JP6618191B2 - Diagnosis and treatment of brain malaria - Google Patents

Description

The present invention relates to the diagnosis and treatment of cerebral malaria (sometimes referred to herein as “CM”).

  Cerebral malaria (CM) is the most deadly complication of Plasmodium falciparum (P. falciparum) infection, characterized by sudden coma, death, or neuropathy. Although the brain is severely affected, the location and spatiotemporal mechanisms of the brain are not well understood as to how CM occurs.

  Cerebral malaria (CM) It is a serious complication of malaria infection in humans caused by falciparum parasites and is characterized by sudden clinical symptoms such as convulsions and coma with high mortality or long-term disability (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.). Since CM presents non-specific symptoms, early diagnosis of CM is not easy, and often symptoms of the disease become apparent when CM treatment is not very effective (Non-Patent Document 3 = Miller, L. H. et al. (2013). Nat Med 19, 156-167.). Therefore, there is a need for early, quick and inexpensive diagnosis of CM that allows timely intervention.

  To understand the pathology of cerebral malaria (CM), A mouse model of CM (known as experimental brain malaria, ECM) using the berghei ANKA (PbA) parasite is widely used (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. In fact, many studies have shown that various cells (mostly white blood cells) accumulate in large numbers in the blood vessels of the brain, where parasites are cross-primed by infected red blood cells (iRBC) and CD8α + dendritic cells (DC). Mention that specific pathogenic CD8 T cells play an important role in the pathogenesis of ECM (non-patent document 5 = Baptista, FG et al. (2010). InfectImmun 78, 4033-4039 .; Patent Document 6 = Haque, A. et al. (2011) .J Immunol 186,6148-6156.; Non-patent Document 7 = Howland, SW et al. (2013). aria.EMBO Mol Med. (May 16, 2013 online version release, doi: 10.1002 / emmm.201202273); Non-patent document 8 = Lundie, R.J. 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)). In addition, recent studies have reported the expression of CD11c in activated CD8 T cells during PbA infection, but their exact role during ECM has not been well studied (12). , T. et al. (2011) .Infect Immun 79, 3947-3956.). BBB dysfunction is a major feature of ECM, and systemic as well as 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.).

Idro, R .; (2010). Pediatr Res 68, 267-274. Taylor, T .; E. (2004). Nat Med 10, 143-145. Miller, L.M. H. (2013). Nat Med 19, 156-167. ). Langhorne, J .; (2011). Malar J 10,23. Baptista, F.M. G. (2010). Infect Immun 78, 4033-4039. Haque, A .; (2011). J Immunol 186, 6148-6156 Howland, S.M. W. (2013). Brain microvessel cross-presentation is a hallmark of experiential cerebral malaria. EMBO Mol Med. (May 16, 2013 online version release, doi: 10.1002 / emmm.201202273) Lundie, R.M. J. et al. (2008). Proc Natl Acad Sci USA 105, 14509-14514. McQuillan, J.M. A. (2011). Int J Parasitol 41,155-163 Renia, L.M. (2012). Viralence 3,193-201 Yui, K .; (2013). EMBO Mol Med. (Online version released on June 5, 2013, doi: 10.1002 / emmm.201302849) Tamura, T .; (2011). Infect Immun 79, 3947-3958. Combes, V.M. (2012). Trends Parasitol 28, 311-319. Nacer, A .; (2012). PLoS Pathog 8, e1002982

We understand whether the initial BBB disruption occurs simultaneously in blood vessels throughout the brain or whether there is a specific place in the brain that makes the BBB tolerant to iRBC and pathogenic immune cells. Research has been advanced. Similarly, extensive research has been conducted to elucidate the spatiotemporal control of immune responses in specific brain regions and / or cells.

  The inventors have used the powerful imaging technique, a combination of ultrahigh magnetic field 11.7T MRI and multiphoton (MP) microscopy, the pathophysiological mechanism and immunology of mouse and non-human primate CM. The spatiotemporal control of the mechanical mechanism was investigated. We have identified PbA (mouse CM model) and P.A. The basic mechanism by which BBB is destroyed and tolerated during systemic infection with coatneyi (monkey CM model) was elucidated. We find that the olfactory bulb (OLF), composed of a unique radial and tangentially dense microcapillary structure, serves as a suitable environment for parasite and cell migration and is the first location to detect malaria infection And found to allow "crosstalk" between the brain and the activated immune system. This links OLF with olfactory loss, hyperthermia, astrocytes, CCL21, CCR7, CXCR3 and CD8 T cells.

In the present specification, 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. Thus, early detection of olfactory loss and immediate disturbance of pathological cell recruitment may provide a novel treatment for ECM. Accordingly, the present invention also provides the following.
(1) A method in which the state of the subject's olfactory bulb (OLF) is used as an index of brain malaria.
(2) The method according to item 1, wherein the state is determined by at least one selected from abnormal olfaction, cell examination, and diagnostic image of the olfactory bulb.
(3) The method according to item 2, wherein the olfactory abnormality is an abnormality in a test selected from the group consisting of a reference olfactory test and a vein olfactory test. (4) The method according to item 2 or 3, wherein the diagnostic image is obtained by nuclear magnetic resonance imaging (MRI).
(5) The method according to item 4, wherein the MRI is taken with a magnetic force of 7 T or more.
(6) The method according to any one of Items 2 to 5, wherein a spot is observed in the diagnostic image is an index of cerebral malaria.
(7) The method according to any one of Items 2 to 6, wherein the cell test is performed by staining or immunohistochemistry.
(8) The detection method according to any one of items 1 to 7, wherein the abnormality of the subject's olfactory bulb is an early indicator of morbidity.
(9) A method of diagnosing cerebral malaria, comprising measuring the state of a subject's olfactory bulb (OLF).
(10) The method according to item 9, wherein the abnormality of the subject's olfactory bulb is an indicator that the subject is suffering from cerebral malaria.
(11) A device for detecting or diagnosing cerebral malaria, including means for measuring the state of the olfactory bulb.
(12) The apparatus according to item 11, wherein the means is a magnetic resonance imaging (MRI) means.
(13) The apparatus according to item 12, wherein the MRI is an MRI of 7T or more.
(14) A preventive or therapeutic agent for cerebral malaria comprising an inhibitor of at least one factor of the CCL21-CXCR3-CCR7 signaling system.
(15) 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.
(16) The inhibitor is a low molecular 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 Item 14 or 15, comprising at least one solvate of a salt acceptable in.
(17) Any of items 14 to 16, wherein the inhibitor comprises at least one antibody 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 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 preventive or therapeutic agent for cerebral malaria according to item 1.
(19) The brain malaria according to any one of items 14 to 18, wherein the inhibitor comprises three types of antibodies, anti-CCL21 antibody, anti-CXCR3 antibody and anti-CCR7 antibody, or fragments or functional equivalents thereof. Preventive or therapeutic agent.
(20) An inhibitor of at least one factor of the CCL21-CXCR3-CCR7 signaling system for the prevention or treatment of cerebral malaria.
(21) The inhibitor according to item 20, further comprising the feature according to any one or more of items 15 to 19.
(22) 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.
(23) A marker for cerebral malaria comprising at least one factor of the CCL21-CXCR3-CCR7 signaling system.
(24) 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. 24. The marker for brain malaria according to item 23.
(25) 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) One inflammatory cytokine and at least one selected from the group consisting of nucleic acids encoding them, their expression products and their derivatives;
(2) CCL21 and / or CCL19
(3) comprising at least one factor selected from the group consisting of: CD8α 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.
(26) (A) Increased expression of CCL21 and / or CCL19 in the olfactory bulb;
(B) Increased expression of IFN-γ in the cortex;
(C) that the CD8α dendritic cells are observed to be primed in a CCR7-dependent manner; and / or (D) that activated CD8 T cells are observed in the brain or spleen. The marker for brain malaria according to any one of items 23 to 25.
(27) The cerebral malaria is 27. The marker for brain malaria according to any one of items 23 to 26, which is caused by a human malaria parasite corresponding to a malaria parasite selected from the group consisting of falciparum.
(28) The marker according to any one of items 23 to 27, wherein the brain malaria is a rodent or a primate.
(29) 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.
(30) (1) CCL21 protein or a nucleic acid encoding the same;
30. The marker according to any one of items 23 to 29, comprising (2) CXCR3 or a nucleic acid encoding the same; and / or (3) CCR7 protein or a nucleic acid encoding the same.
(31) Brain malaria comprising a substance that binds to at least one factor selected from the group consisting of at least one factor of the CCL21-CXCR3-CCR7 signaling system and a nucleic acid encoding them, an expression product thereof, and a derivative thereof. Detection agent or diagnostic agent.
(32) 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.
(33) The detection agent or diagnostic agent according to item 31 or 32, wherein the detection agent or diagnostic agent is labeled.
(34) The detection agent or diagnostic agent according to any one of items 31 to 33, wherein the detection agent or diagnostic agent is a nucleic acid, and the nucleic acid is a probe or a primer.
(35) A method for identifying 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.
(36) If 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 method according to item 35.
(37) The method according to item 35 or 36, wherein the identification is performed using the detection agent or diagnostic agent according to any one of items 31 to 34.

  In the present invention, it is contemplated that the one or more features may be provided in further combinations in addition to the explicit combinations. Still further embodiments and advantages of the present invention will be recognized by those of ordinary skill in the art upon reading and understanding the following detailed description as necessary.

  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. (A) 11.7 T MRI (C57BL / 6, FLASH T2 ^* signal, naïve vs. 6 days after infection) of coronal and sagittal sections of the mouse head. The circle surrounded by the dotted line of the coronal section and the arrow of the sagittal section correspond to the olfactory bulb (OLF). (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 μm. FIGS. 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 11.7T 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 μm from the buffy coat surface and the capillaries (red, less than 5 μm in diameter) in the GL are visualized. Larger blood vessels (superficial arteries and arterioles, approximately 20-40 μm in diameter) are located in the ONL. GL (glomerular layer), ONL (olfactory nerve layer), MCL (mitral cell layer). (B) Representative snapshot MP image from OL of WT mice 5 days after GFP-PbA infection (data not shown but also confirmed by moving images). Arrows indicate GFP-PbA parasites attached / occluded to blood vessels labeled with red TRITC-dextran. The dotted line separates larger blood vessels and capillaries. The scale bar is 50 μm. (C) PbA parasite load in OLF, cortex and cerebellum by q-PCR using PbA 18S rRNA and CD3ε (pan T cell surface marker) specific primers on days 3, 4, 5 and 6 post infection And CD3ε was quantified. Results are expressed as relative mRNA units normalized to the corresponding 18S rRNA (mean ± SD, n = 3 for days 3-5, n = 8 for day 6). ^* P <0.05 and ^** p <0.01 in infected vs. uninfected mice by Mann-Whitney test. (D) New microbleeds in the olfactory bulb. Representative snapshot image on day 6 after infection (time points 0 and 51 minutes). Red indicates a blood vessel labeled with red TRITC-dextran, green (green arrow) indicates a PbA parasite expressing GFP, and blue indicates CD8 T cells (anti-CD8 antibody, white arrow). The red area in the circle indicates the area that has already bleeded, while the yellow arrow indicates the horned blood vessel where new bleeding will occur in the future, and the white arrow in the right image already has new bleeding. 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. The delay in time to find food is shown in seconds (mean ± SD, n = 4-6 for each time point). P <0.05, ^** p <0.01 and ^*** p <0.001 by Student's t-test or Mann-Whitney test. >> indicates that the time was longer than 900 seconds. (B) shows OLF function in naive and P57A- or PyL-infected C57BL / 6, Rag2 ^{− / −} and BALB / c mice as assessed by the food burial test on day 6 after infection (time to find food in seconds Expressed in units, mean ± SD, each group n = 5-8). No statistically significant difference was observed between groups by Student's t test. >> indicates that the time was longer than 900 seconds. (C) To evaluate BBB leakage in naive and infected mice, Evans Blue dye was injected intravenously at the indicated time points. Two hours after dye injection, mice were sacrificed, brains were removed and imaged with a dissecting microscope. The arrow indicates OLF. The scale bar is 1 mm. (D) The indicated point corresponds to the red dot in (E). Here, OLF was extracted and a section for IHC was prepared. These sections were stained with antibodies against tight junction protein ZO-1 (red) and GFP-PbA parasite (green) to visualize nuclei (DAPI, blue). The arrow shows a continuous line of ZO-1 protein around MCL in naïve mice, which was destroyed 6 days after PbA infection (scale bar is 10 μm). (E) Thermal camera monitoring of infected C57BL / 6 mice. Mouse movements and fever were recorded simultaneously in the cage. Absolute value data was exported to an Excel® file and an average exotherm measurement was calculated every 3 hours. The dotted line shows the median fever level of the same mouse before infection. The red dots indicate the daily time points from 0 to 3 am. The data shown is representative of at least 3 infected animals. Fever in (FG) PbA infected Rag2 ^{− / −} mice (F) and PyL infected C57BL / 6 mice (G) was recorded by a thermal camera monitor. Naive and infected mice were housed in cages and fever was continuously monitored. Values were plotted in an Excel® file and average exotherm measurements were calculated every 3 hours. The data shown is representative of at least 3 infected mice in each group. 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 11.7 T 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. (A) C57BL / 6 mice were infected with 10 ⁶ PbA parasites, and brain tissue (OLF-cortex-cerebellum) was excised 3 days after infection. Total RNA was extracted and the expression of a given chemokine / cytokine mRNA was analyzed by real-time q-PCR. Results (mean ± SD) are presented as folds compared to the relative mRNA units of naive mice normalized by the corresponding 18S rRNA levels (n = 3). The dotted line corresponds to 1. ^* P <0.05 and ^** p <0.01 in infected WT vs. uninfected WT mice by Student's t test. (B) OLF sections from infected WT mice (0-3 days after infection) were stained with anti-CCL21 antibody (red). Nuclei were visualized with DAPI (blue). The image is representative of 3 different animals for each time point. The scale bar is 10 μm. (C) Survival curves of WT (C57BL / 6, n = 21), Ccr7 ^+/− (n = 27) and Ccr7 ^{− / −} (n = 23) mice after 10 ⁶ PbA infection. Survival was monitored daily. ^*** p = 0.004 by Log-rank (Mantel-Cox) test. (D) WT and Ccr7 ^{− / −} mice were infected and subjected to food embedding tests to measure OLF function. Time to find food is shown in seconds (mean ± SD, n = 4 for each time point). ^*** p <0.001 by Student's t-test. >> indicates that the time was longer than 900 seconds. (E) Continuous fever monitoring of infected WT and Ccr7 ^{− / −} mice with a thermal camera. Absolute value data was exported to an Excel® file and an average exotherm measurement was calculated every 3 hours. Blue and red dots indicate daily time points from 0 to 3 am. The data shown is representative of at least 3 infected animals in each group. FIG. 5 shows that CCR7 expression is important for CD8c DC priming of CD11c ⁺ CD8 T cells but not for its migration into the brain. (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). Mean ± SD, ^* p <0.05, ^** p <0.01 and ^*** p <0.001) in infected Ccr7 ^{− / −} vs. infected Ccr7 ^+/− mice by Mann-Whitney test. (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-γ secreting CD11c ⁺ CD8 T cells in the spleen of WT mice 6 days after infection. (E) Ccr7 ^{− / −} mice were adoptively immunized with concentrated CD8αDC from infected Rag2 ^{− / −} spleen, infected with PbA, and survival was monitored. The survival curves of Ccr7 ^+/− (n = 9), Ccr7 ^{− / −} (n = 7) and adoptive immunized Ccr7 ^{− / −} (n = 5) mice after infection are shown. Ccr7 ^{− / −} mice adoptively immunized with Ccr7 ^{− / −} vs. CD8α 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. infected Ccr7 ^{− / −} mice and Ccr7 ^{− / −} mice vs. infected CD8α DC adoptive immunized Ccr7 ^{− / −} mice by Student's t-test. FIGS. 5 (F)-(G) show the lack of thermal involvement during the PbA infection day 5 cytokine storm and non-lethal PyNL infection. (F) P.P. recorded by thermal camera monitor. 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. (G) Serum cytokine levels in mice 5 days after PbA infection measured by ELISA are shown (B6, n = 5, average Log ± SD, ^** p <0.01 (Mann-Whitney test)). 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 the 6th and 8th days after PbA infection. Two hours after intravenous injection of Evans Blue, the brain is removed and an image taken with a dissecting microscope is shown (scale bar = 1 mm). 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 μm. (B) Shows how the end feet of astrocytes wrap blood vessels in the GL. OLF GL sections from naive and infected WT mice (day 6) 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 μm. (C) WT mice were treated with recombinant mouse anti-CCL21 antibody and isotype control daily for 3 days from day 0 of infection (50 μg per day) i. v. Injections were monitored for survival. The survival curves of the isotype control treatment group (n = 5) and the anti-CCL21 antibody treatment group (n = 5) of mice after infection with 10 ⁶ PbA are shown. By Log-rank (Mantel-Cox) ^test, ** p <0.01. (D) Survival curves of WT and Ccr7 ^{− / −} mice treated with recombinant anti-CXCR3 antibody and isotype control antibody after infection with 10 ⁶ PbA. Mice were challenged twice daily with 100 μg antibody per mouse on days 4 and 5 after infection, i. v. Injected. By Log-rank (Mantel-Cox) test, P = 0.09 in the isotype control vs. anti-CXCR3 antibody administration group, Ccr7 ^{− / −} isotype control vs. Ccr7 ⁻ / ^{− in the} anti CXCR3 antibody administration group ^** p <0.01 (Each group n = 5). FIGS. 6 (E), (F) and (G) show that CD8α dendritic cells (DC) are required for CD11c + CD8 T cell activation and brain malaria pathogenesis. (E) shows the proportion of CD8α DC in naïve spleen cells of wild type (WT), Ccr7 ^{− / −} and Batf3 ^{− / −} mice determined by FACS analysis (mean ± SD, 3 per group, but Batf3 ⁻ / ⁻ is n = 1). (F) Flow cytometric analysis of whole brain and splenic CD8 T cells 6 days after infection. The numbers inside indicate splenic CD8 T cells and their percentage of CD11c expression. (G) 10 ⁶ after PbA infection Batf3 +/- (n = 3) and BATF - / - shows the survival curve of the (n = 8). Survival was observed daily. ^** p = 0.01, Log rank (Mantel-Cox) test. FIGS. 6 (H) and (I) show flow cytometric analysis 5 days after infection of Rag2 ⁻ / ⁻ mouse spleen CD8α DC. (H) The upper (green) rectangular region (CD8α ⁺ DC) highly expresses CDR7, while the lower (black) rectangular region (CD8α ^- DC) expresses low levels of CCD7. (I) Strategy of enrichment of CD8α DC in Rag2 − / − spleen infected by adoptive transfer The first CD11b ⁺ cells and later CD4 ⁺ cells were passively depleted from spleen cells. CD8α ⁺ purity of the cells was greater than 95%. FIG. 7 shows that the olfactory bulb can be an “entrance / exit” of accumulation of Plasmodium-infected erythrocytes into the brain. Once the host is infected with the PbANKA parasite, immune cells are activated in the periphery such as blood and spleen. CD8α ⁺ DC (1) activated by infection in the spleen acquires 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 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 glomeruli 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).

  Hereinafter, the present invention will be described with reference to the best mode. Throughout this specification, it should be understood that the singular forms also include the plural concept unless specifically stated otherwise. Thus, it should be understood that singular articles (eg, “a”, “an”, “the”, etc. in the case of English) also include the plural concept unless otherwise stated. In addition, it is to be understood that the terms used in the present specification are used in the meaning normally used in the art unless otherwise specified. Thus, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification, including definitions, will control.

  Hereinafter, definitions of terms particularly used in the present specification will be described as appropriate.

  As used herein, “brain malaria” is the most lethal complication of Plasmodium falciparum (P. falciparum) malaria and is attributed to 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. To understand the pathology of cerebral malaria (CM), The mouse model of CM using the berghei ANKA (PbA) parasite has been widely used (Langhorne, J. et al. (2011). Malar J 10, 23.), and the results in this mouse model are It can be extrapolated.

  As used herein, “olfactory bulb (sometimes abbreviated as OLF)” is used in the same meaning as is commonly used in the art, and is a bulge at the front end of the olfactory lobe, located at the front end of the telencephalon and 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.

  In the present specification, “the state of the olfactory bulb” includes an appearance, a structure or a functional state of the olfactory bulb. More specifically, the state of the olfactory bulb can be determined by an olfactory abnormality, a cell examination, a diagnostic image of the olfactory bulb, and the like, but is not limited thereto.

  As used herein, “ultra high magnetic field” nuclear magnetic resonance imaging (MRI) 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.

  In this specification, a “spot” in a diagnostic image means that a spot-like image can be seen in a densely packed image in an olfactory bulb found in a normal state. Dysfunction is presumed.

  In the present specification, “cell examination” has the same meaning as commonly used in the art, and includes staining such as staining (for example, HE staining) and immunohistochemistry.

In this specification, “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 pathogenesis mechanism of brain malaria. Say. The schematic diagram is shown in FIG. Factors of “CCL21-CXCR3-CCR7 signaling system” include CCL21, CXCR3 and CCR7, as well as 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) -γ, KC, MCP-1 (CCR2 ligand), MIP-1α, MIP-1β, and RANTES, CD8α dendritic cells ( DC), CD11c ⁺ CD8 T cells, etc. can also be considered as this factor. Once the host is infected with Plasmodium, immune cells are activated in the periphery such as blood and spleen. CD8α ⁺ 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 glomerulus and their surrounding end feet, thereby It is thought to induce gradual CCL21 secretion from and open the BBB entry / 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.

  As used herein, “CCL21” is an endogenous ligand for 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. This information is also incorporated herein. The CCL 21 can be identified by an accession number with OMIM: 602737. When used for the purposes of this specification, “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. Derivatives thereof, or functionally active fragments thereof, or homologues thereof, or variants encoded by nucleic acids that hybridize to nucleic acids encoding this protein under high or low stringency conditions. , Understood to mean.

  The same applies to all other proteins mentioned in the present invention. Thus, a given protein or nucleic acid name refers not only to a protein or nucleic acid as shown in the sequence listing, but also to 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 said protein under conditions of genency or low stringency, preferably under conditions as described above. As used herein, 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 a nucleic acid encoding such a molecule is moderately stringent under stringent conditions 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.

  Since many mammals other than humans are known to express the CCL21-CXCR3-CCR7 signal transduction system factor (for example, CCL21 protein) of the present invention, these mammals are also of the present invention. It is understood that it falls within the scope.

  As used herein, “functionally active” as used herein refers to the structural function of a protein, such as biological activity, according to the embodiment to which the polypeptide of the invention, ie, fragment or derivative, relates. Refers to a polypeptide, ie fragment or derivative, having a regulatory or biochemical function. In the present invention, 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 a 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 polynucleotide that hybridizes to a polynucleotide of any one of (a) to (e) under stringent conditions and encodes a polypeptide having biological activity; or (g) (a) to The polynucleotide may be a polynucleotide that consists of a base sequence that is at least 70% identical to any one polynucleotide of (e) or a complementary sequence thereof, and encodes a polypeptide having biological activity. Here, biological activity typically refers to the activity 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)-(d) And a polypeptide having biological activity. Here, biological activity typically refers to the activity of CCL21.

  In the present specification, “CXCR3” has the same meaning as commonly used in the art, and is one of the CXC chemokine receptor family that is a G protein-coupled receptor. In addition to G protein-coupled receptor 9 (GPR9) and CD183, CD182; CKR-L2; CMKAR3; IP10-R; Mig-R; 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) and 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. As used herein, “CXCR3” is not only a protein having an amino acid sequence described in a specific SEQ ID NO or accession number (or a nucleic acid encoding it), but also functionally active. Derivatives thereof, or functionally active fragments thereof, or homologues thereof, or variants encoded by nucleic acids that hybridize to nucleic acids encoding this protein under high or low stringency conditions. , 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 polynucleotide that hybridizes to a polynucleotide of any one of (a) to (e) under stringent conditions and encodes a polypeptide having biological activity; or (g) (a) to The polynucleotide may be a polynucleotide that consists of a base sequence that is at least 70% identical to any one polynucleotide of (e) or a complementary sequence thereof, and encodes a polypeptide having biological activity. Here, the biological activity typically means the activity 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 set forth in 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)-(d) And a polypeptide having biological activity. Here, the biological activity typically refers to the activity of CXCR3.

  In the present specification, “CCR7” has the same meaning as commonly used in the art, is an abbreviation for chemokine (C-C motif) receptor 7, and is one of chemokine receptors. There are two types of endogenous ligands in CCR7. One is CCL21, which is found in high endothelial venules (specialized blood vessels through which lymphocytes leave the blood and enter lymph nodes). The other is 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). BLR2; CD197; CDw197; CMKBR7; EBI1, etc. 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. As used herein, “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. Derivatives thereof, or functionally active fragments thereof, or homologues thereof, or variants encoded by nucleic acids that hybridize to nucleic acids encoding this protein under high or low stringency conditions. , Understood to mean.

A typical 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 polynucleotide that hybridizes to a polynucleotide of any one of (a) to (e) under stringent conditions and encodes a polypeptide having biological activity; or (g) (a) to The polynucleotide may be a polynucleotide that consists of a base sequence that is at least 70% identical to any one polynucleotide of (e) or a complementary sequence thereof, and encodes a polypeptide having biological activity. Here, the biological activity typically means the activity of CCR7.

As an 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)-(d) And a polypeptide having biological activity. Here, the biological activity typically means the activity of CCR7.

In addition, there are other factors of the CCL21-CXCR3-CCR7 signaling system, and examples thereof include 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) CD8α dendritic cells and other dendritic cells; and (4) CD8 T cells and other T cells.

  Any of these can be identified using techniques known in the art. Examples of documents that can be referred to as such a technique include documents mentioned elsewhere in this specification.

  In the context of the present invention, “a substance that binds to a factor of the CCL21-CXCR3-CCR7 signaling system” or “an interacting molecule of a factor of the 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). A substance that binds to a CCL21-CXCR3-CCR7 signaling factor (eg, CCL21, CXCR3, CCR7, etc.) is an inhibitor of a CCL21-CXCR3-CCR7 signaling factor (eg, CCL21, CXCR3, CCR7, etc.). Examples thereof may include antibodies, antisense oligonucleotides, siRNA, low molecular weight molecules (LMW), binding peptides, aptamers, ribozymes and peptidomimetics, etc. CCL21-CXCR3 -Activity of factors of CCR21-CXCR3-CCR7 signaling system (for example, CCL21, CXCR3, CCR7, etc.) directed against 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 (for example, CCL21, CXCR3, CCR7, etc.) include, for example, CCL21 gene expression or CCL21-CXCR3-CCR7 signaling system factors (for example, CCL21, CXCR3, CCR7, etc.) Refers to double- or single-stranded DNA or RNA, or a modification or derivative thereof, and includes, without limitation, antisense nucleic acids, aptamers, siRNA (small interfering RNA) and ribozymes. In the present specification, the term “binding protein” or “binding peptide” refers to a factor of the CCL21-CXCR3-CCR7 signaling system with respect to factors of the CCL21-CXCR3-CCR7 signaling system (for example, CCL21, CXCR3, CCR7, etc.). Polyclonal or monoclonal antibodies, antibody fragments and protein backbones that are directed against the type of protein or peptide that binds to CCL21-CXCR3-CCR7 signaling system factors (eg, CCL21, CXCR3, CCR7, etc.) Including but not limited to.

  As used herein, “protein”, “polypeptide”, “oligopeptide” and “peptide” are used interchangeably herein and refer to a polymer of amino acids of any 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. This 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). This definition also includes, for example, polypeptides containing one or more analogs of amino acids (eg, including unnatural amino acids, etc.), peptide-like compounds (eg, peptoids) and other modifications known in the art. Is done. In the present specification, the “amino acid” may be natural or non-natural as long as the object of the present invention is satisfied.

  As used herein, “polynucleotide”, “oligonucleotide” and “nucleic acid” are used interchangeably herein and refer to a polymer of nucleotides 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. Specific examples of such an oligonucleotide 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. Is an N3′-P5 ′ phosphoramidate bond oligonucleotide derivative, an oligonucleotide derivative in which the ribose and phosphodiester bonds in the oligonucleotide are converted to peptide nucleic acid bonds, and the uracil in the 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 Substituted oligonucleotide derivatives, oligonucleotide derivatives in which the cytosine in the oligonucleotide is replaced with phenoxazine-modified cytosine, oligonucleotide derivatives in which the ribose in the DNA is replaced with 2'-0-propylribose Examples thereof include oligonucleotide derivatives in which the ribose in the oligonucleotide is substituted with 2′-methoxyethoxyribose. Unless otherwise indicated, a particular nucleic acid sequence may also be conservatively modified (eg, degenerate codon substitutes) and complementary sequences, as well as explicitly indicated sequences. 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); Rossolini et al., Mol. Cell. Probes). 8: 91-98 (1994)). As used herein, “nucleic acid” is also used interchangeably with gene, cDNA, mRNA, oligonucleotide, and polynucleotide. In the present specification, the “nucleotide” may be natural or non-natural.

  As used herein, “gene” refers to a factor that defines a genetic trait, and “gene” may refer to “polynucleotide”, “oligonucleotide”, and “nucleic acid”.

  As used herein, “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 determined by direct comparison of the sequences 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 have homology. Thus, as used herein, a “homologue” 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 appreciated 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 the generally recognized one letter code. In this specification, the comparison of similarity, identity and homology between amino acid sequences and base sequences is calculated using default parameters using BLAST, which is a sequence analysis tool. The identity search can be performed using, for example, NCBI BLAST 2.2.28 (issued 2013.4.2). In the present specification, 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 taken as the identity value. When identity is evaluated in a plurality of regions, 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.

  In one embodiment of the present invention, “several” may be, for example, 10, 8, 6, 5, 4, 3, or 2, or may be any value or less. Polypeptides that have undergone deletion, addition, insertion, or substitution with other amino acids of one or several amino acid residues are known to retain their biological activity (Market al., Proc Natl. Acad Sci USA 1984 Sep; 81 (18): 5662-5666., Zoller et al., Nucleic Acids Res.1982 Oct 25; 10 (20): 6487-6500., Wang et al., Science.1984. Jun 29; 224 (4656): 1431-1433.). An antibody having a deletion or the like can be produced by, for example, a site-specific mutagenesis method, a random mutagenesis method, or biopanning using an antibody phage library. As a 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 the wild type from mutant antibodies into which deletion or the like has been introduced by performing various characterizations such as FACS analysis and ELISA.

  In one embodiment of the present invention, “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 identical amino acids. Methods for alignment, percentage calculation, comparison methods, and related computer programs are well known in the art (eg, BLAST, GENETYX, etc.). In the present specification, “homology” can be represented by a value measured by BLAST of NCBI unless otherwise specified. Blastp can be used with the default setting for the algorithm when comparing amino acid sequences with BLAST. Measurement results are quantified as Positives or Identities.

  As used herein, “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 0.1 to 2 times the concentration. It means a polynucleotide that can be identified by washing a filter under conditions of 65 ° C. using a SSC (saline-sodium citrate) solution (composition of 1-fold concentration of SSC solution is 150 mM sodium chloride, 15 mM sodium citrate). . As the “stringent conditions”, for example, the following conditions can be adopted. (1) 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.), (2) 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. It should be noted that the formamide concentration may be 50% or higher. The cleaning time may be 5, 15, 30, 60, or 120 minutes, or longer. As factors affecting the stringency of the hybridization reaction, a plurality of factors such as temperature and salt concentration can be considered. , Current Protocols in Molecular Biology, Wiley Interscience Publishers, (1995). Examples of “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. , Current Protocols in Molecular Biology, Supplement 1-38, DNA Cloning 1: Core Techniques, A Practical Approach, Second Edition, Oxford Universe. Here, the 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. 1, 7.42-7.45 Cold Spring Harbor Laboratory Press, 2001, and for nitrocellulose filters, 5 × SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0) pre-wash solution, About 50% formamide at about 40-50 ° C., 2 × SSC-6 × SSC (or other similar, such as Stark's solution in about 50% formamide at about 42 ° C. Hybridization conditions) and washing conditions of about 60 ° C., 0.5 × SSC, 0.1% SDS. Thus, 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.

  As used herein, a “purified” substance or biological factor (such as a nucleic acid or protein) refers to a substance from which at least a part of the factor naturally associated with the substance or biological factor has been removed. . Thus, typically, 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, It means that there is a biological agent of the same type. The substance or biological agent used in the present invention is preferably a “purified” substance. As used herein, an “isolated” substance or biological agent (such as a nucleic acid or protein) 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.

  As used herein, 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 amino acid or nucleotide that has, or is predicted to have, the same action as a nucleotide or moiety, especially in the case of enzyme molecules, present at the same position in the active site and make a similar contribution to catalytic activity It refers to an amino acid, and in the case of a complex molecule, it refers to the corresponding part (eg heparan sulfate). For example, an antisense molecule can be a similar part in an ortholog corresponding to a particular part of the antisense molecule. Corresponding amino acids, for example, cysteinylation, glutathioneation, SS bond formation, oxidation (eg methionine side chain oxidation), formylation, acetylation, phosphorylation, glycosylation, myristylation, etc. Of amino acids. Alternatively, 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.

  As used herein, a “corresponding” gene (eg, a polynucleotide sequence or molecule) has, or is expected to have, in a given species, the same effect as a given gene in the species that is the basis for comparison. A gene (for example, a polynucleotide sequence or molecule) refers to a gene that has the same evolutionary origin when there are a plurality of genes having such an action. Thus, a gene corresponding to a gene can be an ortholog of that gene. Accordingly, human CCL21, CXCR3, and CCR7 can find the corresponding CCL21, CXCR3, and CCR7 in other animals (particularly mammals), respectively. Such corresponding genes can be identified using techniques well known in the art. Therefore, for example, a corresponding gene in an animal (for example, mouse) 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.

  As used herein, “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. For example, 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. In the case of polynucleotides, examples include 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 75, 100 and more nucleotides. Non-integer lengths (eg, 11 etc.) may also be appropriate as a lower limit. In the present specification, such a fragment falls within the scope of the present invention, for example, when 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.

In accordance with the present invention, 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. As used herein, “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. Examples of the antibody include, but are not limited to, generation of specific antibodies, enzyme activity, and imparting resistance. As used herein, “biological activity” refers to the activity that a 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. When two factors interact, 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 an example of a judgment method. For example, when a factor is an enzyme, the biological activity includes the enzyme activity. In another example, when 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. Thus, “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.

  In the present specification, “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. Preferably, a gene, a polynucleotide or the like is transcribed and translated into a polypeptide form. However, transcription and production of mRNA are also an aspect of expression. Accordingly, “expression product” as used herein 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. Specifically, 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, etc. can be determined by the method as described in detail elsewhere in this specification or other methods known in the art.

In this specification, 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. or a variant or modification of the antibody thereof (for example, 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. In the present invention, it is understood that 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. As used herein, “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. Say. 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. Biological 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.

  As a functional equivalent of the present invention, 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. In the present specification, “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, more 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. It may have an amino acid sequence. As used herein, “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. Specific examples include non-polar (hydrophobic) amino acids such as alanine, valine, isoleucine, leucine, proline, tryptophan, phenylalanine, and methionine. Examples of polar (neutral) amino acids include glycine, serine, threonine, tyrosine, glutamine, asparagine, and cysteine. Examples of positively charged (basic) amino acids include arginine, histidine, and lysine. Examples of negatively charged (acidic) amino acids include aspartic acid and glutamic acid.

As used herein, the term “inhibitor” refers to a substance or factor that inhibits the biological action of a target entity (eg, receptor or cell) of the receptor or cell, and is also referred to as an inhibitor 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.
As used herein, the term “agonist” refers to a substance that expresses or enhances the biological action of a target entity (eg, receptor). In addition to natural agonists (also called ligands), synthesized ones and modified ones can be mentioned.
As used herein, the term “antagonist” refers to a substance that suppresses or inhibits the expression of a biological action of a target entity (for example, a receptor). In addition to natural antagonists, those synthesized and modified can be mentioned. In addition to those that inhibit or inhibit competitively with agonists (or ligands), there are those that inhibit or inhibit non-competitively. It can also be obtained by modifying the agonist. 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”.

  In one embodiment of the present invention, “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.

Further, 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, In addition to 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 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 according to one embodiment of the present invention 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, and may be, for example, IgM, IgD, IgG, IgA, IgE, or IgY.

  The anti-CCL21 antibody, anti-CXCR3 antibody, anti-CCR7 antibody and the like according to an embodiment of the present invention may be an antibody fragment having an antigen-binding activity (hereinafter also referred to as “antigen-binding fragment”). In this case, there are effects such as an increase in stability or antibody production efficiency.

  The anti-CCL21 antibody, anti-CXCR3 antibody, anti-CCR7 antibody and the like according to one embodiment of the present invention 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. Here, 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.

  The anti-CCL21 antibody, anti-CXCR3 antibody, anti-CCR7 antibody and the like according to one embodiment of the present invention include, for example, purified CCL21, CXCR3, CCR7, etc., expressed cells such as CCL21, CXCR3, CCR7, 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, CCR7, it is preferable to use cells expressing CCL21, CXCR3, CCR7, etc. for immunization.

  The anti-CCL21 antibody, anti-CXCR3 antibody, anti-CCR7 antibody, etc. according to one embodiment of the present invention 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, CCR7, it is preferable to use cells expressing CCL21, CXCR3, CCR7, etc. for immunization. A CDR set is a set of heavy chain CDRs 1, 2, and 3 and light chain CDRs 1, 2, and 3.

  In one embodiment of the present invention, “expressing cells such as CCL21, CXCR3, CCR7” are expressed by, for example, expressing CCL21, CXCR3, CCR7, etc. after introducing a polynucleotide encoding CCL21, CXCR3, CCR7, etc. May be obtained. Here, CCL21, CXCR3, CCR7 and the like include fragments such as CCL21, CXCR3 and CCR7. In one embodiment of the present invention, “a lipid membrane containing CCL21, CXCR3, CCR7, etc.” may be obtained, for example, by mixing a lipid bilayer with CCL21, CXCR3, CCR7, etc. Here, CCL21, CXCR3, CCR7 and the like include fragments such as CCL21, CXCR3 and CCR7. Further, the anti-CCL21 antibody, anti-CXCR3 antibody, anti-CCR7 antibody and the like according to one embodiment of the present invention 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 according to an embodiment of the present invention may have any binding force as long as the object is achieved, for example, at least 1.0 × 10 ^6. As mentioned above, 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 according to one embodiment of the present invention may have ADCC or CDC activity.

  The anti-CCL21 antibody, anti-CXCR3 antibody, anti-CCR7 antibody, and the like according to one embodiment of the present invention 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.

As used herein, “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 (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. In the modified antibody, 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. In addition, 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. Specifically, 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. In the present invention, 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. In the modified antibody, 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.

  In one embodiment of the present invention, 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). .).

  In one embodiment of the present invention, 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. 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. For example, the monoclonal antibody may be produced by a method similar to the hybridoma method as described in “Kohler G, Milstein C., Nature. 1975 Aug7; 256 (5517): 495-497.”. Alternatively, monoclonal antibodies may be made by methods similar to recombinant methods such as those described in US Pat. No. 4,816,567. Alternatively, monoclonal antibodies can be prepared according to “Clackson et al., Nature. 1991 Aug 15; 352 (6336): 624-628.” Or “Markset al., J Mol Biol. 1991 Dec 5; It may be isolated from a phage antibody library using methods similar to those described in “. Alternatively, it may be prepared by the method described in “Protein Experiment Handbook, Yodosha (2003): 92-96”.

  For the mass production of antibodies, any technique known in the art can be used. For example, the following can be exemplified as the construction of a typical antibody mass production system and antibody production. 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.

  In one embodiment of the present invention, an “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.

  In one embodiment of the present invention, the “Fab antibody” refers to, for example, a fragment obtained by treating an antibody containing a Fab region and an Fc region with the proteolytic enzyme papain, about 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 and the like according to the embodiment of the present invention containing the Fab region and the Fc region with the proteolytic enzyme papain.

In one embodiment of the present invention, “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 an anti-CCL21 antibody, anti-CXCR3 antibody, anti-CCR7 antibody, etc. according to an embodiment of the present invention containing a Fab region and an Fc region with a proteolytic enzyme pepsin. . Further, for example, it can be produced by linking the following Fab ′ with a thioether bond or a disulfide bond.

In one embodiment of the present invention, “Fab ′ antibody” is, for example, an antibody obtained by cleaving a disulfide bond in the hinge region of F (ab ′) ₂ . For example, F (ab ′) ₂ can be obtained by treating with a reducing agent dithiothreitol.

  In one embodiment of the present invention, an “scFv antibody” is an antibody in which VH and VL are linked via an appropriate peptide linker. The 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.

  In one embodiment of the present invention, “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.

  In one embodiment of the present invention, “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. 1994 May; 7 (5): 697-704.). it can.

  In one embodiment of the present invention, an “antigen-binding peptide or polypeptide” is an antibody comprising an 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. For example, DNA encoding a region such as an Fv antibody in the anti-CCL21 antibody, anti-CXCR3 antibody, anti-CCR7 antibody and the like according to an embodiment of the present invention can be incorporated into an expression vector and produced using an expression cell. Alternatively, it may be produced by a chemical synthesis method such as Fmoc method (fluorenylmethyloxycarbonyl method) or tBOC method (t-butyloxycarbonyl method). The antigen-binding fragment according to one embodiment of the present invention may be one or more of the above Fv antibodies.

  In one embodiment of the present invention, a “chimeric antibody” is, for example, a combination of an antibody variable region and an antibody constant region between different organisms, and can be constructed by a gene recombination technique. A mouse-human chimeric antibody can be prepared, for example, by the method described in “Roguska et al., ProcNatl Acad Sci USA 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. To the array to be Alternatively, 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. The fragment of the human antibody constant region can be of any human antibody heavy chain constant region and human antibody light chain constant region, eg, Cγ1, Cγ2, Cγ3 or Cγ4 for human heavy chain, For the light chain, Cλ or Cκ can be mentioned, respectively.

  In one embodiment of the present invention, 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 a variety of techniques known in the art (Almagro et 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. 1994 Feb 1; 91 ( 3): 969-973.), Or FR shuffle (Damschroder et al., Mol Immunol. 2007 Apr; 44 (11): 3049-3060.Epub 2007 Jan 22.). To alter (preferably improve) antigen binding, amino acid residues of the human FR region may be substituted 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.). For example, FR residues important for antigen binding may be identified by modeling the interaction of CDR and FR residues. Alternatively, an unusual FR residue at a particular position may be identified by sequence comparison.

  In one embodiment of the present invention, a “human antibody” is derived from a gene encoding a human immunoglobulin, for example, a variable region and a constant region of a heavy chain, and a variable region and a constant region of a light chain that constitute the antibody. Antibody. The main production methods include a transgenic mouse method for producing human antibodies, a phage display method, and the like. In the transgenic mouse method for producing human antibodies, if a functional human Ig gene is introduced into a mouse in which endogenous Ig has been knocked out, human antibodies having various antigen-binding abilities can be produced instead of the mouse antibody. Furthermore, if this mouse is immunized, a human monoclonal antibody can be obtained by a conventional hybridoma method. For example, it can be produced by the method described in “Lonberg et al., Int Rev Immunol. 1995; 13 (1): 65-93.”. In the 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 “Vaughane et 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. Alternatively, 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. At this time, in order to increase the action efficiency on the target antigen such as anti-CCL21 antibody, anti-CXCR3 antibody, anti-CCR7 antibody, etc., a method known in the art (for example, by randomly mutating the amino acid residue of the antibody, The region excluding the heavy chain CDR or the light chain CDR may be optimized using a method of screening for a higher one or a phage display method. In addition, for example, FR shuffle (Damschroderet al., Mol Immunol. 2007 Apr; 44 (11): 3049-3060.Epub 2007 Jan 22.), or a method of substituting amino acid residues or packaging residues of vernier zones ( The FR region may be optimized using JP-A-2006-241026, or Foote et al., J Mol Biol. 1992 Mar 20; 224 (2): 487-499.).

  In one embodiment of the invention, the “heavy chain” is typically the major 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. Generally, VH has a large specificity and affinity for an antigen. It is known to contribute. For example, in “Reiter et al., J Mol Biol. 1999 Jul 16; 290 (3): 685-98.”, A VH-only molecule was produced, and it was found that it bound specifically to the antigen with high affinity. Has been described. Further, in “Wolfson W, Chem Biol. 2006 Dec; 13 (12): 1243-1244.”, It is found that among the camel antibodies, only a heavy chain antibody having no light chain exists. Has been described.

  In one embodiment of the present invention, a “CDR (complementarity determining region)” is a region in an antibody that is actually in contact with an antigen to form a binding site. Generally, the CDRs are located on the Fv (variable region: including heavy chain variable region (VH) and light chain variable region (VL)) of the antibody. In general, CDRs include CDR1, CDR2, and CDR3 consisting of about 5 to 30 amino acid residues. In particular, it is known that CDRs of heavy chains contribute to the binding of antibodies to antigens. Among CDRs, it is known that CDR3 contributes most to the binding of an antibody to an antigen. For example, “Willy et al., Biochemical and Biophysical Research Communications Volume 356, Issue 1, 27 April 2007, Pages 124-128” describes that the binding ability of the antibody was increased by modifying heavy chain CDR3. . Fv regions other than CDRs are called framework regions (FR) and consist 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.

Several methods have been reported for defining CDRs and their locations. For example, the definition of Kabat (Sequences of Proteins of Immunological Intersect, 5th ed., Public Health Service,
National Institutes of Health, Bethesda, MD. (1991)), or Chothia's definition (Chothia et al., J. Mol. Biol., 1987; 196: 901-917). In one embodiment of the present invention, 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. For example, an overlapping part of CDRs according to each definition or a part including both CDRs according to each definition It can also be a CDR. A specific example of such a method is the method of Martin et al. (Proc. Natl. Acad. Sci. 86: 9268-9272). Such CDR information can be used to produce mutants that can be used in the present invention. In such antibody variants, 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.

  As used herein, “antigen” refers to any substrate that can be specifically bound by an antibody molecule. As used herein, “immunogen” refers to an antigen capable of initiating lymphocyte activation that produces an antigen-specific immune response. As used herein, “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.

  It will be appreciated that antibodies of any specificity may be used as long as false positives are reduced. Therefore, the antibody used in the present invention may be a polyclonal antibody or a monoclonal antibody.

  As used herein, “means” refers to any tool that can achieve a certain purpose (for example, detection, diagnosis, treatment). A means by which one object can be recognized differently from another.

  As used herein, the term “marker (substance or gene)” indicates whether or not a certain state (for example, the level of disease state, disorder state, or malignant state of cerebral malaria, or the risk thereof) 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 the same. As used herein, “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 associated with cerebral malaria can be used as indicators of cerebral malaria.

  In the present specification, the “subject (person)” refers to a subject to be diagnosed or detected or treated according to the present invention (eg, a living organism such as a human or a cell, blood, serum, etc. taken from the living organism). .

  As used herein, “sample” refers to any substance obtained from a subject or the like, and includes, for example, serum and the like. Those skilled in the art can appropriately select a preferable sample based on the description of the present specification.

  In the present specification, “drug”, “agent” or “factor” (both corresponding to “agent” in English) are used interchangeably in a broad sense, and so long as they can achieve their intended purpose. It may also be a substance or other element (eg energy such as light, radioactivity, heat, electricity). Examples of such substances include proteins, polypeptides, oligopeptides, peptides, polynucleotides, oligonucleotides, nucleotides, nucleic acids (eg, DNA such as cDNA and 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, but not limited to. As a factor specific to a polynucleotide, typically, a polynucleotide having a certain sequence homology to the sequence of the polynucleotide (for example, 70% or more sequence identity) and a 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. Alternatively, specific ligands or receptors in the case of ligands, and substrates thereof when the polypeptide is an enzyme include, but are not limited to.

  As used herein, “diagnosis” identifies various parameters associated with 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. By using the methods, devices, and systems of the present invention, conditions in 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. Alternatively, various parameters such as methods can be selected. In this specification, “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, as a general rule, 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. In this specification, in order to clarify that it can be performed away from a medical staff such as a doctor, “predictive diagnosis, pre-diagnosis or diagnosis” may be referred to as “support”.

  In this specification, “detection drug (agent)” or “test drug (agent)” refers to any drug that can detect or test a target object in a broad sense.

  In this specification, the term “diagnostic agent (agent)” refers to any agent that can diagnose a target condition (for example, a disease such as cerebral malaria) in a broad sense.

  As used herein, “treatment” refers to prevention of worsening of a disease or disorder when it becomes such a condition or disease (eg, 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 the patient's disease or disease. Diagnosing in advance and performing appropriate treatment is referred to as “companion treatment”, and the diagnostic agent for this is sometimes referred to as “companion diagnostic agent”.

  In the present specification, the term “therapeutic agent (agent)” broadly refers to any drug that can treat a target condition (for example, a disease such as cerebral malaria), and an inhibitor (for example, provided by the present invention) Antibody). In one embodiment of the present invention, a “therapeutic agent” may be a pharmaceutical composition comprising an active ingredient and one or more pharmaceutically 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. In addition, as long as the therapeutic agent is used for treatment, the form of use is not limited, and the active agent 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).

  As used herein, “prevention” refers to preventing a certain disease or disorder (for example, cerebral malaria) from entering such a state before it enters 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.

  As used herein, the term “prophylactic agent (agent)” refers to any agent that can prevent a target condition (for example, a disease such as cerebral malaria) in a broad sense.

  In this specification, “interaction” refers to two substances, and forces (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.

  As used herein, the term “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.

  Therefore, as used herein, a “factor” (or drug, detection agent, etc.) that interacts (or binds) “specifically” to a biological agent such as a polynucleotide or polypeptide refers to that poly 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.

  As used herein, 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. Thus, when both a substance or factor is a nucleic acid, 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. Also, for example, when 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, interaction by receptor-ligand reaction, enzyme-substrate interaction, and the like. Where the two substances or factors include proteins and nucleic acids, 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. By utilizing such a specific interaction or binding reaction, an object in a sample can be detected or quantified.

  As used herein, “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. Examples of molecular biological measurement methods include Northern blotting, dot blotting, and PCR. Examples of immunological measurement methods include ELISA using a microtiter plate, RIA, fluorescent antibody, luminescence immunoassay (LIA), immunoprecipitation (IP), immunodiffusion (SRID), immunization Examples are turbidimetry (TIA), Western blotting, immunohistochemical staining, and the like. Examples of the quantification 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"). For protein arrays, see NatGenet. 2002 Dec; 32 Suppl: 526-532. 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. Such further analysis methods are described in, for example, Genome Analysis Experimental Method / Yusuke Nakamura Laboratory Manual, Editing / Yusuke Nakamura Yodosha (2002), etc., all of which are incorporated herein by reference. Is done.

  In the present specification, the “expression amount” refers to an amount in which a polypeptide or mRNA is expressed in a target cell, tissue or the like. Such expression level is evaluated by any appropriate method including immunoassay methods such as ELISA, RIA, fluorescent antibody, Western blot, and immunohistochemical staining 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 is 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.

  As used herein, “reduction” or “suppression” or synonyms for activity, expression product (eg, protein, transcript (RNA, etc.)) or a synonym thereof is a decrease in the amount, 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”.

  As used herein, “increase” or “activation” of an activity, expression product (eg, protein, transcript (RNA, etc.)) or a synonym thereof refers to a quantity, quality or effect of a particular activity, transcript or protein. An activity that increases or increases.

  In the present specification, the “(nucleic acid) primer” refers to a substance necessary for the initiation of a reaction of a polymer compound to be synthesized in a polymer synthesizing enzyme reaction. In the synthesis reaction of a nucleic acid molecule, a nucleic acid molecule (eg, DNA or RNA) complementary to a partial sequence of a polymer compound to be synthesized can be used. In the present specification, the primer can be used as a marker detection means.

  As used herein, 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. For example, a nucleic acid molecule containing a specific base sequence or a specific nucleic acid molecule Examples include, but are not limited to, peptides containing amino acid sequences, specific antibodies or fragments thereof. In the present specification, the probe is used as a means for marker detection, inspection or diagnosis.

In this specification, the “label” refers to a presence (for example, a substance, energy, electromagnetic wave, etc.) for distinguishing a target molecule or substance from others. Examples of such a labeling method include RI (radioisotope) method, fluorescence method, biotin method, chemiluminescence method and the like. When the marker of the present invention or a plurality of factors or means for capturing the same are labeled by the fluorescence method, 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. When labeling a ligand, any substance that does not affect the function can be used, but Alexa ^™ Fluor is preferred as the fluorescent substance. Alexa ^™ 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. 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 ^™ 555 and Alexa ^™ 633, a combination of Alexa ^™ 488 and Alexa ^™ 555, and the like. Any nucleic acid can be used as long as it can bind to its base moiety. For example, a cyanine dye (for example, CyDye ^™ series Cy3, Cy5 etc.), rhodamine 6G reagent, 2-acetylaminofluorene ( AAF), AAIF (iodine derivative of AAF) and the like are preferably used. Examples of the fluorescent substance having a fluorescence emission maximum wavelength difference 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. In the present invention, 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.

As used herein, 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 or biotin-streptavidin) and can be included in the category of “label”. Thus, for example, 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. Such 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).

  As used herein, “in vivo” refers to the inside of a living body. In a particular context, “in vivo” refers to the location where the target substance is to be placed.

  As used herein, “in vitro” refers to a state in which a part of a living body is removed or released “in vitro” (eg, in a test tube) for various research purposes. A term that contrasts with in vivo.

  As used herein, “ex vivo” refers to a series of operations ex vivo when a treatment is performed outside the body but is then intended to be returned to the body. Also in the present invention, an embodiment in which cells in vivo are treated with the agent of the present invention and returned to the patient can be envisaged.

  In this specification, 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, instructions, etc.) usually divided into two or more compartments. Say. The form of this kit is preferred when it is intended to provide a composition that should not be provided in admixture because of stability, etc., but preferably used in admixture immediately prior to use. Such kits preferably include instructions or instructions describing how to use the provided parts (eg, how to use test agents, diagnostic agents, therapeutic agents, or how to process the reagents) In the present specification, 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.

  In the present specification, the “instruction” describes a method for using the present invention to a doctor or other user. This instruction describes 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. In addition, the instruction sheet 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 country where the present invention is implemented, and is approved by the supervisory authority. It is clearly stated that it has received. The instruction sheet is a so-called package insert and is usually provided as a paper medium, but is not limited thereto. For example, the instruction sheet may be in a form such as an electronic medium (for example, a home page or e-mail provided on the Internet). Can be provided.

(Preferred embodiment)
Hereinafter, preferred embodiments of the present invention will be described. The embodiments provided below are provided for a better understanding of the present invention, and it is understood that the scope of the present invention should not be limited to the following description. Therefore, it is obvious that those skilled in the art can make appropriate modifications within the scope of the present invention with reference to the description in the present specification. It will also be appreciated that the following embodiments of the invention may be used alone or in combination.

(Diagnosis of brain malaria based on the state of the olfactory bulb)
In one aspect, the present invention provides a method using the state of a subject's olfactory bulb (OLF) as an indicator of brain malaria. Alternatively, the present invention provides a method for diagnosing cerebral malaria comprising the step of determining the state of a subject's olfactory bulb (OLF). Here, it was found in the present invention that 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 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. Further, the determination of the state of the olfactory bulb can be performed by various methods, and the early diagnosis 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.

  In one embodiment, the subject's olfactory bulb (OLF) state determined by the present invention can include, but is not limited to, olfactory abnormalities, cytological examinations, diagnostic images of the olfactory bulb, and the like. Therefore, the diagnostic method of the present invention includes a step of performing at least one selected from olfactory abnormalities, cytological examination, diagnostic images of the olfactory bulb, and the like, and based on the result, the state of the olfactory bulb (OLF) of the subject is determined. It can be used as an indicator of early stage of cerebral malaria.

  In this specification, olfaction includes reactions to foods, reactions to fragrances, (please list other possible tests), and examples of olfaction 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 centometer method, a saline balance method, a three-point comparison odor bag method, and the like.

  In this specification, 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. In the embodiment, 11.7 Tesla is used, and the state of the olfactory bulb can be determined.

  In the determination of the present invention, the observation of spots in the diagnostic image is an indicator of cerebral malaria. Such a spot indicates that there is an abnormality in the olfactory bulb, and it was revealed in the present invention that it is an index of brain malaria infection.

  In the present invention, 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.

  In one aspect, the present invention provides an apparatus for detection or diagnosis of cerebral malaria comprising 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.

  In one embodiment, the means used in the apparatus of the present invention is a magnetic resonance imaging (MRI) means. Preferably, the MRI used in the present invention is an ultra high magnetic field MRI. Ordinary MRI could not detect abnormalities of the olfactory bulb. However, in the present invention, the relationship between abnormalities of the olfactory bulb and brain malaria could be elucidated for the first time by using MRI of an ultrahigh magnetic field. Therefore, it can be said that the present invention is remarkable in that MRI using an ultrahigh magnetic field is used.

(Therapeutic agent based on CCL21-CXCR3-CCR7 signaling system)
In another aspect, the present invention relates to a prophylactic or therapeutic agent for cerebral malaria, a prophylactic or therapeutic agent, a pharmaceutical agent for prevention or treatment, comprising an inhibitor of at least one factor of the CCL21-CXCR3-CCR7 signaling system. Provided are pharmaceutical compositions (these terms are used interchangeably herein unless otherwise indicated) and refer to the same subject. Alternatively, 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 by using this therapeutic or prophylactic agent or a specific therapeutic or prophylactic inhibitor. In addition, 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.

  In one embodiment, 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.

  In a more specific embodiment, 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.

  In a preferred embodiment, the inhibitor used in the present invention comprises at least one antibody 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. . In a more preferred embodiment, the inhibitor used in the present invention comprises at least two antibodies selected from the group consisting of an anti-CCL21 antibody, an anti-CXCR3 antibody and an anti-CCR7 antibody, or fragments or functional equivalents thereof. . Although not wishing to be bound by theory, it has been shown to have a more pronounced effect of brain malaria by exerting an inhibitory effect in two or more of the CCL21-CXCR3-CCR7 signaling systems, and more When an effect is desired, it is assumed that inhibition of two or more types of factors is preferable.

  In yet another embodiment, the inhibitor used in the present invention comprises three antibodies, anti-CCL21 antibody, anti-CXCR3 antibody and anti-CCR7 antibody, or fragments or functional equivalents thereof.

  The anti-CCL21 antibody, anti-CXCR3 antibody, anti-CCR7 antibody and the like according to one embodiment of the present invention include a set of amino acid sequences of heavy chain CDR1, 2, and 3 and light chain CDR1, 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 according to an embodiment of the present invention may be in the form of scFv. It may have an amino acid sequence between As long as the anti-CCL21 antibody, anti-CXCR3 antibody, anti-CCR7 antibody, etc. have a 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 with 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. By using this transformant, 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.

  Examples of the vectors 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 λ 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.

  As a method for introducing the above-described polynucleotide or vector into cells, for example, calcium phosphate method, lipofection method, electroporation method, adenovirus method, retrovirus method, or microinjection can be used (Revised 4th edition) New Genetic Engineering Handbook, Yodosha (2003): 152-179.). As a production method using antibody cells, for example, the method described in “Protein Experiment Handbook, Yodosha (2003): 128-142.” Can be used. In the purification of antibodies, for example, ammonium sulfate, ethanol precipitation, protein A, protein G, gel filtration chromatography, anion, cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyl Apatite chromatography, lectin chromatography or the like can be used (Protein Experiment Handbook, Yodosha (2003): 27-52.).

  In another embodiment, the inhibitor of CCL21-CXCR3-CCR7 signaling system factor is an antibody or 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. For the production of antibodies, etc., embodiments described elsewhere in this specification and / or techniques known in the art can be used. For the purpose of treatment or prevention of the present invention, such an antibody or a fragment or functional equivalent thereof preferably has an inhibitory activity downstream of the CCL21-CXCR3-CCR7 signaling system.

  In order to carry out the present invention, nucleic acid can be selected as an inhibitor of the nucleic acid form of the present invention using antisense activity as an index. Here, “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. Say. As a technique, there are a method for directly introducing an RNA molecule complementary to mRNA produced from a target gene into a cell, and a method for introducing a construction vector capable of expressing RNA complementary to a target gene into the cell. Broadly divided.

  Antisense activity is usually achieved by a nucleic acid sequence of at least 8 contiguous nucleotides that is complementary to the nucleic acid sequence of the gene of interest. Such 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 contiguous nucleotide lengths, 21 contiguous nucleotide lengths, 22 contiguous nucleotide lengths, 23 contiguous nucleotide lengths, 24 contiguous nucleotide lengths, 25 contiguous nucleotide lengths Of 40 consecutive nucleotides of 30 consecutive nucleotide lengths Plastid length of contiguous nucleotides in length 50, may be a nucleic acid sequence. Such 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. Such antisense activity is preferably complementary to a sequence at the 5 'end of the nucleic acid sequence of the gene of interest. Such 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.

  General antisense techniques are described in textbooks (Murray, JAH eds., Antisense RNA and DNA, Wiley-Liss Inc, 1992). Furthermore, the latest research has revealed a phenomenon called RNA interference (RNAi), which has led to the development of antisense technology.

  As used herein, “RNA interference” or “RNAi” is an abbreviation for RNA interference, an organism that is generally known in the art and 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. In this specification, “RNAi” can also be used synonymously with “factor causing RNAi”, “factor causing RNAi”, “RNAi factor” and the like in some cases. For RNAi, see, for example, Zamore and Haley, 2005, Science, 309, 1519-1524; Vaughn and Martinsensen, 2005, Science, 309, 1525-1526; Zamore et al. 2000, Cell, 101, 25-33; Bass, 2001, Nature, 411, 428-429; Elbashirital. , 2001, Nature, 411, 494-498; and Kreutzer et al., WO 00/44895; Zernica-Goetz et al., WO 01/36646; Fire, WO 99/32619; Plaetinck et al., International Publication No. 00/01846; Melo and Fire, International Publication No. 01/29058; Deschamps-Depairlette, International Publication No. 99/07409 and Li et al., International Publication No. 00/44914; Allshire, 2002, Science, 297, 1818-1819; Volpe et al. , 2002, Science, 297, 1833-1837; Jenuwein, 2002, Science, 297, 2215-2218; and Hall et al. , 2002, Science, 297, 2232-2237; Hutvagner and Zamore, 2002, Science, 297, 2056-60; McManus et al. , 2002, RNA, 8, 842-850; Reinhart et al. , 2002, gene & Dev. 16, 1616-1626; and Reinhart & Bartel, 2002, Science, 297, 1831. ). In this specification, the term 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. In the present specification, the “factor causing RNAi” may be any as long as it causes “RNAi”.

  In the present specification, 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.

  The “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.

  In the present invention, in an agent that causes RNAi such as siRNA, the antisense region 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. Here, 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. , 26, 27, 28, 29 or 30 base pairs, but may be longer, 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. Alternatively, 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. , 2002, Molecular Cell, 10, 537-568) and may further comprise a terminal phosphate group such as 5 ', 3'-diphosphate. In certain embodiments, inhibitors of the present invention such as CCL21, CXCR3, CCR7 comprise separate sense and antisense sequences or regions. Here, 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. In certain embodiments, the inhibitor of the present invention, such as CCL21, CXCR3, CCR7, comprises a nucleotide sequence that is complementary to the nucleotide sequence of the target gene. In another embodiment, 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. In the present specification, 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. In certain embodiments, when the invention is a small interfering nucleic acid molecule, it may be devoid of 2'hydroxy (2'-OH) containing nucleotides. In certain embodiments, 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. However, if the presence of a ribonucleotide in an inhibitor such as CCL21, CXCR3, CCR7 is not required to maintain RNAi, 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. In some cases, 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. As used herein, 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).

  As used herein, 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 containing a double-stranded portion of nucleotide length or a variant thereof. Here, the factor preferably comprises a 3 'overhang, more preferably the 3' overhang can be 2 or more nucleotides long DNA (eg 2 to 4 nucleotides long DNA).

  Alternatively, examples of 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).

  Without being bound by theory, one of the possible mechanisms for RNAi to work is that when a molecule that causes RNAi, such as dsRNA, is introduced into a cell, a relatively long (eg, 40 base pairs or more) RNA, helicase In the presence of ATP, an RNaseIII-like nuclease called Dicer having a domain excises the molecule from the 3 ′ end by about 20 base pairs to produce a short dsRNA (also called siRNA). As used herein, “siRNA” is an abbreviation for short interfering RNA, which is artificially chemically synthesized, biochemically synthesized, synthesized in an organism, or about 40 This refers to short double-stranded RNA of 10 base pairs or more formed by decomposing double-stranded RNA of bases or more in the body, and usually has a 5′-phosphate, 3′-OH structure. 'The end protrudes about 2 bases. 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. The relationship between the siRNA sequence and the mRNA sequence cleaved as a target is preferably 100% identical. However, with respect to the base mutation at a position off the center of siRNA, the cleavage activity by RNAi is not completely lost, but a partial activity remains. On the other hand, 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. Utilizing such properties, for mRNA having a mutation, only siRNA having the mutation can be specifically decomposed by synthesizing siRNA having the mutation in the center and introducing it into the cell. Therefore, in the present invention, 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.

  Moreover, although not wishing to be bound by theory, siRNA is synthesized separately from the above pathway, and 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. Thus, in the present invention, siRNA itself and factors that produce siRNA are also useful. In fact, in insects and the like, for example, 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.

  In another embodiment, the factor causing RNAi of the present invention may be a short hairpin structure (shRNA; short hairpin RNA) having an overhang at the 3 'end. As used herein, “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. The above molecules. Such shRNA is artificially chemically synthesized. Alternatively, 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. Although not wishing to be bound by theory, such 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. Here, 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.

  The factor that causes RNAi used in the present invention can also be synthesized in vitro. In this synthesis system, 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. Here, for example, such 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 or 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, proliferation of positive brain malaria cells such as CCL21, CXCR3, and CCR7 can be suppressed. In one embodiment of the present invention, the “polynucleotide” may be a polymer compound having 10 or more nucleotides and in which nucleotides are linearly polymerized.

  In one embodiment of the present invention, 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.

  In one embodiment of the present invention, “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.

  For example, 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.

  In one embodiment of the present invention, “siRNA” includes an RNA strand capable of inducing RNAi. In general, 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. In RNAi research, 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 will become lower.

  In one embodiment of the present invention, “shRNA” includes a single-stranded RNA strand capable of inducing RNAi and forming a hairpin-like folded structure (hairpin-like structure). Typically, 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. However, since many pre-miRNAs that are generally similar in structure and function to shRNA have a length of about 100 nucleotides or more, the length of shRNA is not necessarily 100 bases or less. However, it is thought that it can function as shRNA.

  In one embodiment of the present invention, “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.

  In one embodiment of the present invention, “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, and 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. As long as the base sequence has a desired effect, one or a plurality of base sequences may be deleted, substituted, inserted, or added. In addition, 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.

  In one embodiment of the present invention, the “RNA strand” includes those in which RNA or an equivalent thereof is constituted in a combined form. Further, in one embodiment of the present invention, 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.

  In one embodiment of the present invention, “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. In one embodiment of the present invention, “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.

  In one embodiment of the present invention, “a state in which expression is inhibited” includes a state in which the expression level is significantly reduced as compared with that in a normal state. The expression level may be based on the amount of mRNA or the amount of protein. In one embodiment of the present invention, “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. In one embodiment of the present invention, “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.

  In another aspect, the present invention provides a subject in need of an effective amount of an inhibitor of at least one factor of the CCL21-CXCR3-CCR7 signaling system or a composition or medicament (therapeutic or prophylactic agent) containing the same. There is provided 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. On the other hand, in the present invention, 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 one that is effective 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. When administering an antibody or a polynucleotide, it is effective to use it as an injection. 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.

  In general, a composition, medicament, therapeutic agent, prophylactic agent, etc. of the present invention comprises a therapeutically effective amount of a therapeutic agent or active ingredient, and a pharmaceutically acceptable carrier or excipient. As used herein, “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. As used herein, “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. Preferably, 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. The composition 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). Such 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. In addition to these, for example, 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.

  In one embodiment of the invention, “salts” include, for example, anionic salts formed with any acidic (eg, carboxyl) group, or cationic salts formed with any basic (eg, amino) group. Salts include inorganic or organic salts, see, 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. In one embodiment of the present invention, a “solvate” is a compound formed by a solute and a solvent. For solvates, see, for example, J. Org. Honige et al. , The Van Nostrand Chemist's Dictionary P650 (1953). If 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. In one embodiment of the present invention, “chemical modification” includes, for example, modification with PEG or a derivative thereof, fluorescein modification, biotin modification, or the like.

  When the present invention is administered as a pharmaceutical, 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. It is possible and necessary to administer the medicament by any suitable route, for example by infusion, by bolus injection, by absorption through epithelial or dermal mucosal lining (eg oral, rectal and intestinal mucosa, etc.) Accordingly, 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. When the present invention is 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).

  In certain embodiments where the therapeutic agent is a nucleic acid, it is encoded by in vivo administration of the nucleic acid by constructing the nucleic acid as part of a suitable nucleic acid expression vector and administering it to be present in a cell. 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.

  In a preferred embodiment, 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. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is to be administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.

  It is also possible to mix the composition, medicament, therapeutic agent, or preventive agent of the present invention in a neutral or salt form or other prodrug (eg, 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 that is effective in 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. Further, 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. For example, it may be administered once or twice per 1, 7, 14, 21, or 28 days, or once or twice per any two of these ranges. Also good. 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.

  In one embodiment of the present invention, 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. At this time, 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.

  In certain embodiments, the present invention provides a drug pack or kit comprising one or more containers filled with one or more components of a composition or medicament of the present invention. In some cases, 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.

  In certain embodiments, pharmaceutical compositions comprising the nucleic acids 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.

(Diagnosis of brain malaria based on CCL21-CXCR3-CCR7 signaling system)
In another aspect, the present invention provides a marker for cerebral malaria comprising at least one factor of the CCL21-CXCR3-CCR7 signaling system. Alternatively, 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. Here, when 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. . Such a diagnosis can be performed using a binding agent or a detection agent as described below. Therefore, the method of the present invention includes a step of examining the expression of any of at least one factor of the CCL21-CXCR3-CCR7 signaling system in a subject specimen, and the result may be used as an index of cerebral malaria morbidity. it can.

This CCL21-CXCR3-CCR7 signal transduction system, as described elsewhere in this specification, 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). One inflammatory cytokine; CCL21 and / or CCL19, CD8α 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.

  In a typical embodiment, 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.

In another embodiment, 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. In addition to or alternatively to at least one selected from:
(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) CD8α 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.

In a more detailed embodiment, 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-γ in the cortex;
(C) that the CD8α 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. However, it is not limited to these.

  The brain malaria is Produced by a human malaria parasite corresponding to a malaria parasite selected from the group consisting of falciparum.

  In one particular embodiment, cerebral malaria targeted by the present invention includes rodents or primates.

  In a specific embodiment, 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.

In a particular embodiment, the marker of the present 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.

  In another aspect, 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. Provided is a detection agent or diagnostic agent for cerebral malaria, which comprises 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.

  In the present invention, it has been found that abnormalities (increased expression, etc.) of CCL21-CXCR3-CCR7 signal transduction system factors are indicative of brain malaria. Therefore, according to 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.

  In another aspect, the present invention provides a detection agent, a test agent, or a diagnostic agent for identifying cerebral malaria, including a substance that binds or interacts with a factor of the CCL21-CXCR3-CCR7 signaling system. For such detection, examination or diagnosis, the substance binding is preferably specific.

  As such a detection agent, a test agent, or a diagnostic agent, any substance may be used as long as it can bind to or interact with a factor of the CCL21-CXCR3-CCR7 signaling system. As a specific example, 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.

  In one embodiment, 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) capable of being detected, tested or diagnosed. Alternatively, it may be a complex molecule. As used herein, “complex” or “complex molecule” means any construct comprising two or more moieties. For example, when one part is a polypeptide, the other part may be a polypeptide or other substance (eg, sugar, lipid, nucleic acid, other hydrocarbon, etc.). . In the present specification, 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. When two or more parts are polypeptides, they can also be referred to as chimeric polypeptides. Therefore, in the present specification, 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. As described herein, expression of CCL21-CXCR3-CCR7 signaling factors such as CCL21, CXCR3, CCR7 is an indicator of brain malaria and is useful as an indicator. Thus, the probes and primers according to the invention can be used to identify brain malaria. In one embodiment, 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., and a plurality of deoxyribonucleic acids (DNA) or ribonucleic acids. 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.

  In certain embodiments, the present invention can take the form of a primer. Examples of 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. At least 13 contiguous nucleotides, at least 14 contiguous nucleotides, at least 15 contiguous nucleotides, at least 16 contiguous nucleotides, at least 17 contiguous nucleotides, at least 18 At least 19 contiguous nucleotides, at least 19 contiguous nucleotides, at least 20 contiguous nucleotides, at least 25 contiguous nucleotides, at least 30 contiguous nucleotides, at least 40 Nucleotides long that connection, at least 50 contiguous nucleotides in length, may be a nucleic acid sequence. 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).

  In a particular embodiment, the primer according to the invention can also be used as a primer set consisting of two or more of said primers. In a specific embodiment, 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 PCR. 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. For example, in the PCR method, one of two primers (primer pair) is paired with a plus strand of a double-stranded DNA of a 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. In the LAMP method (WO 00/28082), three regions F3c, F2c and F1c from the 3 ′ end side and three regions B1, B2 and B3 from the 5 ′ end side to the target gene. Each of these six regions can be used to design four types of primers. The primers of the present invention can be chemically synthesized based on the nucleotide sequences disclosed herein. Preparation of the primer is well known, for example, carried out ^{"MolecularCloning, A Laboratory Manual 2 nd} ed." (Cold Spring Harbor Press (1989)), in accordance with "CurrentProtocols in Molecular Biology" (John Wiley & Sons (1987-1997)) can do.

  In certain embodiments, the present invention can take the form of a “probe”. Examples of 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. At least 13 contiguous nucleotides, at least 14 contiguous nucleotides, at least 15 contiguous nucleotides, at least 16 contiguous nucleotides, at least 17 contiguous nucleotides, at least 18 At least 19 contiguous nucleotides, at least 19 contiguous nucleotides, at least 20 contiguous nucleotides, at least 25 contiguous nucleotides, at least 30 contiguous nucleotides, at least 40 Nucleotides long that connection, at least 50 contiguous nucleotides in length, at least the nucleic acid sequence. 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.

  In one embodiment, the detection agent of the present invention can be labeled. Alternatively, 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.

  In one aspect, 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. Provide a method.

  In one embodiment, the method of the present invention uses 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, for example, protein or mRNA, can be detected in vivo. For example, 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.

  In the method of the present invention, CCL21-CXCR3-CCR7 signaling such as CCL21, CXCR3, CCR7, etc., which is a target of interest, is brought into contact with a target sample of the detection agent, test agent or diagnostic agent of the present invention The presence of, or the level or amount of, an expression product of a system factor, such as a protein or mRNA, is determined.

  In the present invention, “contact” means that a plurality of substances are arranged so as to cause interaction or binding between the plurality of substances. In the present invention, a substance that can function as a detection agent, a test agent, or a diagnostic agent. (E.g., 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 protein or mRNA is the method of detecting CCL21, CXCR3, CCR7, etc. in a sample (eg, serum, etc.). 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. Here, the sample used may be any sample that is considered to contain an expression product. For example, serum can be used. Serum can be obtained by a conventional method.

In a particular embodiment, 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, By directly or indirectly detecting the nucleotide duplex, the expression of a CCL21-CXCR3-CCR7 signaling system factor such as CCL21, CXCR3, CCR7, etc. in a cell sample can be detected. For detailed procedures for hybridization ^{techniques, "Molecular Cloning, A Laboratory Manual} 2 nd ed." (Cold Spring Harbor Press (1989), in particular Section9.47-9.58), "Current Protocols in Molecular Biology" (John Wiley & Sons (1987-1997), in particular Section 6.3-6.4), "DNA Cloning 1 :. Core Techniques, A Practical Approach 2 nd ed" (Oxford University (1995), particularly Section 2.10 for conditions ) May be referred to.

Detection of an expression product of a CCL21-CXCR3-CCR7 signaling factor such as CCL21, CXCR3, CCR7 or the like using a hybridization method, for example, mRNA is performed by, for example: (a) a polynucleotide derived from a test sample, and the present invention And (b) detecting the hybridization complex. In step (a), mRNA prepared from the target test sample or complementary DNA (cDNA) transcribed from the mRNA can be contacted with the probe as a polynucleotide derived from the test cell sample. In the detection method using a probe, the probe can be labeled and used. For example, radioactive activity (for example, ³² P, ¹⁴ C, and ³⁵ S), fluorescence (for example, FITC, europium), enzymatic reaction such as chemical coloration (for example, peroxidase, alkaline phosphatase), etc. were used as the label. A label can be mentioned. The detection of the hybridization product can be performed using a known method such as Northern hybridization, Southern hybridization, colony hybridization and the like. The sample in which the hybridization complex was detected indicates that the subject's tissue expresses CCL21-CXCR3-CCR7 signaling factor such as CCL21, CXCR3, CCR7, etc. It can be determined that the possibility of brain malaria is high.

  According to another embodiment of the detection, test or diagnosis according to the present invention, 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. Thus, 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.

  Detection of the expression of 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.

  In step (i), 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. Detection of an amplification product in this sample indicates that the subject's tissue expresses a CCL21-CXCR3-CCR7 signaling factor such as CCL21, CXCR3, CCR7, etc. It can be determined that the possibility of brain malaria is high.

  According to another embodiment of the detection according to the present invention, 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. For example, 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. As 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 contains cells expressing CCL21-CXCR3-CCR7 signaling factors such as CCL21, CXCR3, CCR7, etc. It can be determined that the property is high.

  Each of the detection steps described above can be performed not only once but also by repeating or combining the steps, thereby improving the diagnosis accuracy of cerebral malaria. Therefore, when such an embodiment is adopted, according to the detection, inspection, or diagnosis method of the present invention, cerebral malaria can be diagnosed with higher accuracy by performing the above-described steps twice or more.

  In addition, other marker genes, preferably proliferation marker genes other than 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 By doing so, the diagnostic accuracy of cerebral malaria can be improved.

  Procedures for formulating a diagnostic agent or the like of the present invention as a pharmaceutical or the like are known in the art, and are 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.

  In one embodiment, the concentration of the marker can be measured by mass spectrometry. As ionization methods in this case, both matrix-assisted laser desorption / ionization (MALDI) and electrospray ionization (ESI) can be applied, but production of multivalent ions is small. MALDI is preferred. In particular, according to MALDI-TOF-MS combined with a time-of-flight mass spectrometer (TOF), the concentration of the marker can be measured more accurately. Furthermore, according to MS / MS using two mass spectrometers, the marker concentration can be measured more accurately.

  When measuring the marker concentration 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 darkness and fluorescence intensity of the band corresponding to the target marker. If the separation of the markers is insufficient by SDS-PAGE alone, two-dimensional electrophoresis combined with isoelectric focusing (IEF) can also be used. Furthermore, instead of detecting directly from the gel, Western blotting can be performed to measure the amount of marker on the membrane.

  When measuring the concentration of the marker by chromatography, for example, a method by liquid high performance chromatography (HPLC) 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.

  In one aspect, the present invention provides a kit for detection, testing and / or diagnosis for performing the method for detection, testing and / or diagnosis according to the present invention. This kit contains the detection agent, test agent and / or diagnostic agent of the present invention. As the embodiment, any embodiment described in this specification can be used alone or in combination.

  In one embodiment, 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. Accordingly, 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 according to the present invention 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.

  In another embodiment, 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. Therefore, 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.

  In further embodiments, detection kits according to the present invention include detection kits for carrying out the 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.

  These 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.). Can be used, or a factor that specifically interacts with the marker, or a means for selectively recognizing the marker, and thus only the factors or means specifically described herein. Rather, it is understood that any equivalent factor or means known in the art can be used.

  In one embodiment, 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.). Preferably, 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.

  In a preferred embodiment of the present invention, 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. pI electrophoresis, Southern blotting, two-dimensional electrophoresis), 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 From a combination of It is selected from the group that.

  In a preferred embodiment of the invention, the system or kit of the invention further comprises a marker standard. Such a 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.

  In a preferred embodiment, the present invention may further comprise means for purifying the sample of interest. Examples of such purification means include chromatography. Since purification can increase the accuracy of the diagnosis, it can be used in preferred embodiments, but this is not essential.

  In one embodiment, 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.

  In one embodiment, 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. Such determination means can be realized using a computer.

  In one embodiment, a kit or system of the invention comprises a composition comprising a marker or an agent that specifically interacts with the marker.

  In one aspect, 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. Here, 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. Whether 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. In the detection or diagnosis of the present invention, as 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.

  In one embodiment, when an immunoassay is used as a marker measurement method, it is preferable to use a carrier on which an antibody is immobilized. In this way, an immunoassay system using the antibody immobilized on the carrier as the primary antibody can be easily constructed. For example, 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. In addition, immunoassay systems based on binding inhibition methods and competitive methods can be constructed. Further, when 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.

  On the other hand, in one embodiment, 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. In particular, using a protein chip using a substrate as a carrier, surface-enhanced laser desorption / ionization-time-of-flight mass spectrometry (herein referred to as "SELDI") (Referred to as “-TOF-MS”), the concentration of the marker can be measured more accurately. As the types of substrates that can be used, cation exchange substrates, anion exchange substrates, normal phase substrates, reverse phase substrates, metal ion substrates, antibody substrates, etc. can be used, but cation exchange substrates, particularly weak cation exchanges. A substrate and a metal ion substrate are preferably used.

When the marker is captured on the carrier by ionic bonding, the ion exchanger is immobilized on the carrier. In this case, 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. For example, examples of weak anion exchangers include those having weak anion exchange groups such as dimethylaminoethyl (DE) and diethylaminoethyl (DEAE). Examples of strong anion exchangers include quaternary ammonium (trimethylaminomethyl) (QA), quaternary aminoethyl (diethyl, mono-2-hydroxybutylaminoethyl) (QAE), quaternary ammonium (trimethylammonium). And those having a strong anion exchange group such as (QMA). Examples of weak cation exchangers include those having weak cation exchange groups such as carboxymethyl (CM). Further, examples of the strong cation exchanger include those having a strong cation exchange group such as sulfopropyl (SP). On the other hand, when a marker is captured on a carrier by hydrophobic interaction, a substance having a hydrophobic group is immobilized on the carrier. Examples of hydrophobic groups include C4-C20 alkyl groups, phenyl groups, and the like. Furthermore, 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.

  In one embodiment, well-known materials such as beads, microtiter plates, and resins can be used as examples of carriers to be used. In particular, beads and microtiter plates are conventionally used in immunoassays, and the measurement system can be easily constructed. On the other hand, 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 substrate and an antibody specific for a marker is immobilized in spots on a plurality of spots on the surface.

  The detection, test or diagnostic 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. Preferably, it 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.

(General technology)
Molecular biological techniques, biochemical techniques, and microbiological techniques used in this specification are well known and commonly used in the art, and are described in, for example, Sambrook J. et al. et al. (1989). Molecular Cloning: A Laboratory Manual, Cold Spring Harbor and its 3rd Ed. (2001); Ausubel, F .; M.M. (1987). Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Ausubel, F .; M.M. (1989). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Innis, M .; A. (1990). PCR Protocols: A Guide to Methods and Applications, Academic Press; Ausubel, F .; M.M. (1992). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates; Ausubel, F .; M.M. (1995). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates; Innis, M .; A. et al. (1995). PCR Strategies, Academic Press; Ausubel, F .; M.M. (1999). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Wiley, and annual updates; J. et al. et al. (1999). PCR Applications: Protocols for Functional Genomics, Academic Press, “Experimental Methods for Gene Introduction & Expression Analysis”, Yodosha, 1997, etc. Is incorporated by reference.

  For DNA synthesis technology and nucleic acid chemistry for producing artificially synthesized genes, see, for example, Gait, M. et al. J. et al. (1985). Oligonucleotide Synthesis: A Practical Approach, IRL Press; Gait, M .; J. et al. (1990). Oligonucleotide Synthesis: A Practical Approach, IRL Press; Eckstein, F .; (1991). Oligonucleotides and Analogues: A Practical Approach, IRL Press; Adams, R .; L. et al. (1992). The Biochemistry of the Nucleic Acids, Chapman &Hall; Shabarova, Z .; et al. (1994). Advanced Organic Chemistry of Nucleic Acids, Weinheim; Blackburn, G .; M.M. et al. (1996). Nucleic Acids in Chemistry and Biology, Oxford University Press; Hermanson, G .; T.A. (1996). Bioconjugate Technologies, Academic Press, etc., which are incorporated herein by reference in the relevant part.

  For example, the oligonucleotides of the present invention can be synthesized herein by standard methods known in the art, for example, by using automated DNA synthesizers (such as those commercially available from Biosearch, Applied Biosystems, etc.). is there. For example, phosphorothioate oligonucleotides can be synthesized by the method of Stein et al. (Stein et al., 1988, Nucl. Acids Res. 16: 3209) and controlled 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.

  In this specification, “or” is used when “at least one or more” of the items listed in the text can be adopted. The same applies to “or”. In this specification, when “within the range of two values” is specified, the range includes the two values themselves.

  References such as scientific literature, patents and patent applications cited herein are hereby incorporated by reference in their entirety to the same extent as if each was specifically described.

  The present invention has been described with reference to the preferred embodiments for easy understanding. In the following, the present invention will be described based on examples, but the above description and the following examples are provided only for the purpose of illustration, not for the purpose of limiting the present invention. Accordingly, the scope of the present invention is not limited to the embodiments or examples specifically described in the present specification, but is limited only by the scope of the claims.

Examples are described below. When necessary, the animals used in the following examples were handled according to the Declaration of Helsinki, if necessary, in compliance with the standards stipulated by the National Institute of Pharmaceutical Sciences. Specifically, the products described in the examples were used as reagents, but equivalent products from other manufacturers (Sigma-Aldrich, Wako Pure Chemicals, Nakarai, R & D Systems, USCN Life Science INC, etc.) can be substituted.
(Materials and methods)
P. berghei ANKA (PbA)
Two different PbA lines were used. Details of these PbA strains (with and without GFP) have been previously described (Coban, C. et al. (2007). Int Immunol 19, 67-79 .; Ishino, T. et al. (2006). Mol. Microbiol 59, 1175-1184 .; Zhao, H. et al. (2012). Cell Host Microbe 12, 705-716.).

In vivo chemokine blocking mice with animals and antibodies Age- and sex-matched C57BL / 6 (CLEA, Osaka, Japan) or littermate mice (Ccr7 ^+/- ) were used as wild type (WT) groups. Ccr7 ^{− / −} mice with C57BL / 6 background Miyasaka and M.S. H. Produced by Jang (Osaka University) and described previously (Forster, R. et al. (1999). Cell 99, 23-33.). Batf3 ^{− / −} mice were purchased from Jackson Laboratories and crossed with C57BL / 6 mice to create a ^+/− phenotype.

  Wild type mice were injected intravenously (iv) daily with endotoxin-free anti-CCL21 antibody or isotype control (50 μg per mouse, Peprotech daily) for 3 days from the start of PbA infection. Alternatively, an anti-CXCR3 antibody (LEAF purified anti-mouse CD183 (CXCR3), 100 μg per mouse, Biolegend) was injected twice on days 4 and 5 after infection. 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.

Monkeys A female Japanese macaque (Macacafuscata) weighing 5 kg, as described previously, 1 × 10 ⁹ frozen P. a. Coatneyi infected erythrocytes (CDC line) i. v. Inoculated and followed daily (Kawai, S. and Sugiyama, M. (2010). Acta Trop 114, 152-156.). At the end of the 14th day after infection, when the monkey showed severe malaria symptoms and became unconscious, autopsy was performed after anesthesia by intramuscular injection of ketamine-HCl (15 mg / kg). As a control, P.I. One Japanese macaque infected with knowlesii and one cynomolgus monkey as a non-infected control were used.

  All mouse and monkey experiments were conducted according to the guidelines of the National Institute of Biomedical Innovation and Osaka University, and the guidelines for the use of laboratory animals approved by the Japanese Society for Experimental Zoology.

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. Bleeding was detected using T2 ^* weighted image (FLASH sequence) and diffusion weighted image (DWI; spin echo sequence) with the following parameters: 1) 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. 2) 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. 3) 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.

Thin Cranial Surgery and Multiphoton Imaging To visualize OLF in living mice, mice were previously anesthetized by intramuscular injection of ketamine (0.13 mg / g) and 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. Under a binocular stereomicroscope, the skull on the OLF was carefully thinned (about 20-30 μm) 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. The vasculature of OLF was visualized by intravenous injection of tetramethylrhodamine isothiocyanate (TRITC) -dextran (5 mg / mouse) (T1287, Sigma), and T cells were expressed in TCR-β conjugated with brilliant violet 421 ( Labeled with 10 μg of H57-597, Biolegend), or 5 μg of CD8α (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 μm (x), 507.934 μm (y), 5 μm (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 μm from the buffy coat surface, which corresponded to the glomerular layer (FIGS. 2A-B) (Chaignau, 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.

Measurement of blood vessel diameter The blood vessel diameter in the deep layer near the glomerulus was measured by acquiring a Z image stack and projecting it onto a two-dimensional image. Vessel inner diameter was then measured as previously described (Chaigneau, E. et al. (2003). Proc Natl Acad Sci USA 100, 13081-13086.).
Evaluation of BBB permeability At time points indicated, mice were given i.e. 200 μl 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.

Quantitative real-time reverse transcription PCR analysis Brain samples were homogenized, total RNA was isolated, and q-PCR was performed as previously described (Zhao, H. et al. (2012). Cell Host Microbe 12, 705- 716.). Malaria infection was quantified using primers specific for PbA 18S rRNA. All primers were purchased from Assays on Demand (Applied Biosystems).

Food Embedding Test The food burying test was performed as previously described (Yang, M. and Crawley, JN (2009). Curr Protoc Neurosci Chapter 8, Unit 8 24.). Briefly, mice were left free of food for 18 hours, and then transferred to a new cage with bedding embedded in the random corners of the cage approximately 1 cm below the surface. The time taken for the mouse to find the buried food was recorded. If the mouse did not find any embedded food after 15 minutes, the test was stopped and the time was recorded as 900 seconds (latency score, >>).

Histology Brains were removed and prepared for immunohistochemistry (IHC) as previously reported (Zhao, H. et al. (2012). Cell Host Microbe 12, 705-716.). The following antibodies were used: anti-mouse TER119 antibody (Biolegend), ZO-1 antibody (Invitrogen), CCL21 antibody (Peprotech), PECAM antibody (BD Pharmingen) and GFAP antibody (Dakocytomation). Sections were observed using a fluorescence or LSM 780 confocal laser scanning microscope (Zeiss, Germany).

Temperature monitoring A mouse cage equipped with an in-house thermal monitoring system was developed to record continuous heat generation (Aoshi et al., In preparation for paper submission, see JP2012-231725). The cages were prepared as incubators with a 12-12 hour light / dark cycle and controlled free ambient temperature of 30 ° C. with free access to food and water. The back hair of the mouse was removed with an electric clipper, the skin temperature on the back was continuously recorded at 1 minute intervals by a FLIR b60 thermal camera mounted inside the incubator, and the data was collected using QuickPlot software (FLIR Systems, Inc. .).

Flow cytometric analysis Spleen and brain cells were purified as previously described (Coban, C. et al. (2007). Int Immunol 19, 67-79 .; Zhao, H. et al. (2012). Cell Host. Microbe 12, 705-716.). The cell surface was treated with APC-CD11c (HL3), PE-CD4 (RM4-5), PerCp Cy5.5-CD8α (53-6.7), PE Cy7-CD3 (145-2C11), APC Cy7-TCRβ (H57). -597), APC-CCR7 (4B12), APC-CD44 (IM7), PE CY7-CD11c (HL3) and APC Cy7-CD11b (M1 / 70). These antibodies were purchased from BD Biosciences (San Jose, CA) and BioLegend (San Diego, CA). Samples were taken on a BD LSRFortessa flow cytometer and analyzed using FlowJo software. For intracellular IFNγ staining, splenocytes were incubated for 2 hours in the presence of brefeldin A (10 μg / ml, Sigma), the cells were collected and stained with IFNγ according to the intracellular staining protocol (eBioscience).

Transwell Migration Assay 48 well transwell plates (5 μm pore size, Costar, Corning Inc.) were used for chemotaxis assays as previously described (Rappert, A. et al. (2002). J Immunol. 168, 3221-3226.). Briefly, the lower compartment was filled with recombinant protein CCL21 and / or anti-CCL21 antibody (R & D Systems) diluted in serum-free DMEM at the indicated concentrations. Spleen CD8 T cells enriched after depletion of B cells and CD11b ⁺ cells and CD4 ⁺ cells from infected animals on day 6 after infection were added to the upper wells in triplicate (5 × 10 ⁶ / well). ), And incubated in serum-free DMEM medium at 37 ° C. for 2 hours. Human SDF-1α (Peprotech, Inc.), a potent chemotactic ligand for lymphocytes, was used as a positive control. The migration rate of CD11c ⁺ CXCR3 ⁺ CD8 T cells was calculated by counting total cells with a flow cytometer. Chemokine-induced migration was normalized to that of the unstimulated control group and expressed as a percentage of the control.

Serum cytokines Blood was collected on day 5 after infection, and cytokines and chemokines were detected by mouse Bio-Plex (Bio-rad).

Adoptive immunization experiment Total splenocytes were prepared from Rag2 ^{− / −} mice on the fifth day after PbANKA infection, and spleen CD8α ⁺ 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 CD8α ⁺ 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 CD8α ⁺ 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)
In Example 1, an experiment on the relationship between brain malaria and the olfactory bulb was performed using a mouse model.

(Ultra-high 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.

In order to study changes in the mouse brain and visualize the pathology associated with ECM, we have developed a very high magnetic field 11.7T MRI (Mori, Y. et al. (2011). Magn Reson MedSci 10,219. -227.). Six days after PbA infection in which specific ECM symptoms such as loss of direction and paralysis began, 11.7T MRI showed dark and distinct spots in the bilateral OLF (coronal section in FIG. 1A and FIG. 1E), Or other parts of the brain including the cerebellum did not show such spots (sagittal section in FIG. 1A). Acquiring a diffusion-weighted image (DWI) reveals more detail in the OLF area, which is remarkably similar to the histological detail, where the low density area is the site of bleeding due to hematoxylin and eosin (HE) staining (FIGS. 1B and 1C). The inventors have also demonstrated that by immunohistochemistry (IHC), bleeding (TER119 ⁺ red blood cells) and GFP-PbA parasites are present in the same region of OLF, which is as deep as the granular cell layer (GGL). It was confirmed that there was (FIG. 1D). The inventors additionally performed MRI in the OLF at an earlier time point, but no change was detected before 6 days after infection (FIG. 1F). In addition, several MRI images after the onset of symptoms showed different degrees of microbleeding severity in OLF, possibly causing a progressive disease state (FIGS. 3H, I). In contrast, even if severe bleeding occurred in OLF, MRI showed no clear evidence of microbleeding in other parts of the brain (FIGS. 3H, I). In addition, the lethal parasite P. Mice infected with yoeliiL (PyL) did not detect microbleeds in their OLF by MRI (FIGS. 3H, I). Therefore, instead of the lower magnetic field MRI, our ultra-high magnetic field MRI setting makes the olfactory bulb more susceptible to attack during PbA infection and is more likely to bleed compared to other parts of the brain It is reasonable to think that it was possible to identify.

(In vivo multiphoton imaging of parasites in OLF's fenced microvessels)
We next investigated why and how bleeding from the OLF region occurred during ECM, including the potential involvement of BBB disruption. The OLF is composed of a fence-like microvascular structure with high density and oriented 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.). To investigate whether this unique vascular structure of OLF could be a “fragile” spot for PbA-infected erythrocytes as well as infection-related events, OLF was visualized by in vivo MP microscopy. MP imaging of OLF, in which OLF capillaries as deep as GL (about 150 μm) can be visualized, has been performed with rodents (Chaineau, 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). We performed thin cranial surgery on the dorsal OLF of mice as previously described (Sawada, M. et al., Supra) to keep the tissue intact. Biological images of OLF blood vessels have an anatomically complex capillary structure (diameter <5 μm) (Petzold, et al., Supra) and are expressed in PbA parasites 5 days after infection. It was shown to be suitable for attachment / occlusion of circulating iRBC, which is shown as a GFP signal (also confirmed in FIG. 2B and animation S1 (not shown here)). In the moving image S1, the olfactory bulb of the mouse visualized by an in vivo multiphoton microscope was confirmed. 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). Taken together, these results indicate that OLF is a region that is unique to ECM pathogenesis, possibly due to its fenced 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.). Therefore, we investigated whether CD8 T cell recruitment could be observed by in vivo MP imaging of OLF and / or could be associated with microhemorrhage after PbA infection. . In vivo imaging of labeled CD8 T cells and GFP-expressing PbA parasites in infected mouse OLF clearly showed that microbleeding occurred in the branching capillaries (FIG. 2D, also confirmed by animation S2 (this specification) Not shown in the book)). In 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-CD8α 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. As seen in movie S2, red dextran labeled capillaries were altered during the development of microbleeds (almost red pigment was released into the tissue). Importantly, CD8 T cells are found to “crawl” back and forth within the blood vessels during ECM development, increasing 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 μm). The 3D video showed clearly that CD8 T cells were attached to the blood vessel and actively crawling along the blood vessel wall (video S3 (not shown here)). Movie 3S shows how T cells crawl along microvessels in the olfactory bulb, and shows a 3D constructed multiphoton movie 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-TCRβ antibody are shown. It was found that T cells attached to the wall of microvessels (diameter about 10 μm) and migrated along the wall. The crawl behavior by these T cells was not due to in vivo anti-TCRβ or anti-CD8 antibody labeling (these antibodies had no effect on naive animal T cells) (video) S4 (not shown here), data not shown). On the contrary, fully activated T cells began to accumulate in the OLF capillaries 5 days after infection and greatly increased their number due to crawl movement (movie S4 (not shown here)). Movie S4 shows in vivo olfactory bulb multiphoton imaging of naive mice and mice infected with GFP-PbA parasites 4-6 days after infection, showing T cells activated and crawling inside the olfactory bulb capillaries. Red, red TRITC-dextran-labeled blood vessels; green, GFP-expressing PbA parasites; blue, anti-TCRβ antibody-labeled T cells, but these videos show only the blue channel. Each video was taken for about 1 hour. Blue circles indicate T cells that flow faster in the blood vessel, and red circles indicate T cells that slow down and crawl within the blood vessel. Although it was difficult to capture T cell movement in naive mice, the number of T cells increased with the number of days after infection. The movement shows crawl behavior. In summary, these OLF bio-images show that with increasing numbers of crawling CD8 T cells, the accumulated iRBC must eventually exit the blood vessel due to micro-bleeding of small OLF capillaries. .

  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.). Therefore, we investigated whether CD8 T cell recruitment could be observed by in vivo MP imaging of OLF and / or could be associated with microhemorrhage after PbA infection. . In vivo imaging of labeled CD8 T cells and GFP-expressing PbA parasites in the OLF of infected mice clearly showed that microhemorrhages occurred in branch vessels (FIG. 2C and animation S2 (not shown here)) ). Importantly, CD8 T cells were found to traverse back and forth within the blood vessel as if they “followed” parasites within the OLF blood vessels (FIG. 2D and animation S3 (herein Not shown)). In addition, 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 OLF bioimages show that parasites that have accumulated in the OLF are tracked by CD8 T cells and exit the blood vessel by micro-bleeding of small OLF vessels.

(Confirmation that olfactory function is destroyed during brain malaria)
In view of the above findings that iRBC as well as CD8 T cell accumulation occurs in stages during ECM, leading to microbleeds within the OLF, we have found that OLF is a complex physiology for chemosensitivity. It was hypothesized that the function of OLF (olfaction) is affected by the inclusion of olfactory nerves that form a dynamic synapse. To evaluate the function of OLF, we performed a simple “food burying test” (Yang, M. and Crawley, JN (2009). Curr Protoc Neurosci Chapter 8, Unit 824. .). Briefly, mice were left free of food for 18 hours, and then transferred to a new cage with bedding embedded in the random corners of the cage 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 predictions of ECM manifestation such as bleeding within OLF and potential loss of BBB integrity.

  To assess whether loss of OLF function correlates directly with loss of BBB integrity (BBB leakage), mice were injected with Evans Blue dye at early time points (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 slowed down and stopped in blood vessels, blue dye exudation to brain tissue appeared from OLF as early as 5 days after infection, while the whole brain was 6 to 6 after infection. 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 has been shown to localize in the olfactory epithelium, olfactory neurons, and the olfactory bulb mitral cell layer (Miragal, F. et al. (1994). J Comp Neurol 341, 433-448.). On day 6 post-infection, we observed a significant interruption of ZO-1 immunostaining in OLFMCL, consistent with the accumulation of GFP-expressing parasites (iRBC) (FIG. 3D), indicating that OLF It suggests that disruption of ZO-1 expression in tight junctions may be associated with a loss of BBB integrity in the ECM.

(Example 2: Relationship between brain malaria and chemokines)
Next, in this example, the relationship between brain malaria and chemokines was confirmed, and the relationship between the CCL21-CXCR3-CCR7 signaling system and brain malaria was elucidated.

(High fever and chemokine storms in brain malaria are associated with OLF dysfunction and physical damage)
We next searched for potential factors that contribute to OLF dysfunction during ECM. High fever has been shown to be one of the promoters of BBB loss of function (Kiyatkin, EA and Sharma, HS (2009). Neuroscience 161, 926-939.). Considering that high fever is an important symptom of malarial coma, we developed an experimental system that allows continuous measurement of body temperature in PbA-infected mice using a thermal camera. Continuous and non-invasive measurement of body temperature of infected mice shows that a high fever attack begins 24 hours before the onset of ECM symptoms (near day 5 after PbA infection) and lasts 24 hours, after which heat loss It was revealed that it ended and eventually died within the next 12-24 hours (FIG. 3, animation S5 (not shown here)). In the moving image S5, continuous fever of the infected / non-infected mouse was recorded by the thermal monitor camera. The cage was continuously monitored for 10 days. The motion is filmed for 32 seconds and corresponds to days 5-7 after infection. Uninfected naive control mice (# 1, upper right, yellow), infected mice (# 2, upper left; # 3, lower right; # 4, lower left). 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.
When infected with Rag2 ^{− / −} mice, which are immunodeficient mice that do not develop cerebral malaria, these mice show no signs of hyperthermia throughout the infection and OLF is confirmed by MRI (FIG. 3F and FIG. 3I). In addition, lethal PyL or non-lethal P. Mice infected with yoelii-NL did not develop fever during infection (FIGS. 3G and 5F-G), suggesting that the increase in heat 5 days after infection may be PbA specific. Although the exact mechanism by which high fever triggers and / or promotes BBB collapse and subsequent OLF dysfunction and bleeding is not clear, data show that high fever occurs 24 hours before ECM-related death and OLF is And suggests that it may correlate with BBB leakage after bleeding is promoted. Notably, 5 days after PbA infection, systemic serum cytokine levels (mostly pro-inflammatory cytokines) are elevated, supporting the notion that cytokine storms are associated with high fever (FIGS. 5F-G ).

(CCL21 is highly expressed in OLF at an early stage of infection)
We further investigated factors that may be associated with olfactory loss in the early stages of PbA 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.). Because some chemokines and cytokines have an important role in the development of ECM, we have interferon (IFN) -γ, IP-10 (CXCR3 ligand), MCP-1 (CCR2 ligand) in OLF The expression levels of several cytokine / chemokine mRNAs, including CCL19 and CCL21 (CCR7 ligand), were measured. 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.

From the above results, the present inventors decided to examine whether CCR7 is involved in ECM pathology by recruiting CCR7-expressing cells into the brain via OLF. We infected WT (C57BL / 6) and littermate Ccr7 ^+/− and Ccr7 ^{− / −} mice with PbA and followed their survival. There was a significant increase in survival of Ccr7 ^{− / −} mice and death was ultimately caused by hyperparasemia, but there was no difference in parasite levels between the two groups (FIG. 4C, data shown). ) In order to evaluate whether OLF function in Ccr7 ^{− / −} mice was intact, a food embedding test was performed, and intact OLF function was observed compared to WT mice (FIG. 4D). Interestingly, Ccr7 ^{− / −} mice have a delayed hyperthermia (about 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 CD8α DC activation of CD11c ⁺ CD8 T cells, but not for its movement into the brain)
We next examined the underlying mechanism 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, we examined recruitment of T cells 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). Notably, recent reports indicate that among CD8 T cells recruited into the brain, CD11c ⁺ CD8 T cells are highly activated, presumably producing IFN-γ and granzyme-B. (Tamura, T. et al. (2011). Infect Immun 79, 3947-3956.). Consistent with this report, we found that the majority of CD8 T cells in the brains of WT mice infected with PbA were CD11c ⁺ , and this population was infected with Ccr7 ^{− / −} mice (FIG. 5A; ^* WT and Ccr7 ^{− / −} mice, although percentages and numbers were significantly reduced in ^* p <0.01 and ^*** p <0.001) and spleen (FIG. 5B; ^** p <0.01) In between, the percentage and number of CD8 T cells in the spleen were found to be comparable. Activation of CD11c ⁺ CD8 T cells in the spleen was also impaired in the absence of CCR7 (determined by CD44 expression), with decreased IFN-γ secretion from the same cell population (FIGS. 5C-D). Taken together, these data indicate that CCR7 is involved in the induction and / or migration of activated CD11c ⁺ CD8 T cells, a very specific subpopulation of CD8 T cells, into the PbA-infected brain and in the pathogenesis of ECM It may suggest that it can have an important role. To confirm this, we further determined whether the decrease in the percentage of CD11c ⁺ CD8 T cells in the spleen of Ccr7 ^{− / −} mice during PbA infection was caused by a defect in previous priming in the spleen. It was determined. Since CD8α ⁺ 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.). Consistent with this, we found that mice lacking the basic leucine zipper transcription factor ATF-like-3 (Batf3) without CD8α DC in the spleen (Hildner, 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). On the other hand, considering that Ccr7 ^{− / −} mice had approximately the same number of CD8α DC as in the spleen of WT mice (FIG. 6E), we expressed functional CCR7 expression on CD8α DC. An experiment was designed to understand whether is required for CD8 T cell activation.

(Functional CCR7 is required for priming CD11c ⁺ CD8 T cells in the spleen)
We purified CDR8α DC from infected Rag2 ^{− / −} mice with intact DC but no T / B cells (McLellan, AD et al. (2002). Blood 99, 2084-2093). .). Rag2 ^{- / -} spleen CD8a ⁺ DC from mice, but expressed high levels of CCR7 5 days after infection, CD8a ^- DC did not express it (Figure 6H and I). To purify these DCs, CD8α ⁺ 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 CD8α ⁺ DC population ( ^> 95% purity, FIG. 6B). Subsequently, Ccr7 ^{− / −} mice were adoptively immunized with highly concentrated CCR7 ⁺ CD8α ⁺ 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). Taken together, these findings are important in the pathogenesis of ECM, where high-level functional CCR7 expression on activated CD8α ⁺ DC during PbA infection presumably activates CD11c ⁺ CD8 T cells and their expansion. It suggests having a role. However, CCR7 expression on CD11c ⁺ CD8 T cells is unnecessary for the migration of these pathogenic cells into the brain.

(CCL21 expressed in OLF is consistent with astrocyte activation and may be important for migration of CD11c ⁺ CD8 T cells into OLF)
The finding that CCR7 is not required for migration of CD11c ⁺ CD8 T cells into the OLF implies the importance of CCL21 during priming of CD8 T cells. However, since 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). J Immunol 168, 3221-3226 .; vanWeering, HR et al. ( 2010) .Brain Behav Immun 24,768-775.) And an important chemokine receptor responsible for migration of CD8 T cells into the brain (Campanella, GS et al. (2008). ProcNatl Acad Sci. U S A 105,4814-4819 .; Hansen, DS et al. (2007) .J Immunol 178, 5779-5788.; Van denSteen, PE et al. (2008) .Eur J Immunol 38, 1082-1095. .) Taking into, the present inventors have further analyzed the localization and / or source of CCL21 in infected OLF. Although it was difficult to handle cell types that express CCL21 within OLF, CCL21 staining was limited to inflamed endothelium where astrocytes often co-localize (FIG. 6A). Astrocytes are specialized cells that monitor and sense vascular changes through their end feet, and their redistribution in the retina is associated with ECM (Medana, I. et al. M. et al., (1996). Glia 16, 51-64.). Six days after infection, morphological changes of astrocytes in OLF (eg, poor distribution, thicker and longer protrusions) became apparent, and the correlation between astrocytes and PECAM staining was greatly disturbed (FIG. 6B). Interestingly, CCL21 staining, particularly by its fibrous-like structure, 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, nuclei were visualized with DAPI (blue). Bars are 10 μm.) Arrows indicate CCL21 fibrillar structures that interact with CD8 T cells. To investigate whether there is a chemotactic interaction between CCL21 and CXCR3-expressing CD11c ⁺ CD8 T cells, we next performed an in vitro transwell migration assay. As can be seen in the results, 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. In the omitted figure, migration of purified splenic CD11c ⁺ CXCR3 ⁺ CD8 T cells from infected WT mice in response to increasing concentrations (0, 0.1, 1 and 2 μg / ml) of CCL21 is shown. Human SDF1-α (80 ng / ml) was used as a positive control, and the response to (E) CCL21 (1 μg / ml) and / or anti-CCL21 antibody (1 and 5 μg / 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 (%) And was shown to be specifically blocked by anti-CCL21 antibody.

(Example 3: Prevention and treatment of cerebral malaria)
Next, it was thought that cerebral malaria could be prevented and treated by blocking the CCL21-CXCR3-CCR7 signaling system, and an experiment using an antibody was conducted as an example.

(CCL21-CCR7-CXCR3 axis block is a new strategy for ECM treatment)
Given that the CCL21-CCR7-CXCR3 axis has an important role in CD8α DC-CD8 T cell-mediated ECM immunopathology, we could use 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). However, anti-CCL21 antibody treatment on day 4 after infection had no significant effect on the progression of ECM (data not shown), and involvement of CCL21 during the late phase of ECM is related to other effector mechanisms. It was suggested that it can be impaired by activation. Therefore, we performed a targeted combination of CCR21 and CXCR3. As a proof of concept, blocking CXCR3 with suboptimal doses of antibody (100 μg) on days 4 and 5 post infection resulted in significant survival from ECM in Ccr7 ^{− / −} mice compared to WT matched mice. (Fig. 6D). Similarly, combination antibody treatment with anti-CXCR3 + CCL21 antibody mixture on the day of loss of olfaction (day 4 after infection) resulted in better survival from ECM than anti-CXCR3 antibody alone treatment (not shown). These data suggested that combined blocking of chemokines could be exploited as a novel therapeutic strategy that could lead to escape from ECM. In the abbreviated figure, the survival curve of combined anti-chemokine antibody treatment is shown. A mixture of anti-CXCR3 and anti-CCL21 antibodies was injected 4 days after infection when olfactory loss began. Anti-CCL21 antibody injection was repeated on day 5. ^* P <0.05 in control vs. anti-CXCR3 antibody group and control vs. combination treatment group by Log-rank (Mantel-Cox) test (each group n = 5). The results showed that it resulted in better survival from ECM than anti-CXCR3 antibody monotherapy.

(Example 4: Confirmation with primates: relationship between brain malaria and olfactory bulb)
Next, in this example, an experiment on the relationship between cerebral malaria and the olfactory bulb is performed with primates.

(The olfactory bulb is targeted in a monkey model of brain malaria)
We finally examine whether our findings can be applied to humans by performing MRI of non-human primate OLF. P. of Japanese macaque (Macaca fuscata) Coatneyi infection is a primate model of brain malaria (Kawai, S. and Sugiyama, M. (2010). Acta Trop 114, 152-156.). P. Coatneyi infected red blood cells i. v. Following inoculation, monkeys are tracked daily and parasitemia levels are counted from blood smears. At low levels of parasitemia, possibly isolated in capillaries, the autopsy is performed at the end of the monkey showing typical symptoms and coma. 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. In order to confirm the presence of a bleeding spot, 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, naïve 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.).

  Preliminary experimental results confirm that monkeys have similar results to mice.

(Discussion)
The brain is severely dysfunctional during ECM due to numerous pathological events such as BBB destruction, vascular leakage, immune cell accumulation (especially CD8 T cell infiltration), but the brain is destroyed The exact location is not well understood. In this study, we have identified that the OLF region is susceptible to attack of vascular leakage in the ECM in both mice and non-human primates, but this discovery Could only be possible by using in combination with an MP bioimaging microscope. We further identified the presence of an early olfactory loss in the ECM prior to the onset of coma. Given that detection of malarial coma as early as a day can dramatically increase the success rate of treatment, the previously undetected location of malaria infection that was not previously noticed and the olfactory status Loss detection may provide an early, inexpensive and easy diagnosis of ECM and may allow early treatment before coma and subsequent conditions are established. In exploring the basic mechanism of pathology of ECM via OLF, the present inventors found that CCL21 secreted from OLF astrocytes utilizes pathological CD11c ⁺ CD8 T by utilizing the CXCR3 chemokine axis. It has been found that it may have a role in the recruitment of cells into the brain (FIG. 7). We further move this new understanding to a novel therapeutic strategy by blocking chemokine-receptor interactions with antibody combinations when the loss of olfactory, an early symptom of ECM, becomes apparent. And developed.

  An interesting question is why 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), such a structure comprising a thin end foot of stellate cells surrounding the blood vessel. Presents 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, M. T. (1993) J Comp Neurol 328, 501-526.; Chen, Y. et al. (2013) .J Biomed Opt 18,126012.; Whitman, MC et al. (2009) .J Comp Neurol. 516, 94-104.). In this study, the depth of the OLF nerve layer (ONL), vascular scaffolding around the glomerular layer (GL), and mitral cell layer (MCL) is immunologically related to PbA-infected erythrocytes or parasites. Targeted by the event. Currently, we do not understand which parasites or related factors can contribute to this, but during ECM, the tight junction network (ZO-1) within the OLF is targeted. , Maybe it was destroyed. Even at very low peripheral parasite loads, 3 days post-infection, abundant perivascular astrocyte endofetes in GL and MCL may sense vascular changes (Bailey, M S. and Shipley, MT (1993) J Comp Neurol 328, 501-526. De Saint Jan, D. and Westbrook, GL (2005) J Neurosci 25, 2917-2924. Petzold, GC et al. (2008) Neuron 58, 897-910.). Similar events were reported in the brain after capillary venules and / or arterioles during ECM pathogenesis (Cabrales, P. et al. (2010). Am J Pathol 176, 1306-1315 .; Nacer, A. (2012). PLoS Pathog 8, e1002982.) Needs to be further investigated as to whether different roles exist in different anatomical structures during ECM pathogenesis.

  OLF is known as the OLF neuronal projection, particularly 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.). Recent studies have revealed that neurons migrate from the central nervous system (CNS), via nerves, and along the cerebral blood vessels toward OLF (Bovetti, S. et al. (2007). J Neurosci. 27, 5976-5980.), Suggesting that 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. (Mesholam, RI et al. (1998). ArchNeurol 55, 84-90 .; Perricone, C. et al. (2012). Smell and Autoimmunity: A Comprehensive Review. Clin Rev Alliance Immunol. Edition published, doi: 10.1007 / s12016-012-8343-x); Talamo, BR et al. (1989). Nature 337, 736-739.). Similarly, the present inventors have found that even if iRBC moves inside the blood vessel, these densely structured capillaries are not suitable for the attachment / occlusion of Plasmodium parasites. It means that these consecutive events can be the reason for the loss of olfaction.

This study identified factors that may be involved prior to BBB opening from OLF. One of the factors involved in BBB leakage can be high fever. As with the mouse sepsis model, it is speculated that thermoregulation should be present in the mouse malaria model, but currently no reaction resulting from fever in mouse malaria has been reported (Lamb, TJ et al. (2006). ) Expert RevMol Med 8, 1-22.). Furthermore, mouse brain malaria is thought to cause hypothermia, as opposed to human infection (Grau, GE et al. (1991). Eur J Immunol 21, 2265-2267.). By using a thermal camera, a recently developed but relatively simple technology (Aoshi and Ishii, in preparation for paper submission, see JP2012-231725) is the final manifestation of diseases such as heat loss and death. It was detected that another exothermic period occurred 24 hours before. Importantly, this fever period correlated with severe olfactory loss. Given that the circadian rhythm of mice precludes accurate measurement of fever at a single time point, it is not surprising that previous studies were only able to measure the final heat loss at the final stage of the disease. The inventors have shown that systemic and local cytokines and chemokine storms can cause high fever attacks in mice as well as in human brain malaria, and perhaps play a major role in the loss of BBB integrity It was concluded that Importantly, the absence of hyperthermia in Rag2 ^{− / −} mice and non-lethal and lethal Py infections suggests that fever is associated with ECM and may be associated with BBB leakage during brain malaria. The inventors' knowledge can be confirmed. However, the mediators that cause fever during ECM and its direct role in BBB disruption remain currently unknown. Scientific understanding of the mechanism of fever and its relevance to cytokineemia suggests that LPS is known to correlate well with mostly bacterial products such as LPS and fever with cytokines IL-1β and TNFα. (Netea, MG et al., (2000). Clin Infect Dis 31 Supp15, S178-184.). In contrast, there is no clear information and direct correlation available in mouse malaria that raises exactly the same cytokines and causes malarial fever. Clearly this area needs further consideration.

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.). 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. ( 2008) .J Immunol 180,1217-1230.), And in mice lacking chemokine / chemokine receptors, CCR1 and CCR2 were unnecessary for ECM pathogenesis, whereas CCR5 and CXCR3 Enhanced migration of CD8 T cells into the brain during malaria (Belnoue, E. et al. (2003). Infect Immun 71, 3648-3651 .; Belnoue, E. et al. (2003). Blood 101, 4253-4259 Hansen, DS (2012) PLo S Pathog 8, e1003045 .; Miu, J. et al. (2008). J Immunol 180, 1217-1230.). 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.). Similarly, the inventors have found that the proliferation and migration of effector CD8 T cells expressing CD11c are severely impaired in the spleen and brain in the absence of CCR7. However, from our detailed analysis with CD8α DC from Rag2 ^{− / −} mice, the proliferation of CD11c ⁺ CD8 T cells requires antigenic stimulation from CD8α DC in the spleen and the presence of CCR7 I came to the conclusion that I needed it. It has been suggested that CD8α DC can cross-present antigens, particularly antigens from dead cells, to T cells (Shortman, K. and Heath, WR (2010). Immunol Rev 234, 18- 31.). On the other hand, considering that Batf3 ^{− / −} mice can only partially (about 50%) escape ECM, during ECM, as a result of the combined action of BATF3, BATF and BATF2, in DC development Further consideration is necessary as to whether compensation occurs between members of the BATF family or between other DC types (Edelson, BT et al. (2011). Immunity 35,236. Murphy, TL et al. (2013). Nat Rev Immunol 13, 499-509.). Nevertheless, these results indicate that CD8α DC cross-priming of CD8 T cells during PbA infection requires CCR7 to induce the proliferation of effector CD11c ⁺ CD8 T cells, which these CD11c ⁺ CD8 T cells It has been shown to migrate into the brain and eventually cause ECM. However, activated 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. Although it is clear that 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. In support of our hypothesis, 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.). Furthermore, 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. In vitro transwell migration assays by the inventors support these hypotheses, but due to the limited effectiveness of anti-CCL21 antibody treatment at the onset of OLF dysfunction (day 4), in vivo ECM Compensation by other mechanisms that cause pathology during the effector phase may be suggested. Nonetheless, the inventors herein have described a novel therapeutic approach, CCL21 blocking, anti-CCL21 therapy, and a combination of CCR7-CCL21 axis and CXCR3-CCL21 axis blockade for ECM treatment. Demonstrate a “proof of concept” that can be developed as a new strategy. It should be noted that loss of olfaction can be a new early diagnostic marker for ECM symptoms and allow treatment of mice as early as 4 days after PbA infection.

In summary, this study shows that OLF is a “weakness” for many pathogens to enter the brain in both mice and monkeys, and therefore its complex capillary structure targets the Plasmodium parasite that causes ECM. Demonstrate that it can be. Mouse studies also concluded that immune cells such as pathogenic CD11c ⁺ CD8 T cells enter the brain via microbleeding in OLF. CCL21 in OLF GL early in infection can be one of the fundamental mechanisms of pathogenic CD11c ⁺ CD8 T cell accumulation, and CCL21 expression can be a risk factor for the development of ECM (FIG. 7). These results provide evidence that OLF dysfunction is a useful marker for the onset and early diagnosis of ECM. Currently, it is unclear whether these findings in mice are applicable to humans. Of particular note, the symptoms associated with OLF in humans, such as “Phantom Olfactory” (Perry, TL et al., (2009). Open Med 3, e10-16.) Can be different. What is needed in the future is a new test of olfactory loss and techniques such as improved human OLF MRI imaging (Wang, J. et al. (2011). AJNR Am J Neuroradiol 32, 677-681.) it is obvious. Furthermore, the location of OLF, early BBB leakage and bleeding has proven to be a useful therapeutic target for cerebral malaria and for various neuroimmunological diseases including autoimmune and inflammatory diseases in humans. (Barresi, M. et al. (2012). J Neurol Sci 323, 16-24.).

  As mentioned above, although this invention has been illustrated using preferable embodiment of this invention, it is understood that the scope of this invention should be construed only by the claims. Patents, patent applications, and references cited herein should be incorporated by reference in their entirety as if the contents themselves were specifically described herein. Understood.

  A diagnosis / detection marker for cerebral malaria and a control technique for cerebral malaria are provided, and a technique that can be used in industries (reagents, pharmaceuticals, etc.) related to the diagnosis, treatment, and prevention of cerebral malaria is 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 [Sequence Listing]

Claims (6)

A prophylactic or therapeutic agent for cerebral malaria comprising an inhibitor of at least two factors selected from the group consisting of CCL21, CXCR3 and CCR7, the inhibitor comprising an antibody or a fragment or functional equivalent thereof, siRNA, A prophylactic or therapeutic agent which is shRNA, antisense nucleic acid, aptamer, pharmaceutically acceptable salt or solvate thereof, or solvate of pharmaceutically acceptable salt thereof . The inhibitory Gaizai is, CCL21 protein, including inhibitors of factors selected from the group consisting of CXCR3 protein and CCR7 protein, prophylactic or therapeutic agent for cerebral malaria according to claim 1. 3. The brain malaria according to claim 1, wherein the inhibitor comprises an inhibitor of a factor selected from the group consisting of a nucleic acid encoding a CCL21 protein, a nucleic acid encoding a CXCR3 protein, and a nucleic acid encoding a CCR7 protein. Prophylactic or therapeutic agent. The agent for preventing or treating cerebral malaria according to any one of claims 1 to 3, wherein the inhibitor comprises an antibody. The prevention of cerebral malaria according to claim 1, wherein the inhibitor comprises 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. Or therapeutic agent. The agent for preventing or treating cerebral malaria according to claim 1, wherein the inhibitor comprises three types of anti-CCL21 antibody, anti-CXCR3 antibody and anti-CCR7 antibody, or fragments or functional equivalents thereof.