Opening agent for the nervous system vascular barrier

Abstract

To provide an opening agent for the nervous system vascular barrier. [Solution] A nerve system vascular barrier opening agent is prepared, comprising an antibody capable of binding to a polypeptide consisting of a specific amino acid sequence as an active ingredient. Preferably, the antibody capable of binding to the polypeptide consisting of the specific amino acid sequence is an anti-TRIM21 antibody capable of binding to the polypeptide consisting of the specific amino acid sequence.

Description

Technical Field

[0001] The present invention relates to an opening agent for a nervous system vascular barrier.

Background Art

[0002] In the brain of multicellular organisms including humans, the junctions between vascular endothelial cells form the Blood Brain Barrier (BBB) and blood-retinal barrier (BRB), and function as a checkpoint (tight junction) when various molecules in the blood move from blood vessels to the extravascular cell side. The aforementioned blood-brain barrier is an important mechanism that prevents unwanted substances from being taken up into the brain.

[0003] On the other hand, this nervous system vascular barrier acts as a barrier when it is desired to allow a drug to act on the extravascular cell side. Many attempts have been made to enable drugs to cross the nervous system vascular barrier and reach nervous tissue parenchyma. As one of such methods, there is a technique in which hypertonic mannitol is administered intravascularly, cerebral vascular endothelial cells are deformed by dehydration to physically dissociate the endothelial cells from each other, and then the target therapeutic drug is administered. However, this method causes cell damage and thus cannot be regarded as a useful technique. It is also disclosed that, regarding the blood-brain barrier, proteinases such as matrix metalloproteinases are associated with disruption of the blood-brain barrier (see Non-Patent Document 1).

[0004] In addition to these, despite many studies conducted to date, no useful method for allowing drugs to cross the nervous system vascular barrier and reach nervous tissue parenchyma has been established. Therefore, if a drug or method for artificially opening the closed nervous system vascular barrier with few side effects is developed, a major barrier to drug discovery against neurological diseases will be removed, which will lead to improvement of patient prognosis and further establishment of useful therapeutic methods for complete cure. Furthermore, drugs that open the nervous system vascular barrier are applicable to many intractable neurological diseases in common, and thus can be said to contribute to clinical medicine in an extremely wide range.

[0005] Clinical evidence has shown that blood-brain barrier (BBB) ​​disruption occurs in neuromyelitis optica (NMO)-related diseases. Regarding this blood-brain barrier disruption, anti-GRP78 antibody has been identified as an autoantibody that disrupts the blood-brain barrier in neuromyelitis optica (see Non-Patent Literature 2).

[0006] Incidentally, it has been disclosed that autoantibodies against TRIM21 (Tripartite motif-containing protein 21) are widely found in the serum of autoimmune diseases such as Sjögren's syndrome and systemic lupus erythematosus, and that they can be used for the diagnosis and prediction of the onset of interstitial pneumonia (see Patent Document 1). However, the relationship between TRIM21 and blood-brain barrier disruption has not been described. [Prior art documents] [Patent Documents]

[0007] [Patent Document 1] Japanese Patent Publication No. 2018-77194 [Non-patent literature]

[0008] [Non-Patent Document 1] Pengyu Pan et al., Neuroscience Letters 649(2017); 7-13 [Non-Patent Document 2] Fumitaka Shimizu et al., Sci Transl Med. 2017 July 05; 9(397) [Overview of the project] [Problems that the invention aims to solve]

[0009] The object of the present invention is to provide a novel opening agent for the nervous system vascular barrier. [Means for solving the problem]

[0010] Clinical evidence has shown that the blood-brain barrier opens in neuromyelitis optica-related diseases. The inventors identified GRP78 antibody as an autoantibody that opens the blood-brain barrier in neuromyelitis optica. Next, the inventors attempted to identify novel autoantibodies other than anti-GRP78 antibody that open the blood-brain barrier. As a result, they found that anti-TRIM21 antibody opens the blood-brain barrier, thus completing the present invention.

[0011] In other words, this disclosure is as follows: [1] A neurovascular barrier opener containing an antibody as an active ingredient that can bind to a polypeptide consisting of the amino acid sequence described in any of Sequence ID No. 1 to 3. [2] The neurovascular barrier opener according to [1], characterized in that the antibody capable of binding to a polypeptide consisting of the amino acid sequence described in any of SEQ ID NOs: 1 to 3 is an anti-TRIM21 antibody capable of binding to a polypeptide consisting of the amino acid sequence described in any of SEQ ID NOs: 1 to 3. [3] The anti-TRIM21 antibody is an anti-TRIM21 antibody capable of binding to a polypeptide having the amino acid sequence described in SEQ ID NO: 2, wherein the opening agent for the nervous system vascular barrier according to [2] above. [4] A neurovascular barrier opener according to any of [1] to [3] above, used to promote the passage of a therapeutic drug through the neurovascular barrier when the therapeutic drug for a central nervous system disease is administered to a patient. [5] A neurovascular barrier opener according to any of [1] to [4] above, for use in combination with or sequentially with a therapeutic agent for central nervous system diseases. [6] The neurovascular barrier opener according to [5] above, wherein the central nervous system disease is at least one selected from the group consisting of neurodegenerative diseases, cerebrovascular disorders, neuroimmunological diseases, retinal diseases, psychiatric disorders, central nervous system tumors, and epilepsy.

[0012] Furthermore, other embodiments of the present invention are as follows: (1) A method for delivering at least one therapeutic agent selected from the group consisting of neurodegenerative disease treatment agents, cerebrovascular disease treatment agents, neuroimmunological disease treatment agents, retinal disease treatment agents, psychiatric disease treatment agents, central nervous system tumor treatment agents, and epilepsy treatment agents to the brain of a target patient, comprising the step of administering to the patient, in a combination of a neurovascular barrier opener containing an antibody capable of binding to a polypeptide consisting of an amino acid sequence described in any of SEQ ID NOs: 1 to 3 as an active ingredient, and at least one therapeutic agent selected from the group consisting of neurodegenerative disease treatment agents, cerebrovascular disease treatment agents, neuroimmunological disease treatment agents, retinal disease treatment agents, psychiatric disease treatment agents, central nervous system tumor treatment agents, and epilepsy treatment agents, either simultaneously or sequentially. (2) Use of an antibody capable of binding to a polypeptide consisting of any of the amino acid sequences described in SEQ ID NOs: 1 to 3, for the production of an opener for the nervous system vascular barrier. [Effects of the Invention]

[0013] This disclosure makes it possible to open the neurovascular barrier, and when a therapeutic agent for a central nervous system disease is administered to a patient, it becomes possible to open the neurovascular barrier and facilitate the passage of the therapeutic agent through the neurovascular barrier. [Brief explanation of the drawing]

[0014] [Figure 1] Figure 1 shows the results of examining the expression of TRIM21 in human brain capillary endothelial cells in the example using immunostaining with an anti-TRIM21 antibody. [Figure 2] Figure 2 shows the results of examining TRIM21 expression in human brain capillary endothelial cells using Western blotting with an anti-TRIM21 antibody in the example. [Figure 3] Figure 3 shows the results of immunohistochemical staining to examine the binding of NMO patient IgG (NMO-IgG) to the TRIM21 protein. [Figure 4] Figure 4 shows the results of immunohistochemical staining to investigate the activation of NF-κB nuclear translocation in human brain capillary endothelial cells by anti-TRIM21 antibody in the example. [Figure 5] Figure 5 shows the results of examining the percentage (%) of cells in which NF-κB translocated to the nucleus (NF-κB nuclear positive cells) based on Figure 4 in the Examples. [Figure 6] Figure 6 shows the results of examining the effect of anti-TRIM21 antibody on 10 kDa dextran permeability in the Examples. [Figure 7] Figure 7 shows the results of examining the effect of anti-TRIM21 antibody on 150 kDa dextran permeability in the Examples. [Figure 8] Figure 8 shows the results of examining the effect of removal of TRIM21 antibody on 10 kDa dextran permeability in the Examples. MODE FOR CARRYING OUT THE INVENTION

[0015] The contents described in all patent documents and non-patent documents cited herein are incorporated by reference into the present specification in their entirety.

[0016] The agent for opening a nervous system vascular barrier of the present disclosure is not particularly limited as long as it is an agent for opening a nervous system vascular barrier that contains, as an active ingredient, an antibody capable of binding to a polypeptide consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 1 to 3, and is hereinafter also referred to as "the present agent for opening a nervous system vascular barrier".

[0017] Examples of the nervous system vascular barrier in the present specification include the blood-brain barrier and the blood-retinal barrier. The blood-brain barrier refers to a mechanism that restricts substance exchange between blood and brain tissue fluid. The blood-retinal barrier refers to a mechanism that restricts substance exchange between blood and retinal tissue fluid.

[0018] In this specification, "opening of the nervous system vascular barrier" means a state in which the mechanism that restricts the exchange of substances between the tissue fluid of the nervous system, such as the brain and retina, and the blood is not functioning or is impaired, and the exchange of substances between the tissue fluid of the nervous system, such as the brain and retina, and the blood becomes possible, at least temporarily. Here, the opening includes not only a state in which the nervous system vascular barrier remains open, but also an opening that, after a predetermined period of time, can be restored to a state in which the exchange of substances between the blood and the tissue fluid of the brain is restricted.

[0019] In this specification, "temporary" can refer to a time elapsed since administration of the neurovascular barrier opener of the present invention to a target, ranging from 0.2 to 24 hours, with a lower limit of 0.5 hours, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, or 6 hours, and an upper limit of 20 hours, 18 hours, 16 hours, 12 hours, 10 hours, or 9 hours. Specifically, this can refer to 0.5 to 20 hours, 1 to 18 hours, or 3 to 16 hours.

[0020] The neurovascular barrier opener described herein may be used to facilitate the passage of therapeutic drugs through the neurovascular barrier when a therapeutic drug for a central nervous system disorder is administered to a patient. The neurovascular barrier opener described herein can be administered to a patient concurrently or sequentially with a therapeutic drug for a central nervous system disorder. Therefore, the neurovascular barrier opener described herein is administered concurrently or sequentially to a patient receiving a therapeutic drug for a central nervous system disorder, together with the therapeutic drug for the central nervous system disorder. Here, the term "concurrent administration" means administering two or more drugs to the same subject at the same time. The term "sequential administration" means administering two or more drugs sequentially, i.e., sequentially or individually, at regular intervals, to the same subject.

[0021] When administered sequentially or individually at regular intervals, "regular intervals" can range from 0.5 minutes to 10 hours. Lower limits can include, for example, 1 minute, 2 minutes, 3 minutes, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, and 3 hours, while upper limits can include 8 hours, 6 hours, 5 hours, 4 hours, and 3 hours.

[0022] As a treatment for central nervous system disorders, at least one can be selected from the group consisting of neurodegenerative disease treatments, cerebrovascular disease treatments, neuroimmunological disease treatments, retinal disease treatments, psychiatric disorder treatments, central nervous system tumor treatments, and epilepsy treatments. Neurodegenerative diseases include Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, and spinocerebellar degeneration. Cerebrovascular disorders include cerebral infarction and vascular dementia. Neuroimmunological diseases include multiple sclerosis, autoimmune encephalitis, myasthenia gravis, sarcoidosis, and Behçet's syndrome. Retinal diseases include diabetic retinopathy, age-related macular degeneration, retinal edema, retinal detachment, proliferative vitreoretinopathy, uveitis, eye infections, retinopathy of prematurity, neovascular maculopathy, and chorioretinitis. Psychiatric disorders include depression, schizophrenia, and panic disorder. Examples of central nervous system tumors include gliomas, central nervous system lymphomas, glioblastoma multiforme, and gliosarcomas. Therapeutic agents for these tumors include small molecule compounds, nucleic acid drugs such as antisense oligonucleotides, ribozymes or proteins, polypeptides or peptides, and specifically antibodies against amyloid-beta, tau, alpha-synuclein, and TDP-43, which are pathogenic proteins of amyotrophic lateral sclerosis.

[0023] In one embodiment of the present invention, the opening of the nervous system vascular barrier by the opening agent lasts for 12 hours, 11 hours, 10 hours, 9 hours, 8 hours, 6 hours, 4 hours, 3 hours, 2 hours, 1 hour, 30 minutes, 15 minutes, 10 minutes, or 5 minutes.

[0024] The opening agent for the nervous system vascular barrier in this case only needs to contain an antibody capable of binding to a polypeptide consisting of the amino acid sequence described in any of SEQ ID NOs: 1 to 3 as an active ingredient, and may further contain pharmaceutically acceptable conventional carriers, binders, stabilizers, excipients, diluents, pH buffers, disintegrants, isotonic agents, additives, coatings, solubilizers, lubricants, gliding agents, solubilizers, flavoring agents, sweeteners, solvents, gelling agents, nutrients, and other formulations as needed. Specifically, such formulations include water, physiological saline, animal fats and oils, vegetable oils, lactose, starch, gelatin, crystalline cellulose, gum, talc, magnesium stearate, hydroxypropyl cellulose, polyalkylene glycol, polyvinyl alcohol, and glycerin.

[0025] The types of antibodies used herein may be any type, including human antibodies, chimeric antibodies, or humanized antibodies, as well as antibody fragments such as F(ab')2, Fab, diabody, Fv, ScFv, or Sc(Fv)2. Furthermore, these antibodies may be polyclonal or monoclonal. Chimeric antibodies or humanized antibodies can be produced genetically engineered according to conventional methods. Antibody fragments can be produced by methods such as digesting full-length antibodies with pepsin or papain.

[0026] Furthermore, the above antibodies may be labeled with a labeling substance. Such labeling substances include enzymes such as peroxidase (e.g., horseradish peroxidase), alkaline phosphatase, β-D-galactosidase, glucose oxidase, glucose-6-phosphate dehydrogenase, alcohol dehydrogenase, malate dehydrogenase, penicillinase, catalase, apoglucose oxidase, urease, luciferase or acetylcholinesterase; fluorescent substances such as fluorescein isothiocyanate, phycobiliprotein, rare earth metal chelate, dansil chloride or tetramethylrhodamine isothiocyanate; green fluorescent protein (GFP), cyan fluorescent protein (CFP), blue fluorescent protein (BFP), yellow fluorescent protein (YFP), and red fluorescent protein (Red Fluorescence Protein). Examples include fluorescent proteins such as proteins (RFP), luciferase, radioactive isotopes such as 3H, 14C, 125I, or 131I, metal colloids, nonmetal colloids, dye particles such as dye sols or disperse dyes, latex particles or colored microparticles, biotin, avidin, or chemiluminescent substances.

[0027] The polypeptides consisting of the amino acid sequences described in SEQ ID NOs: 1 to 3 are all partial sequences of the polypeptide of the human TRIM21 protein (NCBI reference sequence: NP_003132.2) consisting of the amino acid sequence described in SEQ ID NOs: 5. Specifically, the polypeptide consisting of the amino acid sequence described in SEQ ID NOs: 1 is the amino acid sequence from positions 463 to 475 of the polypeptide consisting of the amino acid sequence described in SEQ ID NOs: 5, the polypeptide consisting of the amino acid sequence described in SEQ ID NOs: 2 is the amino acid sequence from positions 425 to 475 of the polypeptide consisting of the amino acid sequence described in SEQ ID NOs: 5, and the polypeptide consisting of the amino acid sequence described in SEQ ID NOs: 3 is the amino acid sequence from positions 176 to 475 of the polypeptide consisting of the amino acid sequence described in SEQ ID NOs: 5. Here, TRIM21 is an E3 ubiquitin protein licase and is known to be involved in protein ubiquitination and proteasome degradation. This TRIM21 is sometimes called Ro52. Furthermore, from the viewpoint of permeating larger sized substances, it is preferable that the antibody is capable of binding to the polypeptide consisting of the amino acid sequence described in SEQ ID NOs: 2 or 3, and more preferably that it is capable of binding to the polypeptide consisting of the amino acid sequence described in SEQ ID NOs: 3.

[0028] The antibody capable of binding to a polypeptide consisting of the amino acid sequence described in any of SEQ ID NOs: 1 to 3 (hereinafter also referred to as "the antibody") is preferably an anti-TRIM21 antibody. Here, an anti-TRIM21 antibody capable of binding to a polypeptide consisting of the amino acid sequence described in any of SEQ ID NOs: 1 to 3 means an antibody capable of binding to a polypeptide consisting of the amino acid sequence described in any of SEQ ID NOs: 1 to 3 in TRIM21. Furthermore, the antibody is preferably an antibody that specifically binds to a polypeptide consisting of the amino acid sequence described in any of SEQ ID NOs: 1 to 3.

[0029] The antibody in question, when measured by surface plasmon resonance (SPR), has a mean dissociation constant (KD) of 10. -7 M or less, preferably 10 -8 M or less, more preferably 10 -9M or less, more preferably 10 -10 Examples of antibodies with a molecular weight of M or less that have affinity for polypeptides consisting of any of the amino acid sequences described in SEQ ID NOs: 1 to 3 can be listed.

[0030] The antibody in question may be a commercially available product, or it may be produced by immunizing mice with a polypeptide consisting of the amino acid sequence described in any of SEQ ID NOs: 1 to 3, or a protein containing such a polypeptide. Known methods can be used to determine whether the produced antibody binds to the polypeptide consisting of the amino acid sequence described in any of SEQ ID NOs: 1 to 3, such as ELISA, Western blotting, immunohistochemistry, and flow cytometry.

[0031] The method of administering the neurovascular barrier opening agent is not particularly limited as long as the desired neurovascular barrier opening effect of the present invention is obtained, and can include intravenous administration, oral administration, intravitreous administration, intramuscular administration, subcutaneous administration, transdermal administration, transnasal administration, transpulmonary administration, etc. Furthermore, the dosage of the neurovascular barrier opening agent is not particularly limited and can be appropriately adjusted depending on the recipient's physical condition, disease state, weight, age, sex, etc. As for the dosage, for example, it can be 0.01 μg to 100 g / kg body weight per day, more preferably 0.1 μg to 10 g / kg body weight, and even more preferably 1 μg to 1 g / kg body weight, and may be administered as a single dose or divided into multiple doses (for example, 2 to 4 times) per day.

[0032] There are no particular restrictions on the target population for administration of this neurovascular barrier opener, but mammals such as humans, monkeys, cattle, horses, sheep, pigs, dogs, cats, rats, mice, and hamsters can be mentioned. [Examples]

[0033] The present invention will be described more specifically below with reference to examples, but the technical scope of the present invention is not limited to these examples.

[0034] First, we mixed IgG from patients with neuromyelitis optica (NMO) disease and IgG from healthy individuals with human blood-brain barrier-derived cerebral microvascular endothelial cell line (TY-10) and used immunoprecipitation and proteomic analysis to identify candidate target molecules of autoantibodies in the patient samples. In this process, TRIM21 was identified as a candidate target molecule.

[0035] Therefore, we investigated the effects of anti-TRIM21 antibodies on human blood-brain barrier-derived brain microvascular endothelial cell lines.

[0036] [Example 1] Expression of TRIM21 on the cell surface Immunostaining was performed to confirm the expression of TRIM21 on the cell surface. TY-10, a strain of human brain microvascular endothelial cell (BMEC) immortalized with temperature-sensitive SV-40-LTA (Sano Y, Shimizu F et al., Establishment of a new conditionally immortalized human brain microvascular endothelial cell line retaining an in vivo blood-brain barrier function. J Cell Physiol.2010; 225:519-528.), was cultured in MCDB131 medium (Sigma-Aldrich) supplemented with EGM-2 SingleQuot Kit Supplements and Growth Factors (Lonza) and 20% fetal bovine serum (FBS). The cells were cultured at 33°C under a humidified atmosphere of 5% CO2 / 95% air. Analysis of the TY-10 strain was performed two days after raising the temperature to 37°C.

[0037] The obtained TY-10 strain was fixed by incubation with 4% paraformaldehyde at room temperature for 15 minutes, washed, and treated with 0.3% Triton X-100. After blocking with 5% goat serum, it was treated with anti-TRIM21 antibody (ab4369, 50-fold dilution: Abcam) as the primary antibody for 2 hours, and then labeled with anti-rabbit IgG (Alexa Fluor® 488 goat anti-rabbit IgG (H+L) A11008: 100-fold dilution: Life Technology) as the secondary antibody. The nuclei were stained with DAPI. Observation was performed using an LSM780 confocal microscope with ZEN software version 2010 (Carl Zeiss) or a Leica DM2500 microscope (Leica Microsystems). The results are shown in Figure 1.

[0038] Furthermore, TRIM21 expression in the entire cell or on the cell surface was examined by Western blotting. TM Using the Cell Surface Protein Isolation Kit (89881: Thermo Fisher Scientific), the surface proteins of the TY-10 strain were biotinylated and extracted to obtain the cell membrane fraction. Whole cells or cell membranes were separated by polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride (PVDF) membranes. After blocking with casein solution, anti-TRIM21 antibody (Anti TRIM21, Human (Rabbit) 12108-1-AP: Proteintech Group) was added and incubated overnight at 4°C. After washing, horseradish peroxidase (HRP)-labeled goat anti-human IgG was added and incubated at 25°C for 2 hours. After washing, protein bands were visualized using ImmunoStar LD (FUJIFILM Wako Chemicals) and analyzed using the Quantity One software program (Bio-Rad). The results are shown in Figure 2.

[0039] Figures 1 and 2 confirm that TRIM21 is expressed on the surface of cells in the TY-10 strain.

[0040] [Example 2] Binding of NMO patient IgG (NMO-IgG) to TRIM21 protein First, IgG derived from NMO patients was obtained using the Melon Gel IgG purification kit (Thermo Fisher Scientific). Next, TY-10 cell was incubated with either anti-TRIM21 antibody (ab4369, 50-fold dilution: Abcam) or IgG derived from NMO patients at a concentration of 250 μg / ml for 60 minutes, and the cells were fixed with 4% paraformaldehyde for 15 minutes. Subsequently, permalization was performed with 0.3% Triton-X for 15 minutes, followed by blocking (overnight blocking with 5% FBS in 0.3% Triton-X) for 15 minutes. Secondary antibodies (Goat anti-human IgG (H+L) Secondary Antibody, Alexa Fluor® 488 conjugate, A11013: Life Technology, Inc., and Rabbit anti-Goat IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 568 A-11079: Thermo Fisher Scientific, Inc.) were diluted 200-fold and incubated for 45 minutes. The samples were mounted with ProLong® Gold antifade reagent with DAPI (P36935: Life Technology, Inc.), and observed under a confocal microscope. The binding of the antibody to the protein was confirmed by the overlap of the color development. The results are shown in Figure 3.

[0041] As shown in Figure 3, when the dynamics of NMO-IgG were evaluated after being added to TY10 cells, it was confirmed that NMO-IgG binds to TRIM21.

[0042] [Example 3] Nuclear transfer of NF-κB NF-κB is known to be involved in increasing blood-brain barrier permeability by promoting the expression of cytokines and chemokines. Therefore, we investigated whether the anti-TRIM21 antibody activates NF-κB in the TY-10 strain.

[0043] TY-10 strain was cultured in 96-well plates (Greiner CELLSTAR® 96-well plates: Greiner) with 5000 cells per plate. Analysis of the TY-10 strain was performed two days after raising the temperature to 37°C. TY-10 strain was incubated for 1 hour with TNFα (1000 U / ml), anti-TRIM21 antibody Ab1 (Human TRIM21 / SS-A peptide ab23028: AbAcam), anti-TRIM21 antibody Ab2 (Human TRIM21 / SS-A antibody ab91423: AbAcam), anti-TRIM21 antibody Ab3 (Ag2753: Proteintech), anti-TRIM21 antibody Ab4 (52kDa Ro / SSa(D-12) sc-25351: Santa Cruz Biotechnology), mouse IgG (1:50 Life Technologies), and rabbit IgG (1:100 Santa Cruz Biotechnology). Next, the samples were fixed with paraformaldehyde and treated with NF-kB p65 rabbit monoclonal antibody (1:400 Cell Signaling Technology) as the primary antibody. Alexa Fluor 488 goat anti-rabbit IgG (Alexa Fluor® 488 goat anti-rabbit IgG) was used as the secondary antibody. Table 1 shows the region of the TRIM21 protein recognized by each anti-TRIM21 antibody and its amino acid sequence.

[0044] [Table 1]

[0045] Figure 4 shows the results of images taken from 96 plates using an In Cell Analyzer 2000 (GE Healthcare).

[0046] Furthermore, Figure 5 shows a graph obtained using the In Cell Analyzer software program (Cytiva) to determine the percentage (%) of cells in which NF-κB has migrated to the nucleus (NF-κB nucleus-positive cells), based on Figure 4. In Figure 5, the numbers on the horizontal axis represent the concentration of the added antibody (μg / ml).

[0047] Figures 4 and 5 confirm that among the anti-TRIM21 antibodies, anti-TRIM21 antibody Ab2, anti-TRIM21 antibody Ab3, and anti-TRIM21 antibody Ab4 promote the translocation of NF-κB to the nucleus in a concentration-dependent manner.

[0048] [Example 4] Permeability of substances As demonstrated in the above examples, the anti-TRIM21 antibody was able to activate NF-κB in the TY-10 strain. We investigated whether it affected tight junctions and thus impacted substance permeability in the TY-10 strain.

[0049] The monolayer TY-10 strain described above was cultured on a collagen-coated 24-well Transwell tissue culture insert (pore size 0.4 μm: Corning) at 33°C for 3 days, followed by 37°C for 3 days. Cell monolayers were exposed at 37°C for 24 hours to the following anti-TRIM21 antibodies on the luminal side: Ab1 (Human TRIM21 / SS-A peptide ab4369: Abakam), Ab2 (Human TRIM21 / SS-A antibody ab91423: Abakam), Ab3 (Ag2753: Proteintech), Ab4 (52kDa Ro / SSa(D-12) sc-25351: Santa Cruz Biotechnology), mouse IgG (1:50 Life Technologies), and rabbit IgG (1:100 Santa Cruz Biotechnology) (50 μg / ml). The luminal samples were then collected and replaced with an equal volume of fresh medium. Solute permeability was evaluated using FITC-dextran fluorescence. FITC-dextran fluorescence (10kDa or 15kDa: Sigma-Aldrich) was added to the luminal insert (final concentration, 1 mg / ml), and 5 μl of medium was collected from the luminal chamber over 40 minutes. Aliquots were diluted to 1 ml with PBS, and 100 μl of each was transferred to a 96-well black plate. The fluorescence signal was measured at 490 / 520 nm (absorption / emission) wavelengths using a SpectraMax M3e microplate reader (Molecular Devices). For IgG permeability, each rAb was incubated in the luminal chamber for 18 hours (concentration of each rAb was 50 μg / ml; 100 μl of conditioning medium containing the rAb was incubated in the luminal chamber, and 200 μl of PBS was added to the luminal chamber). Subsequently, PBS was collected from the intracavitary chamber, and the IgG concentration in the intracavitary chamber was measured using the Easy-Titer Human IgG (H+L) Assay Kit (Thermo Fisher Scientific). The results for 10kDa dextran are shown in Figure 6, and the results for 150kDa dextran are shown in Figure 7.

[0050] Figures 6 and 7 show that anti-TRIM21 antibodies Ab1-3 can enhance substance permeability at tight junctions, and in particular, anti-TRIM21 antibody Ab2 was confirmed to enhance permeability to such an extent that even large dextran molecules of 150 kDa could pass through. Furthermore, these results confirm that among anti-TRIM21 antibodies, antibodies that bind to the C-terminus of TRIM21 have a higher ability to enhance substance permeability.

[0051] [Example 5] Activity evaluation by removal of anti-TRIM21 antibody Samples were prepared by removing anti-TRIM21 antibodies from NMO-IgG by adding commercially available TRIM21 protein, and the effect of these samples on permeability was investigated.

[0052] NMO-IgG or healthy control IgG (HC-IgG) (75 μg [250 μg / ml]) derived from healthy individuals was mixed with 2 μg of recombinant protein (Human TRIM21 / RO52 Protein (Recombinant 6His,N-terminus) (Full Length)-LS-G23174). The mixture was incubated by rotation for 1 hour. Gel (EZview) TM 40 μl of Red HIS-Select® HC Nickel Affinity Gel E3528-1ML (Sigma-Aldrich) was added (the gel was washed twice with 500 μl of PBS before adding the gel), and the mixture was incubated by rotation for 1 hour. The mixture was then rotated at 10,000 rpm for 30 seconds, and only the supernatant was collected, leaving the gel intact.

[0053] The four types of samples obtained (NMO-IgG without removal of anti-TRIM21 antibody, NMO-IgG with removal of anti-TRIM21 antibody, HC-IgG without removal of anti-TRIM21 antibody, and HC-IgG with removal of anti-TRIM21 antibody) were applied to TY-10 cells, and changes in 10kDa permeability were observed. The results are shown in Figure 8.

[0054] Figure 8 shows that, under conditions where commercially available TRIM21 protein was used against NMO-IgG, the permeability-increasing effect was reduced by the removal of anti-TRIM21 antibody from NMO-IgG. Therefore, it was confirmed that anti-TRIM21 antibody has a permeability-increasing effect. [Industrial applicability]

[0055] It can be used in the field of drug therapy for conditions that require drug permeability through the nervous system's vascular barrier.

Claims

1. An opener for the nervous system vascular barrier, comprising an antibody as an active ingredient that can bind to a polypeptide consisting of the amino acid sequence described in any of SEQ ID NOs: 1 to 3.

2. The neurovascular barrier opening agent according to claim 1, characterized in that the antibody capable of binding to a polypeptide comprising the amino acid sequence described in any of SEQ ID NOs: 1 to 3 is an anti-TRIM21 antibody capable of binding to a polypeptide comprising the amino acid sequence described in any of SEQ ID NOs: 1 to 3.

3. The neurovascular barrier opening agent according to claim 2, wherein the anti-TRIM21 antibody is an anti-TRIM21 antibody capable of binding to a polypeptide having the amino acid sequence described in SEQ ID NO:

2.

4. An opener for the neurovascular barrier according to any one of claims 1 to 3, used to promote the passage of a therapeutic drug over the neurovascular barrier when the therapeutic drug for a central nervous system disease is administered to a patient.

5. An opener for the nervous system vascular barrier according to any one of claims 1 to 4, for use in combination with or sequentially administered with a therapeutic agent for central nervous system diseases.

6. The neurovascular barrier opening agent according to claim 5, wherein the central nervous system disease is at least one selected from the group consisting of neurodegenerative diseases, cerebrovascular disorders, neuroimmunological diseases, retinal diseases, psychiatric disorders, central nervous system tumors, and epilepsy.

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