  • This detection kit can express markers of brain malaria other than factors of CCL21-CXCR3-CCR7 signal transduction system such as CCL21, CXCR3, CCR7, etc. by any of hybridization, nucleic acid amplification, and antigen-antibody reaction methods. Further detect.
  • detection kits according to the present invention include detection kits for performing detection of further embodiments according to the present invention, specifically, CCL21-CXCR3-CCR7 signals such as CCL21, CXCR3, CCR7, etc.
  • a kit for detecting a protein of a factor of a transmission system which includes at least the antibody according to the present invention. This antibody may be labeled.
  • This detection kit detects the expression of factors of CCL21-CXCR3-CCR7 signaling system such as CCL21, CXCR3, CCR7, etc. by detecting antigen-antibody reaction.
  • the detection method of this embodiment further includes various reagents for carrying out the antigen-antibody reaction, for example, secondary antibodies used in the ELISA method, coloring reagents, buffers, instructions, and / or instruments, if desired. be able to.
  • kits, compositions or systems can be used in samples from any subject as long as they can identify the markers of the present invention (eg, CCL21-CXCR3-CCR7 signaling system factors such as CCL21, CXCR3, CCR7, etc.).
  • markers of the present invention eg, CCL21-CXCR3-CCR7 signaling system factors such as CCL21, CXCR3, CCR7, etc.
  • the factor used in the present invention is selected from the group consisting of nucleic acid molecules, polypeptides, lipids, sugar chains, small organic molecules and complex molecules thereof, preferably the factor is a protein or complex.
  • a molecule eg, glycoprotein, lipid protein, etc.
  • the factor is an antibody (eg, a polyclonal antibody or a monoclonal antibody).
  • Such factors are preferably labeled or labelable. This is because it is easy to diagnose.
  • the means used are mass spectrometer, nuclear magnetic resonance analyzer, X-ray analyzer, SPR, chromatography (eg, HPLC, thin layer chromatography, gas chromatography), immunology (E.g. Western blotting, EIA (enzyme immunoassay), RIA (radioimmunoassay), ELISA (enzyme-linked immunosorbent assay)), biochemical means (e.g.
  • Electrophoresis equipment chemical analysis equipment, fluorescence two-dimensional differential electrophoresis (2DE-DIGE), isotope labeling (ICAT), tandem affinity purification (TAP), physical means, laser microdissection and these Or a combination of It is selected from the group consisting of.
  • the system or kit of the invention further comprises a marker standard.
  • a marker standard may be used to confirm whether a marker detection means (such as a factor that specifically interacts with the marker or a means for selectively recognizing the marker) is functioning normally. preferable.
  • the present invention may further include a means for purifying a target sample.
  • a means for purifying a target sample include chromatography. Since purification can increase the accuracy of the diagnosis, it can be used in preferred embodiments, but this is not essential.
  • the factor or means used in the present invention has the ability to quantify the marker of the present invention.
  • Such quantification may be a means or factor that can draw a calibration curve properly when a standard curve is drawn.
  • Preferable examples include antibodies, mass spectrometry, and chromatographic analysis. Therefore, in one embodiment, the system of the present invention further comprises a quantification means for quantifying the marker.
  • the quantification unit includes a determination unit that compares the standard curve with a measurement result to determine whether the marker is within a normal value range.
  • determination means can be realized using a computer.
  • kits or system of the invention comprises a composition comprising a marker or an agent that specifically interacts with the marker.
  • the present invention provides a level of proliferative ability or differentiation state of a marker in a sample from a subject, a factor that specifically interacts with the marker, or a means for selectively recognizing the marker, or Provided is the use in the manufacture of a medicament for predictive diagnosis, pre-diagnosis, prediction, detection or diagnosis of a disease, disorder or condition related thereto.
  • the sample may be acquired by any means. Usually, when a person in charge other than the doctor is engaged in the measurement, it may have been acquired by the doctor in some form.
  • the determination of the level of proliferative potential or the state of differentiation, or the associated disease, disorder or condition, or whether it is possible, is abnormal compared to the normal value, relative to each marker It can be implemented by determining. It will be appreciated that in the methods of the invention, the markers used, etc. may have any one or more of the features described elsewhere in this specification as long as they do not conflict.
  • a method for measuring the marker concentration a method generally used for protein quantification can be used as it is as long as the marker concentration can be specifically measured. For example, various immunoassays, mass spectrometry (MS), chromatography, electrophoresis and the like can be used.
  • One preferred embodiment in the detection or diagnosis of the present invention is to capture a marker on a carrier and measure the concentration of the captured marker. That is, a substance having affinity for the marker is immobilized on the carrier, and the marker is captured on the carrier via the substance having the affinity. According to this embodiment, the influence of the contaminant contained in the sample can be reduced, and the marker concentration can be measured with higher sensitivity and higher accuracy.
  • an immunoassay when used as a marker measurement method, it is preferable to use a carrier on which an antibody is immobilized.
  • an immunoassay system using the antibody immobilized on the carrier as the primary antibody can be easily constructed.
  • two types of antibodies specific to a marker and having different epitopes are prepared, one is immobilized on a carrier as a primary antibody, and the other is enzyme-labeled as a secondary antibody to construct a sandwich EIA system.
  • immunoassay systems based on binding inhibition methods and competitive methods can be constructed.
  • a substrate is used as a carrier, immunoassay using an antibody chip is possible. According to the antibody chip, the concentration of a plurality of markers can be measured simultaneously, and rapid measurement is possible.
  • the marker when mass spectrometry is used as a method for measuring a marker, the marker can be captured on a carrier by ion binding or hydrophobic interaction in addition to the antibody. Ion binding and hydrophobic interactions are not as specific as bioaffinity such as antigens and antibodies, and substances other than markers are captured, but according to mass spectrometry, they are quantified by a mass spectrometer spectrum that reflects molecular weight. No problem.
  • the concentration of the marker can be measured more accurately.
  • a substrate and a metal ion substrate are preferably used.
  • the ion exchanger When the marker is captured on the carrier by ionic bonding, the ion exchanger is immobilized on the carrier.
  • both an anion exchanger and a cation exchanger can be used as the ion exchanger, and further, a strong anion exchanger, a weak anion exchanger, a strong cation exchanger, and a weak cation exchanger. Any of these can be used.
  • weak anion exchangers include those having weak anion exchange groups such as dimethylaminoethyl (DE) and diethylaminoethyl (DEAE).
  • strong anion exchangers include quaternary ammonium (trimethylaminomethyl) (QA), quaternary aminoethyl (diethyl, mono-2-hydroxybutylaminoethyl) (QAE), and quaternary ammonium (trimethylammonium. And those having a strong anion exchange group such as (QMA).
  • weak cation exchangers include those having weak cation exchange groups such as carboxymethyl (CM).
  • strong cation exchanger include those having a strong cation exchange group such as sulfopropyl (SP).
  • the hydrophobic group examples include a C4 to C20 alkyl group and a phenyl group.
  • the marker can be captured on a carrier on which metal ions such as Cu 2+ , Zn 2+ , Ni 2+ , Ca 2+ , Co 2+ , and Mg 2+ are immobilized.
  • a carrier to be used a known carrier such as a bead, a microtiter plate, or a resin can be used.
  • beads and microtiter plates are conventionally used in immunoassays, and the measurement system can be easily constructed.
  • a carrier having a planar portion such as a substrate can also be used. In this case, it is preferable to immobilize a substance having affinity for the marker in a part of the flat surface portion.
  • An example is a carrier in which a chip is used as a base and an antibody specific for a marker is immobilized in spots on a plurality of spots on the surface.
  • the detection, examination or diagnosis method according to the present invention can be applied to screening of substances effective for the prevention or treatment of cerebral malaria. That is, an effective substance can be screened using the test substance as an index of binding or interaction with a CCL21-CXCR3-CCR7 signaling factor such as CCL21, CXCR3, CCR7, or a nucleic acid molecule encoding the same.
  • Test substances that can be used include synthetic low molecular weight compounds, proteins, synthetic peptides, purified or partially purified polypeptides, antibodies, bacterial release substances (including bacterial metabolites), nucleic acids (antisense, ribozyme, RNAi, etc.), etc.
  • test substance is a compound or a salt thereof, or a solvate (for example, hydrate) thereof, but is not limited thereto.
  • the test substance may be a novel substance or a known substance.
  • the substance identified by the screening method according to the present invention can be used as a substance effective for the treatment or prevention of cerebral malaria.
  • the oligonucleotide of the present invention can be synthesized by a standard method known in the art, for example, by using an automated DNA synthesizer (commercially available from Biosearch, Applied Biosystems, etc.). is there.
  • an automated DNA synthesizer commercially available from Biosearch, Applied Biosystems, etc.
  • it is possible to synthesize phosphorothioate oligonucleotides by the method of Stein et al. (Stein et al., 1988, Nucl. Acids Res. 16: 3209), and control pore glass polymer supports (Sarin et al. , 1988, Proc. Natl. Acad. Sci. USA 85: 7448-7451), etc. can also be used to prepare methylphosphonate oligonucleotides.
  • Wild-type mice were intravenously (iv) daily injected with endotoxin-free anti-CCL21 antibody or isotype control (50 ⁇ g per mouse, Peprotech daily) for 3 days from the start of PbA infection.
  • an anti-CXCR3 antibody LEAF purified anti-mouse CD183 (CXCR3), 100 ⁇ g per mouse, Biolegend
  • CXCR3 LEAF purified anti-mouse CD183
  • the combination treatment was performed using anti-CXCR3 and anti-CCL21 antibody mixture on the 4th day after infection, and repeated injections of anti-CCL21 antibody on the 5th day after infection.
  • MRI brain imaging MRI imaging of the mouse head was performed using an ultra-high magnetic field 11.7T MRI scanner (AVANCE-II 500 WB; Bruker BioSpin).
  • Naive mice were anesthetized with 1.0-1.5% isoflurane during the MRI treatment procedure to obtain an initial image from the living body and an image from a dead mouse with 4% paraformaldehyde (PFA) fixation. Compared. This comparison suggested that there was no significant difference between the two (FIG. 1E). Therefore, in subsequent experiments, infected mice with deep anesthesia were fixed in PFA and visualized by MRI.
  • T2 * weighted image FLASH sequence
  • DWI diffusion weighted image
  • T2 * weighted fine image field of view, 15 mm ⁇ 15 mm; matrix size, 512 ⁇ 512; slice thickness, 0.3 mm; repetition time, 450 ms; echo time, 6.0 ms; average, 64; scan time, 4 hours and 5 minutes.
  • T2 * weighted normal image field of view, 20 mm ⁇ 20 mm; matrix size, 256 ⁇ 256; slice thickness, 0.5 mm; repetition time, 400 ms; echo time, 6.0 ms; average, 16; scan time, 27 minutes.
  • DWI field of view, 15 mm ⁇ 15 mm; matrix size, 512 ⁇ 512; slice thickness, 0.3 mm; repetition time, 6000 ms; echo time, 21.0 ms; b value, 1000 s / mm 2 ; average, 9; scan time 7 hours 41 minutes.
  • mice were previously anesthetized by intramuscular injection of ketamine (0.13 mg / g) and xylazine (0.01 mg / g)
  • ketamine 0.13 mg / g
  • xylazine 0.01 mg / g
  • the surgical “thinned-skull” technique described in S., et al. (Sawada, M. et al., (2011). J Neurosci 31, 11587-11596 .; Wake, H. et al. (2009). J Neurosci 29, 3974-3980.). Briefly, the head of the mouse was fixed on the stereotaxic stage using an ear bar.
  • the skull on the OLF was carefully thinned (approximately 20-30 ⁇ m) using a high-speed drill (Minimo, Miniter) and a scalpel. A metal ring was attached to the skull over that area and the thinned area was kept moist during the experiment. Imaging was performed using a microscope (FV1000MPE, Olympus) equipped with a water immersion objective lens (XLPLN25XWMP, Olympus) at a digital zoom of 1 to 2.5 times.
  • a microscope FV1000MPE, Olympus
  • XLPLN25XWMP water immersion objective lens
  • TRITC tetramethylrhodamine isothiocyanate
  • T1287 T1287, Sigma
  • T cells were expressed in TCR- ⁇ conjugated with brilliant violet 421 ( Labeled with 10 ⁇ g of H57-597, Biolegend), or 5 ⁇ g of CD8 ⁇ (53-6.7, Biolegend).
  • a titanium sapphire laser (MaiTai Hp, Spectral Physics) was tuned to an excitation wavelength of 800 nm for T cells and blood vessels, and a Chameleon laser (Coherent) was tuned to an excitation wavelength of 950 nm for GFP-PbA.
  • Time-lapse imaging of small OLF regions [507.934 ⁇ m (x), 507.934 ⁇ m (y), 5 ⁇ m per 1.1095 seconds] by continuously and repeatedly acquiring a fluorescence image stack containing 30-80 z-planes. z)] (acquisition of one stack image takes about 40 to 90 seconds depending on the number of z planes to be acquired).
  • Typical imaging depths were 80-150 ⁇ m from the buffy coat surface, which corresponded to the glomerular layer (FIGS. 2A-B) (Chaigneau, E. et al. (2003). Proc Natl Acad Sci. U SA 100, 13081-13086.).
  • Intermittent intramuscular injection of a mixture of ketamine and xylazine allowed the animals to be monitored for 1-2 hours. Imaging was performed only once for each mouse. All imaging data was processed and analyzed using Velocity software.
  • mice were given i.e. 200 ⁇ l of 1% Evans Blue dye (Sigma) i. v. Injected. Two hours later, the mice were sacrificed, the brains were removed, washed with PBS, and brain images were taken under a dissecting microscope.
  • splenocytes were prepared from Rag2 ⁇ / ⁇ mice 5 days after PbANKA infection, and spleen CD8 ⁇ + DC were concentrated. Briefly, the CD11b - population was negatively selected using anti-mouse CD11b microbeads (MACS, Miltenyi Biotec., Germany), after which the CD4 + population was removed (L3T4, MACS). The remaining enriched CD8 ⁇ + DC (20 ⁇ 10 6 cells) was administered i.p. to recipient mice on day 0 2 hours prior to PbA infection. v. Injected.
  • the purity and phenotype of the positively selected CD8 ⁇ + DC cell population was assessed by flow cytometry prior to adoptive immunization and was typically found to be more than 95% pure (FIG. 6I).
  • Statistical analysis Differences between the two groups were analyzed for statistical significance using either the Student's t test without two-sided correspondence or the nonparametric Mann-Whitney test (Prism software). For the survival curve, Log-rank (Mantel-Cox) test was performed. p ⁇ 0.05 was considered statistically significant.
  • Example 1 Elucidation of relationship between brain malaria and olfactory bulb
  • ultrahigh magnetic field MRI imaging In the present embodiment, first, ultrahigh magnetic field MRI imaging is performed on the olfactory bulb. identified as the location of microhemorrhages in the brain during cerebral malaria caused by berghei ANKA parasites.
  • the OLF is composed of a fence-like microvascular structure with high density and facing in various directions (radial and tangential directions).
  • Such complex vasculature can act as a suitable scaffolding environment for neuronal migration within tissues, with synaptic interactions within the glomerulus together with neurons and glial cells (Bovetti, S. et al. (2007). J Neurosci 27, 5976-5980 .; Danielyan, L. et al. (2009). Eur J Cell Biol 88, 315-324.).
  • OLF was visualized by in vivo MP microscopy.
  • MP imaging of OLF in which OLF capillaries as deep as GL (about 150 ⁇ m) can be visualized, has been performed with rodents (Chaignau, E. et al., (2003). Proc Natl Acad Sci USA100. Petzold, GC et al., (2008) Neuron 58, 897-910 .; Sawada, M. et al., (2011). J Neurosci 31, 11587-11596.) (FIG. 2A).
  • mice were infected with GFP-PbA parasites. Five days after infection, mice showed no visible ECM symptoms, but blood vessels were labeled with red TRITC-dextran and images were acquired for 30 minutes as shown in FIG. 2A. It was observed that some parasites slowed down and attached to the blood vessels. Although there were five moving images, the same was true for five mice on the fifth day after infection. As can be seen in this animation S1, the rate of some GFP-labeled parasites was reduced and / or stopped, eventually causing occlusion. This was consistent with significantly higher parasite burden as measured by 18S rRNA levels as well as T cell accumulation in OLF at 6 days post infection compared to other parts of the brain (Fig. 2C).
  • OLF is a region that is unique to ECM pathogenesis, possibly due to its flanked capillary structure, whereby circulating iRBCs can slow down, adhere and / or be sequestered. Suggests that eventually it leads to bleeding.
  • CD8 T cell traffic through blood vessels in OLF Intravascular accumulation of CD8 T cells in the brain has been shown to have an important role in the pathogenesis of ECM (Belnoue, E. et al. (2002). J Immunol 169, 6369-6375 .; Miyakoda, M. et al. (2008). J Immunol 181, 1420-1428.).
  • CD8 T cell recruitment could be observed by in vivo MP imaging of OLF and / or could be associated with microbleeding after PbA infection. .
  • In vivo imaging of labeled CD8 T cells and GFP-expressing PbA parasites in the infected mouse's OLF clearly showed that microbleeding occurred in the branching capillaries (FIG.
  • animation S2 (this specification) Not shown in the book)).
  • animation S2 a representative 3D image of a mouse infected with a GFP-PbA parasite showing a new microbleeding site in the olfactory bulb capillary was taken. Infected mice were visualized by in vivo multiphoton microscopy for approximately 1 hour on day 6 after infection. Blood vessels labeled with red, red TRITC-dextran; green, PbA parasite expressing GFP; blue, CD8 T cells labeled with anti-CD8 ⁇ antibody. Some red areas indicate areas that have already bleed that have lost vascular integrity; T cells are crawling through the tissue. Sudden loss of central vascular integrity indicated new bleeding.
  • CD8 T cells are found to “crawl” back and forth within the blood vessels during ECM development and increase in number (FIG. 2D, animated S2, S3 and S4 (not shown here) ) Also confirmed), it was found that some of them attached around the bleeding area (moving image S2 (not shown here)).
  • the behavior of CD8 T cells within these OLFs can passively follow the unstable blood flow at the end of ECM, but was constructed by the inventors on the fifth day after infection in relatively large blood vessels (around 10 ⁇ m).
  • Movie 3S shows how T cells crawl along microvessels in the olfactory bulb, and shows a multiphoton movie constructed in 3D of the olfactory bulb of a mouse infected with a GFP-PbA parasite 5 days after infection. . Blood vessels labeled with red, red TRITC-dextran; green, PbA parasite expressing GFP; blue, T cells labeled with anti-TCR ⁇ antibody are shown. It was found that T cells attached to the walls of microvessels (about 10 ⁇ m in diameter) and migrated along the walls.
  • CD8 T cells Intravascular accumulation of CD8 T cells in the brain has been shown to have an important role in the pathogenesis of cerebral malaria (Belnoue, E. et al. (2002). J Immunol 169, 6369-6375.). We therefore investigated whether CD8 T cell recruitment could be observed by in vivo MP imaging of OLF and / or could be associated with microbleeding after PbA infection. . In vivo imaging of labeled CD8 T cells and GFP-expressing PbA parasites in infected mouse OLF clearly showed that microhemorrhages occurred in branch vessels (FIG. 2C and animation S2 (not shown here)) ).
  • CD8 T cells were found to traverse back and forth within the blood vessel as if they “followed” parasites in the OLF blood vessels (FIG. 2D and animation S3 (in this specification). Not shown)). Furthermore, the constructed 3D animation suggested that CD8 T cells can attach to blood vessels and crawl along the vessel wall (animation S4 (not shown here)). In summary, these biological images of OLF show that parasites accumulated in the OLF are tracked by CD8 T cells and exit from the blood vessel due to micro-bleeding of small OLF blood vessels.
  • mice were left free of food for 18 hours, then transferred to a new cage with food buried in the random corners of the cage, including bedding, approximately 1 cm below the surface. The time taken for the mouse to find the buried food was recorded.
  • OLF function is normal mice that are resistant to ECM, such as BALB / c or Rag2 ⁇ / ⁇ mice or mice infected with lethal PyL parasites, as determined by a delay in the time to find food. Compared with (FIG. 3B), it was significantly impaired as early as 4 days after infection (FIG. 3A). Thus, loss of OLF function may allow prediction of ECM manifestations such as bleeding within OLF and potential loss of BBB integrity.
  • mice were injected with Evans Blue dye at an early time point (days 3-6) after infection, The oozing of blue dye into the tissue was monitored. According to the observation of MP imaging in which iRBC was decelerated and stopped in the blood vessel, the blue dye exudation to the brain tissue appeared from OLF as early as 5 days after infection, while the whole brain was 6 ⁇ It turned blue on day 7 (FIG. 3C). Since BBB is restrictive due to proteins such as tight junctions and tight band-1 (ZO-1), we next examine whether ZO-1 staining in OLF is associated with BBB leakage I checked.
  • ZO-1 tight band-1 staining in OLF is associated with BBB leakage I checked.
  • ZO-1 has been shown to localize in the olfactory epithelium, olfactory neurons, and olfactory bulb mitral cell layer (Miragal, F. et al. (1994). J Comp Neurol 341, 433-448.).
  • OLFMCL GFP-expressing parasites
  • FIG. 3D GFP-expressing parasites
  • Example 2 Relationship between brain malaria and chemokines
  • Infected mouse # 4 eventually lost heat and died early and disappeared from the camera. Infected mouse # 3 died the day after infected mouse # 4 died. The video showed almost the same when taken with at least 10 mice.
  • lethal PyL or non-lethal P Mice infected with yoelii-NL did not fever during infection (FIGS. 3G and 5F-G), suggesting that the increase in heat 5 days after infection may be PbA specific.
  • CCL21 is highly expressed in OLF at an early stage of infection
  • Chemokines are early mediators of infection or inflammation and are increasingly recognized as contributing to the development of fever (Machado, RR et al. (2007). Brain Res 1161, 21-31.). Since some chemokines and cytokines have an important role in the development of ECM, we have interferon (IFN) - ⁇ , IP-10 (CXCR3 ligand), MCP-1 (CCR2 ligand) in OLF
  • IFN interferon
  • CXCR3 ligand IP-10
  • MCP-1 CCR2 ligand
  • CCL21 and CCL19 mRNA and protein are highly expressed in OLF as early as 3 days after infection (FIGS. 4A-B), and early expression of chemokine ligands, particularly CCL21, recruits immune cells to the brain It was suggested that it may be important.
  • Ccr7 ⁇ / ⁇ mice have a delayed hyperthermia (approximately 48 hours) compared to WT mice (24 hours) (FIG. 4E), as determined by MRI and Evans blue staining, There were no signs of bleeding in OLF on day 6 after infection (FIGS. 5H and I). Evans blue staining occurred gradually in about 80% of the brains of Ccr7 ⁇ / ⁇ mice after day 8 (FIGS. 5H and I). These data suggested that CCR7 has a role in the pathogenesis of ECM and also contributes to OLF dysfunction, microhemorrhage and high fever.
  • CCR7 expression is important for CD8 ⁇ DC activation of CD11c + CD8 T cells, but not for its movement into the brain.
  • Ccr7 ⁇ / ⁇ mice We next examined the underlying mechanisms responsible for increasing the survival of Ccr7 ⁇ / ⁇ mice from ECM. Since CCR7 is an important chemokine receptor in the migration of immune cells such as DCs and T cells to secondary lymphoid organs, the inventors investigated T cell recruitment in the brain. Flow cytometric analysis of immune cells obtained from the brain 6 days after PbA infection showed that CD8 T cell accumulation in the brain was reduced by 50% in Ccr7 ⁇ / ⁇ mice compared to WT mice. ( * P ⁇ 0.05; FIG. 5A).
  • CD8 ⁇ + DC can cross-prime the CD8 T cell response, it has been suggested that CD8 T cells are susceptible to disease in the development of mouse brain malaria (Lundie, RJ et al. (2008). Proc. NatlAcad Sci USA 105, 14509-14514 .; Piva, L. et al. (2012). J Immunol 189, 1128-1132.).
  • mice lacking the basic leucine zipper transcription factor ATF-like-3 (Batf3) without CD8 ⁇ DC in the spleen Hudner, K. et al. (2008). Science 322. 1097-1100.
  • the number of activated CD11c + CD8 T cells in the spleen and their accumulation in the brain when infected with PbA is higher in Batf3 ⁇ / ⁇ mice compared to Batf3 +/ ⁇ controls.
  • Significantly impaired Figures 6E and F. It was also confirmed that Batf3 ⁇ / ⁇ mice showed improved survival (FIG. 6G).
  • CD8 ⁇ + DCs are first enriched by negative selection with CD11b + beads, then NK cells, macrophages and granulocytes are removed and purified after further CD4 + cell depletion Obtained a CD8 ⁇ + DC population ( > 95% purity, FIG. 6B). Then, Ccr7 ⁇ / ⁇ mice were adoptively immunized with highly concentrated CCR7 + CD8 ⁇ + DC. Adoptive concentration CD8a + DC is, CCR7 - / - in mouse, activated CD11c + CD8 T cells in the brain (CCR7 - / - of mouse origin) supplemented efficiently revive the accelerated the ECM (FIG. 5E).
  • CCL21 expressed in OLF is consistent with astrocyte activation and may be important for migration of CD11c + CD8 T cells into OLF.
  • CCL21 expression was also observed in PbA-infected OLF from day 3 to the final stage, we further observed that CCL21 is OLF via alternative chemokine receptor interactions other than CCR7. It was hypothesized that it could have an additional role in recruiting CD8 T cells into it.
  • CXCR3 can be an alternative chemokine receptor for CCL21, particularly in microglia and astrocytes (Rappert, A. et al. (2002).
  • CCL21 staining was closely correlated with CD8 T cells in OLF (not shown, in this omitted figure, CD8 T cells are associated with CCL21 in OLF)
  • OLF sections from PbA-infected WT mice, 6 days post infection were stained with anti-CCL21 (green) and anti-CD8 (red) antibodies, and nuclei were visualized with DAPI (blue). Bars are 10 ⁇ m.
  • Arrows indicate CCL21 fibrillar structures that interact with CD8 T cells.
  • CXCR3 + CD11c + CD8 T cells migrated in a dose-dependent manner toward CCL21, but this migration was specifically blocked by anti-CCL21 antibody (the figure is omitted). , Suggesting that it may function as an alternative chemokine ligand for CXCR3 during ECM.
  • migration of purified splenic CD11c + CXCR3 + CD8 T cells from infected WT mice in response to increasing concentrations (0, 0.1, 1 and 2 ⁇ g / ml) of CCL21 is shown.
  • Human SDF1- ⁇ (80 ng / ml) was used as a positive control, and the response to (E) CCL21 (1 ⁇ g / ml) and / or anti-CCL21 antibody (1 and 5 ⁇ g / ml) was examined.
  • Migrated cells are collected from triplicate wells, counted in a flow cytometer, and chemokine-induced migration is normalized to unstimulated control (gray dotted line), as a percentage of unstimulated control migration It was shown as (%) and was shown to be specifically blocked by anti-CCL21 antibody.
  • Example 3 Prevention and treatment of cerebral malaria
  • CCL21-CCR7-CXCR3 axis block is a new strategy for ECM treatment
  • CCL21-CCR7-CXCR3 axis block is a new strategy for ECM treatment
  • the CCL21-CCR7-CXCR3 axis has an important role in CD8 ⁇ DC-CD8 T cell-mediated ECM immunopathology
  • CCL21 as a therapeutic target for the treatment of ECM I evaluated it.
  • In vivo treatment of OPbA infected mice with anti-CCL21 antibody during the first 3 days of infection led to significantly better survival in mice compared to mice treated with isotype control (FIG. 6F, ** p ⁇ 0.01).
  • the head After removing the skull, the head is fixed in 4% paraformaldehyde and the whole head is imaged using 7T MRI.
  • 7TMRI can detect some suspicious bleeding spots in the OLF and check for signs of bleeding in other parts of the brain.
  • the OLF is taken out and imaged by 11.7T MRI.
  • Ultra-high magnetic field imaging is similar to that used for mice. It can be confirmed that bleeding occurs in the monkey OLF after infection with coatneyi. In contrast, na ⁇ ve monkeys and P. aerial malaria model but not brain malaria. It can be confirmed that there is no low density region in OLF from knowlesii-infected monkeys (White, NJ (2008). Clin Infect Dis 46, 172-173.).
  • OLF is the first place to be affected by Plasmodium parasites.
  • the OLF is composed of dense capillaries that are oriented in various directions (radial and tangential directions) (FIGS. 2A-B and movie S1), and such a structure comprises a thin end foot of stellate cells surrounding the blood vessel.
  • FILL radial and tangential directions
  • a network of tight junctions and forms the “guardian” of the BBB. This restricts the flow of substances between blood and nervous tissue, which may probably be through the ability of tight junctions to transmit information between astrocytes (Bailey, MS and Shipley, MT (1993) J Comp Neurol 328, 501-526. Chen, Y. et al. (2013) J Biomed Opt 18, 126012.
  • OLF is known as the OLF neuronal projection, especially the dynamic location of chemosensitivity.
  • the OLF nerve begins with the nasal mucosa and ends with the olfactory bulb via the sieve plate. Lymphatic vessels and blood vessels surround these nerves, through which molecules, cells, and even pathogens can gain access to the brain parenchyma (Danielyan, L. et al. (2009). Eur J CellBiol 88 , 315-324.).
  • CNS central nervous system
  • Bovetti S. et al. (2007). J Neurosci.
  • OLF may be the gateway for dynamic cell migration between the external environment and the CNS. Therefore, it is reasonable for patients suffering from neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease, and autoimmune diseases such as systemic lupus erythematosus (SLE) to experience OLF dysfunction as an initial symptom.
  • neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease
  • autoimmune diseases such as systemic lupus erythematosus (SLE) to experience OLF dysfunction as an initial symptom.
  • SLE systemic lupus erythematosus
  • Astrocytes are common CNS resident cells essential for blood flow regulation and BBB maintenance. Astrocytes also have an important role in the immune defense of the CNS by expressing a wide variety of chemokines during physiological and pathological states (de Haas, AH et al. (2007). Mol Neurobiol 36, 137-151 .; Medana, IM et al. (1996). Glia 16, 51-64.). Furthermore, astrocytes increase CCL21 expression in response to CNS injury and infection (Lalor, SJ and Segal, B.M. (2010). J Neuroimmunol 224, 56-61 .; Noor, S. (2010) .Infect Immun 78, 2257-2263.).
  • Ccr7-deficient mice Because of the increasing gradient of CCL21 expression in OLF, particularly GL where there are a large number of astrocytes, we decided to study Ccr7-deficient mice. This is because CCR7 regulates lymphocyte traffic for immune surveillance of CNS through its interaction with its ligands CCL19 and CCL21 (Noor, S. and Wilson, E. et al. H. (2012) J Neuroinflamation 9, 77.). In addition, previous studies have shown that chemokine receptors such as CCR1, CCR2, CCR5, CCR7, CCR8, CXCR3 and CXCR1 may be important for cell migration into the brain (Miu, J. et al.
  • CCR7 The role of CCR7 in the induction and maintenance of antiviral effector and memory CTL responses has been thoroughly investigated in Ccr7 ⁇ / ⁇ mice (Junt, T. et al. (2004). J Immunol 173, 6684-6669. )), The role of CCR7 in serious malaria models such as ECM has not been studied so far. In a lymphocytic choriomeningitis virus infection model, CCR7 was important for coordination of antiviral effector and memory CTL migration and proliferation (Junt, T. et al. (2004). J Immunol 173, 6684- 6663.).
  • CD8 ⁇ DC can cross-present antigens, particularly antigens from dead cells, to T cells (Shortman, K. and Heath, WR (2010). Immunol Rev 234, 18- 31.).
  • CD11c + CD8 T cells migrate to the brain during the effector phase via multiple molecules including several chemokines and chemokine receptors such as IP-10 and CXCR3.
  • chemokines and chemokine receptors such as IP-10 and CXCR3.
  • CCL21 is involved in priming of CD11c + CD8 T cells
  • the presence of CCL21 in OLF early in the infection 3 days after infection suggests chemotactic support for T cell migration. It meant that relevance.
  • CXCR3 has been recognized as an alternative chemokine receptor for CCL21, particularly in astrocytes and microglia (van Weering, HR et al. (2010). BrainBehave Immuno). 24, 768-775.).
  • CCL21 has been shown to increase rapidly in the brain after Toxoplasma infection and support T cell migration (Wilson, EH et al. (2009). Immunity 30, 300-311.). Therefore, CXCR3 + CD8 T cells may also be able to migrate to OLF by a similar mechanism involving CCL21.
  • Cerebral malaria diagnosis / detection marker and cerebral malaria control technology are provided, and technologies that can be used in industries (reagents, pharmaceuticals, etc.) related to the diagnosis, treatment and prevention of cerebral malaria are provided.
  • SEQ ID NO: 1 Representative nucleic acid sequence of CCL21 SEQ ID NO: 2: Representative amino acid sequence of CCL21 SEQ ID NO: 3: Representative nucleic acid sequence of CXCR3 SEQ ID NO: 4: Representative amino acid sequence of CXCR3 SEQ ID NO: 5: Representative nucleic acid sequence of CCR7 SEQ ID NO: 6: Representative amino acid sequence of CCR7

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Abstract

La présente invention concerne un marqueur de paludisme cérébral et des techniques pour son application. L'invention concerne un marqueur pour identifier le paludisme cérébral, ledit marqueur étant basé sur l'état de bulbe olfactif ou comprenant un facteur dans le système de transduction de signal CCL21-CXCR3-CCR7 tel que CCL21, CXCR3 ou CCR7, ou un produit d'expression de ce dernier, un fragment de ce dernier ou un dérivé de ce dernier ; un agent de détection pour identifier le paludisme cérébral, ledit agent de détection comprenant une substance liée à un facteur dans le système de transduction de signal CCL21-CXCR3-CCR7 tel que CCL21, CXCR3 ou CCR7, ou un produit d'expression de ce dernier ; et une composition médicinale pour prévenir ou traiter le paludisme cérébral, ladite composition médicinale comprenant un inhibiteur d'un facteur dans le système de transduction de signal CCL21-CXCR3-CCR7 tel que CCL21, CXCR3 ou CCR7.
PCT/JP2015/060403 2014-03-27 2015-03-26 Diagnostic et traitement du paludisme cérébral WO2015147335A1 (fr)

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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003072134A1 (fr) * 2002-02-28 2003-09-04 Kyowa Hakko Kogyo Co., Ltd. Diagnostics et therapeutiques pour la pneumonie interstitielle
JP2004517078A (ja) * 2000-12-01 2004-06-10 シェーリング　コーポレイション 哺乳動物遺伝子および関連試薬の使用
JP2008545000A (ja) * 2005-07-06 2008-12-11 ウニベルシダッド・アウトノマ・デ・マドリッド ガンを治療するための抗ｃｃｒ７受容体抗体
JP2009528977A (ja) * 2005-12-23 2009-08-13 ザ リージェンツ オブ ザ ユニバーシティ オブ ミシガン 慢性線維形成性疾患を治療するための物質および方法
JP2012507546A (ja) * 2008-10-31 2012-03-29 レクシコン ファーマシューティカルズ インコーポレイテッド 脳マラリアの治療のためのｓ１ｐ受容体作動薬
WO2012165295A1 (fr) * 2011-05-31 2012-12-06 国立大学法人大阪大学 Circuit à résonance

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2004517078A (ja) * 2000-12-01 2004-06-10 シェーリング　コーポレイション 哺乳動物遺伝子および関連試薬の使用
WO2003072134A1 (fr) * 2002-02-28 2003-09-04 Kyowa Hakko Kogyo Co., Ltd. Diagnostics et therapeutiques pour la pneumonie interstitielle
JP2008545000A (ja) * 2005-07-06 2008-12-11 ウニベルシダッド・アウトノマ・デ・マドリッド ガンを治療するための抗ｃｃｒ７受容体抗体
JP2009528977A (ja) * 2005-12-23 2009-08-13 ザ リージェンツ オブ ザ ユニバーシティ オブ ミシガン 慢性線維形成性疾患を治療するための物質および方法
JP2012507546A (ja) * 2008-10-31 2012-03-29 レクシコン ファーマシューティカルズ インコーポレイテッド 脳マラリアの治療のためのｓ１ｐ受容体作動薬
WO2012165295A1 (fr) * 2011-05-31 2012-12-06 国立大学法人大阪大学 Circuit à résonance

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JP6837242B2 (ja) 2021-03-03
JP2021053427A (ja) 2021-04-08
JP2019136041A (ja) 2019-08-22
JP6618191B2 (ja) 2019-12-11

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