WO2014109289A9 - EPITOPE DES LYMPHOCYTES T CD4+ SPÉCIFIQUE DE Tax, RESTREINT À HLA-DR1 - Google Patents

EPITOPE DES LYMPHOCYTES T CD4+ SPÉCIFIQUE DE Tax, RESTREINT À HLA-DR1 Download PDF

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WO2014109289A9
WO2014109289A9 PCT/JP2014/000053 JP2014000053W WO2014109289A9 WO 2014109289 A9 WO2014109289 A9 WO 2014109289A9 JP 2014000053 W JP2014000053 W JP 2014000053W WO 2014109289 A9 WO2014109289 A9 WO 2014109289A9
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amino acid
acid sequence
htlv
specific
peptide
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WO2014109289A1 (fr
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洋太郎 玉井
温彦 長谷川
真理 神奈木
隆二 田野崎
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国立大学法人東京医科歯科大学
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    • A—HUMAN NECESSITIES
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    • A61K31/00—Medicinal preparations containing organic active ingredients
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    • A—HUMAN NECESSITIES
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    • 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
    • A61K39/12—Viral antigens
    • A61K39/21—Retroviridae, e.g. equine infectious anemia virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • 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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70514—CD4
    • G—PHYSICS
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    • 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
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5047—Cells of the immune system
    • G01N33/505—Cells of the immune system involving T-cells
    • 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
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56966—Animal cells
    • G01N33/56972—White blood cells
    • 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
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • G01N33/56988—HIV or HTLV
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/61—Fusion polypeptide containing an enzyme fusion for detection (lacZ, luciferase)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/14011—Deltaretrovirus, e.g. bovine leukeamia virus
    • C12N2740/14034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/15—Retroviridae, e.g. bovine leukaemia virus, feline leukaemia virus, feline leukaemia virus, human T-cell leukaemia-lymphoma virus

Definitions

  • the present invention relates to an HTLV-I-specific CD4 + T cell-inducing peptide having an activity of inducing CD4 + T cells specific for human T-cell leukemia virus type I (HTLV-I), and an HTLV containing the peptide.
  • HTLV-I human T-cell leukemia virus type I
  • -It relates to an I-specific CTL inducing agent, etc.
  • HTLV-I Human T cell leukemia virus type I
  • ATL adult T cell leukemia / lymphoma
  • CD4 + T cell malignancy non-patented
  • HTLV-I seropositive individuals develop ATL, and 2-3% of others have HTLV-I-associated myelopathy / tropical spastic paraparesis paralysis (HAM / TSP: HTLV-I associated) It develops slowly progressive neurological disease known as myelopathy / tropical spastic paraparesis) and various chronic inflammatory diseases (Non-patent Document 3).
  • HAM / TSP HTLV-I associated
  • myelopathy / tropical spastic paraparesis develops slowly progressive neurological disease known as myelopathy / tropical spastic paraparesis
  • Non-patent Document 3 The vast majority of HTLV-I infected individuals are in asymptomatic carriers throughout their lifetime.
  • ATL is characterized by a very poor prognosis, mainly because ATL inherently has drug resistance to anticancer agents. It has been reported that the prognosis improvement of ATL that could not be achieved by autologous hematopoietic stem cell transplantation was observed in allogeneic hematopoietic stem cell transplantation (allo-HSCT) (Non-patent Documents 4 and 5).
  • Non-patent Document 6 HTLV-I provirus is no longer detected in some ATL patients who have achieved complete remission after allogeneic hematopoietic stem cell transplantation and this undetected condition continues, which makes allogeneic hematopoietic stem cell transplantation an effective treatment for ATL (Non-Patent Documents 6 to 8).
  • HTLV-I-specific CTL-inducing active peptide restricted by human HLA-A11
  • Patent Document 2 CD8 + T cells, particularly CTL, play an important role in the control of viral replication in various infectious diseases involving HIV, hepatitis B virus (HBV), hepatitis C virus (HCV) and the like.
  • HBV hepatitis B virus
  • HCV hepatitis C virus
  • HTLV-I infection it is considered that HTLV-I-specific CD8 + T cells recognize Tax antigen (pX gene product) as a predominant and contribute to the control of infected cells (Non-patent Documents 10 and 11).
  • Tax-specific CD8 + T cells could be detected with high frequency in HAM / TSP patients and some asymptomatic carriers (AC), but in the majority of ATL patients and in a small population of ACs The Tax-specific CD8 + T cell response was significantly reduced (Non-patent Documents 12 and 13). The mechanism by which the HTLV-I specific CD8 + T cell response is suppressed in these patients has not been fully elucidated.
  • Non-patent Documents 14 to 18 In order to induce and maintain virus-specific CTL, a virus-specific CD4 + helper T cell response is required in many viral infections (Non-patent Documents 14 to 18), but HTLV-I-specific helper T cell response There were few reports (Non-Patent Documents 19 to 22). This is probably because HTLV-I-specific helper T cells are susceptible to HTLV-I infection in vivo and in vitro (Non-patent Document 23), and infected cells produce HTLV-I antigens within a few hours after culture. (Non-Patent Documents 24 and 25).
  • Non-Patent Document 26 CD4 + at sites of early progressive inflammatory spinal cord injury with spontaneous production of inflammatory neurotoxic cytokines such as IFN- ⁇ and tumor necrosis factor (TNF) - ⁇ (Non-Patent Document 26). T cells are found in predominant (Non-Patent Documents 27 to 28), suggesting that this is involved in the development of HAM / TSP. However, the exact role of HTLV-I-specific CD4 + T cells in HTLV-I infection has not yet been elucidated.
  • Tsukasaki K Maeda T, Arimura K, et al. Poor outcome of autologous stem cell transplantation for adult T cell leukemia / lymphoma: a case report and review of the literature.
  • Utsunomiya A Miyazaki Y, Takatsuka Y, et al. Iproved outcome of adult T cell leukemia / lymphoma with allogeneic hematopoietic stem cell transplantation.
  • Tanosaki R Uike N, Utsunomiya A, et al.
  • HTLV-I Human T cell lymphotropic virus type I -specific CD4 + T cells: immunodominance hierarchy and preferential infection with HTLV-I. J Immunol. 1735-1743. Hanon E, Hall S, Taylor GP, et al. Abundant tax protein expression in CD4 + T cells infected with human T-cell lymphotropic virus type I (HTLV-I) is prevented by cytotoxic T lymphocytes. Blood. 2000; 95 : 1386-1392. Sakai JA, Nagai M, Brennan MB, Mora CA, Jacobson S. In vitro spontaneous lymphoproliferation in patients with human T-cell lymphotropic virus type I-associated neurologic disease: predominant expansion d : 1506-1511.
  • Umehara F Izumo S, Ronquillo AT, Matsumuro K, Sato E, Osame M. Cytokine expression in the spinal cord lesions in HTLV-I-associated myelopathy. J Neuropathol Exp Neurol. 1994-77 (1) Umehara F, Izumo S, Nakagawa M, et al. Immunocytochemicalunoanalysis of the cellular infiltrate in the spinal cord lesions in HTLV-I-associated myelopathy. J Neuropathol Exp Neurol. 1993; 52 (4): 424-430. Iwasaki Y, Ohara Y, Kobayashi I, Akizuki S. Infiltration of helper / inducer T lymphocytes heralds central nervous system damage in human T-cell leukemia virus infection. Am J Pathol. 1992; 140 (5): 1003-1008.
  • An object of the present invention and HTLV-I-specific CD4 + T cell-inducing activity peptide having an activity to induce CD4 + T cells specific for HTLV-I, HTLV-I-specific CTL-inducing action containing the peptide
  • the object is to provide a potentiator, a vaccine for inducing an HTLV-I-specific immune response using these, a diagnostic agent for immune function testing, and the like.
  • HLA-DR1 human HLA-DRB1 * 0101
  • C When such an HTLV-I-specific CD4 + T cell-inducing activity peptide and an HTLV-I-specific CTL-inducing activity peptide are used in combination, the HTLV-I-specific CTL-inducing activity peptide alone is used. HTLV-I-specific CTLs proliferated significantly.
  • the present invention (1) HTLV-I-specific CD4 + T cell-inducing peptide consisting of the amino acid sequence shown in any of (A) to (E) below:
  • C An amino acid sequence having 80% or more identity to the amino acid sequence shown in (A) or (B) above, wherein the peptide comprising the amino acid sequence is induced to induce HTLV-I-specific CD4 + T cells.
  • Amino acid sequence with activity (D) In the amino acid sequence shown in (A) or (B) above, an amino acid sequence in which one or several amino acids are deleted, substituted or added, and the peptide comprising the amino acid sequence is specific to HTLV-I Amino acid sequence having CD4 + T cell inducing activity: (E) an amino acid sequence consisting of 14 to 30 consecutive amino acids in the amino acid sequence shown in SEQ ID NO: 32, comprising an amino acid sequence of at least amino acid numbers 155 to 167, and an HTLV-I-specific CTL An amino acid sequence combined with the amino acid sequence of the inducing active peptide, (2) The HTLV-I-specific CD4 + T cell-inducing peptide described in (1) above, which is a Tax epitope restricted by HLA-DR1, (3) The HTLV-I-specific CD4 + T cell-inducing peptide according to (1) or (2) above, wherein the CD4 + T cell is a Th1-type helper T cell, (4) a fusion
  • the present invention also provides: (8) HTLV-I-specific CTL inducing action potentiator comprising, as an active ingredient, an HTLV-I-specific CD4 + T cell-inducing peptide consisting of any of the following amino acid sequences (A) to (E): (A) Amino acid sequence represented by any of SEQ ID NOs: 4, 10, 11, 12, 13, 16, 17, 19: (B) an amino acid sequence consisting of 14 to 30 consecutive amino acids in the amino acid sequence shown in SEQ ID NO: 32, and comprising at least the amino acid sequences of amino acid numbers 155 to 167: (C) An amino acid sequence having 80% or more identity to the amino acid sequence shown in (A) or (B) above, wherein the peptide comprising the amino acid sequence is induced to induce HTLV-I-specific CD4 + T cells.
  • A Amino acid sequence represented by any of SEQ ID NOs: 4, 10, 11, 12, 13, 16, 17, 19:
  • B an amino acid sequence consisting of 14 to 30 consecutive amino acids in the amino
  • Amino acid sequence with activity (D) In the amino acid sequence shown in (A) or (B) above, an amino acid sequence in which one or several amino acids are deleted, substituted or added, and the peptide comprising the amino acid sequence is specific to HTLV-I Amino acid sequence having CD4 + T cell inducing activity: (E) an amino acid sequence consisting of 14 to 30 consecutive amino acids in the amino acid sequence shown in SEQ ID NO: 32, comprising an amino acid sequence of at least amino acid numbers 155 to 167, and an HTLV-I-specific CTL An amino acid sequence combined with the amino acid sequence of the inducing active peptide, (9) An expression vector comprising a promoter and a polynucleotide comprising the polynucleotide sequence shown in any of the following (a) to (e), wherein the polynucleotide is operably linked downstream of the promoter.
  • HTLV-I specific CTL inducing action enhancer containing as an active ingredient: (A) a polynucleotide sequence encoding the amino acid sequence represented by any of SEQ ID NOs: 4, 10, 11, 12, 13, 16, 17, 19: (B) a polynucleotide sequence encoding an amino acid sequence comprising 14 to 30 consecutive amino acids in the amino acid sequence represented by SEQ ID NO: 32 and comprising at least the amino acid sequences of amino acid numbers 155 to 167: (C) a polynucleotide sequence having 80% or more identity to the polynucleotide sequence shown in the above (a) or (b), wherein the peptide has HTLV-I-specific CD4 + T cell inducing activity Encoding polynucleotide sequence: (D) a polynucleotide sequence obtained by deleting, substituting, or adding one or several nucleotides in the polynucleotide sequence shown in (a) or (b), wherein the HTLV-I specific
  • the present invention provides (11) below (A) containing - a HTLV-I-specific CD4 + T cell-inducing activity peptide comprising the amino acid sequence shown in any of (E) as an active ingredient, HTLV-I-specific CD4 + T cells Immune function test diagnostic agent to identify: (A) Amino acid sequence represented by any of SEQ ID NOs: 4, 10, 11, 12, 13, 16, 17, 19: (B) an amino acid sequence consisting of 14 to 30 consecutive amino acids in the amino acid sequence shown in SEQ ID NO: 32, and comprising at least the amino acid sequences of amino acid numbers 155 to 167: (C) An amino acid sequence having 80% or more identity to the amino acid sequence shown in (A) or (B) above, wherein the peptide comprising the amino acid sequence is induced to induce HTLV-I-specific CD4 + T cells.
  • A Amino acid sequence represented by any of SEQ ID NOs: 4, 10, 11, 12, 13, 16, 17, 19:
  • B an amino acid sequence consisting of 14 to 30 consecutive amino acids
  • Amino acid sequence with activity (D) In the amino acid sequence shown in (A) or (B) above, an amino acid sequence in which one or several amino acids are deleted, substituted or added, and the peptide comprising the amino acid sequence is specific to HTLV-I Amino acid sequence having CD4 + T cell inducing activity: (E) an amino acid sequence consisting of 14 to 30 consecutive amino acids in the amino acid sequence shown in SEQ ID NO: 32, comprising an amino acid sequence of at least amino acid numbers 155 to 167, and an HTLV-I-specific CTL An amino acid sequence combined with the amino acid sequence of the inducing active peptide, (12) An HTLV comprising, as an active ingredient, a protein-peptide conjugate in which HLA-DR1 and the HTLV-I-specific CD4 + T cell-inducing peptide according to any one of (1) to (3) above are bound Diagnostic reagent for immunological function test for identifying -I specific CD4 + T cells, (13) HLA-DR1 and HTLV-
  • the present invention also provides: (14) HTLV-I-specific CD4 characterized in that HTLV-I-infected T cells from ATL patients before HSCT are used to stimulate PBMCs of the same patients after HSCT from allogeneic HLA-type donors + T cell induction method, (15) HTLV-I specific, characterized by stimulating PBMC of HLA-DR1-positive ATL patients using the HTLV-I specific CTL inducing action enhancer described in (8) or (9) above CD4 + T cell induction method, (16) HTLV-I-specific CD4 + T cell response and HTLV-I-specific, characterized by stimulating PBMC of HLA-DR1-positive ATL patients using the following (X) and (Y) Methods for inducing HTLV-I specific immune responses, including CTL responses: (X) The HTLV-I-specific CTL inducing action enhancer according to (8) or (9) above: (Y) an HTLV-I-specific CTL-inducing active peptide
  • epitopes a minimal epitope of Tax-specific CD4 + T cells restricted by HLA-DR1 was found. Such epitopes have the activity of inducing CD4 + T cells specific for HTLV-I. Therefore, such epitope peptides are useful as HTLV-I specific CD4 + T cell-inducing active peptides.
  • epitopes can be predicted to some extent from amino acid anchor motifs having affinity for each HLA. However, the host immune response to pathogens in vivo is not necessarily consistent with this prediction. The epitope identified according to the present invention is obtained from an infected individual and is recognized with a much stronger selectivity than other epitopes.
  • the present inventors have found a Tax-specific CTL epitope so far, and that such an epitope peptide has HTLV-I-specific CTL inducing activity and can be used as a vaccine for HTLV-I-recognizing CTL induction. I have found it.
  • the HTLV-I-specific CD4 + T cell-inducing activity peptide found by the present inventors is used in combination with the aforementioned HTLV-I-specific CTL-inducing activity peptide, the HTLV-I-specific CTL-inducing activity peptide alone is used.
  • the HTLV-I-specific CD4 + T cell-inducing peptide of the present invention is also useful as an agent for enhancing HTLV-I-specific CTL induction.
  • the HTLV-I specific CD4 + T cell-inducing activity peptide of the present invention, the protein-peptide conjugate in which HLA-DR1 and the peptide of the present invention are bound, and the tetramer of the protein-peptide conjugate are It is also useful as a diagnostic agent for immune function tests for identifying HTLV-I-specific CD4 + T cells.
  • FIG. 6 shows Tax-specific T cell immune responses in ATL patients after allogeneic hematopoietic stem cell transplantation with palliative pretreatment.
  • PBMC from 18 ATL patients 180 days after allogeneic hematopoietic stem cell transplantation with palliative pretreatment or 2 patients 540 days after allogeneic hematopoietic stem cell transplantation with palliative pretreatment (# 350 and # 341) total PBMC and CD8 + cell depleted PBMC (B) in the absence of GST protein (open), in the presence of GST protein (gray), or in the presence of GST-Tax protein (black) After culturing for 4 days, the IFN- ⁇ concentration in the supernatant was quantified by ELISA.
  • the horizontal dotted line in (A) indicates the detection limit (23.5 pg / ml).
  • the error range represents the standard deviation of duplicate measurements.
  • * P ⁇ 0.05 It is a figure which shows the phenotype and function of CD4 ⁇ +> T cell line (T4) produced from patient # 350.
  • A The phenotype on the cell surface of T4 cells was analyzed by flow cytometry.
  • B Total RNA was extracted from LCL- # 350 (lane 1), T4 cells (lane 2), ILT- # 350 (lane 3) and MT-2 (lane 4). Tax mRNA expression of each cell type was analyzed by RT-PCR. GAPDH was used as an internal standard.
  • T4 cells were stimulated for 24 hours in the presence / absence of ILT- # 350 cells or LCL- # 350 cells fixed with formaldehyde.
  • the cytokine concentration in the supernatant was quantified with a cytometry bead array system. It is a figure which shows the identification process of the dominant Tax origin epitope recognized by the established T4 cell.
  • T4 cells and ILT- # 350 in the presence / absence of blocking antibodies (10 ⁇ g / ml anti-human HLA-DR antibody, anti-human HLA-DQ antibody, anti-HLA-class I antibody or isotype control antibody)
  • the IFN- ⁇ produced by T4 cells released into the culture supernatant was quantified by ELISA.
  • the HLA-DR allele of each LCL strain is shown in parentheses.
  • ATL patients (# 350, # 364 and # 341) who received allogeneic hematopoietic stem cell transplantation with palliative pretreatment and whose Major Histocompatibility Complex (MHC) class I and class II are HLA-A24 / HLA-DRB1 * 0101, respectively
  • MHC Major Histocompatibility Complex
  • the obtained PBMC was cultured for 13 days in the presence of 100 nM CTL epitope (Tax301-309) alone, a mixture of Tax301-309 peptide (100 nM) and Tax155-167 peptide (100 nM), or DMSO alone (negative control). did.
  • the data shows the percentage of HLA-A * 2402 / Tax301-309 tetramer positive cells in CD3 + CD8 + T cells.
  • the HTLV-I specific CD4 + T cell-inducing peptide of the present invention that is, the peptide having the activity of inducing CD4 + T cells specific to HTLV-I is any of the following (A) to (E)
  • the peptide is not particularly limited as long as it is an HTLV-I-specific CD4 + T cell-inducing peptide (hereinafter also referred to as “the peptide of the present invention”) having the amino acid sequence shown above.
  • Amino acid sequence with activity (D) In the amino acid sequence shown in (A) or (B) above, an amino acid sequence in which one or several amino acids are deleted, substituted or added, and the peptide comprising the amino acid sequence is specific to HTLV-I Amino acid sequence having CD4 + T cell inducing activity: (E) an amino acid sequence consisting of 14 to 30 consecutive amino acids in the amino acid sequence shown in SEQ ID NO: 32, comprising an amino acid sequence of at least amino acid numbers 155 to 167, and an HTLV-I-specific CTL Amino acid sequence combined with the amino acid sequence of the inducing active peptide:
  • SEQ ID NOs listed in (A) above are HLA-DRB1 * 0101 restricted Tax-specific CD4 + T cell epitopes, and SEQ ID NO: 4 is from 154th to 178th of HTLV-I Tax.
  • SEQ ID NO: 10 is the amino acid sequence consisting of amino acids 151 to 165 of Tax of HTLV-I (Tax151-165)
  • SEQ ID NO: 11 is HTLV- It is an amino acid sequence (Tax154-168) consisting of amino acids 154 to 168 of Tax of I
  • SEQ ID NO: 12 is an amino acid sequence (Tax155-169) consisting of amino acids 155 to 169 of Tax of HTLV-I
  • SEQ ID NO: 13 is an amino acid sequence consisting of amino acids 156 to 170 of Tax of HTLV-I (Tax15 6-170)
  • SEQ ID NO: 16 is an amino acid sequence (Tax155-168) consisting of amino acids 155 to 168 of HTLV-I Tax
  • SEQ ID NO: 17 is 155th to 167 of HTLV-I Tax.
  • the amino acid sequence is the amino acid sequence (Tax155-167) consisting of the first amino acid, and SEQ ID NO: 19 is the amino acid sequence (Tax151-178) consisting of the 151st to 178th amino acids of Tax of HTLV-I.
  • Peptides consisting of these amino acid sequences can be suitably exemplified among the peptides of the present invention in that they are excellent in HTLV-I-specific CD4 + T cell inducing activity.
  • SEQ ID NO: 11 (Tax154-168 ), SEQ ID NO: 12 (Tax155-169), SEQ ID NO: 16 (Tax155-168), SEQ ID NO: 17 (Tax155-167), SEQ ID NO: 4 (Tax154-178), SEQ ID NO: 13 (Tax156-170)
  • SEQ ID NO: 11 (Tax154-168), SEQ ID NO: 12 (Tax155-169), SEQ ID NO: 16 (Tax155-168), and SEQ ID NO: 17 (Tax155-167) are more preferably exemplified.
  • SEQ ID NO: 17 (Tax155-167) is particularly preferably exemplified in that it is a minimal epitope of a HLA-HLA-DR1-restricted Tax-specific CD4 + T cell epitope (hereinafter also simply referred to as “minimal epitope”). can do.
  • minimal epitope a HLA-HLA-DR1-restricted Tax-specific CD4 + T cell epitope
  • HTLV-I-specific CD4 + from the viewpoint of obtaining a suitable induction of T cell
  • HLA-HLA-DRB1 type is HLA-DRB1 * 0101 It is preferable to use for the object which is.
  • the amino acid sequence of (B) above is 14 to 30, preferably 14 to 25, more preferably 14 to 20, and still more preferably 14 to 15 amino acids in the amino acid sequence of Tax (SEQ ID NO: 32).
  • An amino acid sequence comprising at least amino acid sequences of amino acid numbers 155 to 167. Since such an amino acid sequence contains a minimal epitope, it is an HTLV-I-specific CD4 + T cell-inducing active peptide.
  • the amino acid sequence of (C) above is 80% or more, preferably 85% or more, more preferably 90% or more, still more preferably 95% or more, even more than the amino acid sequence shown in (A) or (B) above.
  • it is an amino acid sequence having 98% or more identity
  • the peptide comprising the amino acid sequence is an amino acid sequence having HTLV-I-specific CD4 + T cell inducing activity.
  • Such an amino acid sequence is highly likely to be an HTLV-I-specific CD4 + T cell-inducing peptide.
  • the amino acid sequence (D) is an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in (A) or (B) above, and consists of the amino acid sequence.
  • the peptide is an amino acid sequence having HTLV-I specific CD4 + T cell inducing activity.
  • the “amino acid sequence in which one or several amino acids are deleted, substituted or added” is, for example, 1 to 20, preferably 1 to 15, more preferably 1 to 10, and still more preferably 1.
  • the modified peptide should have the same effect as that having HTLV-I-specific CD4 + T cell inducing activity in the same manner as the peptide consisting of the amino acid sequence listed in (A) above.
  • alteration (mutation) of the amino acid sequence may be caused by, for example, mutation or post-translational modification, but may be artificially altered. In the present invention, all modified peptides having the above characteristics are included regardless of the cause and means of such modification / mutation.
  • the amino acid sequence of (E) above is an amino acid sequence consisting of 14 to 30 consecutive amino acids in the amino acid sequence shown in SEQ ID NO: 32, and comprising at least an amino acid sequence of amino acid numbers 155 to 167, It is an amino acid sequence obtained by binding an amino acid sequence of an HTLV-I-specific CTL-inducing active peptide.
  • the number of consecutive 14 to 30 amino acids in the amino acid sequence (E) is preferably 18 to 30, more preferably 22 to 30.
  • HTLV-I-specific CTL-inducing activity peptide in the present specification is not particularly limited as long as it is a peptide having an activity to induce CTL specific to HTLV-I.
  • Peptides can be preferably exemplified, and among them, HLA-A * 2402 restricted CTL epitope Tax 301-309 (SEQ ID NO: 34) (see Patent Document 1) and the like can be particularly preferably exemplified.
  • a fusion peptide (Helper / Killer-Hybrid Epitope Long Peptide) in which a cancer antigen epitope for CD4 + T cells, which are helper T cells, and a cancer antigen epitope for CD8 + T cells, which are CTLs, are administered is administered to a subject.
  • a cancer antigen epitope for CD4 + T cells which are helper T cells
  • a cancer antigen epitope for CD8 + T cells which are CTLs
  • helper T cell can be illustrated suitably, Th1 type helper T cell can be illustrated more suitably especially.
  • the peptide of the present invention can be produced by chemical or genetic engineering techniques.
  • the chemical method includes peptide synthesis methods by ordinary liquid phase methods and solid phase methods. More specifically, the peptide synthesis method is based on the amino acid sequence information, and a stepwise erosion method in which each amino acid is sequentially linked one by one to extend the chain, and a fragment consisting of several amino acids is synthesized in advance. Then, a fragment condensation method in which each fragment is subjected to a coupling reaction is included.
  • the peptide of the present invention can be synthesized by any of them.
  • the condensation method employed in the peptide synthesis can also follow various known methods. Specific examples thereof include, for example, azide method, mixed acid anhydride method, DCC method, active ester method, redox method, DPPA (diphenylphosphoryl azide) method, DCC + additive (1-hydroxybenzotriazole, N-hydroxysuccinamide) N-hydroxy-5-norbornene-2,3-dicarboximide, etc.), Woodward method and the like. Solvents that can be used in each of these methods can be appropriately selected from general solvents that are well known to be used in this type of peptide condensation reaction.
  • Examples thereof include dimethylformamide (DMF), dimethyl sulfoxide (DMSO), hexaphosphoroamide, dioxane, tetrahydrofuran (THF), ethyl acetate and the like, and mixed solvents thereof.
  • DMF dimethylformamide
  • DMSO dimethyl sulfoxide
  • THF tetrahydrofuran
  • ethyl acetate examples thereof include dimethylformamide (DMF), dimethyl sulfoxide (DMSO), hexaphosphoroamide, dioxane, tetrahydrofuran (THF), ethyl acetate and the like, and mixed solvents thereof.
  • amino acids that do not participate in the reaction and carboxyl groups in the peptide are generally esterified, for example, lower alkyl esters such as methyl ester, ethyl ester, tertiary butyl ester, such as benzyl ester, p- It can be protected as a methoxybenzyl ester, p-nitrobenzyl ester aralkyl ester or the like.
  • an amino acid having a functional group in the side chain for example, a hydroxyl group of Tyr may be protected with an acetyl group, a benzyl group, a benzyloxycarbonyl group, a tertiary butyl group, or the like, but such protection is not necessarily required.
  • the guanidino group of Arg is a suitable protecting group such as nitro group, tosyl group, 2-methoxybenzenesulfonyl group, methylene-2-sulfonyl group, benzyloxycarbonyl group, isobornyloxycarbonyl group, adamantyloxycarbonyl group and the like. Can be protected.
  • the deprotection reaction of these protecting groups in the amino acids having the above-mentioned protecting groups, peptides and finally obtained peptides of the present invention is also carried out by conventional methods such as catalytic reduction, liquid ammonia / sodium, hydrogen fluoride, odor. It can be carried out according to a method using hydrogen fluoride, hydrogen chloride, trifluoroacetic acid, acetic acid, formic acid, methanesulfonic acid or the like.
  • the peptide of the present invention can be obtained by chemical synthesis as described above, and can also be produced by a conventional method using genetic engineering techniques.
  • the peptide of the present invention thus obtained can be obtained according to a conventional method, for example, peptide such as ion exchange resin, partition chromatography, gel chromatography, affinity chromatography, high performance liquid chromatography (HPLC), countercurrent distribution method and the like.
  • the purification can be appropriately performed according to a method widely used in the chemical field.
  • the fusion peptide of the present invention may be any peptide as long as the peptide of the present invention is bound to the marker protein and / or peptide tag, and the marker protein is a conventionally known marker protein. It is not particularly limited as long as it is, for example, alkaline phosphatase, Fc region of antibody, HRP, GFP and the like can be specifically mentioned, and peptide tags include epitope tags such as HA, FLAG, Myc, Specific examples of conventionally known peptide tags such as affinity tags such as GST, maltose-binding protein, biotinylated peptide, oligohistidine and the like can be exemplified. Such a fusion peptide can be prepared by a conventional method.
  • the protein-peptide conjugate of the present invention is not particularly limited as long as it is a conjugate of HLA-DR1 and the peptide of the present invention.
  • the HLA-DR1 molecule and the above (A) to (E) Preferred is a form capable of binding to a CD4 + T cell that recognizes such a conjugate, such as a conjugate with a peptide consisting of any of the amino acid sequences shown.
  • the tetramer of the protein-peptide conjugate of the present invention is not particularly limited as long as it is a tetramer of a protein-peptide conjugate in which HLA-DR1 and the peptide of the present invention are bound.
  • the protein-peptide conjugate can be exemplified by a tetramer having streptavidin as a nucleus.
  • a substrate of the enzyme Bir-A is expressed at the C-terminus of HLA-DR1, and Bir- It can be obtained by mixing HLA-DR1 biotinylated by the A-dependent biotinilation method with phycoerythrin (PE) -labeled deglycosylated avidin at a ratio of 4: 1 (Altman, JD, et al .: Science 274, 94- 96, 1996).
  • PE phycoerythrin
  • HLA protein-peptide conjugates and tetramers thereof are HLA produced by a conventional method using a chemically synthesized peptide of the present invention and a genetic engineering technique using the HLA-DR1 gene (GenBank accession number AF142457).
  • HLA-DR1 GeneBank accession number AF142457
  • the fusion protein of the present invention may be any protein as long as the protein-peptide conjugate or protein-peptide conjugate tetramer is bound to the marker protein and / or peptide tag.
  • the protein is not particularly limited as long as it is a conventionally known marker protein.
  • fluorescent dyes, alkaline phosphatase, antibody Fc region, HRP, GFP and the like can be specifically mentioned.
  • Specific examples of the tag include epitope tags such as HA, FLAG, and Myc, and conventionally known peptide tags such as affinity tags such as GST, maltose-binding protein, biotinylated peptide, and oligohistidine. .
  • Such fusion proteins can be prepared by conventional methods, such as purification of protein-peptide conjugates utilizing the affinity between Ni-NTA and His tags, detection of HTLV-I specific CD4 + T cells, and other such It is also useful as a research reagent in the field.
  • antibodies that specifically bind to the peptide of the present invention include immunospecific antibodies such as monoclonal antibodies, polyclonal antibodies, chimeric antibodies, single chain antibodies, humanized antibodies, and the like.
  • the peptide of the present invention can be prepared by an ordinary method using the antigen as an antigen. Among them, a monoclonal antibody is more preferable in terms of its specificity.
  • Such an antibody that specifically binds to the peptide of the present invention such as a monoclonal antibody is not only useful for diagnosis of, for example, ATL and HAM / TSP, but also HTLV-I specific CD4 + T cells of the peptide of the present invention. It is useful for clarifying the activity mechanism and molecular mechanism of induction.
  • the antibody against the peptide of the present invention can be obtained by subjecting an animal (preferably non-human) to a peptide of the present invention, a complex of the peptide of the present invention and an immunogenic protein,
  • an animal preferably non-human
  • the hybridoma method (Nature 256, 495-497, 1975) is used to produce antibodies produced by continuous cell line cultures. ), Trioma method, human B cell hybridoma method (Immunology Today 4, 72, 1983) and EBV-hybridoma method (MONOCLONAL ANTIBODIES AND CANCER THERAPY, pp.77-96, Alan R.Liss, Inc., 1985)
  • the method can be used.
  • the polynucleotide of the present invention is a polynucleotide encoding an HTLV-I-specific CD4 + T cell-inducing peptide, that is, a polynucleotide comprising the polynucleotide sequence shown in any of the following (a) to (d):
  • the nucleotide is not particularly limited as long as it is a nucleotide (hereinafter also referred to as “polynucleotide of the present invention”).
  • the polynucleotide sequence of (a) is not particularly limited as long as it encodes any of the listed amino acid sequences, but is shown in SEQ ID NO: 24 as a polynucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 4.
  • the polynucleotide sequence shown in SEQ ID NO: 25 can be preferably exemplified as the polynucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 10.
  • polynucleotide sequence shown in SEQ ID NO: 26 can be preferably exemplified as the polynucleotide sequence encoding the amino acid sequence shown, and is shown in SEQ ID NO: 27 as the polynucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 12.
  • a polynucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 16 can be preferably exemplified as the polynucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 13
  • the polynucleotide sequence shown in SEQ ID NO: 29 can be preferably exemplified as the sequence
  • the polynucleotide sequence shown in SEQ ID NO: 30 is preferably exemplified as the polynucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 17. Can do.
  • the polynucleotide sequence of (b) is an amino acid sequence consisting of 14 to 30 consecutive amino acids in the amino acid sequence shown in SEQ ID NO: 32, and comprising an amino acid sequence comprising at least amino acid numbers 155 to 167.
  • the encoding polynucleotide sequence is not particularly limited as long as it encodes the above-mentioned amino acid sequence.
  • a polynucleotide sequence corresponding to the above-mentioned amino acid sequence is preferably exemplified. be able to.
  • Such a polynucleotide sequence can be grasped by comparing SEQ ID NO: 32 showing the amino acid sequence of the entire Tax and SEQ ID NO: 33 showing the polynucleotide sequence of the entire Tax.
  • the polynucleotide sequence (c) is 80% or more, preferably 85% or more, more preferably 90% or more, and still more preferably 95% or more with respect to the polynucleotide sequence shown in (a) or (b). Even more preferably, it is a polynucleotide sequence having 98% or more identity, and encoding a peptide having HTLV-I-specific CD4 + T cell inducing activity.
  • the polynucleotide sequence of (d) is a polynucleotide sequence in which one or several nucleotides are deleted, substituted or added in the polynucleotide shown in (a) or (b) above, and the HTLV-I A polynucleotide sequence encoding a peptide having specific CD4 + T cell inducing activity.
  • the “polynucleotide sequence in which one or several nucleotides are deleted, substituted or added” is, for example, 1 to 20, preferably 1 to 15, more preferably 1 to 10, and still more preferably.
  • the degree of “addition” and the position thereof are derived from HTLV-I-specific CD4 + T cell induction in the same manner as the peptide encoded by the modified polynucleotide sequence is composed of the amino acid sequence listed in (A) above.
  • the nucleotide is not particularly limited as long as it has the same activity, and such nucleotide alteration (mutation) is caused by, for example, mutation or post-translational modification. There is also a, but can also be artificially altered. In the present invention, all modified polynucleotide sequences having the above characteristics are included regardless of the cause and means of such modification / mutation.
  • the polynucleotide sequence of (e) above is an amino acid sequence consisting of 14 to 30 consecutive amino acids in the amino acid sequence shown in column number 32, and comprising at least an amino acid sequence of amino acid numbers 155 to 167, and , A polynucleotide sequence encoding an amino acid sequence obtained by binding an amino acid sequence of an HTLV-I-specific CTL-inducing active peptide.
  • the polynucleotide sequence (e) is a polynucleotide sequence corresponding to the amino acid sequence (E).
  • the polynucleotide of the present invention can be advantageously used when preparing the peptide of the present invention by a conventional method using genetic engineering techniques.
  • the antisense strand of the polynucleotide of the present invention is ATL or the like. It is useful as a diagnostic probe for HTLV-I tumors.
  • the HTLV-I-specific CTL inducing action enhancer or HTLV-I-specific CTL inducing action enhancing composition (preferably pharmaceutical composition) of the present invention includes any of the above amino acids (A) to (E).
  • Such a peptide of the present invention or a peptide of the present invention expressed from the expression vector of the present invention induces HTLV-I specific CD4 + T cells, and such CD4 + T cells induce HTLV-I specific CTL inducing action.
  • “enhancing the HTLV-I-specific CTL inducing action” means that an HTLV-I-specific CTL-inducing active peptide (HTLV-I-specific peptide) is induced through induction of HTLV-I-specific CD4 + T cells. Which has the ability to enhance the HTLV-I-specific CTL inducing action exerted by a peptide having an activity to induce CTL).
  • Whether or not a substance having the ability to enhance the HTLV-I-specific CTL-inducing action exhibited by the HTLV-I-specific CTL-inducing active peptide is determined by, for example, determining the substance and the HTLV-I-specific CTL-inducing active peptide. When used in combination, it can be easily confirmed by investigating whether HTLV-I-specific CTLs are more prominently compared with the case where HTLV-I-specific CTL-inducing activity peptide alone is used. .
  • HTLV-I-specific CTL is more significantly induced when used in combination, the substance has the ability to enhance the HTLV-I-specific CTL inducing action exhibited by the HTLV-I-specific CTL-inducing active peptide. It can be said that it has.
  • the expression vector includes a promoter and a polynucleotide comprising the polynucleotide sequence shown in any of (a) to (e) above, and the polynucleotide is operably linked downstream of the promoter.
  • Any expression vector can be used, and plasmids derived from Escherichia coli (eg, pET28, pGEX4T, pUC118, pUC119, pUC18, pUC19, and other plasmid DNAs), plasmids derived from Bacillus subtilis (Eg, pUB110, pTP5, and other plasmid DNA), yeast-derived plasmids (eg, YEp13, YEp24, YCp50, and other plasmid DNAs), ⁇ phage ( ⁇ gt11, ⁇ ZAP, etc.), mammalian plasmids (pCMV, pSV40) Virus vectors (adenovirus vectors,
  • the promoter is not particularly limited, and a suitable promoter may be selected according to the host, and either a constitutive promoter or an inducible promoter known in the technical field may be used. Specifically, as the promoter, CMV promoter, SV40 promoter, CAG promoter, synapsin promoter, rhodopsin promoter, CaMV promoter, glycolytic enzyme promoter, lac promoter, trp promoter, tac promoter, GAPDH promoter, GAL1 promoter, PH05 promoter, PGK promoter etc. can be mentioned.
  • the expression vector may further contain a terminator downstream of the target polynucleotide.
  • the host cell used when the peptide of the present invention is expressed using the above expression vector may be any host cell as long as the expression vector can express the peptide of the present invention, such as Escherichia coli and Streptomyces.
  • Bacterial prokaryotic cells such as Bacillus subtilis, Streptococcus and Staphylococcus, eukaryotic cells such as yeast and Aspergillus, insect cells such as Drosophila S2 and Spodoptera Sf9, L cells, CHO cells, COS cells, HeLa cells, C127 Examples include cells, BALB / c3T3 cells (including mutants lacking dihydrofolate reductase and thymidine kinase), BHK21 cells, HEK293 cells, Bowes melanoma cells, and oocyte and plant cells.
  • introduction of an expression vector capable of expressing the peptide of the present invention into a host cell is performed by Davis et al. (BASIC METHODS IN MOLECULAR BIOLOGY, 1986) and Sambrook et al. (MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed., Cold Spring). Methods described in many standard laboratory manuals such as Harbor Laboratory, Press, Cold Spring, NY, 1989), eg, calcium phosphate transfection, DEAE-dextran mediated transfection, transvection, microinjection, Cationic lipid-mediated transfection, electroporation, transduction, scrape loading, ballistic introduction, infection, and the like.
  • the HTLV-I specific CTL inducing action enhancer or HTLV-I specific CTL inducing action enhancing composition may contain a pharmaceutically acceptable carrier or diluent, an immunostimulant, an additive, etc.
  • a carrier or diluent include a stabilizer such as SPGA, sorbitol, mannitol, starch.
  • Specific examples include carbohydrates such as sucrose, glucose and dextran, proteins such as albumin and casein, protein-containing substances such as bovine serum and skim milk, and buffers such as phosphate buffer, physiological saline and water. be able to.
  • the immunostimulant examples include cytokines such as interleukin-2 (IL-2), interleukin-12 (IL-12), and tumor necrosis factor ⁇ (THF- ⁇ ).
  • cytokines such as interleukin-2 (IL-2), interleukin-12 (IL-12), and tumor necrosis factor ⁇ (THF- ⁇ ).
  • IL-2 interleukin-2
  • IL-12 interleukin-12
  • TNF- ⁇ tumor necrosis factor ⁇
  • examples include low molecular weight polypeptides (less than about 10 residues), proteins, amino acids, carbohydrates containing glucose or dextran, chelating agents such as EDTA, protein stabilizers, microbial growth inhibitors or inhibitors, and the like. However, it is not limited to these.
  • the HTLV-I-specific CTL inducing action enhancer or HTLV-I-specific CTL inducing action-enhancing composition of the present invention is orally, intravenously, intraperitoneally, intranasally, intradermally.
  • a form that can be administered subcutaneously, intramuscularly, and the like is preferable.
  • the effective amount to be administered can be appropriately determined in consideration of the type and composition of the pharmaceutical or pharmaceutical composition, the administration method, the age and weight of the patient, etc., and these can be administered one to several times per day. preferable.
  • When administered orally it is usually administered in the form of a preparation prepared by mixing with a pharmaceutical carrier.
  • a carrier that can be used in the preparation a substance that is commonly used in the preparation field and does not react with the peptide of the present invention is used.
  • the dosage form include tablets, capsules, granules, powders, syrups, suspensions, suppositories, ointments, creams, gels, patches, inhalants, injections, and the like.
  • These preparations can be prepared according to conventional methods, and in the case of liquid preparations in particular, they can be dissolved or suspended in water or other suitable medium at the time of use. Tablets and granules may be coated by a known method.
  • injection it is prepared by dissolving the peptide of the present invention in water, but it may be dissolved in physiological saline or glucose solution as necessary, and a buffer or preservative may be added. Good. These preparations may also contain other components having therapeutic value.
  • the vaccine for inducing an HTLV-I-specific immune response of the present invention includes the aforementioned HTLV-I-specific CTL inducing action enhancer of the present invention, an HTLV-I-specific CTL-inducing activity peptide, or a polynucleotide encoding the peptide If it contains, it will not restrict
  • the HTLV-I specific CTL inducing peptide is as described above. According to such a vaccine, both HTLV-I-specific CD4 + T cells and HTLV-I-specific CTL can be induced, and the HTLV-I-specific CTL can be significantly induced.
  • the vaccine for inducing immune response of the present invention can be used for treatment of HTLV-I tumors such as ATL. From the viewpoint of obtaining a suitable induction of HTLV-I-specific CTL, it is preferable to use an HTLV-I-specific CTL-inducing active peptide that is suitable for the type of HLA-A to be administered.
  • An HTLV-I specific CD4 + T cell-inducing activity peptide consisting of the amino acid sequence of (E) above or an expression vector containing the polynucleotide of (e) above as an HTLV-I specific CTL inducing action potentiator
  • it When it is contained as an active ingredient, it contains an HTLV-I-specific CTL-inducing active peptide or a polynucleotide encoding the peptide, and need not be separately included.
  • containing the HTLV-I-specific CTL inducing action enhancer of the present invention and an HTLV-I-specific CTL-inducing activity peptide or a polynucleotide encoding the peptide includes the above (E HTLV-I-specific CD4 + T cell-inducing peptide comprising the amino acid sequence of () and an expression vector comprising the polynucleotide (e) are also included for convenience.
  • the vaccine for inducing immune response is more preferably one containing various adjuvants that enhance cellular or local immunity.
  • adjuvants include peptide-specific CD4 + efficiently.
  • an adjuvant when used, it should be used as a recombinant fusion protein or recombinant fusion peptide prepared from a polynucleotide that continuously encodes various bacterial cell components and toxins that serve as adjuvants and the peptide of the present invention. You can also.
  • the immunological function test diagnostic agent of the present invention includes the peptide of the present invention, the expression vector of the present invention, a protein-peptide conjugate in which HLA-DR1 and the peptide of the present invention are bound, or 4 of the protein-peptide conjugate. It is not particularly limited as long as it is capable of examining and diagnosing an immune function, particularly an immune function against HTLV-I, with a monomer as an active ingredient.
  • a radioisotope can be used in addition to the marker protein and the peptide tag.
  • an immune function test diagnostic agent of the present invention an immune function test diagnostic agent for identifying HTLV-I-specific CD4 + T cells can be preferably exemplified.
  • the immune function test diagnostic agent of the present invention is brought into contact with peripheral blood leukocytes (lymphocytes) of a subject subject to recognize an epitope in the peptide or the like of the present invention.
  • peripheral blood leukocytes lymphocytes
  • HTLV-I Tax specific CD4 + T cells can be identified.
  • fluorescent labels such as the above-mentioned protein-peptide conjugate tetramer PE enable detection and quantification of CD4 + T cells by flow cytometry.
  • it is particularly useful for determining the effect of inducing HTLV-I-specific CD4 + T cells.
  • a mononuclear cell fraction is separated from a heparin peripheral blood sample, and a PE-labeled tetramer (protein-peptide conjugate tetramer) and an activation marker antibody such as a CD4 antibody labeled with FITC or PE-Cy5 are used.
  • the immune function of the subject can be examined and diagnosed.
  • the number of tetramer positive cells is often very small in a fresh blood sample, not only a fresh blood sample but also a peptide of the present invention or a cell expressing the same is used for a few days to a week. The same staining analysis can be performed after the culture.
  • HTLV-I-infected T cells derived from an ATL patient prior to HSCT are used to derive from an allogeneic HLA type, that is, an HLA-DR1 type donor.
  • a method of inducing HTLV-I-specific CD4 + T cells characterized by stimulating PBMCs of the same patient after HSCT in vitro, in vivo or ex vivo, and using the peptide of the present invention, HLA-DR1 positive
  • a method of inducing HTLV-I-specific CD4 + T cells characterized by stimulating PBMCs of ATL patients in vitro, in vivo or ex vivo, and using the above-described expression vector of the present invention, for example, to antigen-presenting cells in PBMCs
  • In vitro analysis of PBMCs from HLA-DR1-positive ATL patients such as genetically expressing peptides It is not particularly limited as long as it is a HTLV-I-specific CD4 + T method for inducing cells, characterized by stimulating in vivo or ex vivo, HTLV-I-specific CD4 + T cells obtained by such induction method
  • HTLV-I-specific CTL inducing action characterized by stimulating PB
  • PBMCs of HLA-DR1-positive ATL patients are used as follows: The method is not particularly limited as long as it is a method characterized by stimulation using (X) and (Y).
  • An HTLV-I-specific CD4 + T cell-inducing activity peptide consisting of the amino acid sequence of (E) above or an expression vector containing the polynucleotide of (e) above as an HTLV-I-specific CTL inducing action potentiator
  • it When it is contained as an active ingredient, it contains an HTLV-I-specific CTL-inducing active peptide or a polynucleotide encoding the peptide, so that it is not necessary to prepare it separately and stimulate PBMC with it.
  • Stimulating the PBMC using the above (X) and (Y) includes stimulating PBMC with an HTLV-I-specific CD4 + T cell-inducing activity peptide consisting of the amino acid sequence of (E), Stimulating PBMC with an expression vector containing the polynucleotide of (e) above is also included for convenience.
  • aspects of the present invention include the use of the peptide of the present invention for enhancing the HTLV-I-specific CTL inducing action, and the peptide of the present invention and HTLV-I for inducing an HTLV-I-specific immune response.
  • a characteristic ATL prevention, treatment or amelioration method is also included.
  • Tax-specific CD8 + T cells were examined in a number of ATL patients who received allogeneic hematopoietic stem cell transplantation with palliative pretreatment.
  • Table 1 shows that peripheral blood was collected from 18 ATL patients 180 days after allogeneic hematopoietic stem cell transplantation with palliative pretreatment, and Tax-specific CD8 + T cells (CTL) were obtained by flow cytometry using Tax / HLA tetramer.
  • chimerism (%) indicates the proportion of patient-derived T cell chimerism (indicating the engraftment rate of transplanted cells), and “tetramer-positive cells (%)” are among CD8-positive T cells.
  • the ratio of tetramer positive cells (Tax antigen-specific CTL) is shown.
  • “Infected cell rate” shows the number of infected cells per 1000 peripheral blood mononuclear cells (PBMC) determined quantitatively by nucleic acid.
  • PBMC peripheral blood mononuclear cells
  • PBMCs were isolated by density gradient centrifugation using Ficoll-Paque (registered trademark) PLUS (GE Healthcare UK), and Bambanker stock solution (manufactured by Nippon Genetics Co., Ltd.) PBMCs suspended in) were stored in liquid nitrogen until needed. Some of these stock cells were used to establish IL-2-dependent HTLV-I infected T cell line (ILT) and Epstein-Barr virus (EBV) transformed lymphoblast B cell line (LCL).
  • ILT IL-2-dependent HTLV-I infected T cell line
  • EBV Epstein-Barr virus
  • ILT- # 350 spontaneously immortalized (carcinogenic) by long-term culture of PBMC obtained from patient # 350 before allogeneic hematopoietic stem cell transplantation with 20% fetal calf serum (FCS; manufactured by Sigma Aldrich)
  • FCS fetal calf serum
  • the cells were subcultured with RPMI 1640 (Life Technologies, Inc.) containing 30 U / ml of recombinant human interleukin-2 (rhIL-2; manufactured by Shionogi & Co., Ltd.).
  • LCL- # 307, LCL- # 341, and LCL- # 350 were established from PBMCs obtained from ATL patients # 307, # 341, and # 350 after allogeneic hematopoietic stem cell transplantation, respectively.
  • These PBMCs were subcultured with RPMI 1640 containing 20% FCS, and then infected with B95-8 cell line culture supernatant containing EBV.
  • LCL-Kan was established from
  • A-3 Synthetic peptides In the central region of Tax antigen (residues 103 to 246), a total of 18 overlapping peptides of 12 to 25 mer in length were purchased from Scrum Inc. (see Table 2) for epitope mapping. used. HLA-A * 2402 restricted CTL epitope (Tax301-309) (SEQ ID NO: 34) (Non-patent Document 9) was used for in vitro peptide stimulation of Tax-specific CTL (Hokudo Co., Ltd.).
  • Non-Patent Document 13 Kurihara K, Shimizu Y, Takamori A, et al. Human T-cell leukemia virus type-I (HTLV-I) -specific T-cell responses detected using three-divided glutathione-S -Transfer (GST) -Tax fusion proteins. J Immunol Methods. 2006; 313 (1-2): 61-73.) and the immune response of T cells specific to HTLV-ITax was evaluated.
  • ⁇ ELISA> PBMC (1 ⁇ 10 6 cells / ml) containing 10% FCS in the presence / absence (presence or absence) of a mixture of GST-Tax-A, -B and -C proteins (GST-TaxABC) Incubated with RPMI 1640 (200 ⁇ l). After 4 days, the supernatant was collected, and the IFN- ⁇ concentration in the supernatant was measured using OptiEIA Human IFN- ⁇ ELISA Kit (BD Biosciences). The minimum detectable amount of IFN- ⁇ in this assay was 23.5 pg / ml.
  • CD8 + cells were removed from the cultured PBMC by Dynabeads M-450 CD8 (manufactured by Invitrogen) by the negative selection method according to the manufacturer's protocol.
  • To perform cytokine profiling of the HTLV-I specific CD4 + T cell line the isolated cells were stimulated with ILT- # 350 fixed with formaldehyde for 48 hours. The culture supernatant was collected, and various cytokines were measured using a human Th1 / Th2 / Th17 cytokine kit (BD Biosciecnes) of Cytokine Beads Array.
  • T4 cells Induction of A-5 HTLV-I specific CD4 + T cell line (T4 cells)
  • Patient # 350 PBMC (1 ⁇ 10 6 cells / ml) who achieved complete remission on day 180 after allogeneic hematopoietic stem cell transplantation with palliative pretreatment ) was cultured for 2 weeks in the presence of Tax301-309 peptide (100 nM).
  • Cultured PBMCs were then isolated from CD4 + cells by negative selection using Human CD4 T lymphocyte Enrichment Set-DM (BD Biosciecnes) and contained 20% FCS and rhIL-2 (100 U / ml). Subcultured with RPMI 1640. Every two to three weeks thereafter, isolated CD4 + cells were stimulated with ILT- # 350 fixed with formaldehyde.
  • HTLV-IpX specific primer pX1: 5'-CCA CTT CCC AGG GTT TAG ACA GAT CTT C-3 '(SEQ ID NO: 20) and pX4: 5'-TTC CTT ATC CCT CGA CTC CCC TCC TTC CCC-3 ′ (SEQ ID NO: 21)
  • PCR was performed in 50 ⁇ l of a reaction mixture containing 0.5 ⁇ M of each and 2 ⁇ l of cDNA.
  • GPDH glyceraldehyde-3-phosphate dehydrogenase
  • GAPDH3 ′ 5′-TCC ACC ACC CTG TTG CTG TA-3 ′ (SEQ ID NO: 23)
  • an initial activation step was performed at 94 ° C. for 1 minute, followed by 30 cycles consisting of denaturation (98 ° C., 10 seconds), annealing (60 ° C., 2 minutes) and extension (74 ° C., 30 seconds). Cycled. After electrophoresis on a 2% (w / v) agarose gel, PCR products were visualized with ethidium bromide.
  • CD3-FITC UCHT1, BioLegend
  • CD4-FITC RPA-T4, BioLegend
  • CD8-FITC RPA-T8, BioLegend
  • CD8-PE-Cy5 HIT8a, BD Biosciences
  • PE phycoerythrin
  • HLA-DRB1 * 0101 / Tax155-167 tetramer was newly made by special order from MBL.
  • the PE-labeled Tax / HLA tetramer stained whole blood or cultured cells with a combination of CD3-FITC and CD8-PE-Cy5 or CD4-PE-Cy5.
  • the whole blood sample was lysed with red blood cells, fixed with a BD FACS solution (BD Biosciences), and then washed. Samples were analyzed using FACS Calibur (Becton Dickinson), and FlowJo software (Tree Star, Inc.) was used for data analysis.
  • T4 cells (3 ⁇ 10 5 cells / ml) were treated with LCL- # 350 peptide-stimulated for 1 hour at 37 ° C. under various synthetic peptides and concentration conditions, responder cells (T4) / Stimulation cells (LCL- # 350) were co-cultured for 6 hours at an R / S ratio of 3.
  • ELISA was performed using the collected culture supernatant, and IFN- ⁇ produced by T4 cells by various peptide-specific stimuli was quantified.
  • Tax155-167 peptides were used using various HLA type LCLs (LCL- # 350, LCL- # 341, LCL- # 307 and LCL-Kan) Specific IFN- ⁇ production response was evaluated. These LCLs (1 ⁇ 10 5 cells / ml) were stimulated with 100 ng / ml Tax155-167 peptide for 1 hour, fixed with 2% formaldehyde and then in the presence of T4 cells (3 ⁇ 10 5 cells / ml). Cultured for 6 hours. The culture supernatant was collected, and IFN- ⁇ in the supernatant was measured by ELISA.
  • PBMC proliferation ability
  • B-1 Tax-specific T cell immune response in ATL patients after allogeneic hematopoietic stem cell transplantation with palliative pretreatment Immunoassay using GST-Tax fusion protein (A above) to assess Tax-specific T cell response -4).
  • the immunological function measurement method using GST-Tax fusion protein can analyze both CD4 + T cell response and CD8 + T cell response regardless of HLA type. The results of such an assay are shown in FIG. From the results of 16 patient PBMCs in FIG. 1A, it was revealed that the degree of immune response to Tax protein stimulation based on IFN- ⁇ production was diverse.
  • HTLV-I specific CD4 + T cell line from patient # 350 Allogeneic hematopoietic stem cell transplantation using HTLV-I infected T cell line (ILT- # 350) as antigen-presenting cells and palliative pretreatment HTLV-I specific CD4 + T cells were induced from the same patient # 350 PBMC 180 days later.
  • Peptide stimulation of freshly isolated patient PBMC with Tax301-309 (CTL dominant epitope presented in HLA-A * 2402) for 2 weeks induces Tax-specific CTL and is originally present in patient PBMC HTLV-I infected cells were removed.
  • CD4 + cells were isolated from the cultured cells and stimulated every 2-3 weeks with ILT- # 350 fixed with formaldehyde.
  • T4 cell When the phenotype on the cell surface of the established cell line was analyzed by flow cytometry (see A-7 above), the cells expressed CD3 and CD4 but not CD8 (FIG. 2A). + T cell line was confirmed. Hereinafter, this is called a T4 cell.
  • HTLV-I is known to preferentially infect CD4 + T cells both in vivo and in vitro (Non-patent Document 23).
  • HTLV-I infection in the above-mentioned T4 cells was examined by RT-PCR (see A-7 above), no band was confirmed at the position indicated by pX (FIG. 2B), and HTLV-I was infected. No HTLV-I specific CD4 + T cells were shown.
  • Tax is a pX gene product, and absence of Tax in the cells indicates that HTLV-I is uninfected.
  • the EB virus-transformed B cell line LCL- # 350 was used as a negative control, the HTLV-I-infected T cell line ILT- # 350 and the ATL cell line MT-2 were used as positive controls.
  • T4 cells were stimulated for 24 hours in the presence / absence of ILT- # 350 cells or LCL- # 350 cells fixed with formaldehyde, The cytokine concentration was quantified by a cytometry bead array system. As a result, T4 cells produced large amounts of IFN- ⁇ and TNF- ⁇ in response to ILT- # 350 and produced low amounts of IL-2, IL-4 and IL-10, but LCL- # 350 Production was not observed (FIG. 2C). Thus, it was shown that T4 cells are HTLV-I specific and are a Th1-type CD4 + cell line.
  • T4 cells produced significantly higher levels of IFN- ⁇ for GST-TaxABC and GST-TaxB (TaxB: amino acid sequence of amino acid numbers 113 to 237 of SEQ ID NO: 32) than the GST protein (control).
  • TaxA amino acid sequence of amino acid numbers 1 to 127 of SEQ ID NO: 32
  • GST-TaxC amino acid sequence of amino acid numbers 224 to 353 of SEQ ID NO: 32
  • production is significantly higher.
  • FIG. 3A This indicates that T4 cells recognized mainly the central region of Tax protein as an antigen.
  • Tax154-168 SEQ ID NO: 11
  • Tax155-169 SEQ ID NO: 12
  • Tax156-170 SEQ ID NO: 13
  • Tax155-167 induced the same level of IFN- ⁇ production as Tax155-169 and Tax155-168, but Tax155-166 did not reach the same level (FIG. 3E).
  • IFN- ⁇ production was examined for Tax155-169, Tax155-168, Tax155-167, and Tax146-160 (negative control) by changing their concentration conditions. It was found that the amount of IFN- ⁇ produced in T4 cells in a concentration-dependent manner relative to Tax155-167 was comparable to that produced for Tax155-169 and Tax155-168 (FIG. 3F).
  • B-4 HLA-DRB1 * 0101 restriction of Tax-specific T4 cells
  • anti-HLA-DR In the presence / absence of anti-HLA-DQ and anti-HLA class I blocking antibodies, the immune response of T4 cells stimulated with ILT- # 350 was analyzed (see A-9 above). This epitope was shown to be HLA-DR-restricted because blocking HLA-DR suppressed IFN- ⁇ production of T4 cells upon stimulation from ILT- # 350 (FIG. 4A).
  • HLA-type LCLs that display different HLA-DR were used to examine HLA-DR alleles involved in the presentation of minimal epitopes (see A-9 above).
  • Tax155-167 was presented by autologous LCL- # 350 (DR1 / 14) and allogeneic LCL- # 341 (DR1 / 15), production of IFN- ⁇ by T4 cells was confirmed (FIG. 4B).
  • This result clearly shows that this epitope is presented on antigen-presenting cells by HLA-DRB1 * 0101.
  • the known HLA-DRB1 * 0101 motif (Rammensee HG, Friede T, Stevanoviic S. MHC ligands and peptide motifs: first listing. Immunogenetics. 1995; 41 (4): 178-228.)
  • HLA-DRB1 * 0101 motif FIG. 4C.
  • Tax155-167 specific CD4 + T against Tax specific CTL proliferation in fresh PBMC of patient # 350 Cell helper function was evaluated.
  • PBMCs (A24 / 26, DR1 / 14) that were just isolated from patient # 350 540 days after allogeneic hematopoietic stem cell transplantation with palliative pretreatment were transferred to HLA-A24-restricted CTL epitope Tax301-309 peptide and / or Stimulated for 13 days with Tax155-167 peptide, an HLA-DRB1 * 0101-restricted Th1-type epitope, and the proliferation of Tax-specific CD8 + T cells was measured using the HLA-A * 2402 / Tax301-309 tetramer (A above) See -10).
  • Tax301-309-specific CD8 + T cells proliferated to 9.26% when stimulated with Tax301-309 alone (FIG. 5, upper center panel), whereas both Tax301-309 and Tax155-167 were When stimulated in vitro, Tax-specific CD8 + T cells were significantly increased to 62.3% (upper right panel of FIG. 5). Measurements were performed in the same manner for HLA-DRB1 * 0101 + HTLV-I-infected patients who received allogeneic hematopoietic stem cell transplantation by two other palliative pretreatments.
  • Patient # 364 used PBMC (A24 / 26, DR1 /-) that had just been isolated from the patient 180 days after transplantation, and patient # 341 had PBMC that had been isolated from the patient 360 days after transplantation. (A24 / 33, DR1 / 15) was used.
  • PBMC was stimulated with Tax301-309 alone, an HLA-A24-restricted CTL epitope, Tax301-309-specific CD8 + T cells were 0.85% (patient # 364) and 7.7% (patient # 341). While proliferated (middle middle panel, lower middle panel in FIG. 5), when PBMC were co-stimulated with both Tax301-309 and Tax155-167, Tax301-309 specific CD8 + T cells were 15.5.
  • PBMCs of 3 patients had a detectable level of Tax-specific CD8 + T cells that bind to the tetramer described above, prior to stimulation with the Tax epitope. Were present.
  • Tax155-167 specific CD4 + T cells are present in HLA-DRB1 * 0101 + HTLV-I infected patients who received allogeneic hematopoietic stem cell transplantation by palliative pretreatment, Tax301-309 / Tax155-167 It was shown that the CD8 + T cell response was significantly enhanced by stimulation with mixed epitopes compared to stimulation with Tax301-309 alone.
  • HLA-DRB1 * 0101 + HTLV-I infected individuals maintain Tax155-167 specific CD4 + T cells
  • HLA-DRB1 * 0101 / Tax155-167 tetramers were generated and Tax155-167 specific by detecting the CD4 + T cells directly, from the HLA-DRB1 * 0101 + two patients who received allogeneic hematopoietic stem cell transplantation in the PBMC of freshly isolated, whether there is Tax155-167-specific CD4 + T cells It was confirmed.
  • Patient # 350 used PBMCs that had just been isolated from the patient 540 days after transplantation (A24 / 26, DR1 / 14), and patient # 364 had PBMCs that had been isolated from the patient 180 days after transplantation.
  • PBMC (A24 / 33, DR1 / 15) that had just been isolated from the same patient 360 days after transplantation was used.
  • Tax155-167 specific CD4 + T cells were present at levels detectable (0.11%) without culture or stimulation (ex vivo) (FIG. 6A, upper left panel), Tax155-167. 13 days after stimulation with the peptide, it grew to 11.6% (FIG. 6A, upper right panel).
  • no tax-specific CD4 + T cells were detected ex vivo (FIG. 6A, middle left panel), but proliferated to 0.37% when stimulated in vitro with Tax155-167 peptide ( FIG.
  • Tax-specific CD4 + T cells were not detected ex vivo in HLA-DRB1 * 0101 + seronegative donor # 365 (negative control) (lower left panel in FIG. 6A), 13 days after stimulation with Tax155-167 peptide However, it was not detectable (the right panel at the bottom of FIG. 6A).
  • Tax155-Two in HTLV-I infected individuals carrying HLA-DRB1 * 0101 asymptomatic carrier (AC) # 310 and HTLV-I related myelopathy / tropical spastic paraparesis patient # 294).
  • AC asymptomatic carrier
  • HTLV-I related myelopathy / tropical spastic paraparesis patient # 294 asymptomatic carrier (AC) # 310 and HTLV-I related myelopathy / tropical spastic paraparesis patient # 294).
  • AC asymptomatic carrier
  • tetramer positive cells were detected in peripheral CD4 + T cells, respectively (FIG. 6B).
  • Tax155-167 specific CD4 + T cells were maintained in HTLV-I infected individuals expressing HLA-DRB1 * 0101 alleles regardless of the presence or absence of hematopoietic stem cell transplantation. .
  • the present invention can be used in fields related to the induction of CD4 + T cells specific for human T cell leukemia virus type I (HTLV-I). More specifically, an HTLV-I-specific CTL inducing action enhancer, an HTLV-I-specific immune response inducing vaccine, an immune function test diagnostic agent for identifying HTLV-I-specific CD4 + T cells, It can be suitably used in the field relating to the method for inducing HTLV-I-specific CD4 + T cells.
  • HTLV-I human T cell leukemia virus type I

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Abstract

La présente invention concerne un peptide doté d'une activité d'induction des lymphocytes T CD4+ spécifiques du HTLV-I, lequel peptide est doté d'une activité d'induction d'un lymphocyte T CD4+ spécifique du HTLV-I; une séquence activatrice de l'effet d'induction des lymphocytes T cytotoxiques (CTL) spécifiques du HTLV-I qui comprend le peptide précité; et un vaccin permettant d'induire une réponse immunitaire spécifique du HTLV-I, un diagnostic permettant d'examiner une fonction immune, etc., chacun d'eux faisant appel audit peptide. La présente invention porte sur un peptide ayant une activité d'induction des lymphocytes T CD4+spécifiques du HTLV-I, lequel peptide comprend une séquence d'acides aminés parmi l'une quelconque des séquences (A) à (E), etc. : (A) une séquence d'acides aminés représentée par l'une des séquences SEQ ID NO : 4, 10, 11, 12, 13, 16, 17 et 19; (B) une séquence d'acides aminés constituée de 14 à 30 acides aminés consécutifs dans la séquence d'acides aminés représentée par SEQ ID NO : 32, ladite séquence d'acides aminés comprenant la séquence d'acides aminés d'au moins l'un des acides aminés n° 155 à 167; (C) une séquence d'acides aminés possédant une homogénéité de 80 % ou plus avec la séquence d'acides aminés de (A) ou (B), un peptide comprenant ladite séquence d'acides aminés étant doté d'une activité d'induction des lymphocytes T CDd4+spécifiques du HTLV-I; (D) une séquence d'acides aminés dérivée de la séquence d'acides aminés de (A) ou (B) par délétion, substitution ou ajout d'un ou de plusieurs acides aminés, un peptide comprenant ladite séquence d'acides aminés doté une activité d'induction des lymphocytes T CD4+spécifiques du HTLV-I; et (E) une séquence d'acides aminés comprenant une séquence d'acides aminés constituée de 14 à 30 acides aminés consécutifs dans la séquence d'acides aminés représentée par SEQ ID NO : 32, ladite séquence d'acides aminés comprenant la séquence d'acides aminés d'au moins l'un des acides aminés n° 155 à 167, à laquelle est liée la séquence d'acides aminés d'un peptide doté d'une activité d'induction des lymphocytes T cytotoxiques (CTL) spécifiques du HTLV-I.
PCT/JP2014/000053 2013-01-09 2014-01-09 EPITOPE DES LYMPHOCYTES T CD4+ SPÉCIFIQUE DE Tax, RESTREINT À HLA-DR1 WO2014109289A1 (fr)

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US5717058A (en) * 1993-02-23 1998-02-10 Somatogen, Inc. Peptide inhibitors of tax-dependent transcription
US5420244A (en) * 1993-08-06 1995-05-30 The United States Of America As Represented By The Department Of Health And Human Services Methods and compositions for diagnosing HTLV-I associated myelopathy and adult T-cell leukemia
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