# Patent

## Title
Cancer vaccine composition

## Abstract

A cancer vaccine composition for human leukocyte antigen (HLA)-A*0206-positive persons, comprising a protein product of the tumor suppressor gene WT1 or a partial peptide thereof.

## Background
This application is a Continuation of U.S. application Ser. No. 12/746,257, filed on Jun. 4, 2010, which is a National Stage of PCT/JP2008/072160, filed on Dec. 5, 2008.


TECHNICAL FIELD
The present invention relates to a cancer vaccine composition for human leukocyte antigen (HLA)-A*0206-positive persons, comprising a protein product of the tumor suppressor gene Wilms' tumor 1 (WT1) (hereinafter sometimes abbreviated as WT1 protein) or a partial peptide thereof (hereinafter sometimes abbreviated as WT1 peptide). The present invention also relates to a cancer vaccine composition for HLA-A*0206-positive persons, comprising DNA or RNA encoding the above-mentioned WT1 protein or WT1 peptide, a method for inducing WT1-specific CTLs, a method for inducing dendritic cells that present a cancer antigen, and a method of cancer diagnosis for HLA-A*0206-positive persons, and a method of cancer treatment or prevention in HLA-A*0206-positive persons.
The present invention further relates to a cancer vaccine composition for HLA-A*0201-positive persons, comprising a modified peptide of the WT1 peptide.
BACKGROUND ART
The Wilms' tumor gene WT1 was isolated as a gene associated with tumorigenesis in Wilms' tumor, which is a pediatric renal tumor (see nonpatent literature 1). This gene encodes a zinc finger transcription factor associated with the regulatory mechanism of cell growth and differentiation, and apoptosis and tissue development.
The WT1 gene was originally classified as a tumor suppressor gene. However, based on the recent evidences shown in the following (i) to (iii):

    (i) high expression of the wild-type WT1 gene in various human malignant tumors and solid cancers including hematopoietic malignant tumors such as leukemia and myelodysplastic syndromes (MDS),
    (ii) growth inhibition of human leukemia cells and solid cancer cells treated by WT1 antisense oligonucleotides, and
    (iii) growth promotion and differentiation inhibition of mouse myeloid precursor cells by constitutive expression of the wild-type WT1 gene,

it is suggested that the wild-type WT1 gene exhibits an oncogenic effect rather than a tumor suppressive effect on various malignant diseases (see patent literature 1).



There is also known high expression of the WT1 gene in solid cancers, such as gastric cancer, colon cancer, lung cancer, breast cancer, germ cell cancer, hepatic cancer, skin cancer, bladder cancer, prostate cancer, uterine cancer, cervical cancer and ovarian cancer (see patent literature 2).
In general, the immune system for eliminating foreign substances comprises humoral immunity, in which macrophages, which recognize an antigen and serve as antigen presenting cells, helper T cells, which recognize the antigen presented by the macrophages and produce various lymphokines to activate other T cells, and B lymphocytes, which differentiate into antibody producing cells via the actions of the lymphokines, are involved; and cell-mediated immunity, in which cytotoxic T lymphocytes (CTLs), which are produced through differentiation in response to antigen presentation, attack and destroy target cells.
Currently, it has been considered that cancer immunity is mainly based on cell-mediated immunity in which CTLs are involved. In the CTL-based cancer immunity, precursor T cells recognize a cancer antigen presented in the form of a complex of a major histocompatibility complex (MHC) class I and the cancer antigen, and thereby differentiate and grow into CTLs, which attack and destroy cancer cells. In this case, the cancer cell presents, on the cell surface, a complex of the MHC class I antigen and the cancer antigen, which is the target of the CTLs (see nonpatent literatures 2 to 5). MHC is called as a human leukocyte antigen (HLA) in humans.
It is considered that the above-mentioned cancer antigen, which is presented by an MHC class I antigen on the surfaces of cancer cells, i.e., target cells, is a peptide of about 8 to 12 amino acids produced through intracellular protease-mediated processing of an antigen protein synthesized in cancer cells (see nonpatent literatures 2 to 5). Currently, search for antigen proteins of various cancers is underway, but only a few proteins have been identified as a cancer specific antigen.
The present inventor synthesized polypeptides that each consist of 7 to 30 contiguous amino acids based on the amino acid sequence of the WT1 gene expression product and each contain at least one amino acid presumably serving as an anchor amino acid for binding with HLA-A*2402 or HLA-A*0201, confirmed that these peptides bind with HLA-A*2402 or HLA-A*0201 (these peptides are HLA-A*2402- or HLA-A*0201-restricted), and found that the binding of the peptides with HLA-A*2402 or HLA-A*0201 induces CTLs, resulting in cytotoxic response to target cells (hereinafter abbreviated as CTL response). From this fact, these peptides were identified as a CTL epitope derived from the WT1 gene expression product (WT protein).
At this point, WT1-specific CTL epitopes only for HLA-A*2402 and HLA-A*0201 (see patent literature 3), HLA-A*3303 (see patent literature 4) or HLA-A*1101 are identified (see patent literature 5). It is confirmed that CTL responses induced by the polypeptides disclosed by the above literatures are restricted by HLA-A*2402, HLA-A*0201, HLA-A*3303 and HLA-A*1101.
This indicates a possibility that the protein product of the tumor suppressor gene WT1 is a promising tumor rejection antigen, also called as a tumor associated antigen (TAA). In fact, high levels of WT1-specific CTLs or high-titer anti-WT1 antibodies were observed not in peripheral blood of healthy blood donors, but in that of cancer patients.
However, HLA types are diverse enough to serve as markers for identifying individuals. In the HLAs, MHC class I antigens are classified into HLA-A, HLA-B and HLA-C, and MHC class II antigens are classified into HLA-DP, HLA-DQ and HLA-DR. Each class has several types of antigens. The antigen binding site of each HLA has genetic polymorphism. For example, it is known that HLA-A, HLA-B and HLA-C have 27 or more, 59 or more, and 10 or more kinds of polymorphisms (alleles), respectively.
Therefore, there has been a desire to identify a cancer antigen that binds to other types of HLAs than HLA-A*2402, HLA-A*0201, HLA-A*3303 and HLA-A*1101 and induces a CTL response, and to thereby apply immunotherapy to a wider range of subjects.
Meanwhile, the following three of modified WT1 peptides were reported in two documents:
the WT1187P1Y peptide (YLGEQQYSV; SEQ ID NO: 12), the WT1126P1Y peptide (YMFPNAPYL; SEQ ID NO: 35) (see patent literature 6 for the above two peptides), and the WT1126P9M peptide (RMFPNAPYM; SEQ ID NO: 52) (see patent literature 7).
Further, the following two peptides were reported in the written argument for the examination of European patent No. 1127068:
the WT1126P2L peptide (RLFPNAPYL; SEQ ID NO: 39) and the WT1126P2L&P9V peptide (RLFPNAPYV; SEQ ID NO: 75) (see nonpatent literature 7).
However, it has never been reported whether these WT1 modified peptides serve as a cancer antigen that binds to other types of HLAs than HLA-A*2402, HLA-A*0201, HLA-A*3303 and HLA-A*1101 and induces a CTL response.

    Patent Literature 1: JP-A 9-104629
    Patent Literature 2: JP-A 11-035484
    Patent Literature 3: WO 00/06602 pamphlet
    Patent Literature 4: Japanese Patent Application No. 2006-045287
    Patent Literature 5: Japanese Patent Application No. 2006-355356
    Patent Literature 6: WO 2005/053618 pamphlet
    Patent Literature 7: WO 2007/016466 pamphlet
    Non Patent Literature 1: Gessler, M. et al., Nature, vol. 343, pp. 774-778, 1990
    Non Patent Literature 2: Cur. Opin. Immunol., vol. 5, p. 709, 1993
    Non Patent Literature 3: Cur. Opin. Immunol., vol. 5, p. 719, 1993
    Non Patent Literature 4: Cell, vol. 82, p. 13, 1995
    Non Patent Literature 5: Immunol. Rev., vol. 146, p. 167, 1995
    Non Patent Literature 6: Mol. Cell. Biol., vol. 11, p. 1707, 1991
    Non Patent Literature 7: The written argument for the examination of European patent No. 1127068


SUMMARY OF INVENTION
Problem to be Solved by the Invention
An object of the present invention is to apply, further to HLA-A*0206-positive persons, a method of cancer treatment and/or prevention for patients with malignant tumors including leukemia, the method being based on a protein product of the tumor suppressor gene WT1 (WT1 protein) or a partial peptide thereof (WT1 peptide).
Means for Solving the Problem
The present inventor conducted intensive studies to achieve the above-mentioned object. As a result, he found that the WT1187 peptide (SLGEQQYSV (SEQ ID NO: 2)) and the WT1126 peptide (RMFPNAPYL (SEQ ID NO: 3)) each derived from the human WT1 protein, which were known to induce HLA-A*0201-restricted CTLs only, surprisingly induce HLA-A*0206-restricted CTLs as well. Under the circumstances where only the peptides described in WO 00/06602 pamphlet were known as a WT1 peptide that induces HLA-A*0201-restricted CTLs, the present inventor found that a modified peptide of the WT1187 peptide (also referred to as a modified WT1187 peptide) and a modified peptide of the WT1126 peptide (also referred to as a WT1126 modified peptide) also bind to an HLA-A*0201 molecule. Based on these findings, the present inventor conducted further intensive studies and completed the present invention.
Namely, the present invention relates to the following (1) to (17).

    (1) A cancer vaccine composition for human leukocyte antigen (HLA)-A*0206-positive persons, comprising a protein product of the tumor suppressor gene WT1 or a partial peptide thereof.
    (2) The composition according to the above (1), wherein the protein product of the tumor suppressor gene WT1 is the protein of the following (a) or (b):
    (a) a protein consisting of the amino acid sequence of SEQ ID NO: 1, or
    (b) a protein consisting of an amino acid sequence comprising deletion, substitution or addition of one to several amino acids in the amino acid sequence (a), either of which is immunogenic in HLA-A*0206-positive persons.
    (3) The composition according to the above (1), wherein the partial peptide is
    the WT1187 peptide: Ser Leu Gly Glu Gln Gln Tyr Ser Val (SEQ ID NO: 2),
    the WT1126 peptide: Arg Met Phe Pro Asn Ala Pro Tyr Leu (SEQ ID NO: 3),
    the WT1187P1F peptide: Phe Leu Gly Glu Gin Gln Tyr Ser Val (SEQ ID NO: 11),
    the WT1187P2M peptide: Ser Met Gly Glu Gln Gln Tyr Ser Val (SEQ ID NO: 16),
    the WT1187P3M peptide: Ser Leu Met Glu Gln Gln Tyr Ser Val (SEQ ID NO: 20),
    the WT1126P1F peptide: Phe Met Phe Pro Asn Ala Pro Tyr Leu (SEQ ID NO: 34),
    the WT1126P2L peptide: Arg Leu Phe Pro Asn Ala Pro Tyr Leu (SEQ ID NO: 39),
    the WT1126P3M peptide: Arg Met Met Pro Asn Ala Pro Tyr Leu (SEQ ID NO: 46) or
    the WT1126P9V peptide: Arg Met Phe Pro Asn Ala Pro Tyr Val (SEQ ID NO: 49).
    (4) The composition according to the above (1) to (3), further comprising an adjuvant.
    (5) A cancer vaccine composition for HLA-A*0206-positive persons, comprising DNA encoding a protein product of the tumor suppressor gene WT1 or a partial peptide thereof.
    (6) A cancer vaccine composition for HLA-A*0206-positive persons, comprising RNA encoding a protein product of the tumor suppressor gene WT1 or a partial peptide thereof.
    (7) A method for inducing WT1-specific CTLs, comprising culturing, in the presence of a protein product of the tumor suppressor gene WT1 or a partial peptide thereof, peripheral blood mononuclear cells (PBMCs) derived from an HLA-A*0206-positive person, to obtain WT1-specific CTLs induced therefrom.
    (8) A method for inducing dendritic cells that present a protein product of the tumor suppressor gene WT1 or a partial peptide thereof, comprising culturing, in the presence of the protein product or a partial peptide thereof, immature dendritic cells derived from an HLA-A*0206-positive person, to obtain dendritic cells induced therefrom which present the protein product or a partial peptide thereof.
    (9) A method of cancer diagnosis for HLA-A*0206-positive persons, comprising
    i) a step of detecting or quantifying a protein product of the tumor suppressor gene WT1 or a partial peptide thereof, an antibody thereagainst or WT1-specific CTLs in a sample from an HLA-A*0206-positive person, and a step of comparing the amount of the protein or a partial peptide thereof, an antibody thereagainst or the WT1-specific CTLs, with that in the case where cancer is not developed, or
    ii) a step of administering an HLA-A*0206-positive subject WT1-specific CTLs induced by the method mentioned in the above (7) or dendritic cells induced by the method mentioned in the above (8), and a step of determining the position or region of the CTLs or dendritic cells in the HLA-A*0206-positive subject.
    (10) A cancer vaccine composition for HLA-A*0201-positive persons, comprising the following peptide:
    a modified peptide of the WT1187 peptide: Ser Leu Gly Glu Gln Gln Tyr Ser Val (SEQ ID NO: 2) or the WT1126 peptide: Arg Met Phe Pro Asn Ala Pro Tyr Leu (SEQ ID NO: 3), either of which is a partial peptide of a protein product of the tumor suppressor gene WT1, the modified peptide being immunogenic in HLA-A*0201-positive persons.
    (11) The composition according to the above (10), wherein the modified peptide is
    the WT1187P1F peptide: Phe Leu Gly Glu Gln Gln Tyr Ser Val (SEQ ID NO: 11),
    the WT1187P2M peptide: Ser Met Gly Glu Gln Gln Tyr Ser Val (SEQ ID NO: 16),
    the WT1187P3M peptide: Ser Leu Met Glu Gln Gln Tyr Ser Val (SEQ ID NO: 20),
    the WT1126P1F peptide: Phe Met Phe Pro Asn Ala Pro Tyr Leu (SEQ ID NO: 34),
    the WT1126P2L peptide: Arg Leu Phe Pro Asn Ala Pro Tyr Leu (SEQ ID NO: 39),
    the WT1126P3M peptide: Arg Met Met Pro Asn Ala Pro Tyr Leu (SEQ ID NO: 46) or
    the WT1126P9V peptide: Arg Met Phe Pro Asn Ala Pro Tyr Val (SEQ ID NO: 49).
    (12) A method of cancer treatment or prevention, comprising administering an HLA-A*0206-positive person a composition containing a protein product of the tumor suppressor gene WT1 or a partial peptide thereof.
    (13) A protein product of the tumor suppressor gene WT1 or a partial peptide thereof for cancer treatment or prevention in HLA-A*0206-positive persons.
    (14) A method of cancer treatment or prevention, comprising administering an HLA-A*0201-positive person a composition containing the following peptide:
    a modified peptide of the WT1187 peptide: Ser Leu Gly Glu Gln Gln Tyr Ser Val (SEQ ID NO: 2) or the WT1126 peptide: Arg Met Phe Pro Asn Ala Pro Tyr Leu (SEQ ID NO: 3), either of which is a partial peptide of a protein product of the tumor suppressor gene WT1, the modified peptide being immunogenic in HLA-A*0201-positive persons.
    (15) The following peptide:
    a modified peptide of the WT1187 peptide: Ser Leu Gly Glu Gln Gln Tyr Ser Val (SEQ ID NO: 2) or the WT1126 peptide: Arg Met Phe Pro Asn Ala Pro Tyr Leu (SEQ ID NO: 3), either of which is a partial peptide of a protein product of the tumor suppressor gene WT1, the modified peptide being immunogenic in HLA-A*0201-positive persons,
    for cancer treatment or prevention in HLA-A*0201-positive persons.
    (16) A method of cancer treatment or prevention, comprising introducing RNA encoding a protein product of the tumor suppressor gene WT1 or a partial peptide thereof into dendritic cells of an HLA-A*0206-positive person.
    (17) RNA encoding a protein product of the tumor suppressor gene WT1 or a partial peptide thereof for cancer treatment or prevention in HLA-A*0206-positive persons.


The present invention also relates to use of a protein product of the tumor suppressor gene WT1 or a partial peptide thereof for production of a cancer vaccine composition used for cancer treatment or prevention in HLA-A*0206-positive persons.
The present invention also relates to use of the following peptide:

    a modified peptide of the WT1187 peptide: Ser Leu Gly Glu Gln Gln Tyr Ser Val (SEQ ID NO: 2) or the WT1126 peptide: Arg Met Phe Pro Asn Ala Pro Tyr Leu (SEQ ID NO: 3), either of which is a partial peptide of a protein product of the tumor suppressor gene WT1, the modified peptide being immunogenic in HLA-A*0201-positive persons,
    for production of a cancer vaccine composition used for cancer treatment or prevention in HLA-A*0201-positive persons.


The “cancer vaccine composition” as used herein refers to a medicament used for cancer prevention or treatment via inoculation or administration to an animal including a human. The “treatment” refers to, besides completely curing disease state, stopping progression of disease state by inhibiting progression and/or aggravation of symptoms to some degree even falling short of a complete cure; or improving all or a part of disease state in a direction towards a cure. The “prevention” refers to preventing, inhibiting or delaying disease development.
The following terms: peripheral blood mononuclear cells, immature dendritic cells, WT1-specific CTLs, samples etc. derived from HLA-A*0206-positive or HLA-A*0201-positive persons refer to peripheral blood mononuclear cells, immature dendritic cells, WT1-specific CTLs, biological specimens etc., such as blood, which are isolated or collected from HLA-A*0206-positive or HLA-A*0201-positive persons, respectively. The WT1-specific CTLs derived from HLA-A*0206-positive or HLA-A*0201-positive persons also include CTLs induced from peripheral blood mononuclear cells, immature dendritic cells or biological specimens such as blood, which are isolated or collected from HLA-A*0206-positive or HLA-A*0201-positive persons.
Effect of the Invention
The present invention enables in vivo and in vitro induction of WT1-specific CTLs in HLA-A*0206-positive subjects. Although the subjects of immunotherapy using a vaccine comprising the WT1 protein or WT1 peptide have conventionally been limited to HLA-A*0201-positive patients and HLA-A*2402-positive patients, the present invention can widen the range of the subjects to HLA-A*0206-positive patients. HLA-A2, which is a serotype of HLA class I antigens, is the most frequent in Caucasians (about 50%), and the large majority have HLA-A*0201, while about 4% of Caucasians have HLA-A*0206. On the other hand, HLA-A24 is the most frequent serotype in Japanese people (about 58%), and the large majority have HLA-A*2402. About 42% of Japanese people have HLA-A2. Among them, only about 43% have HLA-A*0201, and the others have HLA-A*0206 or HLA-A*0207. In other words, about 18% of Japanese people have HLA-A*0201, and about 17% of Japanese people have HLA-A*0206. Therefore, the fact that at least an HLA-A*0206-restricted CTL epitope was identified from Japanese people as well as an HLA-A*0201-restricted CTL epitope is significantly useful to widen the subjects of cancer immunotherapy to HLA-A*0206-positive persons. Since 14% of Chinese people and 9% of South Korean people have this allele, it is possible to apply the cancer vaccine composition of the present invention to a further wider range of subjects.
The cancer vaccine composition of the present invention is useful for treatment of WT1-expressing cancers such as hematopoietic tumors and solid cancers in HLA-A*0206-positive persons. The cancer vaccine composition of the present invention is also useful for prevention of cancer development in HLA-A*0206-positive persons.



BRIEF DESCRIPTION OF DRAWINGS
FIG. 1 shows the cytotoxic activity of WT1187 peptide-specific CTLs induced from PBMCs of an HLA-A*0206-positive healthy blood donor. FIG. 1a shows the cytotoxic activity against 51Cr-labeled B-LCLs. FIG. 1b shows that the cytotoxic activity against 51Cr-labeled autologous B-LCLs increases in parallel with the concentration of the WT1187 peptide used to pulse the PBMCs with.
FIG. 2 shows the cytotoxic activity of WT1187 peptide-specific CTLs induced from PBMCs of an HLA-A*0206-positive healthy blood donor. FIGS. 2a and 2b show the respective cytotoxic activities, against 51Cr-labeled B-LCLs, of CTLs separately obtained from two healthy blood donors other than the blood donor of FIG. 1a. 
FIG. 3a shows the cytotoxic activity of WT1187 peptide-specific CTLs against B-LCLs transformed with the WT1 gene, or B-LCLs transformed with a mock vector. FIG. 3b shows the cytotoxic activity of WT1187 peptide-specific CTLs against 0206K562 cells, K562 cells, KH88 cells or JY cells.
FIG. 4 shows the inhibition of the cytotoxic activity of WT1187 peptide-specific CTLs by HLA class I and/or class II antibodies.
FIG. 5 shows the cytotoxic activity, against 51Cr-labeled B-LCLs, of WT1126 peptide-specific CTLs induced from PBMCs of an HLA-A*0206-positive healthy blood donor.
FIGS. 6a and 6b show the respective cytotoxic activities of WT1126 peptide-specific CTLs separately induced from two different donors, against 0206K562 cells, K562 cells, KH88 cells or JY cells.
FIG. 7 shows the results of the flow cytometric analysis of CTLs stained with the HLA tetramer bound to the WT1126 peptide and the anti-CD8 antibody. The CTLs have been induced by stimulation of PBMCs from the HLA-A*0201-positive donor 1 with modified peptides.
FIG. 8 shows the measurement results of the cytotoxic activity of CTLs induced by stimulation of PBMCs from the HLA-A*0201-positive donor 1 with the WT1126P1F peptide.
FIG. 9 shows the measurement results of the cytotoxic activity of CTLs induced by stimulation of PBMCs from the HLA-A*0201-positive donor 1 with the WT1126P2L peptide.
FIG. 10 shows the results of the flow cytometric analysis of CTLs stained with the HLA tetramer bound to the WT1126 peptide and the anti-CD8 antibody. The CTLs have been induced by stimulation of PBMCs from the HLA-A*0201-positive donor 2 with the WT1126P2L peptide.
FIG. 11 shows the measurement results of the cytotoxic activity of CTLs induced by stimulation of PBMCs from the donor 2 with the WT1126P2L peptide.
FIG. 12 shows the measurement results of the cytotoxic activity of CTLs induced by stimulation of PBMCs from the HLA-A*0206-positive donor 3 with modified WT1126 peptides.
FIG. 13 shows the measurement results of the cytotoxic activity of CTLs induced by stimulation of PBMCs from the HLA-A*0206-positive donor 3 with the WT1126P9V peptide.
FIG. 14 shows the measurement results of the cytotoxic activity of CTLs induced by stimulation of PBMCs from the HLA-A*0206-positive donor 4 with modified WT1126 peptides.
FIG. 15 shows the measurement results of the cytotoxic activity of CTLs induced by stimulation of PBMCs from the HLA-A*0206-positive donor 4 with modified WT1126 peptides.
FIG. 16 shows the measurement results of the cytotoxic activity of CTLs induced by stimulation of PBMCs from an HLA-A*0201-positive donor with modified WT1187 peptides.
FIG. 17 shows the measurement results of the cytotoxic activity of CTLs induced by stimulation of PBMCs from an HLA-A*0206-positive donor with the WT1187P1F peptide.
FIG. 18 shows the measurement results of the cytotoxic activity of CTLs induced by stimulation of PBMCs from an HLA-A*0206-positive donor with the WT1187P2M peptide.
FIG. 19 shows the evaluation results of modified WT1187 peptides on the activity of inducing specific cell-mediated immunity.
FIG. 20 shows the evaluation results of modified WT1187 peptides on the activity of inducing specific cell-mediated immunity.
FIG. 21 shows the evaluation results of modified WT1187 peptides on the activity of inducing specific cell-mediated immunity.
FIG. 22 shows the evaluation results of modified WT1187 peptides on the activity of inducing specific cell-mediated immunity.
FIG. 23 shows the evaluation results of modified WT1126 peptides on the activity of inducing specific cell-mediated immunity.
FIG. 24 shows the evaluation results of modified WT1126 peptides on the activity of inducing specific cell-mediated immunity.
FIG. 25 shows the evaluation results of modified WT1126 peptides on the activity of inducing specific cell-mediated immunity.
FIG. 26 shows the evaluation results of modified WT1126 peptides on the activity of inducing specific cell-mediated immunity.
FIG. 27 shows the evaluation results of modified WT1126 peptides on the activity of inducing specific cell-mediated immunity.
FIG. 28 shows the evaluation results of modified WT1187 peptides on the activity of inducing specific cell-mediated immunity.
FIG. 29 shows the evaluation results of modified WT1187 peptides on the activity of inducing specific cell-mediated immunity.
FIG. 30 shows the evaluation results of modified WT1126 peptides on the activity of inducing specific cell-mediated immunity.
FIG. 31 shows the evaluation results of modified WT1126 peptides on the activity of inducing specific cell-mediated immunity.
FIG. 32 shows the evaluation results of modified WT1126 peptides on the activity of inducing specific cell-mediated immunity.



BEST MODE FOR CARRYING OUT THE INVENTION
Hereinafter, the present invention will be illustrated.
The following codes are used when amino acid residues are abbreviated in this description and drawings.

    Ala or A: Alanine residue
    Arg or R: Arginine residue
    Asn or N: Asparagine residue
    Asp or D: Aspartic acid residue
    Cys or C: Cysteine residue
    Gln or Q: Glutamine residue
    Glu or E: Glutamic acid residue
    Gly or G: Glycine residue
    His or H: Histidine residue
    Ile or I: Isoleucine residue
    Leu or L: Leucine residue
    Lys or K: Lysine residue
    Met or M: Methionine residue
    Phe or F: Phenylalanine residue
    Pro or P: Proline residue
    Ser or S: Serine residue
    Thr or T: Threonine residue
    Trp or W: Tryptophan residue
    Tyr or Y: Tyrosine residue
    Val or V: Valine residue


The WT1 protein of the present invention may be a gene product of a zinc finger-type transcription factor isolated as a causative gene of Wilms' tumor, the gene product being capable of binding to an HLA-A*0206 molecule and thereby serving as a target antigen of malignant tumors. More specifically, the WT1 protein of the present invention is preferably the human WT1 protein consisting of 449 amino acids (Sequence list: SEQ ID NO: 1) or a protein which consists of an amino acid sequence comprising deletion, substitution or addition of one to several amino acids (preferably about 2 to 6 amino acids) in the amino acid sequence of the human WT1 protein, and which is immunogenic in HLA-A*0206-positive persons. The amino acid used for addition or substitution may be a non-natural amino acid besides 20 gene-encoded amino acids.
The partial peptide of the WT1 protein (WT1 peptide) refers to a peptide consisting of a part of the amino acid sequence that constitutes the WT1 protein. The WT1 peptide may be a peptide which consists of 8 to 12 amino acids, preferably 8 to 9 amino acids derived from the WT1 protein and which binds to an HLA-A*0206 molecule and thereby induces cytotoxic T cells. Particularly preferred is the WT1187 peptide (Ser Leu Gly Glu Gln Gln Tyr Ser Val; SEQ ID NO: 2) or the WT1126 peptide (Arg Met Phe Pro Asn Ala Pro Tyr Leu; SEQ ID NO: 3), both described in the WO 00/06602 pamphlet.
A modified peptide comprising deletion, substitution or addition of one or several amino acids of the WT1 peptide can also be used as the WT1 peptide of the present invention as long as it is immunogenic in HLA-A*0206-positive persons. Examples of such a modified peptide include a modified WT1187 peptide and a modified WT1126 peptide.
The modified WT1187 peptide is preferably a peptide comprising the same amino acid residues (EQQYS (SEQ ID NO: 76)) at positions 4 to 8 from the N terminus as the WT1187 peptide has at the corresponding positions, and more preferably a peptide comprising the same amino acid residues (EQQYSV (SEQ ID NO: 77)) at positions 4 to 9 from the N terminus as the WT1187 peptide has at the corresponding positions. Such a modified WT1187 peptide is preferably a peptide consisting of any of the following amino acid sequences of SEQ ID NO: 4 to 26 and 54 to 62.









(GLGEQQYSV; SEQ ID NO: 4)



WT1187P1G peptide



 



(ALGEQQYSV; SEQ ID NO: 5)



WT1187P1A peptide



 



(VLGEQQYSV; SEQ ID NO: 6)



WT1187P1V peptide



 



(LLGEQQYSV; SEQ ID NO: 7)



WT1187P1L peptide



 



(ILGEQQYSV; SEQ ID NO: 8)



WT1187P1I peptide



 



(MLGEQQYSV; SEQ ID NO: 9)



WT1187P1M peptide



 



(WLGEQQYSV; SEQ ID NO: 10)



WT1187P1W peptide



 



(FLGEQQYSV; SEQ ID NO: 11)



WT1187P1F peptide



 



(YLGEQQYSV; SEQ ID NO: 12)



WT1187P1Y peptide



 



(SVGEQQYSV; SEQ ID NO: 13)



WT1187P2V peptide



 



(SQGEQQYSV; SEQ ID NO: 14)



WT1187P2Q peptide



 



(SIGEQQYSV; SEQ ID NO: 15)



WT1187P2I peptide



 



(SMGEQQYSV; SEQ ID NO: 16)



WT1187P2M peptide



 



(SLLEQQYSV; SEQ ID NO: 17)



WT1187P3L peptide



 



(SLAEQQYSV; SEQ ID NO: 18)



WT1187P3A peptide



 



(SLVEQQYSV; SEQ ID NO: 19)



WT1187P3V peptide



 



(SLMEQQYSV; SEQ ID NO: 20)



WT1187P3M peptide



 



(SLPEQQYSV; SEQ ID NO: 21)



WT1187P3P peptide



 



(SLWEQQYSV; SEQ ID NO: 22)



WT1187P3W peptide



 



(SLFEQQYSV; SEQ ID NO: 23)



WT1187P3F peptide



 



(SLYEQQYSV; SEQ ID NO: 24)



WT1187P3Y peptide



 



(SLSEQQYSV; SEQ ID NO: 25)



WT1187P3S peptide



 



(SLIEQQYSV; SEQ ID NO: 26)



WT1187P3I peptide



 



(SLGEQQYSL; SEQ ID NO: 53)



WT1187P9L peptide



 



(DLGEQQYSV; SEQ ID NO: 54)



WT1187P1D peptide



 



(ELGEQQYSV; SEQ ID NO: 55)



WT1187P1E peptide



 



(HLGEQQYSV; SEQ ID NO: 56)



WT1187P1H peptide



 



(KLGEQQYSV; SEQ ID NO: 57)



WT1187P1K peptide



 



(NLGEQQYSV; SEQ ID NO: 58)



WT1187P1N peptide



 



(PLGEQQYSV; SEQ ID NO: 59)



WT1187P1P peptide



 



(QLGEQQYSV; SEQ ID NO: 60)



WT1187P1Q peptide



 



(RLGEQQYSV; SEQ ID NO: 61)



WT1187P1R peptide



 



(TLGEQQYSV; SEQ ID NO: 62)



WT1187P1T peptide






The modified WT1126 peptide is preferably a peptide comprising the same amino acid residues (PNAPY (SEQ ID NO: 78)) at positions 4 to 8 from the N terminus as the WT1126 peptide has at the corresponding positions. Such a modified WT1126 peptide is preferably a peptide consisting of any of the following amino acid sequences of SEQ ID NO: 27 to 52 and 63 to 75.









(GMFPNAPYL; SEQ ID NO: 27)



WT1126P1G peptide



 



(AMFPNAPYL; SEQ ID NO: 28)



WT1126P1A peptide



 



(VMFPNAPYL; SEQ ID NO: 29)



WT1126P1V peptide



 



(LMFPNAPYL; SEQ ID NO: 30)



WT1126P1L peptide



 



(IMFPNAPYL; SEQ ID NO: 31)



WT1126P1I peptide



 



(MMFPNAPYL; SEQ ID NO: 32)



WT1126P1M peptide



 



(WMFPNAPYL; SEQ ID NO: 33)



WT1126P1W peptide



 



(FMFPNAPYL; SEQ ID NO: 34)



WT1126P1F peptide



 



(YMFPNAPYL; SEQ ID NO: 35)



WT1126P1Y peptide



 



(RVFPNAPYL; SEQ ID NO: 36)



WT1126P2V peptide



 



(RQFPNAPYL; SEQ ID NO: 37)



WT1126P2Q peptide



 



(RAFPNAPYL; SEQ ID NO: 38)



WT1126P2A peptide



 



(RLFPNAPYL; SEQ ID NO: 39)



WT1126P2L peptide



 



(RIFPNAPYL; SEQ ID NO: 40)



WT1126P2I peptide



 



(RMIPNAPYL; SEQ ID NO: 41)



WT1126P3I peptide



 



(RMLPNAPYL; SEQ ID NO: 42)



WT1126P3L peptide



 



(RMGPNAPYL; SEQ ID NO: 43)



WT1126P3G peptide



 



(RMAPNAPYL; SEQ ID NO: 44)



WT1126P3A peptide



 



(RMVPNAPYL; SEQ ID NO: 45)



WT1126P3V peptide



 



(RMMPNAPYL; SEQ ID NO: 46)



WT1126P3M peptide



 



(RMPPNAPYL; SEQ ID NO: 47)



WT1126P3P peptide



 



(RMWPNAPYL; SEQ ID NO: 48)



WT1126P3W peptide



 



(RMFPNAPYV; SEQ ID NO: 49)



WT1126P9V peptide



 



(RMFPNAPYA; SEQ ID NO: 50)



WT1126P9A peptide



 



(RMFPNAPYI; SEQ ID NO: 51)



WT1126P9I peptide



 



(RMFPNAPYM; SEQ ID NO: 52)



WT1126P9M peptide



 



(DMFPNAPYL; SEQ ID NO: 63)



WT1126P1D peptide



 



(EMFPNAPYL; SEQ ID NO: 64)



WT1126P1E peptide



 



(HMFPNAPYL; SEQ ID NO: 65)



WT1126P1H peptide



 



(KMFPNAPYL; SEQ ID NO: 66)



WT1126P1K peptide



 



(NMFPNAPYL; SEQ ID NO: 67)



WT1126P1N peptide



 



(PMFPNAPYL; SEQ ID NO: 68)



WT1126P1P peptide



 



(QMFPNAPYL; SEQ ID NO: 69)



WT1126P1Q peptide



 



(SMFPNAPYL; SEQ ID NO: 70)



WT1126P1S peptide



 



(TMFPNAPYL; SEQ ID NO: 71)



WT1126P1T peptide



 



(RIFPNAPYI; SEQ ID NO: 72)



WT1126P2I&P9I peptide



 



(RIFPNAPYV; SEQ ID NO: 73)



WT1126P2I&P9V peptide



 



(RLFPNAPYI; SEQ ID NO: 74)



WT1126P2L&P9I peptide



 



(RLFPNAPYV; SEQ ID NO: 75)



WT1126P2L&P9V peptide






Inter alia, the modified WT1187 peptide is preferably the WT1187P1F peptide (SEQ ID NO: 11), the WT1187P2M peptide (SEQ ID NO: 16) or the WT1187P3M peptide (SEQ ID NO: 20), more preferably the WT1187P1F peptide or the WT1187P2M peptide, and still more preferably the WT1187P2M peptide. The modified WT1126 peptide is preferably the WT1126P1F peptide (SEQ ID NO: 34), the WT1126P2L peptide (SEQ ID NO: 39), the WT1126P3M peptide (SEQ ID NO: 46) or the WT1126P9V peptide (SEQ ID NO: 49), more preferably the WT1126P2L peptide, the WT1126P3M peptide or the WT1126P9V peptide, and still more preferably the WT1126P9V peptide.
The WT1 peptide in the cancer vaccine composition of the present invention is preferably the WT1187 peptide, the WT1126 peptide, the WT1187P1F peptide, the WT1187P2M peptide, the WT1187P3M peptide, the WT1126P1F peptide, the WT1126P2L peptide, the WT1126P3M peptide or the WT1126P9V peptide. More preferred is the WT1187 peptide, the WT1126 peptide, the WT1187P1F peptide, the WT1187P2M peptide, the WT1126P2L peptide, the WT1126P3M peptide or the WT1126P9V peptide. Even preferred is the WT1187 peptide, the WT1126 peptide, the WT1187P2M peptide or the WT1126P9V peptide. Particularly preferred is the WT1187 peptide or the WT1126 peptide.
A derivative of the WT1 peptide can also be used as the WT1 peptide. For example, the derivative of the WT1187 or WT1126 peptide may be formed of an amino acid sequence of the above-mentioned 9 contiguous amino acids and various substances bound to the N and/or C terminus thereof. The various substances may be, for example, amino acids, peptides, analogs thereof, etc. Such a substance bound to the WT1187 peptide, the WT1126 peptide or a modified peptide thereof undergoes, for example, in vivo enzyme treatment through intracellular processing etc., and finally the peptide consisting of the above-mentioned 9 amino acids is produced and presented as a complex with an HLA-A*0206 molecule on the cell surface. Thus, a WT1-specific CTL response can be induced in patients with HLA-A*0206.
The WT1 peptide can be prepared by a method usually used in the technical field, such as a peptide synthesis method described in Peptide Synthesis, Interscience, New York, 1966; The Proteins, Vol. 2, Academic Press Inc., New York, 1976; Peptide synthesis, Maruzen Co., Ltd., 1975; Basis and Experiments of Peptide Synthesis, Maruzen Co., Ltd. 1985; the Sequel to Development of Pharmaceuticals, Vol. 14 (peptide synthesis), Hirokawa Publishing Company, 1991; etc.
As a method of screening for the WT1 peptide and a modified peptide thereof, for example, a method involving conducting the IFNγ assay under single stimulation of, with a peptide, PBMCs (peripheral blood mononuclear cells) of some patients having HLA-A*0206, and then selecting a peptide showing a good response, is preferred because of simplicity.
In the present invention, polynucleotides, such as DNA encoding the above-mentioned WT1 protein or WT1 peptide immunogenic in HLA-A*0206-positive persons, can also be used as an active ingredient of the cancer vaccine composition. Namely, by inserting a polynucleotide encoding the WT1 protein or WT1 peptide into a suitable vector, preferably an expression vector, and then administering the vector into animals including humans, cancer immunity can be produced in the living body. Examples of the polynucleotide include DNA, RNA and the like, and preferred is DNA or RNA. The base sequence of the polynucleotide can be determined based on the amino acid sequence of the WT1 protein or WT1 peptide immunogenic in HLA-A*0206-positive persons. The polynucleotide can be prepared by a known DNA or RNA synthesis method, the PCR method, etc. Such a cancer vaccine composition for HLA-A*0206-positive persons, comprising DNA encoding the WT1 protein or WT1 peptide is also one aspect of the present invention. The WT1 protein or WT1 peptide is preferably a WT1 peptide, more preferably the WT1187 peptide, the WT1126 peptide or a modified peptide thereof, and most preferably the WT1187 peptide or the WT1126 peptide. The expression vector used to insert the above-mentioned DNA into is not particularly limited. RNA does not have to be inserted into a vector and can be used as it is as an active ingredient of the composition.
The cancer vaccine composition of the present invention can comprise an adjuvant. The adjuvant is not limited as long as, after administered together with or separately from the WT1 protein or WT1 peptide used as an antigen, it can nonspecifically enhance immunological responses to the antigen. Examples of the adjuvant include precipitating-type adjuvants and oily adjuvants. Examples of the precipitating-type adjuvant include sodium hydroxide, aluminum hydroxide, calcium phosphate, aluminum phosphate, alum, PEPES and carboxyvinyl polymers. A preferable oily adjuvant is one that can form micelles so that oil encloses an aqueous solution of an antigen. Specific examples thereof include liquid paraffin, lanolin, Freund, Montanide ISA-763AVG, Montanide ISA-51, incomplete Freund's adjuvant and complete Freund's adjuvant. These adjuvants can also be used as a mixture of two or more kinds thereof. Preferred is an oily adjuvant.
The amount of the adjuvant in the cancer vaccine composition of the present invention is not particularly limited as long as immunological responses to antigens can be nonspecifically enhanced. The amount thereof may be suitably selected depending on the kind of the adjuvant, etc.
The cancer vaccine composition of the present invention can be administered orally or parenterally (for example, intraperitoneally, subcutaneously, intracutaneously, intramuscularly, intravenously, intranasally, etc.). In the case of parenteral administration, an active ingredient, i.e., the WT1 protein or WT1 peptide, may also be percutaneously absorbed by applying the vaccine composition to the skin, or by attaching to the skin a patch containing the vaccine composition. The vaccine composition of the present invention can also be administered via inhaling etc. The vaccine composition is administered preferably parenterally, and more preferably intracutaneously or subcutaneously. The body part for intracutaneous or subcutaneous administration is preferably the upper arm etc., for example.
The cancer vaccine composition of the present invention can be in various dosage forms depending on its administration route, and exemplary dosage forms thereof include a solid preparation and a liquid preparation. The cancer vaccine composition may be, for example, in the form of a solid or liquid preparation to be used internally for oral administration, an injection for parenteral administration, or the like.
Examples of the solid preparation to be used internally for oral administration include tablets, pills, capsules, powders and granules.
For preparation of the solid preparation to be used internally, the WT1 protein or WT1 peptide is untreated, mixed with an additive, or granulated (according to, for example, stirring granulation, fluidized bed granulation, dry granulation, rolling stirring fluidized bed granulation, etc.), and then is subjected to a usual method. For example, the capsules can be prepared by encapsulation etc. and the tablets can be prepared by tableting etc. One or two kinds or more of the additives may be appropriately incorporated into the solid preparation. Examples of the additive include excipients such as lactose, mannitol, glucose, microcrystalline cellulose and corn starch; binders such as hydroxypropylcellulose, polyvinylpyrrolidone and magnesium aluminometasilicate; dispersing agents such as corn starch; disintegrators such as calcium carboxymethyl cellulose; lubricants such as magnesium stearate; solubilizing agents such as glutamic acid and aspartic acid; stabilizers; water soluble polymers including celluloses such as hydroxypropylcellulose, hydroxypropylmethylcellulose and methylcellulose, and synthetic polymers such as polyethylene glycol, polyvinylpyrrolidone and polyvinyl alcohol; and sweeteners such as white sugar, powder sugar, sucrose, fructose, glucose, lactose, reduced malt sugar syrup (maltitol syrup), reduced malt sugar syrup powder (maltitol syrup powder), high-glucose corn syrup, high-fructose corn syrup, honey, sorbitol, maltitol, mannitol, xylitol, erythritol, aspartame, saccharin and saccharin sodium.
The granules or tablets may be covered with a coating agent etc. if needed, and may be covered with two or more layers thereof. Examples of the coating agent include white sugar, gelatin, hydroxypropyl cellulose and hydroxypropylmethylcellulose phthalate. The capsules can be prepared by mixing the active ingredient with pranlukast hydrate and an excipient appropriately selected from the above excipients, optionally granulating the mixture, and optionally covering the resulting granules with a coating agent, followed by capsule filling. Alternatively, the capsules can be prepared by adding glycerol, sorbitol, etc. to an appropriate capsule base (gelatin etc.) to increase its plasticity, and encapsulating the active ingredient with the resulting base. To the capsule base may be added a colorant or a preservative (sulfur dioxide; and parabens such as methyl parahydroxybenzoate, ethyl parahydroxybenzoate and propyl parahydroxybenzoate) if needed. The capsules include hard capsules and soft capsules.
Examples of the liquid preparation to be used internally for oral administration include waters, suspensions/emulsions, syrups, preparations to be dissolved before use such as dry syrups, and elixirs. For preparation of the liquid preparation to be used internally, the WT1 protein or WT1 peptide is dissolved, suspended or emulsified in a diluent generally used for liquid preparations to be used internally. Examples of the diluent include purified water, ethanol and a mixture thereof. The liquid preparation may further contain a wetting agent, a suspending agent, an emulsifier, a sweetener, a flavoring, a fragrance, a preservative or a buffering agent. The dry syrups can be prepared, for example, by mixing the active ingredient with pranlukast hydrate and an additional ingredient such as white sugar, powder sugar, sucrose, fructose, glucose and lactose. The dry syrups may also be made into granules in a usual manner.
Examples of the dosage form for parenteral administration include injections, ointments, gels, creams, patches, aerosols and sprays. Preferred are injections. For example, the injection preferably contains a conventional carrier with the WT1 protein or WT1 peptide.
The injection for parenteral administration may be an aqueous injection or an oily injection. The aqueous injection can be prepared according to a known method, for example, by appropriately adding a pharmaceutically acceptable additive to an aqueous solvent (water for injection, purified water, etc.) to make a solution, mixing the WT1 protein or WT1 peptide with the solution, filter sterilizing the resulting mixture with a filter etc., and then filling an aseptic container with the resulting filtrate. Examples of the pharmaceutically acceptable additive include the above-mentioned adjuvants; isotonizing agents such as sodium chloride, potassium chloride, glycerol, mannitol, sorbitol, boric acid, borax, glucose and propylene glycol; buffering agents such as a phosphate buffer solution, an acetate buffer solution, a borate buffer solution, a carbonate buffer solution, a citrate buffer solution, a Tris buffer solution, a glutamate buffer solution and an epsilon-aminocaproate solution; preservatives such as methyl parahydroxybenzoate, ethyl parahydroxybenzoate, propyl parahydroxybenzoate, butyl parahydroxybenzoate, chlorobutanol, benzyl alcohol, benzalkonium chloride, sodium dehydroacetate, sodium edetate, boric acid and borax; thickeners such as hydroxyethylcellulose, hydroxypropylcellulose, polyvinyl alcohol and polyethylene glycol; stabilizers such as sodium hydrogen sulfite, sodium thiosulfate, sodium edetate, sodium citrate, ascorbic acid and dibutyl hydroxy toluene; and pH adjusters such as hydrochloric acid, sodium hydroxide, phosphoric acid and acetic acid. The injection may further contain an appropriate solubilizing agent, and examples thereof include alcohols such as ethanol; polyalcohols such as propylene glycol and polyethylene glycol; and non-ionic surfactants such as polysorbate 80, polyoxyethylene hydrogenated castor oil 50, lysolecithin and pluronic polyols. Also, proteins such as bovine serum albumin and keyhole limpet hemocyanin; polysaccharides such as aminodextran; etc. may be contained in the injection. For preparation of the oily injection, for example, sesame oil or soybean oil is used as an oily solvent, and benzyl benzoate or benzyl alcohol may be blended as a solubilizing agent. The prepared injection is usually stored in an appropriate ampule, vial, etc. The liquid preparations, such as injections, can also be deprived of moisture and preserved by cryopreservation or lyophilization. The lyophilized preparations become ready to use by redissolving them in added distilled water for injection etc. just before use.
Another dosage form of the cancer vaccine composition of the present invention may be a liposome containing the WT1 protein or WT1 peptide and, if needed, polysaccharides and/or other ingredients that can be blended into the cancer vaccine composition.
The dose of the cancer vaccine composition of the present invention varies with the kind of the WT1 protein, WT1 peptide or DNA to be used, the age and body weight of the patient, the disease to be treated, etc. For example, in the case of the vaccine composition comprising the WT1 peptide, for example the WT1187 peptide or the WT1126 peptide, the daily dose is preferably about 0.1 μg/kg bw to 1 mg/kg bw as the amount of the WT1 peptide. The dose of the WT1 peptide is usually 0.0001 mg to 1000 mg, preferably 0.01 mg to 1000 mg, and more preferably 0.1 mg to 10 mg. This amount is preferably administered once in several days to several months.
The cancer vaccine composition of the present invention is a cancer vaccine composition for HLA-A*0206-positive persons. The HLA type, which is a measure for selecting HLA-A*0206-positive persons, can be determined from, for example, donors' peripheral blood. Examples of the method of determining the HLA type include known methods, such as the DNA typing method, for example, the SBT (Sequencing Based Typing) method or the SSP method, and the HLA typing method. In the SBT method, the base sequence of a PCR-amplified DNA is compared with the base sequence data of the known alleles to precisely identify the HLA gene type. In the SSP method, after PCR amplification using a variety of primers specific to respective HLA alleles, subsequent electrophoresis is performed to check a positive band. Thus, the HLA gene type can be identified.
When the cancer vaccine composition of the present invention has been administered into an HLA-A*0206-positive person, the HLA-A*0206-restricted WT1 protein or WT1 peptide in the vaccine composition, or the WT1 protein or WT1 peptide expressed from DNA or RNA in the vaccine composition binds to an HLA-A*0206 molecule on the surface of an antigen presenting cell (dendritic cell) of the HLA-A*0206-positive person. This induces specific antitumor immunity, i.e., WT1-specific CTLs, which destroy cancer cells in the subject (HLA-A*0206-positive person). Such antitumor immunity can be checked, for example by the WT1-specific CTL response, the cytotoxicity test against cancer cells (for example, 51Cr release cytotoxicity test), etc. For example, the HLA-A*0201-restricted WT1187 peptide and WT1126 peptide each consisting of 9 amino acids derived from the WT1 protein, which have been reported to be capable of inducing a WT1-specific CTL response, can induce an HLA-A*0206-restricted response. About 17% of Japanese people are HLA-A*0206-positive, while almost the same proportion are HLA-A*0201-positive. In the following Examples 1 to 5, WT1187 peptide-specific CTLs were prepared from PBMCs of three HLA-A*0206-positive blood donors. The induced CTLs showed the cytotoxic effect on WT1-expressing, HLA-A*0206-positive leukemia cells. Since WT1187 peptide- and WT1126 peptide-specific CTL activity can be inhibited by an anti-HLA class I antibody, the activity is found to be exhibited by HLA class I-restricted CTLs. The WT1 protein or WT1 peptide including the WT1187 peptide and/or the WT1126 peptide, or a modified peptide thereof can be a vaccine for HLA-A*0206-positive cancer patients as well as HLA-A*0201-positive cancer patients. Therefore, the immunotherapy based on the WT1 protein or WT1 peptide for patients with malignant tumors, such as hematopoietic tumors and solid cancers, can be applied further to HLA-A*0206-positive cancer patients. The method of cancer treatment and/or prevention in HLA-A*0206-positive persons, comprising administering the cancer vaccine composition of the present invention into an HLA-A*0206-positive person, is one of preferable embodiments of the present invention.
In HLA-A*0206-positive persons, the cancer vaccine composition of the present invention can be used for treatment and/or prevention of cancers accompanied by increased expression of the WT1 gene: for example, hematopoietic tumors such as leukemia, myelodysplastic syndrome, multiple myeloma and malignant lymphoma; and solid cancers such as gastric cancer, colon cancer, lung cancer, breast cancer, germ cell cancer, hepatic cancer, skin cancer, bladder cancer, prostate cancer, uterine cancer, cervical cancer and ovarian cancer.
An exemplary administration method of the cancer vaccine composition of the present invention is a method comprising collecting PBMCs from peripheral blood of an HLA-A*0206-positive patient, extracting dendritic cells from the PBMCs, pulsing the dendritic cells with a peptide, for example the WT1187 peptide or the WT1126 peptide, or a polynucleotide, for example DNA or RNA, contained as an active ingredient in the cancer vaccine composition of the present invention, and returning the dendritic cells to the patient via subcutaneous administration etc. The conditions for pulsing dendritic cells with the WT1 peptide etc. are not particularly limited as long as the effect of the present invention is achieved, and may be ordinary conditions.
In the case where RNA encoding the WT1 protein or WT1 peptide is used for the cancer vaccine composition, it is preferable that the composition is administered so that the RNA is introduced into dendritic cells of an HLA-A*0206-positive person. An exemplary method for introducing RNA into dendritic cells of an HLA-A*0206-positive person is a method comprising collecting dendritic cells from an HLA-A*0206-positive person in the same manner as mentioned above, and introducing RNA into the dendritic cells with an electric pulse. The WT1 protein or WT1 peptide expressed from the introduced RNA in the dendritic cells is allowed to be presented on the surface thereof. By returning the dendritic cells pulsed with the RNA into the HLA-A*0206-positive person, cancer immunity can be quickly produced in the living body. Such a method of cancer treatment or prevention, comprising introducing RNA encoding the WT1 protein or WT1 peptide into dendritic cells of an HLA-A*0206-positive person, is one of preferable embodiments of the present invention.
Another embodiment of the present invention relates to a method for inducing WT1-specific CTLs, by culturing, in the presence of the WT1 protein or WT1 peptide, PBMCs derived from an HLA-A*0206-positive person, to obtain WT1-specific CTLs induced therefrom. The subject from which PBMCs are derived is not particularly limited as long as the subject is HLA-A*0206-positive. Examples of the WT1 protein or WT1 peptide include the WT1187 peptide, the WT1126 peptide and a modified peptide thereof, and preferably the WT1187 peptide and the WT1126 peptide. For example, WT1-specific CTLs can be induced from CTL precursor cells among PBMCs by culturing PBMCs derived from an HLA-A*0206-positive person in the presence of the WT1187 peptide (or WT1126 peptide). The culture conditions for PBMCs derived from an HLA-A*0206-positive person is not particularly limited, and may be ordinary conditions. The thus-obtained CTLs recognize a complex of the WT1187 peptide (or the WT1126 peptide) and an HLA-A*0206 molecule. Therefore, by use of WT1-specific CTLs induced according to the present invention, WT1-highly-expressing tumor cells can be specifically destroyed in an HLA-A*0206-positive person, and thereby hematopoietic tumors and solid cancers in the subject, i.e., an HLA-A*0206-positive person, can be treated and/or prevented. The method for administering such WT1-specific CTLs into an HLA-A*0206-positive subject is not particularly limited, and for example, may be the same as the administration method of the above-mentioned cancer vaccine composition.
Another embodiment of the present invention relates to a kit for inducing WT1-specific CTLs, comprising the HLA-A*0206-restricted WT1 protein or WT1 peptide as an essential constituent. Preferably, the kit is used for the above-mentioned method for inducing WT1-specific CTLs derived from an HLA-A*0206-positive person. Such a kit may comprise, for example, a means for collecting PBMCs, an adjuvant and a reaction container in addition to the HLA-A*0206-restricted WT1 protein or WT1 peptide. By use of the kit, WT1-specific CTLs that recognize a complex of a cancer antigen, such as the WT1187 peptide and the WT1126 peptide, and an HLA-A*0206 molecule can be efficiently induced.
Another embodiment of the present invention relates to a method for inducing dendritic cells that present the WT1 protein or WT1 peptide, by culturing, in the presence of the WT1 protein or WT1 peptide, immature dendritic cells derived from an HLA-A*0206-positive person, to obtain dendritic cells induced therefrom which present the WT1 protein or WT1 peptide. Examples of the WT1 protein or WT1 peptide include the WT1187 peptide, the WT1126 peptide and a modified peptide thereof, and preferably the WT1187 peptide and the WT1126 peptide. The subject from which immature dendritic cells are derived is not particularly limited as long as the subject is HLA-A*0206-positive. Since immature dendritic cells are present among PBMCs etc., PBMCs may also be cultured in the presence of the WT1187 peptide or the WT1126 peptide, for example. By administration of the thus-obtained dendritic cells to an HLA-A*0206-positive person, the above-mentioned WT1-specific CTLs are efficiently induced, and thereby hematopoietic tumors and solid cancers in the subject can be treated and/or prevented. The method for administering such dendritic cells into an HLA-A*0206-positive subject is not particularly limited, and for example, may be the same as the administration method of the above-mentioned cancer vaccine composition.
Another embodiment of the present invention relates to a kit for inducing dendritic cells that present the WT1 protein or WT1 peptide, comprising the HLA-A*0206-restricted WT1 protein or WT1 peptide as an essential constituent. Preferably, the kit is used for the above-mentioned method for inducing dendritic cells. Such a kit may comprise, for example, a means for collecting immature dendritic cells and PBMCs, an adjuvant and a reaction container in addition to the HLA-A*0206-restricted WT1 protein or WT1 peptide. By use of the kit, dendritic cells that present the WT1 protein or WT1 peptide via an HLA-A*0206 molecule can be efficiently induced.
Cancers in HLA-A*0206-positive persons can be diagnosed by use of

    (1) the WT1 protein or WT1 peptide, WT1-specific CTLs induced by the above-mentioned method, or dendritic cells induced by the above-mentioned method, or
    (2) an antibody against the following: the WT1 protein or WT1 peptide, WT1-specific CTLs induced by the above-mentioned method, or dendritic cells induced by the above-mentioned method.

Such a method of cancer diagnosis is also one aspect of the present invention. In the above (1), cancer diagnosis is conducted preferably using WT1-specific CTLs induced by above-mentioned method. Examples of the WT1 protein or WT1 peptide include the WT1187 peptide, the WT1126 peptide and a modified peptide thereof, and preferably the WT1187 peptide and the WT1126 peptide.



According to the present invention, an exemplary method of cancer diagnosis for HLA-A*0206-positive persons comprises a step of detecting or quantifying the WT1 protein or WT1 peptide, an antibody thereagainst or WT1-specific CTLs in a sample from an HLA-A*0206-positive person, and a step of comparing the amount of the protein or a partial peptide thereof, an antibody thereagainst or the WT1-specific CTLs, with that in the case where cancer is not developed.
In a cancer patient sample (for example, blood), the WT1 peptide and/or WT1 protein released from cancer cells is present, and the immunological response against a cancer antigen is enhanced. That is, the cancer patient sample has an increased amount of an antibody against the WT1 peptide or WT1 protein, WT1-specific CTLs, etc. For this reason, when the amount of the WT1 peptide or WT1 protein, an antibody thereagainst or the WT1-specific CTLs in the sample is increased compared with that in the case where cancer is not developed, cancer may have been developed. The amount of the antibody can be measured by the ELISA method, for example. The WT1-specific CTLs can be detected by a method using WT1 multimers such as MHC tetramers described below.
Alternatively, cancer diagnosis can also be performed by incubating the above-mentioned CTLs, dendritic cells or antibody together with a sample from an HLA-A*0206-positive subject, or administering the above-mentioned CTLs, dendritic cells or antibody into an HLA-A*0206-positive subject; and then determining the position, region, amount, etc. of the CTLs, dendritic cells or antibody. Since CTLs and dendritic cells have a property to gather around cancer cells, cancer diagnosis can be performed by administering the CTLs or dendritic cells into the subject, and examining the position or region thereof. A method of cancer diagnosis for HLA-A*0206-positive persons, comprising a step of administering WT1-specific CTLs or dendritic cells induced by the above-mentioned method into an HLA-A*0206-positive subject, and a step of determining the position or region of the CTLs or dendritic cells in the HLA-A*0206-positive subject is also one aspect of the present invention.
Cancer diagnosis can also be performed by incubating CTLs or dendritic cells together with a sample from an HLA-A*0206-positive subject to allow them to react, adding an antibody against the CTLs or dendritic cells, continuing incubation, and detecting or quantifying an antibody-bound complex of the cancer cell and CTLs, antibody-bound dendritic cells, etc. via a label etc. bound to the antibody. When the amount of the antibody-bound complex of the cancer cell and CTLs or the antibody-bound dendritic cells is increased compared with that in the case where cancer is not developed, cancer may have been developed. The above-mentioned CTLs, dendritic cells or antibody may be labeled. The labeling enables the diagnosis to be efficiently performed. Examples of the sample from an HLA-A*0206-positive subject include biological specimens obtained from HLA-A*0206-positive persons, such as urine, blood, tissue extract fluid, saliva, tear and other body fluids, and blood is preferable.
Examples of the method of cancer diagnosis for HLA-A*0206-positive persons using the above-mentioned WT1 protein or WT1 peptide include the MHC tetramer assay, the MHC pentamer assay and the MHC dextramer assay, each of which uses the WT1 peptide as an antigen. For example, in the MHC tetramer assay or MHC pentamer assay using the WT1187 peptide or WT1126 peptide as an antigen peptide, WT1-specific CTLs in HLA-A*0206-positive persons can be detected by use of an MHC/WT1187 peptide complex or an MHC/WT1126 peptide complex as a probe. Since cancer patients show high expression of WT1-specific CTLs, cancer can be diagnosed by measuring the expression of WT1-specific CTLs in HLA-A*0206-positive persons. Since cancer patients manifest an enhanced immunological response against cancer antigens, cancer can be diagnosed also by examining immunological response against the WT1 protein or WT1 peptide in HLA-A*0206-positive persons. Examples of the method of examining immunological response include a method involving measuring an antibody against the WT1 protein or WT1 peptide by ELISA. Such a method of cancer diagnosis for HLA-A*0206-positive persons using a protein product of the tumor suppressor gene WT1 or a partial peptide thereof is also one aspect of the present invention. The MHC tetramer assay and MHC pentamer assay can be performed by a known method using a commercially available kit, for example, “WT1 tetramer” (Medical & Biological Laboratories, Co., Ltd.).
Cancer diagnosis for HLA-A*0206-positive persons can also be performed by a method comprising a step of reacting a sample from an HLA-A*0206-positive subject with an antibody against the following: the WT1 protein or WT1 peptide, WT1-specific CTLs induced by the above-mentioned method or dendritic cells induced by the above-mentioned, and a step of detecting or quantifying a complex of the antibody with the WT1 protein or WT1 peptide, or a complex of the antibody with WT1-specific CTLs or dendritic cells. When the amount of the complex of the antibody with the WT1 protein or WT1 peptide, or the complex of the antibody with WT1-specific CTLs or dendritic cells is increased compared with that in the case where cancer is not developed, cancer may have been developed.
Examples of the antibody against dendritic cells include an antibody which recognizes a WT1 peptide/HLA-A*0206 complex. Since such an antibody can recognize the WT1 peptide and an HLA-A*0206 molecule, the antibody can recognize dendritic cells having the WT1 peptide presented via HLA Class I.
An antibody which recognizes a complex of WT1 peptide/HLA-A*0206/TCR (T cell antigen receptor) of CTLs can also be used as the antibody against dendritic cells. Such an antibody can recognize a complex of a dendritic cell and a CTL, and a complex of a cancer cell and a CTL.
Cancer diagnosis can be performed by incubating such an antibody together with a sample from an HLA-A*0206-positive subject to allow them to form a complex, and detecting or quantifying an antibody-bound complex of the cancer cell and CTLs, antibody-bound dendritic cells presenting the WT1 peptide, or the like via the fluorescence emitted by the antibody. When the amount of the antibody-bound complex of the cancer cell and CTLs, the antibody-bound dendritic cells presenting the WT peptide, or the like is increased compared with that in the case where cancer is not developed, cancer may have been developed.
A method of cancer treatment or prevention, comprising administering a composition containing the WT1 protein or WT1 peptide into an HLA-A*0206-positive person, is also one aspect of the present invention. The composition comprising the WT1 protein or WT1 peptide and preferable embodiments thereof are the same as described regarding the above-mentioned cancer vaccine composition.
Use of the WT1 protein or WT1 peptide for cancer treatment or prevention in HLA-A*0206-positive persons, and use thereof for production of a cancer vaccine composition used for cancer treatment or prevention in HLA-A*0206-positive persons is also one aspect of the present invention. The WT1 protein or WT1 peptide and preferable embodiments thereof are the same as described regarding the above-mentioned cancer vaccine composition.
A cancer vaccine composition for HLA-A*0201-positive persons, comprising a modified peptide of the WT1187 peptide (SEQ ID NO: 2) or the WT1126 peptide (SEQ ID NO: 3), either of which is a partial peptide of a protein product of the tumor suppressor gene WT1, the modified peptide being immunogenic in HLA-A*0201-positive persons, is also one aspect of the present invention.
Examples of a modified WT1187 peptide or a modified WT1126 peptide include peptides comprising deletion, substitution or addition of one or several amino acids of the above-mentioned WT1187 peptide or WT1126 peptide. The modified WT1187 peptide is preferably a peptide comprising the same amino acid residues at positions 4 to 8 from the N terminus as the WT1187 peptide has at the corresponding positions. As such a modified peptide, preferred are the above-mentioned peptides of SEQ ID NOS: 4 to 12, 15 and 16, 18 to 20 and 22 to 25. The WT1187P9L peptide (SLGEQQYSL; SEQ ID NO: 53) is also preferred. The modified WT1126 peptide is preferably a peptide comprising the same amino acid residues at positions 4 to 8 from the N terminus as the WT1126 peptide has at the corresponding positions. For example, preferred are the above-mentioned peptides of SEQ ID NOS: 27 to 37 and 39 to 52.
In yet another preferable embodiment of the present invention, the above-mentioned peptides of SEQ ID NOS: 4 to 26 and 53 to 62 may be used as a modified WT1187 pep tide, and the above-mentioned peptides of SEQ ID NOS: 27 to 52 and 63 to 75 may be used as a modified WT1126 peptide. Among the modified peptides of SEQ ID NOS: 4 to 75, the peptides except the WT1187P1D peptide, the WT1187P1E peptide, the WT1187P1H peptide, the WT1187P1P peptide and the WT1187P2Q peptide; and the WT1126P1D peptide, the WT1126P1E peptide, the WT1126P1P peptide, the WT1126P2A peptide and the WT1126P2Q peptide are preferred.
Inter alia, the modified WT1187 peptide is preferably the WT1187P1F peptide, the WT1187P2M peptide or the WT1187P3M peptide, and more preferably the WT1187P1F peptide or the WT1187P2M peptide. The modified WT1126 peptide is preferably the WT1126P1F peptide, the WT1126P2L peptide, the WT1126P3M peptide or the WT1126P9V peptide, and more preferably the WT1126P1F peptide or the WT1126P2L peptide.
The amount for use of the modified WT1187 pep tide or WT1126 peptide which is immunogenic in HLA-A*0201-positive persons is the same as that of the WT1 peptide in the above-mentioned cancer vaccine composition for HLA-A*0206-positive persons. The other ingredients of the cancer vaccine composition for HLA-A*0201-positive persons and preferable embodiments thereof are the same as those of the above-mentioned vaccine composition for HLA-A*0206-positive persons.
DNA and RNA encoding the above-mentioned modified WT1187 peptide or WT1126 peptide which is immunogenic in HLA-A*0201-positive persons can also be used as an active ingredient of the cancer vaccine composition for HLA-A*0201-positive persons. Such a cancer vaccine composition for HLA-A*0201-positive persons is also one aspect of the present invention.
The other ingredients than the above-mentioned DNA and RNA in the cancer vaccine composition for HLA-A*0201-positive persons and preferable embodiments thereof are the same as those of the above-mentioned cancer vaccine composition for HLA-A*0206-positive persons.
WT1-specific CTLs can be induced from PBMCs derived from an HLA-A*0201-positive person by culturing the PBMCs in the presence of the modified WT1187 peptide or WT1126 peptide which is immunogenic in the above-mentioned HLA-A*0201-positive person. Such a method of inducing WT1-specific CTLs is also one aspect of the present invention.
Preferable examples of the modified WT1187 peptide or WT1126 peptide which is immunogenic in HLA-A*0201-positive persons are the same as used for the above-mentioned cancer vaccine composition for HLA-A*0201-positive persons.
Dendritic cells that present the modified WT1187 peptide or WT1126 peptide can be induced from immature dendritic cells derived from an HLA-A*0201-positive person by culturing the immature dendritic cells in the presence of the modified peptide which is immunogenic in the above-mentioned HLA-A*0201-positive person. Such a method for inducing dendritic cells that present the modified WT1187 peptide or WT1126 peptide is also one aspect of the present invention. Preferable examples of the modified peptide are the same as used for the above-mentioned cancer vaccine composition for HLA-A*0201-positive persons.
Cancers in HLA-A*0201-positive persons can be diagnosed by use of the above-mentioned modified WT1187 peptide or modified WT1126 peptide immunogenic in HLA-A*0201-positive persons, an antibody thereagainst, WT1-specific CTLs induced by the modified peptide or dendritic cells induced by the modified peptide. Such a method of cancer diagnosis for HLA-A 0201-positive persons is also one aspect of the present invention. The method of cancer diagnosis for HLA-A*0201-positive persons and preferable embodiments thereof are the same as the above-mentioned method of cancer diagnosis for HLA-A*0206-positive persons and preferable embodiments thereof.
Examples of the method of cancer diagnosis for HLA-A*0201-positive persons include the MHC tetramer assay, the MHC pentamer assay and the MHC dextramer assay, each of which uses the modified WT1187 peptide or modified WT1126 peptide immunogenic in HLA-A*0201-positive persons as an antigen. Preferable examples of the modified peptide are the same as used for the above-mentioned cancer vaccine composition for HLA-A*0201-positive persons.
Cancers in HLA-A*021-positive persons can be diagnosed by use of an antibody against the following: the above-mentioned modified WT1187 peptide or modified WT1126 peptide immunogenic in HLA-A*0201-positive persons, WT1-specific CTLs induced by the modified peptide or dendritic cells induced by the modified peptide. Such a method of cancer diagnosis for HLA-A*0201-positive persons is also one aspect of the present invention. Preferable examples of the modified peptide are the same as used for the above-mentioned cancer vaccine composition for HLA-A*0201-positive persons. The method of cancer diagnosis for HLA-A*0201-positive persons and preferable embodiments thereof are the same as the above-mentioned method of cancer diagnosis for HLA-A*0206-positive persons and preferable embodiments thereof.
A method of cancer treatment or prevention, comprising administering an HLA-A*0201-positive person a cancer vaccine composition containing the following peptide:
a modified peptide of the WT1187 peptide (SEQ ID NO: 2) or the WT1126 peptide (SEQ ID NO: 3), either of which is a partial peptide of a protein product of the tumor suppressor gene WT1, the modified peptide being immunogenic in HLA-A*0201-positive persons,
is also one aspect of the present invention.
Preferable examples of the modified peptide are the same as used for the above-mentioned cancer vaccine composition for HLA-A*0201-positive persons. The cancer vaccine composition and preferable embodiments thereof are the same as described regarding the above-mentioned vaccine composition for HLA-A*0201-positive persons.
The present invention relates to use of the following peptide:
a modified peptide of the WT1187 peptide (SEQ ID NO: 2) or the WT1126 peptide (SEQ ID NO: 3), either of which is a partial peptide of a protein product of the tumor suppressor gene WT1, the modified peptide being immunogenic in an HLA-A*0201-positive person,

for cancer treatment or prevention in HLA-A*0201-positive persons, and use thereof for production of a cancer vaccine composition used for cancer treatment or prevention in HLA-A*0201-positive persons.

Preferable examples of the modified peptide are the same as used for the above-mentioned cancer vaccine composition for HLA-A*0201-positive persons. The cancer vaccine composition and preferable embodiments thereof are the same as described regarding the above-mentioned vaccine composition for HLA-A*0201-positive persons.
EXAMPLES
Hereinafter, the present invention will be illustrated in more detail by way of examples, but is not limited thereto. Abbreviations in Examples indicate the following meanings. Synthetic peptides were purchased from SIGMA GENOSYS JAPAN.

    DCs: Dendritic cells
    PBMCs: Peripheral blood mononuclear cells
    CD: Cluster of Differentiation (leukocyte differentiation antigen)
    GM-CSF: Granulocyte monocyte colony stimulating factor
    IL: Interleukin
    TNFα: Tumor necrosis factor-α
    PGE: Prostaglandin
    Gy: Gray
    B-LCLs: B-lymphoblastoid cell line
    EB virus: Epstein-Barr virus
    tBu: t-butyl
    Trt: Triphenylmethyl
    Fmoc: 9-fluorenylmethyloxycarbonyl


Example 1
Prediction of HLA Molecules Capable of Binding with the WT1 Peptide
HLA molecules capable of binding with the WT1187 peptide (SEQ ID NO: 2) were predicted using the NetMHC2.0 Server-prediction program.
As a result, the HLA-A*0201-restricted WT1187 peptide capable of inducing WT1-specific CTLs was ranked high in terms of binding affinity to an HLA-A*0206 molecule in the NetMHC2.0 Server-prediction program.
Example 2
Preparation of WT1187 Peptide-specific CTLs from PBMCs of HLA-A*0206-positive Healthy Blood Donors, and Cytotoxicity Test of the CTLs
(1) Separation of PBMCs of HLA-A*0206-positive Healthy Blood Donors, and Preparation of DCs
First, PBMCs were isolated from peripheral blood of each of HLA-A*0206 healthy blood donors (three persons) by Ficoll-Hypaque density gradient centrifugation. Then, CD14-positive cells were selected from the PBMCs using anti-human CD14 Magnetic Particles-DM (manufactured by Becton, Dickinson and company (BD)). In this case, it was considered that a large number of CD14-positive cells are present in the monocyte population. The selected CD14-positive cells were cultured in an X-VIVO15 medium (manufactured by BioWhittaker, Walkersville, Md.) supplemented with 1 v/v % human AB serum, 800 IU/mL GM-CSF (manufactured by Pepro Tech INC. Rocky Hill, N.J.) and 1000 IU/mL IL-4 (manufactured by Pepro Tech INC) to prepare DCs.
(2) Induction of Autologous Mature DCs
The DCs prepared in the above (1) were cultured at 37° C. for 1 day, and then a maturation cytokine cocktail containing 10 ng/mL TNFα (tumor necrosis factor-α; Pepro Tech INC, Rocky Hill, N.J.), 10 ng/mL IL-β, 1000 IU/mL IL-6 and 1 μg/mL PGE2 was added to culture wells containing the DCs. After 24 hour-culture at 37° C., autologous mature DCs were obtained.
(3) Induction of WT1187 Peptide-specific CTLs
The autologous mature DCs were pulsed with the WT1187 peptide, irradiated with 30 Gy of radiation, and co-cultured with CD8-positive T cell-enriched PBMCs obtained from the HLA-A*0206-positive healthy blood donor. The pulsing of the DCs with the WT1187 peptide was performed by culturing the DCs in the presence of 10 μg/mL of the WT1187 peptide at 37° C. for 30 minutes. The CD8-positive T cells were enriched from PBMCs of the HLA-A*0206-positive healthy blood donor using CD8 MicroBeads and MS column (manufactured by Miltenyi Biotec GmbH).
From the second stimulation, autologous PBMCs which had been pulsed with the peptide and then irradiated with radiation were used as selective stimulator cells. Two days after the second stimulation, recombinant IL-2 (provided by Shionogi & Co., Ltd.) and IL-7 (manufactured by Pepro Tech INC) were added to the culture medium at the concentrations of 10 IU/mL and 10 ng/mL, respectively. After the 4th stimulation, the cells were cultured for 10 days at 37° C. and then the resulting cells (CTLs) were collected by centrifugation using a centrifuge. The cytotoxic activity of these cells (CTLs) against target cells was examined by a 51Cr release cytotoxicity test.
(4) Cytotoxicity Test
The cytotoxicity test was performed by a 51Cr release cytotoxicity test. The 51Cr release cytotoxicity test was performed as follows. First, target cells (1×107 cells/mL) were incubated in the presence of 100 μL of 51Cr (specific activity: 1 mCi/ml) in RPMI1640 (manufactured by NIHON PHARMACEUTICAL CO., LTD.) supplemented with 10% fetal bovine serum at 37° C. for 1.5 hours to label the target cells with 51Cr. Then, the 51Cr-labeled target cells were added to wells of 96 round-bottom well plates containing various numbers of CTLs obtained in the above (3) (suspended in 100 μL of an assay medium), mixed with the CTLs and then incubated at 37° C. for 4 hours. These cells were mixed so that the E/T ratio (cell number ratio) was 1:1, 5:1, 20:1 or 25:1, with the proviso that CTLs and the 51Cr-labeled target cells are expressed as “E” and “T”, respectively. After the completion of incubation, 100 μL of the supernatant was collected from each well. The amount of 51Cr release from the labeled cells was determined, and the specific lysis (%) based on the 51Cr release was calculated. The specific lysis (%) was calculated in the following manner.

Specific lysis (%)=(release from a test sample−spontaneous release)/(maximum release−spontaneous release)×100

In the formula, the amount of spontaneous release refers to the amount of fluorescence of culture supernatant in the wells containing target cells only, and the maximum release refers to the amount of fluorescence of culture medium in which the target cells have been completely lysed by treatment with 1 mass % Triton X-100.
The target cells to be used were B-LCLs, K562 cells, JY cells, and KH88 cells, which will be described in detail below. KH88 cells are the same as KH88OF8 cells used in the following Example 9.
B-LCLs, which were established by EB virus-mediated transformation of peripheral blood B lymphocytes obtained from an HLA-A*0206-positive blood donor, do not express WT1.
K562 cells, which were established from a patient with chronic myelogenous leukemia in blastic crisis, are a WT1-expressing, non-HLA class I-expressing cell line. The present inventor was not able to obtain a WT1-expressing, HLA-A*0206-positive wild-type leukemia cell line. For this reason, 0206K562 cells, which were prepared by transformation of K562 cells with HLA-A*0206 genes, were also used. The FACS analysis using an anti-HLA-A2 antibody (cloneBB7.2; manufactured by BD Biosciences Pharmingen) showed that the 0206K562 cells transformed with HLA-A*0206 genes express HLA-A*0206 molecules on the cell surfaces.
The western blot analysis showed that B-LCL cells transformed with the WT1 gene express WT1. B-LCL cells transformed with a mock vector were used as a control.
JY cells are a non-WT1-expressing, HLA-A*0206-negative B cell line established by EB virus-mediated transformation.
KH88 cells are a WT1-expressing, HLA-A*0206-negative leukemia cell line.
Each cell line was cultured in a RPMI1640 culture medium supplemented with 10 v/v % heat-inactivated fetal bovine serum, 50 IU/mL penicillin and 50 mg/mL streptomycin.
(5) Antibody and Flow Cytometry Analysis
Anti-human CD14, CD86, CD80, CD83 and HLA-DR mAbs were purchased from BD. Concentration and maturation of DCs were confirmed by analysis of cell surface antigens using the monoclonal antibodies (mAbs) listed above. Samples were analyzed with a flow cytometer (FACS Calibur; manufactured by BD) using CellQest software.
(6) Results
It was examined whether WT1187 peptide-specific CTLs can be prepared from PBMCs of HLA-A*0206-positive blood donors. The WT1187 peptide-specific cytotoxic activity was examined using the CTLs obtained by repeatedly stimulating the CD8-positive T cell-enriched PBMCs from the HLA-A*0206-positive healthy blood donor with WT1187 peptide-pulsed autologous DCs or PBMCs. The CTLs showed a stronger cytotoxic activity against WT1187 peptide-pulsed autologous B-LCL cells than against non-WT1187 peptide-pulsed B-LCL cells (FIG. 1a). In FIG. 1a, the vertical axis represents the cytotoxic activity, and the horizontal axis represents the ratio of CTLs obtained by peptide stimulation (effector: E) relative to target cells (target: T) (E/T ratio). The closed triangle represents the cells pulsed with 10 μg/mL of the WT1187 peptide, and the closed square represents the cells not pulsed with the WT1187 peptide. The same cytotoxic activity as above was shown also by the CTLs similarly prepared from the PBMCs isolated from the two different HLA-A*0206-positive healthy blood donors (FIGS. 2a and 2b). In FIG. 2, the closed triangle represents the cells pulsed with 10 μg/mL of the WT1187 peptide, and the closed square represents the cells not pulsed with the WT1187 peptide. These results show that each cytotoxic activity is specific to the WT1187 pep tide.
The cytotoxic activity of the CTLs increased in parallel with the concentration of the WT1187 peptide used to pulse the DCs or PBMCs with, and reached the plateau at the peptide concentration of 0.1 μg/mL (FIG. 1b). The half maximum concentration of the WT1187 peptide for specific lysis (half-maximal lysis value) was about 5×10−5 μg/mL. This shows that the affinity of TCRs (T cell antigen receptors) of the CTLs to a WT1187 peptide/HLA-A*0206 complex was relatively high. This result strongly suggests that CTLs induced with the WT1187 peptide can recognize the WT1187 peptide.
In the same manner as above, the cytotoxic activity against various target cells endogenously expressing WT1 was examined using the CTLs obtained by stimulating the CD8-positive T cell-enriched PBMCs from the HLA-A*0206-positive blood donor with WT1187 peptide-pulsed DCs or PBMCs. The results are shown in FIGS. 3a and 3b. The respective cytotoxic activities for the target cells shown in FIGS. 3a and 3b were determined at the same time. FIG. 3a shows the cytotoxic activity of WT1187 peptide-specific CTLs against B-LCLs transformed with the WT1 gene (WT1-expressing, HLA-A*0206-positive; closed triangle), or B-LCLs transformed with a mock vector (non-WT1-expressing, HLA-A*0206-positive; closed square). FIG. 3b shows that the cytotoxic activity of WT1187 peptide-specific CTLs against 0206K562 cells (WT1-expressing, HLA-A*0206-positive; closed square), K562 cells (WT1-expressing, HLA-A*0206-negative; open square), KH88 cells (WT1-expressing, HLA-A*0206-negative; closed circle), or JY cells (non-WT1-expressing, HLA-A*0206-negative; closed triangle).
The CTLs showed a stronger cytotoxic activity against the B-LCLs transformed with WT1 (WT1-expressing, HLA-A*0206-positive) than against the B-LCLs transformed with a mock vector (non-WT1-expressing, HLA-A*0206-positive) (FIG. 3a). Further, as shown in FIG. 3b, the CTLs showed a stronger cytotoxic activity against the 0206K562 cells transformed with HLA-A*0206 (WT1-expressing, HLA-A*0206-positive) than against the K562 cells (WT1-expressing, HLA-A*0206-negative), the KH88 cells (WT1-expressing, HLA-A*0206-negative), or the JY cells (non-WT1-expressing, HLA-A*0206-negative). In other words, the CTLs showed a significant cytotoxic activity against WT1-expressing, HLA-A*0206-positive target leukemia cells, but no cytotoxic activity against non-WT1-expressing and/or HLA-A*0206-negative cells. This result demonstrates that WT1187 peptide-specific CTLs prepared in vitro show the cytotoxic activity against tumor cells endogenously expressing WT1 like leukemia cells and being HLA-A*0206-positive. The result in FIG. 3b strongly suggests that the cytotoxic activity of WT1187 peptide-specific CTLs was restricted by HLA-A class I. This is based on the fact that a stronger cytotoxic activity was observed against 0206K562 cells than against K562 cells.
The above results demonstrate that the above-mentioned cultured CTLs are WT1187 peptide-specific CTLs.
The results of each figure are typical data, and basically reproducible with some variation.
Example 3
Confirmation of the HLA Class by Which WT1187 Peptide-specific CTLs are Restricted
It was examined whether the cytotoxic activity of the WT1187 peptide-specific CTLs obtained in Example 2 was restricted by HLA class I. The 51Cr release cytotoxicity test was performed in the presence or absence of mAbs against HLA class I or HLA class II. Autologous B-LCLs were used as a target cell. In this experiment, the E/T ratio was 5:1.
The results are shown in FIG. 4. FIG. 4a shows the results of the test that was performed using B-LCLs (non-WT1187-expressing, HLA-A*0206-positive) as a target cell in the absence of mAbs against HLA class I (anti-HLA class I mAbs) and mAbs against HLA class II (anti-HLA class II mAbs). FIG. 4b shows the results of the test that was performed using WT1187 peptide-pulsed B-LCLs (WT1187-expressing, HLA-A*0206-positive) as a target cell in the absence of anti-HLA class I mAbs and in the presence of anti-HLA class II mAbs. FIG. 4c shows the results of the test that was performed using WT1187 peptide-pulsed B-LCLs (WT1187-expressing, HLA-A*0206-positive) as a target cell in the presence of anti-HLA class I mAbs and in the absence of anti-HLA class II mAbs. FIG. 4d shows the results of the test that was performed using WT1187 peptide-pulsed B-LCLs (WT1187-expressing, HLA-A*0206-positive) as a target cell in the absence of anti-HLA class I mAbs and anti-HLA class II mAbs.
As shown in FIG. 4, the cytotoxic activity of the WT1187 peptide-specific CTLs was completely inhibited by addition of an anti-HLA class I antibody, not an anti-HLA class II antibody. The result shows that the cytotoxic activity of WT1187 peptide-specific CTLs was restricted by HLA class I as expected.
Example 4
Cytotoxicity Test Against Tumor Cells
The cytotoxicity test against WT1-expressing, HLA-A*0206-positive tumor cells was performed in vitro using the WT1187 peptide-specific CTLs obtained in Example 2. The cytotoxicity test was performed according to the 51Cr release cytotoxicity test described in Example 2. As a result, the WT1187 peptide-specific CTLs showed the cytotoxic activity against WT1-expressing tumor cells (data not shown).
Example 5
Preparation of WT1126 Peptide-specific CTLs, and Cytotoxicity Test of the CTLs
WT1126 peptide-specific CTLs were prepared in the same manner as in Example 2 (3) except that the WT1126 peptide (SEQ ID NO: 3) was used instead of the WT1187 peptide. The cytotoxicity test was performed using these CTLs in the same manner as in Example 2, to determine the WT1126 peptide-specific cytotoxic activity. FIG. 5 shows the cytotoxic activity of WT1126 peptide-specific CTLs induced from PBMCs of the same HLA-A*0206-positive healthy blood donor as in FIG. 2b. In FIG. 5, the closed triangle represents cells pulsed with 10 μg/mL of the WT1126 peptide, and the closed square represents cells not pulsed with the WT1126 peptide. The CTLs showed a stronger cytotoxic activity against the WT1126 peptide-pulsed autologous B-LCL cells than against the non-WT1126 peptide-pulsed B-LCL cells (FIG. 5). This result shows that the cytotoxic activity of the CTLs is specific to the WT1126 peptide.
In the same manner as in Example 2, the cytotoxic activity against various target cells endogenously expressing WT1 was examined using the CTLs prepared by stimulating the CD8-positive T cell-enriched PBMCs from the HLA-A*0206-positive blood donors with WT1126 peptide-pulsed DCs or PBMCs. The cytotoxic activity against each target cell is shown in FIGS. 6a and 6b. The target cells in FIGS. 6a and 6b are 0206K562 cells (WT1-expressing, HLA-A*0206-positive; closed square), K562 cells (WT1-expressing, HLA-A*0206-negative; open square), KH88 cells (WT1-expressing, HLA-A*0206-negative; closed circle), and JY cells (non-WT1-expressing, HLA-A*0206-negative; closed triangle). FIG. 6a shows the cytotoxic activity of WT1126 peptide-specific CTLs induced from PBMCs of the same HLA-A*0206-positive healthy blood donor as in FIG. 2a. FIG. 6b shows the cytotoxic activity of WT1126 peptide-specific CTLs induced from PBMCs of the same HLA-A*0206-positive healthy blood donor as in FIG. 2b. 
Like WT1187 peptide-specific CTLs, the WT1126 peptide-specific CTLs showed a significant cytotoxic activity against WT1-expressing, HLA-A*0206-positive target leukemia cells, but no cytotoxic activity against non-WT1-expressing and/or HLA-A*0206-negative cells. This result demonstrates that WT1126 peptide-specific CTLs prepared in vitro show the cytotoxic activity against tumor cells endogenously expressing WT1 like leukemia cells and being HLA-A*0206-positive. The results of FIGS. 6a and 6b strongly suggest that the cytotoxic activity of WT1126 peptide-specific CTLs was restricted by HLA-A class I. This is based on the fact that a stronger cytotoxic activity was observed against 0206K562 cells than against K562 cells.
The above results demonstrate that the obtained CTLs are WT1126 peptide-specific CTLs.
The results of each figure are typical data, and basically reproducible with some variation.
Example 6
Preparation of Vaccine Compositions
The following cancer vaccine compositions 1 to 8 were prepared. These are only examples of the cancer vaccine composition of the present invention.
Cancer Vaccine Composition 1
















WT1187 peptide
 3 mg



Montanide ISA-51
400 mg



5% glucose in water
400 mg










The above-mentioned ingredients were mixed and the mixture was named cancer vaccine composition 1.
Cancer Vaccine Composition 2
















WT1187 peptide
 1 mg



Montanide ISA-51
400 mg



5% glucose in water
400 mg










The above-mentioned ingredients were mixed and the mixture was named cancer vaccine composition 2.
Cancer Vaccine Composition 3
















WT1187 peptide
0.001 mg  



Montanide ISA-51
400 mg



5% glucose in water
400 mg










The above-mentioned ingredients were mixed and the mixture was named cancer vaccine composition 3.
Cancer Vaccine Composition 4
















WT1187 peptide
 10 mg



Montanide ISA-51
400 mg



5% glucose in water
400 mg










The above-mentioned ingredients were mixed and the mixture was named cancer vaccine composition 4.
Cancer Vaccine Compositions 5 to 8
Cancer vaccine compositions 5 to 8 were prepared in the same manner as in the above-mentioned cancer vaccine compositions 1 to 4 except that the WT1126 peptide was used instead of the WT1187 peptide.
Example 7
Affinity of Modified Peptides to HLA-A*0206 Molecules
As for the WT1187 peptide, the WT1126 peptide, and modified peptides comprising substitution of an amino acid residue at position 1, 2, 3 or 9 from the N terminus of the WT1187 peptide or the WT1126 peptide, the affinity to HLA-A*0206 molecules was analyzed by use of the NetMHC2.0 Server-prediction program. The analysis results of modified WT1187 peptides and modified WT1126 peptides are shown in Tables 1 and 2, respectively. The smaller value (the peptide has a binding ability at a lower concentration) indicates the higher affinity.












TABLE 1






Amino acid
SEQ ID
Predicted
Affinity
Binding


Peptide
sequence
NO
score
(nM)
Strength




















WT1187
SLGEQQYSV
2
0.776
11
Strong







binding







(SB)


 


WT1187P1G
GLGEQQYSV
4
0.756
13
SB


 


WT1187P1A
ALGEQQYSV
5
0.812
7
SB


 


WT1187P1V
VLGEQQYSV
6
0.755
14
SB


 


WT1187P1L
LLGEQQYSV
7
0.810
7
SB


 


WT1187P1I
ILGEQQYSV
8
0.782
10
SB


 


WT1187P1M
MLGEQQYSV
9
0.877
3
SB


 


WT1187P1W
WLGEQQYSV
10
0.876
3
SB


 


WT1187P1F
FLGEQQYSV
11
0.926
2
SB


 


WT1187P1Y
YLGEQQYSV
12
0.896
3
SB


 


WT1187P2V
SVGEQQYSV
13
0.722
20
SB


 


WT1187P2Q
SQGEQQYSV
14
0.824
6
SB


 


WT1187P2I
SIGEQQYSV
15
0.734
17
SB


 


WT1187P2M
SMGEQQYSV
16
0.798
8
SB


 


WT1187P3L
SLLEQQYSV
17
0.865
4
SB


 


WT1187P3A
SLAEQQYSV
18
0.844
5
SB


 


WT1187P3V
SLVEQQYSV
19
0.869
4
SB


 


WT1187P3M
SLMEQQYSV
20
0.896
3
SB


 


WT1187P3P
SLPEQQYSV
21
0.791
9
SB


 


WT1187P3W
SLWEQQYSV
22
0.883
3
SB


 


WT1187P3F
SLFEQQYSV
23
0.864
4
SB


 


WT1187P3Y
SLYEQQYSV
24
0.857
4
SB


 


WT1187P3S
SLSEQQYSV
25
0.801
8
SB


 


WT1187P3I
SLIEQQYSV
26
0.880
3
SB


 


WT1187P9L
SLGEQQYSL
53
0.586
88
weak







binding





















TABLE 2






Amino acid
SEQ ID
Predicted
Affinity
Binding


Peptide
sequence
NO
score
(nM)
Strength




















WT1126
RMFPNAPYL
3
0.83
6
SB


 


WT1126P1G
GMFPNAPYL
27
0.76
14
SB


 


WT1126P1A
AMFPNAPYL
28
0.80
8
SB


 


WT1126P1V
VMFPNAPYL
29
0.75
15
SB


 


WT1126P1L
LMFPNAPYL
30
0.80
8
SB


 


WT1126P1I
IMFPNAPYL
31
0.77
11
SB


 


WT1126P1M
MMFPNAPYL
32
0.86
4
SB


 


WT1126P1W
WMFPNAPYL
33
0.88
3
SB


 


WT1126P1F
FMFPNAPYL
34
0.91
2
SB


 


W71126P1Y
YMFPNAPYL
35
0.88
3
SB


 


WT1126P2V
RVFPNAPYL
36
0.78
11
SB


 


WT1126P2Q
RQFPNAPYL
37
0.85
4
SB


 


WT1126P2A
RAFPNAPYL
38
0.67
35
SB


 


WT1126P2L
RLFPNAPYL
39
0.80
8
SB


 


WT1126P2I
RIFPNAPYL
40
0.78
10
SB


 


WT1126P3I
RMIPNAPYL
41
0.84
5
SB


 


WT1126P3L
RMLPNAPYL
42
0.83
6
SB


 


WT1126P3G
RMGPNAPYL
43
0.71
23
SB


 


WT1126P3A
RMAPNAPYL
44
0.79
9
SB


 


WT1126P3V
RMVPNAPYL
45
0.82
6
SB


 


WT1126P3M
RMMPNAPYL
46
0.86
4
SB


 


WT1126P3P
RMPPNAPYL
47
0.72
21
SB


 


WT1126P3W
RMWPNAPYL
48
0.85
5
SB


 


WT1126P9V
RMFPNAPYV
49
0.91
2
SB


 


WT1126P9A
RMFPNAPYA
50
0.77
12
SB


 


WT1126P9I
RMFPNAPYI
51
0.81
7
SB


 


WT1126P9M
RMFPNAPYM
52
0.65
42
SB









Example 8
Affinity of Modified Peptides to HLA-A*0201 Molecules
As for the WT1187 peptide, the WT1126 peptide, and modified peptides comprising substitution of an amino acid residue at position 1, 2, 3 or 9 from the N terminus of the WT1187 peptide or the WT1126 peptide, the affinity to HLA-A*0201 molecules was analyzed by use of the NetMHC2.0 Server-prediction program. The analysis results of modified WT1187 peptides and modified WT1126 peptides are shown in Tables 3 and 4, respectively. The smaller value indicates the higher affinity.












TABLE 3






Amino acid
SEQ ID
Predicted
Affinity
Binding


Peptide
sequence
NO
score
(nM)
Strength




















WT1187
SLGEQQYSV
2
0.721
20
SB


 


WT1187P1G
GLGEQQYSV
4
0.672
34
SB


 


WT1187P1A
ALGEQQYSV
5
0.648
44
SB


 


WT1187P1V
VLGEQQYSV
6
0.705
24
SB


 


WT1187P1L
LLGEQQYSV
7
0.658
40
SB


 


WT1187P1I
ILGEQQYSV
8
0.698
26
SB


 


WT1187P1M
MLGEQQYSV
9
0.717
21
SB


 


WT1187P1W
WLGEQQYSV
10
0.628
55
SB


 


WT1187P1F
FLGEQQYSV
11
0.824
6
SB


 


WT1187P1Y
YLGEQQYSV
12
0.809
7
SB


 


WT1187P2I
SIGEQQYSV
15
0.556
121
SB


 


WT1187P2M
SMGEQQYSV
16
0.740
16
SB


 


WT1187P3A
SLAEQQYSV
18
0.811
7
SB


 


WT1187P3V
SLVEQQYSV
19
0.766
12
SB


 


WT1187P3M
SLMEQQYSV
20
0.876
3
SB


 


WT1187P3W
SLWEQQYSV
22
0.863
4
SB


 


WT1187P3F
SLFEQQYSV
23
0.852
4
SB


 


WT1187P3Y
SLYEQQYSV
24
0.854
4
SB


 


WT1187P3S
SLSEQQYSV
25
0.793
9
SB


 


WT1187P9L
SLGEQQYSL
53
0.640
49
SB





















TABLE 4






Amino acid
SEQ ID
Predicted
Affinity
Binding


Peptide
sequence
NO
score
(nM)
Strength




















WT1126P1G
GMFPNAPYL
27
0.80
9
SB


 


WT1126P1A
AMFPNAPYL
28
0.81
7
SB


 


WT1126P1V
VMFPNAPYL
29
0.81
8
SB


 


WT1126P1L
LMFPNAPYL
30
0.82
7
SB


 


WT1126P1I
IMFPNAPYL
31
0.81
8
SB


 


WT1126P1M
MMFPNAPYL
32
0.85
4
SB


 


WT1126P1W
WMFPNAPYL
33
0.80
8
SB


 


WT1126P1F
FMFPNAPYL
34
0.91
2
SB


 


WT1126P1Y
YMFPNAPYL
35
0.90
2
SB


 


WT1126P2V
RVFPNAPYL
36
0.55
127
SB


 


WT1126P2Q
RQFPNAPYL
37
0.49
262
SB


 


WT1126P2L
RLFPNAPYL
39
0.78
10
SB


 


WT1126P2I
RIFPNAPYL
40
0.64
48
SB


 


WT1126P3I
RMIPNAPYL
41
0.74
16
SB


 


WT1126P3L
RMLPNAPYL
42
0.78
10
SB


 


WT1126P3G
RMGPNAPYL
43
0.60
73
SB


 


WT1126P3A
RMAPNAPYL
44
0.73
17
SB


 


WT1126P3V
RMVPNAPYL
45
0.68
31
SB


 


WT1126P3M
RMMPNAPYL
46
0.83
6
SB


 


WT1126P3P
RMPPNAPYL
47
0.61
66
SB


 


WT1126P3W
RMWPNAPYL
48
0.83
6
SB


 


WT1126P9V
RMFPNAPYV
49
0.84
5
SB


 


WT1126P9A
RMFPNAPYA
50
0.73
18
SB


 


WT1126P9I
RMFPNAPYI
51
0.79
9
SB


 


WT1126P9M
RMFPNAPYM
52
0.69
29
SB









Example 9
Comparison of HLA-A*0201-restricted CTLs Induced by Various Modified WT1126 Peptides
(1) Purpose
In view of the results of Example 8, the WT1126P1F peptide (SEQ ID NO: 34), the WT1126P2L peptide (SEQ ID NO: 39), the WT1126P3M peptide (SEQ ID NO: 46) and the WT1126P9V peptide (SEQ ID NO: 49) were selected as modified WT1126 peptides to be tested, and the following experiments were conducted to screen for modified WT1126 peptides capable of inducing CTLs having a high cytotoxic activity. The reagents, media, experimental methods, etc. used in Examples 9 to 12 were the same as in Example 1, unless otherwise specified. In Examples 9 to 12, culture was performed at 37° C., unless otherwise specified.
(2) Materials and Methods
From a healthy human donor showing expression of HLA-A*0201 molecules (HLA-A*0201-positive healthy blood donor), PBMCs were isolated, and CD14-positive cells were separated from the PBMCs by use of anti-human CD14 Magnetic Particles-DM. A culture medium was prepared by adding 800 IU/mL GM-CSF and 1000 IU/mL IL-4 to an X-VIVO15 medium supplemented with 1 v/v % human AB serum, and the CD14-positive cells were cultured in the culture medium for 1 day.
To the above culture, a maturation cytokine cocktail containing 10 ng/mL TNFα, 10 ng/mL IL-β, 1000 IU/mL IL-6 and 1 μg/mL PGE2 was added. After additional one day culture, autologous mature DCs were obtained.
The autologous mature DCs were pulsed with 10 μg/mL of a WT1126 modified peptide obtained in Example 13 (the WT1126P1F peptide, the WT1126P2L peptide, the WT1126P3M peptide or the WT1126P9V peptide), cultured for 4 hours, and irradiated with 35Gy of radiation. The thus-obtained cells were used as stimulator cells for CTL induction.
The PBMCs (2×106 cells/well) serving as responder cells and the above-mentioned DCs (2×105 cells/well) were co-cultured in a 24-well plate. One week later, re-stimulation was given by addition of T2 cells which had been pulsed with the peptide and irradiated with 75Gy of radiation. Three days after re-stimulation, 20 IU/mL of IL-2 was added. The same re-stimulation was repeated another 3 times by addition of the peptide-pulsed, irradiated T2 cells, and then CD8-positive cells in the responder cells were enriched.
As for the CD8-positive T cells, the reactivity on an HLA-A*0201 tetramer bound to the WT1126 peptide was analyzed by a flow cytometer, and the cytotoxic activity against various target cells was examined.
The target cells to be used were K562 cells, 0206K562 cells, JY cells, KH88OF8 cells, TF-1 cells and THP-1 cells, which are shown in Table 5. The features of these cells are shown in Table 5. A B-lymphoblastoid cell line (B-LCL) established by EB viral infection from the blood of an HLA-A*0201-positive donor was also used as a target cell.










TABLE 5





Target cell
HLA-A*0201
HLA-A*0206
WT1







K562
negative
negative
expressed


0206K562
negative
positive
expressed


JY
positive
negative
not expressed


KH88OF8
negative
negative
expressed


TF-1
positive
negative
expressed


THP-1
positive
negative
expressed









(3) Results

FIG. 7 shows the results of flow cytometric analysis of PBMCs from the HLA-A*0201-positive donor 1 which were stimulated with different modified WT1126 peptides and then stained with a PE (Phycoerythrin)-labeled HLA-A*0201 tetramer bound to the WT1126 peptide (Medical & Biological Laboratories, Co., Ltd.), and an APC-Cy7-labeled anti-CD8 antibody (APC-Cy7: Allophycocyanin-Cyanine-7). When the PBMCs are stained with the above-mentioned tetramer and anti-CD8 antibody, CTLs induced by stimulation with the modified peptide bind to the tetramer and the anti-CD8 antibody, and thereby, fluorescence emitted by the tetramer and fluorescence emitted by the anti-CD8 antibody can be detected, respectively. In FIGS. 7a to 7e, the vertical axis represents the intensity of fluorescence emitted by the HLA-A*0201 tetramer, and the horizontal axis represents the intensity of fluorescence emitted by the anti-CD8 antibody. Each box in FIGS. 7a to 7e shows the frequency (%) of induced, HLA-A*0201-restricted CTLs capable of recognizing the WT1126 peptide. FIG. 7a shows the analysis result of PBMCs which were not stimulated with any WT1126 modified peptide and stained with the above-mentioned tetramer and anti-CD8 antibody (background). FIG. 7b shows the analysis result of PBMCs which were stimulated with the WT1126P1F peptide and stained. FIG. 7c shows the analysis result of PBMCs which were stimulated with the WT1126P2L peptide and stained. FIG. 7d shows the analysis result of PBMCs which were stimulated with the WT1126P3M peptide and stained. FIG. 7e shows the analysis result of PBMCs which were stimulated with the WT1126P9V peptide and stained.
The frequency of the above-mentioned CTLs induced by stimulation of PBMCs with the WT1126P1F peptide was 0.14% (FIG. 7b). The frequency of the above-mentioned CTLs induced by stimulation of PBMCs with the WT1126P2L peptide was 0.37% (FIG. 7c). The CTLs induced separately with these peptides were HLA tetramer-positive, CD8-positive and capable of binding to the HLA-A*0201 tetramer bound to the WT1126 peptide. These results show that stimulation of PBMCs with the modified WT1126 peptide induced CTLs which can recognize the wild-type peptide (WT1126 peptide).
FIG. 8 shows the measurement results of the cytotoxic activity of CTLs induced by stimulation of PBMCs from the donor 1 with the WT1126P1F peptide. In FIG. 8, the vertical axis represents the cytotoxic activity, and the horizontal axis represents the ratio of CD8-positive T cells obtained by peptide stimulation (effector: E) relative to target cells (target: T) (E/T ratio). The closed diamond represents the group in which JY cells were used as a target cell, and the closed square represents the group in which JY cells pulsed with the WT1126P1F peptide were used as a target cell.
JY cells are HLA-A*0201-positive and WT1-negative. The CTLs showed a stronger cytotoxic activity against the WT1126P1F peptide-pulsed JY cells than against the non-WT1126P1F peptide-pulsed JY cells. This result shows that CTLs which are specific to the peptide used for the above-mentioned stimulation and restricted by HLA-A*0201 were induced.
FIG. 9 shows the measurement results of the cytotoxic activity of CTLs induced by stimulation of PBMCs from the donor 1 with the WT1126P2L peptide. In FIG. 9, the vertical axis represents the cytotoxic activity, and the horizontal axis represents the ratio of CD8-positive T cells obtained by peptide stimulation (effector: E) relative to target cells (target: T) (E/T ratio). In FIG. 9a, the closed diamond represents the group in which JY cells were used as a target cell, and the closed square represents the group in which JY cells pulsed with the WT1126P2L peptide were used as a target cell. In FIG. 9b, the closed diamond represents the group in which TF-1 cells were used as a target cell, the closed square represents the group in which THP-1 cells were used as a target cell, the closed triangle represents the group in which KH88OF8 cells were used as a target cell, and the cross represents the group in which B-LCL cells were used as a target cell.
The induced CTLs showed a stronger cytotoxic activity against the WT1126P2L peptide-pulsed JY cells than against the non-WT1126P2L peptide-pulsed JY cells (FIG. 9a). The induced CTLs showed a stronger cytotoxic activity against the TF-1 cells and THP-1 cells, both of which are HLA-A*0201-positive and WT1-positive, than against the KH88OF8 cells, which are HLA-A*0201-negative and WT1-positive, and the B-LCL cells, which are HLA-A*0201-positive and WT1-negative (FIG. 9b). As is clear from the results, the CTLs induced by stimulation with the WT1126P2L peptide are restricted by HLA-A*0201, and capable of destroying cancer cells endogenously expressing WT1.
FIG. 10 shows the results of flow cytometric analysis of PBMCs from the HLA-A*0201-positive donor 2 which were stimulated with the WT1126P2L peptide and then stained with the PE-labeled HLA-A*0201 tetramer bound to the WT1126 peptide, and the APC-Cy7-labeled anti-CD8 antibody. Namely, FIG. 10 shows the results of the flow cytometric analysis of induced CTLs which were stained with the HLA tetramer bound to the WT1126 peptide, and the anti-CD8 antibody. The vertical axis represents the intensity of fluorescence emitted by the HLA-A*0201 tetramer, and the horizontal axis represents the intensity of fluorescence emitted by the anti-CD8 antibody.
The cells in the upper right area of FIG. 10 are induced CTLs which are restricted by HLA-A*0201 and can recognize the WT1126 peptide. 5.43% of lymphocytes of the PBMCs stimulated with the WT1126P2L peptide were HLA tetramer-positive, CD8-positive CTLs which are capable of binding to the tetramer of HLA-A*0201 bound to the WT1126 peptide. This result shows that stimulation of PBMCs with the modified peptide induced CD8-positive CTLs which can recognize the wild-type peptide.
FIG. 11 shows the measurement results of the cytotoxic activity of the CTLs induced by stimulation of PBMCs from the donor 2 with the WT1126P2L peptide. In FIGS. 11a and 11b, the vertical axis represents the cytotoxic activity, and the horizontal axis represents the ratio of CD8-positive T cells obtained by peptide stimulation (effector: E) relative to target cells (target: T) (E/T ratio). In FIG. 11a, the closed diamond represents the group in which JY cells were used as a target cell, and the closed square represents the group in which cell. In FIG. 11b, the closed diamond represents the group in which JY cells were used as a target cell, and the closed square represents the group in which JY cells pulsed with the WT1126P2L peptide were used as a target cell.
The induced CTLs showed a stronger cytotoxic activity against the WT1126 peptide-pulsed JY cells than against the non-WT1126 peptide-pulsed JY cells (FIG. 11a). The induced CTLs also showed a stronger cytotoxic activity against the WT1126P2L peptide-pulsed JY cells than against the non-WT1126P2L peptide-pulsed JY cells (FIG. 11b). As is clear from the results, the CTLs induced by stimulation with the WT1126P2L peptide can recognize both of the WT1126P2L peptide and the wild-type WT1126 peptide.
Example 10
Comparison of HLA-A*0206-restricted CTLs Induced by Various Modified WT1126 Peptides
(1) Purpose
In view of the results of Example 7, the WT1126P1F peptide, the WT1126P2L peptide, the WT1126P3M peptide and the WT1126P9V peptide were selected as modified WT1126 peptides to be tested, and the following experiments were conducted to screen for modified WT1126 peptides capable of inducing CTLs having a high cytotoxic activity.
(2) Materials and Methods
From a healthy human donor showing expression of HLA-A*0206 molecules (HLA-A*0206-positive healthy blood donor), PBMCs were isolated, and CD14-positive cells were separated from the PBMCs by use of anti-human CD14 Magnetic Particles-DM. A culture medium was prepared by adding 800 IU/mL GM-CSF and 1000 IU/mL IL-4 to an X-VIVO15 medium supplemented with 1 v/v % human AB serum, and the CD14-positive cells were cultured in the culture medium for 1 day.
To the above culture, a maturation cytokine cocktail containing 10 ng/mL TNFα, 10 ng/mL IL-β, 1000 IU/mL IL-6 and 1 μg/mL PGE2 was added. After additional one day culture, autologous mature DCs were obtained.
The autologous mature DCs were pulsed with 10 μg/mL of a WT1126 modified peptide obtained in Example 13 (the WT1126P1F peptide, the WT1126P2L peptide, the WT1126P3M peptide or the WT1126P9V peptide), cultured for 4 hours, and irradiated with 35Gy of radiation. The thus-obtained cells were used as stimulator cells for CTL induction.
CD8-positive T cell-enriched PBMCs (2×106 cells/well) and the above-mentioned DCs (1×105 cells/well) were co-cultured in a 24-well plate. Ten days later, re-stimulation was given by addition of PBMCs which had been pulsed with the peptide and irradiated with 35Gy of radiation. Two days after re-stimulation, 10 IU/mL of IL-2 and 10 ng/mL of IL-7 were added. After the same re-stimulation was repeated another 4 times, CD8-positive T cells were enriched. The CD8-positive T cells were examined for the cytotoxic activity against various target cells.
The target cells to be used were B-LCLs established by EB viral infection from the blood of an HLA-A*0206-positive donor, K562 cells and 0206K562 cells.
(3) Results
FIG. 12 shows the measurement results of the cytotoxic activity of CTLs induced by stimulation of PBMCs from the HLA-A*0206-positive donor 3 with different peptides. FIG. 12a shows the cytotoxic activity of CTLs induced by stimulation with the WT1126P2L peptide. FIG. 12b shows the cytotoxic activity of CTLs induced by stimulation with the WT1126P3M peptide. FIG. 12c shows the cytotoxic activity of CTLs induced by stimulation with the WT1126P9V peptide. In FIGS. 12a to 12c, the vertical axis represents the cytotoxic activity, and the horizontal axis represents the ratio of CD8-positive T cells obtained by peptide stimulation (effector: E) relative to target cells (target: T) (E/T ratio). The closed diamond represents the group in which autologous B-LCL cells were used as a target cell, and the closed square represents the group in which autologous B-LCL cells pulsed with the same modified WT1126 peptide as used for the above-mentioned stimulation were used as a target cell.
The CTLs induced by stimulation with the WT1126P2L peptide showed a stronger cytotoxic activity against the WT1126P2L peptide-pulsed autologous B-LCL cells, which are HLA-A*0206-positive and WT1-negative, than against the non-WT1126P2L peptide-pulsed autologous B-LCL cells (FIG. 12a). The CTLs induced by stimulation with the WT1126P3M peptide showed a stronger cytotoxic activity against the WT1126P3M peptide-pulsed autologous B-LCL cells, which are HLA-A*0206-positive and WT1-negative, than against the non-WT1126P3M peptide-pulsed autologous B-LCL cells (FIG. 12b). The CTLs induced by stimulation with the WT1126P9V peptide showed a stronger cytotoxic activity against the WT1126P9V peptide-pulsed autologous B-LCL cells, which are HLA-A*0206-positive and WT1-negative, than against the non-WT1126P9V peptide-pulsed autologous B-LCL cells (FIG. 12c).
These results show that CTLs which are specific to the peptide used for the above-mentioned stimulation and restricted by HLA-A*0206 were induced.
FIG. 13 shows the measurement results of the cytotoxic activity of CTLs induced by stimulation of PBMCs from the HLA-A*0206-positive donor 3 with the WT1126P9V peptide. In FIG. 13, the vertical axis represents the cytotoxic activity, and the horizontal axis represents the ratio of CD8-positive T cells obtained by peptide stimulation (effector: E) relative to target cells (target: T) (E/T ratio). The closed diamond represents the group in which autologous B-LCL cells were used as a target cell, and the closed square represents the group in which WT1 gene-transfected autologous B-LCL cells were used as a target cell.
In FIG. 13, the CTLs induced by stimulation with the WT1126P9V peptide showed a stronger cytotoxic activity against autologous B-LCL cells made to be WT1-positive by transfection of the WT1 gene into B-LCL cells, which were originally HLA-A*0206-positive and WT1-negative, than against the non-WT1 gene-transfected autologous B-LCL cells (FIG. 12c). As is clear from the result, the CTLs induced by stimulation with the WT1126P9V peptide are restricted by HLA-A*0206, and show the cytotoxic activity by recognizing the wild-type WT1126 peptide presented endogenously.
FIG. 14 shows the measurement results of the cytotoxic activity of CTLs induced by stimulation of PBMCs from the HLA-A*0206-positive donor 4 with different peptides. FIG. 14a shows the cytotoxic activity of CTLs induced by stimulation with the WT1126P2L peptide. FIG. 14b shows the cytotoxic activity of CTLs induced by stimulation with the WT1126P3M peptide. FIG. 14c shows the cytotoxic activity of CTLs induced by stimulation with the WT1126P9V peptide. In FIGS. 14a to 14c, the vertical axis represents the cytotoxic activity, and the horizontal axis represents the ratio of CD8-positive T cells obtained by peptide stimulation (effector: E) relative to target cells (target: T) (E/T ratio). The closed diamond represents the group in which autologous B-LCL cells were used as a target cell, and the closed square represents the group in which autologous B-LCL cells pulsed with the same modified WT1126 peptide as used for the above-mentioned stimulation were used as a target cell.
The CTLs induced by stimulation with the WT1126P2L peptide showed a stronger cytotoxic activity against the WT1126P2L peptide-pulsed autologous B-LCL cells, which are HLA-A*0206-positive and WT1-negative, than against the non-WT1126P2L peptide-pulsed autologous B-LCL cells (FIG. 14a). The CTLs induced by stimulation with the WT1126P3M peptide showed a stronger cytotoxic activity against the WT1126P3M peptide-pulsed autologous B-LCL cells, which are HLA-A*0206-positive and WT1-negative, than against the non-WT1126P3M peptide-pulsed autologous B-LCL cells (FIG. 14b). The CTLs induced by stimulation with the WT1126P9V peptide showed a stronger cytotoxic activity against the WT1126P9V peptide-pulsed autologous B-LCL cells, which are HLA-A*0206-positive and WT1-negative, than against the non-WT1126P9V peptide-pulsed autologous B-LCL cells (FIG. 14c). These results show that CTLs which are specific to the peptide used for the above-mentioned stimulation and restricted by HLA-A*0206 were induced.
FIG. 15 shows the measurement results of the cytotoxic activity of CTLs induced by stimulation of PBMCs from the HLA-A*0206-positive donor 4 with different peptides. The target cells to be used were HLA-A*0206-negative, WT1-positive K562 cells, and K562 cells made to endogenously present WT1 antigen peptides by transfection of the HLA-A*0206 gene thereinto (0206K562 cells). FIG. 15a shows the cytotoxic activity of CTLs induced by stimulation with the WT1126P2L peptide. FIG. 15b shows the cytotoxic activity of CTLs induced by stimulation with the WT1126P3M peptide. FIG. 15c shows the cytotoxic activity of CTLs induced by stimulation with the WT1126P9V peptide. In FIGS. 15a to 15c, the vertical axis represents the cytotoxic activity, and the horizontal axis represents the ratio of CD8-positive T cells obtained by peptide stimulation (effector: E) relative to target cells (target: T) (E/T ratio). The closed diamond represents the group in which K562 cells were used as a target cell, and the closed square represents the group in which 0206K562 cells, i.e., K562 cells made to endogenously present WT1 antigen peptides by transfection of the HLA-A*0206 gene thereinto, were used as a target cell.
In FIGS. 15a to 15c, the CTLs induced by stimulation with the WT1126P2L peptide, the WT1126P3M peptide or the WT1126P9V peptide showed a stronger cytotoxic activity against the 0206K562 cells than against the K562 cells, in each case. As is clear from the results, the CTLs induced by stimulation with any of these modified peptides are restricted by HLA-A*0206, and show the cytotoxic activity by recognizing the wild-type WT1126 peptide presented endogenously.
Example 11
Comparison of HLA-A*0201-restricted CTLs Induced by Various Modified WT1187 Peptides
(1) Purpose
In view of the results of Example 8, the WT1187P1F peptide (SEQ ID NO: 11), the WT1187P2M peptide (SEQ ID NO: 16) and the WT1187P3M peptide (SEQ ID NO: 20) were selected as modified WT1187 peptides to be tested, and the following experiments were conducted to screen for modified WT1187 peptides capable of inducing CTLs having a high cytotoxic activity.
(2) Materials and Methods
From a healthy human donor showing expression of HLA-A*0201 molecules (HLA-A*0201-positive healthy blood donor), PBMCs were isolated, and CD14-positive cells were separated from the PBMCs using anti-human CD14 Magnetic Particles-DM. A culture medium was prepared by adding 800 IU/mL GM-CSF and 1000 IU/mL IL-4 to an X-VIVO15 medium supplemented with 1 v/v % human AB serum, and the CD14-positive cells were cultured in the culture medium for 1 day.
To the above culture, a maturation cytokine cocktail containing 10 ng/mL TNFα, 10 ng/mL IL-β, 1000 IU/mL IL-6 and 1 μg/mL PGE2 was added. After additional one day culture, autologous mature DCs were obtained.
The autologous mature DCs were pulsed with 10 μg/mL of a modified WT1187 peptide obtained in Example 13 (the WT1187P1F peptide, the WT1187P2M peptide or the WT1187P3M peptide), cultured for 4 hours, and irradiated with 35Gy of radiation. The thus-obtained cells were used as stimulator cells for CTL induction.
The PBMCs (2×106 cells/well) serving as responder cells and the above-mentioned DCs (2×105 cells/well) were co-cultured in a 24-well plate. One week later, re-stimulation was given by addition of T2 cells which had been pulsed with the peptide and irradiated with 75Gy of radiation. Three days after re-stimulation, 20 IU/mL of IL-2 was added. The same re-stimulation was repeated another 3 times by addition of the peptide-pulsed, irradiated T2 cells, and then CD8-positive cells in the responder cells were enriched. The CD8-positive T cells were examined for the cytotoxic activity against target cells, i.e., JY cells here.
(3) Results
FIG. 16 shows the measurement results of the cytotoxic activity of CTLs induced by stimulation of PBMCs from the HLA-A*0201-positive donor with the WT1187P1F peptide (FIG. 16a) or the WT1187P2M peptide (FIG. 16b). In FIGS. 16a and 16b, the vertical axis represents the cytotoxic activity, and the horizontal axis represents the ratio of CD8-positive T cells obtained by peptide stimulation (effector: E) relative to target cells (target: T) (E/T ratio). The closed diamond represents the group in which JY cells were used as a target cell, the closed triangle represents the group in which WT1187 peptide-pulsed JY cells were used as a target cell, and the closed square represents the group in which JY cells pulsed with the WT1187 modified peptide (WT1187P1F peptide) that was used for the above-mentioned stimulation were used as a target cell. JY cells are HLA-A*0201-positive and WT1-negative.
The CTLs induced by stimulation with the WT1187P1F peptide showed an equal cytotoxic activity against the WT1187 peptide-pulsed JY cells and the WT1187P1F peptide-pulsed JY cells, and the activity was stronger than that against the non-peptide-pulsed JY cells (FIG. 16a). The CTLs induced by stimulation with the WT1187P2M peptide showed an equal cytotoxic activity against the WT1187 peptide-pulsed JY cells and the WT1187P2M peptide-pulsed JY cells, and the activity was stronger than that against the non-peptide-pulsed JY cells (FIG. 16b). As is clear from the results, the CTLs induced by stimulation with the modified peptide can recognize both of the modified peptide and the wild-type WT1187 peptide.
Example 12
Comparison Of HLA-A*0206-restricted CTLs Induced by Various Modified WT1187 Peptides)
(1) Purpose
In view of the results of Example 7, the WT1187P1F peptide, the WT1187P2M peptide and the WT1187P3M peptide were selected as modified WT1187 peptides to be tested, and the following experiments were conducted to screen for modified WT1187 peptides capable of inducing CTLs having a high cytotoxic activity.
(2) Materials and Methods
From a healthy human donor showing expression of HLA-A*0206 molecules (HLA-A*0206-positive healthy blood donor), PBMCs were isolated, and CD14-positive cells were separated from the PBMCs using anti-human CD14 Magnetic Particles-DM. A culture medium was prepared by adding 800 IU/mL GM-CSF and 1000 IU/mL IL-4 to an X-VIVO15 medium supplemented with 1 v/v % human AB serum, and the CD14-positive cells were cultured in the culture medium for 1 day.
To the above culture, a maturation cytokine cocktail containing 10 ng/mL TNFα, 10 ng/mL IL-β, 1000 IU/mL IL-6 and 1 μg/mL PGE2 was added. After additional one day culture, autologous mature DCs were obtained.
The autologous mature DCs were pulsed with a modified WT1187 peptide obtained in Example 13 (the WT1187P1F peptide, the WT1187P2M peptide or the WT1187P3M peptide), cultured for 4 hours, and irradiated with 35Gy of radiation. The thus-obtained cells were used as stimulator cells for CTL induction.
CD8-positive T cell-enriched PBMCs (2×106 cells/well) and the above-mentioned DCs (1×105 cells/well) were co-cultured in a 24-well plate. Ten days later, re-stimulation was given by addition of PBMCs which had been pulsed with the peptide and irradiated with 35Gy of radiation. Two days after re-stimulation, 10 IU/mL of IL-2 and 10 ng/mL of IL-7 were added. After the same re-stimulation was repeated another 4 times, CD8-positive T cells were enriched. The CD8-positive T cells were examined for the cytotoxic activity against various target cells.
The target cells to be used were B-LCLs established by EB viral infection from the blood of an HLA-A*0206-positive donor, K562 cells and 0206K562 cells.
(3) Results
FIG. 17 shows the measurement results of the cytotoxic activity of CTLs induced by stimulation of PBMCs from the HLA-A*0206-positive donor with the WT1187P1F peptide. In FIG. 17, the vertical axis represents the cytotoxic activity, and the horizontal axis represents the ratio of CD8-positive T cells obtained by peptide stimulation (effector: E) relative to target cells (target: T) (E/T ratio). In FIG. 17a, the closed diamond represents the group in which B-LCL cells were used as a target cell, the closed square represents the group in which WT1187 peptide-pulsed B-LCL cells were used as a target cell, and the closed triangle represents the group in which WT1187P1F peptide-pulsed B-LCL cells were used as a target cell. In FIG. 17b, the closed diamond represents the group in which K562 cells were used as a target cell, and the closed square represents the group in which 0206K562 cells, i.e., K562 cells made to endogenously present WT1 antigen peptides by transfection of the HLA-A*0206 gene thereinto, were used as a target cell.
The CTLs induced by stimulation with the WT1187P1F peptide showed an equal cytotoxic activity against the WT1187 peptide-pulsed B-LCL cells and the WT1187P1F peptide-pulsed B-LCL cells, and the activity was stronger than that against the non-peptide-pulsed B-LCL cells (FIG. 17a). When HLA-A*0206-negative, WT1-positive K562 cells, and K562 cells made to endogenously present WT1 antigen peptides by transfection of the HLA-A*0206 gene thereinto (0206K562 cells) were used as a target cell, the CTLs induced by stimulation with the WT1187P1F peptide showed a stronger cytotoxic activity against the 0206K562 cells than against the K562 cells (FIG. 17b). As is clear from the results, the CTLs induced by stimulation with the WT1187P1F peptide can recognize both of the WT1187P1F peptide and the wild-type WT1187 peptide. Similarly, it was found that the CTLs are restricted by HLA-A*0206, and show the cytotoxic activity by recognizing the wild-type WT1187 peptide presented endogenously.
FIG. 18 shows the measurement results of the cytotoxic activity of CTLs induced by stimulation of PBMCs from the HLA-A*0206-positive donor with the WT1187P2M peptide. In FIG. 18, the vertical axis represents the cytotoxic activity, and the horizontal axis represents the ratio of CD8-positive T cells obtained by peptide stimulation (effector: E) relative to target cells (target: T) (E/T ratio). In FIG. 18a, the closed diamond represents the group in which B-LCL cells were used as a target cell, the closed square represents the group in which WT1187 peptide-pulsed B-LCL cells were used as a target cell, and the closed triangle represents the group in which WT1187P2M peptide-pulsed B-LCL cells were used as a target cell. In FIG. 18b, the closed diamond represents the group in which K562 cells were used as a target cell, and the closed square represents the group in which 0206K562 cells, i.e., K562 cells made to endogenously present WT1 antigen peptides by transfection of the HLA-A*0206 gene thereinto, were used as a target cell.
The CTLs induced by stimulation with the WT1187P2M peptide showed an equal cytotoxic activity against the WT1187 peptide-pulsed B-LCL cells and the WT1187P2M peptide-pulsed B-LCL cells, and the activity was stronger than that against the non-peptide-pulsed B-LCL cells (FIG. 18a). When HLA-A*0206-negative, WT1-positive K562 cells, and K562 cells made to endogenously present WT1 antigen peptides by transfection of the HLA-A*0206 gene thereinto (0206K562 cells) were used as a target cell, the CTLs induced by stimulation with the WT1187P2M peptide showed a stronger cytotoxic activity against the 0206K562 cells than against the K562 cells (FIG. 18b). As is clear from the results, the CTLs induced by stimulation with the WT1187P2M peptide can recognize both of the WT1187P2M peptide and the wild-type WT1187 peptide. Similarly, it was found that the CTLs are restricted by HLA-A*0206, and show the cytotoxic activity by recognizing the wild-type WT1187 peptide presented endogenously.
As is clear from the results of Examples 9 to 12, the CTLs induced by stimulation with the modified WT1126 peptide, i.e., the WT1126P1F peptide, the WT1126P2L peptide, the WT1126P3M peptide or the WT1126P9V peptide, are restricted by HLA-A*0206, and show the cytotoxic activity by recognizing the wild-type WT1126 peptide presented endogenously. Inter alia, the WT1126P9V peptide, the WT1126P2L peptide and the WT1126P3M peptide were highly effective.
As is clear from the above results, the CTLs induced by stimulation with the modified WT1187 peptide, i.e., the WT1187P1F peptide, the WT1187P2M peptide or the WT1187P3M peptide, are restricted by HLA-A*0206, and show the cytotoxic activity by recognizing the wild-type WT1187 peptide presented endogenously. Inter alia, the WT1187P2M peptide and the WT1187P1F peptide were highly effective.
Therefore, it was shown that these modified peptides are effective in treatment and prevention of cancers accompanied by increased expression of the WT1 gene in HLA-A*0206-positive persons.
As is clear from the above results, the CTLs induced by stimulation with the modified WT1126 peptide, i.e., the WT1126P1F peptide, the WT1126P2L peptide, the WT1126P3M peptide or the WT1126P9V peptide, are restricted by HLA-A*0201, and show the cytotoxic activity by recognizing the wild-type WT1126 peptide presented endogenously. Inter alia, the WT1126P1F peptide and the WT1126P2L peptide were highly effective.
As is clear from the above results, the CTLs induced by stimulation with the modified WT1187 peptide, i.e., the WT1187P1F peptide, the WT1187P2M peptide or the WT1187P3M peptide, are restricted by HLA-A*0201, and show the cytotoxic activity by recognizing the wild-type WT1187 peptide presented endogenously. Inter alia, the WT1187P2M peptide and the WT1187P1F peptide were highly effective. Therefore, it was shown that these modified peptides are effective in treatment and prevention of cancers accompanied by increased expression of the WT1 gene in HLA-A*0201-positive persons.
Example 13
Synthesis of WT1187P2V Peptide (SVGEQQYSV; SEQ ID NO: 13; H-Ser-Val-Gly-Glu-Gln-Gln-Tyr-Ser-Val-OH) (SEQ ID NO: 13))
1. Synthesis of Protected Peptide Resin (H-Ser(tBu)-Val-Gly-Glu(OtBu)-Gln(Trt)-Gln(Trt)-Tyr(tBu)-Ser(tBu)-Val-Alko-Resin (SEQ ID NO: 79))
0.4 g of an Fmoc-Val-Alko-resin (Alko is p-alkoxybenzyl alcohol) (manufactured by WATANABE CHEMICAL INDUSTRIES, LTD; 0.80 mmol/g) was placed into the reaction vessel of the ACT496 solid-phase synthesizer manufactured by Advanced ChemTech, washed with DMF(N,N′-dimethylformamide) (Step 1), treated with a 25% solution of piperidine in DMF (5 minutes×1 time, and 30 minutes×1 time) to remove the Fmoc group (Step 2), and was again washed with DMF (Step 3) to give an H-Val-Alko-resin. To this reaction vessel, 0.7 mL of NMP (N-methylpyrrolidinone) and a solution of 121 mg (0.96 mmol) of DIPCI (N,N′-diisopropylcarbodiimide) in 0.9 mL of NMP, and then a solution of 368 mg (0.96 mmol) of Fmoc-Ser(tBu)-OH and 147 mg (0.96 mmol) of HOBT (1-hydroxybenzotriazol) monohydrate in 1.8 mL of NMP were added. Coupling reaction was performed at room temperature for 60 minutes (Step 4). Additional coupling reaction was performed using the same amounts of Fmoc-Ser(tBu)-OH, HOBT monohydrate and DIPCI as above (Step 5). The resulting resin was washed with DMF (Step 6), deprotected (Step 7) and washed again (Step 8) to give an H-Ser(tBu)-Val-Alko-resin. Then, couplings were successively performed by repeating Steps 4 to 8 using Fmoc-Tyr(tBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Gly-OH, Fmoc-Val-OH and Fmoc-Ser(tBu)-OH. The resulting peptide resin was collected from the reaction vessel, washed with ether and then dried in vacuo to give 980 mg of an H-Ser(tBu)-Val-Gly-Glu(OtBu)-Gln(Trt)-Gln(Trt)-Tyr(tBu)-Ser (tBu)-Val-Alko-resin (SEQ ID NO: 79). The outline of the synthesis process mentioned above is shown in Table 6.
<Synthesis Process>










TABLE 6







Repetition
Duration


Step
Reagent
(time)
(min)


















1) washing
DMF 3 mL
5
0.3


2) deprotection
25% piperidine/DMF 3 mL
1
5




1
30


3) washing
DMF 3 mL
5
0.3


4) coupling
Each Fmoc-amino acid (3 Eq),
1
60



HOBT(3 Eq),



DIPCI(3 Eq)/NMP 3.4 mL


5) coupling
Each Fmoc-amino acid (3 Eq),
1
60



HOBT(3 Eq),



DIPCI(3 Eq)/NMP 3.4 mL


6) washing
DMF 3 mL
5
0.3


7) deprotection
25% piperidine/DMF 3 mL
1
5




1
30


8) washing
DMF 3 mL
5
0.3









2. Deprotection of Protected Peptide Resin

To 980 mg of the H-Ser(tBu)-Val-Gly-Glu(OtBu)-Gln(Trt)-Gln(Trt)-Tyr(tBu)-Ser(tBu)-Val-Alko-resin (SEQ ID NO: 79) was added 5 mL of a mixed solution of trifluoroacetic acid/water/triisopropylsilane (95/2.5/2.5 (volume ratio)). The mixture was stirred at room temperature for 2.5 hours. The resin was filtered off and the resulting filtrate was added to ice-cold diethyl ether. The resulting precipitate was collected with a glass filter. The residue was washed with diethyl ether and dried in vacuo to give 268 mg of a crude peptide.
3. Purification of Crude Peptide
268 mg of the obtained crude peptide was dissolved in a 20% aqueous acetic acid solution, and purified by reverse phase liquid chromatography.

    Pump: Shimadzu LC-8A
    Column: YMC-Pack Pro C18 AS12S11-2530WT 3 cmΦ×25 cm
    Eluent 1: H2O/0.1% TFA
    Eluent 2 (the second eluent): CH3CN/0.1% TFA
    low rate: 10 mL/min
    Detection: UV220 nm


After equilibrated with the second eluent at a concentration of 1%, the column was loaded with the crude peptide solution. After that, the concentration of the second eluent was allowed to increase to 8% over 30 minutes and subsequently to 14% over 120 minutes. Fractions containing the objective compound were collected, acetonitrile was evaporated off in vacuo and then the residue was freeze-dried. Thus, 105 mg of the objective WT1187P2V peptide (SVGEQQYSV; SEQ ID NO: 13; H-Ser-Val-Gly-Glu-Gln-Gln-Tyr-Ser-Val-OH (SEQ ID NO: 13)) was obtained.
The conditions used for HPLC analysis and mass spectrometry of the purified peptide are as follows.

    HPLC analysis (Shimadzu LC-10Avp)
    Column: YMC-Pack Pro C18 AS-302 4.6 mmΦ×150 mm
    Eluent 1: H2O/0.1% TFA
    Eluent 2: CH3CN/0.1% TFA
    Gradient: The concentration of eluent 2 was allowed to increase from 10% to 40% over 30 minutes.
    Flow rate: 1 mL/min
    Detection: UV220 nm
    Purity: 97.4%, Retention time: 11.22 minutes
    Amino acid analysis (Hitachi L-8500 Amino acid analyzer)
    Hydrolysis: 1% phenol/6N aqueous hydrochloric acid solution, 110° C., 24 hour
    Analysis method: Ninhydrin method
    Ser:1.78(2) Glx:3.26(3)*Gly:(1) Val:2.04(2) Tyr:1.06(1) *) Gly=standard amino acid, the number in parentheses is a theoretical value.

Mass Spectrometry (Applied Biosystems API 150EX Mass Spectrometer)

    m/z=996.9 [M+1]+ (theoretical value=996.5)

Amino Acid Sequence Analysis (Applied Biosystems 491 Protein Sequencer)



The amino acid sequence was checked sequentially from the N terminal Ser to the C terminal Val.
The peptides shown in Tables 7 and 8 were synthesized in the same manner as above.














TABLE 7









Amount








Amount
of








of crude
purified

HPLC




Amino
SEQ
peptide
peptide
HPLC
retention
Mass



acid
ID
obtained
obtained
purity
time
spectrometry


Peptide
sequence
NO
(mg)
(mg)
(%)
(min)
(m/z)






















WT1187P2Q
SQGEQQYSV
14
251
88
98.2
9.21
1026.0


 


WT1187P2I
SIGEQQYSV
15
244
91
97.6
12.98
1010.7


 


WT1187P2M
SMGEQQYSV
16
260
22
96.9
11.91
1029.1


 


WT1187P3L
SLLEQQYSV
17
238
176
96.8
18.24
1066.9


 


WT1187P3A
SLAEQQYSV
18
268
172
99.1
13.67
1024.8


 


WT1187P3V
SLVEQQYSV
19
267
182
98.7
15.26
1052.8


 


WT1187P3M
SLMEQQYSV
20
280
63
94.8
16.12
1084.8


 


WT1187P3P
SLPEQQYSV
21
227
132
98.1
14.02
1050.8


 


WT1187P3W
SLWEQQYSV
22
248
40
99.4
20.00
1139.5






(of









crude









peptide









100 mg)





 


WT1187P3F
SLFEQQYSV
23
224
110
98.3
19.44
1100.8


 


WT1187P3Y
SLYEQQYSV
24
236
114
98.5
15.76
1116.7


 


WT1187P3S
SLSEQQYSV
25
261
130
99.1
13.33
1040.8


 


WT1187P3I
SLIEQQYSV
26
270
162
97.3
17.51
1066.9























TABLE 8









Amount








Amount
of








of crude
purified

HPLC




Amino
SEQ
peptide
peptide
HPLC
retention
Mass



acid
ID
obtained
obtained
purity
time
spectrometry


Peptide
sequence
NO
(mg)
(mg)
(%)
(min)
(m/z)






















WT1126P2V
RVFPNAPYL
36
253
130
96.5
20.65
1077.1


 


WT1126P2Q
RQFPNAPYL
37
274
87
98.7
18.43
1106.1


 


WT1126P2A
RAFPNAPYL
38
240
63
99.0
19.03
1049.1


 


WT1126P9A
RMFPNAPYA
50
262
139
94.3
16.59
1066.8


 


WT1126P9M
RMFPNAPYM
52
295
167
95.7
19.75
1126.8









The peptides shown in Tables 9 to 12 were similarly synthesized, but the conditions used for HPLC analysis and mass spectrometry of the purified peptides are as follows. HPLC analysis (Agilent HP1100 or Thermo Fisher Scientific Surveyor)

    Column: Thermo Fischer Scientific BioBasic-18 3 mmΦ×250 mm
    Eluent 1: H2O/0.1% TFA
    Eluent 2: CH3CN/0.1% TFA

Gradient: The concentration of eluent 2 was allowed to change to the concentration shown in the table over 20 minutes.

    Flow rate: 0.4 mL/min
    Detection: 215 nm

Mass Spectrometry

Thermo Bioanalysis Dynamo Mass Spectrometer (MALDI-TOF)
















TABLE 9









HPLC








reten-

Mass



Amino
SEQ
HPLC
tion
HPLC
spectrom-



acid
ID
purity
time
gradi-
etry


Peptide
sequence
NO
(%)
(min)
ent
(m/z)





















WT1187P1G
GLGEQQYSV
4
100
12.72
 5-60%
980.7


 


WT1187P1A
ALGEQQYSV
5
98.9
12.00
10-50%
993.0


 


WT1187P1V
VLGEQQYSV
6
98.9
14.98
 5-50%
1022.3


 


WT1187P1L
LLGEQQYSV
7
97.6
12.75
10-65%
1037.6


 


WT1187P1I
ILGEQQYSV
8
98.8
13.46
 5-60%
1037.2


 


WT1187P1M
MLGEQQYSV
9
95.5
14.15
 5-60%
1054.0


 


WT1187P1W
WLGEQQYSV
10
98.9
15.60
 5-60%
1109.9


 


WT1187P1F
FLGEQQYSV
11
95.8
13.14
10-65%
1070.8


 


WT1187P9L
SLGEQQYSL
53
95.4
12.49
10-55%
1024.6






















TABLE 10









HPLC








reten-

Mass



Amino
SEQ
HPLC
tion
HPLC
spectrom-



acid
ID
purity
time
gradi-
etry


Peptide
sequence
NO
(%)
(min)
ent
(m/z)





















WT1126P1G
GMFPNAPYL
27
99.6
15.39
10-70%
1009.5


 


WT1126P1A
AMFPNAPYL
28
98.8
13.79
10-85%
1023.3


 


WT1126P1V
VMFPNAPYL
29
98.4
14.00
10-85%
1052.2


 


WT1126P1L
LMFPNAPYL
30
98.9
14.91
10-80%
1066.3


 


WT1126P1I
IMFPNAPYL
31
99.2
13.93
10-80%
1065.4


 


WT1126P1M
MMFPNAPYL
32
100
13.77
10-80%
1083.5


 


WT1126P1W
WMFPNAPYL
33
97.5
15.64
10-80%
1139.3


 


WT1126P1F
FMFPNAPYL
34
98.8
14.78
10-85%
1099.7


 


WT1126P2L
RLFPNAPYL
39
98.6
13.11
10-80%
1090.9


 


WT1126P2I
RIFPNAPYL
40
100
13.74
10-70%
1090.1


 


WT1126P3I
RMIPNAPYL
41
97.4
14.17
10-70%
1076.7


 


WT1126P3L
RMLPNAPYL
42
100
13.88
10-65%
1076.4


 


WT1126P3G
RMGPNAPYL
43
96.7
12.63
10-65%
1020.9


 


WT1126P3A
RMAPNAPYL
44
95.2
13.95
10-60%
1034.9


 


WT1126P3V
RMVPNAPYL
45
92.9
14.67
10-60%
1062.7


 


WT1126P3M
RMMPNAPYL
46
91.8
14.87
10-60%
1094.8


 


WT1126P3P
RMPPNAPYL
47
95.8
13.56
10-65%
1058.8


 


WT1126P3W
RMWPNAPYL
48
99.6
15.21
10-70%
1149.7


 


WT1126P9V
RMFPNAPYV
49
99.2
13.86
10-60%
1096.5


 


WT1126P9I
RMFPNAPYI
51
99.4
14.00
10-65%
1110.7






















TABLE 11









HPLC








reten-

Mass



Amino
SEQ
HPLC
tion

spec-



acid
ID
purity
time
HPLC
trometry


Peptide
sequence
NO
(%)
(min)
gradient
(m/z)





















WT1187P1D
DLGEQQYSV
54
96.6
13.43
 5-60%
1038.2


 


WT1187P1E
ELGEQQYSV
55
96.4
11.83
10-60%
1052.2


 


WT1187P1H
HLGEQQYSV
56
98.0
15.79
 5-40%
1060.2


 


WT1187P1K
KLGEQQYSV
57
99.0
13.77
 5-45%
1052.2


 


WT1187P1N
NLGEQQYSV
58
95.8
14.34
 5-50%
1037.0


 


WT1187P1P
PLGEQQYSV
59
95.9
14.68
 5-50%
1020.0


 


WT1187P1Q
QLGEQQYSV
60
96.8
13.28
 5-60%
1051.3


 


WT1187P1R
RLGEQQYSV
61
100
13.27
 5-60%
1079.6


 


WT1187P1T
TLGEQQYSV
62
96.7
14.40
 5-50%
1025.0






















TABLE 12









HPLC








reten-

Mass



Amino
SEQ
HPLC
tion

spec-



acid
ID
purity
time
HPLC
trometry


Peptide
sequence
NO
(%)
(min)
gradient
(m/z)





















WT1126P1D
DMFPNAPYL
63
99.2
14.72
10-75%
1067.3


 


WT1126P1E
EMFPNAPYL
64
99.1
15.20
 5-70%
1082.1


 


WT1126P1H
HMFPNAPYL
65
96.7
14.52
10-70%
1089.9


 


WT1126P1K
KMFPNAPYL
66
95.9
13.96
10-75%
1080.0


 


WT1126P1N
NMFPNAPYL
67
99.8
14.69
10-75%
1066.3


 


WT1126P1P
PMFPNAPYL
68
96.9
14.26
10-80%
1049.3


 


WT1126P1Q
QMFPNAPYL
69
95.1
14.94
10-70%
1080.3


 


WT1126P1S
SMFPNAPYL
70
99.8
14.28
10-80%
1040.8


 


WT1126P1T
TMFPNAPYL
71
98.8
13.72
10-85%
1053.5


 


WT1126
RIFPNAPYI
72
97.0
14.23
10-65%
1089.5


P2I&P9I








 


WT1126
RIFPNAPYV
73
100
12.23
10-80%
1077.0


P2I&P9V








 


WT1126
RLFPNAPYI
74
97.7
13.43
10-75%
1090.8


P2L&P9I








 


WT1126
RLFPNAPYV
75
97.0
12.83
10-75%
1076.9


P2L&P9V









Example 14
Evaluation of Modified Peptides on the Activity of Inducing Specific Immune Cells Using HLA-A*0201-expressing Transgenic Mice, and Confirmation of Cross Reactivity of Induced Specific Immune Cells to the Wild-type Peptide
<Methods>
(1) Modified Peptide Candidates
As for the WT1187 peptide, the WT1126 peptide, and modified peptides thereof (peptides comprising substitution of one or two amino acid residues at position 1, 2, 3 and/or 9 from the N terminus of the WT1187 peptide or the WT1126 peptide), the affinity against HLA-A*0201 molecules was analyzed using the known method in the technical field, i.e., the method mediated by the following four computer databases: BIMAS (http://www.mpiib-berlin.mpg.de/MAPPP/binding.html), SYFPEITHI http://www.mpiib-berlin.mpg.de/MAPPP/binding.html), RANLPEP (http://immunax.dfci.harvard.edu/Tools/rankpep.html), and NetMHC3.0 (http://www.cbs.dtu.dk/services/NetMHC/). The analysis results of the WT1187 peptide and its modified peptides are shown in Tables 13 to 16 and 21. The analysis results of the WT1126 peptide and its modified peptides are shown in Tables 17 to 20 and 22 to 23. The predicted affinity is shown in scores.













TABLE 13








Amino
SEQ














Pep-
acid
ID
HLA-A*0201 binding score













tide
sequence
NO
BIMAS
SYFPEITHI
RANKPEP
NetMHC3.0
















WT1187
SLGEQQYSV
2
285
27
96/
0.721







64.43%



 


WT1187
ALGEQQYSV
5
285
27
95/
0.720


P1A




63.76%



 


WT1187
FLGEQQYSV
11
1312
26
82/
0.862


P1F




55.03%



 


WT1187
GLGEQQYSV
4
285
26
86/
0.672


P1G




57.72%



 


WT1187
ILGEQQYSV
8
485
27
90/
0.698


P1I




60.40%



 


WT1187
LLGEQQYSV
7
485
27
89/
0.719


P1L




59.73%



 


WT1187
MLGEQQYSV
9
485
25
92/
0.770


P1M




61.74%



 


WT1187
VLGEQQYSV
6
485
26
92/
0.705


P1V




61.74%



 


WT1187
WLGEQQYSV
10
1312
25
71/
0.693


P1W




47.65%



















TABLE 14








Amino
SEQ



Pep-
acid
ID
HLA-A*0201 binding score













tide
sequence
NO
BIMAS
SYFPEITHI
RANKPEP
NetMHC3.0
















WT1187
SLGEQQYSV
2
285
27
96/
0.721







64.43%



 


WT1187
SIGEQQYSV
15
39
25
85/
0.556


P2I




57.05%



 


WT1187
SMGEQQYSV
16
206
25
84/
0.740


P2M




56.38%



 


WT1187
SQGEQQYSV
14
29
17
52/
0.455


P2Q




34.90%



 


WT1187
SVGEQQYSV
13
25
21
78/
0.461


P2V




52.35%



















TABLE 15








Amino
SEQ




acid
ID
HLA-A*0201 binding score













Peptide
sequence
NO
BIMAS
SYFPEITHI
RANKPEP
NetMHC3.0
















WT1187
SLGEQQYSV
2
285
27
 96/64.43%
0.721


 


WT1187P3A
SLAEQQYSV
18
285
29
110/73.83%
0.811


 


WT1187P3F
SLFEQQYSV
23
1055
28
114/76.51%
0.852


 


WT1187P3I
SLIEQQYSV
26
285
29
115/77.18%
0.817


 


WT1187P3L
SLLEQQYSV
17
1055
29
116/77.85%
0.839


 


WT1187P3M
SLMEQQYSV
20
1055
28
114/76.51%
0.876


 


WT1187P3P
SLPEQQYSV
21
285
27
 95/63.76%
0.748


 


WT1187P3S
SLSEQQYSV
25
285
27
110/73.83%
0.793


 


WT1187P3V
SLVEQQYSV
19
285
27
113/75.84%
0.766


 


WT1187P3W
SLWEQQYSV
22
2367
28
 98/65.77%
0.863


 


WT1187P3Y
SLYEQQYSV
24
913
28
111/74.50%
0.854



















TABLE 16








Amino
SEQ



Pep-
acid
ID
HLA-A*0201 binding score













tide
sequence
NO
BIMAS
SYFPEITHI
RANKPEP
NetMHC3.0
















WT1187
SLGEQQYSV
2
285
27
96/
0.721







64.43%



 


WT1187
SLGEQQYSL
53
88
27
89/
0.640


P9L




59.73%



















TABLE 17








Amino
SEQ



Pep-
acid
ID
HLA-A*0201 binding score













tide
sequence
NO
BIMAS
SYFPEITHI
RANKPEP
NetMHC3.0
















WT1126
RMFPNAPYL
3
314
22
70/
0.802







46.98%



 


WT1126
AMFPNAPYL
28
314
24
77/
0.808


P1A




51.68%



 


WT1126
FMFPNAPYL
34
1444
23
64/
0.909


P1F




42.95%



 


WT1126
GMFPNAPYL
27
314
23
68/
0.795


P1G




45.64%



 


WT1126
IMFPNAPYL
31
534
24
72/
0.802


P1I




48.32%



 


WT1126
LMFPNAPYL
30
534
24
71/
0.819


P1L




47.65%



 


WT1126
MMFPNAPYL
32
534
22
74/
0.852


P1M




49.66%



 


WT1126
VMFPNAPYL
29
534
23
74/
0.804


P1V




49.66%



 


WT1126
WMFPNAPYL
33
1444
22
53/
0.799


P1W




35.57%



















TABLE 18








Amino
SEQ



Pep-
acid
ID
HLA-A*0201 binding score













tide
sequence
NO
BIMAS
SYFPEITHI
RANKPEP
NetMHC3.0
















WT1126
RMFPNAPYL
3
314
22
70/
0.802







46.98%



 


WT1126
RAFPNAPYL
38
6
18
47/
0.376


P2A




31.54%



 


WT1126
RIFPNAPYL
40
60
22
71/
0.640


P2I




47.65%



 


WT1126
RLFPNAPYL
39
435
24
82/
0.784


P2L




55.03%



 


WT1126
RQFPNAPYL
37
44
14
38/
0.485


P2Q




25.50%



 


WT1126
RVFPNAPYL
36
38
18
64/
0.552


P2V




42.95%



















TABLE 19








Amino
SEQ



Pep-
acid
ID
HLA-A*0201 binding score













tide
sequence
NO
BIMAS
SYFPEITHI
RANKPEP
NetMHC3.0
















WT1126
RMFPNAPYL
3
314
22
70/
0.802







46.98%



 


WT1126
RMAPNAPYL
44
85
23
66/
0.734


P3A




44.30%



 


WT1126
RMGPNAPYL
43
85
21
52/
0.602


P3G




34.90%



 


WT1126
RMIPNAPYL
41
85
23
71/
0.741


P3I




47.65%



 


WT1126
RMLPNAPYL
42
314
23
72/
0.781


P3L




48.32%



 


WT1126
RMMPNAPYL
46
314
22
70/
0.834


P3M




46.98%



 


WT1126
RMPPNAPYL
47
85
21
51/
0.613


P3P




34.23%



 


WT1126
RMVPNAPYL
45
85
21
69/
0.680


P3V




46.31%



 


WT1126
RMWPNAPYL
48
2293
22
61/
0.867


P3W




40.94%



















TABLE 20








Amino
SEQ



Pep-
acid
ID
HLA-A*0201 binding score













tide
sequence
NO
BIMAS
SYFPEITHI
RANKPEP
NetMHC3.0
















WT1126
RMFPNAPYL
3
314
22
70/
0.802







46.98%



 


WT1126
RMFPNAPYA
50
73
16
53/
0.731


P9A




35.57%



 


WT1126
RMFPNAPYI
51
153
20
74/
0.790


P9I




49.66%



 


WT1126
RMFPNAPYM
52
73
16
65/
0.686


P9M




43.62%



 


WT1126
RMFPNAPYV
49
1022
22
77/
0.838


P9V




51.68%



















TABLE 21








Amino
SEQ



Pep-
acid
ID
HLA-A*0201 binding score













tide
sequence
NO
BIMAS
SYFPEITHI
RANKPEP
NetMHC3.0
















WT1187
SLGEQQYSV
2
285
27
96/
0.721







64.43%



 


WT1187
DLGEQQYSV
54
21
24
81/
0.252


P1D




54.36%



 


WT1187
ELGEQQYSV
55
21
22
85/
0.366


P1E




57.05%



 


WT1187
HLGEQQYSV
56
10
25
83/
0.610


P1H




55.70%



 


WT1187
KLGEQQYSV
57
998
26
89/
0.745


P1K




59.73%



 


WT1187
NLGEQQYSV
58
285
25
90/
0.613


P1N




60.40%



 


WT1187
PLGEQQYSV
59
6
22
78/
0.287


P1P




52.35%



 


WT1187
QLGEQQYSV
60
285
25
87/
0.630


P1Q




58.39%



 


WT1187
RLGEQQYSV
61
285
25
88/
0.690


P1R




59.06%



 


WT1187
TLGEQQYSV
62
285
25
93/
0.669


P1T




62.42%



















TABLE 22








Amino
SEQ



Pep-
acid
ID
HLA-A*0201 binding score













tide
sequence
NO
BIMAS
SYFPEITHI
RANKPEP
NetMHC3.0
















WT1126
RMFPNAPYL
3
314
22
70/
0.802







46.98%



 


WT1126
DMFPNAPYL
63
24
21
63/
0.353


P1D




42.28%



 


WT1126
EMFPNAPYL
64
24
19
67/
0.501


P1E




44.97%



 


WT1126
HMFPNAPYL
65
11
22
65/
0.747


P1H




43.62%



 


WT1126
KMFPNAPYL
66
1099
23
71/
0.841


P1K




47.65%



 


WT1126
NMFPNAPYL
67
314
22
72/
0.734


P1N




48.32%



 


WT1126
PMFPNAPYL
68
7
19
60/
0.802


P1P




40.27%



 


WT1126
QMFPNAPYL
69
314
22
69/
0.757


P1Q




46.31%



 


WT1126
SMFPNAPYL
70
314
24
78/
0.819


P1S




52.35%



 


WT1126
TMFPNAPYL
71
314
22
75/
0.779


P1T




50.34%



















TABLE 23








Amino
SEQ



Pep-
acid
ID
HLA-A*0201 binding score













tide
sequence
NO
BIMAS
SYFPEITHI
RANKPEP
NetMHC3.0
















WT1126
RMFPNAPYL
3
314
22
70/
0.802







46.98%



 


WT1126
RIFPNAPYI
72
29
20
75/
0.617


P2I&




50.34%



P9I








 


WT1126
RIFPNAPYV
73
195
22
78/
0.722


P2I&




52.35%



P9V








 


WT1126
RLFPNAPYI
74
212
22
86/
0.780


P2L&




57.72%



P9I








 


WT1126
RLFPNAPYV
75
1415
24
89/
0.818


P2L&




59.73%



P9V









A modified peptide which was predicted to have an equal or higher affinity compared with the wild-type peptide (the WT1187 peptide or the WT1126 peptide) in at least one of the databases was selected as a sample to be tested in the following (2) to (4), in addition to wild-type peptides.
(2) Preparation and Administration of Peptide Preparations
A peptide synthesized and freeze-dried in Example 13 was prepared at the concentration of 40 mg/mL in DMSO (manufactured by Nacalai Tesque, Inc.). After that, 32.5 μL of the prepared DMSO solution of the peptide was mixed with 540 μL of distilled water for injection (manufactured by Otsuka Pharmaceutical Factory, Inc.). Next, 550 μL of the mixture was mixed with 700 μL of the Freund's incomplete adjuvant (Montanide ISA-51) using a glass syringe to prepare a water-in-oil emulsion. An HLA-A*0201-expressing transgenic mouse (strain name: HLA-A2+HLA-DR1+/Iaβ° β2m, EMMA ID number EM: 01783) was immunized by subcutaneous administration of 300 μL of the preparation (water-in-oil emulsion) into the base of the tail. The evaluation of each peptide was performed using 2 or 3 mice.
(3) Preparation of Splenic Cells
The spleen was isolated 7 days after immunization. The spleen was smashed by rubbing against the frothed part of a slide glass and then subjected to hemolysis treatment with ACK Lysing Buffer (manufactured by Lonza Co.) to prepare splenic cells. In this experiment, CTM (Complete T-cell Medium: RPMI-1640 medium (manufactured by Invitrogen Corporation) supplemented by 10% FBS, 10 mM HEPES, 20 mM L-glutamine, 1 mM sodium pyruvate, 1 mM MEM non-essential amino acid, 1% MEM vitamin and 55 μM 2-mercaptoethanol with the proviso that these concentrations were all final concentrations) was used as the medium for the splenic cells, and the cell suspension was prepared at the concentration of 5×106 cells/mL.
(4) Elispot Method
Whether the administered peptide has the activity of inducing WT1-specific immune cells was examined by the ELISPOT method using IFNγ as an index. The method was performed according to the attached manual. After the CTM was added in a volume of 50 μL/well into plates for ELISPOT (manufactured by BD Japan, catalog No. 551083), the splenic cell suspension was plated therein in a volume of 100 μL (5×105 cells/well). Further, the administered peptide or the wild-type peptide was added thereto in a volume of 50 μL/well (peptide final concentration: 2 μg/mL). This assay method is known as one of the substitute methods that enable prediction of cytotoxic activity (J. Immunological Methods, 1995, 181, 45-54).
<Results>
The evaluation results of the activity of inducing specific cell-mediated immunity are shown in FIGS. 19 to 22 and 28 to 29 for the modified WT1187 peptides, and in FIGS. 23 to 27 and 30 to 32 for the modified WT1126 peptides. In each of FIGS. 19 to 32, the vertical axis represents the number of antigen peptide-specific responsive cells in 5×105 splenic cells (spots/5×105 cells), and the horizontal axis represents the individual mouse (2 or 3 mice) used for evaluation. The white bar represents the number of the specific immune cells responded under no stimulation with antigen peptides. The gray bar represents the number of the specific immune cells responded to stimulation with the wild-type peptide. The black bar represents the number of the specific immune cells responded to stimulation with the administered (modified) peptide.
FIGS. 19 to 32 show the respective activities of inducing specific cell-mediated immunity regarding the following peptides.
FIG. 19 a: WT1187P1A peptide, b: WT1187P1F peptide, c: WT1187P1G peptide, d: WT1187P1I peptide, e: WT1187P1L peptide, f: WT1187P1M peptide.
FIG. 20 a: WT1187P1V peptide, b: WT1187P1W peptide, c: WT1187P2I peptide, d: WT1187P2M peptide, e: WT1187P2Q peptide, f: WT1187P2V peptide.
FIG. 21 a: WT1187P3A peptide, b: WT1187P3F peptide, c: WT1187P3I peptide, d: WT1187P3L peptide, e: WT1187P3M peptide, f: WT1187 P3P peptide.
FIG. 22 a: WT1187P3S peptide, b: WT1187P3V peptide, c: WT1187P3W peptide, d: WT1187P3Y peptide, e: WT1187P9L peptide.
FIG. 23 a: WT1126P1A peptide, b: WT1126P1F peptide, c: WT1126P1G peptide, d: WT1126P1I peptide, e: WT1126P1L peptide, f: WT1126P1M peptide.
FIG. 24 a: WT1126P1V peptide, b: WT1126P1W peptide, c: WT1126P2A peptide, d: WT1126P2I peptide, e: WT1126P2L peptide, f: WT1126P2Q peptide.
FIG. 25 a: WT1126P2V peptide, b: WT1126P3A peptide, c: WT1126P3G peptide, d: WT1126P3I peptide, e: WT1126P3L peptide, f: WT1126P3M peptide.
FIG. 26 a: WT1126P3P peptide, b: WT1126P3V peptide, c: WT1126P3W peptide, d: WT1126P9A peptide, e: WT1126P9I peptide, fWT1126P9M peptide.
FIG. 27: WT1126P9V peptide.
FIG. 28 a: WT1187P1D peptide, b: WT1187P1E peptide, c: WT1187P1H peptide, d: WT1187P1K peptide.
FIG. 29 a: WT1187P1N peptide, b: WT1187P1P peptide, c: WT1187P1Q peptide, d: WT1187P1R peptide, e: WT1187P1T peptide.
FIG. 30 a: WT1126P1D peptide, b: WT1126P1E peptide, c: WT1126P1H peptide, d: WT1126P1K peptide, e: WT1126P1N peptide, f: WT1126P1P peptide.
FIG. 31 a: WT1126P1Q peptide, b: WT1126P1S peptide, c: WT1126P1T peptide, d: WT1126P2I&P91 peptide, e: WT1126P2I&P9V peptide, f: WT1126P2L&P9I peptide.
FIG. 32: WT1126P2L&P9V peptide.
As shown in these results, when the modified peptides except the WT1187P1D peptide, the WT1187P1E peptide, the WT1187P1H peptide, the WT1187P1P peptide and the WT1187P2Q peptide; the WT1126P1D peptide, the WT1126P1E peptide, the WT1126P1P peptide, the WT1126P2A peptide and the WT1126P2Q peptide were administered into the mice, specific immune cells were remarkably induced in an efficient manner.
Next, the specific immune cells induced by stimulation of the modified peptide were analyzed for the cross reactivity to the wild-type peptide (Tables 24 to 25). The results show that particularly the WT1187P1A peptide, the WT1187P1I peptide, the WT1187P1L peptide, the WT1187P1M peptide, the WT1187P1N peptide, the WT1187P1Q peptide, the WT1187P1T peptide, the WT1187P1V peptide, the WT1187P2V peptide, the WT1187P2M peptide, the WT1187P2I peptide, the WT1187P3A peptide, the WT1187P3F peptide, the WT1187 P3P peptide, the WT1187P3S peptide, the WT1187P3V peptide and the WT1187P9L peptide among the WT1187 modified peptides; and the WT1126P1S peptide, the WT1126P2I peptide, the WT1126P2L peptide, the WT1126P2V peptide, the WT1126P3W peptide, the WT1126P9I peptide, the WT1126P9M peptide and the WT1126P9V peptide among the WT1126 modified peptides, can induce specific immune cells that can efficiently recognize both of the modified peptide and the wild-type peptide.








TABLE 24







Cross
Modified WT1187 peptide











reactivity
modified at
modified at
modified at
modified at


(%)
position 1
position 2
position 3
position 9





80-100
WT1187P1A
WT1187P2V
WT1187P3A
WT1187P9L



WT1187P1N
WT1187P2M
WT1187P3P



WT1187P1Q
WT1187P2I
WT1187P3S



WT1187P1T



WT1187P1V


60-80
WT1187P1I

WT1187P3F



WT1187P1L

WT1187P3V



WT1187P1M


40-60
WT1187P1R

WT1187P3Y


20-40
WT1187P1F

WT1187P3L



WT1187P1W


 0-20
WT1187P1G

WT1187P3I



WT1187P1K

WT1187P3M





WT1187P3W


ND*
WT1187P1D
WT1187P2Q



WT1187P1E



WT1187P1H



WT1187P1P


















TABLE 25









Modified WT1126 peptide












Cross
modified
modified
modified
modified
modified at


reactivity
at
at
at
at
positions


(%)
position 1
position 2
position 3
position 9
2&9





80-100

WT1126P2L
WT1126P3W
WT1126P9M





WT1126P2V

WT1126P9I


60-80
WT1126P1S
WT1126P2I

WT1126P9V


40-60
WT1126P1A



WT1126P1H



WT1126P1K



WT1126P1M



WT1126P1N


20-40
WT1126P1G


WT1126P9A



WT1126P1I



WT1126P1Q



WT1126P1W


 0-20
WT1126P1F

WT1126P3A

WT1126P2I&P9I



WT1126P1L

WT1126P3G

WT1126P2I&P9V



WT1126P1T

WT1126P3I

WT1126P2L&P9I



WT1126P1V

WT1126P3L

WT1126P2L&P9V





WT1126P3M





WT1126P3P





WT1126P3V


ND*
WT1126P1D
WT1126P2Q



WT1126P1E
WT1126P2A



WT1126P1P





ND*: unmeasurable due to no activity shown






INDUSTRIAL APPLICABILITY
The cancer vaccine composition of the present invention is useful as a medicament used for treatment and prevention of WT1-expressing cancers in HLA-A*0206-positive persons. The cancer vaccine composition of the present invention is also useful as a medicament used for treatment and prevention of WT1-expressing cancers in HLA-A*0201-positive persons.

## Claims

1. A method of cancer treatment, comprising administering to an HLA-A*0201-positive person a composition comprising the following peptide:
a modified peptide of the WT1187 peptide: Ser Leu Gly Glu Gln Gln Tyr Ser Val (SEQ ID NO: 2) or the WT1126 peptide: Arg Met Phe Pro Asn Ala Pro Tyr Leu (SEQ ID NO: 3), wherein the modified peptide is a peptide comprising the amino acid residues at position 4 to 8 from the N terminus of the WT1187 or WT1126 peptide and is immunogenic in HLA-A*0201-positive persons.

2. The method of claim 1, wherein the peptide is a modified peptide of WT1187 peptide.

3. The method of claim 1, wherein the peptide is a modified peptide of WT1126 peptide.

4. The method of claim 1, wherein the peptide is SEQ ID NO: 11, 16, 20, 34, 39, 46 or 49.

5. A method of cancer treatment, comprising administering an HLA-A*0201-positive person a composition comprising:
a modified peptide of the WT1187 peptide: Ser Leu Gly Glu Gln Gln Tyr Ser Val (SEQ ID NO: 2) or the WT1126 peptide: Arg Met Phe Pro Asn Ala Pro Tyr Leu (SEQ ID NO: 3), either of which is a partial peptide of a protein which is a gene product of the tumor suppressor gene WT1, the modified peptide being immunogenic in HLA-A*0201-positive persons.