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WO2015083763A1 - Peptide dérivé du vegfr2, et vaccin le contenant - Google Patents

Peptide dérivé du vegfr2, et vaccin le contenant Download PDF

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Publication number
WO2015083763A1
WO2015083763A1 PCT/JP2014/082059 JP2014082059W WO2015083763A1 WO 2015083763 A1 WO2015083763 A1 WO 2015083763A1 JP 2014082059 W JP2014082059 W JP 2014082059W WO 2015083763 A1 WO2015083763 A1 WO 2015083763A1
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Prior art keywords
peptide
present
amino acid
ctl
vegfr2
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Japanese (ja)
Inventor
卓也 角田
龍司 大沢
祥子 山下
朝久 渡辺
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Oncotherapy Science Inc
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Oncotherapy Science Inc
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    • 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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/71—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
    • 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
    • A61K39/0005—Vertebrate antigens
    • 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
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A61K39/001102—Receptors, cell surface antigens or cell surface determinants
    • A61K39/001103—Receptors for growth factors
    • A61K39/001109—Vascular endothelial growth factor receptors [VEGFR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/06—Antipsoriatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • 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
    • A61P37/04—Immunostimulants
    • 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
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • 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
    • G01N33/5005—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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/10—Screening for compounds of potential therapeutic value involving cells

Definitions

  • the present invention relates to a peptide effective for inhibiting angiogenesis and treating and preventing diseases mediated by angiogenesis.
  • the present invention also relates to angiogenesis inhibitors comprising these peptides and pharmaceutical compositions and vaccines for the treatment and / or prevention of diseases mediated by angiogenesis.
  • Tumor growth is generally limited to 1-2 mm 3 in the absence of a blood supply in which blood vessels are born, and angiogenesis has a critical role in tumor invasion, growth, and metastasis (non-patented Reference 1-4). It has also been shown that inhibition of tumor angiogenesis is associated with suppression of tumor progression.
  • VEGF vascular endothelial growth factor
  • VEGFR VEGF receptor
  • These studies have shown that tumor growth can be successfully suppressed in vitro or in vivo using monoclonal antibodies, recombinant receptors, or inhibitors of signaling (Non-Patent Documents 5-10). .
  • these strategies require frequent or continuous administration of reagents at relatively high dose levels, which can be associated with considerable inconvenience and side effects.
  • VEGFR1 is the first identified VEGF receptor (15), VEGF (VEGF-A), and two other members of the VEGF family, VEGF-B (16) and placenta It interacts with growth factor (PlGF) (Non-patent Document 17).
  • PlGF is expected to move VEGF from VEGFR1 so that more VEGF can bind to VEGFR2 and activate VEGFR2, thereby enhancing angiogenesis driven by VEGF (non-patent literature).
  • Other studies have shown that there is a synergy between VEGF and PlGF in vivo, especially in pathological situations, as evidenced by impaired tumorigenesis and vascular leakage in PlGF-/-mice. (Non-patent Document 19).
  • CNV human choroidal neovascularization
  • Non-patent Literature 1-2 Non-Patent Literature 20
  • these peptides are effective for the treatment of diseases mediated by angiogenesis such as tumors (Non-patent Document 21) and macular degeneration (Patent Documents 3-4).
  • the present invention relates to a peptide capable of inducing cytotoxic T cells (CTL) specific to cells expressing VEGFR2.
  • CTL cytotoxic T cells
  • APC antigen-presenting cells
  • HLA human leukocyte antigen
  • CTLs showing cytotoxic activity specific to VEGFR2-expressing cells are induced.
  • the peptides that have been identified so far and have the ability to induce CTL derived from VEGFR2 are HLA-A2 or HLA-A24-restricted peptides, and induce CTL in cells that do not express these HLA I can't. Therefore, conventional peptides are not suitable when immunotherapy is performed in subjects who do not have these HLA.
  • HLA-A11 and HLA-A33 are alleles commonly found in Asians (Sette A, Sidney J., Immunogenetics 1999, 50: 201-12), but in HLA-A11 or HLA-A33 positive subjects In contrast, it is desirable to administer HLA-A11 or HLA-A33 restricted peptides. Therefore, the present invention relates to a VEGFR2-derived peptide restricted to HLA-A11 or HLA-A33 and having a CTL inducing ability. From the results disclosed herein, the peptide of the present invention is an HLA-A11 or HLA-A33-restricted epitope peptide that can induce a strong and specific immune response against cells expressing VEGFR2. Proven.
  • VEGFR2-derived peptide capable of inducing CTL in a HLA-A11 or HLA-A33-restricted manner.
  • These peptides can be used to induce CTL in vitro, ex vivo or in vivo, or can be used to administer to a subject for the purpose of inducing an immune response against vascular endothelial cells expressing VEGFR2.
  • Preferred peptides are those comprising an amino acid sequence selected from SEQ ID NOs: 1, 43, 70 and 89, more preferably nonapeptides or decapeptides, more preferably SEQ ID NOs: 1, 43, 70 and A peptide consisting of an amino acid sequence selected from 89.
  • the peptides of the present invention have one, two, or more amino acids substituted, deleted, inserted and / or added so long as the resulting modified peptide retains the CTL inducibility of the original peptide Also includes peptides.
  • the present invention also provides an isolated polynucleotide that encodes any one of the peptides of the present invention. These polynucleotides, like the peptides of the present invention, can be used to induce APCs with the ability to induce CTLs, or are administered to a subject to induce an immune response against vascular endothelial cells that express VEGFR2. be able to.
  • the present invention also provides an agent or composition comprising one or more peptides of the present invention or one or more polynucleotides encoding one or more peptides of the present invention.
  • the agent or composition of the present invention is preferably a pharmaceutical agent or composition.
  • the peptides of the invention are presented on the surface of the APC, thereby inducing CTL targeting the peptide.
  • a composition for inducing CTL comprising one or more peptides of the present invention or one or more polynucleotides encoding one or more peptides of the present invention This is a further object of the present invention.
  • the present invention further provides one or more peptides of the present invention or one of the present invention formulated for the treatment and / or prevention of diseases mediated by angiogenesis and prevention of its recurrence after surgery.
  • Pharmaceutical compositions comprising one or more polynucleotides encoding one or more peptides are provided.
  • a method for inducing APC having the ability to induce CTL comprising contacting APC with one or more peptides of the present invention, or introducing a polynucleotide encoding any one of the peptides of the present invention into APC It is a further object of the present invention to provide a method comprising the steps of:
  • the present invention also comprises a step of co-culturing a CD8-positive T cell with APC that presents a complex of the HLA antigen and the peptide of the present invention on its surface, and the CD8 positive T cell is converted to an HLA antigen and the peptide of the present invention.
  • Co-cultured with exosomes presenting the complex with exosomes on their surface, or each T cell receptor (TCR) subunit capable of binding to the peptide of the invention presented by the HLA antigen on the cell surface Provided is a method for inducing CTL comprising the step of introducing a vector containing the encoding polynucleotide into CD8-positive T cells.
  • the invention further provides an isolated CTL that targets the peptide of the invention.
  • a method for inducing an immune response against vascular endothelial cells expressing VEGFR2 in a subject comprising the peptide of the present invention or a polynucleotide encoding the peptide, an exosome or APC presenting the peptide, and the peptide on its surface
  • FIG. 1 is composed of photographs (a) and (b) showing the results of an IFN- ⁇ ELISPOT assay in CTL induced with a VEGFR2-derived peptide.
  • a typical example of positive data showing strong IFN- ⁇ production compared to the control is shown in (a).
  • a typical example of negative data that did not show specific IFN- ⁇ production is shown in (b).
  • “+” indicates IFN- ⁇ production for target cells pulsed with an appropriate peptide
  • ⁇ indicates IFN- ⁇ production for target cells not pulsed with any peptide.
  • FIG. 2 is composed of photographs (a) and (b) showing the results of an IFN- ⁇ ELISPOT assay in CTL induced with a VEGFR2-derived peptide.
  • a typical example of positive data showing strong IFN- ⁇ production compared to the control is shown in (a).
  • a typical example of negative data that did not show specific IFN- ⁇ production is shown in (b).
  • “+” indicates IFN- ⁇ production for target cells pulsed with an appropriate peptide
  • ⁇ indicates IFN- ⁇ production for target cells not pulsed with any peptide.
  • FIG. 3 shows VEGFR2-A11-9-319 (SEQ ID NO: 1) (a), VEGFR2-A11-9-863 (SEQ ID NO: 2) (b), VEGFR2-A11-9-521 (SEQ ID NO: 3) ) (C), VEGFR2-A11-9-973 (SEQ ID NO: 4) (d), VEGFR2-A11-9-309 (SEQ ID NO: 5) (e), VEGFR2-A11-9-860 (SEQ ID NO: 9) (f), VEGFR2-A11-9-134 (SEQ ID NO: 15) (g), VEGFR2-A11-9-195 (SEQ ID NO: 26) (h), VEGFR2-A11-9-502 (SEQ ID NO: : 27) (i), VEGFR2-A11-9-1281 (SEQ ID NO: 39) (j), VEGFR2-A11-10-576 (SEQ ID NO: 40) (k), VEGFR2-A11-10-397 (sequence) Number: 41) (l), VEGFR2-A11
  • VEGFR2-A11-9-319 (SEQ ID NO: 1) (a), VEGFR2-A11-9-863 (SEQ ID NO: 2) (b), VEGFR2-A11-9-521 (SEQ ID NO: 3) ) (C), VEGFR2-A11-9-973 (SEQ ID NO: 4) (d), VEGFR2-A11-9-309 (SEQ ID NO: 5) (e), VEGFR2-A11-9-860 (SEQ ID NO: 9) (f), VEGFR2-A11-9-134 (SEQ ID NO: 15) (g), VEGFR2-A11-9-195 (SEQ ID NO: 26) (h), VEGFR2-A11-9-502 (SEQ ID NO: : 27) (i), VEGFR2-A11-9-1281 (SEQ ID NO: 39) (j), VEGFR2-A11-10-576 (SEQ ID NO: 40) (k), VEGFR2-A11-10-397 (sequence) Number: 41) (l), VEGFR2-A11-10-308
  • VEGFR2-A11-9-319 (SEQ ID NO: 1) (a), VEGFR2-A11-9-863 (SEQ ID NO: 2) (b), VEGFR2-A11-9-521 (SEQ ID NO: 3) ) (C), VEGFR2-A11-9-973 (SEQ ID NO: 4) (d), VEGFR2-A11-9-309 (SEQ ID NO: 5) (e), VEGFR2-A11-9-860 (SEQ ID NO: 9) (f), VEGFR2-A11-9-134 (SEQ ID NO: 15) (g), VEGFR2-A11-9-195 (SEQ ID NO: 26) (h), VEGFR2-A11-9-502 (SEQ ID NO: : 27) (i), VEGFR2-A11-9-1281 (SEQ ID NO: 39) (j), VEGFR2-A11-10-576 (SEQ ID NO: 40) (k), VEGFR2-A11-10-397 (sequence) Number: 41) (l), VEGFR2-A11-10-308
  • FIG. 4 shows VEGFR2-A11-9-319 (SEQ ID NO: 1) (a), VEGFR2-A11-9-863 (SEQ ID NO: 2) (b), VEGFR2-A11-9-521 (SEQ ID NO: 3) ) (C), VEGFR2-A11-9-973 (SEQ ID NO: 4) (d), VEGFR2-A11-9-309 (SEQ ID NO: 5) (e), VEGFR2-A11-9-860 (SEQ ID NO: 9) (f), VEGFR2-A11-9-134 (SEQ ID NO: 15) (g), VEGFR2-A11-9-195 (SEQ ID NO: 26) (h), VEGFR2-A11-9-502 (SEQ ID NO: : 27) (i), VEGFR2-A11-9-1281 (SEQ ID NO: 39) (j), VEGFR2-A11-10-576 (SEQ ID NO: 40) (k), VEGFR2-A11-10-397 (sequence) No.
  • VEGFR2-A11-9-319 (SEQ ID NO: 1) (a), VEGFR2-A11-9-863 (SEQ ID NO: 2) (b), VEGFR2-A11-9-521 (SEQ ID NO: 3) ) (C), VEGFR2-A11-9-973 (SEQ ID NO: 4) (d), VEGFR2-A11-9-309 (SEQ ID NO: 5) (e), VEGFR2-A11-9-860 (SEQ ID NO: 9) (f), VEGFR2-A11-9-134 (SEQ ID NO: 15) (g), VEGFR2-A11-9-195 (SEQ ID NO: 26) (h), VEGFR2-A11-9-502 (SEQ ID NO: : 27) (i), VEGFR2-A11-9-1281 (SEQ ID NO: 39) (j), VEGFR2-A11-10-576 (SEQ ID NO: 40) (k), VEGFR2-A11-10-397 (sequence) No.
  • VEGFR2-A11-9-319 (SEQ ID NO: 1) (a), VEGFR2-A11-9-863 (SEQ ID NO: 2) (b), VEGFR2-A11-9-521 (SEQ ID NO: 3) ) (C), VEGFR2-A11-9-973 (SEQ ID NO: 4) (d), VEGFR2-A11-9-309 (SEQ ID NO: 5) (e), VEGFR2-A11-9-860 (SEQ ID NO: 9) (f), VEGFR2-A11-9-134 (SEQ ID NO: 15) (g), VEGFR2-A11-9-195 (SEQ ID NO: 26) (h), VEGFR2-A11-9-502 (SEQ ID NO: : 27) (i), VEGFR2-A11-9-1281 (SEQ ID NO: 39) (j), VEGFR2-A11-10-576 (SEQ ID NO: 40) (k), VEGFR2-A11-10-397 (sequence) No.
  • FIG. 5 shows VEGFR2-A33-9-823 (SEQ ID NO: 87) (a), VEGFR2-A33-9-114 (SEQ ID NO: 89) (b), VEGFR2-A33-9-214 (SEQ ID NO: 90). ) (C), VEGFR2-A33-9-577 (SEQ ID NO: 21) (d), VEGFR2-A33-10-213 (SEQ ID NO: 104) (e), VEGFR2-A33-10-97 (SEQ ID NO: 83) (f), VEGFR2-A33-10-576 (SEQ ID NO: 40) (g), VEGFR2-A33-10-113 (SEQ ID NO: 70) (h) and VEGFR2-A33-10-1046 (SEQ ID NO: 114) Consists of a series of line graphs (a) to (i) showing the results of an IFN- ⁇ ELISA assay that in turn demonstrates IFN- ⁇ production of the CTL line stimulated in (i).
  • FIG. 5 shows VEGFR2-A33-9-823 (SEQ ID NO: 87) (a), VEGFR2-A33-9-114 (SEQ ID NO: 89) (b), VEGFR2-A33-9-214 (SEQ ID NO: 90).
  • VEGFR2-A33-9-577 SEQ ID NO: 21
  • VEGFR2-A33-10-213 SEQ ID NO: 104
  • VEGFR2-A33-10-97 SEQ ID NO: 83
  • VEGFR2-A33-10-576 SEQ ID NO: 40
  • VEGFR2-A33-10-113 SEQ ID NO: 70
  • h VEGFR2-A33-10-1046
  • SEQ ID NO: 114 Consists of a series of line graphs (a) to (i) showing the results of an IFN- ⁇ ELISA assay that in turn demonstrates IFN- ⁇ production of the CTL line stimulated in (i).
  • FIG. 6 shows VEGFR2-A33-9-114 (SEQ ID NO: 89) (a), VEGFR2-A33-9-214 (SEQ ID NO: 90) (b), VEGFR2-A33-9-577 (SEQ ID NO: 21). ) (C), VEGFR2-A33-10-213 (SEQ ID NO: 104) (d), VEGFR2-A33-10-97 (SEQ ID NO: 83) (e), VEGFR2-A33-10-113 (SEQ ID NO: 70)
  • FIG. 7 consists of a series of line graphs (a)-(c) showing specific CTL activity against target cells expressing VEGFR2 and HLA-A * 1101.
  • COS7 cells transfected with HLA-A * 1101 or full-length VEGFR2 gene were prepared as controls.
  • CTL lines or clones established using VEGFR2-A11-9-319 (SEQ ID NO: 1) (a) and VEGFR2-A11-10-159 (SEQ ID NO: 43) (b) are VEGFR2 and HLA- It showed specific CTL activity against COS7 cells transfected with both A * 1101 (black diamonds).
  • FIG. 7 consists of a series of line graphs (a)-(c) showing specific CTL activity against target cells expressing VEGFR2 and HLA-A * 1101. COS7 cells transfected with HLA-A * 1101 or full-length VEGFR2 gene were prepared as controls.
  • VEGFR2-A11-9-319 SEQ ID NO: 1
  • VEGFR2-A11-10-159 SEQ ID NO: 43
  • b VEGFR2 and HLA- It showed specific CTL activity against COS7 cells transfected with both A * 1101 (black diamonds).
  • no significant specific CTL activity was detected against target cells expressing either HLA-A * 1101 (triangle) or VEGFR2 (circle).
  • CTL clones established using VEGFR2-A11-9-863 SEQ ID NO: 2 did not show specific CTL activity (c).
  • FIG. 8 consists of a series of line graphs (a)-(c) showing specific CTL activity against target cells expressing VEGFR2 and HLA-A * 3303.
  • COS7 cells transfected with HLA-A * 3303 or full-length VEGFR2 gene were prepared as controls.
  • CTL lines or CTL clones established using VEGFR2-A33-9-114 (SEQ ID NO: 89) (a) and VEGFR2-A33-10-113 (SEQ ID NO: 70) (b) are VEGFR2 and HLA- It showed specific CTL activity against COS7 cells transfected with both A * 3303 (black diamonds).
  • FIG. 8 consists of a series of line graphs (a)-(c) showing specific CTL activity against target cells expressing VEGFR2 and HLA-A * 3303. COS7 cells transfected with HLA-A * 3303 or full-length VEGFR2 gene were prepared as controls.
  • CTL lines or CTL clones established using VEGFR2-A33-9-114 (SEQ ID NO: 89) (a) and VEGFR2-A33-10-113 (SEQ ID NO: 70) (b) are VEGFR2 and HLA- It showed specific CTL activity against COS7 cells transfected with both A * 3303 (black diamonds). On the other hand, no significant specific CTL activity was detected against target cells expressing either HLA-A * 3303 (triangle) or VEGFR2 (circle). In contrast, although a typical example of negative data, CTL clones established using VEGFR2-A33-9-577 (SEQ ID NO: 21) did not show specific CTL activity (c).
  • the words “a”, “an” and “the” mean “at least one” unless otherwise specified.
  • isolated and purified as used with respect to a substance (eg, peptide, antibody, polynucleotide, etc.) refer to at least one substance that the substance can otherwise be contained in a natural source. Indicates that it does not contain substantially.
  • an isolated or purified peptide is substantially free of cellular material, such as carbohydrates, lipids, or other contaminating proteins from the cell or tissue source from which the peptide is derived, or is chemically synthesized. Refers to a peptide that is substantially free of chemical precursors or other chemicals.
  • substantially free of cellular material includes preparations of a peptide in which the peptide is separated from the cellular components of the cell from which it was isolated or recombinantly produced.
  • a peptide that is substantially free of cellular material is a peptide having less than about 30%, 20%, 10%, or 5% (dry weight basis) of a heterologous protein (also referred to herein as “contaminating protein”).
  • a heterologous protein also referred to herein as “contaminating protein”.
  • the peptide When the peptide is produced by chemical synthesis, the peptide is preferably substantially free of chemical precursors or other chemicals, and the chemical precursors or other chemicals involved in peptide synthesis are included in the peptide preparation. Includes preparations of peptides having less than about 30%, 20%, 10%, 5% (dry weight basis) of volume. Certain peptide preparations contain isolated or purified peptides due to the appearance of a single band after eg sodium dodecyl sulfate (SDS) -polyacrylamide gel electrophoresis and Coomassie brilliant blue staining of the gel. Can show. In preferred embodiments, the peptides and polynucleotides of the invention are isolated or purified.
  • SDS sodium dodecyl sulfate
  • Coomassie brilliant blue staining of the gel Can show.
  • the peptides and polynucleotides of the invention are isolated or purified.
  • polypeptide refers to a polymer of amino acid residues.
  • the term refers to amino acid polymers that are residues in which one or more amino acid residues have been modified or are non-natural residues such as artificial chemical mimetics of the corresponding natural amino acids, and natural amino acids Applied to polymer.
  • amino acid refers to natural and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function similarly to natural amino acids. Natural amino acids are those encoded by the genetic code and amino acids that are post-translationally modified in cells (eg, hydroxyproline, ⁇ -carboxyglutamic acid, and O-phosphoserine).
  • amino acid analog has the same basic chemical structure as a natural amino acid (hydrogen, carboxy group, amino group, and alpha carbon attached to the R group), but has a modified R group or modified backbone. Refers to a compound (eg, homoserine, norleucine, methionine, sulfoxide, methionine methylsulfonium).
  • amino acid mimetic refers to a compound that has a structure that is different from a common amino acid, but that has a similar function.
  • amino acid may be referred to in this specification by a generally known three-letter code or one-letter code recommended by the IUPAC-IUB Biochemical Nomenclature Commission (Biochemical Nomenclature Commission).
  • nucleic acid is used interchangeably herein and are referred to by the generally accepted one-letter code unless otherwise noted.
  • pharmaceutical as in the case of “pharmaceutical agents” and “pharmaceutical compositions” and the active ingredient and carrier
  • pharmaceutical agent and “pharmaceutical composition” refer to a mixture of a molecule or compound of the invention and a pharmaceutically or physiologically acceptable carrier. Refers to any product made.
  • composition is intended to encompass a product containing a specific amount of a specific component and any product that results directly or indirectly from a combination of a specific amount of a specific component.
  • a pharmaceutical composition is one from a product comprising an active ingredient and an inert ingredient that constitutes a carrier, as well as a combination, complexation or aggregation of any two or more ingredients.
  • the pharmaceutical compositions of the present invention encompass any composition made by admixing a compound or cell of the present invention and a pharmaceutically or physiologically acceptable carrier.
  • pharmaceutically acceptable carrier or “physiologically acceptable carrier” includes liquid or solid fillers, diluents, excipients, solvents and encapsulating materials. It means a pharmaceutically or physiologically acceptable material, composition, substance, or vehicle that is not limited thereto.
  • the term “disease mediated by angiogenesis” refers to a disease in which angiogenesis is involved in the development and / or progression of the disease, examples of which are related to angiogenesis in various cancers and choroids.
  • Diseases neovascular macular disease: age-related macular degeneration, myopic macular degeneration, retinitis pigmentosa, central exudative chorioretinopathy, various retinitis pigmentosa, choroidal atrophy, choroideremia, choroidal osteoma Etc.
  • diabetic retinopathy rheumatoid arthritis
  • psoriasis and atherosclerosis. More specifically, it refers to a disease mediated by angiogenesis involving the expression of the VEGFR2 gene. At the diseased sites of these diseases, the VEGFR2 gene is expressed in vascular endothelial cells.
  • cytotoxic T lymphocyte refers to a sub-group of T lymphocytes that can recognize tumor / cancer cells, virus-infected cells) and induce the death of such cells.
  • HLA-A11 refers to the HLA-A11 type, including subtypes such as HLA-A * 1101, HLA-A * 1102, HLA-A * 1103, HLA-A * 1104 and the like.
  • HLA-A33 refers to the HLA-A33 type including subtypes such as HLA-A * 3303, HLA-A * 3301, and HLA-A * 3304.
  • treatment may reduce the expression of the VEGFR2 gene, inhibit angiogenesis, or A treatment is considered “effective” if it provides a clinical benefit, such as a reduction in the symptoms of a disease mediated by angiogenesis.
  • a treatment is considered “effective” if it provides a clinical benefit, such as a reduction in the symptoms of a disease mediated by angiogenesis.
  • “effective” means that the treatment delays or prevents the onset of the disease or prevents or alleviates the clinical symptoms of the disease. Efficacy is determined in connection with any known method for diagnosing or treating a particular disease type.
  • prevention is used herein to reduce the mortality or morbidity burden due to the disease. Including any work to be made. Prevention can take place at “primary, secondary, and tertiary prevention levels”. Primary prevention avoids the development of disease, whereas secondary and tertiary level prevention, in addition to preventing disease progression and the appearance of symptoms, restores function and is disease-related Including the aim of reducing the adverse effects of existing diseases by reducing the complications of Alternatively, prophylaxis can include a wide range of prophylactic treatment aimed at reducing the severity of a particular disorder, eg, reducing tumor growth and metastasis.
  • cancer treatment and / or prevention and / or prevention of its recurrence after surgery includes the following stages: surgical excision of cancer cells, inhibition of cancer cell growth, tumor regression Or any of the stages such as regression, induction of remission and suppression of cancer development, tumor regression, and reduction or inhibition of metastasis.
  • Effective treatment and / or prevention of cancer reduces mortality, improves the prognosis of individuals with cancer, decreases the level of tumor markers in the blood, and detectable symptoms associated with cancer To ease.
  • symptom relief or amelioration constitutes effective treatment and / or prevention, including 10%, 20%, 30%, or more relief or symptom stability.
  • kit as used herein is used in reference to combinations of reagents and other substances. It is contemplated herein that the kit can include a microarray, chip, marker, and the like. The term “kit” is not intended to be limited to a particular combination of reagents and / or substances.
  • antibody refers to immunoglobulins and fragments thereof that react specifically with the designated protein or peptide thereof.
  • Antibodies can include human antibodies, primatized antibodies, chimeric antibodies, bispecific antibodies, humanized antibodies, antibodies fused with other proteins or radiolabels, and antibody fragments.
  • antibody is used in a broad sense, specifically, an intact monoclonal antibody, a polyclonal antibody, a multispecific antibody formed from two or more intact antibodies (for example, a bispecific antibody). And antibody fragments as long as they exhibit the desired biological activity.
  • Antibody refers to all classes (eg, IgA, IgD, IgE, IgG, and IgM).
  • HLA-A11 and HLA-A33 are alleles commonly found in Asians (Sette A, Sidney J., Immunogenetics 1999, 50: 201-12).
  • providing a VEGFR2-derived CTL-inducing peptide that is constrained by HLA-A11 or HLA-A33 can provide many Asians with an effective treatment for diseases mediated by angiogenesis.
  • the present invention provides a VEGFR2-derived peptide that can induce CTLs in a HLA-A11 or HLA-A33-restricted manner.
  • the peptide of the present invention is a VEGFR2-derived peptide capable of inducing CTL in a HLA-A11 or HLA-A33-restricted manner.
  • Peptides capable of inducing CTL in an HLA-A11 restricted manner include peptides having an amino acid sequence selected from SEQ ID NOs: 1-5, 9, 15, 26, 27, and 39-45.
  • a peptide capable of inducing CTL in an HLA-A33-restricted manner a peptide having an amino acid sequence selected from SEQ ID NOs: 21, 40, 70, 83, 87, 89, 90, 104 and 114 Is mentioned.
  • CTLs having cytotoxic activity specific to these peptides can be established by in vitro stimulation of T cells by dendritic cells (DC) pulsed with these peptides. Established CTLs show specific cytotoxic activity against target cells pulsed with each peptide.
  • DC dendritic cells
  • the VEGFR2 gene is a strong target for immunotherapy because it is strongly expressed in vascular endothelial cells at the disease site in diseases mediated by angiogenesis, but is not expressed in most normal organs. Therefore, the peptide of the present invention can be suitably used for immunotherapy of diseases mediated by angiogenesis.
  • a preferred peptide is a nonapeptide (a peptide consisting of 9 amino acid residues) or a decapeptide (a peptide consisting of 10 amino acid residues), and SEQ ID NOs: 1 to 5, 9, 15, 21, 26, 27, 39 to Peptides having an amino acid sequence selected from 45, 70, 83, 87, 89, 90, 104 and 114 are more preferred.
  • a peptide having the amino acid sequence set forth in SEQ ID NO: 1 or 43 is suitable for inducing CTLs that show specific cytotoxic activity against cells expressing HLA-A11 and VEGFR2, and HLA-A11 It can be suitably used for immunotherapy of diseases mediated by angiogenesis in positive patients.
  • the peptide having the amino acid sequence set forth in SEQ ID NO: 70 or 89 is suitable for inducing CTLs that show specific cytotoxic activity against cells expressing HLA-A33 and VEGFR2, and is HLA-A33 positive It can be suitably used for immunotherapy of diseases mediated by angiogenesis in patients.
  • the peptide of the present invention is a peptide consisting of an amino acid sequence selected from SEQ ID NOs: 1, 43, 70 and 89.
  • the peptide of the present invention can be flanked by additional amino acid residues to the amino acid sequence of the peptide of the present invention. Additional amino acid residues can be composed of any type of amino acid as long as they do not impair the ability of the original peptide to induce CTL. Therefore, the peptide of the present invention has an amino acid sequence selected from SEQ ID NOs: 1 to 5, 9, 15, 21, 26, 27, 39 to 45, 70, 83, 87, 89, 90, 104 and 114. Including peptides having the ability to induce CTLs.
  • Such peptides are, for example, less than about 40 amino acids, often less than about 20 amino acids, and usually less than about 15 amino acids. Therefore, if the original peptide is a nonapeptide, the peptide of the present invention includes a peptide having a length of 10 amino acids or a length of 11 to 40 amino acids generated by flanking an additional amino acid to the peptide. In addition, if the original peptide is a decapeptide, it includes a peptide having a length of 11 to 40 amino acids. Such peptides can be, for example, peptides that are 11-20 amino acids in length, and can be peptides that are 11-15 amino acids in length.
  • a preferred example of the additional amino acid residue is an amino acid residue adjacent to the amino acid sequence of the peptide of the present invention in the full-length amino acid sequence of VEGFR2. Therefore, the peptide of the present invention is a peptide derived from VEGFR2, and is SEQ ID NO: 1 to 5, 9, 15, 21, 26, 27, 39 to 45, 70, 83, 87, 89, 90, 104 and 114.
  • modification of one, two, or more amino acids in a peptide does not affect the function of the peptide and in some cases even enhances the desired function of the original peptide.
  • a modified peptide ie, one, two, or a few amino acid residues were modified (ie, substituted, deleted, inserted and / or added) compared to the original reference sequence
  • a peptide composed of an amino acid sequence is known to retain the biological activity of the original peptide (Mark et al., Proc Natl Acad Sci USA 1984, 81: 5662-6; Zoller and Smith, Nucleic Acids) Res 1982, 10: 6487-500; Dalbadie-McFarland et al., Proc Natl Acad Sci USA 1982, 79: 6409-13).
  • the peptide of the invention is selected from among SEQ ID NOs: 1-5, 9, 15, 21, 26, 27, 39-45, 70, 83, 87, 89, 90, 104 and 114
  • this amino acid sequence it may be a peptide comprising an amino acid sequence in which one, two, or several amino acids are substituted, deleted, inserted and / or added, and having CTL inducing ability.
  • amino acid side chain properties that are desirable to preserve include, for example, hydrophobic amino acids (A, I, L, M, F, P, W, Y, V), hydrophilic amino acids (R, D, N, C, E, Q, G, H, K, S, T), as well as side chains having the following functional groups or characteristics in common: aliphatic side chains (G, A, V, L, I, P); hydroxyl group-containing side chains (S, T, Y); sulfur atom-containing side chains (C, M); carboxylic acid and amide-containing side chains (D, N, E, Q); base-containing side chains (R) , K, H); and aromatic-containing side chains (H, F, Y, W).
  • hydrophobic amino acids A, I, L, M, F, P, W, Y, V
  • hydrophilic amino acids R, D, N, C, E, Q, G, H, K, S, T
  • side chains having the following functional groups or characteristics in common aliphatic side chains (G
  • the following eight groups each contain amino acids recognized in the art as conservative substitutions for each other: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), glutamic acid (E); 3) Asparagine (N), glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), leucine (L), methionine (M), valine (V); 6) phenylalanine (F), tyrosine (Y), tryptophan (W); 7) serine (S), threonine (T); and 8) Cysteine (C), methionine (M) (see, for example, Creighton, Proteins 1984).
  • Such conservatively modified peptides are also included in the peptides of the present invention.
  • the peptide of the present invention is not limited to these, and may contain non-conservative modifications as long as the modified peptide retains the CTL-inducing ability of the original peptide.
  • the modified peptides do not exclude polymorphic variants of VEGFR2, interspecies homologs, and CTL inducible peptides from alleles.
  • the term “several” means 5 or fewer amino acids, such as 4 or 3 or fewer.
  • the percentage of amino acids to be modified is preferably 20% or less, more preferably 15% or less, even more preferably 10% or less, or 1-5%.
  • the peptides of the present invention When used in the context of immunotherapy, the peptides of the present invention should be presented on the surface of cells or exosomes, preferably as a complex with the HLA antigen. Therefore, the peptide of the present invention preferably has a high binding affinity for the HLA antigen. Therefore, the peptide may be modified by amino acid residue substitution, deletion, insertion and / or addition to obtain a modified peptide with improved binding affinity.
  • the second amino acid from the N-terminus and the C-terminal amino acid are often anchor residues involved in binding to HLA Class I (Rammensee HG, et al., Immunogenetics. 1995; 41 (4): 178-228.).
  • HLA-A11 threonine, valine, isoleucine, leucine, phenylalanine and tyrosine are the second amino acids from the N-terminus, lysine and arginine are the C-terminal amino acids, and anchor residues with high binding affinity for HLA-A11.
  • HLA-A11 has auxiliary anchor residues at the 3rd and 7th positions from the N-terminus, and the 3rd amino acid from the N-terminus is preferably leucine, phenylalanine, tyrosine, isoleucine, and alanine, It is known that leucine, isoleucine, tyrosine, valine and phenylalanine are preferred as the seventh amino acid from the N-terminal (Falk, et al., Immunogenetics 1994 40 232-41; Chujoh, et al., Tissue Antigens 1998: 52: 501-9).
  • the second amino acid from the N-terminus is replaced with threonine, valine, isoleucine, leucine, phenylalanine or tyrosine, and / or the C-terminal amino acid is lysine.
  • the third amino acid from the N-terminus may be replaced with leucine, phenylalanine, tyrosine, isoleucine, or alanine
  • / or the seventh amino acid from the N-terminus may be replaced with leucine, isoleucine, tyrosine, valine, or phenylalanine. May be desirable.
  • the second amino acid from the N-terminus is threonine, valine, isoleucine, leucine, phenylalanine or tyrosine.
  • the third amino acid from the N-terminus is replaced with leucine, phenylalanine, tyrosine, isoleucine, alanine
  • the seventh amino acid from the N-terminus is replaced with leucine, isoleucine, tyrosine, valine, or phenylalanine
  • a peptide having the ability to induce CTL containing an amino acid sequence in which the C-terminal amino acid is substituted with lysine or arginine is included in the peptide of the present invention.
  • the peptide of the present invention has an amino acid sequence selected from SEQ ID NOs: 1 to 5, 9, 15, 26, 27 and 39 to 45, wherein the second amino acid from the N-terminus is threonine, valine, Substituted with isoleucine, leucine, phenylalanine or tyrosine, the third amino acid from the N-terminus is substituted with leucine, phenylalanine, tyrosine, isoleucine or alanine, the seventh amino acid from the N-terminus is leucine, isoleucine, tyrosine, It may be a peptide having an ability to induce CTL consisting of an amino acid sequence, substituted with valine or phenylalanine, and / or substituted with lysine or arginine at the C-terminal amino acid.
  • the peptide of the present invention is selected from the following (a) to (d) in an amino acid sequence selected from SEQ ID NOs: 1 to 5, 9, 15, 26, 27 and 39 to 45 Including peptides having CTL inducibility, comprising an amino acid sequence having one or more substitutions: (A) the second amino acid from the N-terminus is substituted with threonine, valine, isoleucine, leucine, phenylalanine or tyrosine; (B) the third amino acid from the N-terminus is substituted with leucine, phenylalanine, tyrosine, isoleucine or alanine; (C) The seventh amino acid from the N-terminus is substituted with leucine, isoleucine, tyrosine, valine or phenylalanine; and (d) the C-terminal amino acid is substituted with lysine or arginine.
  • the peptide of the present invention is selected from the above (a) to (d) in an amino acid sequence selected from SEQ ID NOs: 1 to 5, 9, 15, 26, 27 and 39 to 45 It can be a peptide having the ability to induce CTL, consisting of an amino acid sequence having one or more substitutions.
  • the preferred number of substitutions is 1, 2, 3, or 4 substitutions selected from the above (a) to (d).
  • the peptide of the present invention is the amino acid sequence selected from SEQ ID NOs: 1 to 5, 9, 15, 26, 27 and 39 to 45, wherein the second amino acid from the N-terminal is threonine, valine, isoleucine, It can be a peptide capable of inducing CTL, including an amino acid sequence, substituted with leucine, phenylalanine or tyrosine and / or substituted with lysine or arginine at the C-terminal amino acid.
  • the second amino acid from the N-terminal is threonine, valine, isoleucine. , Leucine, phenylalanine or tyrosine, and / or a C-terminal amino acid substituted with lysine or arginine, and a peptide capable of inducing CTL consisting of an amino acid sequence.
  • the peptide of the present invention is selected from the following (a) and (b) in an amino acid sequence selected from SEQ ID NOs: 1 to 5, 9, 15, 26, 27 and 39 to 45
  • a peptide capable of inducing CTLs comprising an amino acid sequence having one or more substitutions: (A) the second amino acid from the N-terminus is substituted with threonine, valine, isoleucine, leucine, phenylalanine or tyrosine; and (b) the C-terminal amino acid is substituted with lysine or arginine.
  • the peptide of the present invention is selected from the above (a) to (b) in an amino acid sequence selected from SEQ ID NOs: 1 to 5, 9, 15, 26, 27 and 39 to 45 It can be a peptide having the ability to induce CTL, consisting of an amino acid sequence having one or more substitutions.
  • the second amino acid from the N-terminus is substituted with threonine, valine, isoleucine or leucine.
  • phenylalanine, tyrosine, alanine, isoleucine, leucine, and valine are the second amino acids from the N-terminus, and arginine and lysine as the C-terminal amino acids have a high binding affinity for HLA-A33.
  • anchor residues Falk, alket al., Immunogenetics 1994 40 232-41; Takiguchi, et al., .Tissue Antigens 2000: 55: 296-302.
  • the first amino acid residue from the N-terminus also functions as an anchor residue, and aspartic acid and glutamic acid are known to be preferred as the first amino acid from the N-terminus.
  • the first amino acid from the N terminus is replaced with aspartic acid or glutamic acid
  • the second amino acid from the N terminus is phenylalanine, tyrosine, alanine, isoleucine
  • the ability to induce CTL including amino acid sequences, in which the second amino acid from the N-terminus is substituted with phenylalanine, tyrosine, alanine, isoleucine, leucine or valine, and / or the C-terminal amino acid is substituted with arginine or lysine
  • the peptide having is included in the peptide of the present invention.
  • the peptide of the present invention has aspartic acid as the first amino acid from the N-terminus in an amino acid sequence selected from SEQ ID NOs: 21, 40, 70, 83, 87, 89, 90, 104 and 114.
  • the second amino acid from the N-terminus is substituted with phenylalanine, tyrosine, alanine, isoleucine, leucine or valine, and / or the C-terminal amino acid is substituted with arginine or lysine, It may be a peptide having the ability to induce CTL consisting of an amino acid sequence.
  • the peptide of the present invention has an amino acid sequence selected from SEQ ID NOs: 21, 40, 70, 83, 87, 89, 90, 104 and 114, from among the following (a) to (c): Includes peptides capable of inducing CTLs, including amino acid sequences having one or more selected substitutions: (A) the first amino acid from the N-terminus is substituted with aspartic acid or glutamic acid; (B) the second amino acid from the N-terminus is substituted with phenylalanine, tyrosine, alanine, isoleucine, leucine or valine; and (c) the C-terminal amino acid is substituted with arginine or lysine.
  • the peptide of the present invention has an amino acid sequence selected from SEQ ID NOs: 21, 40, 70, 83, 87, 89, 90, 104 and 114, and is It may be a peptide having the ability to induce CTL, consisting of an amino acid sequence having one or more selected substitutions.
  • the preferred number of substitutions is one, two or three substitutions selected from the above (a) to (c).
  • the second amino acid from the N-terminus is substituted with phenylalanine or tyrosine.
  • Substitutions can be introduced not only at the terminal amino acid, but also at the potential T cell receptor (TCR) recognition site of the peptide.
  • TCR T cell receptor
  • Some studies have shown that peptides with amino acid substitutions, such as CAP1, p53 (264-272) , Her-2 / neu (369-377) , or gp100 (209-217) , are as active as the original peptide. Have demonstrated or may have superior activity (Zaremba et al. Cancer Res. 57, 4570-7, 1997, TK Hoffmann et al. J Immunol. (2002) Feb 1; 168 (3 ): 1338-47., SO Dionne et al. Cancer Immunol immunother. (2003) 52: 199-206, and SO Dionne et al. Cancer Immunology, Immunotherapy (2004) 53, 307-14).
  • the invention also selects from peptides of the invention (eg, SEQ ID NOs: 1-5, 9, 15, 21, 26, 27, 39-45, 70, 83, 87, 89, 90, 104 and 114) It is contemplated that one, two, or several amino acids can be added to the N-terminal and / or C-terminal of a peptide consisting of the amino acid sequence to be determined. Such modified peptides that retain CTL inducibility are also included in the present invention. For example, a peptide having 1, 2, or several amino acids added to the N-terminal and / or C-terminal of a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 1, 43, 70 or 89 is brought into contact with APC.
  • the peptide is incorporated into APC and processed to become a peptide having the amino acid sequence set forth in SEQ ID NO: 1, 43, 70, or 89. Thereafter, CTL can be induced by presenting on the cell surface of APC via the antigen presentation pathway.
  • the amino acid sequence of the peptide is identical to part of the amino acid sequence of an endogenous or exogenous protein with different functions, side effects such as autoimmune disorders and / or allergic symptoms to certain substances can be induced There is sex. Therefore, in order to avoid a situation where the amino acid sequence of a peptide matches the amino acid sequence of another protein, it is preferable to perform a homology search using an available database. If the homology search reveals that there is not even a peptide that differs by one or two amino acids compared to the peptide of interest, without the risk of such side effects, The peptide of interest can be modified to increase its binding affinity and / or to increase its ability to induce CTLs.
  • the “peptide having the ability to induce CTL” refers to a peptide from which CTL is induced by APC stimulated with the peptide.
  • “Induction of CTL” includes induction of CTL activation, induction of CTL proliferation, induction of target cell lysis by CTL, and induction of increased CTL IFN- ⁇ production.
  • Confirmation of the ability to induce CTL is to induce APC (for example, B lymphocytes, macrophages, and dendritic cells (DC)) carrying HLA antigen, stimulate with peptide, mix with CD8 positive T cells, This can be done by measuring IFN- ⁇ released by CTL against the target cells.
  • APC for example, B lymphocytes, macrophages, and dendritic cells (DC)
  • DC dendritic cells
  • a transgenic animal prepared to express HLA antigen for example, BenMohamed L, Krishnan R, Longmate J, Auge C, Low L, Primus J, Diamond DJ, Hum Immunol 2000 Aug, 61 (8) : 764-79, Related Articles, Books, Linkout Induction of CTL response by a minimal epitope vaccine in HLA A * 0201 / DR1 transgenic mice: those described in dependence on HLA class II restricted T (H) response
  • HLA A * 0201 / DR1 transgenic mice those described in dependence on HLA class II restricted T (H) response
  • the target cells are radiolabeled with 51 Cr or the like, and the cytotoxic activity of CTL induced by the peptide can be calculated from the radioactivity released from the target cells.
  • the ability of CTL to be induced can be measured by measuring IFN- ⁇ produced and released by CTL and visualizing the inhibition zone on the medium using anti-IFN- ⁇ monoclonal antibody. Can be evaluated.
  • the peptides of the present invention can be linked to other peptides as long as the resulting linked peptide retains the ability to induce CTL.
  • suitable peptides to be linked to the peptides of the present invention include CTL inducible peptides derived from tumor associated antigens (TAA).
  • TAA tumor associated antigens
  • the peptides of the present invention can be linked together. Suitable linkers that can be used to link peptides are known in the art, such as AAY (P. M. Daftarian et al., J Trans Med 2007, 5:26), AAA, NKRK (R. P. M . Sutmuller et al., J Immunol.
  • Peptides can be linked in various configurations (eg, linked, overlapping, etc.), and more than two peptides can be linked.
  • the peptide of the present invention can also be linked to other substances as long as the resulting linked peptide retains the ability to induce CTL.
  • suitable substances linked to the peptides of the present invention include, for example, peptides, lipids, sugars or sugar chains, acetyl groups, and natural or synthetic polymers.
  • the peptide of the present invention can be modified such as glycosylation, side chain oxidation, or phosphorylation as long as the CTL inducing ability is not impaired. Such types of modifications can be made to provide additional functions (eg, targeting and delivery functions) or to stabilize the peptide.
  • Peptide stability can be assayed in several ways. For example, peptidases and various biological media such as human plasma and serum can be used to test the stability (see, eg, Verhoef et al., Eur J Drug Metab Pharmacokin 1986, 11: 291-302). I want to be)
  • the modified peptide in which one, two, or several amino acid residues are substituted, deleted, inserted and / or added is the same as or compared with the original peptide. Those having higher activity can be screened or selected. Accordingly, the present invention also provides a method for screening or selecting for a modified peptide having the same or higher activity compared to the original.
  • the present invention provides a method for screening a peptide having the ability to induce CTL, comprising the following steps: (A) SEQ ID NOs: 1 to 5, 9, 15, 21, 26, 27, 39 to 45, 70, 83, 87, 89, 90, 104 and 114, the original amino acid consisting of an amino acid sequence Creating a candidate sequence consisting of an amino acid sequence wherein one, two, or several amino acid residues are substituted, deleted, inserted and / or added to the sequence; (B) selecting a candidate sequence having no significant homology (sequence identity) with any known human gene product other than VEGFR2 from the candidate sequences prepared in (a); (C) contacting the peptide comprising the candidate sequence selected in (b) with APC; (D) contacting the APC of (c) with a CD8-positive T cell; and (e) selecting a peptide having a CTL inducing ability that is equal to or higher than that of the peptide consisting of the original amino acid sequence.
  • the peptide of the present invention is also described as “VEGFR2 peptide” or “VEGFR2 polypeptide”.
  • VEGFR2 peptides The peptides of the present invention can be prepared using well-known techniques. For example, the peptides of the invention can be prepared using recombinant DNA technology or chemical synthesis. The peptides of the present invention can be synthesized individually or as longer polypeptides comprising two or more peptides. After being produced in a host cell using recombinant DNA technology or chemically synthesized, the peptides of the invention can be isolated from the host cell or synthesis reaction. That is, the peptides of the invention can be purified or isolated so that they are substantially free of other host cell proteins and fragments thereof, or any other chemicals.
  • the peptide of the present invention may contain modifications such as glycosylation, side chain oxidation, or phosphorylation, as long as the modification does not impair the biological activity of the original peptide.
  • modifications include the incorporation of D-amino acids or other amino acid mimetics that can be used, for example, to increase the serum half-life of the peptide.
  • the peptide of the present invention can be obtained by chemical synthesis based on the selected amino acid sequence.
  • Examples of conventional peptide synthesis methods that can be adapted for the synthesis include those described in the literature as follows: (I) Peptide Synthesis, Interscience, New York, 1966; (Ii) The Proteins, Vol. 2, Academic Press, New York, 1976; (Iii) “Peptide Synthesis” (Japanese), Maruzen, 1975; (Iv) “Basics and Experiments of Peptide Synthesis” (Japanese), Maruzen, 1985; (V) "Development of drugs” (Japanese), Vol.
  • any known genetic engineering method for producing a peptide can be adapted to obtain a peptide of the invention (eg, Morrison J, J Bacteriology 1977, 132: 349-51; Clark-Curtiss & Curtiss, Methods in Enzymology (Wu et al.) 1983, 101: 347-62).
  • an appropriate vector containing a polynucleotide encoding the peptide of the present invention in an expressible form eg, downstream of a regulatory sequence corresponding to a promoter sequence
  • the host cell is then cultured to produce the peptide of the invention.
  • the peptides of the present invention can be generated in vitro using an in vitro translation system.
  • polynucleotides that encode any of the peptides of the present invention. These include polynucleotides derived from the native VEGFR2 gene (eg, GenBank accession number NM — 002253.2 (SEQ ID NO: 122)) and polynucleotides having conservatively modified nucleotide sequences thereof. As used herein, the phrase “conservatively modified nucleotide sequence” refers to sequences that encode the same or essentially the same amino acid sequence. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any particular protein.
  • the codons GCA, GCC, GCG, and GCU all encode the amino acid alanine.
  • the codon can be changed to any of the corresponding codons without changing the encoded polypeptide.
  • Such nucleic acid mutations are “silent mutations” and are a type of mutation conservatively modified. Every nucleic acid sequence herein that encodes a peptide also represents every possible silent variation of the nucleic acid. Those skilled in the art will be able to modify each codon in the nucleic acid (except AUG, which is usually the only codon for methionine, and TGG, which is usually the only codon for tryptophan) to obtain a functionally identical molecule. You will recognize. Accordingly, each silent variation of a nucleic acid that encodes a peptide is implicitly described in each disclosed sequence.
  • the polynucleotide of the present invention can be composed of DNA, RNA, and derivatives thereof.
  • DNA is suitably composed of bases such as A, T, C and G, and T is replaced by U in RNA.
  • the polynucleotide of the present invention can encode a plurality of peptides of the present invention with or without an intervening amino acid sequence in between.
  • the intervening amino acid sequence can provide a cleavage site (eg, an enzyme recognition sequence) for the polynucleotide or translated peptide.
  • the polynucleotide may comprise any additional sequence to the coding sequence that encodes a peptide of the invention.
  • the polynucleotide may be a recombinant polynucleotide containing regulatory sequences necessary for expression of the peptide, or an expression vector (eg, a plasmid) having a marker gene or the like.
  • such recombinant polynucleotides can be prepared, for example, by manipulating the polynucleotide by conventional recombinant techniques using polymerases and endonucleases.
  • the polynucleotide of the present invention can be produced using any of recombinant techniques and chemical synthesis techniques.
  • a polynucleotide can be made by inserting into an appropriate vector, which can be expressed when transfected into competent cells.
  • polynucleotides can be amplified using PCR techniques or expression in a suitable host (see, eg, Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 1989) I want to be)
  • polynucleotides can be synthesized using the solid phase technique described in Beaucage SL & Iyer RP, Tetrahedron 1992, 48: 2223-311; Matthes et al., EMBO J 1984, 3: 801-5. it can.
  • Exosomes The present invention further provides intracellular vesicles, called exosomes, that present complexes formed between the peptides of the present invention and HLA antigens on their surface. Exosomes can be prepared using, for example, the methods detailed in JP-A-11-510507 and WO99 / 03499, and prepared using APC obtained from a patient to be treated and / or prevented can do. The exosomes of the present invention can be vaccinated as a vaccine in the same manner as the peptides of the present invention.
  • HLA-A11 eg, HLA-A * 1101
  • HLA-A33 eg, HLA-A * 3303
  • these HLA antigen types are Asian It is considered suitable for the treatment of human patients.
  • a clinic has a high level of binding affinity for a specific HLA antigen or through a specific HLA antigen by pre-examining the type of HLA antigen of the patient in need of treatment Appropriate selection of peptides having the ability to induce CTL by presentation is possible.
  • the exosome of the present invention presents a complex of the peptide of the present invention and HLA-A11 or HLA-A33 on its surface.
  • the peptide of the present invention is selected from SEQ ID NOs: 1 to 5, 9, 15, 26, 27, and 39 to 45 It is preferably a peptide having an amino acid sequence or a modified peptide thereof, and is a peptide consisting of an amino acid sequence selected from SEQ ID NOs: 1 to 5, 9, 15, 26, 27 and 39 to 45 or a modified peptide thereof It is more preferable.
  • the peptide of the present invention is selected from among SEQ ID NOs: 21, 40, 70, 83, 87, 89, 90, 104, and 114. It is preferably a peptide having an amino sequence selected or a modified peptide thereof, a peptide comprising an amino sequence selected from SEQ ID NOs: 21, 40, 70, 83, 87, 89, 90, 104 and 114 or More preferably, the modified peptide.
  • the present invention also provides an antigen presenting cell (APC) that presents on its surface the complex formed between the HLA antigen and the peptide of the present invention.
  • the present invention provides an APC having on its cell surface a complex formed between an HLA antigen and a peptide of the present invention.
  • the APC of the present invention can be an isolated APC.
  • isolated when used in reference to a cell (APC, CTL, etc.) refers to the cell being separated from other types of cells.
  • the APC of the present invention may be derived from an APC derived from a patient to be treated and / or prevented, and alone or another drug containing the peptide, exosome, or CTL of the present invention Can be used as a vaccine.
  • the APCs of the present invention are not limited to specific types of cells, including dendritic cells (DCs) that are known to present proteinaceous antigens on their cell surface as recognized by lymphocytes. ), Langerhans cells, macrophages, B cells, and activated T cells. Since DC is a representative APC having the strongest CTL inducing action among APCs, DC can be preferably used as the APC of the present invention. In the present invention, preferred DC is human-derived isolated DC, or a mixture thereof. That is, DCs presenting different peptides as antigens can be mixed and used.
  • DCs dendritic cells
  • the APC of the present invention can be obtained by inducing DCs from peripheral blood monocytes and then stimulating them with the peptides of the present invention in vitro, ex vivo or in vivo.
  • the peptide of the present invention is administered to a subject, APC presenting the peptide of the present invention is induced in the subject's body. Therefore, the APC of the present invention can be obtained by administering the peptide of the present invention to a subject and then recovering the APC from the subject.
  • the APC of the present invention can be obtained by contacting APC recovered from a subject with the peptide of the present invention.
  • the APC of the invention is administered to the subject alone or in combination with other agents, including peptides, exosomes, or CTLs of the invention, to induce an immune response against vascular endothelial cells that express VEGFR2.
  • ex vivo administration may include the following steps: a: recovering APC from the first subject; b: contacting the APC of step a with a peptide; and c: administering the bpc of step b to a second subject.
  • the first object and the second object may be the same individual or different individuals.
  • the HLA of the first object and the second object is preferably the same type.
  • the APC obtained by step b above can be a vaccine for treating and / or preventing diseases mediated by angiogenesis.
  • the APC of the present invention obtained by the method as described above has the ability to induce CTL.
  • CTL inducing ability refers to the ability of APC to induce CTL when contacted with CD8 positive T cells.
  • CTL inducing ability includes APC ability to induce CTL activation, APC ability to induce CTL proliferation, APC ability to promote lysis of target cells by CTL, and IFN- ⁇ production by CTL Includes APC ability to increase.
  • the CTL induced by the APC of the present invention is a CTL specific for VEGFR2, and exhibits a cytotoxic activity specific to VEGFR2-expressing cells.
  • the APC of the present invention can also be prepared by introducing a polynucleotide encoding the peptide of the present invention into APC in vitro.
  • the polynucleotide to be introduced may be in the form of DNA or RNA.
  • introduction methods include, but are not limited to, various methods conventionally practiced in the art, such as lipofection, electroporation, and the calcium phosphate method. More specifically, it is described in Cancer Res 1996, 56: 5672-7; J Immunol 1998, 161: 5607-13; J Exp Med 1996, 184: 465-72; Published Patent Publication No. 2000-509281 Such a method can be used.
  • the polynucleotide By introducing a polynucleotide encoding the peptide of the present invention into APC, the polynucleotide undergoes transcription, translation, etc. in the cell, and then the resulting peptide is processed by MHC class I and passed through the presentation pathway to the main pathway.
  • the peptides of the invention are presented on the cell surface of APC.
  • the APC of the present invention is a complex formed between HLA-A11 (more preferably HLA-A * 1101) or HLA-A33 (more preferably HLA-A * 3303) and the peptide of the present invention.
  • APC presenting the body on its own cell surface.
  • the HLA that forms a complex with the peptide of the present invention is HLA-A11
  • the peptide of the present invention is an amino acid selected from SEQ ID NOs: 1 to 5, 9, 15, 26, 27, and 39 to 45
  • a peptide having a sequence or a modified peptide thereof is preferable, and a peptide consisting of an amino acid sequence selected from SEQ ID NOs: 1 to 5, 9, 15, 26, 27, and 39 to 45 is more preferable.
  • the peptide of the present invention is selected from SEQ ID NOs: 21, 40, 70, 83, 87, 89, 90, 104 and 114.
  • the APC of the present invention is preferably an APC derived by a method comprising the steps described in a or b below: a: contacting APC expressing HLA-A11 (more preferably HLA-A * 1101) or HLA-33 (more preferably HLA-A * 3303) with the peptide of the present invention; b: introducing a polynucleotide encoding the peptide of the present invention into APC expressing HLA-A11 (more preferably HLA-A * 1101) or HLA-A33 (more preferably HLA-A * 3303) .
  • the peptide of the present invention to be contacted with APC expressing HLA-A11 is a peptide having an amino acid sequence selected from SEQ ID NOs: 1 to 5, 9, 15, 26, 27 and 39 to 45 or a modification thereof A peptide is preferable, and a peptide consisting of an amino acid sequence selected from SEQ ID NOs: 1 to 5, 9, 15, 26, 27, and 39 to 45 is more preferable.
  • the peptide of the present invention to be contacted with APC expressing HLA-A33 has an amino sequence selected from SEQ ID NOs: 21, 40, 70, 83, 87, 89, 90, 104 and 114. It is preferably a peptide or a modified peptide thereof, more preferably a peptide consisting of an amino sequence selected from SEQ ID NOs: 21, 40, 70, 83, 87, 89, 90, 104 and 114.
  • the present invention uses of the peptide of the present invention for producing a pharmaceutical composition for inducing APC having CTL inducing ability is provided.
  • the present invention provides a method or process for producing a pharmaceutical composition that induces APC having the ability to induce CTL.
  • the present invention also provides a peptide of the present invention for inducing APC having CTL inducibility.
  • Cytotoxic T lymphocyte (CTL) CTL induced by the peptide of the present invention can be used as a vaccine in the same manner as the peptide of the present invention because it enhances the immune response targeting vascular endothelial cells expressing VEGFR2 in vivo. Accordingly, the present invention provides CTL induced or activated by the peptides of the present invention.
  • the CTL of the present invention is a CTL that targets the peptide of the present invention, and is a CTL that can bind to a complex of the peptide of the present invention and an HLA antigen. Binding of CTL to the complex is performed via a T cell receptor (TCR) present on the cell surface of CTL.
  • TCR T cell receptor
  • the CTL of the present invention can be an isolated CTL.
  • a preferred CTL is an isolated CTL derived from human, or a mixture thereof. That is, CTLs that recognize HLA complexes on which different peptides are presented as antigens can be mixed and used.
  • the CTL of the present invention comprises (1) administering the peptide of the present invention to a subject, or (2) stimulating APC and CD8 positive T cells or peripheral blood mononuclear cells derived from the subject with the peptide of the present invention in vitro. Or (3) contacting CD8 positive T cells or peripheral blood mononuclear cells in vitro with APCs or exosomes that present complexes of HLA antigens and peptides of the invention on their surface, or ( 4) It can be obtained by introducing a vector comprising a polynucleotide encoding each subunit of T cell receptor (TCR) capable of binding to the peptide of the present invention presented by the HLA antigen on the cell surface.
  • TCR T cell receptor
  • exosome and APC used in the above method (2) or (3) can be prepared by the methods described in the sections “V. Exosomes” and “VI. Antigen-presenting cells (APC)”, respectively. Details of the method (4) will be described in the section “VIII. T cell receptor (TCR)”.
  • the CTL of the present invention can be administered alone to a patient to be treated and / or prevented, or used in combination with other drugs including the peptide, APC or exosome of the present invention for the purpose of regulating the effect. Can be administered.
  • the CTL of the present invention can be a CTL derived from a CD8-positive T cell derived from a patient to whom the CTL is administered.
  • the CTL of the present invention specifically acts on a target cell presenting the peptide of the present invention, for example, the same peptide used for induction of the CTL of the present invention.
  • the target cells may be cells that endogenously express VEGFR2, such as endothelial cells of tumor tissue, or cells that have been transfected with the VEGFR2 gene.
  • the target cell of CTL of the present invention is preferably a cell positive for HLA-A11 (more preferably HLA-A * 1101) or HLA-A33 (more preferably HLA-A * 3303).
  • the CTLs of the invention are specific for cells expressing both HLA-A11 (more preferably HLA-A * 1101) or HLA-A33 (more preferably HLA-A * 3303) and VEGFR2.
  • CTL “targets” a cell means that CTL recognizes a cell presenting a complex of HLA and the peptide of the present invention on the cell surface, and cytotoxicity against the cell. Indicates activity. Further, “specifically targeting” means that CTL shows cytotoxic activity against the cell but does not show cytotoxic activity against other cells.
  • the term “recognize a cell” is specific to the cell that binds to the complex of HLA presented on the cell surface and the peptide of the present invention via its TCR. It shows that it shows a cytotoxic activity. Therefore, the CTL of the present invention preferably comprises HLA-A11 (more preferably HLA-A * 1101) or HLA-A33 (more preferably HLA-A * 3303) displayed on the cell surface and the peptide of the present invention. Is a CTL that can be bound to the complex formed between and via TCR.
  • the CTL of the present invention is preferably a CTL induced by a method comprising the steps described in the following a or b: a: A CD8 positive T cell is placed on its surface with a complex of HLA-A11 (more preferably HLA-A * 1101) or HLA-A33 (more preferably HLA-A * 3303) and the peptide of the present invention. Contacting the presenting APC or exosome in vitro; b: binds to the peptide of the present invention presented on CD8 positive T cells by HLA-A11 (more preferably HLA-A * 1101) or HLA-A33 (more preferably HLA-A * 3303) on the cell surface. Introducing a polynucleotide encoding each subunit of the resulting TCR.
  • T cell receptor The present invention also includes a composition comprising a polynucleotide encoding each subunit of a T cell receptor (TCR) capable of binding to the peptide of the present invention presented by the HLA antigen on the cell surface, and a method of using the same I will provide a.
  • the polynucleotide confers CD8 positive T cells with specificity for vascular endothelial cells expressing VEGFR2 by forming a TCR on the cell surface that can bind to the peptides of the present invention presented by the HLA antigen.
  • PCR primers for analysis can be used as a primer set for amplification by combining the following 5′-side primer with the following 3′-side primer, but are not limited thereto.
  • TCR formed by introducing the identified polynucleotide into a CD8-positive T cell can bind with high binding force to a target cell presenting the peptide of the present invention, and presents the peptide of the
  • the polynucleotide encoding each subunit of TCR can be incorporated into an appropriate vector, for example, a retroviral vector. These vectors are well known in the art.
  • the polynucleotide or a vector containing them in an expressible form can be introduced into a CD8-positive T cell, eg, a patient-derived CD8-positive T cell.
  • the present invention makes it possible to quickly and easily produce modified T cells with superior vascular endothelial cell killing properties by rapid modification of the patient's own T cells (or another mammalian T cell).
  • a composition is provided.
  • the specific TCR specifically indicates a complex of the peptide of the present invention and the HLA antigen presented on the surface of a target cell when the TCR is present on the surface of a CD8-positive T cell. It is a TCR that can recognize and confer specific cytotoxic activity on target cells. Specific recognition of the complex can be confirmed by any known method, and preferred examples include HLA multimer staining analysis using HLA molecules and peptides of the present invention, and ELISPOT assay. By performing the ELISPOT assay, it can be confirmed that the T cell introduced with the polynucleotide specifically recognizes the target cell by TCR and that the signal is transmitted intracellularly.
  • confirmation that the TCR can impart a target cell-specific cytotoxic activity to the CD8-positive T cell is also performed by a known method. be able to. Preferred methods include measuring cytotoxic activity against target cells, such as by a chromium release assay.
  • the present invention also relates to each subunit of TCR that binds to a peptide having an amino acid sequence selected from, for example, SEQ ID NOs: 1 to 5, 9, 15, 26, 27, and 39 to 45 in the context of HLA-A11 CTLs prepared by transducing the encoding polynucleotide are provided.
  • the invention in the context of HLA-A33, the invention relates to each sub-unit of TCR that binds to a peptide having an amino acid sequence selected from, for example, SEQ ID NOs: 21, 40, 70, 83, 87, 89, 90, 104 and 114.
  • CTLs prepared by transducing a polynucleotide encoding the unit are provided.
  • Transduced CTL can be homed in vivo and can be propagated by well-known in vitro culture methods (eg, Kawakami et al., J Immunol., 142, 2-3452-61 (1989)).
  • the CTLs of the invention can be used to form immunogenic compositions useful for the treatment or prevention of disease in patients in need of treatment or prevention, the contents of which are hereby incorporated by reference. (See WO2006 / 031221).
  • composition or pharmaceutical composition comprising at least one active ingredient selected from: (A) the peptide of the present invention; (B) a polynucleotide encoding the peptide of the present invention in an expressible form; (C) APC of the present invention; (D) the exosome of the present invention; (E) CTL of the present invention.
  • the pharmaceutical composition of the present invention may contain carriers, excipients and the like that are usually used for pharmaceuticals as necessary, in addition to the above-mentioned active ingredients.
  • carriers that can be used in the pharmaceutical composition of the present invention include sterilized water, physiological saline, phosphate buffer, culture solution, and the like.
  • the present invention also provides a pharmaceutical composition comprising at least one active ingredient selected from the following (a) to (e) and a pharmaceutically acceptable carrier: (A) the peptide of the present invention; (B) a polynucleotide encoding the peptide of the present invention in an expressible form; (C) APC of the present invention; (D) the exosome of the present invention; (E) CTL of the present invention.
  • the pharmaceutical composition of the present invention may contain a stabilizer, a suspension, a preservative, a surfactant and the like, if necessary.
  • VEGFR2 expression is elevated in vascular endothelial cells at disease sites in diseases mediated by angiogenesis compared to normal tissues. Therefore, the peptide of the present invention or a polynucleotide encoding the peptide can be used for treatment and / or prevention of a disease mediated by angiogenesis and / or prevention of its recurrence after surgery.
  • the present invention provides a pharmaceutical composition for the treatment and / or prevention of a disease mediated by angiogenesis and / or prevention of its recurrence after surgery, comprising one kind of the peptide or polynucleotide of the present invention Or the composition which contains multiple types as an active ingredient is provided.
  • the peptides of the invention can be presented on the surface of exosomes or APCs for use as pharmaceutical compositions.
  • a CTL of the present invention that targets any one of the peptides of the present invention can also be used as an active ingredient of the pharmaceutical composition of the present invention.
  • the pharmaceutical composition of the present invention may contain a therapeutically effective amount or a pharmaceutically effective amount of the above-mentioned active ingredient.
  • the pharmaceutical composition of the present invention may also be used as a vaccine.
  • the phrase “vaccine” also referred to as “immunogenic composition” is a composition having the function of inducing an immune response that provides an anti-angiogenic effect when inoculated into an animal. Point to.
  • the pharmaceutical compositions of the present invention can be used to induce an immune response that provides an anti-angiogenic effect.
  • the pharmaceutical composition of the present invention can be used to treat and / or prevent a disease mediated by angiogenesis and / or prevent its recurrence after surgery in a human subject or patient.
  • the pharmaceutical composition of the present invention can be preferably used for HLA-A11 positive or HLA-A33 positive subjects.
  • the present invention also provides the use of an active ingredient selected from among the following in the manufacture of a pharmaceutical composition for treating or preventing a disease mediated by angiogenesis: (A) the peptide of the present invention; (B) a polynucleotide encoding the peptide of the present invention in an expressible form; (C) APC presenting the peptide of the invention on its surface; (D) an exosome presenting a peptide of the invention on its surface; and (e) a CTL of the invention.
  • an active ingredient selected from among the following in the manufacture of a pharmaceutical composition for treating or preventing a disease mediated by angiogenesis: (A) the peptide of the present invention; (B) a polynucleotide encoding the peptide of the present invention in an expressible form; (C) APC presenting the peptide of the invention on its surface; (D) an exosome presenting a peptide of the invention on its surface; and (e) a CTL of the
  • the present invention further provides an active ingredient selected from the following for use in the treatment or prevention of diseases mediated by angiogenesis: (A) the peptide of the present invention; (B) a polynucleotide encoding the peptide of the present invention in an expressible form; (C) APC presenting the peptide of the invention on its surface; (D) an exosome presenting a peptide of the invention on its surface; and (e) a CTL of the invention.
  • the present invention further provides a method or process for producing a pharmaceutical composition for treating or preventing a disease mediated by angiogenesis, comprising at least one active ingredient selected from A method or process comprising formulating a pharmaceutically or physiologically acceptable carrier with: (A) the peptide of the present invention; (B) a polynucleotide encoding the peptide of the present invention in an expressible form; (C) APC presenting the peptide of the invention on its surface; (D) an exosome presenting a peptide of the invention on its surface; and (e) a CTL of the invention.
  • the present invention also provides a method or process for producing a pharmaceutical composition for treating or preventing a disease mediated by angiogenesis, comprising an active ingredient selected from A method or process is provided that comprises mixing with a pharmaceutically or physiologically acceptable carrier: (A) the peptide of the present invention; (B) a polynucleotide encoding the peptide of the present invention in an expressible form; (C) APC presenting the peptide of the invention on its surface; (D) an exosome presenting a peptide of the invention on its surface; and (e) a CTL of the invention.
  • a pharmaceutically or physiologically acceptable carrier comprises mixing with a pharmaceutically or physiologically acceptable carrier: (A) the peptide of the present invention; (B) a polynucleotide encoding the peptide of the present invention in an expressible form; (C) APC presenting the peptide of the invention on its surface; (D) an exosome presenting a peptide of the invention on its surface; and
  • the present invention also provides a method for treating or preventing a disease mediated by angiogenesis, comprising the step of administering to a subject at least one active ingredient selected from the following: I will provide a: (A) the peptide of the present invention; (B) a polynucleotide encoding the peptide of the present invention in an expressible form; (C) APC presenting the peptide of the invention on its surface; (D) an exosome presenting a peptide of the invention on its surface; and (e) a CTL of the invention.
  • a peptide having an amino acid sequence selected from SEQ ID NOs: 1 to 5, 9, 15, 26, 27 and 39 to 45 is capable of inducing a strong and specific immune response. It was found as a sex epitope peptide.
  • a peptide having an amino acid sequence selected from SEQ ID NOs: 21, 40, 70, 83, 87, 89, 90, 104 and 114 is capable of inducing a strong and specific immune response. It was found as a sex epitope peptide.
  • the pharmaceutical composition of the present invention comprising at least one peptide having an amino acid sequence selected from among SEQ ID NOs: 1 to 5, 9, 15, 26, 27 and 39 to 45 is HLA antigen as HLA antigen.
  • composition of the present invention comprising at least one peptide having an amino acid sequence selected from SEQ ID NOs: 21, 40, 70, 83, 87, 89, 90, 104, and 114 is an HLA antigen.
  • HLA-A33 eg, HLA-A * 3303.
  • compositions containing the targeted CTL ie, the CTLs of the present invention. That is, a pharmaceutical composition comprising an active ingredient related to a peptide having an amino acid sequence selected from SEQ ID NOs: 1 to 5, 9, 15, 26, 27 and 39 to 45 is a subject having HLA-A11 (Ie, suitable for administration to HLA-A11 positive subjects).
  • a pharmaceutical composition comprising an active ingredient related to a peptide having an amino acid sequence selected from SEQ ID NOs: 21, 40, 70, 83, 87, 89, 90, 104 and 114 is HLA-A33.
  • Suitable for administration to a subject having a HLA-A33 positive ie, HLA-A33 positive subject.
  • the pharmaceutical composition of the invention is a pharmaceutical composition comprising at least one peptide having an amino acid sequence selected from SEQ ID NOs: 1, 43, 70 and 89.
  • Diseases to be treated and / or prevented by the pharmaceutical composition of the present invention are not particularly limited, and various cancers, diseases related to angiogenesis in the choroid (neovascular maculopathy: age-related macular degeneration, myopia) Macular degeneration, retinitis pigmentosa, central exudative retina choroidopathy, various retinal pigment epitheliopathy, choroidal atrophy, choroideremia, choroidal osteoma, etc.), diabetic retinopathy, rheumatoid arthritis, psoriasis, or atheroma Includes all types of angiogenesis-mediated diseases involving VEGFR2, including atherosclerosis and the like.
  • angiogenesis at the tumor site is also closely associated with solid tumor growth and metastasis (Folkman, J. (1995) NatureN Med. 1: 27-31; Bicknell et al., (1996) Curr. Opin . Oncol. 8: 60-5). Therefore, the pharmaceutical composition of the present invention can also be used to suppress the growth and / or metastasis of cancer (solid tumor).
  • the pharmaceutical composition of the present invention comprises, in addition to the above-mentioned active ingredient, other peptides having the ability to induce CTLs against vascular endothelial cells at disease sites (for example, peptide CTL-inducing peptides derived from VEGFR1), the other peptides Other polynucleotides encoding the other, other cells presenting the other peptides, and the like.
  • the pharmaceutical composition of the present invention when the pharmaceutical composition of the present invention is particularly for treating and / or preventing cancer, the pharmaceutical composition of the present invention has the ability to induce CTL against cancer cells.
  • a peptide for example, a cancer antigen-derived CTL-inducing peptide
  • a polynucleotide encoding the peptide, a cell presenting the peptide, and the like may be included.
  • the pharmaceutical composition of the present invention may optionally contain other therapeutic substances as an active ingredient as long as the anti-angiogenic effect of the active ingredient such as the peptide of the present invention is not inhibited.
  • the pharmaceutical composition of the present invention may optionally include an anti-inflammatory composition, an analgesic, a chemotherapeutic agent and the like.
  • the pharmaceutical composition of the present invention may also be administered sequentially or simultaneously with one or more other pharmaceutical compositions. it can.
  • the dosage of the pharmaceutical composition of the present invention and other pharmaceutical compositions depends, for example, on the type of pharmaceutical composition used, the disease to be treated, and the schedule and route of administration.
  • compositions of the invention may also contain other ingredients customary in the art in view of the type of formulation. Should.
  • the present invention also provides a product or kit comprising the pharmaceutical composition of the present invention.
  • the product or kit of the present invention may comprise a container containing the pharmaceutical composition of the present invention.
  • suitable containers include, but are not limited to, bottles, vials, and test tubes.
  • the container can be formed from a variety of materials such as glass or plastic.
  • a label may be attached to the container, and the label can describe a disease or a disease state in which the pharmaceutical composition of the present invention is to be used.
  • the label may also indicate directions for administration and the like.
  • the product or kit of the present invention may optionally further comprise a second container containing a pharmaceutically acceptable diluent in addition to the container containing the pharmaceutical composition of the present invention.
  • the product or kit of the present invention further includes other materials desirable from a commercial and user standpoint, such as other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use. May be included.
  • the pharmaceutical composition of the present invention can be provided in a pack or dispenser device that may contain one or more unit dosage forms containing the active ingredient.
  • the pack may include metal foil or plastic foil, such as a blister pack.
  • the pack or dispenser device can be accompanied by instructions for administration.
  • composition containing a peptide as an active ingredient can be formulated by a conventional formulation method, if necessary.
  • the pharmaceutical composition of the present invention can contain carriers, excipients and the like that are commonly used in pharmaceuticals as needed without particular limitation. Examples of carriers that can be used in the pharmaceutical composition of the present invention include sterilized water, physiological saline, phosphate buffer, culture solution, and the like.
  • the pharmaceutical composition of the present invention may contain a stabilizer, a suspension, a preservative, a surfactant and the like, if necessary. Since the pharmaceutical composition of the present invention can induce specific immunity against vascular endothelial cells expressing VEGFR2, it can be used for anti-angiogenic purposes.
  • the pharmaceutical composition of the present invention can also contain a combination of two or more kinds of the peptides of the present invention.
  • the peptide combination may take the form of a cocktail in which the peptides are mixed, or the peptides may be linked together using standard techniques.
  • the peptides may be chemically conjugated or expressed as a single fusion polypeptide sequence.
  • APC eg, DC
  • APCs that present any of the peptides of the invention on their cell surface.
  • APCs can be re-administered to a subject to induce CTL in the subject, resulting in increased aggression against vascular endothelial cells that express VEGFR2.
  • the pharmaceutical composition of the present invention may also include an adjuvant known to efficiently establish cellular immunity.
  • An adjuvant refers to a compound that, when administered with (or sequentially) an antigen having immunological activity, enhances the immune response to the antigen.
  • the adjuvant for example, known ones described in documents such as Clin Microbiol Rev 1994, 7: 277-89 can be used.
  • suitable adjuvants include aluminum phosphate, aluminum hydroxide, alum, cholera toxin, salmonella toxin, incomplete Freund's adjuvant (IFA), complete Freund's adjuvant (CFA), ISCOMatrix, GM-CSF, CpG, oil-in-water type Although emulsion etc. are included, it is not limited to these.
  • the adjuvant may be contained in a container separate from the pharmaceutical composition containing the peptide of the present invention.
  • the adjuvant and the pharmaceutical composition may be administered to the subject sequentially or may be mixed immediately before administration to the subject.
  • a kit containing a pharmaceutical composition containing such a peptide of the present invention and an adjuvant is also provided by the present invention.
  • the pharmaceutical composition of the present invention is a liposome preparation encapsulating the peptide of the present invention, a granule preparation in which the peptide is bound to beads having a diameter of several micrometers, or a preparation in which a lipid is bound to the peptide. Also good.
  • the peptide of the invention may also be administered in the form of a pharmaceutically acceptable salt.
  • the salt include a salt with an alkali metal, a salt with a metal, a salt with an organic base, a salt with an amine, an organic acid (acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid, tartaric acid, And salts with citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid and the like, and salts with inorganic acids (hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid, nitric acid, etc.).
  • the phrase “pharmaceutically acceptable salt” retains the biological effectiveness and properties of the compound, and includes hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid. , Methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid and the like such salts obtained by reaction with inorganic bases. Accordingly, pharmaceutical compositions comprising pharmaceutically acceptable salts of the peptides of the present invention are also encompassed by the present invention.
  • the pharmaceutical composition of the present invention may further comprise an ingredient that stimulates CTL.
  • Lipids have been identified as substances that can stimulate CTLs in vivo against viral antigens.
  • palmitic acid residues can be attached to the ⁇ and ⁇ amino groups of lysine residues and then linked to the peptides of the invention.
  • the lipidated peptide can then be administered directly in the form of micelles or particles, administered in liposomes, or emulsified in an adjuvant.
  • Another example of lipid stimulation of the CTL response is when E.
  • coli lipoproteins such as tripalmitoyl-S-glycerylcysteinyl-seryl-serine (P3CSS) are used when covalently bound to the appropriate peptide.
  • P3CSS tripalmitoyl-S-glycerylcysteinyl-seryl-serine
  • Suitable methods of administration of the peptides or pharmaceutical compositions of the invention include oral, intradermal, subcutaneous, intramuscular, intraosseous, peritoneal, intravenous injection, and the like, and systemic administration or local to the target site It may be administration (ie direct injection). Among them, a common administration method is, for example, subcutaneous injection. Administration can be by a single dose or can be boosted by multiple doses.
  • the peptide of the present invention is a pharmaceutically effective amount for treating a disease mediated by angiogenesis or a pharmaceutically effective amount for inducing immunity (more specifically, CTL) against vascular endothelial cells expressing VEGFR2. Can be administered to a subject.
  • the dose of the peptide of the present invention can be appropriately adjusted according to the disease to be treated, the age, body weight, administration method, etc. of the patient, and this is usually 0.001 mg to 1000 mg, for example 0.01 mg, for each peptide of the present invention. It can be ⁇ 100 mg, such as 0.1 mg to 30 mg, such as 0.1 mg to 10 mg, for example 0.5 mg to 5 mg. Further, the administration interval can be once every several days to several months. For example, administration can be performed once a week. Those skilled in the art can appropriately select an appropriate dose (dose).
  • composition comprising polynucleotide as active ingredient
  • the pharmaceutical composition of the present invention may also comprise a polynucleotide encoding the peptide of the present invention in a form capable of expression.
  • the phrase “in an expressible form” means that the peptide of the invention is expressed when the polynucleotide is introduced into a cell.
  • the sequence of the polynucleotide of the invention includes regulatory elements necessary for expression of the peptide of the invention.
  • the polynucleotides of the invention can be provided with the sequences necessary to achieve stable insertion into the genome of the target cell (for a description of homologous recombination cassette vectors, see eg, Thomas KR & Capecchi MR, Cell 1987, 51: 503-12). See, for example, Wolff et al., Science 1990, 247: 1465-8; US Pat. Nos. 5,580,859; 5,589,466; 5,804,566; 5,739,118; 5,736,524; 5,679,647; and WO98 / 04720 I want to be.
  • DNA-based delivery technologies include “naked DNA”, facilitated (bupivacaine, polymer, peptide-mediated) delivery, cationic lipid complexes, and particle-mediated (“gene gun”) or pressure-mediated Delivery is included (see, eg, US Pat. No. 5,922,687).
  • the peptide of the present invention can also be expressed by a viral vector or a bacterial vector.
  • expression vectors include attenuated viral hosts such as vaccinia virus or fowlpox virus.
  • vaccinia virus can be used as a vector for expressing the peptide of the present invention.
  • vaccinia virus When introduced into a host, recombinant vaccinia virus expresses an immunogenic peptide, thereby eliciting an immune response.
  • Vaccinia vectors and methods useful for immunization protocols are described, for example, in US Pat. No. 4,722,848.
  • Another vector is BCG (Bacilli Calmette Guerin). BCG vectors are described in Stover et al., Nature 1991, 351: 456-60.
  • adenovirus vectors and adeno-associated virus vectors such as adenovirus vectors and adeno-associated virus vectors, retrovirus vectors, Salmonella typhi vectors, detoxified anthrax toxin vectors, etc.
  • retrovirus vectors such as Salmonella typhi vectors, detoxified anthrax toxin vectors, etc.
  • Salmonella typhi vectors such as Salmonella typhi vectors, detoxified anthrax toxin vectors, etc.
  • Delivery of the polynucleotide of the present invention into a patient may be direct, and in this case, the patient can be directly exposed to a vector carrying the polynucleotide of the present invention.
  • it may be indirect, in which case the cells are first transformed in vitro with a vector carrying the polynucleotide of the invention and then the cells are transplanted into the patient.
  • Each of these two approaches is known as in vivo and ex vivo gene therapy.
  • polynucleotide administration may be by oral, intradermal, subcutaneous, intravenous, intramuscular, intraosseous and / or peritoneal injection, etc., and systemic administration or near the target site Topical administration of will find use. Administration can be by a single dose or can be boosted by multiple doses.
  • the polynucleotide of the present invention has a pharmaceutically effective amount for treating a disease mediated by angiogenesis or a pharmaceutically effective amount for inducing immunity (more specifically, CTL) against vascular endothelial cells expressing VEGFR2. Can be administered to the subject.
  • the dosage of the polynucleotide in a suitable carrier, or the dosage of the polynucleotide in a cell transformed with a polynucleotide encoding a peptide of the invention will depend on the disease being treated, the age, weight of the patient, method of administration, etc. This can usually be from 0.001 mg to 1000 mg, such as from 0.01 mg to 100 mg, such as from 0.1 mg to 30 mg, such as from 0.1 mg to 10 mg, such as from 0.5 mg to 5 mg.
  • the dosing interval can be once every few days to once every several months, for example, once a week. Those skilled in the art can appropriately select an appropriate dose (dose).
  • peptides and polynucleotides of the present invention can be used to induce APCs and CTLs.
  • CTLs can also be induced using the exosomes and APCs of the present invention.
  • the peptides, polynucleotides, exosomes, and APCs of the present invention can be used in combination with any other compound as long as their ability to induce CTLs is not inhibited. Therefore, a CTL of the present invention can be induced using a pharmaceutical composition comprising any of the peptide, polynucleotide, APC and exosome of the present invention.
  • APC of this invention can be induced
  • the present invention provides a method for inducing APC having CTL inducing ability using the peptide or polynucleotide of the present invention.
  • the method of the invention comprises contacting APC with a peptide of the invention in vitro, ex vivo or in vivo.
  • a method of contacting APC with the peptide ex vivo may comprise the following steps: a: recovering APC from the subject; and b: contacting the APC of step a with the peptide of the invention.
  • the APC is not limited to a particular type of cell, but is known to present proteinaceous antigens on its cell surface as recognized by lymphocytes, DCs, Langerhans cells, macrophages, B cells, and Activated T cells can be used. Since DC has the strongest CTL inducing ability among APCs, DC can be preferably used.
  • any peptide of the invention can be used alone or with other peptides of the invention.
  • the peptide of the present invention can be used in combination with another CTL-inducing peptide (for example, a VEGFR1-derived CTL-inducing peptide or a cancer antigen-derived CTL-inducing peptide).
  • the methods of the invention can include the step of administering to the subject a peptide of the invention.
  • the polynucleotide of the present invention is administered to a subject in an expressible form, the peptide of the present invention is expressed in vivo, which comes into contact with APC in vivo, and as a result, APC having high CTL inducing ability is Induced in the subject's body.
  • the present invention can also include the step of administering a polynucleotide of the present invention to a subject.
  • the present invention can also include the step of introducing the polynucleotide of the present invention into APC in order to induce APC having CTL inducing ability.
  • the method may include the following steps: a: recovering APC from the subject; and b: introducing a polynucleotide encoding the peptide of the present invention into the APC of step a.
  • Step b can be performed as described above in section "VI. Antigen-presenting cells (APC)".
  • the present invention provides a method for inducing APC having CTL inducing ability, comprising the following steps a or b: a: contacting APC with a peptide of the invention; b: introducing a polynucleotide encoding the peptide of the present invention into APC.
  • the present invention also provides a method for preparing APC having CTL inducing ability, comprising the following steps a or b: a: contacting APC with a peptide of the invention; b: introducing a polynucleotide encoding the peptide of the present invention into APC.
  • the above method can be performed in vitro, ex vivo, or in vivo, but is preferably performed in vitro or ex vivo.
  • the APC used in the above method may be derived from a subject to whom administration of induced APC is scheduled, but may be derived from a different subject.
  • APC derived from a subject (donor) that is different from the subject to be administered, the HLA type of the subject and the donor needs to be the same.
  • the peptide of the present invention when a peptide having an amino acid sequence selected from SEQ ID NOs: 1 to 5, 9, 15, 26, 27 and 39 to 45 or a modified peptide thereof is used as the peptide of the present invention, administration
  • the HLA type of the subject and the donor is preferably HLA-A11 (more preferably HLA-A * 1101).
  • the APC used in the above method is preferably an APC expressing HLA-A11 (more preferably HLA-A * 1101).
  • the donor HLA is preferably HLA-A33 (more preferably HLA-A * 3303).
  • the APC used in the above method is preferably an APC expressing HLA-A33 (more preferably HLA-A * 3303).
  • APC can be prepared from blood collected from a donor by specific gravity centrifugation or the like, and then prepared from the PBMC using a known method.
  • the present invention also provides a pharmaceutical composition for inducing ACTL having the ability to induce CTL, comprising the peptide of the present invention or a polynucleotide encoding the peptide.
  • the present invention further provides use of the peptide of the present invention or a polynucleotide encoding the peptide in the manufacture of a pharmaceutical composition for inducing APC having CTL inducing ability.
  • the present invention further provides a peptide of the present invention or a polynucleotide encoding the peptide for use in the induction of APC having the ability to induce CTL.
  • the present invention further relates to a method or process for producing a pharmaceutical composition for inducing APC comprising a peptide of the present invention or a polynucleotide encoding the peptide, pharmaceutically or physiologically.
  • a method or process is provided that includes the step of formulating an acceptable carrier.
  • the present invention also provides a method or process for producing a pharmaceutical composition for inducing APC having CTL inducing ability, comprising the peptide of the present invention or a polynucleotide encoding the peptide.
  • a method or process is provided that comprises admixing with a pharmaceutically or physiologically acceptable carrier.
  • APC induced by the method of the present invention can induce a CTL specific for VEGFR2 (ie, the CTL of the present invention).
  • the present invention also provides a method for inducing CTL using the peptide, polynucleotide, exosome, or APC of the present invention.
  • the invention also provides one or more polynucleotides that encode a polypeptide (ie, TCR subunit) that can form a T cell receptor (TCR) that can recognize a complex of a peptide of the invention and an HLA antigen.
  • TCR T cell receptor
  • the method of inducing CTL comprises at least one step selected from the following: a. Contacting a CD8 positive T cell with an antigen presenting cell that presents a complex of the HLA antigen and a peptide of the invention on its surface; b.
  • CD8 positive T cell Contacting a CD8 positive T cell with an exosome that presents a complex of HLA antigen and a peptide of the invention on its surface; and c. Introducing one or more polynucleotides encoding a polypeptide capable of forming a TCR capable of recognizing a complex of the peptide of the present invention and an HLA antigen into CD8 positive T cells.
  • the methods of the invention can include administering to the subject a peptide, polynucleotide, APC, or exosome of the invention.
  • CTLs can be induced by using them in vitro or ex vivo.
  • the method of the present invention may include the following steps: a. Recovering APC from the subject, b. Contacting the APC of step a with a peptide of the invention, and c. Co-culturing the APC of step b with CD8 positive T cells. The induced CTL may then be returned to the subject.
  • APC co-cultured with CD8-positive T cells in the above step c can be obtained by introducing a polynucleotide encoding the peptide of the present invention into APC as described above in the section “VI. Antigen-presenting cells (APC)”. It can also be prepared.
  • APC Antigen-presenting cells
  • the APC used in the method of the present invention is not limited to this, and any APC that presents a complex of the HLA antigen and the peptide of the present invention on its surface can be used.
  • an exosome that presents a complex of the HLA antigen and the peptide of the present invention on its surface can be used instead of such APC. That is, the method of the present invention can include the step of co-culturing exosomes that present a complex of the HLA antigen and the peptide of the present invention on its surface.
  • exosomes can be prepared by the methods described above in the section “V. Exosomes”.
  • CTL can be induced by introducing a vector containing a polynucleotide encoding each subunit of TCR capable of binding to the peptide of the present invention presented by the HLA antigen on the cell surface into CD8-positive T cells. it can.
  • Such transduction can be performed as described above in the section “VIII. T Cell Receptor (TCR)”.
  • the present invention provides a method for inducing CTL comprising the step selected from: a: co-culturing CD8 positive T cells with APC presenting a complex of HLA antigen and peptide of the invention on its surface; b: co-culturing CD8 positive T cells with exosomes presenting a complex of HLA antigen and a peptide of the invention on its surface; and c: of the invention presented by HLA antigen on the cell surface Introducing a vector comprising a polynucleotide encoding each subunit of TCR capable of binding to a peptide into a CD8-positive T cell.
  • the above method can be performed in vitro, ex vivo, or in vivo, but is preferably performed in vitro or ex vivo.
  • the APC or exosome and CD8 positive T cell used in the above method may be derived from a subject to whom the induced CTL is to be administered, or may be derived from a different subject. Good.
  • APC or exosome derived from a subject (donor) different from the subject to be administered, and CD8 positive T cells the HLA type of the subject and the donor needs to be the same.
  • the administration subject and donor The HLA type is preferably HLA-A11 (more preferably HLA-A * 1101).
  • the APC or exosome used in the above method is HLA-A11 (more preferably HLA-A * 1101) and the peptide of the present invention (SEQ ID NOs: 1 to 5, 9, 15, 26, 27 and 39 to 45). It is preferably an APC or exosome that presents a complex with a peptide having an amino acid sequence selected from the above or a modified peptide thereof on its surface.
  • the induced CTL exhibits specific cytotoxic activity against cells that present a complex of HLA-A11 and the peptide of the present invention (for example, HLA-A11 positive cells expressing VEGFR2).
  • HLA-A11 positive cells expressing VEGFR2
  • a peptide having an amino acid sequence selected from SEQ ID NOs: 21, 40, 70, 83, 87, 89, 90, 104 and 114 or a modified peptide thereof is used as the peptide of the present invention
  • the donor HLA is preferably HLA-A33 (more preferably HLA-A * 3303).
  • the APC or exosome used in the above method is HLA-A33 (more preferably HLA-A * 3303) and the peptide of the present invention (SEQ ID NOs: 21, 40, 70, 83, 87, 89, 90, 104).
  • the induced CTL exhibits specific cytotoxic activity against cells that present a complex of HLA-A33 and the peptide of the present invention (for example, HLA-A33 positive cells that express VEGFR2). .
  • the present invention also provides a composition or pharmaceutical composition for inducing CTL comprising at least one active ingredient selected from the following: (A) the peptide of the present invention; (B) a polynucleotide encoding the peptide of the present invention in an expressible form; (C) APC presenting the peptides of the present invention on its surface; and (d) exosomes presenting the peptides of the present invention on its surface.
  • the present invention also provides the use of an active ingredient selected from among the following in the manufacture of a composition or pharmaceutical composition for inducing CTL: (A) the peptide of the present invention; (B) a polynucleotide encoding the peptide of the present invention in an expressible form; (C) APC presenting the peptides of the present invention on its surface; and (d) exosomes presenting the peptides of the present invention on its surface.
  • an active ingredient selected from among the following in the manufacture of a composition or pharmaceutical composition for inducing CTL: (A) the peptide of the present invention; (B) a polynucleotide encoding the peptide of the present invention in an expressible form; (C) APC presenting the peptides of the present invention on its surface; and (d) exosomes presenting the peptides of the present invention on its surface.
  • the present invention further provides an active ingredient selected from among the following for use in the induction of CTL: (A) the peptide of the present invention; (B) a polynucleotide encoding the peptide of the present invention in an expressible form; (C) APC presenting the peptides of the present invention on its surface; and (d) exosomes presenting the peptides of the present invention on its surface.
  • the present invention further relates to a method or process for producing a composition or pharmaceutical composition for inducing CTLs, comprising an active ingredient selected from the following, pharmaceutically or physiologically
  • a method or process comprises formulating an acceptable carrier with: (A) the peptide of the present invention; (B) a polynucleotide encoding the peptide of the present invention in an expressible form; (C) APC presenting the peptides of the present invention on its surface; and (d) exosomes presenting the peptides of the present invention on its surface.
  • the present invention also provides a method or process for producing a composition or pharmaceutical composition for inducing CTL, wherein an active ingredient selected from the following is pharmaceutically or physiologically
  • a method or process comprising the step of admixing with a chemically acceptable carrier is provided: (A) the peptide of the present invention; (B) a polynucleotide encoding the peptide of the present invention in an expressible form; (C) APC presenting the peptides of the present invention on its surface; and (d) exosomes presenting the peptides of the present invention on its surface.
  • Suitable diseases are those mediated by angiogenesis, including various cancers, diseases related to angiogenesis in the choroid (neovascular macular disease: age-related macular degeneration, myopic macular degeneration, retina) Pigment streaks, central exudative chorioretinopathy, various retinal pigment epitheliopathy, choroidal atrophy, choroideremia, choroidal osteoma), diabetic retinopathy, rheumatoid arthritis, psoriasis, and atherosclerosis Including, but not limited to.
  • the present invention also provides a method for inducing an immune response against vascular endothelial cells expressing VEGFR2.
  • VEGFR2 is found to be strongly expressed in vascular endothelial cells at the disease site. Therefore, when an immune response against vascular endothelial cells expressing VEGFR2 is induced, angiogenesis at the disease site is inhibited as a result.
  • the present invention also provides a method of inhibiting angiogenesis at a disease site of a disease mediated by angiogenesis.
  • the method of the present invention may comprise the step of administering a composition comprising any of the peptides of the present invention or a polynucleotide encoding them.
  • the methods of the present invention also contemplate administration of exosomes or APCs that present any of the peptides of the present invention.
  • exosomes and APCs that can be used in the methods of the invention to induce an immune response include the aforementioned “V. exosomes”, “VI. Antigen presenting cells (APCs)”, and “X. It is described in detail in the sections (1) and (2) of “Method Using Exosome, APC, and CTL”.
  • the invention also provides a pharmaceutical composition or vaccine for inducing an immune response against a disease site of a disease mediated by angiogenesis, comprising an active ingredient selected from:
  • a pharmaceutical composition or vaccine is provided: (A) the peptide of the present invention; (B) a polynucleotide encoding the peptide of the present invention in an expressible form; (C) APC presenting the peptide of the invention on its surface; (D) an exosome presenting a peptide of the invention on its surface; and (e) a CTL of the invention.
  • the present invention further relates to a pharmaceutical composition or vaccine for inducing an immune response against vascular endothelial cells expressing VEGFR2, which comprises an active ingredient selected from the following: I will provide a: (A) the peptide of the present invention; (B) a polynucleotide encoding the peptide of the present invention in an expressible form; (C) APC presenting the peptide of the invention on its surface; (D) an exosome presenting a peptide of the invention on its surface; and (e) a CTL of the invention.
  • a pharmaceutical composition or vaccine for inducing an immune response against vascular endothelial cells expressing VEGFR2 which comprises an active ingredient selected from the following: I will provide a: (A) the peptide of the present invention; (B) a polynucleotide encoding the peptide of the present invention in an expressible form; (C) APC presenting the peptide of the invention on its surface; (D) an exo
  • the present invention further relates to a pharmaceutical composition or vaccine for inhibiting angiogenesis at a disease site of a disease mediated by angiogenesis, comprising an active ingredient selected from the following: Provide a composition or vaccine: (A) the peptide of the present invention; (B) a polynucleotide encoding the peptide of the present invention in an expressible form; (C) APC presenting the peptide of the invention on its surface; (D) an exosome presenting a peptide of the invention on its surface; and (e) a CTL of the invention.
  • a composition or vaccine for inhibiting angiogenesis at a disease site of mediated by angiogenesis, comprising an active ingredient selected from the following: Provide a composition or vaccine: (A) the peptide of the present invention; (B) a polynucleotide encoding the peptide of the present invention in an expressible form; (C) APC presenting the peptide of the invention on its surface; (D) an exosome
  • the present invention also provides the use of an active ingredient selected from among the following in the manufacture of a pharmaceutical composition or vaccine for inducing an immune response against a disease site of a disease mediated by angiogenesis: provide: (A) the peptide of the present invention; (B) a polynucleotide encoding the peptide of the present invention in an expressible form; (C) APC presenting the peptide of the invention on its surface; (D) an exosome presenting the peptide of the present invention in its own surface; and (e) a CTL of the present invention.
  • the present invention further provides the use of an active ingredient selected from the following in the manufacture of a pharmaceutical composition or vaccine for inducing an immune response against vascular endothelial cells expressing VEGFR2: (A) the peptide of the present invention; (B) a polynucleotide encoding the peptide of the present invention in an expressible form; (C) APC presenting the peptide of the invention on its surface; (D) an exosome presenting a peptide of the invention on its surface; and (e) a CTL of the invention.
  • an active ingredient selected from the following in the manufacture of a pharmaceutical composition or vaccine for inducing an immune response against vascular endothelial cells expressing VEGFR2: (A) the peptide of the present invention; (B) a polynucleotide encoding the peptide of the present invention in an expressible form; (C) APC presenting the peptide of the invention on its surface; (D) an exosome presenting a peptid
  • the present invention further provides the use of an active ingredient selected from among the following in the manufacture of a pharmaceutical composition or vaccine for inhibiting angiogenesis at a disease site of a disease mediated by angiogenesis: : (A) the peptide of the present invention; (B) a polynucleotide encoding the peptide of the present invention in an expressible form; (C) APC presenting the peptide of the invention on its surface; (D) an exosome presenting a peptide of the invention on its surface; and (e) a CTL of the invention.
  • an active ingredient selected from among the following in the manufacture of a pharmaceutical composition or vaccine for inhibiting angiogenesis at a disease site of a disease mediated by angiogenesis: : (A) the peptide of the present invention; (B) a polynucleotide encoding the peptide of the present invention in an expressible form; (C) APC presenting the peptide of the invention on its surface; (D) an exosome presenting
  • the invention also includes a method or process for producing a pharmaceutical composition that induces an immune response, comprising mixing or formulating a peptide or polynucleotide of the invention with a pharmaceutically acceptable carrier. Provide a way to get.
  • the present invention is for inhibiting angiogenesis at a disease site of a disease mediated by angiogenesis, comprising administering to a subject a vaccine or pharmaceutical composition comprising an active ingredient selected from the following:
  • a method or method of inducing an immune response against a disease site of a disease mediated by angiogenesis is provided: (A) the peptide of the present invention; (B) a polynucleotide encoding the peptide of the present invention in an expressible form; (C) APC presenting the peptide of the invention on its surface; (D) an exosome presenting a peptide of the invention on its surface; and (e) a CTL of the invention.
  • diseases mediated by angiogenesis associated with VEGFR2 expression can be treated by administering the peptides, polypeptides, APCs, exosomes and / or CTLs of the present invention.
  • administration of the peptides, polypeptides, APCs, exosomes and / or CTLs of the present invention can induce an immune response against the disease site of a disease mediated by angiogenesis associated with VEGFR2 expression.
  • diseases include various cancers, diseases related to angiogenesis in the choroid (neovascular macular disease: age-related macular degeneration, myopic macular degeneration, retinitis pigmentosa, central exudation Retinopathy of the retina, various retinitis pigmentosa, choroidal atrophy, choroideremia, choroidal osteoma, etc.), diabetic retinopathy, rheumatoid arthritis, psoriasis, and atherosclerosis.
  • neovascular macular disease age-related macular degeneration, myopic macular degeneration, retinitis pigmentosa, central exudation Retinopathy of the retina, various retinitis pigmentosa, choroidal atrophy, choroideremia, choroidal osteoma, etc.
  • diabetic retinopathy rheumatoid arthritis
  • psoriasis psoriasis
  • atherosclerosis by administering the
  • the invention provides a method of treating a disease in a patient in need of treatment of a disease mediated by angiogenesis associated with VEGFR2 expression, such method comprising the following steps: i) measuring the expression level of VEGFR2 in a biological sample obtained from a disease site of a subject having a disease to be treated; ii) comparing the expression level of VEGFR2 with a normal control; and iii) overexpressing VEGFR2 compared to the normal control with at least one component selected from the group consisting of (a) to (e) above Administering to a subject having a disease.
  • the present invention also comprises at least one active ingredient selected from the group consisting of (a) to (e) above for administration to a subject having a disease mediated by angiogenesis associated with VEGFR2 expression.
  • a vaccine or pharmaceutical composition comprising is provided.
  • the present invention further relates to a method for identifying a subject to be treated with at least one active ingredient selected from the group consisting of (a) to (e) above, wherein the expression level of VEGFR2 in a biological sample derived from the subject is determined.
  • the subject is treated with at least one active ingredient selected from the group consisting of (a)-(e) above, wherein the level is elevated compared to the normal control level. Methods are provided that are identified as being obtained.
  • VEGFR2 expression in a biological sample can be measured by a known method, for example, a method of detecting a transcript of the VEGFR2 gene by a probe or PCR method (for example, cDNA microarray method, Northern blot method, RT-PCR method, etc.)
  • a method for detecting a translation product of the VEGFR2 gene with an antibody for example, Western blotting, immunostaining, etc.
  • the expression level of the VEGFR2 gene in cells that overexpress VEGFR2 is increased by, for example, 10%, 25% or 50% from the control level (e.g., levels in normal cells) or more than 1.1 fold It can be determined that it has risen if it rises above 1.5 times, above 2.0 times, above 5.0 times, above 10.0 times, or more.
  • Control levels should be determined by using previously collected and stored samples from subjects with known angiogenesis-related disease states (with or without the disease) associated with VEGFR2 expression Can do.
  • normal cells obtained from areas that do not require treatment of the organ having the disease to be treated may be used as normal controls.
  • the control level may be determined by statistical methods based on results obtained by analyzing a predetermined expression level of the VEGFR2 gene in a sample derived from a subject with known disease state. Good.
  • the control level can be derived from a database of expression patterns from previously tested cells.
  • the expression level of the VEGFR2 gene in the biological sample may be compared to a plurality of control levels determined from a plurality of reference samples.
  • a control level determined from a reference sample derived from a tissue type similar to that of the subject-derived biological sample is used.
  • a reference value for the expression level of the VEGFR2 gene in a population whose disease state is known can be obtained by any method known in the art. For example, an average value +/ ⁇ 2 S.D. or a range of average value +/ ⁇ 3 S.D. can be used as the reference value.
  • control nucleic acid whose expression level has been found not to vary depending on the state of the disease mediated by angiogenesis associated with VEGFR2 expression, for example It can be normalized to the expression level of the housekeeping gene.
  • control genes include, but are not limited to, ⁇ -actin, glyceraldehyde 3-phosphate dehydrogenase, and ribosomal protein P1.
  • an HLA-A11 positive subject is selected as an administration subject of an active ingredient related to a peptide having an amino acid sequence selected from SEQ ID NOs: 1 to 5, 9, 15, 26, 27 and 39 to 45 It is preferable to do.
  • an administration target of an active ingredient related to a peptide having an amino acid sequence selected from SEQ ID NOs: 21, 40, 70, 83, 87, 89, 90, 104 and 114 an HLA-A33 positive subject Is preferably selected.
  • the present invention also provides a complex of the peptide of the present invention and HLA.
  • the complex of the present invention may be a monomer or a multimer.
  • the number of polymerizations is not particularly limited, and can be a multimer of any number of polymerizations. Examples include, but are not limited to, tetramer, pentamer, hexamer and the like.
  • Dextramers WO2002 / 072631
  • streptamers KnabelnaM et al.,. Nat Med. 2002 Jun; 8 (6): 631-7.
  • the complex of the peptide of the present invention and HLA can be prepared according to a known method (for example, Altman JD et al., Science.1996,274 (5284): 94-6, WO2002 / 072631, WO2009 / 003492 Knabel M et al., Nat Med. 2002 Jun; 8 (6): 631-7 etc.).
  • the complex of the present invention can be used, for example, for quantification of CTL specific for the peptide of the present invention.
  • a blood sample is collected from a subject administered with the pharmaceutical composition of the present invention, PBMCs are separated, CD4 negative cells are prepared, and the complex of the present invention conjugated with a fluorescent dye and the CD4 negative cells Make contact.
  • the ratio of CTL specific for the peptide of the present invention can be measured by analyzing by flow cytometry. For example, by measuring CTL specific for the peptide of the present invention before, during and / or after administration of the pharmaceutical composition of the present invention, the immune response inducing effect of the pharmaceutical composition of the present invention can be increased. Can be monitored.
  • the present invention further provides antibodies that bind to the peptides of the present invention.
  • Preferred antibodies specifically bind to peptides of the invention and do not bind (or bind weakly) to non-peptides of the invention.
  • such antibodies may include antibodies that recognize peptides in the context of HLA molecules, ie, antibodies that bind to peptide-MHC complexes.
  • the binding specificity of the antibody can be confirmed by an inhibition test. That is, if the binding between the antibody to be analyzed and the full-length VEGFR2 polypeptide is inhibited in the presence of the peptide of the invention, this indicates that the antibody specifically binds to the peptide of the invention.
  • the antibodies against the peptides of the present invention can be used in disease diagnosis and prognostic assays, as well as in the selection of subjects for administration of the pharmaceutical compositions of the present invention and monitoring of the pharmaceutical compositions of the present invention.
  • the present invention also provides various immunological assay methods for detecting and / or quantifying the peptides of the present invention or fragments thereof.
  • immunological assays include, but are not limited to, radioimmunoassay, immunochromatography, enzyme-linked immunosorbent assay (ELISA), enzyme-linked immunofluorescence assay (ELIFA), etc. It is performed within a variety of immunological assay formats well known in the art.
  • the antibodies of the present invention can be used in immunological imaging methods that can detect diseases expressing VEGFR2, examples of which include radioscintigraphic imaging methods using labeled antibodies of the present invention.
  • Such assays are used clinically in the detection, monitoring, and prognosis of diseases that express VEGFR2, and examples of such diseases include diseases associated with various cancers, angiogenesis in the choroid (neoplasia)
  • diseases associated with various cancers angiogenesis in the choroid (neoplasia)
  • Vascular macular degeneration age-related macular degeneration, myopic macular degeneration, retinitis pigmentosa, central exudative retinochoroidosis, various retinal pigment
  • the antibody of the present invention can be used in any form such as a monoclonal antibody or a polyclonal antibody, for example, antiserum obtained by immunizing animals such as rabbits with the peptide of the present invention, all classes of polyclonal antibodies and monoclonal antibodies. It may further include antibodies, human antibodies, and humanized antibodies produced by genetic recombination.
  • the peptide of the present invention or a fragment thereof used as an antigen for obtaining an antibody can be obtained by chemical synthesis or genetic engineering techniques based on the amino acid sequence disclosed in the present specification.
  • the peptide used as an immunizing antigen may be a peptide of the present invention or a fragment of the peptide of the present invention.
  • KLH keyhole limpet hemocyanin
  • Methods for conjugating KLH and peptides are also well known in the art.
  • Rodentia Lagomorpha, or Primate animals
  • Rodent animals include, for example, mice, rats, and hamsters.
  • Rabbits include, for example, rabbits.
  • Primatological animals include, for example, monkeys of the Catarrhini (Old World monkey) such as cynomolgus monkeys (Macaca fascicularis), rhesus monkeys, baboons, and chimpanzees.
  • antigen Intraperitoneal or subcutaneous injection of antigen is a standard method for immunizing mammals. More specifically, the antigen is diluted with an appropriate amount of phosphate buffered saline (PBS), physiological saline or the like and suspended. If desired, the antigen suspension can be mixed with an appropriate amount of a standard adjuvant, such as Freund's complete adjuvant, emulsified and administered to a mammal. Thereafter, the antigen mixed with an appropriate amount of Freund's incomplete adjuvant is preferably administered several times every 4 to 21 days. A suitable carrier may be used for immunization. After immunization as described above, serum can be examined by standard methods for increasing amounts of the desired antibody.
  • PBS phosphate buffered saline
  • a suitable carrier may be used for immunization. After immunization as described above, serum can be examined by standard methods for increasing amounts of the desired antibody.
  • Polyclonal antibodies against the peptides of the present invention can be prepared by recovering blood from the immunized mammal examined for an increase in the desired antibody in the serum and separating the serum from the blood by any conventional method.
  • Polyclonal antibodies may include serum containing polyclonal antibodies, and fractions containing polyclonal antibodies may be isolated from the serum.
  • Immunoglobulin G or M is obtained from a fraction that recognizes only the peptide of the present invention by using, for example, an affinity column to which the peptide of the present invention is bound, and further using this protein A or protein G column. It can be purified and prepared.
  • immune cells are collected from a mammal immunized with an antigen, and after confirming an increase in the level of a desired antibody in serum as described above, it is subjected to cell fusion.
  • the immune cells used for cell fusion can be preferably obtained from the spleen.
  • Other preferred parental cells to be fused with the above immune cells include, for example, mammalian myeloma cells, and more preferably myeloma cells that have acquired properties for selection of fusion cells with drugs.
  • the above immune cells and myeloma cells can be fused.
  • Hybridomas resulting from cell fusion can be selected by culturing them in a standard selective medium such as HAT medium (medium containing hypoxanthine, aminopterin, and thymidine). Cell culture typically continues in HAT medium for days to weeks, a period sufficient for all other cells (non-fused cells) except the desired hybridoma to die. Thereafter, standard limiting dilution can be performed to screen and clone hybridoma cells producing the desired antibody.
  • HAT medium medium containing hypoxanthine, aminopterin, and thymidine
  • human lymphocytes such as lymphocytes infected with EB virus can be transformed with peptides, cells expressing the peptides, or lysates thereof. It is also possible to immunize in vitro. Subsequently, the lymphocyte after immunization is fused with an infinitely-dividable human-derived myeloma cell such as U266 to obtain a hybridoma that produces a desired human antibody that can bind to the peptide (Japanese Patent Application Laid-Open No. Sho A). 63-17688).
  • the obtained hybridoma is transplanted into the abdominal cavity of the mouse, and ascites is extracted.
  • the obtained monoclonal antibody can be purified by, for example, ammonium sulfate precipitation, protein A or protein G column, DEAE ion exchange chromatography, or an affinity column to which the peptide of the present invention is bound.
  • immune cells that produce antibodies can be immortalized by oncogenes and used to prepare monoclonal antibodies.
  • the monoclonal antibodies thus obtained can also be prepared recombinantly using genetic engineering techniques (see, for example, Borrebaeck and Larrick, Therapeutic Monoclonal Antibodies, published in the UK by MacMillan Publishers LTD (1990)).
  • Wanna For example, DNA encoding an antibody is cloned from an immune cell such as an antibody-producing hybridoma or immunized lymphocyte, inserted into an appropriate vector, and then introduced into a host cell to prepare a recombinant antibody. Can do.
  • the present invention also provides a recombinant antibody prepared as described above.
  • the antibody of the present invention may be an antibody fragment or a modified antibody as long as it binds to the peptide of the present invention.
  • the antibody fragment desirably contains an antigen-binding site of an antibody.
  • the antibody fragment may be Fab, F (ab ′) 2 , Fv, or a single chain Fv (scFv) in which Fv fragments derived from H and L chains are linked by an appropriate linker.
  • an antibody fragment can be prepared by treating an antibody with an enzyme such as papain or pepsin.
  • a gene encoding the antibody fragment can be constructed, inserted into an expression vector, and expressed in a suitable host cell (eg, Co et al., J Immunol 152: 2968-76 (1994); Better and Horwitz, Methods Enzymol 178: 476-96 (1989); Pluckthun and Skerra, Methods Enzymol 178: 497-515 (1989); Lamoyi, Methods Enzymol 121: 652-63 (1986); Rousseaux et al., Methods Enzymol 121: 663-9 (1986); see Bird and Walker, Trends Biotechnol 9: 132-7 (1991)).
  • Antibodies can be modified by binding with various molecules such as polyethylene glycol (PEG).
  • PEG polyethylene glycol
  • the present invention provides such modified antibodies.
  • Modified antibodies can be obtained by chemically modifying antibodies. These modification methods are routine in the art.
  • the antibody of the present invention is a chimeric antibody between a variable region derived from a non-human antibody and a constant region derived from a human antibody, or a complementarity determining region (CDR) derived from a non-human antibody and a human antibody. It can also be obtained as a humanized antibody comprising a framework region (FR) derived from and a constant region.
  • Such antibodies can be prepared according to known techniques. Humanization can be performed by replacing the corresponding sequence of a human antibody with a rodent CDR or CDR sequence (see, for example, Verhoeyenyet al., Science 239: 1534-6 (1988)). . Accordingly, such humanized antibodies are chimeric antibodies in which substantially less than a human variable domain has been replaced by the corresponding sequence from a non-human species.
  • human antibodies containing human variable regions can also be used.
  • Such antibodies can be generated using various techniques known in the art. For example, in vitro methods include the use of recombinant libraries of human antibody fragments displayed on bacteriophages (eg, Hoogenboom & Winter, J. Mol. Biol. 227: 381 1991 (1991)).
  • human antibodies can be made by introducing human immunoglobulin loci into transgenic animals, such as mice, in which the endogenous immunoglobulin genes are partially or completely inactivated. This approach is described, for example, in US Pat. Nos. 6,150,584, 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425;
  • the antibody obtained as described above may be purified to homogeneity.
  • separation and purification of antibodies can be performed according to separation and purification methods used for general proteins. For example, but not limited to, appropriately selecting and combining the use of column chromatography such as affinity chromatography, filters, ultrafiltration, salting out, dialysis, SDS polyacrylamide gel electrophoresis, and isoelectric focusing
  • column chromatography such as affinity chromatography, filters, ultrafiltration, salting out, dialysis, SDS polyacrylamide gel electrophoresis, and isoelectric focusing
  • Protein A and protein G columns can be used as affinity columns.
  • Exemplary protein A columns to be used include, for example, HyperD, POROS, and Sepharose F. F. (Pharmacia) is included.
  • Exemplary chromatography includes, in addition to affinity chromatography, for example, ion exchange chromatography, hydrophobic chromatography, gel filtration, reverse phase chromatography, adsorption chromatography, etc. (Strategies for Protein Purification and Characterization: A Laboratory Course Manual. Ed Daniel R. Marshak et al., Cold Spring Harbor Laboratory Press (1996)). Chromatographic procedures can be performed by liquid phase chromatography such as HPLC and FPLC.
  • the antigen-binding activity of the antibody of the present invention using absorbance measurement, enzyme-linked immunosorbent assay (ELISA), enzyme-linked immunosorbent assay (EIA), radioimmunoassay (RIA), and / or immunofluorescence.
  • ELISA enzyme-linked immunosorbent assay
  • EIA enzyme-linked immunosorbent assay
  • RIA radioimmunoassay
  • ELISA the antibody of the present invention is immobilized on a plate, the peptide of the present invention is added to the plate, and then a sample containing a desired antibody such as a culture supernatant of antibody-producing cells or a purified antibody is added.
  • a secondary antibody that recognizes the primary antibody and is labeled with an enzyme such as alkaline phosphatase is then added and the plate is incubated.
  • an enzyme substrate such as p-nitrophenyl phosphate is added to the plate, the absorbance is measured, and the antigen binding activity of the sample is evaluated.
  • a peptide fragment such as a C-terminal or N-terminal fragment may be used as an antigen.
  • BIAcore Pharmacia
  • the peptide of the present invention can be obtained by the above method. Can be detected or measured. Since the method for detecting or measuring a peptide according to the present invention can specifically detect or measure a peptide, this method can be used in various experiments using the peptide.
  • the antibodies of the present invention can also be used to detect the peptides of the present invention present in a blood sample (eg, a serum sample) of interest. Alternatively, conversely, the antibody of the present invention present in the blood sample (eg, serum sample) of the subject can be detected using the peptide of the present invention.
  • the result of measuring the peptide of the present invention or the antibody of the present invention in the blood sample of the subject can be useful for selecting the administration target of the pharmaceutical composition of the present invention or monitoring the effect of the pharmaceutical composition of the present invention. it can.
  • the present invention also provides a vector comprising a polynucleotide encoding the peptide of the present invention and a host cell into which the vector has been introduced.
  • the vectors of the present invention can be used to retain a polynucleotide of the present invention in a host cell, to express a peptide of the present invention in a host cell, or to administer a polynucleotide of the present invention for gene therapy. .
  • E. coli When E. coli is the host cell and the vector is amplified in large quantities by amplification in E. coli (eg, JM109, DH5 ⁇ , HB101, or XL1Blue), the vector is a “replication origin” for amplification in E. coli, It is necessary to have a marker gene (for example, a drug resistance gene selected by a drug such as ampicillin, tetracycline, kanamycin, chloramphenicol) for selecting the transformed E. coli.
  • a marker gene for example, a drug resistance gene selected by a drug such as ampicillin, tetracycline, kanamycin, chloramphenicol
  • M13 vectors, pUC vectors, pBR322, pBluescript, pCR-Script and the like can be used.
  • an expression vector can be used.
  • an expression vector to be expressed in E. coli must have the above characteristics in order to amplify in E. coli.
  • E. coli such as JM109, DH5 ⁇ , HB101, or XL1 Blue is used as a host cell
  • the vector may be a promoter that can efficiently express a desired gene in E.
  • the vector may contain a signal sequence for peptide secretion.
  • An exemplary signal sequence that causes the peptide to be secreted into the periplasm of E. coli is the pelB signal sequence (Lei et al., J Bacteriol 169: 4379 (1987)).
  • Means for introducing the vector into the target host cell include, for example, the calcium chloride method and the electroporation method.
  • expression vectors derived from mammals for example, pcDNA3 (Invitrogen), and pEGF-BOS (Nucleic Acids Res 18 (17): 5322 (1990)
  • pEF pCDM8
  • insect cell-derived expression Vectors eg, “Bac-to-BAC baculovirus expression system” (GIBCO BRL), pBacPAK8)
  • plant-derived expression vectors eg, pMH1, pMH2
  • animal virus-derived expression vectors eg, pHSV, pMV, pAdexLcw
  • Retrovirus-derived expression vectors eg, pZIpneo
  • yeast-derived expression vectors eg, “Pichia expression kit” (Invitrogen), pNV11, SP-Q01
  • Bacillus subtilis Expression vectors eg, pPL608, pKTH50
  • pPL608, pKTH50 Bacillus subtilis Expression vectors
  • the vector In order for a vector to be expressed in animal cells such as CHO, COS, or NIH3T3 cells, the vector must be a promoter required for expression in such cells, such as the SV40 promoter (Mulligan et al., Nature 277: 108 (1979 )), MMLV-LTR promoter, EF1 ⁇ promoter (Mizushima et al., Nucleic Acids Res 18: 5322 (1990)), CMV promoter and the like, and preferably a marker gene (eg, drug (eg , Neomycin, G418).
  • a promoter required for expression in such cells such as the SV40 promoter (Mulligan et al., Nature 277: 108 (1979 )), MMLV-LTR promoter, EF1 ⁇ promoter (Mizushima et al., Nucleic Acids Res 18: 5322 (1990)), CMV promoter and the like, and preferably a marker gene (eg, drug
  • a peptide of less than 15 amino acids having an ability to induce cytotoxic T cells comprising an amino acid sequence selected from the following group: (A) an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-5, 9, 15, 21, 26, 27, 39-45, 70, 83, 87, 89, 90, 104 and 114; and (b ) SEQ ID NOs: 1-5, 9, 15, 21, 26, 27, 39-45, 70, 83, 87, 89, 90, 104, and 114 in the amino acid sequence selected from the group An amino acid sequence in which one or several amino acids are substituted, deleted, inserted and / or added.
  • a composition comprising a pharmaceutically acceptable carrier and at least one active ingredient selected from the group consisting of the following (a) to (e): (A) one or more peptides according to any one of [1] to [4]; (B) one or a plurality of polynucleotides encoding the peptide of any one of [1] to [4] in a form capable of being expressed; (C) an antigen-presenting cell (APC) that presents a complex of the peptide according to any one of [1] to [4] and an HLA antigen on its cell surface; (D) an exosome that presents a complex of the peptide according to any one of [1] to [4] and an HLA antigen on its cell surface; and (e) any one of [1] to [4] CTL targeting the peptide according to one item.
  • APC antigen-presenting cell
  • D an exosome that presents a complex of the peptide according to any one of [1] to [4] and an HLA
  • a composition for inducing CTL comprising at least one active ingredient selected from the group consisting of the following (a) to (d): (A) one or more peptides according to any one of [1] to [4]; (B) one or a plurality of polynucleotides encoding the peptide of any one of [1] to [4] in a form capable of being expressed; (C) an antigen-presenting cell (APC) that presents a complex of the peptide according to any one of [1] to [4] and an HLA antigen on its cell surface; and (d) [1] to [ [4] An exosome that presents a complex of the peptide of any one of [4] and an HLA antigen on its cell surface.
  • APC antigen-presenting cell
  • composition according to [6] which is a pharmaceutical composition.
  • the group consisting of (i) treatment of diseases mediated by angiogenesis, (ii) prevention of diseases mediated by angiogenesis, and (iii) prevention of postoperative recurrence of diseases mediated by angiogenesis The composition according to [8], which is a pharmaceutical composition for one or more uses selected from the above.
  • Diseases mediated by angiogenesis are cancer, diseases associated with angiogenesis in the choroid (neovascular macular disease), diabetic retinopathy, rheumatoid arthritis, psoriasis, and atherosclerosis.
  • Diseases related to angiogenesis in the choroid are age-related macular degeneration, myopic macular degeneration, retinitis pigmentosa, central exudative reticulochoroidosis, various retinal pigment epitheliopathy, choroidal atrophy
  • the composition according to [11] which is choroideremia or choroidal osteoma.
  • a method for inducing APC having CTL inducing ability comprising a step selected from the group consisting of: (A) contacting APC with the peptide according to any one of [1] to [4] in vitro, ex vivo or in vivo; and (b) any one of [1] to [4] Introducing a polynucleotide encoding the peptide of APC into APC.
  • a method of inducing CTL comprising a step selected from the group consisting of: (A) co-culturing CD8-positive T cells with APC that presents a complex of the HLA antigen and the peptide according to any one of [1] to [4] on its surface; (B) co-culturing a CD8-positive T cell with an exosome that presents a complex of the HLA antigen and the peptide of any one of [1] to [4] on its surface; and (c) A polynucleotide encoding each subunit of a T cell receptor (TCR) capable of binding to the peptide according to any one of [1] to [4] presented on the cell surface by an HLA antigen is a CD8 positive T cell Stage to introduce.
  • TCR T cell receptor
  • APC that presents a complex of the HLA antigen and the peptide according to any one of [1] to [4] on its surface.
  • Angiogenesis at a disease site of a disease mediated by angiogenesis comprising administering to a subject a composition comprising at least one active ingredient selected from the group consisting of the following (a) to (e): How to inhibit: (A) one or more peptides according to any one of [1] to [4]; (B) one or a plurality of polynucleotides encoding the peptide of any one of [1] to [4] in a form capable of being expressed; (C) an antigen-presenting cell (APC) that presents a complex of the peptide according to any one of [1] to [4] and an HLA antigen on its cell surface; (D) an exosome that presents a complex of the peptide according to any one of [1] to [4] and an HLA antigen on its cell surface; and (e) any one of [1] to [4] CTL targeting the peptide according to one item.
  • APC antigen-presenting cell
  • D an exosome that presents
  • Treatment and / or prevention of a disease mediated by angiogenesis comprising the step of administering to a subject a composition comprising at least one active ingredient selected from the group consisting of (a) to (e) below: How to do and / or prevent its recurrence after surgery: (A) one or more peptides according to any one of [1] to [4]; (B) one or a plurality of polynucleotides encoding the peptide of any one of [1] to [4] in a form capable of being expressed; (C) an antigen-presenting cell (APC) that presents a complex of the peptide according to any one of [1] to [4] and an HLA antigen on its cell surface; (D) an exosome that presents a complex of the peptide according to any one of [1] to [4] and an HLA antigen on its cell surface; and (e) any one of [1] to [4] CTL targeting the peptide according to one item.
  • APC anti
  • a method for screening a peptide having CTL inducing ability comprising the following steps: (A) SEQ ID NOs: 1 to 5, 9, 15, 21, 26, 27, 39 to 45, 70, 83, 87, 89, 90, 104 and 114, the original amino acid consisting of an amino acid sequence Creating a candidate sequence consisting of an amino acid sequence in which one, two, or several amino acid residues are substituted, deleted, inserted and / or added to the sequence; (B) selecting a candidate sequence having no significant homology (sequence identity) with any known human gene product other than VEGFR2 from the candidate sequences prepared in (a); (C) contacting the peptide comprising the candidate sequence selected in (b) with APC; (D) contacting the APC of (c) with a CD8-positive T cell; and (e) selecting a peptide having a CTL inducing ability that is equal to
  • APC antigen-presenting cell
  • D an exosome that presents a complex
  • [26] The group consisting of the following (a) to (e) in the manufacture of a pharmaceutical composition for the treatment and / or prevention of a disease mediated by angiogenesis and / or prevention of its recurrence after surgery: Use of at least one active ingredient selected from: (A) one or more peptides according to any one of [1] to [4]; (B) one or a plurality of polynucleotides encoding the peptide of any one of [1] to [4] in a form capable of being expressed; (C) an antigen-presenting cell (APC) that presents a complex of the peptide according to any one of [1] to [4] and an HLA antigen on its cell surface; (D) an exosome that presents a complex of the peptide according to any one of [1] to [4] and an HLA antigen on its cell surface; and (e) any one of [1] to [4] CTL targeting the peptide according to one item.
  • APC antigen-
  • [27] Use of at least one active ingredient selected from the group consisting of the following (a) to (e) for inhibiting angiogenesis at a disease site of a disease mediated by angiogenesis: (A) one or more peptides according to any one of [1] to [4]; (B) one or a plurality of polynucleotides encoding the peptide of any one of [1] to [4] in a form capable of being expressed; (C) an antigen-presenting cell (APC) that presents a complex of the peptide according to any one of [1] to [4] and an HLA antigen on its cell surface; (D) an exosome that presents a complex of the peptide according to any one of [1] to [4] and an HLA antigen on its cell surface; and (e) any one of [1] to [4] CTL targeting the peptide according to one item.
  • APC antigen-presenting cell
  • D an exosome that presents a complex of the peptide according
  • Use of two active ingredients: (A) one or more peptides according to any one of [1] to [4]; (B) one or a plurality of polynucleotides encoding the peptide of any one of [1] to [4] in a form capable of being expressed; (C) an antigen-presenting cell (APC) that presents a complex of the peptide according to any one of [1] to [4] and an HLA antigen on its cell surface; (D) an exosome that presents a complex of the peptide according to any one of [1] to [4] and an HLA antigen on its cell surface; and (e) any one of [1] to [4] CTL targeting the peptide according to one item.
  • APC antigen-presenting cell
  • D an exo
  • a method for inducing cytotoxic activity against cells expressing VEGFR2 comprising the step of administering to a subject a composition comprising at least one active ingredient selected from the group consisting of the following (a) to (e): : (A) one or more peptides according to any one of [1] to [4]; (B) one or a plurality of polynucleotides encoding the peptide of any one of [1] to [4] in a form capable of being expressed; (C) an antigen-presenting cell (APC) that presents a complex of the peptide according to any one of [1] to [4] and an HLA antigen on its cell surface; (D) an exosome that presents a complex of the peptide according to any one of [1] to [4] and an HLA antigen on its cell surface; and (e) any one of [1] to [4] CTL targeting the peptide according to one item.
  • APC antigen-presenting cell
  • D an exosome that
  • Cell line HLA-A and HLA-B negative human B lymphoblastoid cell line C1R, and African green monkey kidney cell line COS7 were purchased from ATCC.
  • HLA-A * 1101 or HLA-A * 3303 Creating stably expressing stimulator cells with HLA-A * 1101 or HLA-A * 3303 the C1R stably expressing (C1R-A11 and C1R-A33) is Used as stimulator cells.
  • CDNA encoding the open reading frame of HLA-A * 1101 or HLA-A * 3303 was amplified by PCR and cloned into an expression vector.
  • C1R cells were transformed with the above expression vector using Neon transfection system (Invitorogen) and then selected for 2 weeks using G418 (Invitrogen). G418-selected cells were seeded into the wells of a 96-well plate containing a culture medium supplemented with G418. Expression of exogenous HLA-A * 1101 or HLA-A * 3303 in C1R cells was confirmed by flow cytometry analysis.
  • Tables 1 and 2 show the HLA-A11 binding 9mer peptide and 10mer peptide of VEGFR2 in descending order of binding affinity. A total of 86 peptides with potential HLA-A11 binding ability were selected.
  • Table 3 and Table 4 show the HLA-A33-binding 9mer peptide and 10mer peptide of VEGFR2 in descending order of binding affinity. A total of 51 peptides with potential HLA-A33 binding ability were selected.
  • CTL-derived monocyte-derived dendritic cells were used as antigen-presenting cells to induce cytotoxic T lymphocyte (CTL) responses to peptides presented on human leukocyte antigen (HLA).
  • CTL cytotoxic T lymphocyte
  • HLA human leukocyte antigen
  • DCs were generated in vitro as described elsewhere (Nakahara S et al., Cancer Res 2003, 63 (14): 4112-8).
  • peripheral blood mononuclear cells isolated from healthy volunteers positive for HLA-A * 1101 or HLA-A * 3303
  • Ficoll-Paque plus (Pharmacia) solution were transformed into plastic tissue culture dishes (Becton Dickinson) and separated as monocyte fractions.
  • AIM-V medium Invitrogen
  • AS heat-inactivated autoserum
  • a population enriched for monocytes was cultured in the presence of 4 (R & D System). After 7 days of culture, cytokine-induced DCs were pulsed with 20 ⁇ g / ml of each synthetic peptide in AIM-V medium for 3 hours at 37 ° C. in the presence of 3 ⁇ g / ml ⁇ 2-microglobulin.
  • the generated cells appeared to express DC related molecules such as CD80, CD83, CD86, and HLA class II on their cell surface (data not shown).
  • DC related molecules such as CD80, CD83, CD86, and HLA class II on their cell surface (data not shown).
  • These peptide-pulsed DCs were then inactivated by X-ray irradiation (20 Gy) and mixed with autologous CD8 + T cells obtained by positive selection using CD8 Positive Isolation Kit (Dynal) at a ratio of 1:20 . These cultures were seeded in 48-well plates (Corning). Each well was filled with 1.5 x 10 4 peptide-pulsed DC, 3 x 10 5 CD8 + T cells and 10 ng / ml IL-7 (R & D System) in 0.5 ml AIM-V / 2% AS medium. ) Included.
  • IL-2 (CHIRON) was added to these cultures to a final concentration of 20 IU / ml.
  • T cells were further stimulated with peptide-pulsed autologous DCs.
  • DC was prepared each time by the same method as above.
  • CTL clones Dilution was carried out so that the number of CTL was 0.3, 1, and 3 / well in a 96 round bottom microtiter plate (Nalge Nunc International).
  • CTL was combined with two human B lymphoblastoid cell lines, 1 x 10 4 cells / well, 30 ng / ml anti-CD3 antibody, and 125 IU / ml IL-2 for a total of 150 ⁇ l / well of 5% AS
  • the cells were cultured in a containing AIM-V medium. Ten days later, 50 ⁇ l / well of IL-2 was added to the medium so that the final concentration of IL-2 reached 125 IU / ml.
  • an interferon (IFN) - ⁇ enzyme-linked immunospot (ELISPOT) assay and an IFN- ⁇ enzyme-linked immunosorbent assay (ELISA) were performed. Specifically, peptide-pulsed C1R-A11 or C1R-A33 (1 ⁇ 10 4 cells / well) was prepared as stimulating cells. Cultured cells in 48 well plates were used as responder cells. IFN- ⁇ ELISPOT and IFN- ⁇ ELISA assays were performed according to the manufacturer's procedures.
  • VEGFR2-A11-9-319 (SEQ ID NO: 1) (a), VEGFR2-A11-9-863 (SEQ ID NO: 2) (b), VEGFR2-A11-9-521 (SEQ ID NO: 3) (c) , VEGFR2-A11-9-973 (SEQ ID NO: 4) (d), VEGFR2-A11-9-309 (SEQ ID NO: 5) (e), VEGFR2-A11-9-860 (SEQ ID NO: 9) (f ), VEGFR2-A11-9-134 (SEQ ID NO: 15) (g), VEGFR2-A11-9-195 (SEQ ID NO: 26) (h), VEGFR2-A11-9-502 (SEQ ID NO: 27) ( i), VEGFR2-A11-9-1281 (SEQ ID NO: 39) (j), VEGFR2-A11-10-576 (SEQ ID NO: 40) (k), VEGFR2-A11-10-397 (SEQ ID NO: 41) (L), VEGFR2-A11-10-308 (SEQ
  • VEGFR2-A11-9-319 (SEQ ID NO: 1) (a), VEGFR2-A11-9-863 (SEQ ID NO: 2) (b), VEGFR2-A11-9-521 (SEQ ID NO: 3) (c) , VEGFR2-A11-9-973 (SEQ ID NO: 4) (d), VEGFR2-A11-9-309 (SEQ ID NO: 5) (e), VEGFR2-A11-9-860 (SEQ ID NO: 9) (f ), VEGFR2-A11-9-134 (SEQ ID NO: 15) (g), VEGFR2-A11-9-195 (SEQ ID NO: 26) (h), VEGFR2-A11-9-502 (SEQ ID NO: 27) ( i), VEGFR2-A11-9-1281 (SEQ ID NO: 39) (j), VEGFR2-A11-10-576 (SEQ ID NO: 40) (k), VEGFR2-A11-10-397 (SEQ ID NO: 41) (L), VEGFR2-A11-10-308 (SEQ
  • VEGFR2-A33-9-823 (SEQ ID NO: 87) (a), VEGFR2-A33-9-114 (SEQ ID NO: 89) (b), VEGFR2-A33-9-214 (SEQ ID NO: 90) (c) , VEGFR2-A33-9-577 (SEQ ID NO: 21) (d), VEGFR2-A33-10-213 (SEQ ID NO: 104) (e), VEGFR2-A33-10-97 (SEQ ID NO: 83) (f ), VEGFR2-A33-10-576 (SEQ ID NO: 40) (g), VEGFR2-A33-10-113 (SEQ ID NO: 70) (h) and VEGFR2-A33-10-1046 (SEQ ID NO: 114) ( The CTL line induced in i) showed strong IFN- ⁇ production against target cells pulsed with the corresponding peptide compared to target cells not pulsed with the peptide (FIG.
  • VEGFR2-A33-9-114 (SEQ ID NO: 89) (a), VEGFR2-A33-9-214 (SEQ ID NO: 90) (b), VEGFR2-A33-9-577 (SEQ ID NO: 21) (c) , VEGFR2-A33-10-213 (SEQ ID NO: 104) (d), VEGFR2-A33-10-97 (SEQ ID NO: 83) (e), VEGFR2-A33-10-113 (SEQ ID NO: 70) (f ) And VEGFR2-A33-10-1046 (SEQ ID NO: 114) (g) stimulated strong IFN- ⁇ production from CTL clones (FIG. 6).
  • VEGFR2-A11-9-319 SEQ ID NO: 1
  • VEGFR2-A11-10-159 SEQ ID NO: 43
  • HLA-A * 1101 Showed strong CTL activity against COS7 cells expressing both.
  • no significant specific CTL activity was detected with respect to the control cells (FIG. 7).
  • VEGFR2-A33-9-114 (SEQ ID NO: 89) (a) and VEGFR2-A33-10-113 (SEQ ID NO: 70) (b) are expressed in COS7 cells expressing both VEGFR2 and HLA-A * 3303 On the other hand, it showed strong CTL activity. On the other hand, no significant specific CTL activity was detected with respect to the control cells (FIG. 8).
  • VEGFR2-A33-9-114 (SEQ ID NO: 89) and VEGFR2-A33-10-113 (SEQ ID NO: 70) are peptides generated by endogenous processing of VEGFR2 and HLA-A * It is clearly demonstrated that it is presented on target cells with the 3303 molecule and recognized by CTL.
  • the present invention can induce a strong and specific immune response against vascular endothelial cells at a disease site with abnormal angiogenesis, and has applicability to a wide variety of diseases mediated by angiogenesis. Obtained is a novel peptide derived from VEGFR2.
  • Such peptides may be useful as peptide vaccines against diseases mediated by angiogenesis associated with VEGFR2, and examples of such diseases include various cancers, diseases associated with angiogenesis in the choroid (neovascular Macular dysfunction: age-related macular degeneration, myopic macular degeneration, retinitis pigmentosa, central exudative retina choroidopathy, various retinal pigment epitheliopathy, choroidal atrophy, choroideremia, choroidal osteoma, etc.), diabetes Include, but are not limited to, retinopathy, rheumatoid arthritis, psoriasis, and atherosclerosis.

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Abstract

Cette invention concerne un épitope peptidique qui est dérivé du VEGFR2 et qui peut induire des lymphocytes T cytotoxiques (CTL). Cette invention concerne en outre un polynucléotide codant pour ledit peptide, une cellule présentatrice d'antigène qui présente ledit peptide, un CTL qui cible ledit peptide, et un procédé pour induire ladite cellule présentatrice d'antigène et lesdits CTL. Une composition et une composition médicamenteuse les contenant à titre de principes actifs sont en outre décrites. De plus, cette invention concerne une méthode destinée à traiter et/ou à prévenir les maladies médiées par l'angiogenèse et/ou leur récurrence après une chirurgie, ladite méthode utilisant le peptide, le polynucléotide, la cellule présentatrice d'antigène, le CTL ou la composition médicamenteuse selon l'invention. Une méthode destinée à inhiber l'angiogenèse aux sièges des maladies médiées par l'angiogenèse est en outre décrite.
PCT/JP2014/082059 2013-12-06 2014-12-04 Peptide dérivé du vegfr2, et vaccin le contenant Ceased WO2015083763A1 (fr)

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JP2013253000A JP2017042047A (ja) 2013-12-06 2013-12-06 Ｖｅｇｆｒ２由来ペプチドおよびそれを含むワクチン
JP2013-253000 2013-12-06

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