US20080070835A1 - Hla-Dr-Binding Antigen Peptide Derived From Wt1 - Google Patents

Hla-Dr-Binding Antigen Peptide Derived From Wt1 Download PDF

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US20080070835A1
US20080070835A1 US10/578,183 US57818304A US2008070835A1 US 20080070835 A1 US20080070835 A1 US 20080070835A1 US 57818304 A US57818304 A US 57818304A US 2008070835 A1 US2008070835 A1 US 2008070835A1
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peptide
cells
amino acid
hla
cancer
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Haruo Sugiyama
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International Institute of Cancer Immunology 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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • 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
    • A61K39/001152—Transcription factors, e.g. SOX or c-MYC
    • A61K39/001153—Wilms tumor 1 [WT1]
    • 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
    • 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
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • 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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4748—Tumour specific antigens; Tumour rejection antigen precursors [TRAP], e.g. MAGE
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to HLA-DRB1*0405-binding antigen peptides derived from WT1.
  • WT1 gene (Wilms' tumor gene 1) has been identified as one of causative genes of Wilms' tumor that is a childhood renal tumor ( Cell 60: 509, 1990, Nature 343: 774, 1990). WT1 gene encodes the transcription factor WT1 which plays an important role in many processes such as proliferation, differentiation and apoptosis of cells, and development of tissues ( Int. Rev. Cytol. 181: 151, 1998). WT1 gene was originally defined as a tumor suppressor gene. However, subsequent studies revealed that WT1 gene is highly expressed in leukemia and various solid cancers including lung cancer and breast cancer, indicating that WT1 gene rather exerts an oncogenic function that promotes cancer growth.
  • helper T cells specific to cancer antigen is essential for effective induction of CTLs ( Cancer. Res. 62: 6438, 2002).
  • Helper T cells (CD4-positive T cells) are induced (made proliferate) and activated when they recognize a complex of MHC class II molecule and antigen peptide on antigen-presenting cells.
  • the activated helper T cells produce cytokines such as IL-2, IL-4, IL-5, IL-6, and/or interferons and mediate the growth, differentiation, and maturation of B cells.
  • the activated helper T cells also function to promote the growth, differentiation or maturation of other subsets of T cells such as Tc and TD cells.
  • the activated helper T cells can activate the immune system through the promotion of growth and activation of B and T cells.
  • helper T cells being under the influence of MHC-class II-binding antigen peptide (also referred to as “helper peptide”), whereby efficacy (potency) of cancer vaccine in cancer immunotherapy (cancer vaccine therapy) is increased ( J. Immunother., 24:195, 2001).
  • helper peptide MHC-class II-binding antigen peptide
  • WT1-derived peptides only one antigen peptide is known to bind to a subtype of MHC class II molecule, i.e., HLA-DRB1*0401 ( Cancer Immunol. Immunother. 51:271, 2002). There are no WT1-derived peptides which have been reported to bind to different subtypes.
  • the purpose of the present invention is to provide HLA-DRB1*0405-binding antigen peptides derived from WT1, and use of the peptide as an enhancer of cancer vaccine efficacy (an agent for enhancing efficacy of cancer vaccine).
  • WT1-derived antigen peptides (“helper peptides”) having an activity of binding to MHC class II antigen and enhancing the cancer vaccine efficacy (potency) in cancer immunotherapy.
  • WT1 contains an antigen peptide portion(s) which has an activity of binding to HLA-DRB1*0405 among a number of MHC class II subclasses and inducing helper T cells. This finding led to the development of a novel therapeutic method by which WT1-specific helper T cells are induced and enhanced in HLA-DRB1*0405-positive cancer patients.
  • helper peptides which are helper peptides capable of binding to plural HLA-class II molecules and inducing helper CD4-positive T cells ( British J cancer, 85(10), p1527-1534 (2001); J. Immunol., 169, p557-565 (2002)).
  • WT1 332-347 is one of the above-described HLA-DRB1*0405-binding antigen peptides (helper peptides), to elucidate whether or not it is potentially a promiscuous helper peptide.
  • the WT1 332-347 peptide of the present invention is a helper peptide applicable to patients having HLA-DRB1*1502 as well as those having HLA-DRB1*0405.
  • WT1 contains an antigen peptide portion(s) capable of binding to HLA-DRB1*1502, one of a number of MHC class II subclasses, and inducing helper T cells for the first time.
  • the present invention has been established on the basis of these findings.
  • the present invention encompasses the followings.
  • a peptide of 10-25 amino acids which comprises an amino acid sequence wherein the amino acid residue at position 1, 4, 6 and/or 9 of an amino acid sequence set forth in any one of SEQ ID NOS: 2-23 is substituted by another amino acid residue, and which binds to an HLA-DRB1*0405 and induces helper T cells.
  • a peptide comprising a peptide described in any one of (1) to (9) above together with a cancer antigen peptide.
  • a process for producing a peptide described in any one of (1) to (10) above which comprises culturing the cell described in (13) above under the condition where the peptide can be expressed.
  • a pharmaceutical composition which comprises a peptide described in any one of (1) to (10) above, an expression vector described in (12) above or a cell described in (13) above, in association with a pharmaceutically acceptable carrier.
  • composition of (16) above which is a therapeutic or preventive agent for cancer, and which comprises a peptide described in (10) above; an expression vector described in (12) related to a peptide of (10) above; or a cell described in (13) above related to a peptide of (10) above, in association with a pharmaceutically acceptable carrier.
  • a method of treating or preventing cancer which comprises administering a peptide described in any one of (1) to (10) above, an expression vector described in (12) above or a cell described in (13) above to a subject in need thereof.
  • a pharmaceutical composition which comprises a peptide described in any one of (1) to (9) above in combination with a cancer antigen peptide.
  • a kit for treating or preventing cancer which comprises a pharmaceutical composition comprising a peptide of any one of (1) to (9) above in association with a pharmaceutically acceptable carrier, and a pharmaceutical composition comprising a cancer antigen peptide in association with a pharmaceutically acceptable carrier.
  • a method of treating or preventing cancer which comprises administering a peptide of any one of (1) to (9) above in combination with a cancer antigen peptide to a subject in need thereof.
  • the present invention provides an HLA-DRB1*0405-binding antigen peptide derived from WT1, a polynucleotide encoding the peptide, an inducer of helper T cells (“helper T cell inducer”) comprising said peptide or polynucleotide, and the like.
  • helper T cell inducer of the present invention is useful as an enhancer of cancer vaccine efficacy.
  • the enhancer of cancer vaccine efficacy of the present invention is applicable to many HLA-DRB1*0405-positive patients, and is particularly useful for enhancing efficacy of WT1 vaccine.
  • FIG. 1 shows the results of examination into responsiveness of CD4-positive T cells (helper T cells) stimulated with a WT1-derived WT1 332-347 peptide to various dendritic cells.
  • “Untreated” represents the responsiveness to dendritic cells not pulsed with a peptide;
  • “PHA” the results of examination wherein CD4 positive T cells were treated with PHA instead of dendritic cells, “WT1 172-186 pulse” the responsiveness to dendritic cells pulsed with WT1 172-186 peptide, “WT1 225-243 pulse” the responsiveness to dendritic cells pulsed with WT1 225-243 peptide, and “WT1 332-347 pulse” the responsiveness to dendritic cells pulsed with “WT1 332-347 peptide.
  • the vertical axis indicates the amount of [ 3 H]-thymidine uptake (cpm) by CD4-positive T cells.
  • FIG. 2 shows the results of examination into responsiveness of G2 cell lines to dendritic cells pulsed with a WT1-derived WT1 332-347 peptide.
  • “Untreated” represents the results obtained using dendritic cells not pulsed with a peptide; and “WT1 332-347 pulse” to the results obtained using dendritic cells pulsed with WT1 332-347 .
  • the vertical axis indicates the amount of [ 3 H]-thymidine uptake (cpm) by G2 cell lines.
  • FIG. 3 shows the results of examination into responsiveness of G2 cell line to B-LCL(+) cells expressing WT1 gene.
  • B-LCL( ⁇ ) represents the results obtained using B-LCL( ⁇ ) cells not-expressing WT1 gene
  • B-LCL(+) the results obtained using B-LCL(+) cells expressing WT1 gene
  • B-LCL(+)+anti-HLA-DR antibody to the results obtained using B-LCL(+) cells treated with anti-HLA-DR antibody.
  • the vertical axis indicates the amount of [ 3 H]-thymidine uptake (cpm) by G2 cell lines.
  • FIG. 4 shows the results of examination into responsiveness of E04.1 cell line to dendritic cells pulsed with a WT1-derived WT1 332-347 peptide.
  • ⁇ represents the results obtained using dendritic cells not pulsed with a peptide
  • 332 the results obtained using dendritic cells pulsed with WT1 332-347 peptide.
  • the vertical axis indicates the amount of [ 3 H]-thymidine uptake (cpm) by E04.1 cell lines.
  • FIG. 5 shows the results of examination into responsiveness of E04.1 cell line to stimulated cells which have been pulsed with WT1-derived WT1 332-347 peptide and then treated with various anti-HLA inhibitory antibodies.
  • the stimulated cell used is B-LCL( ⁇ ) cell which is a B cell line established from blood of a healthy volunteer positive for HLA-DRB1*0405 as shown herein below in Example 3.
  • ⁇ represents the results obtained using stimulated cells not pulsed with a peptide
  • “332” the results obtained using stimulated cells pulsed with WT1 332-347 peptide.
  • “332+ ⁇ -classI” represents the results obtained using stimulated cells treated with WT1 332-347 peptide and anti-HLA-class I antibody
  • “332+ ⁇ -DR” to the results obtained using stimulated cells treated with the WT1 332-347 peptide and anti-HLA-DR antibody
  • “332+ ⁇ -DQ” to the results obtained using stimulated cells treated with WT1 332-347 peptide and anti-HLA-DQ antibody.
  • “332+mIgG” represents the results obtained using stimulated cells treated with WT1 332-347 peptide and anti-mouse-IgG antibody as a negative control for inhibitory antibody.
  • the vertical axis indicates the amount of [ 3 H]-thymidine uptake (cpm) by E04.1 cell lines.
  • FIG. 6 shows the results of examination into responsiveness of E04.1 cell line to HLA-DRB1*0405-positive or -negative PBMC pulsed with WT1-derived WT1 332-347 peptide.
  • “ ⁇ ” represents the results obtained using PBMC not pulsed with a peptide
  • “332” the results obtained using PBMC pulsed with WT1 332-347 peptide.
  • HLA-DRB1 genotypes of respective donors are as follows. Donor 1 (HLA-DRB1*0405/0803), Donor 2 (HLA-DRB1*0405/0101), Donor 3 (HLA-DRB1*0101/1001) and Donor 4 (HLA-DRB1*1201/0802).
  • the vertical axis indicates the amount of [ 3 H]-thymidine uptake (cpm) by E04.1 cell lines.
  • FIG. 7 shows the results of examination into responsiveness of E04.1 cell line to B-LCL(+) cells expressing WT1 gene.
  • B-LCL( ⁇ ) represents the results obtained using B-LCL( ⁇ ) cell not-expressing WT1 gene as stimulated cells
  • B-LCL(+) the results obtained using B-LCL(+) cells expressing WT1 gene as stimulated cells.
  • the vertical axis indicates the amount of [ 3 H]-thymidine uptake (cpm) by E04.1 cell lines.
  • FIG. 8 shows the results of examination into responsiveness of E04.1 cell line to dendritic cells pulsed with B-LCL(+) cells in which apoptosis has been induced.
  • apoptotic B-LCL (+) represents the results obtained using dendritic cells pulsed with B-LCL(+) cells expressing WT1 gene and having been induced apoptosis
  • apoptotic B-LCL ( ⁇ ) represents the results obtained using dendritic cells pulsed with B-LCL( ⁇ ) cells not-expressing WT1 gene and having been induced apoptosis.
  • E04.1+ represents the results obtained by cocultivation of E04.1 cells with dendritic cells
  • E04.1 ⁇ the results obtained by cocultivation without E04.1 cells.
  • the vertical axis indicates the amount of [ 3 H]-thymidine uptake (cpm) by E04.1 cell lines.
  • FIG. 9 shows the results of examination into cytokine production of E04.1 cell lines to dendritic cells pulsed with WT1-derived WT1 332-347 peptide.
  • ⁇ represents the results obtained using dendritic cells not pulsed with a peptide
  • 332 the results obtained using dendritic cells pulsed with WT1 332-347 peptide.
  • the vertical axis indicates the percentage (%) of E04.1 cells showing the production of IL-4 (blank bar) or IFN- ⁇ (filled bar).
  • FIG. 10 shows the results of analysis of E04.1 cell line stained with anti-CD4 antibody and anti-CXCR3 antibody by flow cytometer.
  • the horizontal and vertical axes indicate cells positive for CD4 and for CXCR3, respectively.
  • the percentage of cells positive for both CD4 and CXCR3 was 90.1%.
  • FIG. 11 shows the results of examination into influence of WT1-derived WT1 332-347 peptide on the induction of WT1-specific CTLs.
  • PBMCs originated in a healthy volunteer (HLA-A*2402/1101, DRB1*0405/0803) were cultured for 7 days under the stimulation conditions of (A) WT1 235-243 peptide, (B) WT1 235-243 peptide+WT1 332-347 peptide, (C) WT1 235-243 peptide+E04.1 cell and (D) WT1 235-243 peptide+WT1 332-347 peptide+E04.1 cell.
  • the horizontal and vertical axes indicate the percentage of cells positive for CD8 and for WT1 235-243 peptide/HLA-A*2402, respectively.
  • FIG. 12 shows the results of examination into influence of WT1-derived WT1 332-347 peptide on the activation of WT1-specific CTLs. Another half of the cells recovered in the experiment mentioned in FIG. 11 were stimulated with WT1 235-243 peptide for 6 hours, and intracellular IFN- ⁇ was stained. The vertical and horizontal axes indicate the cells positive for intracellular IFN- ⁇ and for anti-mouse IgG antibody, respectively.
  • the figure shows the results of stimulation with (E) WT1 235-243 peptide, (F) WT1 235-243 peptide+WT1 332-347 peptide, (G) WT1 235-243 peptide+E04.1 cell, (H) WT1 235-243 peptide+WT1 332-347 peptide+E04.1 cell.
  • FIG. 13 shows the results of examination into responsiveness of CD4-positive T cells stimulated with WT1-derived WT1 332-347 peptide with various dendritic cells.
  • ⁇ represents the responsiveness with dendritic cells not pulsed with a peptide
  • 332 the responsiveness with dendritic cells pulsed with WT1 332-347 peptide
  • 172 the responsiveness with dendritic cells pulsed with WT1 172-186 peptide
  • 225 the responsiveness with dendritic cells pulsed with WT1 225-243 peptide.
  • the vertical axis indicates the amount of [ 3 H]-thymidine uptake (cpm) by CD4-positive T cells.
  • the symbol “**” and “n.s.” mean that the difference in the test groups is statistically significant or is not, respectively.
  • FIG. 14 shows the results of T-cell repertoire analysis of CD4-positive T cells stimulated with WT1-derived WT1 332-347 peptide.
  • the cells were stained with different antibodies specific for respective V ⁇ chains of TCR and analyzed by flow cytometry.
  • the cell population in the lower right portion of quartered area represents V ⁇ 3-positive cells.
  • the cell population in the lower right portion of quartered area represents V ⁇ 20-positive cells.
  • FIG. 15 shows the results of examination into the responsiveness of E15.1 cell line or E15.2 cell line to autologous PBMCs pulsed with WT1-derived WT1 332-347 peptide.
  • ⁇ represents the results obtained by using autologous PBMCs not pulsed with a peptide
  • 332 the results obtained using autologous PBMCs pulsed with WT1 332-347 peptide.
  • the vertical axis indicates the amount of [ 3 H]-thymidine uptake (cpm) by separated cell lines.
  • A) and B) show the results obtained using E15.1 cell line and E15.2 cell line, respectively.
  • the symbol “**” means that the difference in the test groups is statistically significant.
  • FIG. 16 shows the results of examination into cytokine production of E15.2 cell line to autologous PBMCs pulsed with WT1-derived WT1 332-347 peptide.
  • ⁇ represents the results obtained using dendritic cells not pulsed with a peptide
  • 332 the results obtained using autologous PBMCs pulsed with WT1 332-347 peptide.
  • the vertical axis indicates the percentage (%) of E15.2 cells showing the production of IL-4 (blank bar) or IFN- ⁇ (filled bar).
  • FIG. 17 shows the results of examination into the relation between the concentration of WT1-derived WT1 332-347 peptide pulsed into autologous PBMCs and the responsiveness of E15.2 cell line.
  • the vertical axis indicates the amount of [ 3 H]-thymidine uptake (cpm) by E15.1 cell lines.
  • the horizontal axis indicates the concentration of WT1 332-347 peptide pulsed into autologous PBMC.
  • FIG. 18 shows the results of examination into responsiveness of E15.2 cell line to PBMCs positive or negative for HLA-DRB1*1502 pulsed with WT1-derived WT1 332-347 peptide.
  • “ ⁇ ” represents the results obtained by using PBMCs not pulsed with the WT1 332-347 peptide
  • “332” the results obtained using PBMCs pulsed with the peptide.
  • A) shows the results obtained using PBMCs from an HLA-DRB1*1502-positive healthy volunteer and B) the results obtained using PBMCs from an HLA-DRB1*1502-negative healthy volunteer.
  • the vertical axis indicates the amount of [ 3 H]-thymidine uptake (cpm) by E15.2 cell lines.
  • the symbol “**” and “n.s.” mean that the difference in the test groups is statistically significant or not, respectively.
  • the present invention provides a peptide consisting of 10-25 contiguous amino acids in the amino acid sequence of human WT1 set forth in SEQ ID NO: 1, said peptide binding to HLA-DRB1*0405 and inducing helper T cells.
  • the present invention encompasses peptides wherein the N-terminal and/or C-terminal amino acid residue is modified or those wherein a particular amino acid residue(s) is altered.
  • helper T cells a peptide that induces helper T cells (or a peptide that induces CD4-positive T cells)” may be referred to as “a helper peptide”.
  • amino acid sequence of human WT1 set forth in SEQ ID NO: 1 is a known sequence as described in Cell, 60:509, 1990, and NCBI data base (Accession Nos. XP — 034418 and P19544).
  • the peptide of the present invention is a partial peptide which consists of 10-25 contiguous amino acids present in the amino acid sequence of human WT1 set forth in SEQ ID NO: 1.
  • the definition of “10-25 amino acids” is based on the facts that peptides having an activity of binding to MHC class II generally consist of 10 to 25 amino acids ( Immunogenetics, 41: 178-228, 1995, Biochimica et Biophysica Acta 1316, 85-101 (1996), Immunology, 96, 1-9 (1999), Peptides, Vol. 19, 179-198 (1998), Immunobiology, 5th Edt., 116-117, Garland Publishing (2001)).
  • Preferred peptides are those consisting of 13-17 contiguous amino acids in the amino acid sequence of human WT1.
  • the peptide of the present invention can be identified by synthesizing a peptide (candidate peptide) consisting of 10-25 contiguous amino acids in the amino acid sequence set forth in SEQ ID NO: 1, and assaying whether or not the peptide is capable of binding to HLA-DRB1*0405 and inducing helper T cells.
  • a peptide can be synthesizing a peptide (candidate peptide) consisting of 10-25 contiguous amino acids in the amino acid sequence set forth in SEQ ID NO: 1, and assaying whether or not the peptide is capable of binding to HLA-DRB1*0405 and inducing helper T cells.
  • a peptide can be conducted according to processes generally used in the field of peptide chemistry. Such a method can be found in literatures including Peptide Synthesis, Interscience, New York, 1966; The Proteins, Vol. 2, Academic Press Inc., New York, 1976; Peptide Synthesis, Maruzen, Inc., 1975; Peptide - Gosei no Kiso to Jikken, Maruzen, Inc., 1985; and Iyakuhin no Kaihatsu (Zoku), Vol. 14, Peptide Synthesis, Hirokawa-syoten, 1991.
  • dendritic cells are prepared by isolating peripheral blood mononuclear cells (PBMCs) from a human subject positive for HLA-DRB1*0405, and removing non-adherent cells.
  • helper T cells CD4-positive T cells are prepared from the same HLA-DRB1*0405-positive subject by density gradient centrifugation with Ficoll-Paque, and so on.
  • the above-described dendritic cells are cultured after addition of a candidate peptide, and further cultured with the above-described helper T cells.
  • the helper T cells are then recovered and stimulated several times with dendritic cells pulsed with the candidate peptide in a similar manner. It is possible to evaluate whether or not helper T cells are induced (activated) in response to stimulation with a peptide by measuring, for example, (1) growth activity of helper T cells or (2) cytokine-producing-activity of helper T cells.
  • the growth activity (1) can be examined by measuring the amount of [ 3 H]-thymidine uptake by helper T cells.
  • the cytokine-producing activity (2) can be examined by measuring the amount of cytokine such as IFN- ⁇ produced by activated helper T cells by enzyme enzyme-linked immunosorbent assay (ELISA) or the like.
  • ELISA enzyme-linked immunosorbent assay
  • the amino acid sequence of antigen peptides binding to MHC class I or MHC class II molecule and being presented obeys a certain rule (binding motif).
  • binding motif There are terminal amino acid residues at the both ends of peptides binding to MHC class I molecule which play a significant role in the binding with MHC class I molecule; however, there are no such amino acids at either end of peptides binding to MHC class II molecule, and the terminal amino acids do not bind to MHC class II molecule.
  • Such a peptide rather is accommodated and immobilized (fixed) in a peptide-binding groove longitudinally.
  • the immobilization of a peptide in a peptide-binding groove can be achieved through the binding of the side chains of amino acids constituting the peptide to the peptide-binding groove and the binding of the main chain of the peptide to the side chains of amino acids well-preserved in the entire peptide-binding groove for MHC class II molecules.
  • a peptide-binding groove has small or large pockets and there is amino acid polymorphism in the amino acid residues constituting the pockets depending on the MHC class II molecule.
  • the amino acid motif of peptides binding to a pocket(s) of peptide-binding groove can be estimated by analyzing the pattern of amino acid residues commonly found in the binding peptides for respective MHC class II molecules originated in different alleles. It is considered that, because peptides of about 9 amino acids having such a motif is accommodated in the peptide-binding groove in such a manner that the both termini protrude from the both sites of groove, there are basically no limitations regarding the length of peptides which can bind to MHC class II molecules. However, in many cases, a long peptide is cleaved into peptides of 13-17 amino acid length by peptidases ( Immunobiology, 5th Edt., 116-117, Garland Publishing (2001)).
  • the amino acids at positions 1, 4, 6 and 9 in the HLA(MHC)-binding domain consisting of 9-amino acids are expected to have the following regularities (motifs) ( Immunogenetics, 41, 178-228 (1995), Biochimica et Biophysica Acta 1316, 85-101 (1996)).
  • the present invention is based on the finding that WT1 (SEQ ID NO: 1) contains antigen peptide portions which bind to HLA-DRB1*0405 (a kind of MHC class II) and induce helper T cells for the first time.
  • WT1 SEQ ID NO: 1
  • HLA-DRB1*0405 a kind of MHC class II
  • Examples of putative HLA-DRB1*0405-binding 9-amino-acid portions in the amino acid sequence of said WT1 include the 9-amino-acid portions derived from WT1 as shown in SEQ ID NOS: 2-23.
  • the present invention provides a peptide which comprises an amino acid sequence set forth in any one of SEQ ID NOS: 2-23, and which binds to HLA-DRB1*0405 and induces helper T cells.
  • a peptide of such a peptide is a partial peptide of WT1 and comprises an amino acid sequence set forth in any one of SEQ ID NOS: 2-23, and has an activity of binding to HLA-DRB1*0405 and inducing helper T cells.
  • a peptide of about 9 amino acids with a binding motif can accommodate in a peptide-binding groove with the both ends extruding from the both sites of groove, and hence there are essentially no limitations regarding the length of the peptides capable of binding to MHC class II molecule.
  • the MHC class II-binding peptides having been reported so far are about 10-25 amino acids in length ( Immunogenetics, 41, 178-228 (1995), Biochimica et Biophysica Acta 1316, 85-101 (1996), Immunology, 96, 1-9 (1999), Peptides, Vol. 19, 179-198 (1998), immunobiology, 5th Edt., 116-117, Garland Publishing (2001)).
  • the peptides of the present invention preferably consist of about 10-25 amino acids, and more preferably about 13-17 amino acids.
  • examples of a preferred embodiment of the peptide of the present invention which comprises an amino acid sequence set forth in any one of SEQ ID NOS: 2-23 include partial peptides derived from WT1 consisting of 10-25 amino acids (preferably, 13-17 amino-acids), which comprise an amino acid sequence set forth in any one of SEQ ID NOS: 2-23, and which have an activity of binding to HLA-DRB1*0405 and inducing helper T cells.
  • Examples of more preferred embodiments include partial peptides derived from WT1 consisting of 10-25 amino acids (preferably, 13-17 amino-acids), which comprise an amino acid sequence set forth in SEQ ID NO: 12, and which have an activity of binding to HLA-DRB1*0405 and inducing helper T cells.
  • Examples of still more preferred embodiments include partial peptides derived from WT1 consisting of 16-25 amino acids (preferably, 16-17 amino-acids), which comprise the amino acid sequence set forth in SEQ ID NO: 24, and which have an activity of binding to HLA-DRB1*0405 and inducing helper T cells.
  • the amino acid sequence set forth in SEQ ID NO: 24 represents a 16-amino-acid partial peptide derived from WT1 and includes the amino acid sequence set fort in SEQ ID NO: 12.
  • Further preferred embodiment is the peptide consisting of the amino acid sequence set forth in SEQ ID NO: 24.
  • the peptides of the present invention may be altered as appropriate within a range that the activity is maintained.
  • alteration of amino acid residue means substitution, deletion and/or addition of amino acid residue(s) (the addition is inclusive of addition of amino acid(s) at the N- and/or C-terminus of a peptide).
  • the substitution of amino acid residue(s) is preferred.
  • substitution involves substitution of an amino acid residue(s)
  • any number of amino acid residues at any position can be replaced on the condition that the activity as a helper peptide is retained.
  • the alteration is preferably involves one to several amino acids.
  • amino acid residue at position 1, 4, 6 and/or 9 of a 9 amino acid peptide having the structure for the binding motif of HLA-DRB1*0405 is substituted.
  • substitution-related peptides of the present invention include peptides of 10-25 amino acids, which comprise an amino acid sequence wherein the amino acid residue at position 1, 4, 6 and/or 9 of an amino acid sequence set forth in any one of SEQ ID NOS: 2-23 is substituted by another amino acid residue, and which bind to an HLA-DRB1*0405 and induce helper T cells.
  • Preferred examples include peptides of 10-25 amino acids, which comprise an amino acid sequence wherein the amino acid residue at position 1, 4, 6 and/or 9 of an amino acid sequence set forth in any one of SEQ ID NOS: 2-23 is substituted by an amino acid residue selected from the following amino acids:
  • substitution of amino acid residue at position 1, 4, 6 and/or 9 can be conducted for the purpose of improving the binding activity to HLA-DRB1*0405 or enhancing the activity of the above-mentioned natural-type helper peptides of the present invention which consist of partial sequences of WT1.
  • the parts other than the substituted amino acid at position 1, 4, 6 and/or 9 of a peptide may remain to have the natural-type sequence (i.e., being kept to have the partial sequence of WT1), or may be further altered as far as the activity is retained.
  • More preferred examples include peptides of 10-25 amino acids, which comprise an amino acid sequence wherein the amino acid residue at position 1, 4, 6 and/or 9 of the amino acid sequence set forth in SEQ ID NO: 12 is substituted by an amino acid residue selected from the following amino acids:
  • Still more preferred examples include peptides wherein the amino acid residue at position 3, 6, 8 and/or 11 of the 16-amino-acid partial peptide derived from WT1 set forth in SEQ ID NO: 24, which comprises the amino acid sequence set forth in SEQ ID NO: 12, is substituted by an amino acid residue selected from the following amino acids:
  • the present invention also provides a peptide (so-called an epitope peptide) comprising a helper peptide (natural- or altered-peptide) of the present invention together with a cancer antigen peptide.
  • helper T cells CD4-positive T cells activated by a helper peptide exert various activities including induction of differentiation and maintenance of CTLs, and activation of effectors such as macrophages, etc, and hence are considered to enhance the CTL-induction by cancer antigens.
  • the peptides of the present invention also include epitope peptides comprising a helper peptide of the present invention and a cancer antigen peptide.
  • cancer antigen peptide any of known cancer antigen peptides can be used; however, it is preferred to use a cancer antigen peptide derived from WT1 (natural or altered peptide).
  • WT1-derived peptides restricted to HLA-A1, -A0201, -A0204, -A0205, -A0206, -A0207, -A11, -A24, -A31, -A6801, -B7, -B8, -B2705, -B37, -Cw0401, -Cw0602, and the like.
  • WT1-derived cancer antigen peptides include those listed in Table II-Table XLVI of WO2000/18795 and altered peptides thereof which have the activity as a cancer antigen peptide (an activity of binding to an HLA antigen and inducing CTLs).
  • WT1-derived cancer antigen peptides include the followings.
  • Abu refers to “ ⁇ -aminoacetic acid.
  • the peptides set forth in SEQ ID NOS: 27 and 29 are HLA-A24 antigen- and HLA-A2 antigen-binding peptides
  • the peptides set forth in SEQ ID NOS: 44 and 45 are HLA-A2 antigen-binding peptides.
  • the other peptides are HLA-A24 antigen-binding peptides.
  • Preferred cancer antigen peptide is the one set forth in SEQ ID NO: 27, 28, 29, 30, 44 or 45.
  • epitope peptides of the present invention include those which comprise a helper peptide that is a WT1-derived partial peptide of 10-25 amino acids comprising an amino acid sequence set forth in any one of SEQ ID NOS: 2-23 and has an activity of binding to HLA-DRB1*0405 and inducing helper T cells, together with a cancer antigen peptide set forth in any one of SEQ ID NOS: 27-45 above.
  • Epitope peptides preferably comprise a helper peptide consisting of the amino acid sequence set forth in SEQ ID NO: 24 together with a cancer antigen peptide set forth in any one of SEQ ID NOS: 27-45.
  • epitope peptides comprise a helper peptide consisting of the amino acid sequence set forth in SEQ ID NO: 24 together with a cancer antigen peptide set forth in any one of SEQ ID NOS: 27-30, 44 and 45.
  • the epitope peptides can be prepared by aforementioned usual method for peptide synthesis. It can also be prepared by a usual method for DNA synthesis and genetic engineering on the basis of sequence information of a polynucleotide encoding an epitope peptide wherein multiple epitopes are ligated. Specifically, an epitope peptide wherein a multiple epitopes are ligated can be prepared by inserting a polynucleotide encoding the peptide into a known expression vector, transforming a host cell with the resultant recombinant expression vector, culturing the transformants, and recovering the objective peptide from the culture.
  • the activity of said epitope peptide as a helper peptide can be confirmed according to the above-mentioned method. Further, the activity of said epitope peptide as a cancer antigen peptide can be confirmed by subjecting the peptide to model animals for human described in WO02/47474 or Int J. Cancer: 100, 565-570, 2002.
  • An epitope peptide of the present invention is considered to be useful to establish more efficient treatment or prevention of cancer, because a helper peptide portion in the helper epitope peptide can activate helper T cells (CD4-positive T cells) to give activated helper T cells which exert various activities including induction of differentiation and maintenance of CTLs and activation of effectors such as macrophages, whereby it enhances CTL-induction by cancer antigen peptides further.
  • helper T cells CD4-positive T cells
  • amino group of the N-terminal amino acid or the carboxyl group of the C-terminal amino acid of the above-described peptide of the present invention may be modified.
  • the peptides wherein the N-terminal and/or C-terminal amino acid residue is modified fall within the scope of the peptide of the present invention.
  • Examples of a group usable in the modification of amino group of the N-terminal amino acid include 1 to 3 groups selected from C 1-6 alkyl group, phenyl group, cycloalkyl group and acyl group.
  • Acyl groups include C 1-6 alkanoyl group, C 1-6 alkanoyl group substituted by phenyl group, carbonyl group substituted by C 5-7 cycloalkyl group, C 1-6 alkylsulfonyl group, phenylsulfonyl group, C 2-6 alkoxycarbonyl group, alkoxycarbonyl group substituted by phenyl group, carbonyl group substituted by C 5-7 cycloalkoxy group, phenoxycarbonyl group, and the like.
  • esters and amides examples include esters and amides.
  • Esters specifically include C 1-6 alkyl esters, C 0-6 alkyl esters substituted by phenyl group, C 5-7 cycloalkyl esters, and the like.
  • Amides specifically include amides, amides substituted by one or two C 1-6 alkyl groups, amides substituted by one or two C 0-6 alkyl groups that are substituted by phenyl group, amides forming 5- to 7-membered azacycloalkane inclusive of nitrogen atom of amide group, and the like.
  • the present invention also provides a polynucleotide encoding the above-mentioned peptide (natural-, altered- or epitope-peptide) of the present invention.
  • the polynucleotide encoding a peptide of the present invention may be in the form of DNA or RNA.
  • the polynucleotides of the present invention can be easily prepared on the basis of information about amino acid sequence of the present peptide or polynucleotide sequence of DNA encoding the same. Specifically, synthesis can be carried out using usual method of DNA synthesis or amplification by PCR.
  • polynucleotides include those encoding the above-mentioned epitope peptides. More specifically, examples of polynucleotide include those encoding epitope peptides which comprise a helper peptide that is a WT1-derived partial peptide of 10-25 amino acids comprising an amino acid sequence set forth in any one of SEQ ID NOS: 2-23 and has an activity of binding to HLA-DRB1*0405 and inducing helper T cells, together with a cancer antigen peptide set forth in any one of SEQ ID NOS: 27-45 above.
  • a helper peptide that is a WT1-derived partial peptide of 10-25 amino acids comprising an amino acid sequence set forth in any one of SEQ ID NOS: 2-23 and has an activity of binding to HLA-DRB1*0405 and inducing helper T cells, together with a cancer antigen peptide set forth in any one of SEQ ID NOS: 27-45 above.
  • Preferred examples include a polynucleotide encoding an epitope peptide which comprises a helper peptide consisting of the amino acid sequence set forth in SEQ ID NO: 24 and a cancer antigen peptide set forth in any one of SEQ ID NOS: 27-45.
  • More preferred examples include a polynucleotide encoding an epitope peptide which comprise a helper peptide consisting of the amino acid sequence set forth in SEQ ID NO: 24 and a cancer antigen peptide set forth in any one of SEQ ID NOS: 27-30, 44 and 45.
  • polynucleotide encoding the peptide of the present invention encompasses polynucleotides that can hybridize under the stringent conditions to the complementary sequence of the said polynucleotide and that encode peptides which have activities equivalent to the peptide of the present invention.
  • hybridize under the stringent condition the “hybridization” herein used can be carried out according to conventional method described in, for example, Sambrook J., Frisch E. F., Maniatis T. ed. Molecular Cloning 2nd edition, Cold Spring Harbor Laboratory press.
  • a recombinant expression vector for expressing the peptide of the present invention can be constructed by incorporating a polynucleotide prepared above into an expression vector.
  • expression vectors usable herein include plasmids, phage vectors, virus vectors, and the like.
  • examples of vector include plasmid vectors such as pUC118, pUC119, pBR322, pCR3, and the like; and phage vectors such as ⁇ ZAPII, ⁇ gt11, and the like.
  • examples of vector include pYES2, pYEUra3, and the like.
  • examples of vector include pAcSGHisNT-A, and the like.
  • vectors When the host is animal cells, examples of vector include plasmid vectors such as pKCR, pCDM8, pGL2, pcDNA3.1, pRc/RSV, pRc/CMV, and the like; and virus vectors such as retrovirus vector, adenovirus vector, adeno-associated virus vector, and the like.
  • the expression vector may optionally contain a factor(s) such as promoter capable of inducing expression, a gene encoding a signal sequence, a marker gene for selection, terminator, and the like.
  • a factor(s) such as promoter capable of inducing expression, a gene encoding a signal sequence, a marker gene for selection, terminator, and the like.
  • the expression vector may contain an additional sequence for allowing the peptide to express as a fusion protein with thioredoxin, His tag, GST (glutathione S-transferase), or the like, so as to facilitate the isolation and purification.
  • Vectors usable in such a case include GST fusion protein vectors containing an appropriate promoter (lac, tac, trc, trp, CMV, SV40 early promoter, etc) that functions in host cells, such as pGEX4T; vectors containing Tag sequence (Myc, His, etc) such as pcDNA3.1/Myc-His; and vectors capable of expressing a fusion protein between thioredoxin and His tag such as pET32a.
  • Transformed cells containing the vector of the present invention can be prepared by transforming host cells with an expression vector obtained in the above.
  • Host cells usable herein include Escherichia coli, yeast, insect cells and animal cells.
  • Escherichia coli include strains of E. coli K-12 such as HB101, C600, JM109, DH5 ⁇ and AD494 (DE3).
  • yeast include Saccharomyces cerevisiae.
  • animal cells include L929, BALB/c3T3, C127, CHO, COS, Vero and Hela cells.
  • insect cells include sf9.
  • Introduction of an expression vector into host cells can be done using a conventional method suited for the respective host cells above. Specifically, introduction can be done using calcium phosphate method, DEAE-dextran method, electroporation method, and a method using lipid for gene transfer (Lipofectamine Lipofectin; Gibco-BRL). Following the introduction, the cells are cultured in a conventional medium containing a selection marker, whereby transformants containing the expression vector can be selected.
  • the peptide of the present invention can be produced by culturing the transformed cells under appropriate conditions (conditions under which peptides can be expressed).
  • the resultant peptide may be further isolated and purified according to standard biochemical purification procedures.
  • the purification procedures include salting out, ion exchange chromatography, absorption chromatography, affinity chromatography, gel filtration chromatography, etc.
  • the polypeptide of the present invention has been expressed as a fusion peptide with thioredoxin, His tag, GST, or the like, as mentioned above, the peptide can be isolated and purified by appropriate purification procedures making use of the characteristics of the fusion protein or tags.
  • the present invention provides an antibody which specifically binds to a peptide of the present invention.
  • the antibody of the present invention is not restricted to any form and may be polyclonal or monoclonal antibody raised against a peptide of the present invention as an antigen.
  • the antibody of the present invention on the condition that it specifically binds to a peptide of present invention.
  • preferred antibody include those specifically bind to a helper peptide that is a WT1-derived partial peptide of 10-25 amino acids comprising an amino acid sequence set forth in any one of SEQ ID NOS: 2-23 and has an activity of binding to HLA-DRB1*0405 and inducing helper T cells.
  • Antibodies specifically bind to a helper peptide consisting of the amino acid sequence set forth in SEQ ID NO: 24 is more preferred.
  • antibodies of the present invention can be obtained by immunizing a non-human animal such as rabbit using a peptide of the present invention as an antigen, and recovering the antibodies from serum of the immunized animal in a conventional manner.
  • monoclonal antibodies they can be obtained by immunizing a non-human animal such as mouse with a peptide of the present invention, subjecting the resultant splenocytes to cell fusion with myeloma cells, and recovering monoclonal antibodies from the resultant hybridoma cells ( Current protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley and Sons. Section 11.4-11.11).
  • the antibodies against the peptide of the present invention can also be produced while enhancing the immunological response using different adjuvants depending on the host.
  • adjuvants include Freund adjuvants; mineral gels such as aluminium hydroxide; surfactants such as lysolecithin, Pluronic® polyol, polyanion, peptide, oil emulsion, keyhole limpet hemocyanin and dinitorophenol; human adjuvants such as BCG (Bacille de Calmette-Guerin) or Corynebacterium, etc.
  • antibodies that recognize a peptide of the present invention and antibodies that neutralize the activity thereof can easily be prepared by immunizing an animal in a conventional manner.
  • the antibodies may be used in affinity chromatography, immunological diagnostic method, and the like.
  • Immunological diagnostic method may be selected as appropriate from immunoblotting, radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), a fluorescent or luminescent assay, and the like.
  • the immunological diagnostic method is effective in the diagnosis of cancer expressing WT1 gene such as gastric cancer, colon cancer, lung cancer, breast cancer, embryonal cancer, skin cancer, bladder cancer, prostate cancer, uterine cancer, cervical cancer, ovarian cancer, and the like.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a peptide (natural-, altered-, and epitope-type peptides) of the present invention, an expression vector containing a polynucleotide of the present invention or a cell containing an expression vector of the present invention, in association with a pharmaceutically acceptable carrier.
  • the pharmaceutical composition can be used effectively as an inducer of helper T cells or an enhancer of cancer vaccine efficacy, as described in detail below.
  • the peptide of the present invention has an activity of inducing helper T cells, and the induced helper T cells in turn are able to enhance the CTL-inducing activity, which is the effects of cancer vaccine, through the induction of differentiation and maintenance of CTLs and the activation of effectors such as macrophages.
  • the present invention provides an enhancer of cancer vaccine efficacy comprising as an active ingredient a peptide of the present invention (pharmaceutical composition as an agent for enhancing efficacy of cancer vaccine).
  • the enhancer of the present invention When the enhancer of the present invention is administered to an HLA-DRB1*0405-positive and WT1-positive patient, the peptide is presented to HLA-DRB1*0405 antigen of an antigen-presenting cell; specific helper T cells (CD4-positive T cells) recognizing a complex of the peptide and HLA-DRB1*0405 antigen are induced and activated; and the activated helper T cells can exert the activity concerning induction of differentiation and maintenance of CTLs and activation of effectors such as macrophages. In this manner, the activity of activating and inducing CTLs as the effect of cancer vaccine is enhanced.
  • specific helper T cells CD4-positive T cells recognizing a complex of the peptide and HLA-DRB1*0405 antigen are induced and activated; and the activated helper T cells can exert the activity concerning induction of differentiation and maintenance of CTLs and activation of effectors such as macrophages. In this manner, the activity of
  • the enhancer of cancer vaccine efficacy of the present invention can be used in the prevention or treatment of cancer accompanied by elevated expression level of WT1 gene, for example, blood cancers such as leukemia, myelodysplastic syndrome, multiple myeloma and malignant lymphoma, and solid cancers such as gastric cancer, colon cancer, lung cancer, breast cancer, embryonal cancer, hepatic cancer, skin cancer, bladder cancer, prostate cancer, uterine cancer, cervical cancer, and ovarian cancer.
  • blood cancers such as leukemia, myelodysplastic syndrome, multiple myeloma and malignant lymphoma
  • solid cancers such as gastric cancer, colon cancer, lung cancer, breast cancer, embryonal cancer, hepatic cancer, skin cancer, bladder cancer, prostate cancer, uterine cancer, cervical cancer, and ovarian cancer.
  • the enhancer of cancer vaccine efficacy of the present invention can be administered concurrently with, before or after the administration of cancer vaccine.
  • the enhancer of cancer vaccine efficacy comprising as an active ingredient a peptide of the present invention may contain as an active ingredient a helper peptide(s) or an epitope peptide wherein a peptide(s) is ligated with a cancer antigen peptide(s) (CTL epitope(s))
  • CTL epitope(s) cancer antigen peptide(s)
  • helper peptides When an epitope peptide of this form is administered, said peptide is incorporated into antigen-presenting cells; among the antigen peptides generated by intracellular degradation, helper peptides bind to MHC class II antigen (HLA-DRB1*0405) while cancer antigen peptides to MHC class I antigen; and respective complexes thus formed are presented on the surface of antigen-presenting cells in high density.
  • helper T cells recognize the complex of HLA-DRB1*0405 antigen and helper peptide, the CTL-inducing activity that is the effect of cancer vaccine is further enhanced as a result of induction of differentiation and maintenance of CTLs and the activation of effectors such as macrophages.
  • the pharmaceutical composition of the present invention comprising as an active ingredient an epitope peptide of the present invention can be used as a cancer vaccine per se, as well as an enhancer of cancer vaccine efficacy.
  • the enhancer of cancer vaccine efficacy which comprises as an active ingredient a peptide of the present invention may be administered together with a pharmaceutically acceptable carrier, for example, an appropriate adjuvant, or in the form of particles so that the cellular immunity can be established effectively.
  • a pharmaceutically acceptable carrier for example, an appropriate adjuvant, or in the form of particles so that the cellular immunity can be established effectively.
  • an adjuvant those described in a literature ( Clin. Microbiol. Rev., 7:277-289, 1994), and the like are applicable. Concrete examples include microorganism-derived components, cytokines, plant-derived components, marine organism-derived components, mineral gels such as aluminium hydroxide, surfactants such as lysolecithin and Pluronic® polyols, polyanions, peptides, oil emulsion (emulsion preparations) and the like. Liposomal preparations, particulate preparations in which the ingredient is bound to beads having a diameter of several ⁇ m, preparations in which the ingredient is
  • Administration may be achieved, for example, intradermally, subcutaneously, intramuscularly, or intravenously.
  • dosage of the peptide of the present invention in the formulation may be adjusted as appropriate depending on, for example, the disease to be treated, the age and the body weight of a patient, it is usually within the range of 0.0001 mg-1000 mg, preferably 0.001 mg-1000 mg, more preferably 0.1 mg-10 mg, which can be preferably administered once in every several days to every several months.
  • the present invention also provides a pharmaceutical composition comprising a combination of a peptide of the present invention and a cancer antigen peptide.
  • a cancer antigen peptide as cancer vaccine i.e., activity of inducing and activating CTLs
  • the treatment or prevention of cancer can be achieved more effectively.
  • ком ⁇ онент encompasses the both forms where a peptide of the present invention and a cancer antigen peptide are administered in a mixed form or discrete forms.
  • Administration of the peptides in the mixed form can be conducted using a previously prepared formulation containing the peptides as a mixture, or combining previously prepared formulations each comprising the respective peptides before use.
  • the discrete formulations may be administered successively with a time-interval, or administered concurrently.
  • a peptide of the present invention (enhancer of cancer vaccine efficacy) and a cancer antigen peptide (cancer vaccine) may be administered in this order or in reverse order.
  • An embodiment of the combination of a peptide of the present invention and a cancer antigen peptide includes a kit.
  • any cancer antigen peptides conventionally known can be used, and examples include WT1-derived cancer antigen peptides (natural-, altered-type). Concrete examples include a cancer antigen peptide set forth in any one of SEQ ID NOS: 27-45, preferably, SEQ ID NOS: 27-30, 44 and 45.
  • an expression vector containing a polynucleotide encoding a peptide of the present invention similar to the above-mentioned peptide of the present invention, has an activity of inducing helper T cells and is useful as an active ingredient of an enhancer of cancer vaccine efficacy of the present invention.
  • the present invention provides an enhancer of cancer vaccine efficacy (i.e., a pharmaceutical composition as an agent enhancing efficacy of cancer vaccine) comprising as an active ingredient an expression vector containing a polynucleotide encoding a peptide of the present invention.
  • CTL epitope cancer antigen peptide
  • helper peptide helper epitope
  • an active ingredient of enhancer of cancer vaccine efficacy can be obtained by incorporating a polynucleotide encoding the above-described epitope peptide of present invention into an appropriate expression vector.
  • any means including those utilizing viral vectors or other methods are applicable ( Nikkei - Science, April, 1994, p20-45; Gekkan - Yakuji, 36(1), p23-48 (1994); Jikken - Igaku - Zokan, 12(15), 1994, and references cited therein).
  • Examples of means utilizing a viral vector include those wherein a DNA of the present invention is incorporated into DNA or RNA virus such as retrovirus, adenovirus, adeno-associated virus, herpes virus, vaccinia virus, poxvirus, poliovirus, or Sindbis virus, and then introduced into cells. Above all, a method utilizing retrovirus, adenovirus, adeno-associated virus, or vaccinia virus, or the like, is particularly preferred.
  • DNA vaccination examples include those wherein an expression vector is directly injected intramuscularly (DNA vaccination), liposome method, Lipofectin method, microinjection, calcium phosphate method and electroporation. DNA vaccination and liposome method are particularly preferred.
  • the expression vector of the present invention act as a medicament in practice
  • an in vivo method wherein the expression vector is directly introduced into the body
  • an ex vivo method wherein the expression vector is introduced extracorporeally into a certain cells removed from human, and the cells are reintroduced into the body
  • the in vivo method is more preferred.
  • the administration can be effected through any appropriate routes depending on the disease and symptoms to be treated.
  • it may be administered via intravenous, intraarterial, subcutaneous, intracutaneous, intramuscular route, or the like.
  • the compositions may be administered in various forms such as solution, and are typically formulated, for example, in the form of injection containing, as an active ingredient, an expression vector of the present invention to which conventional carriers may also be added, if necessary.
  • liposomes or membrane-fused liposomes such as Sendai virus (HVJ)-liposomes
  • they may be in the form of liposomal formulation such as suspension, frozen drug, centrifugally-concentrated frozen drug, or the like.
  • an expression vector in a formulation may be adjusted as appropriate depending on, for example, the disease to be treated, age and body weight of a patient, usually, 0.0001 mg-100 mg, preferably 0.001 mg-10 mg of an expression vector of the present invention can be administered once in every several days to every several months.
  • the peptide of the present invention is presented to HLA-DRB1*0405 antigen of an antigen-presenting cell; specific helper T cells (CD4-positive T cells) recognizing a complex of the peptide and HLA-DRB1*0405 antigen are induced and activated; the activated helper T cells can exert the activity concerning induction of differentiation and maintenance of CTLs and activation of effectors such as macrophages. In this manner, the activity of inducing CTLs as an effect of cancer vaccine is enhanced.
  • the enhancer of cancer vaccine efficacy comprising as an active ingredient an expression vector containing a polynucleotide of the present invention can be used in the prevention or treatment of cancer accompanied by elevated expression level of WT1 gene, for example, blood cancers such as leukemia, myelodysplastic syndrome, multiple myeloma and malignant lymphoma, and solid cancers such as gastric cancer, colon cancer, lung cancer, breast cancer, embryonal cancer, hepatic cancer, skin cancer, bladder cancer, prostate cancer, uterine cancer, cervical cancer, and ovarian cancer.
  • blood cancers such as leukemia, myelodysplastic syndrome, multiple myeloma and malignant lymphoma
  • solid cancers such as gastric cancer, colon cancer, lung cancer, breast cancer, embryonal cancer, hepatic cancer, skin cancer, bladder cancer, prostate cancer, uterine cancer, cervical cancer, and ovarian cancer.
  • antigen presenting cells incorporate the same and generate antigen peptides through intracellular degradation, of which helper peptides and cancer antigen peptides bind to MHC class II antigen (HLA-DRB1*0405) and MHC class I antigen, respectively, to form complexes.
  • MHC class II antigen HLA-DRB1*0405
  • MHC class I antigen MHC class I antigen
  • the pharmaceutical composition of the present invention comprising as an active ingredient an expression vector containing a polynucleotide encoding an epitope peptide of the present invention can be used as a cancer vaccine per se, as well as an enhancer of cancer vaccine efficacy.
  • the present invention also provides a peptide consisting of 10-25 contiguous amino acids in the amino acid sequence of human WT1 set forth in SEQ ID NO: 1, said peptide binding to HLA-DRB1*1502 and inducing helper T cells.
  • the present invention encompasses antigen peptides binding to HLA-DRB1*1502 wherein the N-terminal and/or C-terminal amino acid residue is modified or those wherein a particular amino acid residue(s) is altered.
  • the synthesis and determination of activity of antigen peptides binding to HLA-DRB1*1502 of the present invention can be carried out in a manner similar to that described in relation to the above-mentioned antigen peptides binding to HLA-DRB1*0405 of the present invention.
  • the HLA-DRB1*1502-binding antigen peptides of the present invention is a partial peptide which consists of 10-25 contiguous amino acids in the amino acid sequence of human WT1 set forth in SEQ ID NO: 1.
  • Preferred peptides are those consisting of 13-17 contiguous amino acids in the amino acid sequence of human WT1.
  • the present invention is based on the finding that human WT1 contains an antigen peptide portion having activity of binding to HLA-DRB1*1502 and inducing helper T cells.
  • Search for 9-amino-acid portions potentially binding to HLA-DRB1*1502 (9-amino-acid portions capable of being accommodated in a peptide-binding groove of MHC class II molecule) was conducted using a software for predicting MHC class II binding peptides (Propred, Bioinformatics 17: 1236, 2001). Examples of the identified 9-amino-acid portions of WT1 are shown in SEQ ID NOS: 46-56.
  • HLA-DRB1*1502-binding antigen peptides of the present invention include a peptide which comprises an amino acid sequence set forth in any one of SEQ ID NOS: 46-56, and which binds to HLA-DRB1*1502 and induces helper T cells.
  • the said peptides preferably consist of about 10-25 amino acids, and more preferably about 13-17 amino acids.
  • Examples of more preferred embodiments include partial peptides derived from WT1 consisting of 10-25 amino acids (preferably, 13-17 amino-acids), which comprise the amino acid sequence set forth in SEQ ID NO: 50, and which have an activity of binding to HLA-DRB1*1502 and inducing helper T cells.
  • Examples of still more preferred embodiments include partial peptides derived from WT1 consisting of 16-25 amino acids (preferably, 16-17 amino-acids), which comprise the amino acid sequence set forth in SEQ ID NO: 24, and which have an activity of binding to HLA-DRB1*1502 and inducing helper T cells.
  • the amino acid sequence set forth in SEQ ID NO: 24 represents a 16-amino-acid partial peptide derived from WT1 and includes the amino acid sequence set fort in SEQ ID NO: 50.
  • WT1 332-347 peptide consisting of the amino acid sequence set forth in SEQ ID NO: 24.
  • WT1 332-347 peptide is a promiscuous helper peptide which binds not only to HLA-DRB1*0405 molecule but also to HLA-DRB1*1502 molecule. Accordingly, WT1 332-347 is a helper peptide applicable to patients having HLA-DRB1*0405 and those having HLA-DRB1*1502 as well, and hence is useful from the viewpoint of wide application range of patients.
  • the epitope peptide, polynucleotide, antibody, and pharmaceutical composition regarding the HLA-DRB1*1502-binding antigen peptide of the present invention can be made and use (put into effect) in a manner similar to above-mentioned HLA-DRB1*0405-binding antigen peptide of the present invention.
  • PBMCs Peripheral blood mononuclear cells
  • Blood obtained from the same healthy volunteer as in (1) above was used. Blood was diluted by 2-times with RPMI medium. To about 100 ml of the diluted blood was added antibody cocktail, RosetteSepTM (Stemcell) for separation of CD4-positive T cells, and the mixture was left to stand at room temperature for 20 minutes. CD4-positive T cells were then collected by density gradient centrifugation with Ficoll-Paque.
  • WT1 protein Amino acid sequence of WT1 protein (NCBI database, Accession No. P19544, XP — 034418, SEQ ID NO: 1) was searched for peptides potentially binding to HLA-DRB1*0405 using a prediction program (Propred, Bioinformatics 17: 1236, 2001). Three peptides were selected and synthesized. These peptides have the same amino acid sequences as those present at the following positions of WT1:
  • Position 172-186 PNHSFKHEDPMGQQG (WT1 172-186, SEQ ID NO: 25); Position 225-243: NLYQMTSQLECMTWNQMNL (WT1 225-243, SEQ ID NO: 26); and Position 332-347: KRYFKLSHLQMHSRKH (WT1 332-347, SEQ ID NO: 24).
  • Dendritic cells prepared in (1) above were seeded in 24-well culture plate at 3 ⁇ 10 5 cells/well, and a peptide of SEQ ID NO: 24 was added up to 50 ⁇ g/ml. After 4-hour-cultivation, cell growth was stopped by X-ray irradiation (25Gy). CD4-positive cells prepared in (2) above were added to each well at 3 ⁇ 10 6 cells/well and cocultured with dendritic cells. As medium, X-VIVO 15TM medium containing 1% AB serum was used. After the cultivation was started, one half of medium was replaced with fresh medium every 2 days and IL-2 was added up to 20 U/ml.
  • T cells were collected and seeded in 24-well plate at 3 ⁇ 10 6 cells/well, and thereto were added 3 ⁇ 10 5 dendritic cells having been pulsed with 20 ⁇ g/ml of a peptide (SEQ ID NO: 24) and undergone X-ray irradiation (25Gy). The cells were then cocultured.
  • X-VIVO 15TM medium containing 1% AB serum and 20 U/ml IL-2 was used.
  • T cells were recovered and seeded in 96-well plate at 3 ⁇ 10 4 cells/well.
  • Dendritic cells having been pulsed with 20 ⁇ g/ml of a peptide (SEQ ID NO: 24) and undergone X-ray irradiation (25Gy) were added at 3 ⁇ 10 4 cells/well, followed by coculture.
  • As a negative control group T cells were cocultured with dendritic cells not pulsed with the peptide, and as a positive control group, 0.2% PHA was added instead of dendritic cells.
  • [ 3 H]-thymidine 37 kBq/well was added, and the cells were cultured for another 16 hours.
  • [ 3 H]-thymidine incorporated by cells was then measured using ⁇ -scintillation counter. The results are shown in FIG. 1 .
  • CD4-positive T cells stimulated with a peptide at position 332-347 of WT1 (WT1 332-347 , SEQ ID NO: 24) showed proliferative response when cocultured with dendritic cells pulsed with WT1 332-347 .
  • CD4-positive T cells did not show proliferative response when cocultured with dendritic cells not pulsed with the peptide, or dendritic cells pulsed with a peptide having amino acid sequence of SEQ ID NO: 25 or 26 which is different from that of SEQ ID NO: 24.
  • SEQ ID NO: 24 induces specific CD4-positive T cells as an antigen peptide.
  • Dendritic cells prepared in a manner similar to Example 1 were seeded in 96-well plate at 10 4 cells/well, and then CD4-positive T cells induced by WT1 332-347 peptide (SEQ ID NO: 24) were seeded at 10 3 cells/well.
  • As medium X-VIVO 15TM medium containing 1% AB serum, 20 U/ml IL-2 and 5 ⁇ g/ml PHA was used.
  • CD4-positive T-cell line was established by continued cultivation and named as “G2 cell line”. Responsiveness of G2 cell line to dendritic cells pulsed with a peptide was measured by a similar method to Example 1. The results are shown in FIG. 2 .
  • G2 cell line showed proliferative response when cocultured with dendritic cells pulsed with WT1 332-347 peptide, but did not when cocultured with dendritic cells not-pulsed with the peptide.
  • G2 cell line is a CD4-positive T-cell line specific for WT1 332-347 peptide.
  • PBMCs Peripheral blood mononuclear cells
  • PBMCs Peripheral blood mononuclear cells
  • B-LCL( ⁇ ) cells were adjusted to 3 ⁇ 10 7 cells/mL, and thereto were added medium containing virus expressing WT1 gene and then polypropylene (final concentration, 8 ⁇ g/mL), and the mixture was added to a 24-well plate at 1 ml/well. After 16-hour-cultivation, 1 ml of fresh medium was added to each well, and cultivation was continued. To each well was added G418 (neomycin) up to 0.7 ⁇ g/mL, and the plate was cultured for 5 to 7 days, when cells into which the gene was introduced were selected. The selected B-cell line expressing WT1 was named as “B-LCL(+) cell”.
  • the amount of WT1 gene expressed by B-LCL( ⁇ ) and B-LCL(+) cells was measured by RT-PCR technique according to the method described in Blood, 89:1405, 1997. The measurements were converted by assuming the expression amount of K562 cell line as the positive control to be 1. The resulting value for B-LCL( ⁇ ) cells was 1.6 ⁇ 10 ⁇ 4 while that for B-LCL(+) cells 3.2, indicating that WT1 gene is highly expressed. Responsiveness of G2 cells to B-LCL(+) cells was examined in a manner similar to Example 2. In a test group, B-LCL(+) cells were treated with anti-HLA-DR antibody before mixing with G2 cells to confirm the HLA-DR-restriction.
  • Dendritic cells were prepared using blood isolated from an HLA-DRB1*0405-positive healthy volunteer in a manner similar to Example 1 except that the final concentration of TNF- ⁇ added on day 6 was 200 IU/ml.
  • CD4-positive T cells were prepared using blood obtained from the same healthy volunteer as that used for the preparation of dendritic cells. CD4-positive T cells were separated according to the instructions of RosetteSep (StemCell) for separation of CD4-positive T cells.
  • the above-described dendritic cells and CD4-positive T cells were used to induce CD4-positive T cells specific for a WT1 peptide (SEQ ID NO: 24, WT1 332-347 ) in a manner similar to Example 1.
  • the resultant CD4-positive T cells specific for WT1 332-347 peptide were cultured continuously by limiting dilution technique to establish CD4-positive T-cell line E04.1.
  • As feeder cells in the limiting dilution technique PBMCs prepared in a manner similar to Example 1 and treated by X-ray irradiation were seeded at 1 ⁇ 10 5 cells/well.
  • As medium X-VIVO 15TM medium containing 20 IU/ml IL-2 and 5 ⁇ g/ml PHA was used.
  • E04.1 cell line to dendritic cells pulsed with WT1 332-347 peptide was measured by a similar method to Example 1 except that cultivation was continued for 18 hours after adding [ 3 H]-thymidine. The results are shown in FIG. 4 .
  • E04.1 cells showed proliferative response when cocultured with dendritic cells pulsed with WT1 332-347 peptide but did not when cocultured with dendritic cells not-pulsed with the peptide. These results demonstrate that E04.1 cell is a CD4-positive T-cell line specific for WT1 332-347 peptide.
  • E04.1 cells established in Example 4 were seeded in 96-well culture plate at 1 ⁇ 10 4 cells/well.
  • B cell line B-LCL ( ⁇ ) cells established from blood of an HLA-DRB1*0405-positive healthy volunteer in Example 3 were pulsed with WT1 332-347 peptide at a concentration of 20 ⁇ g/ml and treated by X-ray irradiation, seeded in 96-well plate at 3 ⁇ 10 4 cells/well, and cocultured with E04.1 cells.
  • B-LCL ( ⁇ ) cells not pulsed with the peptide were cocultured with E04.1 cells.
  • B-LCL ( ⁇ ) cells having been pulsed with WT1 332-347 peptide and undergone X-ray irradiation were treated with 20 ⁇ g/ml of anti-HLA-DR antibody (G46.6, BD ParMingen), anti-H LA-class I antibody (G46-2.6, BD ParMingen), or anti-HLA-DQ antibody (SPVL3, Immunotech) for 30 minutes, and cocultured with E04.1 cells to confirm HLA-DR restricted nature.
  • anti-HLA-DR antibody G46.6, BD ParMingen
  • anti-H LA-class I antibody G46-2.6, BD ParMingen
  • SPVL3, Immunotech anti-HLA-DQ antibody
  • E04.1 cells showed proliferative response when cocultured with B-LCL ( ⁇ ) cells pulsed with WT1 332-347 peptide. However, when WT1 332-347 peptide-pulsed B-LCL ( ⁇ ) cells were treated with anti-HLA-DR antibody, growth of cells was inhibited. Further, E04.1 cells showed proliferative response to WT1 332-347 -peptide-pulsed B-LCL ( ⁇ ) cells treated with other antibody, but did not show proliferative response to B-LCL ( ⁇ ) cells not pulsed with the peptide. These results demonstrate that WT1 332-347 peptide specifically binds to HLA-DR among HLA molecules, and induces growth of CD4-positive E04.1 cell line specific for WT1 332-347 peptide.
  • PBMCs were prepared from blood of HLA-DRB1*0405-positive or -negative healthy volunteer in a manner similar to Example 4. PBMCs were then pulsed with 20 ⁇ g/ml of WT1 332-347 peptide and subjected to X-ray irradiation, and seeded in 96-well plate at 3 ⁇ 10 4 cells/well. E04.1 cells were then seeded into the 96-well plate at 1 ⁇ 10 4 cells/well, and the cells were cocultured. As a negative control group, PBMCs not pulsed with the peptide and E04.1 cells were cocultured.
  • Donor 1 HLA-DRB1*0405/0803
  • Donor 2 HLA-DRB1*0405/0101
  • E04.1 cells cocultured with PBMCs isolated from each donor and pulsed with WT1 332-347 peptide showed proliferative response.
  • Donor 3 HLA-DRB1*0101/1001
  • Donor 4 HLA-DRB1*1201/0802
  • E04.1 cells cocultured with PBMCs isolated from each donor and pulsed with WT1 332-347 peptide did not show proliferative response. Further, in all cases, no proliferative response was observed when PBMCs not-pulsed with the peptide were used.
  • WT1 332-347 peptide specifically binds to HLA-DRB1*0405 among HLA-DRB1 molecules showing polymorphism, and induce the growth of WT1 332-347 -specific CD4-positive cell line E04.1.
  • B-LCL( ⁇ ) cells B-cell line
  • B-LCL (+) cells B-cell line expressing WT1
  • B-cell line expressing WT1 B-cell line expressing WT1
  • E04.1 cells were seeded into each well at 1 ⁇ 10 4 cells and cocultured. Growth response of E04.1 cells was then measured in a manner similar to Example 4. The results are shown in FIG. 7 .
  • E04.1 cells showed proliferative response when cocultured with B-LCL (+) cells expressing WT1, but not when cocultured with B-LCL ( ⁇ ) cells not-expressing WT1.
  • B-LCL( ⁇ ) or B-LCL (+) cells (1 ⁇ 10 5 cells each) having been induced apoptosis were cocultured for 16 hours with dendritic cells (3 ⁇ 10 4 cells) prepared from blood of an HLA-DRB1*0405-positive healthy volunteer in a manner similar to Example 4, seeded in well of 96-well plate, and cocultured with E04.1 cells (1 ⁇ 10 4 cells). Growth response of E04.1 cells was then measured in a manner similar to Example 1. The results are shown in FIG. 8 .
  • E04.1 cells showed proliferative response when cocultured with dendritic cells pulsed with apoptosis-induced B-LCL (+) cells, but did not when cocultured with dendritic cells pulsed with apoptosis-induced B-LCL ( ⁇ ) cells. These results indicate that WT1 332-347 peptide is first generated through the degradation of WT1 protein in B-LCL (+) cells, then presented to HLA-DRB1*0405 and induce proliferation of E04.1 cells.
  • Induction of apoptosis in B-LCL ( ⁇ ) and B-LCL (+) cells was conducted by osmotic shock. Namely, 1 ⁇ 10 6 cells were suspended in 500 ⁇ l of a hyperosmotic medium (RPMI medium containing 0.5 M sucrose, 10% w/v polyethylene glycol 1000 and 10 mM HEPES, pH 7.2), and left to stand at 37° C. for 10 minutes. The culture was then diluted by 30-times with a hypoosmotic medium (60% RPMI, 40% water) previously adjusted to 37° C., and left to stand at 37° C. for 2-3 minutes. Cells were collected by centrifuging at room temperature for 5 minutes and used as apoptosis-induced cells. Induction of apoptosis was confirmed by a fluorescent dye for staining dead cells (Propidium Iodide, and AnnexinV, i.e., a phosphatidyl serine-binding reagent).
  • RPMI medium containing 0.5
  • Dendritic cells prepared from blood of an HLA-DRB1*0405-positive healthy volunteer in a manner similar to Example 4 were pulsed with WT1 332-347 peptide, mixed with E04.1 cells, and cultured for 24 hours. As a negative control group, dendritic cells not pulsed with the peptide were mixed with E04.1 cells. After 24-hour-cultivation, Brefeldin A was added to a final concentration of 10 ⁇ g/ml to inhibit exocytosis of E04.1 cells. Further, CD4-positive T cells were recovered after culturing for another 6 hours, and fixed with PBC containing 2% formaldehyde, and treated by permeabilization solution containing 0.1% saponin to increase the cell membrane permeability of antibody.
  • the treated cells were then intracellularly stained by treating with PE-labeled anti-IFN- ⁇ antibody (BD PharMingen) and FITC-labeled anti-IL-4 antibody (BD PharMingen), and analyzed using a flow cytometer.
  • the results are shown in FIG. 9 . It was revealed that E04.1 cells, when cocultured with dendritic cells pulsed with WT1 332-347 peptide, were induced strongly to produce IFN- ⁇ which is a Th-1-type cytokine, but not to produce IL-4 which is a Th-2-typ cytokine.
  • E04.1 cells were stained by treating with anti-CD4 antibody and anti-CXCR3 antibody, and analyzed by flow cytometer. The results are shown in FIG. 10 . It was revealed that more than 90% of E04.1 cells are CD4-positive T cells of Th-1 type which are positive for CD4 and CXCR3. CXCR3 is known to be a chemokine receptor highly expressed on Th-1 type immunocytes.
  • WT1 332-347 peptide activate E04.1 cells, WT1 332-347 -specific CD4-positive cell line, and induce the cells to produce IFN- ⁇ which is a Th-1 type cytokine. These results demonstrate that WT1 332-347 peptide activates and makes CD4-positive T cells differentiate into Th-1 type.
  • PBMCs were prepared using blood of the same healthy volunteer (HLA-A*2402/1101, DRB1*0405/0803) as that used for establishment of E04.1 cells in a manner similar to Example 4, and seeded in 24-well culture plate at 3 ⁇ 10 4 cells/well. To the well were added WT1 235-243 peptide (SEQ ID NO: 27) and E04.1 cells in the following manners, and the plate was cultured at 37° C. for 7 days.
  • WT1 235-243 peptide (20 ⁇ g/ml); WT1 235-243 peptide (20 ⁇ g/ml)+WT1 332-347 peptide (20 ⁇ g/ml); WT1 235-243 peptide (20 ⁇ g/ml)+E04.1 cells (1.5 ⁇ 10 6 cells/well); or WT1 235-243 peptide (20 ⁇ g/ml)+WT1 332-347 peptide (20 ⁇ g/ml)+E04.1 cells (1.5 ⁇ 10 6 cells/well).
  • As medium X-VIVO 15TM medium containing 10% AB serum was used.
  • WT1 235-243 peptide used here is a cancer antigen peptide having activity of inducing HLA-A*2402-restricted CTLs (WO2004/024175).
  • Another half of the recovered cells (3 ⁇ 10 5 cells) were cocultured for 6 hours with dendritic cells pulsed with WT1 235-243 peptide and undergone X-ray irradiation (30 Gy).
  • WT1 235-243 peptide and undergone X-ray irradiation (30 Gy).
  • Brefeldin A was added to inhibit exocytosis of cells.
  • Cultivation was continued for another 5 hours and the cells were stained with anti-CD8 antibody and WT1 235-243 peptide/HLA-A*2402-specific PE-labeled tetramer.
  • the cells were fixed and treated with a permeabilization solution to increase cell membrane permeability, and intracellularly stained by PE-labeled anti-IFN- ⁇ antibody in a manner similar to Example 8.
  • As a negative control group cells were stained with APC-labeled anti-mouse IgG antibody (BD PharMingen), and cell populations positive for both IFN- ⁇ and mouse IgG were excluded as
  • FIG. 12 The results are shown in FIG. 12 (A-D).
  • WT1 235-243 peptide-specific CTL precursors are stimulated with WT1 235-243 peptide for 6 hours, CTLs which are specific for activated WT1 235-243 peptide and are positive for CD8, WT1 235-243 peptide/HLA-A*2402 and IFN- ⁇ are induced.
  • stimulation was conducted using WT1 235-243 peptide alone, the percentage of CTLs specific for activated WT1 235-243 peptide was 17.0% ( FIG. 12-A ).
  • WT1 332-347 peptide is a helper peptide which enhances the induction and activation of WT1-specific CTL precursors. It was also revealed that E04.1 cell is a helper T cell which enhances the activation of WT1-specific CTLs and that its helper function is increased by WT1 332-347 peptide and the activation of WT1-specific CTLs is enhanced.
  • WT1 332-347 peptide was examined whether it also can bind to HLA-DRB1*1502 molecule, which is said that many Japanese possess, and induce WT1 332-347 -specific CD4-positive T cells as a promiscuous helper peptide.
  • PBMCs Peripheral blood mononuclear cells
  • HLA-DRB1*1502/1403 a healthy volunteer
  • X-VIVO15TM medium a medium containing 1000 IU/ml IL-4 (PeproTech), 1000 IU/ml GM-CSF (PeproTech), 1% AB-type serum, and X-VIVO 15TM medium.
  • IL-4 and GM-CSF were added, and on day 6, TNF- ⁇ was added up to 100 IU/ml to make dendritic cells mature.
  • CD4-positive T cells were isolated from blood of the same volunteer using RosetteSep (StemCell) for separating CD4-positive T cells.
  • the resulting CD4-positive T cells were seeded in 24-well plate at 3 ⁇ 10 6 cells/well, and stimulated with autologous dendritic cells (3 ⁇ 10 5 cells) pulsed with WT1 332-347 peptide (20 ⁇ g/ml) and undergone radiation irradiation (25Gy).
  • IL-2 was added up to 20 IU/ml.
  • the stimulated CD4-positive T cells were stimulated every one week with dendritic cells pulsed with WT1 332-347 peptide (20 ⁇ g/ml). Further, medium change was conducted using IL-2-containing medium on every other day following the second stimulation.
  • CD4-positive T cells induced by 3 times of stimulation in total.
  • CD4-positive T cells were cocultured with stimulators for 2 hours, and thereto was added Brefeldin A. Four hours later, cells were recovered, subjected to treatment for fixation and permeation, stained with FITC-labeled anti-IL-4 antibody (BD Pharmingen) and PE-labeled anti-IFN- ⁇ antibody (BD Pharmingen), and analyzed by flow cytometry.
  • FITC-labeled anti-IL-4 antibody BD Pharmingen
  • PE-labeled anti-IFN- ⁇ antibody BD Pharmingen
  • PBMCs (6 ⁇ 10 5 cells) pulsed or not pulsed with WT1 332-347 peptide were treated by radiation irradiation (25Gy), and each group of cells was cocultured with WT1 332-347 -induced CD4-positive T cells (6 ⁇ 10 5 cells). After 72-hour-cultivation, the supernatant was recovered, and 300 ⁇ l of the solution was used for measurement of IL-4 and IFN- ⁇ .
  • TCR T-cell receptor
  • CD4-positive T cells isolated from blood of an healthy volunteer were stimulated three times in total with autologous dendritic cells pulsed with WT1 332-347 peptide.
  • the so induced CD4-positive T cells were examined for the peptide specificity by growth assay using each of WT1 172-186 , WT1 225-243 and WT1 332-347 peptides.
  • CD4-positive T cells induced by WT1 332-347 peptide did not proliferate in the absence of the peptide or under the stimulation with WT1 172-186 or WT1 225-243 , but proliferated to become about 10-times as much when stimulated with WT1 332-347 ( FIG. 13 ). From these results, the induced CD4-positive T cells have specificity for WT1 332-347 .
  • the WT1 332-347 peptide-induced CD4-positive T cells were subjected to TCR repertoire assay. The results are shown in FIG. 14 . Cells having V ⁇ 3 and those having V ⁇ 20 were dominant and each accounted for 10% of the total.
  • E15.1 subline the cell line having V ⁇ 3 was named as E15.1 subline, while the one having V ⁇ 20 as E15.2 subline.
  • E15.2 subline showed higher responsiveness to WT1 332-347 peptide ( FIG. 15 ).
  • E15.2 subline was then stimulated with WT1 332-347 peptide, and the secreted IL-4 and IFN- ⁇ were measured by Intracellular stain method. As a result, it was revealed that IFN- ⁇ (Th-1-type cytokine) and not IL-4 (Th-2-type cytokine) is dominantly produced by said cell line ( FIG. 16 ). It was also revealed that the WT1 332-347 -specific proliferative-response of E15.2 subline depends on the concentration of WT1 332-347 ( FIG. 17 ). These results indicated that E15.2 subline is a Th-1-type CD4-positive T cell line specific for WT1 332-347 .
  • Th-1-type CD4-positive T cell lines specific for WT1 332-347 from CD4-positive T cells derived from a healthy volunteer who is positive for HLA-DRB1*1502 molecule but negative for HLA-DRB1*0405 molecule. Based on these results, it is considered that said CD4-positive T cells participate in cellular immunity and can activate CTLs through the secretion of cytokines. Thus, it was shown that antitumor effects can be further enhanced by using WT1 332-347 in combination with HLA-class I-restricted WT1 peptide (cancer antigen peptide) capable of activating CTLs.
  • WT1 332-347 in combination with HLA-class I-restricted WT1 peptide (cancer antigen peptide) capable of activating CTLs.
  • PBMCs derived from an HLA-DRB1*1502-positive healthy volunteer (1502/0901) or an HLA-DRB1*1502-negative healthy volunteer (1302/0803) were pulsed with WT1 332-347 peptide to obtain stimulators. Each of stimulators was cocultured with E15.2 subline, and analyzed for WT1 332-347 -specific proliferation by growth assay. The results are shown in FIG. 18 . In an HLA-DRB1*1502-positive healthy volunteer, WT1 332-347 -specific proliferation was observed but in an HLA-DRB1*1502-negative healthy volunteer, no proliferation was observed ( FIG. 18 ). This indicated that the WT1 332-347 -specific proliferation of E15.2 subline is restricted to HLA-DRB1*1502.
  • WT1 332-347 is a promiscuous helper peptide which binds not only to HLA-DRB1*0405 molecule but also to HLA-DRB1*1502 molecule which molecules are found in the first and the third frequencies, respectively, among Japanese.
  • the present invention provides a WT1-derived HLA-DRB1*0405-binding antigen peptide, a polynucleotide encoding said peptide, a helper T cell inducer comprising said peptide or polynucleotide, and the like.
  • the helper T cell inducer of the present invention is useful as an enhancer of cancer vaccine efficacy.
  • the enhancer of cancer vaccine efficacy of the present invention is applicable to many cancer patients positive for HLA-DRB1*0405, and particularly useful as an enhancer of WT1 vaccine efficacy.
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