WO2022168861A1 - 自己免疫疾患治療剤 - Google Patents

自己免疫疾患治療剤 Download PDF

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WO2022168861A1
WO2022168861A1 PCT/JP2022/004026 JP2022004026W WO2022168861A1 WO 2022168861 A1 WO2022168861 A1 WO 2022168861A1 JP 2022004026 W JP2022004026 W JP 2022004026W WO 2022168861 A1 WO2022168861 A1 WO 2022168861A1
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cells
nrp1
mice
amino acid
foxp3
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French (fr)
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伸司 大木
隆 山村
ベンジャミン ジョセフ エドワード レイバニー
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National Center of Neurology and Psychiatry
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/65—Peptidic linkers, binders or spacers, e.g. peptidic enzyme-labile linkers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • C12Q1/06—Quantitative determination
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor

Definitions

  • the present invention relates to a therapeutic agent for autoimmune diseases.
  • systemic lupus erythematosus is a typical systemic autoimmune disease, characterized by chronic inflammation with fever, malaise, joint pain, muscle pain, and fatigue.
  • the pathogenesis of SLE is believed to be related to the production of autoantibodies against a wide variety of endogenous antigens.
  • Most autoantibodies are high-affinity IgGs produced by autoreactive B cells that have undergone somatic hypermutation and class switching by interacting with autoreactive Th cells.
  • Tfh cells follicular helper T cells
  • GCs germinal centers
  • Tfh cells are identified by co-expression of PD-1, which is important for Tfh cell activation, and CXCR5, which forces Tfh cells to migrate to B-cell follicles in lymphoid tissue, and secrete interleukin-21 (IL-21).
  • autoreactive Th cells are envisioned as potential targets for SLE therapy, it is still difficult to distinguish between autoreactive and non-autoreactive Th cells.
  • BxSB-Yaa mice have a severe spontaneous SLE-like disease characterized by lymphoid hyperplasia, monocytosis, high antibody production including autoantibodies such as antinuclear antibodies (ANA), and immune complex-mediated glomerulonephritis. develop disease. Because this SLE-like disease is enhanced by the presence of the Yaa locus (a Y-linked autoimmune-promoting locus), pathogenic self-responses occur predominantly in male mice. IL-21 and Th cells, especially IL-21-producing ICOS-expressing Tfh cells, are believed to be important for the development of SLE-like disease in BXSB mice.
  • NR4A2 affects pathogenic responses to autoantigens in BxSB mice by regulating autoreactive Th cells.
  • T cells that escape central tolerance allow peripheral expression of autoantigen-specific T cell receptors (TcRs) that can become autoreactive antigens. So far, the study of T cells responding to autoantigens has been hampered by the inability to distinguish such autoreactive T cells from the useful T cell pool.
  • TcRs autoantigen-specific T cell receptors
  • an object of the present invention is to provide a therapeutic agent for autoimmune diseases that targets autoreactive Th cells.
  • the present invention provides the following [1] to [23].
  • [1] A polypeptide comprising an amino acid sequence capable of binding to NRP1 and an amino acid sequence capable of inducing apoptosis.
  • the polypeptide of [1], wherein the amino acid sequence capable of binding to NRP1 comprises the amino acid sequence shown in SEQ ID NO:1.
  • the polypeptide of [1] or [2], wherein the apoptosis-inducible amino acid sequence comprises the amino acid sequence shown in SEQ ID NO:2.
  • a therapeutic or preventive agent for autoimmune diseases comprising the polypeptide of any one of [1] to [3].
  • [5] A pharmaceutical composition containing the polypeptide of any one of [1] to [3].
  • a method for treating or preventing an autoimmune disease which comprises administering a polypeptide comprising an amino acid sequence capable of binding to NRP1 and an amino acid sequence capable of inducing apoptosis to a subject in need thereof.
  • the method for treating or preventing an autoimmune disease of [7] wherein the amino acid sequence capable of binding to NRP1 comprises the amino acid sequence shown in SEQ ID NO:1.
  • the method for treating or preventing an autoimmune disease of [7] or [8], wherein the apoptosis-inducible amino acid sequence comprises the amino acid sequence shown in SEQ ID NO:2.
  • [10] Use of a polypeptide comprising an amino acid sequence capable of binding to NRP1 and an amino acid sequence capable of inducing apoptosis for the treatment or prevention of autoimmune diseases.
  • Use of the polypeptide of [10] or [11], wherein the apoptosis-inducible amino acid sequence comprises the amino acid sequence shown in SEQ ID NO:2.
  • Markers for detecting autoimmune reactions including anti-NRP1 antibodies.
  • a diagnostic agent for autoimmune diseases comprising an anti-NRP1 antibody.
  • a composition for diagnosing an autoimmune disease comprising an anti-NRP1 antibody.
  • An autoimmune disease marker including NRP1.
  • a separation step of separating TcR ⁇ + CD4 + Th cells from collected blood, a measurement step of measuring the ratio of NRP1 + PD-1 + cells in the TcR ⁇ + CD4 + Th cells, and NRP1 obtained in the measurement step A method for determining the onset or progression of an autoimmune disease, comprising: comparing the proportion of + PD-1 + cells to a preset threshold, and determining progression of the autoimmune disease if it is higher than the threshold.
  • a separation step of separating TcR ⁇ + CD4 + Th cells from collected blood a measurement step of measuring the ratio of NRP1 + PD-1 + cells in the TcR ⁇ + CD4 + Th cells, and NRP1 obtained in the measurement step diagnosing the onset or progression of the autoimmune disease based on the proportion of + PD-1 + cells.
  • therapeutic agents for autoimmune diseases that target autoreactive Th cells can be provided.
  • conventional non-specific chemotherapy eg, steroids, immunosuppressants
  • cytokine-targeted chemotherapy eg, TNF, IL-6
  • T cell-targeted chemotherapy e.g., natalizumab, fingolimod
  • autoreactive T cells can specifically suppress immune responses while maintaining immune function against other antigens, resulting in more side effects.
  • a controlled treatment or prophylaxis can be provided.
  • (a) is a graph showing the percentage of CD45 + cells in control and NR4A2cKO mice, and the percentage of cells expressing CD11b, CD11c, Ly6G, Ly6C, and NK1.1 therein.
  • (b) is a graph showing the percentage of Ly6G ⁇ Ly6C + cells (top) and dendritic cells (bottom) in male and female control and NR4A2cKO mice.
  • (a) is a cytogram for FAS and GL-7 of CD19 + B cells from NR4A2cKO and control mice.
  • (b) is a graph showing the number of GL7 + GC B cells in NR4A2cKO mice and control mice (male and female).
  • (a) is a graph showing the percentage of GC and MZ B cells in B cells from NR4A2cKO and control mice.
  • (b) is a graph showing CD69 expression of CD19 + B cells and GL7 + GC B cells. Graph showing total IgG (upper left), IgGl (lower left), IgG2a (upper right) and IgG2b (lower right) levels in serum.
  • (a) is a graph comparing ANA levels in NR4A2cKO and control mice.
  • (b) is a graph showing the correlation between serum total IgG (upper left), IgG2a (upper right), ANA concentration (lower) and spleen weight.
  • (a) is a photograph showing the results of immunoblotting using control mouse serum on protein lysates extracted from 11 different tissues.
  • (b) is a photograph showing the results of immunoblotting using serum from NR4A2cKO and control mice on protein lysates extracted from mouse liver. It is a micrograph showing the stained area of each kidney section.
  • HE indicates hematoxylin and eosin staining
  • PAS indicates periodic acid/Schiff reagent staining
  • MTC indicates Masson's trichrome staining.
  • FIG. 2 is a graph showing the percentage of PD-1 + cells in .
  • Left cytogram of CD4 + T cells separated by PD-1 and CXCR5.
  • On the right is a graph showing a comparison of cell numbers of PD-1 ⁇ T cells, Tph cells and TfH cells.
  • (a) is a graph showing the number of Tph cells in each group of mice.
  • (b) is a graph showing the number of Tfh cells in each group of mice.
  • 1 is a graph showing the relationship between the percentage of Tph cells in splenic Th cells and immunoglobulins (IgG1, IgG2a, IgG2b) or ANA.
  • 1 is a graph showing the relationship between the ratio of Tfh cells in splenic Th cells and immunoglobulins (IgG1, IgG2a, IgG2b) or ANA. Graphs plotting the percentage of ANA, Tph cells and Tfh cells, IgG2a titers against the percentage of Th cell subsets in the blood.
  • (a) is a graph showing IL-21 expression in each subset.
  • (b) is a graph showing the expression level of the il21 gene in PD-1 ⁇ cells, Tph cells and Tfh cells.
  • (c) is a graph showing the relative expression level of each gene (bcl6, blimp1, maf).
  • FIG. 10 is a graph showing high- and low-affinity NP-specific IgG1 and IgG2a responses by NP-KLH immunization (NP(4) or NP(31)) and comparing the levels of the responses.
  • NP(4) or NP(31) Photomicrographs showing splenic sections after NP-KLH immunization in NR4A2cKO and control mice.
  • Fig. 3 is a graph showing fecal IgA levels.
  • (a) is a graph showing the Shannon index of CDR3 sequences.
  • (b) is a graph showing the 100 CDR3 sequences ranked by number of reads, and (c) shows the cumulative percentage of reads across all unique sequences ranked by the most common CDR3 sequences. is a graph.
  • (a) and (b) sort the unique TcR sequences in order of most common based on read number and calculate the percentage of self-promoting and self-restricting clones relative to the top 100 clones of each genotype. It is a graph showing the number and cumulative number of reads.
  • (a) is a plot showing the percentage of self-promoting and self-limiting clones for the top 50 clones of each genotype.
  • (b) is a heatmap showing the number of reads for the top 25, 50, 100 and 200 clones of the most common sequences.
  • (a) is a cytogram showing the expression of NRP1 and PD-1 in Foxp3 ⁇ CD4 + Th cells.
  • (b) is a graph showing the proportion of NRP1/PD-1-expressing Th cells in Foxp3 ⁇ Th cells.
  • (a) is a graph showing the percentage of NRP1 + PD-1 + cells in TcR ⁇ + CD4 + Foxp3 + cells.
  • (b) is a graph showing the ratio of NRP1 + subsets in Foxp3 ⁇ Th cells and Foxp3 + Th cells of each autoimmune disease model mouse.
  • (a) is a cytogram showing Foxp3 ⁇ CD4 + Th cells and Foxp3 + CD4 + Th cells in C57BL/6 background mice.
  • (b) is a graph showing the number of Th cells expressing NRP1 and/or PD-1 in each of Foxp3 + CD4 + Th cells and Foxp3 ⁇ CD4 + Th cells. Graphs plotting the percentage of Foxp3 ⁇ NRP1 + PD-1 + cells on the vertical axis versus spleen weight (top), serum total IgG (middle) or autoantibody ANA (bottom) on the horizontal axis.
  • (a) is a graph showing the cumulative percentage of total TCR sequence reads to common unique sequences and (b) is a graph showing the Shannon index.
  • (c) is a graph showing the number of promoting reads in NRP1/PD-1 expressing Th cells.
  • (b) is a graph quantifying the percentage of PD-1 and GITR in Foxp3 ⁇ CD4 + T cells. Cytogram for PD-1 and GITR in C57BL/6 background mice.
  • (a) is a graph showing the percentage of Foxp3 + and Foxp3 ⁇ cells among CD4 + T cells.
  • (b) is a graph showing the percentage of PD-1 + GITR lo cells and PD-1 ⁇ GITR hi cells. Graph showing mean fluorescence intensity (MFI) of CD5 expression of each T cell.
  • (a) is a graph showing the ratio of Tph cells or Tfh cells in Th cells.
  • (b) is a graph showing the expression of Foxp3 and GITR in CD4 + Th cells.
  • (a) is a graph showing the number of dividing PD-1 + Th cells by PD-L1 treatment.
  • (b) is a graph showing photographs and weights of spleens removed from the RPA peptide-administered group and the control peptide-administered group; It is a photograph.
  • FIG. 1 is a graph showing serum total IgG and ANA concentrations in an RPA peptide-administered group and a control peptide-administered group.
  • (a) is a cytogram of an RPA peptide-administered group and a control peptide-administered group.
  • (b) is a graph quantifying the percentage of NRP1 + PD-1 + Th cells.
  • (c) is a graph showing the ratio of Tph and Tfh in TcR ⁇ + CD4 + Th cells.
  • (a) is a graph showing the ratio of Foxp3 + cells in TcR ⁇ + CD4 + Th cells in the RPA peptide administration group and the control peptide administration group, and the expression of NRP1 and PD-1 in TcR ⁇ + CD4 + Foxp3 + Th cells. It is a graph showing.
  • (b)-(d) are graphs showing spleen weights, serum total IgG levels and ANA levels, respectively.
  • (e) is a graph showing the percentage of NRP1 ⁇ PD-1 ⁇ cells and NRP1 + PD-1 + Th cells among Foxp3 ⁇ CD4 + cells.
  • (a) is a graph showing changes over time in the body weight of MRL-lpr mice in the RPA peptide-administered group and the control group.
  • (b) is a graph showing spleen weights and
  • (c) is a graph showing serum IgG and anti-dsDNA antibody levels. Cytograms (top) and quantified graphs (bottom) for NRP1/PD-1 in MRL-lpr mice.
  • (a) is a cytogram of CD4 + CD3 + Th cells from the peripheral blood of SLE patients.
  • (b) is a graph showing the ratio of Nrp-1 positive cells to Th cells in peripheral blood derived from healthy subjects or SLE patients.
  • (c) is a graph showing the ratio of Nrp-1-positive cells to CD127-positive Th cells in peripheral blood derived from healthy subjects or SLE patients.
  • a first embodiment of the present invention is a polypeptide comprising an amino acid sequence capable of binding to NRP1 and an amino acid sequence capable of inducing apoptosis.
  • an amino acid sequence capable of binding to NRP1 and an amino acid sequence capable of inducing apoptosis may be bound via a linker sequence, and the form thereof may be linear, part or all of which may be A ring may be formed.
  • the polypeptide may also contain D amino acids among its constituent amino acids.
  • the polypeptide can bind to NRP1-expressing T cells via an amino acid sequence capable of binding to NRP1 and induce apoptosis of the T cells.
  • polypeptide is a polymer formed by peptide bonds of multiple amino acids, and is also referred to as a protein.
  • the polypeptide is not particularly limited as long as it contains an amino acid sequence capable of binding to NRP1 and an amino acid sequence capable of inducing apoptosis.
  • the polypeptide according to this embodiment is composed of 10 or more amino acid residues, preferably a polypeptide composed of 10 to 200 amino acid residues, 10 to 150, 0 to 100, 10 ⁇ 90, 10-80, 10-70, 10-60, 10-50, 10-45, 10-40, 10-35, 10-30, 10-25, 12 More preferred are polypeptides composed of ⁇ 30, 12-25, 14-30, or 14-25 amino acid residues.
  • the molecular weight of the polypeptide according to this embodiment may be 750 daltons or more, 800 daltons or more, 900 daltons or more, 1000 daltons or more, 1100 daltons or more, 1200 daltons or more, 1300 daltons or more, 1400 daltons or more, or 1500 Daltons or more are preferred.
  • the molecular weight of the polypeptide according to this embodiment may be 10000 daltons or less, 9000 daltons or less, 8000 daltons or less, 7000 daltons or less, 6500 daltons or less, 6000 daltons or less, 5500 daltons or less, 5000 daltons or less, 4500 daltons or less. , 4000 Daltons or less, 3500 Daltons or less, or 3000 Daltons or less.
  • NRP1 neurotrophilin 1, also called CD304
  • CD304 is a transmembrane protein with a molecular weight of about 130 kDa, and acts as a VEGF-A receptor in vascular endothelial cells, tumor cells (e.g., non-small cell lung cancer, glioblastoma, colorectal cancer). cancer, prostate cancer, ovarian cancer).
  • tumor cells e.g., non-small cell lung cancer, glioblastoma, colorectal cancer. cancer, prostate cancer, ovarian cancer.
  • NRP1 enhances the kinase activity of VEGF2 when bound to VEGF-A, promoting angiogenesis, angiogenesis, and vascular hyperpermeability.
  • the amino acid sequence capable of binding to NRP1 is not particularly limited as long as it is an amino acid sequence capable of binding to NRP1.
  • the amino acid sequence capable of binding to NRP1 may be, for example, an antigen-binding fragment of an anti-NRP1 antibody.
  • the antigen-binding fragment may be any antibody fragment containing the antigen-binding site of the anti-NRP1 antibody, and examples thereof include Fab, Fab', F(ab')2, scFv, diabodies and the like.
  • Amino acid sequences capable of binding to NRP1 are preferably RPARPAR (SEQ ID NO: 1), CNGRCGG (SEQ ID NO: 2), WIFPWIQL (SEQ ID NO: 3), CGRDKRLYDC (SEQ ID NO: 4), CRGDKGPDC (SEQ ID NO: 5), CRGDKRLYDC (SEQ ID NO: 5) 6), RLLRLLRLLR (SEQ ID NO: 7), CRGDKGG (SEQ ID NO: 8), RGDRGDRLLR (SEQ ID NO: 9).
  • the amino acid sequence capable of binding to NRP1 may be the anti-NRP1 antibody itself.
  • the polypeptide according to this embodiment is an aspect in which an anti-NRP1 antibody and an amino acid sequence capable of inducing apoptosis are bound via a linker sequence or not.
  • the position to which the apoptosis-inducible amino acid sequence binds may be a position that does not inhibit antigen binding of the anti-NRP1 antibody.
  • the anti-NRP1 antibody may be a monoclonal antibody or a polyclonal antibody.
  • Anti-NRP1 antibodies may be mouse, rat, guinea pig, hamster, rabbit, monkey, dog, chimeric, humanized or human antibodies.
  • the anti-NRP1 antibody may be chemically modified in order to improve physical properties such as blood retention.
  • anti-NRP1 antibodies may be conjugated with radionuclides, toxins, etc., in order to enhance therapeutic effects.
  • the polypeptide according to this embodiment is composed of 1000 or more amino acid residues, and is composed of 1000 to 4000 amino acid residues.
  • Peptides are preferred, 1000-3900, 1000-3800, 1000-3700, 1000-3600, 1000-3500, 1000-3400, 1000-3300, 1000-3200, 1000-3100, 1000 More preferred are polypeptides composed of ⁇ 3000, 1000-2900, 1000-2800, 1000-2700, 1200-2700, 1200-2700, or 1200-2700 amino acid residues.
  • the molecular weight of the polypeptide according to this embodiment may be 10,000 Daltons or more, 20,000 Daltons or more, 30,000 Daltons or more, 40,000 Daltons or more, 50,000 Daltons or more, 60,000 Daltons or more, or 70,000 Daltons. Above, 80,000 daltons or more, or 90,000 daltons or more are preferable.
  • the molecular weight of the polypeptide according to this embodiment may be 300,000 daltons or less, 280,000 daltons or less, 260,000 daltons or less, 240,000 daltons or less, 220,000 daltons or less, 200,000 daltons or less, 190,000 daltons or less, 180,000 daltons or less, 170,000 daltons or less, 160,000 daltons or less, 150,000 daltons or less, or 140,000 daltons or less are preferable.
  • Anti-NRP1 antibodies include, for example, EPR3113 (ab81321) (trade name, manufactured by Abcam), neuropilin 1 antibody 60067-1-Ig (trade name, manufactured by Proteintech Japan), anti-NRP1 antibody (trade name, Funakoshi company).
  • the amino acid sequence capable of inducing apoptosis is not particularly limited as long as it is an amino acid sequence capable of inducing cell apoptosis.
  • the apoptosis-inducible amino acid sequence may be, for example, KLLNLISKLF (SEQ ID NO: 10), KLAKLAK (SEQ ID NO: 11), or repeats thereof.
  • the amino acid sequence capable of inducing apoptosis is preferably KLAKLAKKLAKLAK (SEQ ID NO: 12).
  • the apoptosis-inducible amino acid sequence contains at least one D-amino acid. At least one amino acid in the above amino acid sequences may be a D-amino acid.
  • a more preferred amino acid sequence capable of inducing apoptosis is KLAKLAKKLAKLAK (both of which are D amino acids, that is, D-Lys-D-Leu-D-Ala-D-Lys-D-Leu-D-Ala-D-Lys- D-Lys-D-Leu-D-Ala-D-Lys-D-Leu-D-Ala-D-Lys-D-Leu-D-Ala-D-Lys).
  • an amino acid sequence capable of binding to NRP1 and an amino acid sequence capable of inducing apoptosis may be bound via a linker sequence.
  • the linker sequence may be an amino acid sequence that does not suppress or inhibit NRP1-binding activity and apoptosis-inducing activity, and is, for example, an amino acid sequence composed of 1 to 20 amino acids. Specific examples of linker sequences include GG and CGG.
  • polypeptides according to this embodiment are shown in Table 1.
  • a preferred polypeptide has an amino acid sequence in the order from the N-terminal side, an amino acid sequence capable of binding to NRP1 and an amino acid sequence capable of inducing apoptosis.
  • a most preferred polypeptide is RPARPAR-CGG- D (KLAKLAK) 2 , in which KLAKLAK is composed of D amino acids.
  • a first embodiment is a method for treating or preventing a central demyelinating disease, comprising administering a polypeptide comprising an amino acid sequence capable of binding to NRP1 and an amino acid sequence capable of inducing apoptosis to a human subject in need thereof. It also has the aspect of
  • a second embodiment of the present invention is a therapeutic or preventive agent for autoimmune diseases containing the polypeptide according to the first embodiment.
  • the content of the polypeptide in the therapeutic or preventive agent for autoimmune diseases according to the second embodiment is not particularly limited, and is, for example, 0.001 to 100 based on the total amount of the therapeutic or preventive agent for autoimmune diseases. % by mass.
  • the therapeutic or prophylactic agent for autoimmune diseases according to the second embodiment may be composed only of the polypeptide according to the first embodiment, and in addition to the above polypeptides, excipients commonly used in the technical field of formulations, Additives such as buffers, stabilizers, antioxidants, binders, disintegrants, fillers, emulsifiers and flow additive modifiers may be included.
  • the therapeutic or preventive agent for autoimmune diseases according to the second embodiment may be administered orally or parenterally.
  • a specific dosage for example, when administered to an adult male human (body weight 60 kg), the daily dosage of the therapeutic or preventive agent for central demyelinating diseases is usually, in terms of the amount of active ingredient, 0.0001 ⁇ g to 10000 mg/day/person.
  • the autoimmune disease therapeutic or preventive agent according to this embodiment may be administered alone, and existing autoimmune disease therapeutic agents (e.g., steroids, immunosuppressants, TNF, IL-6, natalizumab, fingolimod) may be administered in combination with existing autoimmune disease therapeutic agents (e.g., steroids, immunosuppressants, TNF, IL-6, natalizumab, fingolimod) may be administered in combination with existing autoimmune disease therapeutic agents (e.g., steroids, immunosuppressants, TNF, IL-6, natalizumab, fingolimod) may be administered in combination with
  • the second embodiment also has an aspect of a method for treating or preventing an autoimmune disease, which comprises administering a composition containing the above polypeptide to a human subject in need thereof.
  • the second embodiment also has an aspect of using a polypeptide comprising an amino acid sequence capable of binding to NRP1 and an amino acid sequence capable of inducing apoptosis for the treatment or prevention of autoimmune diseases.
  • a third embodiment of the invention is a marker for detecting an autoimmune reaction comprising an anti-NRP1 antibody.
  • Preferred markers include anti-NRP1 antibodies and anti-PD-1 antibodies.
  • T cells that exhibit autoimmune reactions express NRP1 on their cell surfaces.
  • the anti-NRP1 antibody as a marker, only autoimmune reactions can be detected, distinguishing them from other immune reactions.
  • T cells exhibiting an autoimmune reaction co-express NRP1 and PD-1, the combination of anti-NRP1 antibody and anti-PD-1 antibody enables more accurate detection. .
  • the marker according to the present embodiment has an aspect of being a diagnostic agent for autoimmune diseases containing anti-NRP1 antibodies.
  • an immunoassay method using an anti-NRP1 antibody By applying an immunoassay method using an anti-NRP1 antibody to the collected blood, it is possible to determine whether the Th cells in the blood of the subject express NRP1 on their cell surfaces.
  • anti-NRP1 antibody and anti-PD-1 antibody in combination By using anti-NRP1 antibody and anti-PD-1 antibody in combination, more accurate detection becomes possible.
  • This embodiment also has an aspect of an autoimmune disease diagnostic agent containing an anti-NRP1 antibody.
  • the autoimmune disease diagnostic agent may further contain stabilizers, solvents and the like.
  • the content of the anti-NRP1 antibody may be 0.1 to 100% by mass with respect to the total mass of the autoimmune disease diagnostic agent.
  • a fourth embodiment of the present invention comprises a separation step of separating TcR ⁇ + CD4 + Th cells from collected blood, a measurement step of measuring the ratio of NRP1 + PD-1 + cells in the TcR ⁇ + CD4 + Th cells, A determination step of comparing the proportion of NRP1 + PD-1 + cells obtained in the measurement step with a preset threshold value, and determining the onset or progression of the autoimmune disease if it is higher than the threshold value. It is a judgment method.
  • the blood used for determination can be blood collected from a patient (human or animal) suspected of developing or progressing an autoimmune disease.
  • the blood may be previously collected and stored blood.
  • the separation step is a step of separating TcR ⁇ + CD4 + Th cells from blood collected from humans or animals. Separation means can use methods well known to those skilled in the art. For example, collected blood is subjected to Ficoll density gradient centrifugation to isolate peripheral blood mononuclear cells (PBMC) and prepare a single cell suspension.
  • PBMC peripheral blood mononuclear cells
  • the measuring step is to measure the proportion of NRP1 + PD-1 + cells in TcR ⁇ + CD4 + Th cells.
  • the measuring step can apply means known to those skilled in the art, such as flow cytometry.
  • a primary antibody anti-NRP1 antibody, anti-PD-1 antibody, etc.
  • a fluorescently labeled secondary antibody that binds to the primary antibody.
  • they are subjected to a flow cytometer to generate cytograms for NRP1 and PD-1.
  • the results are also used to calculate the percentage of NRP1 + PD-1 + cells in Th cells.
  • the ratio of NRP1 + PD-1 + cells obtained in the measurement step is compared with a preset threshold value, and if the ratio is higher than the threshold value, it is determined that an autoimmune disease has developed. Since T cells in which NRP1 and PD-1 are co-expressed show a positive correlation with autoimmune reactions, it is possible to distinguish them from other immune diseases and determine whether or not they exhibit autoimmune reactions.
  • the threshold value may be a value calculated from blood collected from the subject himself/herself before the onset or progression of autoimmune disease is suspected, or an average value calculated from multiple patients (human or animal) of the same age. may When the threshold value is a value calculated from the blood sampled from the subject himself/herself, the degree of progress of the autoimmune disease can also be determined.
  • a fifth embodiment of the present invention comprises a separation step of separating TcR ⁇ + CD4 + Th cells from collected blood, a measurement step of measuring the proportion of NRP1 + PD-1 + cells in the TcR ⁇ + CD4 + Th cells, a diagnostic step of diagnosing the onset or progression of the autoimmune disease based on the ratio of NRP1 + PD-1 + cells obtained in the measuring step.
  • the diagnosis step refer to the ratio of NRP1 + PD-1 + cells obtained in the measurement step, and diagnose the autoimmune disease based on the measurement results of other autoimmune disease-related parameters, the knowledge and / or experience of the doctor.
  • NRP1 can be used as a diagnostic marker for detecting autoimmune diseases.
  • NR4A2cKO BxSB mice BxSB mice develop spontaneous SLE-like disease due to responses to a range of self-antigens. Therefore, this model may be more representative of the natural course of systemic autoimmune disease than many artificial models of autoimmunity directed against a single antigen. The disease is manifested by extreme lymphocytosis, hypertrophy of secondary lymphoid organs, and high levels of pathogenic autoreactive antibodies.
  • the development of systemic autoimmunity in BxSB mice requires IL-21 produced by Th cells to influence the differentiation of autoreactive B cells.
  • NR4A2 regulates IL-21 secretion by Th cells in models of organ-specific autoimmunity.
  • NR4A2 also plays an essential role in IL-21-dependent systemic autoimmune diseases
  • BxSB mice with NR4A2 gene deletion in T cells were generated.
  • T-specific NR4A2-deficient (Cre-CD4 +/- NR4A2 fl/fl BxSB, NR4A2cKO) and their littermates were generated by backcrossing CD4-CreTg /+ NR4A2 fl/fl mice (see Non-Patent Document 1) to the BxSB background.
  • BxSB mice with both offspring (NR4A2 fl/fl BxSB, control mice) were obtained.
  • Control mice developed severe splenomegaly by 16-20 weeks of age, similar to wild-type BxSB mice, whereas NR4A2cKO littermates were significantly protected from lymphatic expansion.
  • Cre-CD4 NR4A2 fl/fl mice backcrossed with BxSB-Yaa mice were maintained under SPF conditions until 8, 16, 20 weeks of age.
  • Spleens from co-housed littermate NR4A2 fl/fl BxSB (control) and Cre-CD4NR4A2 fl/fl BxSB (NR4A2cKO) mice were harvested. Spleens were frozen at ⁇ 80° C. in OTC and then sectioned in a Microm-HM-525 cryostat. Tissue sections were fixed in acetone for 5 minutes and blocked with 3% BSA/PBS.
  • Sections were stained overnight with conjugated antibodies (FITC-conjugated anti-B220 or anti-GL7), biotinylated anti-CD4 (all from Biolegend) and secondary staining with AlexaFluor-594 conjugated streptavidin (Thermo Life Technologies). dyed. Sections were mounted with Fluoromount (Southern Biotech) and photographed with a fluorescence BZ-X850 microscope equipped with BZ-X software (Keyence). Spleen weights and total cell counts were then determined.
  • conjugated antibodies FITC-conjugated anti-B220 or anti-GL7
  • biotinylated anti-CD4 all from Biolegend
  • AlexaFluor-594 conjugated streptavidin Thermo Life Technologies
  • Fig. 1(a) is a micrograph of the NR4A2cKO mouse spleen.
  • Male mice lacking NR4A2 also lack the Yaa locus and maintain normal spleen weight and cell status similar to age-matched disease-free female mice (2-3).
  • Inguinal lymph nodes of 16-week-old male NR4A2cKO BxSB mice and control mice were also imaged. Spleens of male and female mice were mechanically disrupted through a 70 mm cell strainer (BDBiosciences) and single cell suspensions were made by lysing red blood cells with ACK lysing solution. Cell suspensions were counted by flow cytometry. Male mouse non-T and B cells were then further fractionated by staining with CD11b, CD11c, Ly6G, Ly6C, and NK1.1. CD45 + CD11b + Ly6G ⁇ Ly6C + cells (Ly6G ⁇ Ly6C + cells) and CD45 + CD11c + cells (dendritic cells) were also evaluated using male and female mice.
  • FIG. 1(b) is a photomicrograph showing inguinal lymph nodes of NR4A2cKO BxSB mice and control mice. Control mice had increased numbers of various major immune cell populations including T cells, B cells and monocytes. In NR4A2cKO mice, on the other hand, the increase was significantly suppressed and cell populations remained at pre-disease levels and similar to female mice.
  • FIG. 3 is a graph showing cell numbers of TcR ⁇ + T cells, CD19 + B cells, and scatter-gated CD45 + CD19 ⁇ TcR ⁇ NK1.1 ⁇ monocytes.
  • FIG. 1(b) is a photomicrograph showing inguinal lymph nodes of NR4A2cKO BxSB mice and control mice. Control mice had increased numbers of various major immune cell populations including T cells, B cells and monocytes. In NR4A2cKO mice, on the other hand, the increase was significantly suppressed and cell populations remained at pre-disease levels and similar to female mice.
  • FIG. 4(a) is a graph showing the percentage of CD11b, CD11c, Ly6G, Ly6C, and NK1.1 expressing cells in CD45 + cells from control and NR4A2cKO mice.
  • FIG. 4(b) is a graph showing the percentage of Ly6G ⁇ Ly6C + cells and dendritic cells in male and female control and NR4A2cKO mice.
  • GC B cells splenic germinal center B cells
  • MZ marginal zone
  • FIG. 5(a) is a cytogram for FAS and GL-7 of CD19 + B cells from NR4A2cKO and control mice.
  • NR4A2cKO mice had increased FAS ⁇ GL7 + B cells but decreased FAS + GL7 + B cells compared to control mice.
  • Male NR4A2cKO mice had significantly reduced numbers of GL7 + B cells.
  • Figures 6(a) and (b) are graphs showing the percentage of GC B cells and MZ B cells in B cells from NR4A2cKO mice and control mice.
  • NR4A2cKO mice maintained pre-disease B-cell proportions and exhibited significantly lower levels of B-cell activation than diseased mice, as indicated by CD69 levels (Fig. 6).
  • NR4A2 deficiency is T-cell specific, the differences observed in non-T cells suggest that the expansion of these populations is due to T-cell-associated changes.
  • IgG Levels and Antinuclear Antibody Levels in NR4A2cKO Mice Spleens and sera were prepared from 16-week-old male and female control or NR4A2cKO mice. Spleens were weighed and serum antibody levels were determined by mouse IgG1, IgG2a and IgG2b Ready-set-Go! An ELISA kit (eBioscience) was used according to the manufacturer's instructions and measured using a standard ELISA with a secondary antibody. Serum prepared from 8-, 13-, 16-, and 20-week-old male and female mice was also evaluated for IgG1, IgG2a, IgG2b, and antinuclear antibody (ANA) levels by the same method.
  • ANA antinuclear antibody
  • FIG. 7 is a graph showing total IgG, IgG1, IgG2a and IgG2b levels in serum.
  • Serum IgG levels in male control mice were elevated after disease onset, but no corresponding antibody response was seen in NR4A2cKO mice or female littermates.
  • Most of the NR4A2-dependent increase in serum IgG levels reflected increases in IgG2a and IgG2b, while IgG1 levels remained low.
  • ANA is the major pathogenic effector autoantibody in SLE and increased with age in male control but not NR4A2cKO mice, as shown in Figure 8(a).
  • the serum total IgG concentration, serum IgG2a concentration, and serum ANA concentration of 16-week-old mice were compared with spleen weight, and Spearman's R correlation test was performed. showed a significant correlation.
  • the solid line indicates the results of regression analysis, and the dotted line indicates the 95% confidence interval.
  • FIG. 9(a) is a photograph showing the results of immunoblotting for protein lysates extracted from 11 different tissues. Serum IgG from control mice was found to be highly reactive to multiple protein extracts, particularly from liver. FIG. 9(b) is a photograph showing the binding of serum antibodies to protein lysates extracted from mouse liver. Serum IgG from NR4A2cKO mice or control mice (females) showed little reaction to liver-derived proteins.
  • kidneys were removed from NR4A2cKO mice and control mice (24-week-old, male), and sections were prepared. The kidney sections obtained were stained with hematoxylin & eosin (HE), periodic acid/Schiff's reagent (PAS) or Masson's trichrome (MTC) and observed under a microscope.
  • HE hematoxylin & eosin
  • PAS periodic acid/Schiff's reagent
  • MTC Masson's trichrome
  • FIG. 10 is a micrograph showing the stained area of each kidney section (scale bar: 100 ⁇ m). Multiple pathologies were observed in the kidneys of control mice (aged, male), including glomerular basement membrane hyperplasia, intense perivascularitis, focal glomerular fibrosis, and glycoprotein deposition, whereas NR4A2cKO mice (litter pups), these pathologies were milder.
  • CD4 + Th cells ie, TcR ⁇ + CD4 + CD8 ⁇ cells
  • CD8 + Tc cells ie, TcR ⁇ + CD4 ⁇ CD8 + cells
  • CD4 + T cells ie, TcR ⁇ + CD4 + cells
  • FIG. 11(a) is a cytogram of CD4 + T cells separated by PD-1 and ICOS
  • FIG. 12 left is a cytogram of CD4 + T cells separated by PD-1 and CXCR5.
  • PD-1 ⁇ CXCR5 ⁇ T cells are referred to as “PD-1 ⁇ T cells”, PD-1 + CXCR5 ⁇ T cells as “Tph cells”, and PD-1 + CXCR5 + T cells as “TfH cells”.
  • Tph cells are known to promote autoantibody production in mouse models of rheumatoid arthritis and in human SLE.
  • the right side of FIG. 12 is a graph showing a comparison of the numbers of PD-1 ⁇ T cells, Tph cells, and TfH cells (error bars: SEM, **P ⁇ 0.01, *P ⁇ 0.05).
  • Control mice (16-week-old, male) showed a marked increase in the number of Tph and TfH cells, whereas NR4A2cKO mice showed a mild increase.
  • NR4A2cKO mice showed a mild increase.
  • a relative decrease in PD-1 ⁇ T cells was observed with an increase in the ratio of Tph cells and TfH cells.
  • ELISA kit (eBioscience) was used according to the manufacturer's instructions and measured using a standard ELISA with a secondary antibody. Antinuclear antibodies were measured using an ANA ELISA kit (US Biological) according to the manufacturer's instructions. Serum IgG1, IgG2a, IgG2b and ANA levels were compared with the percentage of Tph and Tfh cells in splenic Th cells.
  • FIG. 13 is a graph comparing the percentage of Tph and Tfh cells in splenic Th cells with spleen weight.
  • Tph cells were substantially present in male and female mice at 8 weeks of age, as shown in Figures 12-13. However, they were found to predominate over Tfh cells at the onset of systemic autoimmunity and to be maintained throughout the disease.
  • FIG. 14 is a graph showing the numbers of Tph cells and Tfh cells in each group of mice (*: p ⁇ 0.05, **: p ⁇ 0.01, ***: p ⁇ 0.001).
  • Tph and Tfh cells There was a significant increase in the number of Tph and Tfh cells in 16 and 20 week old male control mice. Interestingly, as shown in Figures 13, 15 and 16, splenic Tph cell levels were significantly more correlated with splenomegaly than Tfh cells, and correlations were also obtained between Tph cells and serum ANA and IgG2a levels. However, serum IgG1, IgG2a and IgG2b levels were less correlated with either cell subset.
  • circulating Th cell subsets behaved differently than the increase in the proportion of circulating Tfh cells, the increase in circulating Tph cells occurred early and reflected increased serum ANA and IgG2a levels.
  • NR4A2cKO mice increases in circulating Tfh and Tph cells were suppressed, and increases in ANA production and IgG2a titers were suppressed at all time points.
  • FIG. 18 is a graph plotting the ratio of Tph cells in blood and Tph cells in the spleen, and a graph plotting the ratio of Tfh cells in blood and Tfh cells in the spleen.
  • Blood Tph cell levels correlated well with levels in the spleen, but for Tfh cells the correlation between sites was lower.
  • Expression of CXCR5 presumably targets Tfh cells to the spleen.
  • Tph and Tfh cells produced IL-21 upon stimulation, but Tph cells had a significantly higher percentage of IL-21 + cells (***: p ⁇ 0.0001, **: p ⁇ 0.01 , *: P ⁇ 0.05).
  • IL-21 production was significantly reduced in Tph cells from NR4A2cKO mice compared to Tph cells from control mice.
  • total RNA was extracted from flow cytometrically sorted spleen cell populations using the RNeasy Mini Kit (Qiagen) according to the manufacturer's instructions, followed by first strand cDNA.
  • cDNA was prepared using a kit (manufactured by Takara).
  • FIG. 19(b) is a graph showing the expression level of the il21 gene in PD-1-cells, Tph cells and Tfh cells.
  • FIG. 19(c) is a graph showing the relative expression level of each gene (bcl6, blimp1, maf).
  • Tph and Tfh cells from control mice showed increased expression of the il21 gene, whereas Tph and Tfh cells from NR4A2cKO mice expressed less il21.
  • Transcripts of Tfh-related markers behaved similarly between Tfh and Tph cells, with increased expression in control mice and decreased expression in NR4A2cKO mice.
  • FIG. 19(b) is a graph showing the expression level of the il21 gene in PD-1-cells, Tph cells and Tfh cells.
  • FIG. 19(c) is a graph showing the relative expression level of each gene (bcl6, blimp1, maf).
  • Tph and Tfh cells from control mice showed increased expression of the il
  • FIGS. 20(a) and (b) there was no significant difference in CXCR4 expression between Tph and Tfh cells derived from control mice and NR4A2cKO mice.
  • ELISA kit (eBioscience) was used according to the manufacturer's instructions and measured using a standard ELISA with a secondary antibody.
  • the ratio of low to high affinity antibodies was calculated for anti-IgG2a and anti-IgG1 antibodies.
  • NR4A2cKO mice or their wild-type littermate C57BL/6 mice were immunized twice with NP-KLH emulsified in alum. Two weeks after immunization, spleens were harvested and 7 ⁇ m spleen sections were prepared and stained with Alexa-Fluor-594-CD4 (developed red) and FITC-B220 or FITC-GL7 (developed green). dyed.
  • FIG. 22(a) is a photomicrograph showing splenic sections after immunization in NR4A2cKO mice and control mice (scale bar: 100 nm).
  • control mice When C57BL/6 mice that do not develop spontaneous autoimmune disease (control mice) were immunized with NP-KLH, similar levels of The formation of lymphoid follicular structures and germinal centers was induced, accompanied by the induction of anti-NP antibodies.
  • fecal IgA levels produced in response to bacterial antigens were measured (see Non-Patent Document 7). Specifically, using stool samples from control mice or NR4A2cKO mice, fecal IgA antibodies were measured by ELISA (manufactured by Affymetrix) according to the manufacturer's instructions.
  • Unbiased TCR repertoire analysis Spleens were collected from 20-week-old control mice or NR4A2cKO mice, and splenic memory T cells (TcR ⁇ + CD4 + CD44 hi CD62L lo cells) were sorted. The resulting T cells were subjected to unbiased TCR repertoire analysis using next-generation sequencing. Next-generation sequence analysis was performed using unbiased TCR repertoire analysis technology (manufactured by Repertoire Genesis Inc.). Unbiased adapter ligation PCR was performed according to Immunogenetics. 2000 Nov;52(1-2):35-45. After PCR amplification, index (barcode) sequences were added by amplification using the next generation XTindex kit v2 set A (Illumina).
  • the indexed amplicon products were mixed at equimolar concentrations and quantified by a Qubit 2.0 fluorometer (Thermo Fisher Scientific). Sequencing was performed on the Illumina Miseq paired-end platform (2 x 300 bp). All paired-end reads were sorted by index sequence. Sequence assignments were made by determining the highest identity sequences in a dataset of reference sequences from the International ImMunoGenetics information system (IMGT) database (http://www.imgt.org). Data processing, allocation and data aggregation were performed automatically using repertoire analysis software (Repertoire Genesis).
  • IMGT International ImMunoGenetics information system
  • the nucleotide sequence of the CDR3 region ranges from the conserved cysteine at position 104 (Cys104) in IMGT nomenclature to the conserved phenylalanine at position 118 (Phe118), translating the next glycine (Gly119) into the amino acid sequence was estimated.
  • a unique sequence read (USR) was defined as a sequence read that had no identity in the deduced amino acid sequences of TRV, TRJ and CDR3 with any other sequence read. The number of copies of the same USR was counted automatically by the RG software in each sample and then ranked in order of copy number.
  • FIG. 24(a) is a graph showing 100 reads of the CDR3 sequence in descending order of the number of reads, where the horizontal axis indicates the rank and the vertical axis indicates the number of reads.
  • FIG. 24(c) is a graph showing the cumulative percentage of reads across all unique sequences ranked by the most common CDR3 sequences.
  • the leads are then further classified based on residues at positions 6 and 7 of CDR3 (i.e., hydrophobic P6-7 doublet (promoting self-reactivity), hydrophilic P6-7 doublet (restricting self-reaction)). did.
  • Figures 25(a) and (b) sort the unique TcR sequences in order of most prevalence based on read number and show the percentage of self-promoting and self-restricting clones relative to the top 100 clones of each genotype. , the number of clones and the number of cumulative reads.
  • FIG. 25(c) shows the percentage of total reads covered by the population of top clones (25th, 50th, 100th or 200th clones from the most common clone).
  • the most common TcR ⁇ CDR3 sequences represent the most expanded Th clones, and these clones represent the Th cell repertoire most relevant to ongoing disease. For example, the top 50 clones represent at least 25% of the memory cell pool.
  • FIG. 26(a) shows the percentage of self-promoting and self-limiting clones for the top 50 clones of each genotype
  • FIG. 26(b) shows the top 25,50 most common sequences.
  • FIG. 26(b) is a heatmap showing the number of reads for 100 and 200 clones.
  • Autoreactive TcRs among the 50 most common clones were greatly reduced in NR4A2cKO mice.
  • FIG. 26(b) in NR4A2cKO mice, the reduction of autoreactive clones was observed in the top 25, 100 and 200 clones (covering approximately 15%, 40% and 50% of the repertoire reads, respectively). ), but the levels of self-restricted clones were similar to control mice. This result suggested selective loss of autoreactive T cells from the periphery in the absence of NR4A2.
  • NRP1 and PD-1 Spleens were harvested from 10-week-old and 18-week-old control mice (BxSB mice) or NR4A2cKO mice, and expression of NRP1 and PD-1 in Foxp3 ⁇ CD4 + Th cells was analyzed by flow analysis. Assessed by cytometry.
  • FIG. 27(a) is a cytogram showing the expression of NRP1 and PD-1 in Foxp3 ⁇ CD4 + Th cells. Comparing the cytograms of 10-week-old mice and 18-week-old mice, the ratio of NRP1 + PD-1 + cells is markedly increased in control mice at 18 weeks of age, whereas the ratio of NRP1 + PD-1 + cells is significantly increased in NR4A2cKO mice at 18 weeks of age. However, the increase in NRP1 + PD-1 + cells was suppressed.
  • DhiTh cells Thi cells co-expressing NRP1 and PD-1, NRP1 hi PD-1 hi cells
  • NRP1 + PD The percentage of ⁇ 1 + Foxp3 ⁇ Th cells was assessed.
  • FIG. 27(b) is a graph showing the proportion of NRP1/PD-1-expressing Th cells in Foxp3 ⁇ Th cells (**: p ⁇ 0.01, *: p ⁇ 0.05). It was found that the ratio of NRP1/PD-1-expressing Th cells significantly increased from 18 weeks of age in DhiTh cells derived from control mice, whereas it significantly decreased in DhiTh cells derived from NR4A2cKO mice.
  • FIG. 28(b) is a graph showing the proportion of NRP1 + subsets in Foxp3 ⁇ Th cells and Foxp3 + Th cells of each autoimmune disease model mouse (error bars: SD, ***: p ⁇ 0. 0001, ***: p ⁇ 0.001, **: p ⁇ 0.01).
  • Aberrant increases in NRP1/PD-1 co-expressing Th cells in Foxp3 ⁇ Th cells can also be significantly increased in other animal models prone to develop systemic autoimmune diseases compared to C57/BL6 mice. all right.
  • FIG. 29(a) is a cytogram of TcR ⁇ + CD4 + Foxp3 ⁇ T cells (Foxp3 ⁇ CD4 + Th cells) and TcR ⁇ + CD4 + Foxp3 + T cells (Foxp3 + CD4 + Th cells). About 81% of Foxp3 + CD4 + Th cells expressed NRP1, whereas Foxp3 ⁇ CD4 + Th cells did not express NRP1 and PD-1.
  • FIG. 29(b) is a graph showing the number of Th cells expressing NRP1 and/or PD-1 in each of Foxp3 + CD4 + Th cells and Foxp3 ⁇ CD4 + Th cells. In C57BL/6 mice without autoimmune disease, NRP1/PD-1 co-expressing Th cells were restricted to the Treg population and few NRP-1 expressing Th cells.
  • FIG. 30 is a graph plotting the percentage of Foxp3 ⁇ NRP1+PD-1+ cells on the vertical axis and spleen weight, serum total IgG or autoantibodies (ANA) on the horizontal axis.
  • ANA autoantibodies
  • Th cell diversity was assessed by CDR3 sequencing. After alignment, unique CDR3 sequences were identified and the number of reads for each TcR clone was calculated. Diversity was also assessed by calculating the Shannon index.
  • NRP1/PD-1 co-expressing Th cells have a more restricted repertoire than Th cells that do not express PD-1, with higher CDR3 sequence coverage and reduced diversity of the most frequent TcRs. rice field.
  • repertoire diversity was demonstrated by calculating the cumulative proportion of all TCR sequence reads to the declining common unique sequences, showing distribution skew.
  • the TCR repertoires of NRP1 + PD-1 + Th cells and NRP1 ⁇ PD-1 ⁇ Th cells were also analyzed using spleens harvested from 16-week-old control and NR4A2cKO mice. Read numbers for clones with autoreactive TcRs were calculated for the top 1000 clones.
  • the autoreactive P6-7 doublet of CDR3 of TcR ⁇ was increased in NRP1/PD-1-expressing Th cells compared to NRP1 ⁇ PD-1 ⁇ Th cells.
  • GITR-expressing PD-1 + GITR hi Treg subsets in BxSB mice have been reported to contain a T cell population with autoreactive TcRs (9).
  • GITR also called TNFRSF18
  • TNFRSF18 is one of the tumor necrosis factor receptor superfamily members and can regulate regulatory T cells.
  • FIG. 32(a) is a cytogram for Foxp3 and TcR ⁇ (left) and a cytogram for PD-1 and CITR (right).
  • Foxp3 ⁇ CD4 + T cells from control mice had 54.6% PD-1 + GITR hi Th cells and 17.5% PD-1 ⁇ GITR lo Th cells, compared to NR4A2cKO mice.
  • the derived Foxp3 ⁇ CD4 + T cells were 15.1% PD-1 + GITR hi Th cells and 64.9% PD-1 ⁇ GITR lo Th cells.
  • FIG. 32(b) is a graph quantifying this.
  • FIG. 34(a) is a graph showing the percentage of Foxp3 + and Foxp3 ⁇ cells among CD4 + T cells.
  • FIG. 34(b) is a graph showing the percentage of PD-1 + GITR lo cells and PD-1 ⁇ GITR hi cells. Little PD-1 + GITR hi was observed among conventional non-Treg Th cells in NR4A2cKO littermates (see Figure 32(b)) or in C57BL/6 mice that do not spontaneously develop autoimmune disease (see Figure 33). This suggested that the proliferation of Foxp3 ⁇ PD-1 + GITR hi Th cells was dependent on NR4A2 and associated with the development of autoreactive responses.
  • CD5 expression can result from continuous exposure to autoantigens in vivo, and cells exhibiting autoantigen-specific TcR expression are known to be associated with upregulation of CD5. (See Non-Patent Documents 10-11).
  • CD5 expression in BxSB mice Foxp3 ⁇ Th cell subsets (Foxp3 ⁇ CD4 + cells, NRP1 + PD-1 + Foxp3 ⁇ cells, NRP1 ⁇ PD-1 - Foxp3 - cells), CD5 expression was measured by flow cytometry using a fluorescently labeled anti-CD5 antibody.
  • FIG. 35 is a graph showing the mean fluorescence intensity (MFI) of each T cell (**: p ⁇ 0.01, ***: p ⁇ 0.001, ***: p ⁇ 0.0001). .
  • MFI mean fluorescence intensity
  • Tph cells and Tfh cells were separated from 18-week-old control mice (BxSB mice) or NR4A2cKO mice, respectively, and the ratio to CD4 + Th cells was calculated.
  • GITR hi cells were found in both NRP1 + Tph and NRP1 + Tfh cells, but a stronger suppression of GITR hi cells was observed in NRP1 + Tph cells compared to NRP1 + Tfh cells in NR4A2cKO mice.
  • NR4A2 expression in T cells is potentially involved in the proliferation of Th cells with autoreactive TcR.
  • intrinsically autoreactive Th cells share their phenotype with Treg cells, and expansion of NR4A2-dependent NRP1/PD-1 co-expressing Th cells is highly associated with the development of autoimmune diseases. it is conceivable that.
  • NRP1/PD-1 co-expressing Th cells are thought to be capable of inducing SLE-like diseases. tried to apply.
  • BxSB-Yaa mice received 200 ⁇ M of RPA peptide (RPARPAR-CGG- D (KLAKLAK) 2 ) or control peptide.
  • RPA peptide RPA peptide
  • a PBS solution of (GAGAGAG-CGG- D (KLAKLAK) 2 ) was administered intraperitoneally (n 4).
  • spleens and inguinal lymph nodes were removed from mice and weighed. Total IgG and ANA concentrations in serum were measured. Serum antibody levels were measured by mouse total IgG1, IgG2a and IgG2b Ready-set-Go!
  • ELISA kit (eBioscience) was used according to the manufacturer's instructions and measured using a standard ELISA with a secondary antibody. Antinuclear antibodies were measured using an ANA ELISA kit (US Biological) according to the manufacturer's instructions. In addition, NRP1/PD-1 subpopulations were calculated for CD4 + Foxp3 ⁇ Th cells by intracellular flow cytometry.
  • FIG. 37(b) is a photograph of the excised spleen and a graph showing its weight (*: P ⁇ 0.05).
  • FIG. 37(c) is a photograph of an excised inguinal lymph node.
  • Figure 38 is a graph showing total IgG and ANA concentrations in serum. Spleen size, serum total IgG, and ANA levels were all smaller in the RPA peptide-administered group (denoted as RPA in the figure) than in the control peptide-administered group (denoted as PBS in the figure). .
  • FIG. 39(a) is a cytogram of the RPA peptide-administered group and the control peptide-administered group.
  • the ratio of NRP1 + PD-1 + Th cells accounted for approximately 50%
  • the percentage of NRP1 + PD-1 + Th cells was suppressed to approximately 12%.
  • FIG. 39(b) is a graph quantifying the percentage of NRP1 + PD-1 + Th cells.
  • FIG. 39(c) is a graph showing the ratio of Tph and Tfh in TcR ⁇ + CD4 + Th cells. Administration of both RPA peptides tended to reduce Tph and Tfh cells.
  • NRP1 and PD-1 co-expression in TcR ⁇ + CD4 + Foxp3 + Th cells Control mice or NR4A2cKO mice at 10, 12 and 14 weeks of age were administered RPA peptide intraperitoneally. Spleens were removed from mice at 16 weeks of age and TcR ⁇ + CD4 + Th cells were isolated. The percentage of Foxp3 + cells among TcR ⁇ + CD4 + Th cells was measured. NRP1 + PD-1 ⁇ Th cells, NRP1 ⁇ PD-1 + Th cells, and NRP1 + PD-1 + Th cells in TcR ⁇ + CD4 + Foxp3 + Th cells were also measured.
  • FIG. 40(a) is a graph showing the ratio of Foxp3 + cells in TcR ⁇ + CD4 + Th cells, and a graph showing the expression of NRP1 and PD-1 in TcR ⁇ + CD4 + Foxp3 + Th cells.
  • Administration of RPA peptide reduced the proliferation of NRP1/PD-1 co-expressing Th cells and also reduced the numbers of Tph and Tfh cells, but Foxp3 + Treg cells were unaffected, suggesting a distinct NRP1-expressing subset. were suggested to have different sensitivities to RPA peptides.
  • BxSB-Yaa mice were injected with 500 ⁇ M of the RPA peptide (RPARPAR-CGG- D (KLAKLAK) 2 ) or control peptide (GAGAGAG).
  • -CGG- D (KLAKLAK) 2 ) in PBS was administered intraperitoneally.
  • spleens were removed from mice and serum antibody levels were determined by mouse IgG1, IgG2a and IgG2b Ready-set-Go!
  • An ELISA kit (eBioscience) was used according to the manufacturer's instructions and measured using a standard ELISA with a secondary antibody. Antinuclear antibodies were measured using an ANA ELISA kit (US Biological) according to the manufacturer's instructions.
  • the percentage of spleen-derived TcR ⁇ + CD4 + Th cells was determined by NRP1/PD-1 staining.
  • FIG. 40(b) is a graph showing spleen weight.
  • Figures 40(c) and (d) are graphs showing serum total IgG and ANA levels.
  • the increase in spleen weight was suppressed and the ANA level was reduced as compared with the control peptide-administered group.
  • NRP1 + PD-1 + Foxp3 - CD4 + Th cells were slightly decreased by administration of RPA peptide after onset, but remained the major disease-related cells. indicators were declining.
  • MRL-lpr mice (Fas lpr mice) lack CD95 (Fas ligand) and are known as a systemic autoimmune disease model because they induce spontaneous autoimmunity. It is Mutations in the CD95 gene fas lpr cause massive lymphadenopathy, weight loss, glomerulonephritis, induction of anti-dsDNA antibodies.
  • FIG. 41(a) is a graph showing changes in body weight over time in the RPA peptide-administered group and control group mice.
  • the mice in the control peptide-administered group showed a moderate weight gain, whereas the RPA peptide-administered group tended to gain more in body weight.
  • Figures 41(b) and (c) are graphs showing spleen weight, serum IgG and anti-dsDNA antibody levels (*: P ⁇ 0.05).
  • FIG. 42 is a cytogram and a quantified graph for NRP1/PD-1.
  • Administration of RPA peptide markedly decreased the proportion of NRP1 + Th cells.
  • the proportion of NRP1 + PD-1 + cells among CD4 + Th cells was decreased by administration of RPA peptide.
  • Nrp1-positive Th cells Frequency of Nrp1-positive Th cells in peripheral blood of systemic lupus erythematosus (SLE) patients The number of Nrp1-positive Th cells detected in MS patients was limited. Therefore, targeting SLE patients who are expected to have a larger pool size, peripheral blood was collected from SLE patients and healthy adults, and after isolating CD4 + CD3 + Th cells, Nrp1-positive Th cells were analyzed by flow cytometry. Cell frequencies were compared.
  • FIG. 43(a) is a cytogram of CD4 + CD3 + Th cells derived from the peripheral blood of SLE patients.
  • peripheral blood derived from healthy adults almost no Nrp-1 positive cells were observed in CD127 positive Th cells (corresponding to human effector Th cells) (about 0.69%), but SLE patient-derived cells In peripheral blood, Nrp-1-positive cells were clearly increased (about 3.39%) among CD127-positive Th cells.
  • Figure 43 (b) is a graph showing the ratio of Nrp-1-positive cells to Th cells in peripheral blood from healthy adults or SLE patients
  • Figure 43 (c) shows Nrp-1-positive to CD127-positive Th cells.
  • Fig. 3 is a graph showing percentage of cells; In the peripheral blood of healthy adults, the ratio of Nrp-1-positive cells to Th cells was low in all cases, and the ratio of Nrp-1-positive cells to CD127-positive Th cells was also low.
  • the peripheral blood derived from SLE patients although the variation was large, the overall trend was that the percentage of Nrp-1 positive cells was significantly higher than that of healthy adults.

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